{"paper_id":"0ee34ccd-d44f-4e39-a33e-6f5e7e277b10","body_text":"Distinct subnetworks of the mouse anterior thalamic nuclei | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Distinct subnetworks of the mouse anterior thalamic nuclei Houri Hintiryan, Mitchell Rudd, Sumit Nanda, Adriana Gutierrez, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4356188/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Despite significant advancements in identifying cell types in the mouse cerebral cortex, the classification of neuron types in the mouse thalamus remains largely incomplete. Specifically, the anterior thalamic nuclei (ATN), an integral component of the Papez circuit, encompass the anterodorsal (AD), anteroventral (AV), and anteromedial (AM) thalamic nuclei. Structurally, the ATN serve as a hub to facilitate communication among the neocortex, hippocampus, amygdala, and hypothalamus. Functionally, they play pivotal roles in regulating learning, memory, spatial navigation, and goal-directed behaviors. Thus, the ATN provide a promising avenue to investigate the relationship between structural and functional complexity with neuron type diversity. Our comprehensive and systematically collected macroscale pathway tracing data revealed several connectionally unique cell populations within the AM, AV, and AD that suggest several disparate parallel subnetworks run through each nucleus. Further, we applied genetic sparse labeling, brain clearing, 3D microscopic imaging, and computational informatics to catalog neuron types across the ATN, ascertained their brain-wide connectivity profile at the single neuron and synaptic resolutions, and characterized their morphological features. This study provides insights into how the prefrontal cortex, hippocampus, and amygdala interact through neuron type-specific ATN subnetworks to coordinate and synchronize both cognitive and emotional aspects of goal-directed behavior, resolving longstanding controversies surrounding the validity of the Papez circuit and its structural and functional roles. Biological sciences/Neuroscience/Neural circuits Health sciences/Anatomy/Nervous system/Brain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Background The classic Papez circuit, comprising the hippocampal formation, mammillary bodies, anterior thalamic nuclei, cingulate cortex, and parahippocampal cortices, was proposed as a foundation for emotional expression 1 . Building upon this, the concept of the limbic system or visceral brain was introduced, which incorporated additional structures like the amygdala, hypothalamus, and septum. Since then, this model has been revised to underscore the cognitive roles of these structures in episodic memory, attention, and spatial processes 2 , 3 . Within this structural framework, the anterior thalamic nuclei (ATN) serve as a multifunctional hub, supporting diverse aspects of cognition in both animal models and humans 2 . The ATN consists of the anterodorsal (AD), anteroventral (AV), and anteromedial (AM) thalamic nuclei, the latter of which is further divided into dorsal (AMd) and ventral (AMv) compartments 4 . Together, the ATN support functions of memory and spatial navigation 5 . Pathology of the entire ATN culminates in diencephalic amnesia 6 , 7 , inactivation of the nuclei manifest in perturbations of spatial functions 8 – 10 , and elevated ATN activity can rescue spatial memory loss sustained following mammillothalamic tract insult 11 . Increasing evidence also suggests the ATN's contribution to age-related cognitive changes 12 . Further, many hippocampal-dependent memory processes are shown to require a functional ATN emphasizing the role of the ATN in the hippocampal-diencephalic-cingulate network 9 , 13 , 14 . Based on their unique connections, three parallel hippocampal ATN streams have been suggested 15 . Generally, the AD is implicated in head directionality because it harbors the largest number of head direction cells within the thalamus 16 – 19 and is nestled in a network whose components exhibit head direction specificity like the lateral mammillary nucleus (LM), dorsal tegmental nucleus (DTN), and the postsubiculum (POST) (DTN→LM→AD↔POST) 17 , 20 . The AV exhibits theta rhythm oscillations and is interconnected with structures also implicated in theta rhythmicity imperative for spatial and non-spatial mnemonic functions 21 – 23 . These structures include the medial mammillary nucleus (lateral part) (MMl), ventral tegmental nucleus (VTN), and the subiculum (SUB) (VTN→MMl→AV↔SUB) 20 . More recent studies in mice have shown that through their connections with the retrosplenial cortical area (RSP), the AD and AV are involved in contextual memory and memory specificity, respectively 24 . Importantly, the AV is implicated in age related decline in working memory that can be ameliorated through AV activation 25 . Alternatively, the AM has been proposed to serve as a transmitter of integrated hippocampal-diencephalic information to the cortex for higher order processing 15 , 26 and in goal-directed behaviors 27 given its unique connections with medial prefrontal cortical areas like the prelimbic (PL) and infralimbic (ILA) areas 28 . The AM in fact regulates emotional learning since its manipulation affects contextual fear responses 29 – 31 , suggesting more than just a relay role of the ATN nuclei 32 . Together, these studies stress the importance of attaining an intricate and complete connectivity roadmap of the ATN to reveal more granular, potentially functionally disparate, subnetworks. Although studies in rats have shown some segregation within the AM 33 , 34 and AV 35 , a systematic and extensive examination of ATN connectivity that can reveal the exceedingly granular subnetworks to account for ATN functional diversity is lacking. To achieve this, we traced approximately 200 sets of cortical, thalamic, hippocampal, amygdalar, striatal, midbrain, and hindbrain pathways in mice, constructing a comprehensive ATN wiring diagram. We identified connectionally unique cell types within the ATN, associated with seven parallel subnetworks within the AMd, four within the AV, and two within the AD, providing a structural basis for understanding the ATN’s functional role as a network hub among the neocortex, hippocampus, amygdala, and hypothalamus. Combining genetic sparse labeling, brain clearing, and 3D microscopic imaging with advanced computational informatics, we systematically reconstructed and cataloged ATN neuron types based on their detailed neuronal morphology. To our knowledge, single-cell morphological assessments have not been systematically conducted for the ATN in any species. Finally, we validated synaptic connections of ATN neurons that facilitate communication among the MPF, SUBv, and BLAa. Results Characterizing ATN domain connectivity To assess ATN connectivity, anterograde (Phal, AAV) and retrograde (CTB, FG, AAVretro-Cre) pathway tracers were placed across different cortical, hippocampal, amygdalar, thalamic, striatal, midbrain, and hindbrain areas (Table 1; see Methods for details regarding each of these approaches and Discussion section, Caveats to tracing experiments , regarding unique tracing and neurotropism characteristics of the tracers used). To determine the structure and approximate boundaries of domains within the AD, AV, and AM, representative experimental cases with distinct labeling in each domain were selected and sections across the ATN were annotated and analyzed. The sections were processed through our proprietary software Connection Lens 36 – 39 . Individual sections were matched and warped to their corresponding atlas level of the Allen Reference Atlas (ARA 4 ) and subsequently the tracer labels were segmented (Fig. 1 a-b). Grid-based overlap annotation was performed in which the AD, AV, and AM were divided into 105 x 105-pixel square grids (equivalent to 63 µm 2 ) to tabulate labeling within distinct domains of the ATN nuclei (Fig. 1 b). The case specific annotations were then aggregated into a single matrix and Louvain community detection was conducted (Fig. 1 c). Grids were color-coded according to community assignment and reordered such that the resulting Louvain clusters were placed along the diagonal of the visualized matrix (Fig. 1 d). To validate domain-specific connections, Cre-dependent anterograde and TVA receptor mediated rabies tracing strategies were applied utilizing connectionally guided delivery of Cre. For these cases, sections spanning the whole brain were registered (warped) and tracer labels were segmented and annotated based on ROI. The annotated data underwent normalization and 2D hierarchical clustering to ascertain and visualize the distinct whole-brain projection patterns resulting from neurons traced in different ATN domains. A total of ~ 200 injections were used to generate connectivity diagrams and a global wiring diagram of the ATN (Fig. 1 e; https://ucla-brain.github.io/atn/ ). Reported connections underwent validation through at least one of the following methods. Injections targeting different regions were duplicated, and consistency across label patterns was manually evaluated. The Cre-dependent anterograde and TVA receptor mediated rabies also validated the connections. Further, retrograde tracers were introduced into regions displaying anterograde terminal labeling to confirm anterograde connections, whereas anterograde tracers were administered into the sites of retrogradely-labeled projection cells to validate retrograde injection data. Single neuron tracing experiments provided additional validation of select connections. General ATN connections with the neocortex and subicular complex The AD, AV, and AM each are reported to have select connections with the cortex and hippocampus 40 . Multiple tracer injections placed within a single brain clearly demonstrate these segregated connections (Fig. 1 f). Reciprocal communication between the medial prefrontal cortex (MPF) and ATN are specifically through the AM, while projections from ATN to the post- (POST), pre- (PRE), and para (PAR) subiculum are exclusively through the AD and AV, input from SUBd is exclusively to AV, and output to the striatum is solely through the interanterodorsal thalamic nucleus (IAD; Fig. 1 f). See Table 2 for a list of structure abbreviations. Regarding laminar specific connections, typically, ATN thalamocortical neurons are reported to show projections to cortical layers I, IV, and V and to all subicular layers 34 , 41 . In turn, layer 6 corticothalamic neurons and deeper subiculo-thalamic neurons 42 , 43 project back to ATN. Our data also show this canonical corticothalamic and thalamocortical patterns of labeling (Fig. 1 g). An anterograde and retrograde tracer cocktail placed in the AM show layer I, IV, and V anterograde tracer labeling in the MPF, while layer VI neurons in the same regions are retrogradely labeled (Fig. 1 g). A similar injection in the AV shows similar laminar specific connections with the ventral retrosplenial cortical area (RSPv). Our data also show that projections from ATN to POST, PRE, and PAR spread across all 3 layers, while ATN projecting neurons in those same regions are in deep layer 3 (Fig. 1 g). AD subnetworks Within the AD, we identified the AD.medial (ADm) and AD.lateral (ADl) domains (Fig. 2 a). The AD projects to the dorsal (RSPd) and ventral (RSPv) retrosplenial cortical areas and to the POST, PRE, and PAR (Extended data Fig. 1 a). AD neurons projecting to POST, PRE, and PAR are located primarily in the AD.medial (AD.medial→PRE/PAR/POST), while those projecting to the RSPd/v predominate the AD.lateral (AD.lateral→RSPd/v) (Fig. 2 b-c; Extended Data Fig. 1 b-d). The clear segregation of the AD.medial and AD.lateral neuronal populations is evident in a case in which retrograde tracers were placed in the RSPv and the PRE in the same animal (Fig. 2 d). While AD.lateral connections with RSP are reciprocal (AD.lateral↔RSPd/v) (Fig. 2 c; Extended Data Fig. 1 b), AD.medial→POST/PRE/PAR connections are unidirectional (Fig. 2 e). This is clearly demonstrated with an anterograde and retrograde tracer co-injection in the PRE which results in retrogradely labeled neurons, but not anterogradely labeled axonal terminals, in the AD.medial (Fig. 2 f; Extended Data Fig. 1 c). No connection between the AD and SUB proper were detected (Fig. 2 g-h). Instead, those subicular connections were with distinct AV regions (Fig. 2 g; see next section for details). This data updates the canonical AD circuits that connect with the RSP and subiculum (Fig. 2 i). AV subnetworks Four domains with distinct connectional properties were identified within the AV. These were the AV.dorsal, AV.lateral, AV.medial, and AV.medial tip (Fig. 3 a). The AV shares strong reciprocal connections with the POST, PRE, and PAR (Fig. 3 b1, 3c1; Extended Data Fig. 2 a). Our data show that these connections are specifically with the AV.dorsal (AV.dorsal↔POST/PRE/PAR) since anterograde and retrograde tracers in the POST, PRE, and PAR label terminals and neurons only this domain (Fig. 3 b2, 3c2; Extended Data Fig. 2 b-d). Note the labeling in the mammillary bodies and the lateral dorsal thalamic nucleus supporting the precise location of the ATN and subicular injections (Fig. 3 b). The AV is also reciprocally connected with the RSPd, RSPagl, and RSPv (Extended Data Fig. 2 e). The connections with the RSPd and RSPagl are through the AV.dorsal domain (AV.dorsal↔RSPd/agl; Fig. 3 d), while those with RSPv are through the AV.medial domain (AV.medial↔RSPv; Fig. 3 e; Extended Data Fig. 2 f). These domain-specific connections of the AV.dorsal and AV.medial are clearly demonstrated in several ways. First, retrograde tracers placed in the PRE and RSPv in the same animal show segregated AV→PRE neuron population in the AV.dorsal and the AV→RSPv neuron types in the AV.medial (Fig. 3f1). Second, multiple tracer injections also demonstrate the approximate border between the AV.medial and its dorsally adjacent AV.dorsal, and ventrally adjacent AV.medial tip, domains (Fig. 3f2). Third, when retrograde tracer injections were made primarily in the AV.dorsal or AV.medial, labeled neurons in the POST/PRE/PAR and RSPd/agl were observed when the CTB injection was in the AV.dorsal, but not the AV.medial (Fig. 3 g). A different AV domain, the AV.medial tip, receives input from the SUBv and deeper layers of RSPv (SUBv/RSPv_6→AV.medial tip; Fig. 3 h-i), which is validated with a retrograde injection that also involves the AV.medial tip (Fig. 3 g). Finally, the AV.lateral is the domain where inputs from the SUBd predominantly target (SUBd→AV.lateral; Fig. 3 j; Extended Data Fig. 2 g). Double anterograde injections in the SUBd and SUBv in the same animal validate these connections and show the distinction between the AV.medial tip and AV.lateral (Fig. 3 k; Extended Data Fig. 2 h-i). AV neurons that project back to the SUB were difficult to reveal given the sparse AV→SUB projections to a very specific region of the caudal SUB (Fig. 3l1). Many retrograde tracer injections made across the SUB did not label neurons in the AV. Only one in the caudal SUBv labeled neurons in the AV.lateral, highlighting the specific AV→SUB connection (Fig. 3l2). All the connections of the AV domains are summarized in a wiring diagram (Fig. 3 m) and the unique connections of the ATN with the SUB are presented (Fig. 3 n). AM subnetworks Anterograde and retrograde tracers placed across different cortical, amygdalar, and hippocampal regions revealed segregated neuron populations within the AM and the connectional distinctions between the AMd and AMv (Fig. 4 a). Computational analysis of the AMd connectivity data revealed 5 domains within the AMd (Fig. 4 b-c), namely the medial (AMd.m), dorsomedial (AMd.dm), dorsal (AMd.d), dorsolateral (AMd.dl), and lateral (AMd.l). The AM connects strongly with the dorsal (ACAd) and ventral (ACAv) anterior cingulate cortices (Fig. 4 d). The AMd.dorsomedial domain (AMd.dm) contains neurons that project to the rostral parts of the ACAd and ACAv (AMd.dorsomedial→rostral ACAd/ACAv) (Fig. 4 e; Extended Data Fig. 3 a-b 1 ). The AMd.dorsomedial also receives input from the rostral ACAd/ACAv, but this rostral ACAv/ACAd→AMd.dorsomedial connection is far weaker than the converse pathway (Fig. 4 e). These AMd.dorsomedial connections were validated with repeated rostral ACA injections (Extended Data Fig. 3b1). Note that the AMv also shares reciprocal connections with the rostral ACAd/ACAv (AMv↔rostral ACAd/ACAv) (Fig. 4 e; Extended Data Fig. 3 b-c). The AMd.dorsal (AMd.d) shares the strongest connections with the caudal ACAd/ACAv (AMd.dorsal↔caudal ACAd/ACAv) (Fig. 4 f). These connections were validated with repeated caudal ACA injections (Extended Data Fig. 3b2), but also through a Cre-dependent anterograde AAV injection that traced the output of mostly AMd.dorsal neurons (Fig. 4 g-h) that showed labeled terminals in the caudal ACA, but none in the rostral ACA. No connections were detected between the caudal ACAd/ACAv and the AMv (Fig. 4 f; Extended Data Fig. 3b2). Multiple tracer injections made in the same animal highlight the distinct AMd.dorsomedial and AMd.dorsal domains (Fig. 4 i ; see Fig. 4 j for a summary of AMd.dorsomedial and AMd.dorsal connections). Further, connections with the ACA in general are unique to the AM and no connection between the ACA and the AV or AD were identified (Fig. 4 k-l; Extended Data Fig. 3 c). Some connections from the AMd to the RSP were detected (Fig. 5 a) and these connections were specifically through the AMd.dorsolateral domain (AMd.dl), which houses the neurons that project to the RSPv (AMd.dorsolateral→RSPv) (Fig. 5 b). This AMd.dorsolateral→RSPv connection was validated with repeated RSP injections (Extended Data Fig. 3 d) and through a Cre-dependent anterograde injection that traced the output of primarily AMd.dorsolateral neurons (Fig. 5 c-d). No projections back to this domain, nor to any other AMd domain, from the RSPv were identified (Fig. 5 e). Similarly, no connections between the AMv and RSP were detected (Extended Data Fig. 3 d). The AMd is bidirectionally connected with the posterior parietal cortex (PTLp; Fig. 5 f). The AMd.lateral domain (AMd.l) is shown to project to and receive input from the PTLp, specifically the caudal medial part (AMd.lateral↔PTLp caudal medial) (Fig. 5 g; see Extended Data Fig. 3 e for repeated PTLp injections). A Cre-dependent AAV anterograde injection that primarily traced the neurons of AMd.lateral neurons shows projections to the PTLp (caudal, medial), while a similar injection in the opposite side of the AMd shows no projections to the PTLp (Fig. 5 h-i), validating this connectional specificity. The PTLp is not connected with AMv, the AD, nor AV (Fig. 5 j). The AMd.lateral also receives input from the SUBv (AMd.lateral←SUBv) (Fig. 5 k-l). Neurons in the AMd that project back to the SUB were difficult to find given the sparse AM→SUB projections and given that these projections are to a very specific region within the caudal SUBv (Extended Data Fig. 4 a). A CTB retrograde tracer injection in this caudal SUBv region labeled neurons predominantly in the AMd.lateral (Extended Data Fig. 4 b). Multiple tracer injections in the same brain highlight the AMd.lateral and AMd.dorsolateral domains relative to other AMd domains (Fig. 5 m). The fifth AM domain identified is the AMd.medial (Fig. 6 a). Although undetected through the modularity maximization algorithm, there were two distinct zones within the AMd.medial domain: (1) the AMd.medial tip (AMd.mt) and (2) AMd.ventromedial (AMd.vm) (Fig. 6 a). The AM shares strong connections with the MPF, SUBv, and amygdala as shown by anterograde and retrograde AM injections (Fig. 6 b, d), which all occur either through the AMd.mt, AMd.vm, or the AMv. The AD and AV are not connected with the MPF nor the amygdala (Fig. 1 f). The AMd.medial tip receives inputs from the SUBv and some light input from PL and ILA (SUBv→AMd.medial tip; PL/ILA→AMd.medial tip), but strongly projects to the PL (AMd.medial tip→PL) and to the anterior basolateral amygdala (AMd.medial tip→BLAa) (Fig. 6 b-e). These AMd.mt outputs were validated with a Cre-dependent anterograde AAV injection that primarily traced neurons in this domain (Fig. 6 f), while their inputs were validated through TVA receptor mediated rabies tracing (Extended Data Fig. 4 f; see also Extended Data Fig. 4 c-e for repeated injections). The AMd.ventromedial domain also receives light input from PL and ILA (none from the SUB) (PL/ILA→AMd.ventromedial) (Fig. 6 c), has reciprocal connections with the medial (ORBm) and ventrolateral (ORBvl) ORB (AMd.ventromedial↔ORBm/vl) (Fig. 6 g-h), and strongly projects back to ILA (AMd.ventromedial→ILA) (Fig. 6 e; Extended Data Fig. 4 e). Multiple injections made into the ILA and PL illustrate the distinct AMd.mt and AMd.vm neuronal populations (Fig. 6 i; Extended Data Fig. 4 d-e) and quantification of the Cre-dependent tracing from each of these populations validates the stronger AMd.mt→PL and AMd.vm→ILA connections (Fig. 6 j). The strongest input from the PL, ILA, and ORB to the AM are to the AMv (PL/ILA/ORB→AMv) (Fig. 6 c; Extended Data Fig. 4 d-e) and the AMv has strong projections back to the PL (AMv→PL) (Fig. 6 e; Extended Data Fig. 4 d-e). These output and input AMv connections were also validated with Cre-dependent AAV and TVA receptor mediated rabies tracing (Extended Data Fig. 4 g-i). Quantification of this data clearly shows the distinct AMv connections (Extended Data Fig. 4 h, j). Schematic summaries of these connections are provided (Fig. 6 k; Extended Data Fig. 4 k). A final AMd domain not identified through our computational analysis, but that was apparent from the tracing data, was the AMd.core. All domains discussed thus far predominate the peripheral region of the AMd, generally leaving its core region unoccupied by label (Fig. 4 a-c). This peripheral-core organization is most apparent with multiple tracer injections made in the same brain (Fig. 7 a). The AMd.core shows reciprocal connections with the secondary motor cortex (MOs), specifically with the frontal eye field region ( AMd.core↔MOs.fef ) (ACAd adjacent 44 ) (Fig. 7 b-d). Cre-dependent AAV tracing of these AMd.c neuron outputs confirm strong AMd.core→MOs-fef connections (Fig. 7 e). The AMd.core also houses neurons that project to the lateral entorhinal cortex (AMd.core→ENTl) (Fig. 7 f-h). The AMd.core→MOs.fef and ENTl connections are unique to the AMd, and the AMd.core→ENTl projections are to a very specific region of the ENTl layer V (Fig. 7 f-g). No connections with the medial entorhinal cortex (ENTm) were detected (Fig. 7 f-g). Exclusive projections from the AMd.core were also observed to the deep layers (V/VI) of the caudal ectorhinal and perirhinal cortical areas (AMd.core→ECT/PERI caudal) (Fig. 7 f-g, i). The data from the domain-specific Cre-dependent anterograde tracing from the AMd.dorsal/dorsolateral, AMd.ventromedial, AMd.medial tip, AMd.core, and AMv were grouped and subjected to a 2D hierarchical clustering algorithm to show the specificity of connections between these domains with the (1) MOs, (2) ORB, (3) PL, (4) ILA, (5) RSP, and (6) PTLp (Fig. 7 l). The output showed that neurons that project most strongly to the PL are in the AMd.medial tip, while those that project most strongly to the ILA are in the AMd.ventromedial domain validating those connections. The data nicely show the projections from the AMv to the PL, ILA, ORBm, ORBvl and show no projections from AMv to the ORBl. The data also show the projections from the AMd.core to the MOs and from the AMd.dorsolateral to the RSPv (Fig. 7 l). Similar patterns emerge when data from injections administered in the broad AMd medial area compared to those in the AMd lateral area are analyzed (Fig. 7 j), showing a clear distinction between the AMd ventral-medial domains and dorsal-lateral domains, with the medial domains connected more with limbic network structures and the lateral domains with visual processing network areas (Fig. 7 k). Finally, axon terminations of singly traced neurons in the AMd.medial, AMd.dorsal/dorsolateral, and AMd.core also support the domain-specific results (Fig. 7 m). A singly traced neuron from the AMd.medial showed greater axonal terminals in PL and ILA than neurons located more dorsal/dorsolaterally in the AMd. On the other hand, a neuron located more dorsal/dorsolaterally in the AMd showed more axonal terminations in the RSPv compared to the neurons located more medially and in the core region. Finally, only the neuron located more in the AMd.core showed axon terminations in the MOs compared to the neurons located more in the medial and dorsal/dorsolateral parts of the AMd (Fig. 7 m). AMd.medial and AMv serve as subnetwork hubs for subiculum and medial prefrontal cortical areas As reported above, the AM domains connect with a specific set of cortical structures, including the SUBv and BLAa, suggesting a critical role of these AM domains as network hubs for integrating and transmitting information among the cortex, hippocampus, and amygdala. For example, the AMd.medial tip domain receives input from the SUBv, and in turn, projects to the PL and BLAa. This suggests that the AMd.medial tip domain regulates communication between (1) the SUBv and the BLAa via a SUBv→AMd.medial tip→BLAa and (2) the SUBv and PL through a SUBv→AMd.medial tip→PL circuit. We utilized an AAV1-Cre based transsynaptic circuit mapping method 45 combined with a genetic sparse labeling reporter line, MORF3 46 to validate these disynaptic circuits. In MORF3 mice, neurons stochastically and Cre-dependently express a membrane-targeted V5 spaghetti monster protein, which can be visualized via immunostaining. This method reveals the complete detailed neuronal morphology of MORF3-labeled neurons including dendritic arborizations and spines. Therefore, in MORF3 mice, an AAV viral tracer expressing synaptophysin tagged with RFP was injected into the SUBv and an AAVretro-Cre injection was made into either the BLAa or the PL (Fig. 8 a, h). In this strategy, Cre is retrogradely transported from injection sites to the AMd-medial tip to trigger MORF3 expression, consequently revealing the detailed dendritic morphology of the AMd-medial tip neurons in their entirety, including dendritic spines. Meanwhile, the synaptophysin-tagged AAV labels the synaptic terminals of fibers originating in the SUBv and terminating in the AMd.medial tip (Fig. 8 b, i-k). As such, this strategy reveals SUBv fibers potentially synapsing onto BLAa- or PL-projecting AMd.medial tip neurons. In both cases, putative contacts can be clearly seen from the SUBv onto PL- and BLAa-projecting neurons in the AMd.medial tip via the close apposition of terminals and dendrites (Fig. 8 e, l). Synapse reconstructions further validated some of the potential contacts, substantiating the disynaptic circuits (Fig. 8 f-g, m). The same strategy was applied to illustrate the disynaptic ILA→AMv→PL circuit (Fig. 8 n-o). Altogether, these data suggest that these AM domains serve as network hubs to bridge communication among the MPF, ventral hippocampus, and amygdala. ATN connections with the mammillary body and brainstem structures The ATN receive inputs from the mammillary nuclei in a topographically arranged manner: the lateral mammillary nucleus (LM) projects to the AD, the lateral part of the medial mammillary nucleus (MMl) to the AV, and the medial part of the MM (MMm) to the AMd and AMv (Fig. 9 a-b; Extended Data Fig. 5 a). Notably, the MMme targets specifically the IAD, a ventral extension of the AD situated dorsal to the AMd (Fig. 9a1). Projections from the LM and MMme to the AD and IAD are bilateral, whereas those from the MMm to the AV or AM are ipsilateral (Fig. 8 c-d). Projections from the mammillary nuclei to the ATN appear non-specific with respect to the newly identified domains. We applied Cre-dependent TVA receptor-mediated rabies tracing, which validated these connections and demonstrated the AMd- and AMv bi-synaptic connections (Fig. 9 e). Specifically, a large AAVretro-Cre injection was made into the MPF that retrogradely delivered Cre to the AM. The AMd or AMv was then injected with a Cre-dependent TVA helper and EnVA-pseudotyped rabies viruses to trace their monosynaptic inputs (Fig. 9 e). Dense rabies labeling is shown in the MMm, confirming a bi-synaptic MMm→AMd/AMv→MPF pathway (Fig. 9 e). It is well established that the AD is embedded within a network processing head directionality, including the dorsal tegmental nucleus (DTN) pathway: DTN→LM→AD↔POST/PRE/PAR. In contrast, the AV is part of a network involved in theta rhythmicity and the ventral tegmental nucleus (VTN): VTN→MMl→AV↔SUB. Our anterograde tracer injections in the DTN and VTN confirmed their established connections with the LM (DTN→LM) and MM (VTN→MMl), while also revealing unexpected direct connections from the VTN and DTN to the AD/AV (Fig. 8 f-g), which were validated through retrograde tracing (Fig. 8 h-i). These findings summarized in Fig. 8 j, provide novel insights into a potential crosstalk between two relatively segregated subnetworks: one involved in head directionality and visual-spatial processing (peach), and the other associated with theta rhythmicity (blue) (Fig. 8 k). We also identified a less-documented direct projection from the dorsal raphe nucleus (DR) to the AD and AV (DR→AD/AV), providing serotonergic input to the ATN (Extended Data Fig. 5 b-c). Combining CTB AD/AV retrograde tracing and immunostaining, we confirmed that the majority of AD/AV projecting DR neurons co-localize with tryptophan hydroxylase 2 (TPH2), a protein marker of serotonigic neurons (Extended Data Fig. 5 d). The AV and AM, but not AD, also receive cholinergic inputs from the laterodorsal tegmental nucleus (LDT→AV/AM) (Extended Data Fig. 5 e-f). Lastly, our tracing revealed an unreported neural network involving the IAD. Anterograde tracer injections in the AMd occasionally labeled axon terminals in the dorsomedial caudoputamen (CP) (Extended Data Fig. 6 a-b). Retrograde tracer injections into the dorsomedial CP labeled neurons exclusively in the IAD, not the AM (Extended Data Fig. 6 a-c), indicating strong IAD→CP connections. This was further validated by Cre-dependent anterograde tracers injected into the AMd: one involving the IAD and one excluding it. Only the injection that traced IAD neurons labeled the dorsomedial CP (Extended Data Fig. 6 d-e). Projections from the IAD to the CP are to specific domains that integrate visual and spatial information 39 (Extended Data Fig. 6 f). Sparse connections also were observed between the superior colliculus and the IAD (SC→IAD) (Extended Data Fig. 6 g). As aforementioned, the IAD also receives inputs from MMme (Fig. 8a4). Together, these data suggest that the IAD