Development of feedforward and feedback connections between the dorsal lateral geniculate nucleus and the thalamic reticular nucleus

Development of feedforward and feedback connections between the dorsal lateral geniculate nucleus and the thalamic reticular nucleus | 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 Research Article Development of feedforward and feedback connections between the dorsal lateral geniculate nucleus and the thalamic reticular nucleus Peter W Campbell, Gubbi Govindaiah, William Guido This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4014221/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The thalamic reticular nucleus (TRN) serves as an important node between the thalamus and neocortex, regulating thalamocortical rhythms and sensory processing in a state dependent manner. Disruptions in TRN circuitry also figures prominently in several neurodevelopmental disorders including epilepsy, autism, and attentional defects. An understanding of how and when connections between TRN and 1 st order thalamic nuclei, such as the dorsal lateral geniculate nucleus (dLGN), develop is lacking. We used the mouse visual thalamus as a model system to study the organization, pattern of innervation and functional responses between TRN and the dLGN. Genetically modified mouse lines were used to visualize and target the feedforward and feedback components of these intra-thalamic circuits and to understand how peripheral input from the retina impacts their development. Retrograde tracing of thalamocortical (TC) afferents through TRN revealed that the modality-specific organization seen in the adult, is present at perinatal ages and seems impervious to the loss of peripheral input. To examine the formation and functional maturation of intrathalamic circuits between the visual sector of TRN and dLGN, we examined when projections from each nuclei arrive, and used an acute thalamic slice preparation along with optogenetic stimulation to assess the maturation of functional synaptic responses. Although thalamocortical projections passed through TRN at birth, feedforward axon collaterals determined by vGluT2 labeling, emerged during the second postnatal week, increasing in density through the third week. Optogenetic stimulation of TC axon collaterals in TRN showed infrequent, weak excitatory responses near the end of week 1. During weeks 2-4, responses became more prevalent, grew larger in amplitude and exhibited synaptic depression during repetitive stimulation. Feedback projections from visual TRN to dLGN began to innervate dLGN as early as postnatal day 2 with weak inhibitory responses emerging during week 1. During week 2-4, inhibitory responses continued to grow larger, showing synaptic depression during repetitive stimulation. During this time TRN inhibition started to suppress TC spiking, having its greatest impact by week 4-6. Using a mutant mouse that lacks retinofugal projections revealed that the absence of retinal signaling led to an acceleration of TRN innervation of dLGN but had little impact on the development of feedforward projections from dLGN to TRN. Together, these experiments reveal how and when intrathalamic connections emerge during early postnatal ages and provide foundational knowledge to understand the development of thalamocortical network dynamics as well as neurodevelopmental diseases that involve TRN circuitry. dorsal lateral geniculate nucleus thalamic reticular nucleus development mouse 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 thalamic reticular nucleus (TRN) is a shell-like structure that surrounds the dorsal and lateral aspects of the thalamus [ 1 – 5 ]. Comprised entirely of GABAergic neurons, the TRN serves as an important nexus for thalamocortical (TC) and corticothalamic (CT) communication. [ 4 , 6 – 9 ]. This nucleus receives excitatory input from thalamocortical and corticothalamic axon collaterals, which in turn serve to activate GABAergic inhibitory feedback to many thalamic nuclei. This pattern of connectivity is arranged in a sectorial manner that allows for both modality-specific and network-wide interactions (Fig. 1 ). The TRN plays a key role in TC function by modulating sensory signaling during different behavioral states, participating in the generation and propagation of thalamocortical rhythms during sleep and wakefulness [ 3 , 10 , 11 ]. Additionally, a disruption in connectivity or in the intrinsic membrane properties of TRN neurons has been implicated in a number of neurodevelopmental diseases including epilepsy, autism spectrum disorder and attention hyperactivity disorder [ 9 , 12 – 20 ]. Although the organization of TRN and its impact on state dependent behavior has been studied extensively, we still lack a fundamental understanding of how and when these intrathalamic connections develop and become operational. Studies in rodent have begun to characterize early postnatal TC network activity [ 21 – 23 ], however, the underlying circuitry linking the TRN to first-order thalamic nuclei remain unexplored. The dLGN of the mouse has emerged as a model system to study thalamic circuit development [ 24 ]. Much of our present understanding is based on the study of the retinogeniculate pathway [ 24 – 26 ]. Studies have also delineated when and how input from nonretinal sources, such as those arising from layer VI of visual cortex, as well as those originating from cholinergic nuclei of the brainstem, innervate dLGN and form functional connections with thalamocortical relay neurons [ 27 – 30 ]. Taken together, these studies reveal that circuit assembly is dLGN is a highly orchestrated event, with retinogeniculate connections emerging first, followed by CT innervation during the second postnatal week, and then finally by input from cholinergic brainstem areas which proceeds slowly through the end of the first postnatal month. Interestingly, the arrival of retinal afferents plays a key role in the timing of nonretinal innervation. For example, the absence or elimination of retinal signaling accelerates the arrival of CT input by disrupting the expression of aggrecan, a repulsive CPSG that normally inhibits cortical axons from entering dLGN prematurely [ 28 ]. However, the emergence of connections between dLGN and TRN have yet to explored in this context. More specifically, what is lacking is an understanding about how and when the connections linking dLGN and TRN occur, and whether the absence of retinal input affects the development of these feedforward and feedback loops. To accomplish this, we made use of specific Cre-driver, reporter, and transgenic lines that allow for the visualization and interrogation of intrathalamic feedforward and feedback circuits. To study the impact of retinal signaling, we took a loss of function approach and employed a novel form of genetic deafferentation by utilizing a Math5 null (Math 5 −/− ) mouse. This mutant lacks the transcription factor Math5, which is essential for the differentiation of retinal progenitors into retinal ganglion cells. As a result, Math5 −/− mice exhibit a wholesale loss (> 95%) of retinal ganglion cells, a failure to develop an optic nerve, and a brain that is devoid of retinofugal projections [ 31 – 34 ]. Finally, because our focus is on visual intrathalamic circuits, we also assessed whether the modality-specific sectors of TRN were present at birth and if such an arrangement was altered by visual deafferentation. Materials and Methods Subjects Experiments were conducted in mice P0-P46 of either sex. We used the GAD65 EGFP transgenic strain that expresses enhanced green fluorescent protein (EGFP) in TRN [ 27 , 35 , 36 ]. We also utilized two Cre-driver lines, somatostatin-Cre (SST-Cre; Jax stock no. 013044, RRID: IMSR_JAX:013044) and corticotropin releasing hormone-Cre (CRH-Cre, MMRRC no. 030850-UCD, RRID: MMRRC_030850-UCD), to target reporter expression within TRN (SST-Cre) or thalamocortical neurons (CRH-Cre). The Cre-driver lines were crossed with reporters to enable Cre-dependent expression of either tdTomato (Ai9; Jax stock no. 007909; RRID: IMSR_JAX:007909) or channelrhodopsin 2 – EYFP (ChR2; Ai32; Jax stock no. 012569; RRID: IMSR_JAX:012569). All lines were crossed onto a Math 5 −/− background in order to study TRN and dLGN circuit assembly in the absence of retinal input [ 31 , 33 , 37 ]. All breeding and experimental procedures were approved by the University of Louisville Institutional Animal Care and Use Committee. Cholera toxin subunit B injections Cholera toxin subunit B injections To retrogradely label TC projections in TRN, P1 CRH-Cre or CRH-Cre x Math 5 −/− mice were deeply anesthetized using isoflurane vapors. The skull was pierced with a sterile needle and then a glass pipette (10-20um tip diameter) filled with a 1% solution of Cholera toxin subunit B (CTB) conjugated to different Alexa Fluors (488, 546 or 647; Invitrogen) dissolved in distilled water was lowered into the targeted region. The pipette was attached to a picospritzer and ~ 2uL of CTB was injected into visual cortex or somatosensory cortex. After a 24-hr survival period, animals were deeply anesthetized by isoflurane vapors and transcardially perfused with PBS followed by 4% paraformaldehyde in 0.1M phosphate buffer (4% PFA). Brains were removed and post-fixed for 24hr in 4% PFA and then transferred to phosphate buffered saline [PBS: 0.01 M phosphate buffer (PB) with 0.9% NaCl]. To verify the injection site, the cortical surface of excised brains was imaged using a stereomicroscope (Olympus SZX2-ILLB) with fluorescence illumination (Prior Scientific Lumen 200). Immunohistochemistry To visualize thalamocortical axon collateral terminals in TRN we used vesicular glutamate transporter 2 (vGluT2) labeling [ 38 – 40 ]. At least three coronal sections (35um) containing the visual sector of TRN from at least two different mice were collected from CRH-Cre and CRH-Cre x Math 5 −/− at ages that ranged from P5-P28. Sections were blocked (10% normal goat serum [NGS; Vector Labs, S-1000, RRID: AB_2336615] and 0.3% Triton X-100 in PBS) for 1hr and then incubated overnight in rabbit anti-vGluT2 (SysSys, AB135403 1:500, RRID: AB_887883; diluted 1:100 with 10% NGS in PBS). After a wash in PBS, a secondary antibody, goat anti-rabbit 488 (ThermoFisher, A11034, RRID: AB_2576217) was applied for 1hr. Sections were washed, then immunolabeled to visualize neurons, and incubated overnight in mouse NeuN (Milipore, MAB377, RRID: AB_2298772). After a wash, sections were incubated for 1hr with biotinylated goat anti-mouse (Vector Labs, BA9200, RRID: AB_2336171) and then labeled with streptavidin-AF647 (ThermoFisher, S21374, RRID: AB_2336066, 1:100 diluted in PBS). Sections were mounted with Prolong Gold (Invitrogen, P36931), coverslipped, imaged using confocal microscopy, and analyzed using methods described below. For all in vitro recordings, biocytin (0.5%, Sigma) was included in the internal pipette solution for intracellular filling and 3D neuron reconstruction using confocal microscopy [ 33 , 41 , 42 ]. Following the completion of the recording session, slices were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), then washed with PBS and incubated overnight with AlexaFluor 647-conjugated streptavidin (Invitrogen, S21374) in a PBS solution containing Triton X-100 (0.1%). Image acquisition and analysis To visualize TRN terminals in dLGN, and TC axons in TRN, fixed brains from GAD-65 EGFP and CRH-Cre XAi9 mice were cut in the coronal plane (70uM) and mounted on slides using ProLong mounting medium containing DAPI (Life Technologies P36931). Sections containing TRN or dLGN were imaged using a multiphoton laser scanning confocal microscope (Olympus, model no. FV1200BX61) equipped with a 20x (0.75 NA) objective. Fluorophores were excited using Ar (488nm) and HeNe (635nm) lasers, and Z-stacked images (1.26um optical sections) were acquired using Fluoview software at a scanning resolution of 1600 x 1600 pixels. To quantify the spatial extent of innervation or the density of VGlut2 staining at different postnatal ages, we analyzed at least three coronal sections through the middle of TRN