transmits spatial (MM) and visual (SC) information to the dorsomedial CP (MMme/SC→IAD→CPi.dm/CPc.d), which also receives direct inputs from visual, spatial, entorhinal cortices implicated in coordinating eye, head and neck movement for goal directed behavior 39 (Extended Data Fig. 6 h). ATN dendritic morphology Dendrites are crucial in how neurons receive and integrate signals from other cells. The shape and structure of dendrites influence signal processing and determine connectivity patterns within neural circuits, while the scope of dendritic arbors defines the size of a neuron's receptive field. Dendritic morphology is also a key characteristic that distinguishes different neuronal types, although this information is limited for ATN. We integrated MORF3 genetic sparse labeling with brain clearing and three-dimensional microscopic imaging to obtain brain images through the ATN. These images showcased sparsely labeled excitatory ATN projection neurons with intricately detailed dendritic morphology (Fig. 10 a-d), which were then digitally reconstructed (Fig. 10 e). This approach enabled us to systematically characterize the morphological attributes of AD, AV, and AM domain neurons using computational methods (Fig. 10 e-h). Statistical comparisons of dendritic morphological features were carried out for a total of 109 neurons (10 for AD, 17 for AV, and 82 for AM). The results showed that AM dendritic arbors had greater dendritic length and higher number of branches than both AD (Wilcoxon signed rank test, p < 0.001 for both length and number of branches) and AV dendritic arbors (Wilcoxon signed rank test, p < 0.001 for length and p < 0.01 for number for branches) (Extended Data Fig. 7 a-b). While AD and AV neurons did not differ much in length, AD neurons had greater branching asymmetry (i.e., disproportionate distribution of dendritic tips between the two daughter branches at branch points) than both AM and AV neurons (Extended Data Fig. 7 c). While AD neurons had reduced average branch order (a measure of complexity, where the branch order of every compartment in a dendritic tree is averaged) compared to AV neurons (p < 0.05), AD neurons had increased height (p < 0.05) and increased maximum dendritic path length (p < 0.05) compared to neurons from AV (Extended Data Fig. 7 d-f). Due to this asymmetric dendritic extension, the AD neurons matched AM neurons in height and maximum path distance, even though AM neurons were much larger and more complex (Extended Data Fig. 7 e-f). The average Sholl intersection profile also showed that the AV neurons had a higher number of intersections closer to soma compared to the AD neurons which extended out to a greater path distance away from the soma, matching the AM neurons in extension while being much lower in the number of intersections per concentric circle drawn at 50 µm intervals (Extended Data Fig. 7 g). Comparative morphometrics suggest that AD neurons send out their dendritic arbor over specific directions in an asymmetric manner, whereas the AV neurons have a higher branch density closer to the cell-body and sample the dendritic field more uniformly. We also classified all neurons from ATN based on dendritic arbor size (total dendritic length) and complexity (total number of branches). All the ATN neurons could be distinguished into two morphological types using K-means clustering. Type 1 neurons (84 out of 109) included relatively smaller neurons with lower numbers of branches (average dendritic length 3645 ± 1428 µm, average number of branches: 51 ± 16) (Extended Data Fig. 7 h). Type 2 (25 neurons) included larger and more branched neurons (average dendritic length 8976 ± 1998 µm, average number of branches: 126 ± 27) (Extended Data Fig. 7 h). Previous studies have identified two types of thalamic projection neurons based on prominent morphological features such as size and complexity. The two morphological types are bushy and radiate neurons 47 – 50 . Comparing previous neuron types with the current study suggests that radiate neurons with reduced numbers of branches could be considered analogous to the Type 1 neurons. The bushy neurons, with greater length and more branches are like the Type 2 morphology. All Type 2 neurons belonged to the AM region, whereas Type 1 neurons were from all three ATN nuclei (AD, AM, and AV). Finally, we also observed the grape-like appendages on the projection neurons that have been reported in the literature 48 , 51 – 53 (Extended Data Fig. 7 i). Altogether, these data suggests that neurons within each of the ATN display distinct morphological features. Edge versus non-edge AMd neuron morphology We initially observed that neurons with somas lying predominantly on the edges of the AMd displayed a distinct morphological appearance (Fig. 10 i). The edge neurons displayed relatively less dendritic wiring and the dendrites seemed to have an orientation bias towards the center of the AMd (center-tropism). To quantify and test these qualitative observations, we first assigned each neuron as edge or non-edge based on their soma location within the AMd region. We then compared the quantified morphological features between the two groups. We observed that edge neurons are generally smaller (reduced dendritic length, t-test, p = 0.022) than the neurons with somas located towards the center (non-edge) of the AMd (Extended Data Fig. 7 j). To evaluate and quantify the orientation bias (towards the AMd center) of the dendritic arbors, we calculated two new features: (a) average angular deviation from the AMd center (Fig. 10 j-l) and (b) proportion of dendrites closer to the AMd center than the soma (Fig. 10 m). We measured each dendritic compartment’s angular deviation relative to the local AMd center vector (i.e., the vector connecting the compartment’s origin point to the AMd center, see Method for details) (Fig. 10 j). Averaging that value across all dendritic compartments gives us the average angular deviation of the whole neuron. The inverse of the average angular deviation gives us an estimate of the neuron's AMd bias toward the center (Fig. 10 k). In other words, the smaller the average angular deviation, the greater the orientation bias towards the AMd center. We observed that edge neurons had significantly lower average angular deviation compared to the non-edge neurons (t-test, p > 0.0001) (Fig. 10 l). We also measured, for each neuron, the proportion of its total dendritic wiring that is closer to the AMd center than the neuron’s soma (see Method for details). Similarly, as the branch orientation result suggested, the edge neurons had a higher proportion of dendritic wiring closer to the center than the soma compared to the non-edge neurons (t-test, p > 0.0001), demonstrating an AMd center bias (Fig. 10 m). Overall, we observed that the average angular deviation of neurons is inversely correlated with the distance from the AMd center (Pearson’s correlation coefficient=-0.65, p < 0.0001), i.e., neurons lying further away from the AMd center demonstrated greater tropism towards the AMd center (Fig. 10 k). AMd domain level morphological comparison Neurons from the five AMd domains (core, dorsal, dorsomedial, lateral and medial) also demonstrated distinct levels of center bias, both in terms of average angular deviation (omnibus Kruskal-Wallis test, p = 0.012) and proportion of dendrites closer to core than soma (omnibus Kruskal-Wallis test, p = 0.015) (Extended Data Fig. 7 k,l). Pairwise comparison between domains showed that AMd.core neurons demonstrated a higher average angular deviation (lower AMd center-bias) relative to their local AMd center vectors compared to AMd.dorsomedial and AMd.medial domains (FDR-corrected Wilcoxon signed rank test, p < 0.05) (Extended Data Fig. 7 k). Both the AMd.core and the AMd.dorsal neurons had a relatively lower proportion of dendrites closer to the center (lower AMd center-bias) compared to the AMd.medial neurons (FDR-corrected Wilcoxon signed rank test, p < 0.05; Extended Data Fig. 7 l). AMd domains also demonstrated distinct levels of average arbor height (omnibus Kruskal-Wallis test, p = 0.004) and maximum path length (omnibus Kruskal-Wallis test, p = 0.0004). AMd.dorsomedial neurons had reduced height, compared to core, lateral, and medial neurons. Medial neurons also showed greater height than dorsal neurons (FDR-corrected Wilcoxon signed rank test, p < 0.05; Extended Data Fig. 7 m). Both medial and lateral neurons had greater maximum dendritic path distance (path distance of the dendritic terminal that is furthest away from the soma) than both dorsal and dorsomedial neurons (FDR-corrected Wilcoxon signed rank test, p < 0.05) (Extended Data Fig. 7 n). The fact that medial and lateral domain neurons that showed greater bias toward the center (i.e., showed lower angular deviation and higher proportion of dendrites closer to the soma) also tended to have greater height and maximum path length, corroborated the initial qualitative observation that neurons lying away from the AMd center tend to look stretched. Like AM edge and non-edge neurons, AD and AV neurons whose somas were located at the edges of the nuclei qualitatively displayed a morphological appearance (i.e., stretched) that differed from their non-edge counterparts (Fig. 10 g-h). These distinct features were not quantified due to the low numbers of reconstructed neurons in each category for each nucleus. Discussion Summary of findings and overview of the ATN functional neural network The ATN are a key component of the classic Papez circuit (Fig. 11a), which has influenced our understanding of the neural basis of emotion and memory. Over time, the Papez circuit has evolved from a simple model of emotional processing to a more complex framework that integrates cognitive functions. Modern research has emphasized the critical roles of these structures in not only emotion, but also episodic memory, attention, and spatial processes (for review, see 2,3 ). Accordingly, we present an updated and expanded version of the Papez circuit that subserves cognition and emotion (Fig. 11a, c). Specifically, we show that distinct ATN domains act as network hubs, bridging communications between (1) the cortico-basal ganglia system, which controls spatial orientation, navigation, and goal-directed behaviors 44 , and (2) the classic limbic system (such as the SUBv, amygdala, MPF), which regulates neuroendocrine, autonomic and social behavior associated with emotion 54,55 . These interactions are mediated through several major pathways, as discussed below, and depicted in a wiring diagram in Fig. 11c. Both AD domains, the AD.medial and AD.lateral, receive direct inputs from the LM and DTN. While the AD.lateral shares reciprocal connections with the RSPd and RSPv, the AD.medial densely projects to the PRE, POST, and PAR, which, in turn, project back to the LM, to complete a circuit loop with the AD and DTN. Each structure within this network houses head-direction cells 17 , pivotal for spatial orientation. The AV subdivisions (AV.lateral, AV.dorsal, AV.medial, and AV.medial tip) are densely connected with the MM and VTN, forming a central network that regulates theta rhythmicity, to synchronize activity across structures to facilitate communication relevant for learning and memory 22,56 . The AV.dorsal shares reciprocal connections with the subicular complex (PRE, PAR, POST) and with RSPd and RSPagl. The AV.lateral receives input from the SUBd, but generates projections to the SUBv, which in turn projects back to the AV.medial and AV.medial tip. These latter AV domains share reciprocal connections with the RSPv, a critical component of the medial cortical network 44 that also receives direct inputs from the SUBd. The AV also receives input from the LDT nucleus, implicated in REM sleep, attention, and reward processes. We identified five distinct subdomains within the AMd, each also densely interconnected with parts of the medial cortical network 44 , including the RSPv, PTLp, ACAv, ACAd and its adjacent MOs-fef. Thus, information processed regarding head direction, theta rhythm, attention, and REM sleep from the ATN converge onto the medial cortical network , within which this information is integrated with other external environmental cues including visual, auditory, and somatosensory inputs, and higher-order associative cortical information 44 . These medial neural network cortical areas project densely to the SC, zona incerta (ZI), and CPdm, which projects to the ventromedial division of the substantia nigra pars reticulata (SNr), and the SNr in turn projects to the medial SC 37,57 . Together, these regions establish a core neural network regulating eye, head, and neck movements 36 that are important for attention, spatial orientation, navigation, and exploratory behavior. Further, the AMd.medial and AMv are closely linked with the MPF (PL, ILA), SUBv, and BLAa—three limbic cortical areas that regulate social behavior and emotion-related activities through their projections to: (1) The medial amygdalar nucleus (MEA) and the posterior part of the bed nuclei of the stria terminalis (BSTp), which generate dense projections to the hypothalamic medial behavioral control column composed of the anterior hypothalamic (AHN), ventromedial hypothalamic (VMH), and dorsal premammillary nuclei (PMd) 58 . This hypothalamic subnetwork projects extensively to the dorsal PAG (PAGd), governing goal-directed behaviors with strong emotional components, such as hunting and attacking 58 , which require attention and navigation during their execution. Notably, the PMd, which functions as a threat detector by sensing dynamic changes under threatening conditions as the animal approaches and avoids the threatening source 59 , projects densely back to the AMv, which is crucial for updating memory processes to adapt to changes under threatening conditions. (2) The central amygdalar nucleus (CEA) and anterior BST (BSTa), which are involved in the regulation of autonomic and neuroendocrine activities 58 . (3) The nucleus accumbens (ACB), which, along with the ventral tegmental area (VTA), controls reward mechanisms and addiction. AMd.medial and AMv as communication subnetworks for MPF, HPF, and amygdala A network of structures including the cortex, hippocampus, and thalamus are critical for the formation of new memories. The ATN play a critical role in this network given their strong connections with the cortex and hippocampus. This is especially true for the AM since this is the only ATN nucleus connected with both the hippocampus and MPF. The AMd also connects with the BLA, shown here to be specifically the BLAa (Fig. 6f). Importantly, cortical, hippocampal, and amygdalar connections are relevant for the formation of emotional memories including fear-related behavior 60 . In fact, AM lesions disrupt contextual fear responses 29,30 . We found a specific subnetwork within the AMd through which these cortico-hippocampal-amygdalar structures communicate. These were namely the SUBv→AMd.medial→PL/ILA and SUBv→AMd.medial→BLAa (Fig. 8a, h). Completing this network are the strong PL/ILA↔BLAa reciprocal connections 38 . These connections clearly show AMd.medial as a hub for cortico-hippocampal-amygdalar interactions. In addition, we show that the AMv is strongly connected with MPF areas like the rostral ACA, PL, ILA, and ORB (Fig. 6k) and the ILA→AMv→PL circuit was specifically shown at the synaptic level (Fig. 8n). These AMv connections are also likely relevant in the formation of emotional memories given the demonstrated role of corticotropin-releasing factor containing AMv neurons in the regulation of fear conditioning 31 . Notably, AM–MPF connections are linked to goal directed behaviors involving intracranial self-stimulation 27 . Specifically, stimulation of MPF terminals in the AM activates midbrain dopaminergic neurons in the ventral tegmental area (VTA) and reinforces intracranial self-stimulation. A similar outcome results from stimulation of AM terminals in the MPF. Inhibition of these VTA neurons reduces the self-stimulation facilitated by the MPF-AM connections. This finding is interesting in the context of a wider AM network, specifically for the AMd.medial subnetworks (Fig. 11a). The projections from the AMd.medial tip to the BLAa are to its caudal domain (BLA.ac) (Fig. 6f), which is shown to be in a network of structures involved in drug seeking behavior 38 (Fig. 11b). These structures include the SUBv, ILA/PL, medial accumbens (ACB), medial olfactory tubercle (OT), rostral paraventricular thalamic nucleus (PVT), and CA3. Fig. 11c also demonstrates the wider network of the MPF and AMd.medial that lead to the VTA. Direct connections between the ATN, DTN, and VTN Two parallel circuits DTN↔LM→AD and the VTN↔MM→AM/AV are respectively involved in the head direction system and in theta rhythm activity. A surprising finding was direct connections between the midbrain nuclei and the AD and AV. We found that the DTN and VTN show slight connections with both the AD and AV, connections that were validated with multiple retrograde tracer injections placed across the ATN (Fig. 8f-g). Given the reported highly segregated tegmental-mammillary pathways, this result was unexpected and suggests some level of convergence across the two processing streams. Within the context of confluence, these direct connections are less surprising since the idea of head directionality and theta rhythmicity coalescing with the ATN is also suggested by the presence of head direction cells in the AV 16,18 . Interactions between head directionality and theta rhythmicity also seem intuitive: the synchronization of activity across regions involved in head directionality would facilitate an animal’s ability to learn and retain spatially pertinent information essential for navigation. While no direct connections between ATN and VTN/DTN have been officially documented, there is a study in humans that potentially lends support for this 61 . ATN connectivity reported in our findings and the current body of literature Studies across different species have identified mammillary body and hippocampal connections primarily exclusive to AM, AD, and AV that form parallel, segregated loops across the Papez circuit subserving distinct functions in navigation 1,9 . Projections from distinct regions of the medial (MM) and lateral (LM) mammillary nucleus to each of the ATN nuclei are well documented in rats and non-human primates, with the MMm projecting mostly to AM 62,63 , the MMl to AV 64 , and the LM to AD 63,65 . Projections from the MM medianus (MMme) to IAM are also reported 15 . Our data show these precise MB→ATN connections, although we also show MMme→IAD projections (Fig. 9a-e). Aside from the mammillary body projections to ATN, many inconsistencies are reported regarding ATN connectivity, likely due to the different methods, species, injection sizes, injection site locations, and even different anatomic nomenclature that have been utilized across the studies. Hippocampal connections with individual ATN nuclei are similarly segregated with many conflicting connections reported. Some show AD projecting to postsubiculum (POST) and AV and AM projecting to proximal (near CA1) and distal subiculum, respectively 66,67 , while others show PRE/SUB→AM/AV and POST/PAR↔AV 15 . POST/PRE/PAR↔AV/AD, AM→PRE, and AD↔hippocampus connections also have been reported 68 . Our collective data of ~200 traced pathways showed specifically (1) AD.medial→POST/PRE/PAR with no projections back to AD (Fig. 2b, e-f); (2) AV.dorsal↔POST/PRE/PAR (Figs. 2f; 3b-d); and (3) no connections between the AM and POST, PRE, and PAR (Fig. 1f). Regarding the SUB, we showed (1) no connections between SUB and AD (Fig. 2g); (2) SUBv→AV.medial tip (Fig. 3k) and SUBd→AV.lateral→SUBv (Fig. 3j-k); and (3) SUBv→AMd.medial (Fig. 5b-c). No connection between the ATN and the CA1, CA2, CA3 were found. Different combinations of connections have been reported between the ATN and ENT like ENT→AM 42 , AM→ENT 33,34 , and AD/AV/AM→ENT 28 . We only found connections between the AM and ENT. We placed many anterograde and retrograde injections across the ENTl and ENTm (Table 1). The large majority of ENTl injections did not produce any labeling in the AM. This was because the AMd projects to layer 5 of a very specific ENTl region (Fig. 7f-g). Once a retrograde tracer injection was successfully placed in that ENTl region, some AMd.core neurons were labeled (Fig. 7f). Our anterograde and retrograde injections in the AM did not produce any labeling in the ENTm despite reports of these connections in the literature 33 . ATN connections with cortical regions are reported to be separable with the ventral (granular) RSP (RSPv or RSPg) connecting mostly with AD and AV, the dorsal or agranular RSP (RSPd or RSPagl) with the AM 67,69,70 and the prelimbic area (PL) and orbital (ORB) with the AM 28,34 . Our data showed (1) RSPd/v↔AD.lateral (Fig. 2c-d); (2) RSPd/agl↔AV.dorsal (Fig. 3g; Extended Data Fig. 2e) (3) RSPv↔AV.medial (Fig. 3e, g); and (4) RSPv←AMd.dorsolateral (Fig. 5a). Sparse RSP-AM connections were detected and AM connections with ACA were exclusive despite reported ACA connections with AV and AD (Fig. 4k) 28,42,71 . Specifically, AMd.dorsomedial/AMv↔rostral ACA and AMd.dorsal↔caudal ACA (Fig. 4k; Extended Data Fig. 3b) were found. The exclusive AM-ACA connections reinforce the AM’s role in emotional learning given that the ACA is involved in emotional processing, fear acquisition memory, and in the control of innate fear responses 72-74 . Further, a rostral versus caudal distinction of the ACA is generally accepted, with distinct respective roles in emotional versus cognitive processing 75-77 . Our data generally agree with the literature regarding connections of the PL, ILA and ORB being exclusive to AM; however, our data greatly expand upon this by demonstrating AM domain level connections with these medial prefrontal cortical areas, and also the dissociable connections of the AMd and AMv, which have not been extensively reported 31 . We show (1) AMd.medial tip/AMv↔PL; (2) AMd.ventromedial/AMv↔ILA; and (3) AMd.ventromedial/AMv↔ORBm/vl (Fig. 6b-d; g-h. We did not find any significant AMv projections to BLA, RSP, ECT, PERI, ENTl, nor to CA1, which have been reported for AMv CRH neurons 31 , and to our knowledge a study dissociating the brain-wide inputs to the AMv has not been conducted. Regarding ATN connections with secondary motor cortical areas (MOs), our data showed selective reciprocal connections with the AMd.core (Fig. 7b-e), and although these connections have been reported for the AM 33 , AV↔MOs connections also are reported elsewhere 28 . The MOs is a large, undivided structure in most rodent atlases, and it is possible this discrepancy is due to differences in MOs injection site locations. We placed injections across the entire MOs, and only the injections specifically in the MOs-fef region (adjacent to the ACAd) 39,44 produced labeling in the AM. Notably, this MOs-fef region projects to the CP dorsomedial region, which integrates information from a variety of visual areas including VISam, VISal, ACAv, and PTLp caudal medial 39 (Extended Data Fig. 6f). We also show AM→caudal ECT/PERI connections (Fig. 7f-g), both of which heavily connect with cortical areas involved in visual and auditory processing 44 . Finally, despite documented connections between the internal part of the globus pallidus with the AD 78 , these connections were not found through our dataset. Caveats to tracing experiments Our large dataset used for the current project utilized a variety of chemical and viral tracers. Importantly, each anterograde and retrograde tracer has distinct characteristics and exhibits varied neurotropism that can meaningfully affect connectivity results. For example, anterograde AAVs label fibers of passage, while Phal does not. AAV-retro preferentially labels the cortex, while rabies preferentially labels hypothalamic neurons 79 . These differences underscore the importance of data validation. In this work, the connectivity data were validated in multiple ways to ensure the reliability of the results. Retrograde tracers were placed in regions of anterograde terminations, while anterograde tracers were placed in regions of retrogradely labeled cells. For example, anterograde tracer injections in the AM and AV show labeled terminals in the SUBv, but primarily in the caudal regions (Extended Data Fig. 4a). Retrograde tracers in the rostral SUBd, rostral SUBv, and the caudal SUBv confirm that only the caudal SUBv projects to the AM and AV (Extended Data Fig. 4b). Our data was further validated with Cre-dependent tracing methods and with repeated injections made in each ROI (e.g., Extended Data Fig. 3b, 4c). Together, these validation studies increase the confidence in the reported connections and mitigate potential issues arising from individual tracer characteristics. Notably, we have previously shown that repeated injections even in the smallest of ROIs produce similar brainwide labeling patterns regardless of the tracer used 38 . On a final note, the accurate assessment of injection site location is also a critical component of connectomics. Generally, the exposure times used to image and capture labeled fibers and cells typically oversaturate the injection sites. As such, we reimage injection sites with lower exposure parameters to gauge its location and spread more accurately. The reimaging along with all the data validation experiments facilitate the accurate assessment of injection site location. For instance, an anterograde AAV AM tracer injection labeled fibers and terminals in the CP and the ACA (Extended Data Fig. 6a-b). Retrograde tracer injections in the CP placed precisely in the region of the AAV labeled fibers, back labeled neurons in the adjacent IAD and not AM (Extended Data Fig. 6a-b) suggesting an IAD→CP connection and not an AM→CP connection. Retrograde ACA tracer injections on the other hand showed labeled neurons in the AM (Fig. 4e-f). ATN morphology Identification of the morphological neuron types based on their prominent dendritic features is necessary to understand the input-output relations within the ATN. Across the thalamus, the somatodendritic morphology of projection neurons and interneurons have been identified. Generally, four thalamic projection neurons (TPNs) or Golgi Type I neurons, whose axons extend outside their native thalamic nucleus to innervate the cortex, striatum, and amygdala have been reported. The first are the bushy tufted TPNs, which are large with a high number of branches whose dendrites profusely arborize, are intertwined, and are covered in spines, which give them their bush-like appearance 50 . The second are the radial (or stellate) TPNs, whose dendrites arborize in a radial fashion and compared to the tufted TPNs display relatively reduced wiring with shorter branches, particularly in the distal dendrites 48 . These radiate cells often also display grape-like appendages close to the initial branch points of the primary dendrites 47,48,80 , although these appendages are also reported for thalamic interneurons 51,80 . The tufted and radiate TPNs are found throughout the thalamus, while the third category of TPNs, the diffuse or reticulated, have been reported for primarily the parafascicular (PF) 47,81-84 , ethmoid-limitans 47 , and paralaminar group (medial division of the medial geniculate nucleus, posterior intralaminar, suprageniculate, peripeduncular) 85 . Diffuse TPNs are characterized by few, poorly ramifying primary dendrites that spread across long distances and have many spines. Although ATN somatodendritic morphology has not been systematically examined in any species, some investigations have been made in the AM and AV of the cat 86 , camel 51 , and human 52 through Golgi staining, and have identified both bushy and radiated TPNs and interneurons. Our k-mean analysis on the total branch number and total dendritic length identified two clusters of neuron types in the ATN: one larger with more complex branching and a second that are smaller and less complex. These two clusters potentially correlate to the bushy and radiate cells identified in other thalamic nuclei (Extended Data Fig. 7h). Visually, no reticulated cells were identified in the ATN, and grape-like appendages were observed in some instances (Extended Data Fig. 7i). In addition to TPNs, the thalamus contains interneurons or Golgi Type II, neurons whose axons do not extend out of the parent thalamic nucleus and make local connections. These interneurons generally are smaller and have fewer primary dendrites that poorly ramify but are covered in spines. Due to our method, we did not label any ATN interneurons (e.g., AAVretro-Cre injections in MORF3 mice to label TPNs). Morphology and function Dendritic topological differences between bushy and radiate thalamic neurons are likely indicative of functional variability. For example, two types of neurons in the motor thalamic VA and VL nuclei have been identified. Neurons that receive predominantly inhibitory inputs display significantly fewer dendrites and their axons project to layer 1 of cortex and to the striatum 87 . The neurons that receive predominantly excitatory inputs have more dense dendritic trees and their axons project solely to layers II-V of cortex. Presumably, the former neurons correspond to radiate TPNs, while the latter to tufted ones 88,89 . The tufted and radiate cells of the medial geniculate (MG) have been similarly functionally differentiated 90 . Larger dendritic arbors allow for a greater number of presynaptic partners for a neuron. We observed that dendritic arbors from the AM region were larger and more complex compared to AV and AD regions. We also observed that based on our morphological classification (using arbor length and number of branches as the two determining classifiers), all the Type 2 neurons (large and complex, analogous to bushy neurons) belonged to the AM regions. Greater length of the AM dendritic arbors would in principle allow them to form many more synapses than the AV and AD neurons. This suggests greater level of synaptic integration (particularly from excitatory long-range axons) occurring in AM compared to AV or AD. Similarly, for the AMd regions, the non-edge neurons had larger dendritic arbors than edge neurons, indicating a greater level of axonal input for the non-edge neurons. Apart from topological properties (such as length and number of branches), the spatial orientation of neuronal arbors have been shown to be relevant to their functional roles 91,92 and AMd edge neurons demonstrate an orientation bias towards the AMd center that increases as the distance from the center also increases. The higher overall length of dendritic arbors at the AMd center region, combined with the preferential orientation of the edge neuron’s dendritic arbors towards the center suggests a functional role, where individual neurons are attempting to maximize their presynaptic input from the central parts of the AMd region. It also suggests an attempt to make well-defined, separate channels of information processing with some spillover across border regions. The anterograde/retrograde tracing data of the ATN tend to show a high degree of topographic specificity, in many instances precisely innervating right up to the border of the target nucleus with minimal crossover (e.g., Fig. 4a). Simultaneously, the dendrites of the edge neurons likewise conform to this specificity, minimizing their spread into adjacent nuclei (Fig. 10e). The result is that each subnucleus has a particular combination of information it preferentially integrates, and the dendritic morphology of the neurons helps to maintain that segregation of information processing. To our knowledge, this type of morphological organization has not been reported. Declarations Animal Ethics Statement All procedures were conducted in compliance with regulatory standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the animal protocols were approved by the IACUCs at USC (our previous affiliation) and UCLA (our current affiliation: protocol number ARC 2020-113). Data and code availability The data and code for this project are available upon request. Author contribution H.H., H.