or dLGN [ 29 , 30 , 43 ]. Z-stacks of each image were generated and imported into Photoshop (Adobe). Images were binarized using a threshold that clearly distinguished signal and background fluorescence. Binarized images were imported into ImageJ (NIH, RRID: SCR_003070) in order to count the number of fluorescent pixels as well as the total number of pixels within a nucleus. These values were used to calculate the percent area that contained fluorescent pixels. At each postnatal age, values were obtained from 3–28 hemispheres taken from > 2 mice. For summary statistics, each hemisphere served as a unit of observation and the median value with a 95% confidence interval were plotted. In vitro slice preparation and whole cell recording Acutely prepared, thalamic brain slices were made from mice that were deeply anesthetized and rapidly decapitated. The brain was excised and placed into 4°C oxygenated cutting solution (in mM): Sucrose 234, glucose 11, NaHCO3 26, MgSO4 10, KCl 2.5, CaCl2 0.5, NaH2PO4 1.25. Bran slices containing thalamus were cut in the coronal plane (270 µm thick) on a vibratome (Leica), placed in a chamber and bathed for 30 min in warm (32°C) oxygenated artificial cerebrospinal fluid (aCSF) (in mM: 126 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4). Recordings were conducted in a chamber mounted on a perfused continuously with 32°C aCSF at a rate of 2–3 mL/min. Thalamic nuclei were visualized on an upright microscope (Olympus BX51WI) with DIC optics and fluorescent filters (GFP: Chroma 49002; tdTomato: Chroma 49005) using a 10x or 60x water immersion objective. A vertical puller (Narashige PC-10) was used to pull patch electrodes from borosilicate glass. For voltage clamp recordings, the electrode solution contained (in mM) 117 Cs gluconate, 11.0 CsCl, 1.0 MgCl2, 1.0 CaCl2, 0.1 EGTA, 10.0 HEPES, 2 Na2-ATP, 0.4 Na2-GTP. For current clamp recordings, the electrode internal solution contained (in mM) 117 K-gluconate, 13 KCl, 1.0 MgCl2 1, 0.07 CaCl2, 0.1 EGTA, 10 HEPES. A 0.5% The final electrode tip resistance was 4–7 M Ohms. Whole cell recordings were made in current or voltage clamp mode using an amplifier (Multiclamp 700B, Molecular Devices), filtered at 3–10 kHz, and digitized (Digidata 1440A) at 20 kHz. Pipette capacitance, series resistance, input resistance, and whole-cell capacitance were monitored throughout the recording session. Inhibitory postsynaptic currents were measured by holding the neuron at 0mV using a cesium-containing electrode while excitatory currents were measured at -70mV using a potassium-containing electrode. Optogenetic stimulation and analysis We took an optogenetic approach to assess functional connections between TRN and dLGN. By crossing CRH-Cre [ 44 ] or SST-Cre [ 36 ] with an Ai32 mouse [ 45 – 47 ], we were able to selectively stimulate feedforward or feedback projections in thalamic slices in WT and Math 5 −/− backgrounds. Light-gated postsynaptic responses were evoked using a light emitting diode (LED, Prizmatix) that delivered blue light through a 60x objective. Blue light pulses were 0.3mm in diameter with a light power of 525mW/mm2 and with a pulse width of 1ms. Trains of 10 blue light pulses were presented at different temporal frequencies (0.25, 0.5, 1, 5, 10 and 50 Hz). The incidence of optically evoked postsynaptic responses was assessed by determining if the amplitude was > 2 x RMS (root mean squared) of baseline [ 43 ]. The peak amplitude was measured relative to baseline levels obtained for 1s just prior to photostimulation. To examine the changes in the synaptic response to repetitive stimulation, paired pulse ratios were calculated by comparing the amplitude of the initial response to that evoked by the nth pulse (EPSCn/EPSC1) as well as the 10th pulse (EPSC10/ EPSC). To examine how TRN stimulation affects spiking activity, dLGN neurons were held at -70 mV and injected with a 1.5-s square wave depolarizing current pulse of sufficient strength to evoke a steady train of spike firing 5 Hz [ 48 ]. Changes in spike firing were then calculated by comparing equivalent periods (0.5 s) of activity in the presence or absence of blue light stimulation (2, 5, 10, 20, and 50 Hz). Typically, the measurements described above were based on the average of four stimulus presentations. All traces reflect the averaged responses of individual trials. All statistical tests were performed using Prism 10.1.1 (Graphpad Software, La Jolla, CA) and reported in the results section. Results Development of the Sectorial Arrangement of TRN The TRN is comprised of nonoverlapping, modality-specific sectors that are defined by the source of their ascending thalamocortical (TC) and descending corticothalamic (CT) collateral inputs [ 3 , 43 , 49 – 51 ]. While TC and CT projections pass through TRN at perinatal ages, [ 52 – 57 ], it remains unclear whether they innervate the TRN in a segregated, modality-specific manner. To address this, we made injections of a retrograde tracer (CTB conjugated to either 488 or 647 Alexa dyes) into visual (Fig. 2 , blue) or somatosensory cortex (Fig. 2 , yellow) in WT mice at postnatal day 1 (n = 6). As early as P2, CTB injections into visual cortex (Fig. 2 A) resulted in retrograde labeling of TC neurons in dLGN as well as the pulvinar, but not in ventrobasal complex (VB) (Fig. 2 B). In all six cases, the labeled axons within TRN were restricted to the head (i.e., dorsal-caudal region) of the nucleus (Fig. 2 C, see Fig. 1 B). Moreover, we failed to detect any axonal labeling ventral to the apex of TRN, a boundary that delineates visual and nonvisual sectors of the TRN in the adult mouse [ 43 ]. A similar modality specific patterning was evident following CTB injections into somatosensory cortex (Fig. 2 D). These injections labeled TC neurons that were restricted to somatosensory thalamic nuclei such as the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei (i.e., ventrobasal complex (VB), Fig. 2 E). In TRN, the axons from somatosensory nuclei were restricted to the ventro-medial region at or below the apex (Fig. 2 F) adjacent to VB. While TRN sectors are established at perinatal ages, it is unclear whether this arrangement is preserved in the absence of peripheral sensory input. To test for this, we made similar CTB injections in Math 5 −/− mice, a mutant mouse that lacks > 95% of retinal ganglion cells and that is completely devoid of retinal input to the brain [ 31 , 33 , 37 ]. Similar to WT, injections into visual cortex of Math 5 −/− mice (Figs. 2 G and H) led to retrograde labeling in the head of TRN above the apex (Fig. 2 I, blue, n = 3), while injections into somatosensory cortex (Figs. 2 J and K) labeled axons in the ventromedial region of TRN at or below the apex (Fig. 2 L, yellow, n = 2). Taken together, these results reveal that the sensory specific organization of TRN is established by perinatal ages and that such organization is retained in the absence of sensory stimulation. These results also allowed for us to examine the formation of visual feedforward and feedback connections by targeting the dorsal caudal aspect (head) of TRN (see Fig. 1 B). dLGN innervation of TRN To visualize excitatory feedforward projections from dLGN to TRN, we crossed corticotropin releasing hormone-Cre (CRH-Cre) mice with an Ai9 reporter line [ 43 , 58 , 59 ] in order to express tdTomato in TC neurons of 1st order thalamic nuclei such as the dLGN and VB complex [ 43 , 44 , 58 ]. Figure 3 provides examples of coronal sections of labeled TC axons at different postnatal ages in both WT (Fig. 3 A) and Math 5 −/− (Fig. 3 B) mice. For both groups, as early as P0, TC axons coursed through TRN on a trajectory from the medial aspect of the nucleus to the lateral edge where they aggregate to form the internal capsule. At later ages, axons within TRN were arranged into fascicles forming a reticular configuration. This arrangement emerged as early as P7 and was readily apparent within the head of vTRN by P14. It is important to note that the projections from dLGN to TRN involve fine caliber axon collaterals while thalamocortical axons are much larger and bundled into fascicles [ 3 ]. Indeed, these processes are difficult to resolve at the light microscopic level. Thus, to visualize terminal boutons of thalamocortical axon collaterals in TRN we used an antibody against a vesicular glutamate transporter 2 (vGluT2) [ 38 – 40 , 60 , 61 ]. Figure 4 A illustrates the punctate vGluT2 labeling of TC axon collaterals (blue) in relation to TC tdTomato axonal labeling (purple) and TC somatic NeuN labeling (yellow) in the same coronal section through the TRN of an adult mouse. To determine when TC axon collateral terminals appear in vTRN and whether they are affected by the removal of retinal signaling, we examined vGluT2 labeling in coronal sections from age-matched WT (Fig. 4 B, top) and Math 5 −/− (Fig. 4 B, bottom) mice. To analyze the rate of TRN innervation by TC axon collaterals, we quantified the percentage of vTRN area covered by vGluT2 puncta across different postnatal ages and plotted the median values (Fig. 4 C; [ 43 ]). Measurements were confined to the head of TRN above the apex (Fig. 1 B.) In WT, the area covered by vGluT2 puncta increased with age (filled circles, n = 104 sections, Kruskal-Wallis: p < 0.0001). During the first postnatal week (P5-P7) vGluT2 labeling was sparse but showed a progressive increase, eventually covering nearly half of vTRN by P10 (P5 and P7 vs P10, Dunn’s multiple comparison test, p < 0.01) and then all of it by P21 (all comparisons between P10, P14 and P21, p < 0.01). At P21, thalamic innervation of visual TRN plateaued and did not differ from P28 (p = 0.3063). Thus, innervation of vTRN by TC axon collaterals occurred largely during the second postnatal week (P7-P14) and encompassed the entire visual sector by the end of the third postnatal week (P21). Similar to WT, there was an age-related increase in vGluT2 puncta in the vTRN of Math 5 −/− mice (Fig. 4 C, open circles, n = 72 sections, Kruskal-Wallis: p < 0.0001). Moreover, the age-related increase in vGluT2 labeling had a similar trajectory in WT and Math 5 −/− mice (two-way ANOVA, F genotype (1,164) = 0.5503, p = 0.4593). Together, these data demonstrate that while TC axons course through TRN at birth, the appearance of axon collaterals increases during the second postnatal week and reaches adult-like levels by the end of the third postnatal week (P21). Furthermore, the absence of retinal inputs does not alter the time course of TC innervation of visual TRN. TRN axon innervation of dLGN To visualize inhibitory feedback projections from TRN to dLGN we used GAD65-EGFP mice [ 27 , 35 , 36 ], which labels TRN neurons with EGFP as early as P0 (Fig. 5 A). We also crossed Math 5 −/− mutants with GAD65-EGFP mice to assess whether the absence of retinal signaling disrupted the timing of feedback innervation. Figure 5 provides coronal sections of dLGN from early postnatal GAD65 and GAD65 x Math 5 −/− mice. In WT (Fig. 5 B, top row), TRN fibers were absent from dLGN at birth, but began to innervate the nucleus at P2 from the medial-ventral border. By the end of the first postnatal week TRN input encompassed all of dLGN in a dense plexus of terminals. We analyzed the rate of TRN innervation by quantifying the spatial extent of terminals expressed as a percentage of dLGN area [ 29 , 30 ] and plotted the median values (Fig. 5 C) for WT and Math 5 −/− groups. In WT (filled circles, n = 47 sections), TRN terminals arrived in dLGN at P2 and showed a progressive increase with age (Kruskal-Wallis, p < 0.0001). Most of the projections arrived between P2-4 and showed nearly a 3-fold increase over this period (median values, P2 = 22.40 vs P4 = 61.34). By P6, TRN innervation was complete, forming a dense but diffuse network that spanned the entire nucleus (median values, P6 = 83.87, P9 = 82.67, P18 = 86.08). In Math 5 −/− (n = 59 sections). The absence of retinal signaling appeared to accelerate TRN innervation of dLGN (Fig. 5 B bottom row). At P0, TRN axons could be seen innervating the ventral medial border, and by P2 they nearly encompassed all of dLGN