-W.D. conceived and designed the project. H.H. managed the project and conducted the data analysis, H.H., H.-W.D. wrote the manuscript, and prepared the figures for publication. M.R. conducted all of sectioning, staining, clearing, and imaging of the thick tissue and the manual mapping of the AM labeling. S.N. mapped registered axonal projections onto the Allen Brain Common Coordinate Framework (CCF) and calculated axonal projections to individual brain regions. S.N. developed analysis code , performed all dendritic morphological analyses, generated all the corresponding graphs, and wrote the corresponding portions of the paper. A.G., H.-S.M., Q.X. carried out the dendritic reconstructions of neurons. H-S.M. mapped the reconstructed neurons onto the atlas template. L.Garcia., I.B. managed and executed the analysis of the 2D dataset (community detection, hierarchical clustering) and generated their corresponding visualizations. D.L. , T.B. performed all the post-image processing for the 2D datasets in Connection Lens. J.S. performed the synapse reconstructions. C.E., S.Y. assisted with the online visualization of the wiring diagram. Y.Y. managed the breeding of all the MORF3 mice. L.Gou, B.Z. performed tracer injection surgeries, while C.C., J.G. , H.X ., I.Y. processed and imaged the sections. M.Z. generated the code for hierarchical clustering. I.B., K.M., S.N., A.D. developed code for image processing and digital morphological reconstructions. Q.Z. assisted with high resolution imaging of neurons. L.L., X.C., Z.Y. performed the neuron axonal reconstructions, while H.P. managed that portion of the project. N.N.F. assisted in the design of experiments, with figure arrangements, and contributed to the editing of the manuscript. Ethics and inclusion statement We are committed to promoting ethical research practices and ensuring the welfare of all animals involved in our studies. All procedures adhered to regulatory standards as delineated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as well as institutional guidelines set forth by the Institutional Animal Care and Use Committees at both the University of Southern California (USC) and the University of California, Los Angeles (UCLA). We affirm our dedication to fostering an inclusive research environment that values the contributions of all individuals, regardless of race, ethnicity, gender identity, sexual orientation, disability, or other characteristics. We actively strive to create a welcoming and respectful atmosphere for all our research team members. Acknowledgements Funding for this project was provided by NIH Grants U01MH114829 (H.-W.D.) and 1R01NS133744-01 (H.-W. D.). Methods Subjects One hundred fifty 8-week-old C57Bl/6J (Jackson Laboratories) male mice were used to trace approximately 200 pathways (see Table 1). Animals were housed in pairs in a room with controlled temperature (21–22 °C), humidity (51%), and lighting (12-hour light:12-hour dark). Prior to stereotaxic surgeries for tracer delivery, mice were given at least 1 week to acclimate to their environment. Throughout the experiments, subjects had unrestricted access to tap water and mouse chow. All procedures were conducted in compliance with regulatory standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and institutional guidelines established by the Institutional Animal Care and Use Committee at the University of Southern California (USC) and at the University of California, Los Angeles (UCLA). Seventeen male MORF3 mice from our established breeding colony at UCLA (state were used (1) to sparsely label neurons in AD, AV, and AM (including AM domains) for morphometric analysis and (2) to reveal the disynaptic circuitry through the AMd.medial tip (SUBv→AMd.medial tip→PL/BLAa) and AMv (PL→AMv→ILA). MORF3 mice (C57BL/6-Gt(ROSA)26Sortm3(CAG-sfGFP*)Xwy/J), developed by Dr. X. William Yang's lab at UCLA 46 , are a Cre reporter mouse line engineered to utilize a mononucleotide repeat frameshift (MORF) for in vivo cell labeling. These mice express a Cre-dependent tandem \"spaghetti monster\" fluorescent protein with 20 V5 epitopes (smFP-V5), preceded by a polycytosine repeat (C22) MORF switch, all under the control of a CAG promoter. Through Cre recombination and a spontaneous frameshift mechanism, MORF3 mice enable sparse and stochastic labeling of neural cells, which can be visualized using V5 antibody staining. Neural tracer injections Anterograde and retrograde tracers were administered to anatomically defined regions throughout the brain to investigate their connectivity patterns. Stereotaxic surgeries for tracer infusions were conducted under isoflurane anesthesia. Initially, mice were anesthetized in an induction chamber containing isoflurane and then secured to the stereotaxic apparatus, where they remained under anesthesia via a vaporizer. Isoflurane was vaporized and mixed with oxygen (0.5 L/min), maintaining the percentage of isoflurane in the gas mixture between 2 and 2.5. Buprenorphine SR (1 mg/kg) was administered at the beginning of the surgery as an analgesic and ophthalmic ointment was applied to the eyes for protection from light. Tracers were delivered either iontophoretically (10 min 5 µAmp, 7-second alternating current) or via pressure injection (20-80 nl) using glass micropipettes with outside tip diameters measuring approximately 10-30 µm. Tracers The tracers used to determine afferent and efferent connections included phaseolus vulgaris leucoagglutinin (Phal, 2.5%; Iontophoresis, 10 min; Vector Laboratories); AAV-GFP (AAV1-hSyn-EGFP-WPRE-bGH; 2.7 x 10^13; Iontophoresis, 3 min; Addgene); AAV-RFP (AAV1-CAG-tdTomato-WPRE-SV40; 2.0 x 10^13; Iontophoresis, 3 min Addgene); Glycoprotein-deleted rabies (RVΔG) (Gdel-RV-4tdTomato and Gdel-RV-4eGFP; 9.6 x 10^10; Pressure, 50 nl; Ian Wickersham laboratory at MIT); Fluorogold (FG, 1%; Iontophoresis, 3 min; Fluorochrome); Cholera toxin subunit B-Alexa Fluor 488, 555, 647 conjugates (CTB, 0.1–0.2%; Pressure, 50 nl; Invitrogen); AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute). Typically, in a single brain, 2-4 of these tracers were injected in different combinations. For example, in the triple anterograde tracing method, 3 anterograde tracers were injected in 3 different brain regions to reveal topographic projections. In the quadruple retrograde design, four retrograde tracers are injected in 4 different brain regions. These multiple injections/brain assisted in revealing, validating, and clearly visualizing the ATN domains (e.g., Figs. 4h; 5b). In the double co-injection design, two co-injections of an anterograde/retrograde tracer cocktail are injected into 2 different regions to assess input/output of different regions and to assess the interaction of the two regions injected. For Cre-dependent anterograde tracing, the following tracers were used: AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute); Cre-dependent AAV-FLEX-GFP (AAV1-CAG-Flex-eGFP-WPRE-bGH; 2.0 x 10^13; Iontophoresis, 5 min; Addgene); Cre-dependent AAV-FLEX-RFP (AAV1-CAG-Flex-tdTomato-WPRE-bGH; 1.1 x 10^13; Iontophoresis, 5 min; Addgene). For Cre-dependent TVA receptor mediated rabies tracing (or TRIO tracing) the tracers used included AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute); AAV1-hSyn-FLEX-TVA-2A-GFP-2A-G (2.3 x 10^13; Pressure, 80 nl; Addgene); and EnvA G-deleted rabies dsRedXpress(2.26 x 10^10; Pressure, 40 nl; Ian Wickersham lab, MIT). To determine synaptic circuitry in MORF3 mice, the tracers AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute) and AAV1-hSyn-DIO-mGFP-2A-SypRuby-WPRE (2.7 x 10^13; Pressure, 100 nl: Addgene) were used. Finally, to unlock MORF expression in MORF3 mice to reveal the morphological details of ATN neurons, the main tracer used was AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute). Tracing strategies Cre-dependent anterograde. To validate the exclusive brain-wide output of AMd domain neurons, a Cre-dependent anterograde tracing method was applied. One case (n=1) was used per AM domain. In brief, AAVretro-Cre was injected into a downstream target of an AMd domain (e.g., PL) to deliver Cre to the AMd.medial. Next, a Cre-dependent AAV expressing either GFP or tdTomato was injected in the AMd. This method reveals the brain-wide output of AMd.medial→PL projecting neurons. Cre-dependent TVA receptor mediated rabies tracing method. To identify mono-synaptic inputs to neuronal populations defined by projections in the AMd (e.g., brain-wide input to AMd.medial→PL neurons), we employed a Cre-dependent TVA receptor mediated rabies tracing strategy sometimes referred to as TRIO (tracing the relationship between input and output) tracing. These cases were typically used to validate brain-wide input to the AM domains and one case (n=1) was used per AM domain. For example, an AAVretro-Cre injection was made into a downstream projection target of AMd.medial (e.g., PL), which delivered Cre to AMd.medial neurons. Next, a Cre-dependent TVA- and RG-expressing helper virus (AAV8-hSyn-FLEX-TVA-P2A-GFP-2A-oG) and an mCherry-expressing G-deleted rabies virus were injected into the AM thereby revealing brain-wide ROIs→AMd.medial→PL connections. Tissue processing and imaging in 2D Tracer transport was allowed for either one (Phal, CTB, FG, RVΔG) or three (AAVs/viral tracers) weeks before animals were perfused, and their brains were extracted. For all experiments except those involving MORF3 mice to assess morphology, a 2D tissue processing workflow was employed. After administering an overdose injection of sodium pentobarbital (Euthasol, 2 mg/kg, intraperitoneal injection), each animal underwent transcardial perfusion with 50 ml of 0.9% NaCl followed by 50 ml of 4% paraformaldehyde solution (PFA; pH 9.5). Brains were post-fixed in 4% PFA for 24–48 h at 4 °C before being embedded in 3% Type I-B agarose for sectioning. Coronal sections, each 50-µm thick, were sliced into four equivalent series using a vibratome. One of the four series of sections underwent immunostaining for the antigen of interest (Phal or AAVretro-Cre) using the free-floating method. Sections were first transferred to a blocking solution containing normal donkey serum and Triton X-100 for 1 h. After three 5-minute rinses, sections were incubated in a KPBS solution with donkey serum, Triton, and the appropriate antibody [1:1000 rabbit anti-Phal antibody (Vector Laboratories, #AS-2300), 1:4000 mouse anti-Cre recombinase antibody (EMD Millipore, #MAB3120), or 1:500 rabbit anti-tryptophan hydroxylase (TPH2) to identify serotonin positive neurons (ThermoFisher Scientific, #PA1-778] for 48–72 h at 4 °C. Following three KPBS rinses, sections were soaked for 3 h in the secondary antibody solution, which contained donkey serum, Triton, and a 1:500 concentration of anti-rabbit IgG conjugated with Alexa Fluor® 488 or 647 (Invitrogen, 488: #A-21206; 647: #A-31573) for Phal and serotonin staining. For Cre recombinase staining, the secondary solution contained donkey serum, Triton, and a 1:500 concentration of anti-mouse IgG conjugated with Alexa Fluor® 488 or 647 (Life Technology, 488: #A-21202; 647: #A-31571). After three KBS rinses, the sections were counterstained with a fluorescent Nissl stain, NeuroTrace® 435/455 (NT; 1:500; Invitrogen, #N21479), mounted, and coverslipped using 65% glycerol. Finally, the sections were scanned at 10x magnification as high-resolution, multichannel virtual slide image (VSI) files using an Olympus VS120, with identical exposure parameters maintained across all cases. 2D post-image processing workflow To accurately compare connectivity tracer signals across experiments, we utilized our high throughput image processing workflow, Connection Lens, to achieve (1) high-quality image registration, (2) tracer signal segmentation, and (3) tracer signal quantification. Connection Lens provides a user-friendly interactive interface and deploys all processes on a high-performance computational infrastructure that allows high throughput processing of large TB-sized datasets. This post-image processing Connection Lens pipeline is summarized as follows: Each 50 µm-thick section was initially matched and subsequently aligned (registered) to its corresponding atlas level in the Allen Reference Atlas (ARA; available at http://mouse.brain-map.org/static/atlas) (Fig. 1a-c). Our semi-automated registration pipeline applies a diffeomorphic registration approach allowing iterative modifications based on user feedback, enhancing the accuracy of registered images. Next, threshold parameters were individually adjusted for each case and tracer signal. Conspicuous artifacts in the threshold output were filtered. The final overlap processing step generates a file with annotated (quantified) values: pixel density for anterograde tracers and cell counts for retrograde tracers. 2D data analysis Identifying AD, AV, AM domains and their relative boundaries. Our pipeline conducted connectivity analysis for the AV, AD and AM. After segmenting tracer labeling, grid-based overlap annotation was performed in which the AD, AV, and AM were divided into 105 x 105 pixel square grids (equivalent to 63 µm 2 ) to tabulate labeling within distinct domains of the ATN nuclei. This was done for ARA atlas levels 61 and 63 for the AD and ARA level 61 for the AV and AM. In the given analysis, anterograde and retrograde were combined. The case specific annotations were then aggregated into a single matrix and Louvain community detection was conducted with gamma 0.75. Grid cells were color-coded according to community assignment and reordered such that the resulting Louvain clusters were placed along the diagonal of the visualized matrix (Fig. 1b-d). Identifying brain-wide input/output connectivity of AM domains . To assess domain-specific AM connections, Cre-dependent anterograde and retrograde tracing strategies were applied utilizing connectionally guided delivery of Cre. For each case, sections spanning the whole brain were registered (warped) and tracer labels were segmented and annotated based on ROI. The annotated data (pixel intensity for anterograde, cell counts for retrograde) were subsequently normalized. Neuroanatomical tracing experiments across the entire brain entail numerous sources of variation, including tracer injection volume, quality of the collected tissue, variations in the plane of sectioning and immunostaining quality, accuracy of tissue registration to the atlas template, as well as microscopic imaging settings, among others. Conducting meaningful inter-experimental data comparisons necessitates the standardization of these experimental variations. Given that tissue sections within a single specimen typically experience consistent experimental conditions, we utilize a self-normalization method to derive whole-brain connectivity fractions for each brain specimen. The connectivity fraction characterizes the proportion of connectivity found in a region out of all connectivity found in the entire specimen. The fraction calculation performs a total intensity normalization within each brain specimen, effectively normalizing away the effects of experimental condition variations. The fraction calculation was as follows: f =Count/Sum of all ROI count across the case, where count is number of labeled pixels (pixel count for anterograde, cell count for retrograde). We also define a connectivity density vector d that approximates the density of connectivity at each gray matter region. The density vector is derived from the fraction vector and represents the projection densities at each brain region of each specimen. It maintains the total counts of segmented pixels (for anterograde tracers) or cells (for retrograde tracers) constant across all specimens. This density measure offers an intuitive understanding of the connectivity data, as connectivity densities are commonly discussed in neuroanatomical literature, are easily understood, and considers ROI sizes. The density calculation was as follows: d= (Fraction/area)/maximum density value. Density values were used to generate the bar graphs depicting the connectivity of AM domains (e.g., Fig. 6h, j). They were also used to perform hierarchical 2D clustering of the AM domain-specific tracing data to determine and visualize the similarity of whole brain projection patterns of the different injections (e.g., Fig. 1f). 3D tissue fixation, clearing, staining, and imaging pipeline Following 4% PFA perfusion, 250 µm-thick (for synaptic connectivity experiments) and 800 µm-thick (for morphological assessments) tissue samples were drop fixed in 4% PFA at 4°C for up to two weeks, then transferred into PBSN (1x PBS + 0.02% sodium azide and stored at 4°C until SHIELD epoxy fixation. For SHIELD epoxy fixation, samples were incubated in the SHIELD OFF solution at 4°C for three days with agitation, then transferred into pre-heated SHIELD ON solution and incubated at 37°C overnight with agitation. After the SHIELD ON step, samples were either transferred into PBSN and stored at 4°C until the delipidation step or washed 2x for 2 hours and moved onto the delipidation step. When ready for delipidation, samples were put into 10 mL LifeCanvas’s Active delipidation buffer and incubated overnight at 45°C with agitation. The next day, samples began active delipidation using LifeCanvas’s SmartBatch+ pre-installed active clearing protocol (40V, 30 hours). Following active delipidation, samples were washed 2x 2 hours with wish 1x PBSN and stored in 1x PBSN at 4°C until ready for staining. When samples were ready for staining, they were incubated in a 10 mL primary sample buffer with 5% NDS overnight at 37°C. Replacing this solution once in the morning, samples were then put in the SmartBatch+ to begin the active primary V5 staining protocol [1:1000 anti-V5, Fortis, #A190-119A (goat) or #A190-120A (rabbit)]. Sample solution contained primary antibody added to the primary sample buffer + 5% NDS, and LifeCanvas’s primary conduction buffer was used (18–22 hours). Primary staining concluded when the sample buffer reached < pH 8.0. Following the primary stain, samples were washed 2x 2 hours in 1xPBSN then incubated in 4% PFA overnight at 4°C to prevent primary antibody dissociation. After 4% PFA incubation, samples were washed in 1x PBSN at RT for 2 hours then 10 mL secondary sample buffer + 5% NDS at 37°C for 2 hours with agitation. Samples were then put in the SmartBatch+ for one 2-hour active secondary sample buffer + 5% NDS wash using the active secondary protocol pre-installed in the SmartBatch+. Active secondary protocol was run with secondary antibodies (1:1000 in house conjugated anti-goat and anti-rabbit Fab-Setau-647, see following section for details regarding secondary antibody conjugation procedure) in the secondary sample buffer + 5% NDS for 12 hours. The next morning, samples underwent 2x 2 hours active washes with only the secondary sample buffer. Samples were then washed 2x 2 hours in 1x PBSN and then incubated in 4% PFA overnight at 4°C to fix secondary antibodies in place. The next morning, samples were washed 2x 2 hours in 1x PBSN and then were either placed into storage in 1x PBSN at 4°C or moved onto the light sheet or spinning disc confocal preparation. Tissue being processed for the LifeCanvas SmartSPIM light sheet microscope at 15x (for morphological assessment) were moved into 10mL delipidation buffer containing 1:500 of a nucleic acid stain [Syto13 (ThermoFisher Scientific, #S7575) or Propidium Iodide (ThermoFisher Scientific, #P1304MP)] and incubated overnight at 37°C with agitation. The following morning, samples were washed 2x 2 hours in 1x PBSN then incubated in 4% PFA at 4°C overnight. The next morning, samples were washed 2x 2 hours in 1x PBSN then either stored in 1x PBSN at 4°C, or immediately moved into 50% EasyIndex and incubated overnight at 37°C then put into 100% EasyIndex and incubated overnight at 37°C. The next morning, samples were mounted onto a sample holder using 1.5% agarose and superglue and were put into the light sheet chamber for imaging. Tissues processed for Olympus Dragonfly spinning disk confocal imaging were sectioned at either 800 µm for 20x imaging (for morphological assessment) or 250 µm for 60x imaging (for synaptic connectivity experiments). Sections were then incubated in 1:500 DAPI (ThermoFisher Scientific, #D1306) in 1x PBSN solution or immediately moved into 50% EasyIndex and incubated overnight at RT. The next day samples were then put into 100% EasyIndex and incubated for at least 3 hours or were held in a parafilm wrapped 24-well plate and protected from light until ready for slide mounting. Sections were mounted onto plain microscope slides with Sunjin spacers and coverslipped with 1.5H coverslips that were held in place with superglue. SeTau-647 secondary antibody conjugation For optimal signal retention of MORF3 V5 tagged neurons in 3D imaging, we chose to conjugate SeTau-647-NHS ester (Setabiomedicals, #K9-4149) to AffiniPure Fab fragments (Fab donkey anti-rabbit, Jackson Immunoresearch, #705-007-003; Fab donkey anti-goat, Jackson Immunoresearch, #711-007-003) because of the intensity of emission and its resilience to photobleaching. Fab’s were conjugated to SeTau-647-NHS ester using a 1:2 molar ratio (Fab:Dye). 1M sodium bicarbonate, at 10% of the volume of the Fab:Dye solution, was added to the Fab:Dye solution (e.g., if Fab:Dye solution is 100 µl, then add 10 µl of sodium bicarbonate for a final solution volume of 110 µl) and was agitated at 500rpms at room temperature for 1 hour, then excess dye was removed using size exclusion columns (Zeba™ spin desalting columns and plates, 40K MWCO, 0.5 mL, ThermoFisher Scientific, #A57760). 3D neuron reconstruction and mapping To accurately select the neurons from the regions of interest (i.e., AD, AV, and AM domains), the Orthoslicer and FilamentTracer tools within Imaris (BITPLANE, RRID:SCR_007370) were used to manually identify the (somatic) location of each neuron. The Orthoslicer thickness of the tissue was adjusted to be between 10-30 µm. The cell body of each neuron was ensured to be within the selected region’s border in each of the coronal views of the tissue, located between ARA 58-66 on the Allen Reference Atlas. The identified neurons were scaled from voxel to micron dimensions (and scaled back to voxel dimensions if required) using a custom python script and were manually reconstructed within the Terafly program in Vaa3d 93 . For precise quality check of the reconstructions, the neurons were opened and edited in neuTube 94 to ensure correct branch typing, location of branch points and proper connections of the nodes. Each digital SWC reconstruction is a tree structure constituting a series of connected frustums/compartments, where a compartment is represented by a single row (containing seven columns) of an SWC file 95 . The compartment's id, type (dendrite, axon, or soma), end point coordinates (X, Y, Z location) and the end point thickness are represented by the first six columns. The 7th column of the SWC row has the id of the compartment’s origin point/parent compartment (for additional details, see swc-specification.readthedocs.io). Imaris was once more used to visualize, and further quality check the neuron morphology and the location markers such as identifying neurons lying closer to the borders of the regions of interest (edge) versus the ones that are more within the center of the AM (non-edge). A total of 109 neural reconstructions were analyzed for this study that included 10 AD, 82 AM, and 17 AV neurons. Out of the 82 AM neurons, 72 neurons were from the AMd region and the remaining 10 were from the AMv region. The AMd neurons were subdivided into five domains: (i) AMd.core (12 neurons), (ii) AMd.dorsal (12 neurons combined from AMd.dorsal and AMd.dorsolateral domains), (iii) AMd.dorsomedial (11 neurons), (iv) AMd.lateral (20 neurons), and (v) AMd.medial (17 neurons from both the AMd.medial tip and AMd.ventromedial domains). To reconstruct the full 3-D morphology of neurons, including both dendrites and axons, we utilized Vaa3D (http://vaa3d.org 95-97 along with its recent successor, Collaborative Augmented Reconstruction (CAR, https://github.com/neurogeom/CAR 98 . This approach was applied to fMOST images obtained through the collaboration 99 . To guarantee the precision of the neuron locations and morphologies, we conducted human inspection of the somas and their 3D structures. Statistical analyses of morphometrics We first quantified basic morphometric features that demonstrated distinct and complementary aspects of dendritic architecture. These features included total wiring (total dendritic length), total complexity (number of dendritic branches), arbor height, maximum path distance, and average branch order. We noticed distinct morphological features for AMd neurons whose somas were on the border of the AMd (edge, n=27) compared to those whose somas were located more internally (non-edge, n=45). To investigate this, we performed quantification of two new attributes for these edge or non-edge AMd neurons, specifically to measure the orientation bias (towards the AMd center) of a neuron’s overall dendritic architecture. The first feature is the average angular deviation relative to the local core vector (Fig. 8). The deviation of every dendritic compartment vector relative to its local core vector (where the local core vector of a compartment is a straight line connecting the compartment’s origin point to the AMd center) is averaged across all compartments for a collective average deviation for that neuron. An acute compartment deviation (deviation of less than 90°) would mean that the dendritic compartment’s end point is closer to the AMd center than its origin point. An obtuse compartment deviation on the other hand, would mean that the compartment’s end point is further away from the center compared to the compartment’s origin point. Neuronal nodes are resampled for this measurement so that each compartment (i.e., internode distance) is approximately 1 micron in length. Therefore, a neuron with a lower average angular deviation (from the AMd center) would have greater tropism/bias towards the AMd center. The second feature measured the proportion of total dendrite that lay closer to the AMd core than the neuron’s cell body. Hence, neurons with a higher proportion of dendrites closer to the center also demonstrate greater tropism/bias towards the AMd center. We carried out a comparative morphometric study on three levels of groupings due to smaller sample sizes for both AD (n=10) vs AM (n=82) vs AV (n=17) comparison as well as for the AMd domain comparisons. The domains included the AMd.core (n=12), AMd.dorsal (AMd.dorsal and AMd.dorsolateral combined, n=12), AMd.dorsomedial (n=11), AMd.lateral (n=20), and AMd.medial (AMd.medial tip and AMd.ventromedial combined, n=17). Because of smaller sample sizes, the omnibus Kruskal-Wallis test was carried out to identify significant differences across multiple groups, followed by Wilcoxon rank sum test for each pair (three pairs in AD vs. AM vs. AV comparison and a total of 10 pairs for the five AMd domains. For the AMd edge vs non-edge analysis, a t-test was carried out since the sample size of both groups were higher and the distributions were normal. Synapse reconstructions Soma, dendrites, and spines were reconstructed using the “Soma”, \"Tree\", and “Spine” module respectively in Neurolucida 360 software, while axon boutons that are adjacent to the soma, dendrites, and spines were reconstructed with the \"Puncta\" module. Briefly, soma was created using the default setting, and dendrites were created using the user-guided mode with the Rayburst Crawl mode. 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Gou\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lin\",\"middleName\":\"\",\"lastName\":\"Gou\",\"suffix\":\"\"},{\"id\":434340474,\"identity\":\"2facc518-cb65-416a-91c8-09e4da09a94d\",\"order_by\":14,\"name\":\"Chunru Cao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chunru\",\"middleName\":\"\",\"lastName\":\"Cao\",\"suffix\":\"\"},{\"id\":434340475,\"identity\":\"630a892a-8d0e-477e-a9af-17b5770de923\",\"order_by\":15,\"name\":\"Jennifer Gonzalez\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jennifer\",\"middleName\":\"\",\"lastName\":\"Gonzalez\",\"suffix\":\"\"},{\"id\":434340476,\"identity\":\"24aacc16-8135-4f87-a18f-6c1c0368f10a\",\"order_by\":16,\"name\":\"Keivan Moradi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"David Geffen School of Medicine at UCLA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Keivan\",\"middleName\":\"\",\"lastName\":\"Moradi\",\"suffix\":\"\"},{\"id\":434340477,\"identity\":\"cfd3886f-95a5-411a-8082-e3f3e6cb3454\",\"order_by\":17,\"name\":\"Qiuying Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qiuying\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"},{\"id\":434340478,\"identity\":\"9bb0a511-fd36-4b11-a52a-44fae4fdc6b1\",\"order_by\":18,\"name\":\"Inga Yenokian\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Inga\",\"middleName\":\"\",\"lastName\":\"Yenokian\",\"suffix\":\"\"},{\"id\":434340479,\"identity\":\"531eb0a8-450d-4a4c-a8ba-488898b6939f\",\"order_by\":19,\"name\":\"Aishwarya Dev\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Aishwarya\",\"middleName\":\"\",\"lastName\":\"Dev\",\"suffix\":\"\"},{\"id\":434340480,\"identity\":\"1755b19c-5214-45a1-9bf0-50ac0e429223\",\"order_by\":20,\"name\":\"Brian Zingg\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Brian\",\"middleName\":\"\",\"lastName\":\"Zingg\",\"suffix\":\"\"},{\"id\":434340481,\"identity\":\"7ed3962f-d271-47d3-b608-7649f3f512ef\",\"order_by\":21,\"name\":\"Hanpeng Xu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hanpeng\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"},{\"id\":434340482,\"identity\":\"46074243-0dca-4a03-bbbd-d86f2db42a12\",\"order_by\":22,\"name\":\"Qing Xue\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qing\",\"middleName\":\"\",\"lastName\":\"Xue\",\"suffix\":\"\"},{\"id\":434340483,\"identity\":\"9da9634e-dda3-4597-9150-fa0d8bc49285\",\"order_by\":23,\"name\":\"Muye Zhu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"David Geffen School of Medicine at UCLA\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Muye\",\"middleName\":\"\",\"lastName\":\"Zhu\",\"suffix\":\"\"},{\"id\":434340484,\"identity\":\"0eda6b0b-32a5-43dd-874d-bd5ce448c05e\",\"order_by\":24,\"name\":\"Lijuan Liu\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-9548-6183\",\"institution\":\"Southeast University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lijuan\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":434340485,\"identity\":\"cd53e38f-46db-455a-b4ee-f1d7e5247f6e\",\"order_by\":25,\"name\":\"Xin Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute for Brain and Intelligence, Southeast University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xin\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":434340486,\"identity\":\"5200a4f5-f078-4884-9304-80a3fe173982\",\"order_by\":26,\"name\":\"Zhixi Yun\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute for Brain and Intelligence, Southeast University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhixi\",\"middleName\":\"\",\"lastName\":\"Yun\",\"suffix\":\"\"},{\"id\":434340487,\"identity\":\"4d6f354c-3b0a-48c1-a79d-7963b42fc82a\",\"order_by\":27,\"name\":\"Hanchuan Peng\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-3478-3942\",\"institution\":\"Fudan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hanchuan\",\"middleName\":\"\",\"lastName\":\"Peng\",\"suffix\":\"\"},{\"id\":434340488,\"identity\":\"081d59db-cd3b-485d-a54a-56fe469e0ba2\",\"order_by\":28,\"name\":\"Nicholas Foster\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0003-1740-9788\",\"institution\":\"Center for Integrative Connectomics, USC Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, University of Southern California\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nicholas\",\"middleName\":\"\",\"lastName\":\"Foster\",\"suffix\":\"\"},{\"id\":434340489,\"identity\":\"6cfd4686-f0d8-4fac-a7e6-f2a078abaeab\",\"order_by\":29,\"name\":\"Hong-Wei Dong\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-9972-3177\",\"institution\":\"University of California Los Angeles\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hong-Wei\",\"middleName\":\"\",\"lastName\":\"Dong\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-05-01 23:50:22\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4356188/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4356188/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41467-025-60774-6\",\"type\":\"published\",\"date\":\"2025-07-01T04:00:00+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":81964575,\"identity\":\"4b8aeddd-ef17-435d-8c57-5491e9e90481\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:25:50\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":16887270,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eApproach\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eTissue sections from tracer injection cases are imported into Connection Lens, matched to their corresponding ARA atlas template level and fiducial markers placed within the tissue Nissl channel are aligned with those in the atlas template. Tissue images are then deformably warped (e.g., registered) and \\u003cstrong\\u003eb,\\u003c/strong\\u003e the tracer labeling is segmented (e.g., thresholded). The ATN is subdivided into 105 px\\u003csup\\u003e2\\u003c/sup\\u003e grid cells and the axon (pixels) or cell (cell count) labeling within each grid square is quantified.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ec.\\u003c/strong\\u003e The aggregated quantified data from all injections to all the grid cells is then analyzed using a modularity maximization algorithm to group injection sites with terminations in common grid cells. The darker shading indicates stronger connections. \\u003cstrong\\u003ed.\\u003c/strong\\u003e The output is visualized in a color-coded matrix to illustrate the subnetworks within each nucleus. \\u003cstrong\\u003ee. \\u003c/strong\\u003eThe generated data was used to create the whole brain wiring diagram of the AD, AV, and AM and their identified domains.\\u003cstrong\\u003e f. \\u003c/strong\\u003eThe overall organization of the ATN nuclei, followed by the unique connections of each nucleus, e.g., SUBd→AV, AD/AV→PRE, IAD→CP, PL↔AM, RSPv→AD/AV, and AM→ACAv.\\u003cstrong\\u003e g.\\u003c/strong\\u003e The laminar organization of thalamo-cortical projections and corticothalamic projection neurons. An anterograde and retrograde tracer co-injection in the AM shows labeled thalamocortical projections to layers I-V and layer VI corticothalamic cells. The bottom left panel shows a similar laminar organization of thalamocortical projections from the AV. Right panels show laminar organization of hippocampal-thalamic projection neurons and thalamo-hippocampal projection terminals. A CTB retrograde tracer injection in the AV shows layer III labeled POST, PRE, and PAR projection neurons. An anterograde Phal injection in the AD shows projections to layers I-III of POST, PRE, and PAR. See Table 2 for list of structure abbreviations.\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/4895d8a5fe7b91fde2847358.png\"},{\"id\":81963305,\"identity\":\"ba130627-d1ed-415e-aef4-7d93bea40463\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:17:49\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14173827,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConnectivity of the AD\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eBased on network analysis of their connectivity, the AD was subdivided into two domains: the AD.medial and AD.lateral, across ARA 61 and 63.\\u003cstrong\\u003e \\u003c/strong\\u003eThe matrices represent the percentage of a specified grid covered by axons or cells from each injection site. These data were subject to a modularity maximization algorithm that grouped injection sites (rows) with labels in common grid cells (columns) within the AD. The matrix was reordered to present the identified groups (i.e., domains) along the diagonal. Domain names are listed above the matrices. Finally, each AD pixel grid was recolored according to their community structure to visualize the domains on the atlas AD. A total of 6 cases (6 sections with AD labels at ARA 61 and another 6 sections at ARA 63) were included in the analysis. Injection ROIs are indicated in the rows of the matrix.\\u003cstrong\\u003e b. \\u003c/strong\\u003eCTB retrograde tracer injections in the PRE and PAR show labeled neurons specifically in the AD.medial at ARA levels 61 and 63. \\u003cstrong\\u003ec. \\u003c/strong\\u003eAnterograde (Phal) and retrograde (FG) injections in the RSPv label mostly the AD.lateral at ARA 61 and 63. \\u003cstrong\\u003ed. \\u003c/strong\\u003eDouble retrograde injections in the PRE and RSPv in the same animal clearly demonstrate the AD.medial and AD.lateral distinction. \\u003cstrong\\u003ee. \\u003c/strong\\u003eNumerous anterograde tracer injections placed across the POST, PRE, and PAR did not label axons in the AD. \\u003cstrong\\u003ef. \\u003c/strong\\u003eAD.medial and AV.dorsal project to PRE (AD.medial/AV.dorsal→PRE), but PRE projects back only to AV (AV←PRE). A co-injection of an anterograde and retrograde into the PRE retrogradely labels AD.medial and AV.dorsal neurons, but anterogradely labeled terminals are present only in the AV.dorsal.\\u003cstrong\\u003e g. \\u003c/strong\\u003eNo connections were detected between the SUB and the AD. Anterograde tracers in the SUBd and SUBv do not label terminals in the AD. \\u003cstrong\\u003eh. \\u003c/strong\\u003eAnterograde and retrograde injections show no labeling in the AD.\\u003cstrong\\u003e i. \\u003c/strong\\u003eLeft is a canonical schematic of connections among the SUB, AD, RSP, and LM with the AD. Right is an updated version of those connections with the medial and lateral subnetworks running through the AD.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/18c76e31e9e3f2198b63a3e3.png\"},{\"id\":81963324,\"identity\":\"cfd25107-b4c6-46f0-ad82-5d8e7ffc81f5\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:17:49\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":18478784,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConnectivity of the AV\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eThe AV is subdivided into 4 domains, AV.medial tip, AV.medial, AV.dorsal, and AV.lateral based on their connectivity (top). The matrix below presents the Louvain-identified AV domains and shows the injection sites that are grouped together based on the similarity of axonal or cell labeling they produce at each AV grid box (ARA 61). Multiple injections per brain region (e.g., SUBd) were included in the analysis to show they group together in the community analysis. The recolored AV grids visualize the domains on the atlas AV and the ROIs that each domain connects with are listed. A total of 19 cases (19 sections with AV labeling at ARA 61) were included in the analysis. Injection ROIs are indicated in the rows of the matrix.\\u003cstrong\\u003e \\u003c/strong\\u003eInjection ROIs are indicated in the rows of the matrix.\\u003cstrong\\u003e b1. \\u003c/strong\\u003eCTB in the AV backlabels neurons in deep layers of the POST/PRE/PAR. Labeled cells in the MMl shown as verification of injection location. \\u003cstrong\\u003eb2.\\u003c/strong\\u003e Anterograde tracers in the POST and PRE (labeling in the LM and LD as injection location verification) show POST/PRE/PAR (layer III)→AV.dorsal domain. Note the absence of labels in the AD suggesting no inputs to AD from POST, PRE, and PAR. \\u003cstrong\\u003ec1. \\u003c/strong\\u003ePhal injection in AV labels all layers of POST/PRE/PAR (AV→POST/PRE/PAR). \\u003cstrong\\u003ec2.\\u003c/strong\\u003e Retrograde injections in the POST, PRE, and PAR in a single animal reveals this as AV.dorsal→POST/PRE/PAR. \\u003cstrong\\u003ed.\\u003c/strong\\u003e Tracer injections in the RSPd and RSPagl show their connections with the AV.dorsal (RSPv/RSPagl↔AV.dorsal). \\u003cstrong\\u003ee.\\u003c/strong\\u003e Injections in the RSPv show their connections with the AV.lateral (RSPv↔AV.lateral). \\u003cstrong\\u003ef.\\u003c/strong\\u003e Multiple tracers per brain show the distinction among the AV domains. \\u003cstrong\\u003eg.\\u003c/strong\\u003e Retrograde tracer injection in the AV.dorsal (+AV.lateral) validates the POST/PRE/PAR→AV.dorsal and the RSPagl→AV.dorsal connection. A retrograde tracer primarily in the AV.medial (+medial tip) labels the SUBv (not the POST/PRE/PAR) and the RSPv (not RSPagl) corroborating the distinct SUBv/RSPv→AV.medial tip connections. \\u003cstrong\\u003eh.\\u003c/strong\\u003e AV.medial tip domain receives inputs from the SUBv (label in MM confirms SUBv injection site) and \\u003cstrong\\u003ei, \\u003c/strong\\u003efrom deep layers of RSPv. \\u003cstrong\\u003ej.\\u003c/strong\\u003e The SUBd provides input to the AV.lateral domain. SUBd→MM projections verifies the SUB injection location. \\u003cstrong\\u003ek.\\u003c/strong\\u003e Cre-dependent AAV injections in the SUBd (red) and SUBv (green) show their projections to distinct subregions of the AV (SUBd→AV.lateral; SUBv→AV.medial tip). AAVretro-Cre injection was in the AV. Projections from the SUBd and SUBv to the MM are shown to validate their injection locations\\u003csup\\u003e54\\u003c/sup\\u003e. l1. Phal AV injection labels caudal parts of the SUBv (AV→caudal SUBv), while l2, a retrograde tracer in the caudal SUBv validates the connection and demonstrates it to be specifically with AV.lateral (AV.lateral→caudal SUBv). \\u003cstrong\\u003em.\\u003c/strong\\u003e Wiring diagram summarizing connections of the AV domains. \\u003cstrong\\u003en.\\u003c/strong\\u003e Wiring diagram showing connections of the ATN with the SUB. See Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/21247ff81afadaa09d8c32aa.png\"},{\"id\":81964592,\"identity\":\"8a7ea806-c086-4a89-bcb7-fc135c43e8e4\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:25:50\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":18031658,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConnectivity of the AMd.dorsomedial and AMd.dorsal domains\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eTracer injections across the cortex and hippocampus label different domains within the AMd (ARA 61). Tracer labels were manually mapped onto the atlas template to highlight their distinction locations.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eb. \\u003c/strong\\u003eThe AM grids recolored to visualize the domains identified by the Louvain are shown, and the ROIs that each domain connects with are listed. Note the common connections of the AMd.medial and AMv with MPF areas, also evident in the raw data. \\u003cstrong\\u003ec. \\u003c/strong\\u003eThe matrix presents the identified Louvain groups (i.e., domains) and shows the injection sites that are grouped together based on the similarity of labeling they produce at each AM (ARA 61) grid box. A total of 16 cases (16 sections with AM labeling at ARA 61) were included in the analysis. Injection ROIs are indicated in the rows of the matrix. \\u003cstrong\\u003ed.\\u003c/strong\\u003eA Phal/CTB AM co-injection labels cells (layer VI) and terminals (layers I-V) in the rostral (left) and caudal (right) ACAv/ACAd suggesting a rostral/caudal ACA↔AMd connections. Dashed circles denote location of co-injections in e and f. \\u003cstrong\\u003ee. \\u003c/strong\\u003eA Phal/CTB co-injection made in the rostral ACA labels terminals and cells specifically in the AMd.dorsomedial domain (rostral ACA↔AMd.dorsomedial) and the AMv (rostral ACA↔AMv). \\u003cstrong\\u003ef. \\u003c/strong\\u003eA Phal/CTB co-injection made in the caudal ACA labels terminals and cells specifically in the AMd.dorsal domain (rostral ACA↔AMd.dorsal) and does not label AMv neurons.\\u003cstrong\\u003e g. \\u003c/strong\\u003eA Cre-dependent AAV injection that traces mostly AMd.dorsal (and dorsolateral) neurons (AAVretro-Cre injection made in ACA/RSP) shows projections only in the caudal ACA, confirming the caudal ACA↔AMd.dorsal connection.\\u003cstrong\\u003e h. \\u003c/strong\\u003eQuantified projections to the ACAd, ACAv, PL, and ILA from traced neurons in the AMd.dorsal domain (in g) validate their stronger projections to the ACAv compared to other ROIs like the PL and ILA.\\u003cstrong\\u003e i. \\u0026nbsp;\\u003c/strong\\u003eMultiple tracer injections across different ROIs in the same brain showcase the distinct AMd domains.\\u003cstrong\\u003e j. \\u003c/strong\\u003eSummarized connections of the AMd.dorsomedial (left) and AMd.dorsal (right).\\u003cstrong\\u003e k.\\u003c/strong\\u003e Rostral and caudal ACAv injections show that ACA connectivity is selectively with AM and not AV nor AD.\\u003cstrong\\u003e l.\\u003c/strong\\u003e This selective AM-ACA connection is also shown with tracer injections made in the AV and AMd of the same brain. Neither anterograde nor retrograde AV injections produce labels in the ACA, while the retrograde injection in the AMd labels neurons in the deep layers of ACA.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/b674f64464dbe811866c5875.png\"},{\"id\":81967581,\"identity\":\"263cf90f-c069-4cb9-a748-b91ee67d8479\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:41:49\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":10357471,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConnectivity of the AMd.dorsolateral and AMd.lateral domains\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eA Phal/CTB AM injection labels only fibers in the RSPv. Dashed circle denotes the location of a Phal/CTB co-injection in the RSPv in b. \\u003cstrong\\u003eb.\\u003c/strong\\u003e Phal/CTB co-injection back labels cells in the AMd.dorsolateral domain (AMd.dorsolateral→RSPv). Note the absence of fibers in the AMv. \\u003cstrong\\u003ec.\\u003c/strong\\u003e A Cre-dependent anterograde injection that tracers AMd.dorsolateral neurons labels the RSPv. \\u003cstrong\\u003ed.\\u003c/strong\\u003e Quantified projections to the PL, ILA, RSPv, PTLp, and MOs from these traced neurons in the AMd.dorsolateral domain show strongest projections to the RSPv compared to other regions. \\u003cstrong\\u003ee.\\u003c/strong\\u003e Wiring diagram of the AMd.dorsolateral domain. \\u003cstrong\\u003ef.\\u003c/strong\\u003eA Phal/CTB anterograde and retrograde co-injection labels terminals and cells in the PTLp. Dashed circle denotes location of co-injection in g. \\u003cstrong\\u003eg.\\u003c/strong\\u003e A Phal/CTB injection in the PTLp (caudal, medial part) labels terminals and cells in the AMd.lateral domain. \\u003cstrong\\u003eh. \\u003c/strong\\u003eCre-dependent AAV injections made in the AMd.medial tip (left: AAVretro-Cre in PL/ILA) and in the AMd.lateral (right; AAVretro-Cre in PTLp). Only traced neurons in the AMd.lateral label the PTLp, specifically the caudal medial part (AMd.lateral↔PTLp caudal medial). \\u003cstrong\\u003ei.\\u003c/strong\\u003e Bar graph shows the quantification of these projections to the PTLp, which are greater from traced AMd.lateral neurons versus those traced from the AMd.medial. \\u003cstrong\\u003ej.\\u003c/strong\\u003e Connections with the PTLp are exclusively through the AM. \\u003cstrong\\u003ek.\\u003c/strong\\u003eWiring diagram of the AMd.lateral connections. \\u003cstrong\\u003el. \\u003c/strong\\u003eAn AM CTB injection labels SUBv (caudal) cells. Dashed circle denotes location of an AAV injection made in the same SUBv caudal region that labels terminals in AMd.lateral (SUBv→AMd.lateral). \\u003cstrong\\u003em.\\u003c/strong\\u003e Multiple tracer injections in a single brain show the distinct AMd.lateral and AMd.dorsolateral domains. See Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/cc2c5753ca71dfcf125bf528.png\"},{\"id\":81965872,\"identity\":\"b127fd11-fe3a-4551-8131-9fd71cd6b122\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:33:49\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14728640,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConnectivity of the AMd. medial tip and AMd.ventromedial domains\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea.\\u003c/strong\\u003e The AMd.medial can be divided into the AMd.medial tip and AMd.ventromedial domains, each of which show distinct connections. \\u003cstrong\\u003eb.\\u003c/strong\\u003eAn AM CTB injection labels cell in the SUBv (top), PL (middle), and ILA (bottom). Dashed circles denote locations of Phal injections in c. \\u003cstrong\\u003ec.\\u003c/strong\\u003ePhal injections in the SUBv (top), PL (middle), and ILA (bottom) show their distinct connections with the AMd.medial tip and AMv (SUBv, PL, ILA→AMd.medial tip; PL/ILA→AMv). \\u003cstrong\\u003ed.\\u003c/strong\\u003eAn AM AAV injection labels fibers in the PL (top), ILA (middle), and BLA (bottom). Dashed circles denote location of retrograde tracer injections in e. \\u003cstrong\\u003ee.\\u003c/strong\\u003eRetrograde tracer injections in the PL (top), ILA (middle), and BLA (bottom) show their connections with the AMd.medial tip, AMd. ventromedial and the AMv (AMd.medial tip/AMv→PL; AMd.medial tip/ventromedial/AMv→ILA; AMd.medial tip→BLA). \\u003cstrong\\u003ef. \\u003c/strong\\u003eCre-dependent AAV injections traced AMd.medial tip neurons (green; AAVretro-Cre in MPF) or avoided those neurons and traced only those in the AMd.core (red; AAVretro-Cre in ACA/MOs). Only the AMd.medial tip traced neurons labeled terminals in the PL, ILA, and BLA validating those connections, but also showing the specificity of the domain level connections. \\u003cstrong\\u003eg.\\u003c/strong\\u003e A CTB AM injection (left) labels neurons in the ORBm. Dashed circle denotes location of the Phal and CTB ORBm injections in the right, which show labeled fibers and cells in the AMd.ventromedial domain (AMd.ventromedial↔ORBm). \\u003cstrong\\u003eh.\\u003c/strong\\u003e A Cre-dependent AAV injection that traces primarily AMd.ventromedial neurons (AAVretro-Cre made in the MPF) labels terminals in the ORBm and ILA validating those projections. \\u003cstrong\\u003ei.\\u003c/strong\\u003eDouble retrograde tracer injections in the PL and ILA in the same animal shows (1) the distinct AMd.medial tip and AMd.ventromedial domains; (2) the select AMd.ventromedial/AMv→PL; and (3) less selective AMd.medial tip/AMd.ventromedial/AMv→ILA projections. \\u003cstrong\\u003ej.\\u003c/strong\\u003eQuantification of projections to the PL and ILA from the Cre-dependent AAV injections that traced neurons in either the AMd.medial tip or the AMd.ventromedial validate the stronger AMd.medial tip→PL and the AMd.ventromedial→ILA connections. \\u003cstrong\\u003ek.\\u003c/strong\\u003e Wiring diagrams summarizing the connections of the AMd.ventromedial (top), AMd.medial tip (middle), and AMv (bottom) domains. See Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/7ee2334221bf21c3c21fe5de.png\"},{\"id\":81967580,\"identity\":\"f818aa00-916b-4617-9bfa-fddb4731737b\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:41:49\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":12068276,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConnectivity of the AMd.core domain, domain networks, and single neuron validations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eMultiple tracer injections per brain show the periphery/core organization of the AMd. \\u003cstrong\\u003eb. \\u003c/strong\\u003eConnectivity wiring diagram of the AMd.core domain. \\u003cstrong\\u003ec. \\u003c/strong\\u003eAnterograde and retrograde labels are present in the MOs-fef following AAV (left) and CTB (right) AM injections, suggesting AMd↔MOs-fef connections.\\u003cstrong\\u003e d.\\u003c/strong\\u003e A co-injection of Phal/CTB in the MOs-fef confirms the AMd↔MOs-fef reciprocity and reveals the connection to be specific to the AMd.core domain. Also note the periphery/core organization of the AMd. \\u003cstrong\\u003ee. \\u003c/strong\\u003eA Cre-dependent AAV tracer was used to trace mostly AMd.core neurons, which further validated its projections to the MOs-fef (left) (AAVretro-Cre made in ACA). Comparisons of projections from traced neurons in the AMd.core, AMd.ventromedial, AMd.medial tip/dorsomedial, and AMv shows strongest projections to the MOs are from the AMd.core injection (right). \\u003cstrong\\u003ef. \\u003c/strong\\u003eA Phal AM injection labels terminals in the ECT, PERI, and ENTl that are specifically to layer 5. This laminar specific projection is consistent across ARA levels (ARA 93 and 97 are shown). A retrograde tracer injection placed in layer 5 ENTl (dashed circle at ARA 97) back labels neurons in the AMd.core region validating the connection, but also showing that the AMd projections to ENTl are from AMd.core. \\u003cstrong\\u003eg.\\u003c/strong\\u003e A Cre-dependent AAV tracer injection in the AM that traces mostly AMd.core neurons (AAVretro-Cre made in ACA) shows similar layer 5 projections to ECT, PERI, and ENTl at ARA 95 and ARA 98. At ARA 95, the boxed region is enlarged to the right. \\u003cstrong\\u003eh. \\u003c/strong\\u003eA Cre-dependent AAV tracer injection that does not trace AMd.core neurons does not show labeling in the ECT, PERI, ENTl, validating the specificity of domain connections. \\u003cstrong\\u003ei. \\u003c/strong\\u003eQuantification of projections to ENTl, PERI, ECT, and ENTm from injections that trace mostly AMd.core, AMv, and AMd.dorsal/dorsolateral neurons. Note the strongest projections to ENTl, PERI, and ECT from AMd.core. Also note the lack of projections to ENTm. \\u003cstrong\\u003ej. \\u003c/strong\\u003eHierarchical clustering of projections from Cre-dependent AAV injections made in the medial (n=1) versus lateral (n=1) parts of the AMd validate their distinct connections. Note the strongest projections to ILA and PL are from the medial injection, while strongest projections to the RSPv (also RSPd, PTLp) are from the lateral injection.\\u003cstrong\\u003e k. \\u003c/strong\\u003eTop: Schematic of AM connections with ROIs that process visual/spatial information. The dashed gray line is an approximate division between the ventral-medial AMd domains (AMd.m) that connect with the more limbic regions like MPF, HPF, and amygdala (bottom left) and the visual dorsal-lateral AMd domains that connect with the visual/spatial processing areas like ACA, RSP, and PTLp (bottom right).\\u003cstrong\\u003e l. \\u003c/strong\\u003eThe results of hierarchical clustering of normalized ROI-based quantified data for Cre-dependently traced pathways from AMd.dorsal/dorsolateral, AMd.ventromedial, AMd.medial tip, AMd.core, and AMv (n=1 each) . The result is visualized in a matrix to show strength of AMd domain connections with namely, MOs, ORBl, PL, PTLp, ORBm, ORBvl, ILA, and RSPv. \\u003cstrong\\u003em. \\u003c/strong\\u003eAxon reconstructions of neurons in the AMd.medial, AMd.dorsal/dorsolateral, and AMd.core were performed and their normalized projections to the cortex are visualized. Of the three neurons traced, the AMd.medial neuron shows strongest projections to the PL and ILA, the AMd.dorsal/dorsolateral shows strongest projections to ACAv and RSPv, while the AMd.core neuron is the only one that projects to the MOs. These single neuron reconstructions correlate with the mesoscale connectivity data presented for each of those AMd domains.\\u003cstrong\\u003e \\u003c/strong\\u003eSee Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/24eaabc8a712beb398443a24.png\"},{\"id\":81963350,\"identity\":\"37b66da0-50dd-4b31-88d1-5a25718aada4\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:17:50\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":19579183,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMedial prefrontal, hippocampal, and amygdalar synaptic circuits through AMd.medial tip and AMv\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eInjection strategy used in MORF3 mice to validate the SUBv→AMd.medial→BLA disynaptic circuit. \\u003cstrong\\u003eb. \\u003c/strong\\u003eA synaptophysin tagged anterograde AAV injection made in the SUBv labels terminals in the AMd.medial tip, while an AAVretro-Cre injection in the BLA back labels neurons in the AMd.medial tip. \\u003cstrong\\u003ec. \\u003c/strong\\u003e60x magnification of a BLA projecting AMd.medial tip neuron. \\u003cstrong\\u003ed. \\u003c/strong\\u003eThe same AMd.medial tip→BLA neuron in c merged with labeled synaptic terminals from the SUBv. \\u003cstrong\\u003ee. \\u003c/strong\\u003eNote the close apposition of the neuron processes and terminals suggesting putative synaptic contacts of SUBv terminals onto the BLA projecting AMd.medial tip neuron. \\u003cstrong\\u003ef-g. \\u003c/strong\\u003ePutative contacts were reconstructed and validated via Neurolucida (see Methods for details). \\u003cstrong\\u003eh.\\u003c/strong\\u003e Injection strategy used in MORF3 mice to validate the SUBv→AMd.medial tip→PL disynaptic circuit. \\u003cstrong\\u003ei. \\u003c/strong\\u003eA synaptophysin tagged anterograde AAV injection made in the SUBv labels terminals in the AMd.medial tip, while an AAVretro-Cre injection in the PL back labels neurons in the AMd.medial tip. \\u003cstrong\\u003ej. \\u003c/strong\\u003e60x magnification of a PL projecting AMd.medial tip neuron. \\u003cstrong\\u003ek. \\u003c/strong\\u003eThe same AMd.medial→PL neuron in j merged with labeled synaptic terminals from the SUBv. \\u003cstrong\\u003el. \\u003c/strong\\u003eClose apposition of neuron processes and terminals suggests putative synaptic contacts of SUBv terminals onto the BLA projecting AMd.medial tip neuron. \\u003cstrong\\u003em. \\u003c/strong\\u003e\\u0026nbsp;Putative contacts were validated via Neurolucida same as in f-g. \\u003cstrong\\u003en. \\u003c/strong\\u003eThe same injection strategy in a and h was applied in MORF3 mice to validate the ILA→AMv→PL circuit. \\u003cstrong\\u003eo. \\u003c/strong\\u003eSynaptic contacts of ILA projections onto PL-projecting AMv neurons were reconstructed and validated. See Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/37a184833216056c6300607f.png\"},{\"id\":81964586,\"identity\":\"24666fc4-36f3-4d68-8719-b2e6b9b123a4\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:25:50\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":15769302,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eATN connections with hypothalamus, midbrain, and hindbrain\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea. \\u003c/strong\\u003eProjections from the mammillary bodies to the ATN. \\u003cstrong\\u003ea1. \\u003c/strong\\u003e\\u0026nbsp;A CTB injection in the AD labels neurons in the LM (and in the MMl from leakage of injection into the AV) to reveal the LM→AD connection. A Phal injection in the LM labels the AD validating the connection. \\u003cstrong\\u003ea2. \\u003c/strong\\u003eA CTB injection in the AV labels neurons in the MMl showing the MMl→AV connection, which is validated by an AAV injection in the MMl. \\u003cstrong\\u003ea3. \\u003c/strong\\u003eA CTB injection in the AM labels MMm neurons showing the MMm→AM connection, which is validated by an AAV injection in the MMm that labels the AMd and AMv. \\u003cstrong\\u003ea4. \\u003c/strong\\u003eA retrograde CTB injection in the IAD labels neurons in MM median (MMme) to reveal MMme→IAD connection (top). A Phal anterograde tracer injection in the MMme (middle) labels the IAD (bottom) validating the connection. \\u003cstrong\\u003eb. \\u003c/strong\\u003eSchematic summarizing ATN, MM, and LM connectivity. \\u003cstrong\\u003ec. \\u003c/strong\\u003ePhal and AAV tracers in the MMm and LM in the same brain show the MMm→AM and LM→AD connection and show the bilateral LM→AD compared to the unilateral MMm→AM projection. \\u003cstrong\\u003ed. \\u003c/strong\\u003eA Phal injection in the MMme shows bilateral projections to IAD (MMme→IAD). \\u003cstrong\\u003ee. \\u003c/strong\\u003eCre-dependent TVA receptor mediated rabies tracing of AMv and AMd.medial neurons retrogradely label the MMm validating the MMm→AMd/AMv connection shown in a3. Bottom panel is the quantification of retrogradely labeled cells in the MM versus LM following the rabies injections in the AMd.medial and AMv. In both cases, AAVretro-Cre was injected into the MPF to retrogradely deliver Cre only to the AM. The Cre-dependent TVA helper and the EnVA pseudotyped rabies viruses were injected into the AMd and AMv to trace their inputs (see Methods for details). \\u003cstrong\\u003ef. \\u003c/strong\\u003eAAV in the DTN (top), confirmed by the strong label in the LM (middle), labels axon terminals in the AD and AV (bottom). \\u003cstrong\\u003eg.