with some axons extending close to the dorsal lateral border just beneath the optic tract. TRN terminals showed a two-fold increase in innervation between P0 and P2, (open circles n = 59) which rose steadily between P4-6 (median values: P0 = 26.18, P2 = 51.02, P4 = 61.28). This pattern of innervation was significantly different from WT mice showing greater innervation between P0-3 (multiple K-S tests, for P0, P2 and P3, D = 1.000, adjusted p-value = < 0.05). Together, these data demonstrate that TRN projections arrive in dLGN during the first postnatal week and that loss of retinal signaling accelerates innervation but does not seem to alter the overall density or pattern of innervation. Development of feedforward synaptic connections between dLGN and TRN We adopted an optogenetic approach to assess when functional feedforward connections between dLGN and TRN neurons emerge and to test whether this time course was influenced by the loss of retinal signaling. To photoactivate TC axon collateral terminals and record postsynaptic responses in TRN neurons, we used CRH-Cre x Ai32 (ChR2-EYFP) mice and crossed these onto a Math 5 −/− background. To determine if ChR2 expression in TC neurons was sufficient to drive postsynaptic activity in TRN at early postnatal ages, we recorded light evoked activity directly from TC neurons. At P4, ChR2-EYFP was evident in TC neurons, and blue light stimulation with brief pulses (1ms) led to light evoked depolarizations and spike firing (Figure S1 A-B). To examine feedforward excitatory postsynaptic activity in TRN, we presented repetitive trains of blue light at different temporal frequencies (0.25Hz, 0.5Hz, 1Hz, 5Hz, 10Hz, 50Hz) and conducted voltage clamp recordings using a potassium-based internal solution while holding vTRN neurons at -70mV. We recorded from 186 WT and 114 Math 5 −/− neurons between P5-28 that were verified to be in vTRN based on their biocytin filled reconstructions. Examples of the light evoked responses to blue light pulse trains (1ms, 20 pulses) trains presented at 0.5Hz, 5Hz, and 10Hz are shown in Fig. 6 . During the first postnatal week (P5-7), photoactivation of TC axon collaterals in visual TRN evoked little postsynaptic activity. EPSC activity was rare, of low amplitude and could only follow low rates of stimulation (e.g., 0.5Hz). Between the second and third postnatal weeks in both WT and Math 5 −/− mice, the incidence of light evoked EPSC activity was more prevalent, greater in amplitude and capable of following higher rates of stimulation (e.g., 10 and 50Hz by week 3). A similar profile was observed during the fourth postnatal week with robust excitatory responses recorded throughout the stimulus train. Of notable significance was the emergence of synaptic depression when higher rates of stimulation were used. For example, by week 3, at 5 Hz and 50 Hz the amplitude of EPSCs began to attenuate after the initial pulse but then stabilized to values that were about half the amplitude of the first response. This form of synaptic depression can be quantified by generating paired pulse ratios where the amplitude of the N th response within a stimulus train can be is divided by the initial response (EPSCn/EPSC1; see Fig. 7 C-D). A summary of these age-related changes for WT and Math 5 −/− mice are shown in Fig. 7 , where we plot the incidence of responsive neurons (Fig. 7 A), the amplitude of the initial (and maximal) response (Fig. 7 B), and the paired pulse ratios (Fig. 7 C EPSCn/EPSC1; Fig. 7 D EPSC10/ESPC1). For both WT and Math 5 −/− , during the first postnatal week, only a few neurons showed weak light evoked responses (P5-7, WT: 3/18, 17%; Math 5 −/− : 0/8, 0%). However, between P11-13, the incidence of light evoked EPSC activity showed about a three-fold increase (P11-13, WT: 25/38, 66%; Math 5 −/− : 20/26, 77%). The incidence of light evoked activity continued to rise so that by P21 nearly all neurons tested were responsive (P19-21, WT: 28/31, 90%; Math 5 −/− : 16/18, 89%). This progression was unaffected by the loss of retinal signaling with Math 5 −/− mice showing a similar trajectory as WT (Fisher’s exact test, p > 0.05). Consistent with the increase in incidence, both groups showed an age-related increase in peak amplitude. Overall, amplitude increased with age (Fig. 7 B, two-way ANOVA, F age(2,138) = 5.743, p = 0.0040) and was unaffected by the loss of retinal signaling (F genotype(1,138) = 0.2572, p = 0.6128). In WT and Math 5 −/− , peak amplitude increased during the second postnatal week but was stable between weeks 3 and 4 (Tukey’s multiple comparison test, week 2 vs week 3: p = 0.0174; week 2 vs week 4: p = 0.0096; week 3 vs week 4: p = 0.7982). To estimate the degree of synaptic depression, we computed paired pulse ratios (Fig. 7 C EPSCn/EPSC1; Fig. 7 D EPSC10/ESPC1). A summary of these ratios for each stimulus pulse (EPSCn/EPSC1) for a 1 Hz train for both WT and Math 5 −/− is shown in Fig. 7 C. For both groups, paired pulse ratios exhibited a progressive decline after the initial response that stabilized by the 5th pulse (two-way ANOVAs with repeated measures, F stimulus (1.415, 167.0) = 637.8, p < 0.0001). However, the magnitude of depression was significantly greater during the second postnatal week (red circles) than either the third (green squares) or fourth (blue triangles) weeks (two-way ANOVAs, WT: F age(2,79) = 13.63, p < 0.0001, Math 5 −/− : F age(2,39) = 11.98, p < 0.0001; Tukey’s multiple comparison tests: week 2 vs week 3 or 4: p < 0.0001 in both WT and Math 5 −/− ). To quantify the degree of synaptic depression at different temporal frequencies (0.25, 0.50, 1, 5, 10, and 50Hz) we calculated the paired pulse ratios between the 10th and 1st response (Fig. 7 D). For both groups, there was an age dependent depression with higher temporal frequencies exhibiting the greatest decrements (two-way ANOVAs with Tukey’s multiple comparison testing, WT: F week(2,410) = 6.131 p = 0.0024, F frequency(5,410) = 92.47, p < 0.0001; Math 5 −/− : F week(2,208) = 21.26, p < 0.0001, F frequency(5,208) = 115.1, p < 0.0001, for