\\u003c/strong\\u003e AAV in the VTN (top), confirmed by the strong label in MM (middle), labels axons in the AD and AV (bottom). \\u003cstrong\\u003eh.\\u003c/strong\\u003e Validation of the DTN/VTN→AD/AV connections. CTB injection in AM/AD labels cells in the VTN, while a FG AM/AV injection, validated by the MM labeled cells, shows labeled cells in DTN and LDT. \\u003cstrong\\u003ei.\\u003c/strong\\u003eDouble retrograde injections in AV (CTB, pink) and MM (CTB, green) both show labeled cells in the VTN (VTN→AV and MM) and in SUBv (SUBv→AV and MM). Note the labeled cells in the MMl, which validates the AV injection. Also, note laminar specific arrangement of the SUBv→AV and SUBv→MM projecting neurons. \\u003cstrong\\u003ej. \\u003c/strong\\u003eSchematic of connections among the ATN, DTN, and VTN. \\u003cstrong\\u003ek. \\u003c/strong\\u003eSchematic showing ROIs generally involved in the head directionality/visual-spatial processing (peach) and those involved in theta rhythmicity (blue). Generally, the two show segregated networks, e.g., AV←MM↔VTN (theta rhythm) and AD←LM↔DTN (head directionality). Our data show direct connections between VTN and AD/AV and DTN and AD/AV suggesting that these structures are in an interconnected network rather than segregated circuits. See Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/d740b71ee26808cf986ee876.png\"},{\"id\":81964570,\"identity\":\"bd2931f4-611c-40f3-b17a-c2c6af493f81\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:25:49\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14901467,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDendritic morphology analysis of ATN neurons\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRepresentative images of \\u003cstrong\\u003ea\\u003c/strong\\u003e AM, \\u003cstrong\\u003eb\\u003c/strong\\u003e AD, and \\u003cstrong\\u003ec\\u003c/strong\\u003e AV neurons within their demarcated borders (left panels) and their corresponding representative digital reconstructions (right panels). \\u003cstrong\\u003ed.\\u003c/strong\\u003e Example of MORF expressing neurons from the ATN neurons expressing various levels of signal brightness. \\u003cstrong\\u003ee.\\u003c/strong\\u003e Overall shape of the ATN with all the regions delimited by dashed borders. Representative dendritic arbors from edge (left panels) and non-edge (right panels) neurons in the \\u003cstrong\\u003ef,\\u003c/strong\\u003eAM, \\u003cstrong\\u003eg,\\u003c/strong\\u003e AD, and \\u003cstrong\\u003eh,\\u003c/strong\\u003e AV. \\u003cstrong\\u003ei.\\u003c/strong\\u003e Location and orientation of the edge (in bright green) and non-edge neurons (in aqua blue) from the AMd regions. \\u003cstrong\\u003ej.\\u003c/strong\\u003e Graphical definition of a compartment’s angular orientation relative to the local AMd core vector (a local AMd core vector is essentially the dashed straight lines that connect a compartment’s origin point to the AMd center). A decrease in the angle between a dendrite’s orientation and its local core vector means there is an increase in its tropism (or bias) toward the center of the AMd. \\u003cstrong\\u003ek.\\u003c/strong\\u003e Compartment deviation angle from the AMd center as a function of the somatic distance from the AMd center. The greater the distance of a neuron’s soma from the AMd center, the lesser its average compartment deviation (R = -0.63). The edge AMd neurons are at a greater distance from the AMd center compared to the non-edge neurons. \\u003cstrong\\u003el\\u003c/strong\\u003e. Hence, the edge neurons (n=27) showed reduced angular deviation from the AMd center compared to the AMd non-edge (n=45) neurons (t-test, p\\u0026lt;0.0001). Edge neurons have a higher proportion of dendritic compartments oriented toward the AMd center. Red colored dendrites have an acute angular deviation and are oriented towards the AMd center as opposed to the blue dendrites that have an obtuse angular deviation and are oriented away from the AMd center (right panel). \\u003cstrong\\u003em.\\u003c/strong\\u003e The edge neurons also had a higher proportion of (total) dendrite closer to the center than their individual cell bodies. Closer dendrites are colored in Pink, as opposed to blue dendrites that are further away (right panel). Taken together this suggests that the dendritic arbors of edge neurons demonstrate bias/tropism towards the AMd center, whereas those off the edge (more towards the AMd center) of the AMd do not show this tropism. For the boxplots, the line inside of each box is the sample median. The top and bottom edges of each box are the upper and lower quartiles, respectively. The distance between the top and bottom edges is the interquartile range (IQR). The upper quartile corresponds to the 0.75 quantile and the lower quartile corresponds to the 0.25 quantile. Outliers are values that are more than 1.5 · IQR away from the top or bottom of the box. Boxplot displays each outlier using an 'o' symbol. The whiskers are lines that extend above and below each box. One whisker connects the upper quartile to the nonoutlier maximum (the maximum data value that is not an outlier), and the other connects the lower quartile to the nonoutlier minimum (the minimum data value that is not an outlier).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/afe263390902b3169e41de79.png\"},{\"id\":81963314,\"identity\":\"84bca92a-93fe-4a52-ad66-8044a4610535\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:17:49\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":898157,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunctional ATN networks\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea.\\u003c/strong\\u003e Depiction of the classic Papez circuit. \\u003cstrong\\u003eb.\\u003c/strong\\u003e Network of structures involved in drug seeking behavior centered around the AMd.medial domains. Projections from the AM→BLAa are to a specific domain, BLA.ac, which is also connected with regions shown to be involved in drug seeking behavior (see discussion). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ec.\\u003c/strong\\u003e Functional neural network of the ATN. An extended ATN wiring diagram showing how each of the newly identified ATN domains connects with different areas within the medial cortical subnetwork\\u003csup\\u003e44\\u003c/sup\\u003e, which controls movements and the orientation of the eyes, head, and neck for attention, navigation, and exploratory behavior via the cortico-basal ganglia (SNr serves as the output portal) or through its cortico-tectal projections (SC serves as the output portal). Meanwhile, several ATN subnuclei (especially AMv, AMd.m, AMd.l, AVl, AVm and AVmt) connect with the MPF (ILA, PL, DP), BLAa, and SUBv. These three cortical areas are highly interconnected and generate massive descending projections to the medial amygdalar nucleus (MEA), central amygdalar nucleus (CEA), anterior (BSTa) and posterior (BSTp) bed nuclei of the stria terminalis, hypothalamus, and PAG to regulate neuroendocrine, autonomic, and goal-directed behavior associated with emotion (e.g., hunting or attacking). Note that the dorsal premammillary nucleus (PMd) generates dense projections back to the AMv\\u003csup\\u003e59\\u003c/sup\\u003e. Finally, these three cortical areas also generate dense projections to the ACB, which, together with the VTA, are essential for reward and addiction. The structures within the dashed box are included in the classic Papez circuit. As illustrated here, the ATN serves as a critical network hub, bridging communication between the medial cortical subnetwork and the \\\"emotional\\\" network to control goal-directed behavior. See Table 2 for structure abbreviations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"11.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/eb435a1feaf4b9f24d8f673a.png\"},{\"id\":85829546,\"identity\":\"ca3187b1-5a31-4dda-9a42-c73df38c7ed8\",\"added_by\":\"auto\",\"created_at\":\"2025-07-02 07:37:55\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":142866049,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/d45e9fac-1973-4ad7-83af-5dc93d500bfb.pdf\"},{\"id\":81963300,\"identity\":\"67b09b7c-b94f-4b72-967d-de536a1e52b3\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:17:49\",\"extension\":\"xlsx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":15215,\"visible\":true,\"origin\":\"\",\"legend\":\"Table 1\",\"description\":\"\",\"filename\":\"Table1.InjectiontableforATNconnectome.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/9fa006aab1d377aa2716e293.xlsx\"},{\"id\":81964560,\"identity\":\"665be741-cd57-4a5a-aba4-2381b559b23c\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:25:49\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":74391,\"visible\":true,\"origin\":\"\",\"legend\":\"Table 2\",\"description\":\"\",\"filename\":\"Table2.ListofabbreviationsATN.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/62cd5b47fae06489f2732c61.xlsx\"},{\"id\":81963313,\"identity\":\"08db7a36-e8cc-4037-9500-ea576adcd9c2\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:17:49\",\"extension\":\"pdf\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2408366,\"visible\":true,\"origin\":\"\",\"legend\":\"Reporting Summary\",\"description\":\"\",\"filename\":\"NCOMMS2463320Ars.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/dd694826f0daacd8552f7afd.pdf\"},{\"id\":81964567,\"identity\":\"d8909853-159a-460f-a938-f2a83badc719\",\"added_by\":\"auto\",\"created_at\":\"2025-05-05 11:25:49\",\"extension\":\"docx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":7379003,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ExtendedDataFigs.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4356188/v1/3c78d157add4ff04a00cbf2a.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Distinct subnetworks of the mouse anterior thalamic nuclei\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eThe classic Papez circuit, comprising the hippocampal formation, mammillary bodies, anterior thalamic nuclei, cingulate cortex, and parahippocampal cortices, was proposed as a foundation for emotional expression\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Building upon this, the concept of the limbic system or visceral brain was introduced, which incorporated additional structures like the amygdala, hypothalamus, and septum. Since then, this model has been revised to underscore the cognitive roles of these structures in episodic memory, attention, and spatial processes\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. Within this structural framework, the anterior thalamic nuclei (ATN) serve as a multifunctional hub, supporting diverse aspects of cognition in both animal models and humans\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe ATN consists of the anterodorsal (AD), anteroventral (AV), and anteromedial (AM) thalamic nuclei, the latter of which is further divided into dorsal (AMd) and ventral (AMv) compartments\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Together, the ATN support functions of memory and spatial navigation\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Pathology of the entire ATN culminates in diencephalic amnesia\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e, inactivation of the nuclei manifest in perturbations of spatial functions\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e, and elevated ATN activity can rescue spatial memory loss sustained following mammillothalamic tract insult\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Increasing evidence also suggests the ATN's contribution to age-related cognitive changes\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. Further, many hippocampal-dependent memory processes are shown to require a functional ATN emphasizing the role of the ATN in the hippocampal-diencephalic-cingulate network\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eBased on their unique connections, three parallel hippocampal ATN streams have been suggested\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e. Generally, the AD is implicated in head directionality because it harbors the largest number of head direction cells within the thalamus\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR17 CR18\\\" citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e and is nestled in a network whose components exhibit head direction specificity like the lateral mammillary nucleus (LM), dorsal tegmental nucleus (DTN), and the postsubiculum (POST) (DTN\\u0026rarr;LM\\u0026rarr;AD\\u0026harr;POST)\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. The AV exhibits theta rhythm oscillations and is interconnected with structures also implicated in theta rhythmicity imperative for spatial and non-spatial mnemonic functions\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR22\\\" citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. These structures include the medial mammillary nucleus (lateral part) (MMl), ventral tegmental nucleus (VTN), and the subiculum (SUB) (VTN\\u0026rarr;MMl\\u0026rarr;AV\\u0026harr;SUB)\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. More recent studies in mice have shown that through their connections with the retrosplenial cortical area (RSP), the AD and AV are involved in contextual memory and memory specificity, respectively\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. Importantly, the AV is implicated in age related decline in working memory that can be ameliorated through AV activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. Alternatively, the AM has been proposed to serve as a transmitter of integrated hippocampal-diencephalic information to the cortex for higher order processing\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e and in goal-directed behaviors\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e given its unique connections with medial prefrontal cortical areas like the prelimbic (PL) and infralimbic (ILA) areas\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. The AM in fact regulates emotional learning since its manipulation affects contextual fear responses\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR30\\\" citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e, suggesting more than just a relay role of the ATN nuclei\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eTogether, these studies stress the importance of attaining an intricate and complete connectivity roadmap of the ATN to reveal more granular, potentially functionally disparate, subnetworks. Although studies in rats have shown some segregation within the AM\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e and AV\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e, a systematic and extensive examination of ATN connectivity that can reveal the exceedingly granular subnetworks to account for ATN functional diversity is lacking. To achieve this, we traced approximately 200 sets of cortical, thalamic, hippocampal, amygdalar, striatal, midbrain, and hindbrain pathways in mice, constructing a comprehensive ATN wiring diagram. We identified connectionally unique cell types within the ATN, associated with seven parallel subnetworks within the AMd, four within the AV, and two within the AD, providing a structural basis for understanding the ATN\\u0026rsquo;s functional role as a network hub among the neocortex, hippocampus, amygdala, and hypothalamus. Combining genetic sparse labeling, brain clearing, and 3D microscopic imaging with advanced computational informatics, we systematically reconstructed and cataloged ATN neuron types based on their detailed neuronal morphology. To our knowledge, single-cell morphological assessments have not been systematically conducted for the ATN in any species. Finally, we validated synaptic connections of ATN neurons that facilitate communication among the MPF, SUBv, and BLAa.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCharacterizing ATN domain connectivity\\u003c/h2\\u003e \\u003cp\\u003eTo assess ATN connectivity, anterograde (Phal, AAV) and retrograde (CTB, FG, AAVretro-Cre) pathway tracers were placed across different cortical, hippocampal, amygdalar, thalamic, striatal, midbrain, and hindbrain areas (Table\\u0026nbsp;1; see Methods for details regarding each of these approaches and Discussion section, \\u003cem\\u003eCaveats to tracing experiments\\u003c/em\\u003e, regarding unique tracing and neurotropism characteristics of the tracers used). To determine the structure and approximate boundaries of domains within the AD, AV, and AM, representative experimental cases with distinct labeling in each domain were selected and sections across the ATN were annotated and analyzed. The sections were processed through our proprietary software Connection Lens\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR37 CR38\\\" citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Individual sections were matched and warped to their corresponding atlas level of the Allen Reference Atlas (ARA\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e) and subsequently the tracer labels were segmented (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea-b). Grid-based overlap annotation was performed in which the AD, AV, and AM were divided into 105 x 105-pixel square grids (equivalent to 63 \\u0026micro;m\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e) to tabulate labeling within distinct domains of the ATN nuclei (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb). The case specific annotations were then aggregated into a single matrix and Louvain community detection was conducted (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec). Grids were color-coded according to community assignment and reordered such that the resulting Louvain clusters were placed along the diagonal of the visualized matrix (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed).\\u003c/p\\u003e \\u003cp\\u003eTo validate domain-specific connections, Cre-dependent anterograde and TVA receptor mediated rabies tracing strategies were applied utilizing connectionally guided delivery of Cre. For these cases, sections spanning the whole brain were registered (warped) and tracer labels were segmented and annotated based on ROI. The annotated data underwent normalization and 2D hierarchical clustering to ascertain and visualize the distinct whole-brain projection patterns resulting from neurons traced in different ATN domains. A total of ~\\u0026thinsp;200 injections were used to generate connectivity diagrams and a global wiring diagram of the ATN (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee; \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://ucla-brain.github.io/atn/\\u003c/span\\u003e\\u003cspan address=\\\"https://ucla-brain.github.io/atn/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eReported connections underwent validation through at least one of the following methods. Injections targeting different regions were duplicated, and consistency across label patterns was manually evaluated. The Cre-dependent anterograde and TVA receptor mediated rabies also validated the connections. Further, retrograde tracers were introduced into regions displaying anterograde terminal labeling to confirm anterograde connections, whereas anterograde tracers were administered into the sites of retrogradely-labeled projection cells to validate retrograde injection data. Single neuron tracing experiments provided additional validation of select connections.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eGeneral ATN connections with the neocortex and subicular complex\\u003c/h3\\u003e\\n\\u003cp\\u003eThe AD, AV, and AM each are reported to have select connections with the cortex and hippocampus\\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. Multiple tracer injections placed within a single brain clearly demonstrate these segregated connections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef). Reciprocal communication between the medial prefrontal cortex (MPF) and ATN are specifically through the AM, while projections from ATN to the post- (POST), pre- (PRE), and para (PAR) subiculum are exclusively through the AD and AV, input from SUBd is exclusively to AV, and output to the striatum is solely through the interanterodorsal thalamic nucleus (IAD; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef). See Table\\u0026nbsp;2 for a list of structure abbreviations.\\u003c/p\\u003e \\u003cp\\u003eRegarding laminar specific connections, typically, ATN thalamocortical neurons are reported to show projections to cortical layers I, IV, and V and to all subicular layers\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. In turn, layer 6 corticothalamic neurons and deeper subiculo-thalamic neurons\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e project back to ATN. Our data also show this canonical corticothalamic and thalamocortical patterns of labeling (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg). An anterograde and retrograde tracer cocktail placed in the AM show layer I, IV, and V anterograde tracer labeling in the MPF, while layer VI neurons in the same regions are retrogradely labeled (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg). A similar injection in the AV shows similar laminar specific connections with the ventral retrosplenial cortical area (RSPv). Our data also show that projections from ATN to POST, PRE, and PAR spread across all 3 layers, while ATN projecting neurons in those same regions are in deep layer 3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg).\\u003c/p\\u003e\\n\\u003ch3\\u003eAD subnetworks\\u003c/h3\\u003e\\n\\u003cp\\u003eWithin the AD, we identified the AD.medial (ADm) and AD.lateral (ADl) domains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea). The AD projects to the dorsal (RSPd) and ventral (RSPv) retrosplenial cortical areas and to the POST, PRE, and PAR (Extended data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). AD neurons projecting to POST, PRE, and PAR are located primarily in the AD.medial (AD.medial\\u0026rarr;PRE/PAR/POST), while those projecting to the RSPd/v predominate the AD.lateral (AD.lateral\\u0026rarr;RSPd/v) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb-c; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb-d). The clear segregation of the AD.medial and AD.lateral neuronal populations is evident in a case in which retrograde tracers were placed in the RSPv and the PRE in the same animal (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed). While AD.lateral connections with RSP are reciprocal (AD.lateral\\u0026harr;RSPd/v) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb), AD.medial\\u0026rarr;POST/PRE/PAR connections are unidirectional (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee). This is clearly demonstrated with an anterograde and retrograde tracer co-injection in the PRE which results in retrogradely labeled neurons, but not anterogradely labeled axonal terminals, in the AD.medial (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec). No connection between the AD and SUB proper were detected (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg-h). Instead, those subicular connections were with distinct AV regions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg; see next section for details). This data updates the canonical AD circuits that connect with the RSP and subiculum (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ei).\\u003c/p\\u003e\\n\\u003ch3\\u003eAV subnetworks\\u003c/h3\\u003e\\n\\u003cp\\u003eFour domains with distinct connectional properties were identified within the AV. These were the AV.dorsal, AV.lateral, AV.medial, and AV.medial tip (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea). The AV shares strong reciprocal connections with the POST, PRE, and PAR (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb1, 3c1; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea). Our data show that these connections are specifically with the AV.dorsal (AV.dorsal\\u0026harr;POST/PRE/PAR) since anterograde and retrograde tracers in the POST, PRE, and PAR label terminals and neurons only this domain (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb2, 3c2; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb-d). Note the labeling in the mammillary bodies and the lateral dorsal thalamic nucleus supporting the precise location of the ATN and subicular injections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb). The AV is also reciprocally connected with the RSPd, RSPagl, and RSPv (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee). The connections with the RSPd and RSPagl are through the AV.dorsal domain (AV.dorsal\\u0026harr;RSPd/agl; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed), while those with RSPv are through the AV.medial domain (AV.medial\\u0026harr;RSPv; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef). These domain-specific connections of the AV.dorsal and AV.medial are clearly demonstrated in several ways. First, retrograde tracers placed in the PRE and RSPv in the same animal show segregated AV\\u0026rarr;PRE neuron population in the AV.dorsal and the AV\\u0026rarr;RSPv neuron types in the AV.medial (Fig.\\u0026nbsp;3f1). Second, multiple tracer injections also demonstrate the approximate border between the AV.medial and its dorsally adjacent AV.dorsal, and ventrally adjacent AV.medial tip, domains (Fig.\\u0026nbsp;3f2). Third, when retrograde tracer injections were made primarily in the AV.dorsal or AV.medial, labeled neurons in the POST/PRE/PAR and RSPd/agl were observed when the CTB injection was in the AV.dorsal, but not the AV.medial (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eg). A different AV domain, the AV.medial tip, receives input from the SUBv and deeper layers of RSPv (SUBv/RSPv_6\\u0026rarr;AV.medial tip; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eh-i), which is validated with a retrograde injection that also involves the AV.medial tip (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eg).\\u003c/p\\u003e \\u003cp\\u003eFinally, the AV.lateral is the domain where inputs from the SUBd predominantly target (SUBd\\u0026rarr;AV.lateral; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ej; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg). Double anterograde injections in the SUBd and SUBv in the same animal validate these connections and show the distinction between the AV.medial tip and AV.lateral (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ek; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eh-i). AV neurons that project back to the SUB were difficult to reveal given the sparse AV\\u0026rarr;SUB projections to a very specific region of the caudal SUB (Fig.\\u0026nbsp;3l1). Many retrograde tracer injections made across the SUB did not label neurons in the AV. Only one in the caudal SUBv labeled neurons in the AV.lateral, highlighting the specific AV\\u0026rarr;SUB connection (Fig.\\u0026nbsp;3l2). All the connections of the AV domains are summarized in a wiring diagram (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003em) and the unique connections of the ATN with the SUB are presented (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003en).\\u003c/p\\u003e\\n\\u003ch3\\u003eAM subnetworks\\u003c/h3\\u003e\\n\\u003cp\\u003eAnterograde and retrograde tracers placed across different cortical, amygdalar, and hippocampal regions revealed segregated neuron populations within the AM and the connectional distinctions between the AMd and AMv (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea). Computational analysis of the AMd connectivity data revealed 5 domains within the AMd (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb-c), namely the medial (AMd.m), dorsomedial (AMd.dm), dorsal (AMd.d), dorsolateral (AMd.dl), and lateral (AMd.l).\\u003c/p\\u003e \\u003cp\\u003eThe AM connects strongly with the dorsal (ACAd) and ventral (ACAv) anterior cingulate cortices (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed). The AMd.dorsomedial domain (AMd.dm) contains neurons that project to the \\u003cem\\u003erostral\\u003c/em\\u003e parts of the ACAd and ACAv (AMd.dorsomedial\\u0026rarr;rostral ACAd/ACAv) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea-b\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The AMd.dorsomedial also receives input from the rostral ACAd/ACAv, but this rostral ACAv/ACAd\\u0026rarr;AMd.dorsomedial connection is far weaker than the converse pathway (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee). These AMd.dorsomedial connections were validated with repeated rostral ACA injections (Extended Data Fig.\\u0026nbsp;3b1). Note that the AMv also shares reciprocal connections with the rostral ACAd/ACAv (AMv\\u0026harr;rostral ACAd/ACAv) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb-c).\\u003c/p\\u003e \\u003cp\\u003eThe AMd.dorsal (AMd.d) shares the strongest connections with the \\u003cem\\u003ecaudal\\u003c/em\\u003e ACAd/ACAv (AMd.dorsal\\u0026harr;caudal ACAd/ACAv) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef). These connections were validated with repeated caudal ACA injections (Extended Data Fig.\\u0026nbsp;3b2), but also through a Cre-dependent anterograde AAV injection that traced the output of mostly AMd.dorsal neurons (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eg-h) that showed labeled terminals in the caudal ACA, but none in the rostral ACA. No connections were detected between the caudal ACAd/ACAv and the AMv (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef; Extended Data Fig.\\u0026nbsp;3b2). Multiple tracer injections made in the same animal highlight the distinct AMd.dorsomedial and AMd.dorsal domains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ei ; see Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ej for a summary of AMd.dorsomedial and AMd.dorsal connections). Further, connections with the ACA in general are unique to the AM and no connection between the ACA and the AV or AD were identified (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ek-l; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec).\\u003c/p\\u003e \\u003cp\\u003eSome connections from the AMd to the RSP were detected (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea) and these connections were specifically through the AMd.dorsolateral domain (AMd.dl), which houses the neurons that project to the RSPv (AMd.dorsolateral\\u0026rarr;RSPv) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). This AMd.dorsolateral\\u0026rarr;RSPv connection was validated with repeated RSP injections (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed) and through a Cre-dependent anterograde injection that traced the output of primarily AMd.dorsolateral neurons (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec-d). No projections back to this domain, nor to any other AMd domain, from the RSPv were identified (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ee). Similarly, no connections between the AMv and RSP were detected (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed).\\u003c/p\\u003e \\u003cp\\u003eThe AMd is bidirectionally connected with the posterior parietal cortex (PTLp; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ef). The AMd.lateral domain (AMd.l) is shown to project to and receive input from the PTLp, specifically the caudal medial part (AMd.lateral\\u0026harr;PTLp caudal medial) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eg; see Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee for repeated PTLp injections). A Cre-dependent AAV anterograde injection that primarily traced the neurons of AMd.lateral neurons shows projections to the PTLp (caudal, medial), while a similar injection in the opposite side of the AMd shows no projections to the PTLp (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eh-i), validating this connectional specificity. The PTLp is not connected with AMv, the AD, nor AV (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ej). The AMd.lateral also receives input from the SUBv (AMd.lateral\\u0026larr;SUBv) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ek-l). Neurons in the AMd that project back to the SUB were difficult to find given the sparse AM\\u0026rarr;SUB projections and given that these projections are to a very specific region within the caudal SUBv (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea). A CTB retrograde tracer injection in this caudal SUBv region labeled neurons predominantly in the AMd.lateral (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb). Multiple tracer injections in the same brain highlight the AMd.lateral and AMd.dorsolateral domains relative to other AMd domains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003em).\\u003c/p\\u003e \\u003cp\\u003eThe fifth AM domain identified is the AMd.medial (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). Although undetected through the modularity maximization algorithm, there were two distinct zones within the AMd.medial domain: (1) the AMd.medial tip (AMd.mt) and (2) AMd.ventromedial (AMd.vm) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea).\\u003c/p\\u003e \\u003cp\\u003eThe AM shares strong connections with the MPF, SUBv, and amygdala as shown by anterograde and retrograde AM injections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb, d), which all occur either through the AMd.mt, AMd.vm, or the AMv. The AD and AV are not connected with the MPF nor the amygdala (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef). The AMd.medial tip receives inputs from the SUBv and some light input from PL and ILA (SUBv\\u0026rarr;AMd.medial tip; PL/ILA\\u0026rarr;AMd.medial tip), but strongly projects to the PL (AMd.medial tip\\u0026rarr;PL) and to the anterior basolateral amygdala (AMd.medial tip\\u0026rarr;BLAa) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb-e). These AMd.mt outputs were validated with a Cre-dependent anterograde AAV injection that primarily traced neurons in this domain (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ef), while their inputs were validated through TVA receptor mediated rabies tracing (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef; see also Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec-e for repeated injections).\\u003c/p\\u003e \\u003cp\\u003eThe AMd.ventromedial domain also receives light input from PL and ILA (none from the SUB) (PL/ILA\\u0026rarr;AMd.ventromedial) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec), has reciprocal connections with the medial (ORBm) and ventrolateral (ORBvl) ORB (AMd.ventromedial\\u0026harr;ORBm/vl) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eg-h), and strongly projects back to ILA (AMd.ventromedial\\u0026rarr;ILA) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ee; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee). Multiple injections made into the ILA and PL illustrate the distinct AMd.mt and AMd.vm neuronal populations (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ei; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed-e) and quantification of the Cre-dependent tracing from each of these populations validates the stronger AMd.mt\\u0026rarr;PL and AMd.vm\\u0026rarr;ILA connections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ej).\\u003c/p\\u003e \\u003cp\\u003eThe strongest input from the PL, ILA, and ORB to the AM are to the AMv (PL/ILA/ORB\\u0026rarr;AMv) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed-e) and the AMv has strong projections back to the PL (AMv\\u0026rarr;PL) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ee; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed-e). These output and input AMv connections were also validated with Cre-dependent AAV and TVA receptor mediated rabies tracing (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eg-i). Quantification of this data clearly shows the distinct AMv connections (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eh, j). Schematic summaries of these connections are provided (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ek; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ek).\\u003c/p\\u003e \\u003cp\\u003eA final AMd domain not identified through our computational analysis, but that was apparent from the tracing data, was the AMd.core. All domains discussed thus far predominate the peripheral region of the AMd, generally leaving its core region unoccupied by label (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea-c). This peripheral-core organization is most apparent with multiple tracer injections made in the same brain (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ea). The AMd.core shows reciprocal connections with the secondary motor cortex (MOs), specifically with the frontal eye field region \\u003cb\\u003e(\\u003c/b\\u003eAMd.core\\u0026harr;MOs.fef\\u003cb\\u003e)\\u003c/b\\u003e (ACAd adjacent\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eb-d). Cre-dependent AAV tracing of these AMd.c neuron outputs confirm strong AMd.core\\u0026rarr;MOs-fef connections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ee). The AMd.core also houses neurons that project to the lateral entorhinal cortex (AMd.core\\u0026rarr;ENTl) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ef-h). The AMd.core\\u0026rarr;MOs.fef and ENTl connections are unique to the AMd, and the AMd.core\\u0026rarr;ENTl projections are to a very specific region of the ENTl layer V (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ef-g). No connections with the medial entorhinal cortex (ENTm) were detected (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ef-g). Exclusive projections from the AMd.core were also observed to the deep layers (V/VI) of the caudal ectorhinal and perirhinal cortical areas (AMd.core\\u0026rarr;ECT/PERI caudal) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ef-g, i).\\u003c/p\\u003e \\u003cp\\u003eThe data from the domain-specific Cre-dependent anterograde tracing from the AMd.dorsal/dorsolateral, AMd.ventromedial, AMd.medial tip, AMd.core, and AMv were grouped and subjected to a 2D hierarchical clustering algorithm to show the specificity of connections between these domains with the (1) MOs, (2) ORB, (3) PL, (4) ILA, (5) RSP, and (6) PTLp (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003el). The output showed that neurons that project most strongly to the PL are in the AMd.medial tip, while those that project most strongly to the ILA are in the AMd.ventromedial domain validating those connections. The data nicely show the projections from the AMv to the PL, ILA, ORBm, ORBvl and show no projections from AMv to the ORBl. The data also show the projections from the AMd.core to the MOs and from the AMd.dorsolateral to the RSPv (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003el). Similar patterns emerge when data from injections administered in the broad AMd medial area compared to those in the AMd lateral area are analyzed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ej), showing a clear distinction between the AMd ventral-medial domains and dorsal-lateral domains, with the medial domains connected more with limbic network structures and the lateral domains with visual processing network areas (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ek).\\u003c/p\\u003e \\u003cp\\u003eFinally, axon terminations of singly traced neurons in the AMd.medial, AMd.dorsal/dorsolateral, and AMd.core also support the domain-specific results (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003em). A singly traced neuron from the AMd.medial showed greater axonal terminals in PL and ILA than neurons located more dorsal/dorsolaterally in the AMd. On the other hand, a neuron located more dorsal/dorsolaterally in the AMd showed more axonal terminations in the RSPv compared to the neurons located more medially and in the core region. Finally, only the neuron located more in the AMd.core showed axon terminations in the MOs compared to the neurons located more in the medial and dorsal/dorsolateral parts of the AMd (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003em).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAMd.medial and AMv serve as subnetwork hubs for subiculum and medial prefrontal cortical areas\\u003c/h2\\u003e \\u003cp\\u003eAs reported above, the AM domains connect with a specific set of cortical structures, including the SUBv and BLAa, suggesting a critical role of these AM domains as \\u003cem\\u003enetwork hubs\\u003c/em\\u003e for integrating and transmitting information among the cortex, hippocampus, and amygdala. For example,\\u003c/p\\u003e \\u003cp\\u003ethe AMd.medial tip domain receives input from the SUBv, and in turn, projects to the PL and BLAa. This suggests that the AMd.medial tip domain regulates communication between (1) the SUBv and the BLAa via a SUBv\\u0026rarr;AMd.medial tip\\u0026rarr;BLAa and (2) the SUBv and PL through a SUBv\\u0026rarr;AMd.medial tip\\u0026rarr;PL circuit.\\u003c/p\\u003e \\u003cp\\u003eWe utilized an AAV1-Cre based transsynaptic circuit mapping method\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e combined with a genetic sparse labeling reporter line, MORF3\\u003csup\\u003e46\\u003c/sup\\u003e to validate these disynaptic circuits. In MORF3 mice, neurons stochastically and Cre-dependently express a membrane-targeted V5 spaghetti monster protein, which can be visualized via immunostaining. This method reveals the complete detailed neuronal morphology of MORF3-labeled neurons including dendritic arborizations and spines. Therefore, in MORF3 mice, an AAV viral tracer expressing synaptophysin tagged with RFP was injected into the SUBv and an AAVretro-Cre injection was made into either the BLAa or the PL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ea, h). In this strategy, Cre is retrogradely transported from injection sites to the AMd-medial tip to trigger MORF3 expression, consequently revealing the detailed dendritic morphology of the AMd-medial tip neurons in their entirety, including dendritic spines. Meanwhile, the synaptophysin-tagged AAV labels the synaptic terminals of fibers originating in the SUBv and terminating in the AMd.medial tip (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eb, i-k). As such, this strategy reveals SUBv fibers potentially synapsing onto BLAa- or PL-projecting AMd.medial tip neurons. In both cases, putative contacts can be clearly seen from the SUBv onto PL- and BLAa-projecting neurons in the AMd.medial tip via the close apposition of terminals and dendrites (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ee, l). Synapse reconstructions further validated some of the potential contacts, substantiating the disynaptic circuits (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ef-g, m). The same strategy was applied to illustrate the disynaptic ILA\\u0026rarr;AMv\\u0026rarr;PL circuit (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003en-o). Altogether, these data suggest that these AM domains serve as network hubs to bridge communication among the MPF, ventral hippocampus, and amygdala.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eATN connections with the mammillary body and brainstem structures\\u003c/h3\\u003e\\n\\u003cp\\u003eThe ATN receive inputs from the mammillary nuclei in a topographically arranged manner: the lateral mammillary nucleus (LM) projects to the AD, the lateral part of the medial mammillary nucleus (MMl) to the AV, and the medial part of the MM (MMm) to the AMd and AMv (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003ea-b; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). Notably, the MMme targets specifically the IAD, a ventral extension of the AD situated dorsal to the AMd (Fig.\\u0026nbsp;9a1). Projections from the LM and MMme to the AD and IAD are bilateral, whereas those from the MMm to the AV or AM are ipsilateral (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ec-d). Projections from the mammillary nuclei to the ATN appear non-specific with respect to the newly identified domains. We applied Cre-dependent TVA receptor-mediated rabies tracing, which validated these connections and demonstrated the AMd- and AMv bi-synaptic connections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003ee). Specifically, a large AAVretro-Cre injection was made into the MPF that retrogradely delivered Cre to the AM. The AMd or AMv was then injected with a Cre-dependent TVA helper and EnVA-pseudotyped rabies viruses to trace their monosynaptic inputs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003ee). Dense rabies labeling is shown in the MMm, confirming a bi-synaptic MMm\\u0026rarr;AMd/AMv\\u0026rarr;MPF pathway (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003ee).\\u003c/p\\u003e \\u003cp\\u003eIt is well established that the AD is embedded within a network processing head directionality, including the dorsal tegmental nucleus (DTN) pathway: DTN\\u0026rarr;LM\\u0026rarr;AD\\u0026harr;POST/PRE/PAR. In contrast, the AV is part of a network involved in theta rhythmicity and the ventral tegmental nucleus (VTN): VTN\\u0026rarr;MMl\\u0026rarr;AV\\u0026harr;SUB. Our anterograde tracer injections in the DTN and VTN confirmed their established connections with the LM (DTN\\u0026rarr;LM) and MM (VTN\\u0026rarr;MMl), while also revealing unexpected direct connections from the VTN and DTN to the AD/AV (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ef-g), which were validated through retrograde tracing (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eh-i). These findings summarized in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ej, provide novel insights into a potential crosstalk between two relatively segregated subnetworks: one involved in head directionality and visual-spatial processing (peach), and the other associated with theta rhythmicity (blue) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ek).\\u003c/p\\u003e \\u003cp\\u003eWe also identified a less-documented direct projection from the dorsal raphe nucleus (DR) to the AD and AV (DR\\u0026rarr;AD/AV), providing serotonergic input to the ATN (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb-c). Combining CTB AD/AV retrograde tracing and immunostaining, we confirmed that the majority of AD/AV projecting DR neurons co-localize with tryptophan hydroxylase 2 (TPH2), a protein marker of serotonigic neurons (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed). The AV and AM, but not AD, also receive cholinergic inputs from the laterodorsal tegmental nucleus (LDT\\u0026rarr;AV/AM) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ee-f).\\u003c/p\\u003e \\u003cp\\u003eLastly, our tracing revealed an unreported neural network involving the IAD. Anterograde tracer injections in the AMd occasionally labeled axon terminals in the dorsomedial caudoputamen (CP) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea-b). Retrograde tracer injections into the dorsomedial CP labeled neurons exclusively in the IAD, not the AM (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea-c), indicating strong IAD\\u0026rarr;CP connections. This was further validated by Cre-dependent anterograde tracers injected into the AMd: one involving the IAD and one excluding it. Only the injection that traced IAD neurons labeled the dorsomedial CP (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed-e). Projections from the IAD to the CP are to specific domains that integrate visual and spatial information\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ef). Sparse connections also were observed between the superior colliculus and the IAD (SC\\u0026rarr;IAD) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eg). As aforementioned, the IAD also receives inputs from MMme (Fig.\\u0026nbsp;8a4). Together, these data suggest that the IAD transmits spatial (MM) and visual (SC) information to the dorsomedial CP (MMme/SC\\u0026rarr;IAD\\u0026rarr;CPi.dm/CPc.d), which also receives direct inputs from visual, spatial, entorhinal cortices implicated in coordinating eye, head and neck movement for goal directed behavior\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eh).\\u003c/p\\u003e\\n\\u003ch3\\u003eATN dendritic morphology\\u003c/h3\\u003e\\n\\u003cp\\u003eDendrites are crucial in how neurons receive and integrate signals from other cells. The shape and structure of dendrites influence signal processing and determine connectivity patterns within neural circuits, while the scope of dendritic arbors defines the size of a neuron's receptive field. Dendritic morphology is also a key characteristic that distinguishes different neuronal types, although this information is limited for ATN. We integrated MORF3 genetic sparse labeling with brain clearing and three-dimensional microscopic imaging to obtain brain images through the ATN. These images showcased sparsely labeled excitatory ATN projection neurons with intricately detailed dendritic morphology (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ea-d), which were then digitally reconstructed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ee). This approach enabled us to systematically characterize the morphological attributes of AD, AV, and AM domain neurons using computational methods (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ee-h).\\u003c/p\\u003e \\u003cp\\u003eStatistical comparisons of dendritic morphological features were carried out for a total of 109 neurons (10 for AD, 17 for AV, and 82 for AM). The results showed that AM dendritic arbors had greater dendritic length and higher number of branches than both AD (Wilcoxon signed rank test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 for both length and number of branches) and AV dendritic arbors (Wilcoxon signed rank test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 for length and p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 for number for branches) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ea-b).\\u003c/p\\u003e \\u003cp\\u003eWhile AD and AV neurons did not differ much in length, AD neurons had greater branching asymmetry (i.e., disproportionate distribution of dendritic tips between the two daughter branches at branch points) than both AM and AV neurons (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ec). While AD neurons had reduced average branch order (a measure of complexity, where the branch order of every compartment in a dendritic tree is averaged) compared to AV neurons (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), AD neurons had increased height (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and increased maximum dendritic path length (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) compared to neurons from AV (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ed-f). Due to this asymmetric dendritic extension, the AD neurons matched AM neurons in height and maximum path distance, even though AM neurons were much larger and more complex (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ee-f). The average Sholl intersection profile also showed that the AV neurons had a higher number of intersections closer to soma compared to the AD neurons which extended out to a greater path distance away from the soma, matching the AM neurons in extension while being much lower in the number of intersections per concentric circle drawn at 50 \\u0026micro;m intervals (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eg). Comparative morphometrics suggest that AD neurons send out their dendritic arbor over specific directions in an asymmetric manner, whereas the AV neurons have a higher branch density closer to the cell-body and sample the dendritic field more uniformly.\\u003c/p\\u003e \\u003cp\\u003eWe also classified all neurons from ATN based on dendritic arbor size (total dendritic length) and complexity (total number of branches). All the ATN neurons could be distinguished into two morphological types using K-means clustering. Type 1 neurons (84 out of 109) included relatively smaller neurons with lower numbers of branches (average dendritic length 3645\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1428 \\u0026micro;m, average number of branches: 51\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;16) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eh). Type 2 (25 neurons) included larger and more branched neurons (average dendritic length 8976\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1998 \\u0026micro;m, average number of branches: 126\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;27) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eh). Previous studies have identified two types of thalamic projection neurons based on prominent morphological features such as size and complexity. The two morphological types are bushy and radiate neurons\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR48 CR49\\\" citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e. Comparing previous neuron types with the current study suggests that radiate neurons with reduced numbers of branches could be considered analogous to the Type 1 neurons. The bushy neurons, with greater length and more branches are like the Type 2 morphology. All Type 2 neurons belonged to the AM region, whereas Type 1 neurons were from all three ATN nuclei (AD, AM, and AV). Finally, we also observed the grape-like appendages on the projection neurons that have been reported in the literature\\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR52\\\" citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ei). Altogether, these data suggests that neurons within each of the ATN display distinct morphological features.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEdge versus non-edge AMd neuron morphology\\u003c/h2\\u003e \\u003cp\\u003eWe initially observed that neurons with somas lying predominantly on the edges of the AMd displayed a distinct morphological appearance (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ei). The edge neurons displayed relatively less dendritic wiring and the dendrites seemed to have an orientation bias towards the center of the AMd (center-tropism). To quantify and test these qualitative observations, we first assigned each neuron as edge or non-edge based on their soma location within the AMd region. We then compared the quantified morphological features between the two groups.\\u003c/p\\u003e \\u003cp\\u003eWe observed that edge neurons are generally smaller (reduced dendritic length, t-test, p\\u0026thinsp;=\\u0026thinsp;0.022) than the neurons with somas located towards the center (non-edge) of the AMd (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ej). To evaluate and quantify the orientation bias (towards the AMd center) of the dendritic arbors, we calculated two new features: (a) average angular deviation from the AMd center (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ej-l) and (b) proportion of dendrites closer to the AMd center than the soma (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003em). We measured each dendritic compartment\\u0026rsquo;s angular deviation relative to the local AMd center vector (i.e., the vector connecting the compartment\\u0026rsquo;s origin point to the AMd center, see Method for details) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ej). Averaging that value across all dendritic compartments gives us the average angular deviation of the whole neuron. The inverse of the average angular deviation gives us an estimate of the neuron's AMd bias toward the center (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ek). In other words, the smaller the average angular deviation, the greater the orientation bias towards the AMd center. We observed that edge neurons had significantly lower average angular deviation compared to the non-edge neurons (t-test, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.0001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003el).\\u003c/p\\u003e \\u003cp\\u003eWe also measured, for each neuron, the proportion of its total dendritic wiring that is closer to the AMd center than the neuron\\u0026rsquo;s soma (see Method for details). Similarly, as the branch orientation result suggested, the edge neurons had a higher proportion of dendritic wiring closer to the center than the soma compared to the non-edge neurons (t-test, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.0001), demonstrating an AMd center bias (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003em). Overall, we observed that the average angular deviation of neurons is inversely correlated with the distance from the AMd center (Pearson\\u0026rsquo;s correlation coefficient=-0.65, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), i.e., neurons lying further away from the AMd center demonstrated greater tropism towards the AMd center (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ek).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAMd domain level morphological comparison\\u003c/h2\\u003e \\u003cp\\u003eNeurons from the five AMd domains (core, dorsal, dorsomedial, lateral and medial) also demonstrated distinct levels of center bias, both in terms of average angular deviation (omnibus Kruskal-Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.012) and proportion of dendrites closer to core than soma (omnibus Kruskal-Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.015) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ek,l). Pairwise comparison between domains showed that AMd.core neurons demonstrated a higher average angular deviation (lower AMd center-bias) relative to their local AMd center vectors compared to AMd.dorsomedial and AMd.medial domains (FDR-corrected Wilcoxon signed rank test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003ek). Both the AMd.core and the AMd.dorsal neurons had a relatively lower proportion of dendrites closer to the center (lower AMd center-bias) compared to the AMd.medial neurons (FDR-corrected Wilcoxon signed rank test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003el).\\u003c/p\\u003e \\u003cp\\u003eAMd domains also demonstrated distinct levels of average arbor height (omnibus Kruskal-Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.004) and maximum path length (omnibus Kruskal-Wallis test, p\\u0026thinsp;=\\u0026thinsp;0.0004). AMd.dorsomedial neurons had reduced height, compared to core, lateral, and medial neurons. Medial neurons also showed greater height than dorsal neurons (FDR-corrected Wilcoxon signed rank test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003em). Both medial and lateral neurons had greater maximum dendritic path distance (path distance of the dendritic terminal that is furthest away from the soma) than both dorsal and dorsomedial neurons (FDR-corrected Wilcoxon signed rank test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003en). The fact that medial and lateral domain neurons that showed greater bias toward the center (i.e., showed lower angular deviation and higher proportion of dendrites closer to the soma) also tended to have greater height and maximum path length, corroborated the initial qualitative observation that neurons lying away from the AMd center tend to look stretched.\\u003c/p\\u003e \\u003cp\\u003eLike AM edge and non-edge neurons, AD and AV neurons whose somas were located at the edges of the nuclei qualitatively displayed a morphological appearance (i.e., stretched) that differed from their non-edge counterparts (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eg-h). These distinct features were not quantified due to the low numbers of reconstructed neurons in each category for each nucleus.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003e\\u003cu\\u003eSummary of findings and overview of the ATN functional neural network\\u0026nbsp;\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe ATN are a key component of the classic Papez circuit (Fig. 11a), which has influenced our understanding of the neural basis of emotion and memory. Over time, the Papez circuit has evolved from a simple model of emotional processing to a more complex framework that integrates cognitive functions. Modern research has emphasized the critical roles of these structures in not only emotion, but also episodic memory, attention, and spatial processes (for review, see \\u003csup\\u003e2,3\\u003c/sup\\u003e).\\u0026nbsp;Accordingly, we present an updated and expanded version of the Papez circuit that subserves cognition and emotion (Fig. 11a, c). Specifically, we show that distinct ATN domains act as network hubs, bridging communications between (1) the cortico-basal ganglia system, which controls spatial orientation, navigation, and goal-directed behaviors\\u003csup\\u003e44\\u003c/sup\\u003e, and (2) the classic limbic system (such as the SUBv, amygdala, MPF), which regulates neuroendocrine, autonomic and social behavior associated with emotion\\u003csup\\u003e54,55\\u003c/sup\\u003e. These interactions are mediated through several major pathways, as discussed below, and depicted in a wiring diagram in Fig. 11c.