both genotypes, 0.25Hz or 0.5Hz vs all other stimulation rates: p < 0.0001, 10Hz vs 50Hz WT: p = 0.1822, Math 5 −/− : p = 0.2735). Additionally, we noted that the degree of synaptic depression weakened with age with weeks 3 and 4 exhibiting larger paired pulse ratios than week 2 (WT: week 2 vs week 3: p = 0.0015, week 2 vs week 4: p = 0.0449, week 3 vs week 4: p = 0.8169; Math 5 −/− : week 2 vs week 3 or 4: p < 0.0001, week 3 vs week 4: p = 0.4586) Development of feedback synaptic connections between TRN and dLGN To examine the emergence of feedback inhibitory activity from TRN to dLGN, we conducted whole cell recordings from SST-Cre x ChR2 mice and ones crossed on a Math 5 −/− background. To determine if ChR2 expression in TRN neurons was sufficient to drive postsynaptic activity in dLGN at early postnatal ages, we recorded light evoked activity from vTRN neurons (supplemental Fig. 1C). As early as P2, ChR2-EYFP was evident in TRN and photoactivation with brief pulses (1ms) of blue light led to reliable depolarizations and spike firing (supplemental Fig. 1D). To investigate TRN-mediated inhibition of TC neurons in dLGN, we conducted voltage clamp recordings using a cesium-based internal solution while holding neurons at 0 mV [ 62 , 63 ]. We recorded 352 WT and 195 Math 5 −/− neurons in dLGN between postnatal ages P2-P35. Examples of the light evoked responses at different postnatal weeks to trains of blue light pulses (1ms) presented at different temporal frequencies (0.5Hz, 5Hz, and 10Hz) are shown in Fig. 8 . In both WT and Math 5 −/− dLGN, during the first postnatal week, light evoked inhibitory activity was weak and infrequent. When present, it was typically limited to the first pulse of a stimulus train. By the second postnatal week, inhibitory activity was stronger and more prevalent, but responses to repetitive stimulus trains were limited to low rates of stimulation (e.g., 0.5Hz, 1Hz and 5Hz). By the start of the third postnatal week, all TC neurons exhibited light evoked inhibition that was also accompanied by a form of synaptic depression. At temporal frequencies ≥ 5Hz, IPSC activity began to diminish after the initial pulse but stabilized midway through the stimulus train to values that were about half the amplitude of the first response. A summary of these age-related inhibitory responses for WT and Math 5 −/− mice are depicted in Fig. 9 where we plot the incidence of light evoked responses, (Fig. 9 A), the amplitude of the initial evoked (and maximal) IPSC (Fig. 9 B), and paired pulse ratios (Fig. 9 C EPSCn/EPSC1; Fig. 9 D EPSC10/ESPC1). For both groups, the incidence of light evoked responses followed a similar time course (Fisher’s exact test, p > 0.05 at all ages) with IPSC activity emerging as early as P4 (P4 WT: 5/14, 36%, Math 5 −/− : 33%) and increasing rapidly thereafter so that by the end of the first postnatal week virtually all TC neurons exhibited responses (P7 WT: 18/21, 86%; Math 5 −/− : 8/9, 89%, P10-12 WT: 18/18, 100%, Math 5 −/− : 11/11, 100%; >P12 WT: 198/198, 100%, Math 5 −/− : 116/121, 96%). Both groups showed a comparable age-related increase in IPSC amplitude (Fig. 9 B; two-way ANOVA with Tukey multiple comparison testing, F age(4,156) = 30.26, p < 0.0001; F genotype(1,156) = 0.1095, p = 0.7412; ). Between weeks 1–3, IPSC amplitude exhibited a progressive increase (WT or Math 5 −/− : week 1 vs week 3, 4 or 5: p < 0.01, week 2 vs week 4 or 5: p 0.05). For both groups, measurements of paired pulse ratios taken for a 1Hz stimulus train (Fig. 9 C EPSC n /EPSC 1 ) or across a range of temporal frequencies (0.25, 0.50, 1, 5, 10 and 50Hz; Fig. 9 D) reflected a sustained synaptic depression. At 1 Hz, ratios computed for each pulse within the stimulus train show an initial reduction that remained constant throughout stimulation with the greatest attenuation seen prior to the third postnatal week (Fig. 9 C; two-way ANOVA with repeated measures, F age(7,115) = 11.31, p < 0.0001). However there was no difference between WT (solid symbols) and Math 5 −/− (open symbols; two-way ANOVAs, week 2: F genotype (1,41) = 0.0003, p = 0.9866; week3: F genotype (1,32) = 0.0620, p = 0.8049; week 4: F genotype (1,26) = 2.911, p = 0.0999; week 5: F genotype (1,16) = 0.0048, p = 0.9455). When examined across a range of temporal frequencies, paired pulse ratios based on the 10th pulse revealed a sustained depression, with higher temporal frequencies exhibiting the largest decline (two-way ANOVAs WT: F frequency(5,231) = 60.54, p < 0.0001; Math 5 −/− : F frequency(5,385) = 187.0, p < 0.0001; Tukey multiple comparison testing in both WT and Math 5 −/− : 0.25Hz, 0.5Hz or 1Hz vs 5Hz, 10Hz or 50Hz: p < 0.0005). Such depression weakened with age (two-way ANOVAs WT: F week(4,231) = 24.03, p < 0.0001; Math 5 −/− : F week(4, 385) = 61.23, p < 0.0001; within each stimulus and genotype, week 1 or week 2 vs weeks 3–5: p 0.05) but still remained robust for both groups. Finally, we assessed how the emergence of feedback inhibition from TRN influenced spike activity of dLGN TC neurons. We conducted current clamp recordings (WT n = 52, Math 5 −/− n = 47 cells) and photoactivated TRN terminals while injecting a depolarizing current pulse to trigger a steady train spiking in TC neurons. Changes in spike firing were then calculated by comparing equivalent periods (0.5 s) of activity in the presence or absence of blue light stimulation (10 or 50 Hz). Examples for WT (top) and Math 5 −/− (bottom) neurons recorded at 2 and 4 weeks are shown in Fig. 10 A in which a square current pulse (gray, 1500ms) evoked tonic firing before (left) and during blue light stimulation (500ms, blue) at 10Hz (middle) and 50Hz (right; see also supplemental Fig. 2 for recordings at week 1–4, and 6). For both groups, during postnatal weeks 1 and 2, activation of TRN terminals at 10Hz or 50Hz had little impact on spike firing. However, by week 3, TRN activation began to reduce TC spiking, and by week 4, it led to an almost complete suppression of activity. These data are summarized in Fig. 10 B which plots the average firing