\\u003c/p\\u003e\\n\\u003cp\\u003eBoth AD domains, the AD.medial and AD.lateral, receive direct inputs from the LM and DTN. While the AD.lateral shares reciprocal connections with the RSPd and RSPv, the AD.medial densely projects to the PRE, POST, and PAR, which, in turn, project back to the LM, to complete a circuit loop with the AD and DTN. Each structure within this network houses head-direction cells\\u003csup\\u003e17\\u003c/sup\\u003e, pivotal for spatial orientation.\\u003c/p\\u003e\\n\\u003cp\\u003eThe AV subdivisions (AV.lateral, AV.dorsal, AV.medial, and AV.medial tip) are densely connected with the MM and VTN, forming a central network that regulates theta rhythmicity, to synchronize activity across structures to facilitate communication relevant for learning and memory\\u003csup\\u003e22,56\\u003c/sup\\u003e. The AV.dorsal shares reciprocal connections with the subicular complex (PRE, PAR, POST) and with RSPd and RSPagl. The AV.lateral receives input from the SUBd, but generates projections to the SUBv, which in turn projects back to the AV.medial and AV.medial tip. These latter AV domains share reciprocal connections with the RSPv, a critical component of the \\u003cem\\u003emedial cortical network\\u003c/em\\u003e\\u003csup\\u003e44\\u003c/sup\\u003e that also receives direct inputs from the SUBd. \\u0026nbsp;The AV also receives input from the LDT nucleus, implicated in REM sleep, attention, and reward processes.\\u003c/p\\u003e\\n\\u003cp\\u003eWe identified five distinct subdomains within the AMd, each also densely interconnected with parts of the \\u003cem\\u003emedial cortical network\\u003c/em\\u003e\\u003csup\\u003e44\\u003c/sup\\u003e, including the RSPv, PTLp, ACAv, ACAd and its adjacent MOs-fef. Thus, information processed regarding head direction, theta rhythm, attention, and REM sleep from the ATN converge onto the\\u003cem\\u003e\\u0026nbsp;medial cortical network\\u003c/em\\u003e, within which this information is integrated with other external environmental cues including visual, auditory, and somatosensory inputs, and higher-order associative cortical information\\u003csup\\u003e44\\u003c/sup\\u003e. These \\u003cem\\u003emedial neural network\\u003c/em\\u003e cortical areas project densely to the SC, zona incerta (ZI), and CPdm, which projects to the ventromedial division of the substantia nigra pars reticulata (SNr), and the SNr in turn projects to the medial SC\\u003csup\\u003e37,57\\u003c/sup\\u003e. Together, these regions establish a core neural network regulating eye, head, and neck movements\\u003csup\\u003e36\\u003c/sup\\u003e that are important for attention, spatial orientation, navigation, and exploratory behavior.\\u003c/p\\u003e\\n\\u003cp\\u003eFurther, the AMd.medial and AMv are closely linked with the MPF (PL, ILA), SUBv, and BLAa—three limbic cortical areas that regulate social behavior and emotion-related activities through their projections to: (1) The medial amygdalar nucleus (MEA) and the posterior part of the bed nuclei of the stria terminalis (BSTp), which generate dense projections to the hypothalamic medial behavioral control column composed of the anterior hypothalamic (AHN), ventromedial hypothalamic (VMH), and dorsal premammillary nuclei (PMd)\\u003csup\\u003e58\\u003c/sup\\u003e. This hypothalamic subnetwork projects extensively to the dorsal PAG (PAGd), governing goal-directed behaviors with strong emotional components, such as hunting and attacking\\u003csup\\u003e58\\u003c/sup\\u003e, which require attention and navigation during their execution. Notably, the PMd, which functions as a threat detector by sensing dynamic changes under threatening conditions as the animal approaches and avoids the threatening source\\u003csup\\u003e59\\u003c/sup\\u003e, projects densely back to the AMv, which is crucial for updating memory processes to adapt to changes under threatening conditions. (2) The central amygdalar nucleus (CEA) and anterior BST (BSTa), which are involved in the regulation of autonomic and neuroendocrine activities\\u003csup\\u003e58\\u003c/sup\\u003e. (3) The nucleus accumbens (ACB), which, along with the ventral tegmental area (VTA), controls reward mechanisms and addiction.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eAMd.medial and AMv as communication subnetworks for MPF, HPF, and amygdala\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA network of structures including the cortex, hippocampus, and thalamus are critical for the formation of new memories. The ATN play a critical role in this network given their strong connections with the cortex and hippocampus. This is especially true for the AM since this is the only ATN nucleus connected with both the hippocampus and MPF. The AMd also connects with the BLA, shown here to be specifically the BLAa (Fig. 6f). Importantly, cortical, hippocampal, and amygdalar connections are relevant for the formation of emotional memories including fear-related behavior\\u003csup\\u003e60\\u003c/sup\\u003e. In fact, AM lesions disrupt contextual fear responses\\u003csup\\u003e29,30\\u003c/sup\\u003e. We found a specific subnetwork within the AMd through which these cortico-hippocampal-amygdalar structures communicate. These were namely the SUBv→AMd.medial→PL/ILA and SUBv→AMd.medial→BLAa (Fig. 8a, h). Completing this network are the strong PL/ILA↔BLAa reciprocal connections\\u003csup\\u003e38\\u003c/sup\\u003e. These connections clearly show AMd.medial as a hub for cortico-hippocampal-amygdalar interactions.\\u003c/p\\u003e\\n\\u003cp\\u003eIn addition, we show that the AMv is strongly connected with MPF areas like the rostral ACA, PL, ILA, and ORB (Fig. 6k) and the ILA→AMv→PL circuit was specifically shown at the synaptic level (Fig. 8n). These AMv connections are also likely relevant in the formation of emotional memories given the demonstrated role of corticotropin-releasing factor containing AMv neurons in the regulation of fear conditioning\\u003csup\\u003e31\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eNotably, AM–MPF connections are linked to goal directed behaviors involving intracranial self-stimulation\\u003csup\\u003e27\\u003c/sup\\u003e. Specifically, stimulation of MPF terminals in the AM activates midbrain dopaminergic neurons in the ventral tegmental area (VTA) and reinforces intracranial self-stimulation. A similar outcome results from stimulation of AM terminals in the MPF. Inhibition of these VTA neurons reduces the self-stimulation facilitated by the MPF-AM connections. This finding is interesting in the context of a wider AM network, specifically for the AMd.medial subnetworks (Fig. 11a).\\u0026nbsp;The projections from the AMd.medial tip to the BLAa are to its caudal domain (BLA.ac) (Fig. 6f), which is shown to be in a network of structures involved in drug seeking behavior\\u003csup\\u003e38\\u003c/sup\\u003e (Fig. 11b). These structures include the SUBv, ILA/PL, medial accumbens (ACB), medial olfactory tubercle (OT), rostral paraventricular thalamic nucleus (PVT), and CA3. Fig. 11c also demonstrates the wider network of the MPF and AMd.medial that lead to the VTA. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eDirect connections between the ATN, DTN, and VTN\\u0026nbsp;\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwo parallel circuits DTN↔LM→AD and the VTN↔MM→AM/AV are respectively involved in the head direction system and in theta rhythm activity. A surprising finding was direct connections between the midbrain nuclei and the AD and AV. We found that the DTN and VTN show slight connections with both the AD and AV, connections that were validated with multiple retrograde tracer injections placed across the ATN (Fig. 8f-g). Given the reported highly segregated tegmental-mammillary pathways, this result was unexpected and suggests some level of convergence across the two processing streams. Within the context of confluence, these direct connections are less surprising since the idea of head directionality and theta rhythmicity coalescing with the ATN is also suggested by the presence of head direction cells in the AV\\u003csup\\u003e16,18\\u003c/sup\\u003e. Interactions between head directionality and theta rhythmicity also seem intuitive: the synchronization of activity across regions involved in head directionality would facilitate an animal’s ability to learn and retain spatially pertinent information essential for navigation. \\u0026nbsp;While no direct connections between ATN and VTN/DTN have been officially documented, there is a study in humans that potentially lends support for this\\u003csup\\u003e61\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eATN connectivity reported in our findings and the current body of literature\\u0026nbsp;\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStudies across different species have identified mammillary body and hippocampal connections primarily exclusive to AM, AD, and AV that form parallel, segregated loops across the Papez circuit subserving distinct functions in navigation\\u003csup\\u003e1,9\\u003c/sup\\u003e. Projections from distinct regions of the medial (MM) and lateral (LM) mammillary nucleus to each of the ATN nuclei are well documented in rats and non-human primates, with the MMm projecting mostly to AM\\u003csup\\u003e62,63\\u003c/sup\\u003e, the MMl to AV\\u003csup\\u003e64\\u003c/sup\\u003e, and the LM to AD\\u003csup\\u003e63,65\\u003c/sup\\u003e. Projections from the MM medianus (MMme) to IAM are also reported\\u003csup\\u003e15\\u003c/sup\\u003e. Our data show these precise MB→ATN connections, although we also show MMme→IAD projections (Fig. 9a-e).\\u003c/p\\u003e\\n\\u003cp\\u003eAside from the mammillary body projections to ATN, many inconsistencies are reported regarding ATN connectivity, likely due to the different methods, species, injection sizes, injection site locations, and even different anatomic nomenclature that have been utilized across the studies. Hippocampal connections with individual ATN nuclei are similarly segregated with many conflicting connections reported. Some show AD projecting to postsubiculum (POST) and AV and AM projecting to proximal (near CA1) and distal subiculum, respectively\\u003csup\\u003e66,67\\u003c/sup\\u003e,\\u0026nbsp;while others show PRE/SUB→AM/AV and POST/PAR↔AV\\u003csup\\u003e15\\u003c/sup\\u003e. POST/PRE/PAR↔AV/AD, AM→PRE, and AD↔hippocampus connections also have been reported\\u003csup\\u003e68\\u003c/sup\\u003e.\\u0026nbsp;Our collective data of ~200\\u0026nbsp;traced pathways showed specifically (1) AD.medial→POST/PRE/PAR with no projections back to AD (Fig. 2b, e-f); (2) AV.dorsal↔POST/PRE/PAR (Figs. 2f; 3b-d); and (3) no connections between the AM and POST, PRE, and PAR (Fig. 1f). Regarding the SUB, we showed (1) no connections between SUB and AD (Fig. 2g); (2) SUBv→AV.medial tip (Fig. 3k) and SUBd→AV.lateral→SUBv (Fig. 3j-k); and (3) SUBv→AMd.medial (Fig. 5b-c). No connection between the ATN and the CA1, CA2, CA3 were found.\\u003c/p\\u003e\\n\\u003cp\\u003eDifferent combinations of connections have been reported between the ATN and ENT like ENT→AM\\u003csup\\u003e42\\u003c/sup\\u003e, AM→ENT\\u003csup\\u003e33,34\\u003c/sup\\u003e, and AD/AV/AM→ENT\\u003csup\\u003e28\\u003c/sup\\u003e. We only found connections between the AM and ENT. We placed many anterograde and retrograde injections across the ENTl and ENTm (Table 1). The large majority of ENTl injections did not produce any labeling in the AM. This was because the AMd projects to layer 5 of a very specific ENTl region (Fig. 7f-g). Once a retrograde tracer injection was successfully placed in that ENTl region, some AMd.core neurons were labeled (Fig. 7f). Our anterograde and retrograde injections in the AM did not produce any labeling in the ENTm despite reports of these connections in the literature\\u003csup\\u003e33\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eATN connections with cortical regions are reported to be separable with the ventral (granular) RSP (RSPv or RSPg) connecting mostly with AD and AV, the dorsal or agranular RSP (RSPd or RSPagl) with the AM\\u003csup\\u003e67,69,70\\u003c/sup\\u003e and the prelimbic area (PL) and orbital (ORB) with the AM\\u003csup\\u003e28,34\\u003c/sup\\u003e. Our data showed (1) RSPd/v↔AD.lateral (Fig. 2c-d); (2) RSPd/agl↔AV.dorsal (Fig. 3g;\\u0026nbsp;Extended Data Fig. 2e) (3) RSPv↔AV.medial (Fig. 3e, g); and (4) RSPv←AMd.dorsolateral (Fig. 5a). Sparse RSP-AM connections were detected and AM connections with ACA were exclusive despite reported ACA connections with AV and AD (Fig. 4k)\\u003csup\\u003e28,42,71\\u003c/sup\\u003e. Specifically, AMd.dorsomedial/AMv↔rostral ACA and AMd.dorsal↔caudal ACA (Fig. 4k; Extended Data Fig. 3b) were found. The exclusive AM-ACA connections reinforce the AM’s role in emotional learning given that the ACA is involved in emotional processing, fear acquisition memory, and in the control of innate fear responses\\u003csup\\u003e72-74\\u003c/sup\\u003e. Further, a rostral versus caudal distinction of the ACA is generally accepted, with distinct respective roles in emotional versus cognitive processing\\u003csup\\u003e75-77\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eOur data generally agree with the literature regarding connections of the PL, ILA and ORB being exclusive to AM; however, our data greatly expand upon this by demonstrating AM domain level connections with these medial prefrontal cortical areas, and also the dissociable connections of the AMd and AMv, which have not been extensively reported\\u003csup\\u003e31\\u003c/sup\\u003e. We show (1) AMd.medial tip/AMv↔PL; (2) AMd.ventromedial/AMv↔ILA; and (3) AMd.ventromedial/AMv↔ORBm/vl (Fig. 6b-d; g-h. We did not find any significant AMv projections to BLA, RSP, ECT, PERI, ENTl, nor to CA1, which have been reported for AMv CRH neurons\\u003csup\\u003e31\\u003c/sup\\u003e, and to our knowledge a study dissociating the brain-wide inputs to the AMv has not been conducted.\\u003c/p\\u003e\\n\\u003cp\\u003eRegarding ATN connections with secondary motor cortical areas (MOs), our data showed selective reciprocal connections with the AMd.core (Fig. 7b-e), and although these connections have been reported for the AM\\u003csup\\u003e33\\u003c/sup\\u003e, AV↔MOs connections also are reported elsewhere\\u003csup\\u003e28\\u003c/sup\\u003e. The MOs is a large, undivided structure in most rodent atlases, and it is possible this discrepancy is due to differences in MOs injection site locations. We placed injections across the entire MOs, and only the injections specifically in the MOs-fef region (adjacent to the ACAd)\\u003csup\\u003e39,44\\u003c/sup\\u003e produced labeling in the AM. Notably, this MOs-fef region projects to the CP dorsomedial region, which integrates information from a variety of visual areas including VISam, VISal, ACAv, and PTLp caudal medial\\u003csup\\u003e39\\u003c/sup\\u003e (Extended Data Fig. 6f). We also show AM→caudal ECT/PERI connections (Fig. 7f-g), both of which heavily connect with cortical areas involved in visual and auditory processing\\u003csup\\u003e44\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Finally, despite documented connections between the internal part of the globus pallidus with the AD\\u003csup\\u003e78\\u003c/sup\\u003e, these connections were not found through our dataset.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eCaveats to tracing experiments\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOur large dataset used for the current project utilized a variety of chemical and viral tracers. Importantly, each anterograde and retrograde tracer has distinct characteristics and exhibits varied neurotropism that can meaningfully affect connectivity results. For example, anterograde AAVs label fibers of passage, while Phal does not. AAV-retro preferentially labels the cortex, while rabies preferentially labels hypothalamic neurons\\u003csup\\u003e79\\u003c/sup\\u003e. These differences underscore the importance of data validation. In this work, the connectivity data were validated in multiple ways to ensure the reliability of the results. Retrograde tracers were placed in regions of anterograde terminations, while anterograde tracers were placed in regions of retrogradely labeled cells. For example, anterograde tracer injections in the AM and AV show labeled terminals in the SUBv, but primarily in the caudal regions (Extended Data Fig. 4a). Retrograde tracers in the rostral SUBd, rostral SUBv, and the caudal SUBv confirm that only the caudal SUBv projects to the AM and AV (Extended Data Fig. 4b). Our data was further validated with Cre-dependent tracing methods and with repeated injections made in each ROI (e.g., Extended Data Fig. 3b, 4c). Together, these validation studies increase the confidence in the reported connections and mitigate potential issues arising from individual tracer characteristics. Notably, we have previously shown that repeated injections even in the smallest of ROIs produce similar brainwide labeling patterns regardless of the tracer used\\u003csup\\u003e38\\u003c/sup\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eOn a final note, the accurate assessment of injection site location is also a critical component of connectomics. Generally, the exposure times used to image and capture labeled fibers and cells typically oversaturate the injection sites. As such, we reimage injection sites with lower exposure parameters to gauge its location and spread more accurately. The reimaging along with all the data validation experiments facilitate the accurate assessment of injection site location. \\u0026nbsp;For instance, an anterograde AAV AM tracer injection labeled fibers and terminals in the CP and the ACA (Extended Data Fig. 6a-b). Retrograde tracer injections in the CP placed precisely in the region of the AAV labeled fibers, back labeled neurons in the adjacent IAD and not AM (Extended Data Fig. 6a-b) suggesting an IAD→CP connection and not an AM→CP connection. Retrograde ACA tracer injections on the other hand showed labeled neurons in the AM (Fig. 4e-f). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eATN morphology\\u0026nbsp;\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIdentification of the morphological neuron types based on their prominent dendritic features is necessary to understand the input-output relations within the ATN. Across the thalamus, the somatodendritic morphology of projection neurons and interneurons have been identified. Generally, four thalamic projection neurons (TPNs) or Golgi Type I neurons, whose axons extend outside their native thalamic nucleus to innervate the cortex, striatum, and amygdala have been reported. The first are the bushy tufted TPNs, which are large with a high number of branches whose dendrites profusely arborize, are intertwined, and are covered in spines, which give them their bush-like appearance\\u003csup\\u003e50\\u003c/sup\\u003e. The second are the radial (or stellate) TPNs, whose dendrites arborize in a radial fashion and compared to the tufted TPNs display relatively reduced wiring with shorter branches, particularly in the distal dendrites\\u003csup\\u003e48\\u003c/sup\\u003e. These radiate cells often also display grape-like appendages close to the initial branch points of the primary dendrites\\u003csup\\u003e47,48,80\\u003c/sup\\u003e, although these appendages are also reported for thalamic interneurons\\u003csup\\u003e51,80\\u003c/sup\\u003e. The tufted and radiate TPNs are found throughout the thalamus, while the third category of TPNs, the diffuse or reticulated, have been reported for primarily the parafascicular (PF)\\u003csup\\u003e47,81-84\\u003c/sup\\u003e, ethmoid-limitans\\u003csup\\u003e47\\u003c/sup\\u003e, and paralaminar group (medial division of the medial geniculate nucleus, posterior intralaminar, suprageniculate, peripeduncular)\\u003csup\\u003e85\\u003c/sup\\u003e. Diffuse TPNs are characterized by few, poorly ramifying primary dendrites that spread across long distances and have many spines. Although ATN somatodendritic morphology has not been systematically examined in any species, some investigations have been made in the AM and AV of the cat\\u003csup\\u003e86\\u003c/sup\\u003e, camel\\u003csup\\u003e51\\u003c/sup\\u003e, and human\\u003csup\\u003e52\\u003c/sup\\u003e through Golgi staining, and have identified both bushy and radiated TPNs and interneurons. Our k-mean analysis on the total branch number and total dendritic length identified two clusters of neuron types in the ATN: one larger with more complex branching and a second that are smaller and less complex. These two clusters potentially correlate to the bushy and radiate cells identified in other thalamic nuclei (Extended Data Fig. 7h). Visually, no reticulated cells were identified in the ATN, and grape-like appendages were observed in some instances (Extended Data Fig. 7i). \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn addition to TPNs, the thalamus contains interneurons or Golgi Type II, neurons whose axons do not extend out of the parent thalamic nucleus and make local connections. These interneurons generally are smaller and have fewer primary dendrites that poorly ramify but are covered in spines. Due to our method, we did not label any ATN interneurons (e.g., AAVretro-Cre injections in MORF3 mice to label TPNs).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eMorphology and function\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDendritic topological differences between bushy and radiate thalamic neurons are likely indicative of functional variability. For example, two types of neurons in the motor thalamic VA and VL nuclei have been identified. Neurons that receive predominantly inhibitory inputs display significantly fewer dendrites and their axons project to layer 1 of cortex and to the striatum\\u003csup\\u003e87\\u003c/sup\\u003e. The neurons that receive predominantly excitatory inputs have more dense dendritic trees and their axons project solely to layers II-V of cortex. Presumably, the former neurons correspond to radiate TPNs, while the latter to tufted ones\\u003csup\\u003e88,89\\u003c/sup\\u003e. The tufted and radiate cells of the medial geniculate (MG) have been similarly functionally differentiated\\u003csup\\u003e90\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eLarger dendritic arbors allow for a greater number of presynaptic partners for a neuron. We observed that dendritic arbors from the AM region were larger and more complex compared to AV and AD regions. We also observed that based on our morphological classification (using arbor length and number of branches as the two determining classifiers), all the Type 2 neurons (large and complex, analogous to bushy neurons) belonged to the AM regions. Greater length of the AM dendritic arbors would in principle allow them to form many more synapses than the AV and AD neurons. This suggests greater level of synaptic integration (particularly from excitatory long-range axons) occurring in AM compared to AV or AD. Similarly, for the AMd regions, the non-edge neurons had larger dendritic arbors than edge neurons, indicating a greater level of axonal input for the non-edge neurons. Apart from topological properties (such as length and number of branches), the spatial orientation of neuronal arbors have been shown to be relevant to their functional roles\\u003csup\\u003e91,92\\u003c/sup\\u003e and AMd edge neurons demonstrate an orientation bias towards the AMd center that increases as the distance from the center also increases. The higher overall length of dendritic arbors at the AMd center region, combined with the preferential orientation of the edge neuron’s dendritic arbors towards the center suggests a functional role, where individual neurons are attempting to maximize their presynaptic input from the central parts of the AMd region. It also suggests an attempt to make well-defined, separate channels of information processing with some spillover across border regions. The anterograde/retrograde tracing data of the ATN tend to show a high degree of topographic specificity, in many instances precisely innervating right up to the border of the target nucleus with minimal crossover (e.g., Fig. 4a). Simultaneously, the dendrites of the edge neurons likewise conform to this specificity, minimizing their spread into adjacent nuclei (Fig. 10e). The result is that each subnucleus has a particular combination of information it preferentially integrates, and the dendritic morphology of the neurons helps to maintain that segregation of information processing. To our knowledge, this type of morphological organization has not been reported.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch5\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAnimal Ethics Statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAll procedures were conducted in compliance with regulatory standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the animal protocols were approved by the IACUCs at USC (our previous affiliation) and UCLA (our current affiliation: protocol number ARC 2020-113).\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003c/h5\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData and code availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data and code for this project are available upon request.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eH.H., H.-W.D.\\u003c/strong\\u003e conceived and designed the project. \\u003cstrong\\u003eH.H.\\u003c/strong\\u003e managed the project and conducted the data analysis, \\u003cstrong\\u003eH.H., H.-W.D.\\u003c/strong\\u003e wrote the manuscript, and prepared the figures for publication. \\u003cstrong\\u003eM.R.\\u0026nbsp;\\u003c/strong\\u003econducted all of sectioning, staining, clearing, and imaging of the thick tissue and the manual mapping of the AM labeling. \\u003cstrong\\u003eS.N.\\u003c/strong\\u003e mapped registered axonal projections onto the Allen Brain Common Coordinate Framework (CCF) and calculated axonal projections to individual brain regions.\\u003cstrong\\u003e\\u0026nbsp;S.N.\\u0026nbsp;\\u003c/strong\\u003edeveloped analysis code\\u003cstrong\\u003e,\\u0026nbsp;\\u003c/strong\\u003eperformed all dendritic morphological analyses, generated all the corresponding graphs, and wrote the corresponding portions of the paper. \\u003cstrong\\u003eA.G., H.-S.M., Q.X.\\u003c/strong\\u003e carried out the dendritic reconstructions of neurons. \\u003cstrong\\u003eH-S.M.\\u0026nbsp;\\u003c/strong\\u003emapped the reconstructed neurons onto the atlas template. \\u003cstrong\\u003eL.Garcia., I.B.\\u003c/strong\\u003e managed and executed the analysis of the 2D dataset (community detection, hierarchical clustering) and generated their corresponding visualizations. \\u003cstrong\\u003eD.L.\\u003c/strong\\u003e, \\u003cstrong\\u003eT.B.\\u003c/strong\\u003e performed all the post-image processing for the 2D datasets in Connection Lens. \\u003cstrong\\u003eJ.S.\\u003c/strong\\u003e performed the synapse reconstructions. \\u003cstrong\\u003eC.E., S.Y.\\u003c/strong\\u003e assisted with the online visualization of the wiring diagram. \\u003cstrong\\u003eY.Y.\\u003c/strong\\u003e managed the breeding of all the MORF3 mice. \\u003cstrong\\u003eL.Gou, B.Z.\\u003c/strong\\u003e performed tracer injection surgeries, while \\u003cstrong\\u003eC.C., J.G.\\u003c/strong\\u003e, \\u003cstrong\\u003eH.X\\u003c/strong\\u003e., \\u003cstrong\\u003eI.Y.\\u003c/strong\\u003e processed and imaged the sections. \\u003cstrong\\u003eM.Z.\\u003c/strong\\u003e generated the code for hierarchical clustering. \\u003cstrong\\u003eI.B., K.M., S.N., A.D.\\u0026nbsp;\\u003c/strong\\u003edeveloped code for image processing and digital morphological reconstructions. \\u003cstrong\\u003eQ.Z.\\u003c/strong\\u003e assisted with high resolution imaging of neurons. \\u003cstrong\\u003eL.L., X.C., Z.Y.\\u003c/strong\\u003e performed the neuron axonal reconstructions, while \\u003cstrong\\u003eH.P.\\u003c/strong\\u003e managed that portion of the project. \\u003cstrong\\u003eN.N.F.\\u003c/strong\\u003e assisted in the design of experiments, with figure arrangements, and contributed to the editing of the manuscript. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics and inclusion statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe are committed to promoting ethical research practices and ensuring the welfare of all animals involved in our studies. All procedures adhered to regulatory standards as delineated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as well as institutional guidelines set forth by the Institutional Animal Care and Use Committees at both the University of Southern California (USC) and the University of California, Los Angeles (UCLA). We affirm our dedication to fostering an inclusive research environment that values the contributions of all individuals, regardless of race, ethnicity, gender identity, sexual orientation, disability, or other characteristics. We actively strive to create a welcoming and respectful atmosphere for all our research team members.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFunding for this project was provided by\\u0026nbsp;NIH Grants U01MH114829 (H.-W.D.) and 1R01NS133744-01 (H.-W. D.).\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e\\u003cu\\u003eSubjects\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOne hundred fifty 8-week-old C57Bl/6J (Jackson Laboratories) male mice were used to trace approximately 200 pathways (see Table 1). Animals were housed in pairs in a room with controlled temperature (21–22 °C), humidity (51%), and lighting (12-hour light:12-hour dark). Prior to stereotaxic surgeries for tracer delivery, mice were given at least 1 week to acclimate to their environment. Throughout the experiments, subjects had unrestricted access to tap water and mouse chow. All procedures were conducted in compliance with regulatory standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and institutional guidelines established by the Institutional Animal Care and Use Committee at the University of Southern California (USC) and at the University of California, Los Angeles (UCLA). \\u003c/p\\u003e\\n\\u003cp\\u003eSeventeen male MORF3 mice from our established breeding colony at UCLA (state were used (1) to sparsely label neurons in AD, AV, and AM (including AM domains) for morphometric analysis and (2) to reveal the disynaptic circuitry through the AMd.medial tip (SUBv→AMd.medial tip→PL/BLAa) and AMv (PL→AMv→ILA). MORF3 mice (C57BL/6-Gt(ROSA)26Sortm3(CAG-sfGFP*)Xwy/J), developed by Dr. X. William Yang's lab at UCLA\\u003csup\\u003e46\\u003c/sup\\u003e, are a Cre reporter mouse line engineered to utilize a mononucleotide repeat frameshift (MORF) for \\u003cem\\u003ein vivo\\u003c/em\\u003e cell labeling. These mice express a Cre-dependent tandem \\\"spaghetti monster\\\" fluorescent protein with 20 V5 epitopes (smFP-V5), preceded by a polycytosine repeat (C22) MORF switch, all under the control of a CAG promoter. Through Cre recombination and a spontaneous frameshift mechanism, MORF3 mice enable sparse and stochastic labeling of neural cells, which can be visualized using V5 antibody staining.