rate (left) observed during control and photostimulation epochs. In the absence of photostimulation, both WT and Math 5 −/− groups showed comparable firing rates (three-way ANOVA, mixed effects analysis of control epoch: F week(4,90) = 7.626, p < 0.0001, F genotype(1,90) = 0.06348, p = 0.8017). During photostimulation, there was an age-related suppression of TC spiking that emerged at week 3, continued to decline through week 4 and then led to a nearly complete suppression by week 6 (three-way ANOVA, mixed effects analysis: F week(4,90) = 13.73, p < 0.0001; Tukey multiple comparison testing: week 1 or 2 vs week 4 or 6: p < 0.0005). A similar pattern emerged when firing rates are converted to percent change (stim-control/control) in activity (Fig. 10 B, right) with values approaching 100% reduction at 6 weeks of age (mean +/- SEM: WT 10Hz: 90.107 +/- 4.893, 50Hz: 92.755 +/- 3.916, Math 5 −/− 10Hz: 87.517 +/- 5.035, 50Hz: 88.483 +/- 8.670). Discussion These studies provide new information about the postnatal development of the feedforward and feedback projections between dLGN and TRN. Our retrograde tracing experiments reveal that thalamocortical axons passed through TRN in an orderly, modality-specific manner that resembles the arrangement noted in adult mice. As early as P2 there was a well-defined and non-overlapping organization of sensory afferents, with visual TC axons restricted to a region that is dorsal to the apex and in the head of TRN while nonvisual ones pass through the “tail”, ventral to the apex [ 43 , 64 , 65 ]. Moreover this sectorial arrangement persists even in the absence of retinal signaling (see also [ 66 , 67 ]), suggesting that the organization of TRN is determined largely by intrinsic guidance cues and seems impervious to alterations in signaling from the periphery [ 52 , 68 ]. Our anatomical and electrophysiological experiments in strains that isolate the reciprocal connections between TRN and dLGN revealed that feedback connections from TRN to dLGN were established before feedforward TC collateral input onto TRN neurons. (Fig. 11 ). TRN axons passed through the ventral-medial border of dLGN at birth and began to innervate dLGN at P2 (Fig. 11 , green), fully encompassing the nucleus by P7. At this time, optogenetic stimulation of TRN terminals in dLGN led to weak, light-evoked inhibitory postsynaptic activity among TC neurons. Over the course of the second and third postnatal weeks, responses increased in amplitude and showed some form of synaptic suppression during repetitive stimulation. During weeks 3–5 this depression remained relatively strong, especially during high rates of repetitive stimulation. Interestingly, TRN mediated inhibition had little impact on TC firing during weeks 1–2, perhaps because synaptic currents remained relatively weak. However, the continued age-related increase in current strength between weeks 3–5 led to a concomitant reduction in TC spiking. Indeed, by week 4 TRN stimulation led to an almost complete suppression of TC spiking, especially at high rates of stimulation [ 48 ]. The synaptic depression noted during weeks 1–2 may in part reflect the immature state of a developing synapse since even low rates of stimulation evoked responses that showed substantial fatigue. However, the depression noted in subsequent weeks is likely to reflect the emergence of an adult-like property that is consistent with their ultrastructural synaptic profile [ 48 ]. By contrast, excitatory feedforward connections between TC collaterals and TRN neurons appear later. Although TC axons passed through visual TRN at birth, the labeling of axon collaterals with vGluT2 was not detected until the second week (P10) and did not fully encompass visual TRN until the third week. The onset of synaptic activity followed a similar trajectory. Optogenetic stimulation of TC collaterals in TRN evoked weak excitatory responses during the second week which peaked in amplitude by the end of the third week. Accompanying this increase in synaptic strength was a frequency dependent depression, suggesting that excitatory TC input to TRN is driver-like in nature [ 69 , 70 ]. While retinal afferents are the first to arrive to dLGN and make functional connections with TC neurons [ 71 – 73 ], TRN feedback connections seem to precede the arrival and establishment of other nonretinal inputs [ 24 ]. For example, descending CT projections from layer 6 begin to arrive in force and make connections with TC neurons during the second postnatal week [ 29 , 74 ], while those from brainstem cholinergic nuclei take weeks to arrive and fully innervate dLGN [ 43 ]. Indeed, feedback inhibition from TRN to dLGN also occurs prior to the onset of feedforward inhibition from GABAergic intrinsic interneurons [ 41 ], which form connections with TC neurons near the end of the second week [ 72 ]. The latter sequence has important implications for understanding the source of thalamic inhibition as a contributing factor underlying the maturation of TC network dynamics. The emergence of thalamic inhibition has been postulated to account for the transformation of network activity from an immature state dominated by weak intrinsic oscillatory activity to a more mature one that is comprised of stimulus evoked events and sleep/wake EEG patterns [ 21 – 23 ]. The early postnatal arrival of TRN feedback inhibition appears to roughly coincide with the maturation of TC network dynamics and thus makes for a suitable candidate mechanism. Finally, it is important to note the role of retinal signaling in intrathalamic circuit formation. Our results in Math5 −/− mice, a mutant that is devoid of retinofugal projections [ 31 , 33 ], indicate that the loss of retinal signaling accelerates TRN innervation of dLGN, but has little or no impact on the development of dLGN input to TRN. The premature innervation of dLGN by TRN afferents is consistent with several other studies that underscore the role of retinal signals in regulating the timing of non-retinal innervation of dLGN [ 24 ]. Indeed, in the absence of retinal