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eNeural tracer injections\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAnterograde and retrograde tracers were administered to anatomically defined regions throughout the brain to investigate their connectivity patterns. Stereotaxic surgeries for tracer infusions were conducted under isoflurane anesthesia. Initially, mice were anesthetized in an induction chamber containing isoflurane and then secured to the stereotaxic apparatus, where they remained under anesthesia via a vaporizer. Isoflurane was vaporized and mixed with oxygen (0.5 L/min), maintaining the percentage of isoflurane in the gas mixture between 2 and 2.5. Buprenorphine SR (1 mg/kg) was administered at the beginning of the surgery as an analgesic and ophthalmic ointment was applied to the eyes for protection from light. Tracers were delivered either iontophoretically (10 min 5 µAmp, 7-second alternating current) or via pressure injection (20-80 nl) using glass micropipettes with outside tip diameters measuring approximately 10-30 µm. \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eTracers\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe tracers used to determine afferent and efferent connections included \\u003cem\\u003ephaseolus vulgaris\\u003c/em\\u003e leucoagglutinin (Phal, 2.5%; Iontophoresis, 10 min; Vector Laboratories); AAV-GFP (AAV1-hSyn-EGFP-WPRE-bGH; 2.7 x 10^13; Iontophoresis, 3 min; Addgene); AAV-RFP (AAV1-CAG-tdTomato-WPRE-SV40; 2.0 x 10^13; Iontophoresis, 3 min Addgene); Glycoprotein-deleted rabies (RVΔG) (Gdel-RV-4tdTomato and Gdel-RV-4eGFP; 9.6 x 10^10; Pressure, 50 nl; Ian Wickersham laboratory at MIT); Fluorogold (FG, 1%; Iontophoresis, 3 min; Fluorochrome); Cholera toxin subunit B-Alexa Fluor 488, 555, 647 conjugates (CTB, 0.1–0.2%; Pressure, 50 nl; Invitrogen); AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute). Typically, in a single brain, 2-4 of these tracers were injected in different combinations. For example, in the triple anterograde tracing method, 3 anterograde tracers were injected in 3 different brain regions to reveal topographic projections. In the quadruple retrograde design, four retrograde tracers are injected in 4 different brain regions. These multiple injections/brain assisted in revealing, validating, and clearly visualizing the ATN domains (e.g., Figs. 4h; 5b). In the double co-injection design, two co-injections of an anterograde/retrograde tracer cocktail are injected into 2 different regions to assess input/output of different regions and to assess the interaction of the two regions injected.\\u003c/p\\u003e\\n\\u003cp\\u003eFor Cre-dependent anterograde tracing, the following tracers were used: AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute); Cre-dependent AAV-FLEX-GFP (AAV1-CAG-Flex-eGFP-WPRE-bGH; 2.0 x 10^13; Iontophoresis, 5 min; Addgene); Cre-dependent AAV-FLEX-RFP (AAV1-CAG-Flex-tdTomato-WPRE-bGH; 1.1 x 10^13; Iontophoresis, 5 min; Addgene).\\u003c/p\\u003e\\n\\u003cp\\u003eFor Cre-dependent TVA receptor mediated rabies tracing (or TRIO tracing) the tracers used included AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute); AAV1-hSyn-FLEX-TVA-2A-GFP-2A-G (2.3 x 10^13; Pressure, 80 nl; Addgene); and EnvA G-deleted rabies dsRedXpress(2.26 x 10^10; Pressure, 40 nl; Ian Wickersham lab, MIT).\\u003c/p\\u003e\\n\\u003cp\\u003eTo determine synaptic circuitry in MORF3 mice, the tracers AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute) and AAV1-hSyn-DIO-mGFP-2A-SypRuby-WPRE (2.7 x 10^13; Pressure, 100 nl: Addgene) were used.\\u003c/p\\u003e\\n\\u003cp\\u003eFinally, to unlock MORF expression in MORF3 mice to reveal the morphological details of ATN neurons, the main tracer used was AAVretro-Cre (AAVretro-EF1a-Cre; 2.4 x 10^13; Iontophoresis, 5 min; Salk Institute).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eTracing strategies\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eCre-dependent anterograde.\\u003c/em\\u003e To validate the exclusive brain-wide output of AMd domain neurons, a Cre-dependent anterograde tracing method was applied. One case (n=1) was used per AM domain. In brief, AAVretro-Cre was injected into a downstream target of an AMd domain (e.g., PL) to deliver Cre to the AMd.medial. Next, a Cre-dependent AAV expressing either GFP or tdTomato was injected in the AMd. This method reveals the brain-wide output of AMd.medial→PL projecting neurons. \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eCre-dependent TVA receptor mediated rabies tracing method.\\u003c/em\\u003e To identify mono-synaptic inputs to neuronal populations defined by projections in the AMd (e.g., brain-wide input to AMd.medial→PL neurons), we employed a Cre-dependent TVA receptor mediated rabies tracing strategy sometimes referred to as TRIO (tracing the relationship between input and output) tracing. These cases were typically used to validate brain-wide input to the AM domains and one case (n=1) was used per AM domain. For example, an AAVretro-Cre injection was made into a downstream projection target of AMd.medial (e.g., PL), which delivered Cre to AMd.medial neurons. Next, a Cre-dependent TVA- and RG-expressing helper virus (AAV8-hSyn-FLEX-TVA-P2A-GFP-2A-oG) and an mCherry-expressing G-deleted rabies virus were injected into the AM thereby revealing brain-wide ROIs→AMd.medial→PL connections.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eTissue processing and imaging in 2D \\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTracer transport was allowed for either one (Phal, CTB, FG, RVΔG) or three (AAVs/viral tracers) weeks before animals were perfused, and their brains were extracted. For all experiments except those involving MORF3 mice to assess morphology, a 2D tissue processing workflow was employed. After administering an overdose injection of sodium pentobarbital (Euthasol, 2 mg/kg, intraperitoneal injection), each animal underwent transcardial perfusion with 50 ml of 0.9% NaCl followed by 50 ml of 4% paraformaldehyde solution (PFA; pH 9.5). Brains were post-fixed in 4% PFA for 24–48 h at 4 °C before being embedded in 3% Type I-B agarose for sectioning. Coronal sections, each 50-µm thick, were sliced into four equivalent series using a vibratome.\\u003c/p\\u003e\\n\\u003cp\\u003eOne of the four series of sections underwent immunostaining for the antigen of interest (Phal or AAVretro-Cre) using the free-floating method. Sections were first transferred to a blocking solution containing normal donkey serum and Triton X-100 for 1 h. After three 5-minute rinses, sections were incubated in a KPBS solution with donkey serum, Triton, and the appropriate antibody [1:1000 rabbit anti-Phal antibody (Vector Laboratories, #AS-2300), 1:4000 mouse anti-Cre recombinase antibody (EMD Millipore, #MAB3120), or 1:500 rabbit anti-tryptophan hydroxylase (TPH2) to identify serotonin positive neurons (ThermoFisher Scientific, #PA1-778] for 48–72 h at 4 °C. Following three KPBS rinses, sections were soaked for 3 h in the secondary antibody solution, which contained donkey serum, Triton, and a 1:500 concentration of anti-rabbit IgG conjugated with Alexa Fluor® 488 or 647 (Invitrogen, 488: #A-21206; 647: #A-31573) for Phal and serotonin staining. For Cre recombinase staining, the secondary solution contained donkey serum, Triton, and a 1:500 concentration of anti-mouse IgG conjugated with Alexa Fluor® 488 or 647 (Life Technology, 488: #A-21202; 647: #A-31571). After three KBS rinses, the sections were counterstained with a fluorescent Nissl stain, NeuroTrace® 435/455 (NT; 1:500; Invitrogen, #N21479), mounted, and coverslipped using 65% glycerol. Finally, the sections were scanned at 10x magnification as high-resolution, multichannel virtual slide image (VSI) files using an Olympus VS120, with identical exposure parameters maintained across all cases.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003e2D post-image processing workflow\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo accurately compare connectivity tracer signals across experiments, we utilized our high throughput image processing workflow, Connection Lens, to achieve (1) high-quality image registration, (2) tracer signal segmentation, and (3) tracer signal quantification. Connection Lens provides a user-friendly interactive interface and deploys all processes on a high-performance computational infrastructure that allows high throughput processing of large TB-sized datasets. This post-image processing Connection Lens pipeline is summarized as follows: Each 50 µm-thick section was initially matched and subsequently aligned (registered) to its corresponding atlas level in the Allen Reference Atlas (ARA; available at http://mouse.brain-map.org/static/atlas) (Fig. 1a-c). Our semi-automated registration pipeline applies a diffeomorphic registration approach allowing iterative modifications based on user feedback, enhancing the accuracy of registered images. Next, threshold parameters were individually adjusted for each case and tracer signal. Conspicuous artifacts in the threshold output were filtered. The final overlap processing step generates a file with annotated (quantified) values: pixel density for anterograde tracers and cell counts for retrograde tracers. \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003e2D data analysis\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eIdentifying AD, AV, AM domains and their relative boundaries.\\u003c/em\\u003e Our pipeline conducted connectivity analysis for the AV, AD and AM. After segmenting tracer labeling, grid-based overlap annotation was performed in which the AD, AV, and AM were divided into 105 x 105 pixel square grids (equivalent to 63 µm\\u003csup\\u003e2\\u003c/sup\\u003e) to tabulate labeling within distinct domains of the ATN nuclei. This was done for ARA atlas levels 61 and 63 for the AD and ARA level 61 for the AV and AM. In the given analysis, anterograde and retrograde were combined. The case specific annotations were then aggregated into a single matrix and Louvain community detection was conducted with gamma 0.75. Grid cells were color-coded according to community assignment and reordered such that the resulting Louvain clusters were placed along the diagonal of the visualized matrix (Fig. 1b-d). \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eIdentifying brain-wide input/output connectivity of AM domains\\u003c/em\\u003e. To assess domain-specific AM connections, Cre-dependent anterograde and retrograde tracing strategies were applied utilizing connectionally guided delivery of Cre. For each case, sections spanning the whole brain were registered (warped) and tracer labels were segmented and annotated based on ROI. The annotated data (pixel intensity for anterograde, cell counts for retrograde) were subsequently normalized. Neuroanatomical tracing experiments across the entire brain entail numerous sources of variation, including tracer injection volume, quality of the collected tissue, variations in the plane of sectioning and immunostaining quality, accuracy of tissue registration to the atlas template, as well as microscopic imaging settings, among others. Conducting meaningful inter-experimental data comparisons necessitates the standardization of these experimental variations.\\u003c/p\\u003e\\n\\u003cp\\u003eGiven that tissue sections within a single specimen typically experience consistent experimental conditions, we utilize a self-normalization method to derive whole-brain connectivity fractions for each brain specimen. The connectivity fraction characterizes the proportion of connectivity found in a region out of all connectivity found in the entire specimen. The fraction calculation performs a total intensity normalization within each brain specimen, effectively normalizing away the effects of experimental condition variations. The fraction calculation was as follows: \\u003cstrong\\u003e\\u003cem\\u003ef\\u003c/em\\u003e\\u003c/strong\\u003e=Count/Sum of all ROI count across the case, where count is number of labeled pixels (pixel count for anterograde, cell count for retrograde).\\u003c/p\\u003e\\n\\u003cp\\u003eWe also define a connectivity density vector \\u003cstrong\\u003e\\u003cem\\u003ed\\u003c/em\\u003e\\u003c/strong\\u003e that approximates the density of connectivity at each gray matter region. The density vector is derived from the fraction vector and represents the projection densities at each brain region of each specimen. It maintains the total counts of segmented pixels (for anterograde tracers) or cells (for retrograde tracers) constant across all specimens. This density measure offers an intuitive understanding of the connectivity data, as connectivity densities are commonly discussed in neuroanatomical literature, are easily understood, and considers ROI sizes. The density calculation was as follows: \\u003cstrong\\u003e\\u003cem\\u003ed=\\u003c/em\\u003e\\u003c/strong\\u003e(Fraction/area)/maximum density value.\\u003c/p\\u003e\\n\\u003cp\\u003eDensity values were used to generate the bar graphs depicting the connectivity of AM domains (e.g., Fig. 6h, j). They were also used to perform hierarchical 2D clustering of the AM domain-specific tracing data to determine and visualize the similarity of whole brain projection patterns of the different injections (e.g., Fig. 1f).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003e3D tissue fixation, clearing, staining, and imaging pipeline\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing 4% PFA perfusion, 250 µm-thick (for synaptic connectivity experiments) and 800 µm-thick (for morphological assessments) tissue samples were drop fixed in 4% PFA at 4°C for up to two weeks, then transferred into PBSN (1x PBS + 0.02% sodium azide and stored at 4°C until SHIELD epoxy fixation. For SHIELD epoxy fixation, samples were incubated in the SHIELD OFF solution at 4°C for three days with agitation, then transferred into pre-heated SHIELD ON solution and incubated at 37°C overnight with agitation. After the SHIELD ON step, samples were either transferred into PBSN and stored at 4°C until the delipidation step or washed 2x for 2 hours and moved onto the delipidation step. When ready for delipidation, samples were put into 10 mL LifeCanvas’s Active delipidation buffer and incubated overnight at 45°C with agitation. The next day, samples began active delipidation using LifeCanvas’s SmartBatch+ pre-installed active clearing protocol (40V, 30 hours). Following active delipidation, samples were washed 2x 2 hours with wish 1x PBSN and stored in 1x PBSN at 4°C until ready for staining. When samples were ready for staining, they were incubated in a 10 mL primary sample buffer with 5% NDS overnight at 37°C. Replacing this solution once in the morning, samples were then put in the SmartBatch+ to begin the active primary V5 staining protocol [1:1000 anti-V5, Fortis, #A190-119A (goat) or #A190-120A (rabbit)]. Sample solution contained primary antibody added to the primary sample buffer + 5% NDS, and LifeCanvas’s primary conduction buffer was used (18–22 hours). Primary staining concluded when the sample buffer reached \\u0026lt; pH 8.0. Following the primary stain, samples were washed 2x 2 hours in 1xPBSN then incubated in 4% PFA overnight at 4°C to prevent primary antibody dissociation. After 4% PFA incubation, samples were washed in 1x PBSN at RT for 2 hours then 10 mL secondary sample buffer + 5% NDS at 37°C for 2 hours with agitation. Samples were then put in the SmartBatch+ for one 2-hour active secondary sample buffer + 5% NDS wash using the active secondary protocol pre-installed in the SmartBatch+. Active secondary protocol was run with secondary antibodies (1:1000 in house conjugated anti-goat and anti-rabbit Fab-Setau-647, see following section for details regarding secondary antibody conjugation procedure) in the secondary sample buffer + 5% NDS for 12 hours. The next morning, samples underwent 2x 2 hours active washes with only the secondary sample buffer. Samples were then washed 2x 2 hours in 1x PBSN and then incubated in 4% PFA overnight at 4°C to fix secondary antibodies in place. The next morning, samples were washed 2x 2 hours in 1x PBSN and then were either placed into storage in 1x PBSN at 4°C or moved onto the light sheet or spinning disc confocal preparation.\\u003c/p\\u003e\\n\\u003cp\\u003eTissue being processed for the LifeCanvas SmartSPIM light sheet microscope at 15x (for morphological assessment) were moved into 10mL delipidation buffer containing 1:500 of a nucleic acid stain [Syto13 (ThermoFisher Scientific, #S7575) or Propidium Iodide (ThermoFisher Scientific, #P1304MP)] and incubated overnight at 37°C with agitation. The following morning, samples were washed 2x 2 hours in 1x PBSN then incubated in 4% PFA at 4°C overnight. The next morning, samples were washed 2x 2 hours in 1x PBSN then either stored in 1x PBSN at 4°C, or immediately moved into 50% EasyIndex and incubated overnight at 37°C then put into 100% EasyIndex and incubated overnight at 37°C. The next morning, samples were mounted onto a sample holder using 1.5% agarose and superglue and were put into the light sheet chamber for imaging.\\u003c/p\\u003e\\n\\u003cp\\u003eTissues processed for Olympus Dragonfly spinning disk confocal imaging were sectioned at either 800 µm for 20x imaging (for morphological assessment) or 250 µm for 60x imaging (for synaptic connectivity experiments). Sections were then incubated in 1:500 DAPI (ThermoFisher Scientific, #D1306) in 1x PBSN solution or immediately moved into 50% EasyIndex and incubated overnight at RT. The next day samples were then put into 100% EasyIndex and incubated for at least 3 hours or were held in a parafilm wrapped 24-well plate and protected from light until ready for slide mounting. Sections were mounted onto plain microscope slides with Sunjin spacers and coverslipped with 1.5H coverslips that were held in place with superglue.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eSeTau-647 secondary antibody conjugation\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor optimal signal retention of MORF3 V5 tagged neurons in 3D imaging, we chose to conjugate SeTau-647-NHS ester (Setabiomedicals, #K9-4149) to AffiniPure Fab fragments (Fab donkey anti-rabbit, Jackson Immunoresearch, #705-007-003; Fab donkey anti-goat, Jackson Immunoresearch, #711-007-003) because of the intensity of emission and its resilience to photobleaching. Fab’s were conjugated to SeTau-647-NHS ester using a 1:2 molar ratio (Fab:Dye). 1M sodium bicarbonate, at 10% of the volume of the Fab:Dye solution, was added to the Fab:Dye solution (e.g., if Fab:Dye solution is 100 µl, then add 10 µl of sodium bicarbonate for a final solution volume of 110 µl) and was agitated at 500rpms at room temperature for 1 hour, then excess dye was removed using size exclusion columns (Zeba™ spin desalting columns and plates, 40K MWCO, 0.5 mL, ThermoFisher Scientific, #A57760).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003e3D neuron reconstruction and mapping\\u003cbr\\u003e\\u003c/u\\u003e To accurately select the neurons from the regions of interest (i.e., AD, AV, and AM domains), the Orthoslicer and FilamentTracer tools within Imaris (BITPLANE, RRID:SCR_007370) were used to manually identify the (somatic) location of each neuron. The Orthoslicer thickness of the tissue was adjusted to be between 10-30 µm. The cell body of each neuron was ensured to be within the selected region’s border in each of the coronal views of the tissue, located between ARA 58-66 on the Allen Reference Atlas. The identified neurons were scaled from voxel to micron dimensions (and scaled back to voxel dimensions if required) using a custom python script and were manually reconstructed within the Terafly program in Vaa3d\\u003csup\\u003e93\\u003c/sup\\u003e. For precise quality check of the reconstructions, the neurons were opened and edited in neuTube\\u003csup\\u003e94\\u003c/sup\\u003e to ensure correct branch typing, location of branch points and proper connections of the nodes. Each digital SWC reconstruction is a tree structure constituting a series of connected frustums/compartments, where a compartment is represented by a single row (containing seven columns) of an SWC file\\u003csup\\u003e95\\u003c/sup\\u003e. The compartment's id, type (dendrite, axon, or soma), end point coordinates (X, Y, Z location) and the end point thickness are represented by the first six columns. The 7th column of the SWC row has the id of the compartment’s origin point/parent compartment (for additional details, see swc-specification.readthedocs.io). Imaris was once more used to visualize, and further quality check the neuron morphology and the location markers such as identifying neurons lying closer to the borders of the regions of interest (edge) versus the ones that are more within the center of the AM (non-edge). A total of 109 neural reconstructions were analyzed for this study that included 10 AD, 82 AM, and 17 AV neurons. Out of the 82 AM neurons, 72 neurons were from the AMd region and the remaining 10 were from the AMv region. The AMd neurons were subdivided into five domains: (i) AMd.core (12 neurons), (ii) AMd.dorsal (12 neurons combined from AMd.dorsal and AMd.dorsolateral domains), (iii) AMd.dorsomedial (11 neurons), (iv) AMd.lateral (20 neurons), and (v) AMd.medial (17 neurons from both the AMd.medial tip and AMd.ventromedial domains). \\u003c/p\\u003e\\n\\u003cp\\u003eTo reconstruct the full 3-D morphology of neurons, including both dendrites and axons, we utilized Vaa3D (http://vaa3d.org\\u003csup\\u003e95-97\\u003c/sup\\u003e along with its recent successor, Collaborative Augmented Reconstruction (CAR, https://github.com/neurogeom/CAR\\u003csup\\u003e98\\u003c/sup\\u003e. This approach was applied to fMOST images obtained through the collaboration\\u003csup\\u003e99\\u003c/sup\\u003e. To guarantee the precision of the neuron locations and morphologies, we conducted human inspection of the somas and their 3D structures.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eStatistical analyses of morphometrics\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe first quantified basic morphometric features that demonstrated distinct and complementary aspects of dendritic architecture. These features included total wiring (total dendritic length), total complexity (number of dendritic branches), arbor height, maximum path distance, and average branch order. \\u003c/p\\u003e\\n\\u003cp\\u003eWe noticed distinct morphological features for AMd neurons whose somas were on the border of the AMd (edge, n=27) compared to those whose somas were located more internally (non-edge, n=45). To investigate this, we performed quantification of two new attributes for these edge or non-edge AMd neurons, specifically to measure the orientation bias (towards the AMd center) of a neuron’s overall dendritic architecture. The first feature is the average angular deviation relative to the local core vector (Fig. 8). The deviation of every dendritic compartment vector relative to its local core vector (where the local core vector of a compartment is a straight line connecting the compartment’s origin point to the AMd center) is averaged across all compartments for a collective average deviation for that neuron. An acute compartment deviation (deviation of less than 90°) would mean that the dendritic compartment’s end point is closer to the AMd center than its origin point. An obtuse compartment deviation on the other hand, would mean that the compartment’s end point is further away from the center compared to the compartment’s origin point. Neuronal nodes are resampled for this measurement so that each compartment (i.e., internode distance) is approximately 1 micron in length. Therefore, a neuron with a lower average angular deviation (from the AMd center) would have greater tropism/bias towards the AMd center. The second feature measured the proportion of total dendrite that lay closer to the AMd core than the neuron’s cell body. Hence, neurons with a higher proportion of dendrites closer to the center also demonstrate greater tropism/bias towards the AMd center. \\u003c/p\\u003e\\n\\u003cp\\u003eWe carried out a comparative morphometric study on three levels of groupings due to smaller sample sizes for both AD (n=10) vs AM (n=82) vs AV (n=17) comparison as well as for the AMd domain comparisons. The domains included the AMd.core (n=12), AMd.dorsal (AMd.dorsal and AMd.dorsolateral combined, n=12), AMd.dorsomedial (n=11), AMd.lateral (n=20), and AMd.medial (AMd.medial tip and AMd.ventromedial combined, n=17). Because of smaller sample sizes, the omnibus Kruskal-Wallis test was carried out to identify significant differences across multiple groups, followed by Wilcoxon rank sum test for each pair (three pairs in AD vs. AM vs. AV comparison and a total of 10 pairs for the five AMd domains. For the AMd edge vs non-edge analysis, a t-test was carried out since the sample size of both groups were higher and the distributions were normal. \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cu\\u003eSynapse reconstructions\\u003c/u\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSoma, dendrites, and spines were reconstructed using the “Soma”, \\\"Tree\\\", and “Spine” module respectively in Neurolucida 360 software, while axon boutons that are adjacent to the soma, dendrites, and spines were reconstructed with the \\\"Puncta\\\" module. Briefly, soma was created using the default setting, and dendrites were created using the user-guided mode with the Rayburst Crawl mode. Spines were then automatically segmented with the following detection settings: outer range set to 5 µm, minimum height set to 0.3 µm, detector sensitivity set to 120%, and minimum count set to 10 voxels. Axons were created using the machine learning method with the maximum distance to soma, dendrites, or spines set to 1 µm. Data analysis was performed in Neurolucida Explorer software. For quantifying the number of synapses, the overlap percent to colocalize was set to 10%.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003ePapez, J. W. A proposed mechanism of emotion. 1937. \\u003cem\\u003eJ Neuropsychiatry Clin Neurosci\\u003c/em\\u003e\\u003cstrong\\u003e7\\u003c/strong\\u003e, 103-112, doi:10.1176/jnp.7.1.103 (1995).\\u003c/li\\u003e\\n \\u003cli\\u003eAggleton, J. P., Nelson, A. J. D. \\u0026amp; O\\u0026apos;Mara, S. M. 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Collaborative Augmented Reconstruction for Scaled Production of 3D Neuron Morphology in Mouse and Human Brains. \\u003cem\\u003ebioRxiv\\u003c/em\\u003e, doi: https://doi.org/10.1101/2023.10.06.561172 (2023).\\u003c/li\\u003e\\n \\u003cli\\u003ePeng, H.\\u003cem\\u003e\\u0026nbsp;et al.\\u003c/em\\u003e Morphological diversity of single neurons in molecularly defined cell types. \\u003cem\\u003eNature\\u003c/em\\u003e\\u003cstrong\\u003e598\\u003c/strong\\u003e, 174-181, doi:10.1038/s41586-021-03941-1 (2021).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTables 1 and 2 are available in the Supplementary Files section\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4356188/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4356188/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eDespite significant advancements in identifying cell types in the mouse cerebral cortex, the classification of neuron types in the mouse thalamus remains largely incomplete. Specifically, the anterior thalamic nuclei (ATN), an integral component of the Papez circuit, encompass the anterodorsal (AD), anteroventral (AV), and anteromedial (AM) thalamic nuclei. Structurally, the ATN serve as a hub to facilitate communication among the neocortex, hippocampus, amygdala, and hypothalamus. Functionally, they play pivotal roles in regulating learning, memory, spatial navigation, and goal-directed behaviors. Thus, the ATN provide a promising avenue to investigate the relationship between structural and functional complexity with neuron type diversity. Our comprehensive and systematically collected macroscale pathway tracing data revealed several connectionally unique cell populations within the AM, AV, and AD that suggest several disparate parallel subnetworks run through each nucleus. Further, we applied genetic sparse labeling, brain clearing, 3D microscopic imaging, and computational informatics to catalog neuron types across the ATN, ascertained their brain-wide connectivity profile at the single neuron and synaptic resolutions, and characterized their morphological features. This study provides insights into how the prefrontal cortex, hippocampus, and amygdala interact through neuron type-specific ATN subnetworks to coordinate and synchronize both cognitive and emotional aspects of goal-directed behavior, resolving longstanding controversies surrounding the validity of the Papez circuit and its structural and functional roles.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Distinct subnetworks of the mouse anterior thalamic nuclei\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-05-05 11:17:44\",\"doi\":\"10.21203/rs.3.rs-4356188/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"9754c459-67b3-4e79-afe8-3be67e30e706\",\"owner\":[],\"postedDate\":\"May 5th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":46249637,\"name\":\"Biological sciences/Neuroscience/Neural circuits\"},{\"id\":46249638,\"name\":\"Health sciences/Anatomy/Nervous system/Brain\"}],\"tags\":[],\"updatedAt\":\"2025-07-02T07:36:51+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4356188\",\"link\":\"https://doi.org/10.1038/s41467-025-60774-6\",\"journal\":{\"identity\":\"nature-communications\",\"isVorOnly\":false,\"title\":\"Nature Communications\"},\"publishedOn\":\"2025-07-01 04:00:00\",\"publishedOnDateReadable\":\"July 1st, 2025\"},\"versionCreatedAt\":\"2025-05-05 11:17:44\",\"video\":\"\",\"vorDoi\":\"10.1038/s41467-025-60774-6\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41467-025-60774-6\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4356188\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4356188\",\"identity\":\"rs-4356188\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}