signaling, intrinsic interneurons fail to target dLGN appropriately, leading to a loss of their input onto TC neurons [ 41 , 75 ]. It also results in a delay and misrouting of brainstem cholinergic input to dLGN [ 43 ], as well as an acceleration of descending layer VI innervation of dLGN [ 29 ]. The latter is mediated by the early degradation of aggrecan an extracellular matrix molecule that serves as a repellent for CT innervation of dLGN [ 28 ]. However, in the absence of retinal input this repulsive molecule is nearly absent at perinatal ages due to abnormally high levels of degradative proteases that cleave it. Whether aggrecan also regulates the timing of TRN innervation remains untested. Interestingly, the absence of retinal input to dLGN did not appear to alter the timing or nature of TRN synapse formation. The incidence, strength, and degree of synaptic suppression were unaffected. Therefore, the presence of retinal inputs at early ages, while altering the rate of TRN innervation, does not appear to have an impact on TRN synapse formation or maturation. This would suggest that these developmental steps in circuit assembly (innervation and synapse formation) are separable and regulated by independent mechanisms. Conclusion The TRN is organized into modality specific sectors at or near birth. The feedback connections from TRN to dLGN are established before feedforward TC collateral input onto TRN neurons. While TC axons pass through TRN at birth, their collateral sprouting and synaptic innervation of TRN neurons occurs largely during weeks 2-3. Feedback projections from TRN to dLGN begins at P2, with terminals encompassing most of dLGN by the end of the first week and functional inhibitory responses emerging soon after, reaching an adult-like state by 3 weeks of age. There is an age-related increase in TRN mediated inhibition which is accompanied by a progressive increase in the suppression of TC spiking. The absence of retinal signaling, leads largely to an acceleration of TRN innervation of dLGN but has little impact on the development of feedforward projections from dLGN to TRN. Together, these studies provide a foundation to investigate the development of TC network dynamics as well as a reference to understand neurodevelopmental diseases that implicate TRN circuitry. Declarations Ethics approval and consent to participate: N/A Consent for publication: N/A Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: None Funding: This work was supported by EY12716 (WG) and EY026792 (PC). Authors' contributions: P.W.C., G.G., and W.G. conceived and designed research; P.W.C., G.G., and W.G. performed experiments; P.W.C. and W.G. analyzed data; P.W.C., and W.G. prepared figures; P.W.C. and W.G. drafted manuscript. Acknowledgements: We thank B. O’Steen for expert technical support. References Halassa MM, Sherman SM. Thalamocortical Circuit Motifs: A General Framework. Neuron. 2019;103:762–70. Jones EG. The thalamus. Secondedition. Cambridge University Press; 2007. Pinault D. The thalamic reticular nucleus: Structure, function and concept. Brain Res Brain Res Rev. 2004;46:1–31. Mitrofanis J, Guillery RW. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4014221","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276822242,"identity":"3df7c39d-2725-47e9-8848-035ef995ab1d","order_by":0,"name":"Peter W Campbell","email":"","orcid":"","institution":"University of Louisville","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"W","lastName":"Campbell","suffix":""},{"id":276822243,"identity":"42d97bc9-1ba0-4743-980f-1100e80e9582","order_by":1,"name":"Gubbi Govindaiah","email":"","orcid":"","institution":"University of Louisville","correspondingAuthor":false,"prefix":"","firstName":"Gubbi","middleName":"","lastName":"Govindaiah","suffix":""},{"id":276822244,"identity":"62e33a3e-fd16-49c2-a28d-94af9980fec8","order_by":2,"name":"William Guido","email":"data:image/png;base64,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","orcid":"","institution":"University of Louisville","correspondingAuthor":true,"prefix":"","firstName":"William","middleName":"","lastName":"Guido","suffix":""}],"badges":[],"createdAt":"2024-03-04 19:49:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4014221/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4014221/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52139734,"identity":"dec92f0c-7e68-4315-9299-7980ef40c093","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Wiring diagram illustrating the pattern of connectivity within the visual thalamus. Retinal projections (blue) innervate the dorsal lateral geniculate nucleus (dLGN) and provide excitatory drive for intrinsic interneurons (black) and thalamocortical relay neurons (R, purple). Thalamocortical relay neurons convey excitatory signals to the visual cortex (yellow). Their axons pass through the visual sector of the thalamic reticular nucleus (TRN, green) and give off axon collaterals that excite TRN neurons (feedforward excitation). Neurons from visual TRN project back to dLGN relay neurons to suppress thalamocortical transmission (feedback inhibition). \u003cstrong\u003eB. \u003c/strong\u003eNeuN staining in a coronal section from an adult mouse illustrates the cytoarchitectural borders (dashed lines) of the TRN. The apex (dotted line) is a landmark that delineates the division between visual and non-visual sectors of TRN [43,64].\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/cd4259fe067d79e42a03ac63.jpg"},{"id":52139735,"identity":"72ee0a15-bc32-4d83-aaa4-a4c7c68c601b","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90802,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of TRN sensory sectors in P2 WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e mice. \u003cstrong\u003eA.\u003c/strong\u003e An image of the cortical surface of a P2 brain illustrating the location of the CTB injection into the visual cortex. Coronal sections from this case are shown in B and C. Dotted line depicts the longitudinal fissure. Abbreviations: CB: cerebellum, OB: olfactory blub, A: anterior, P: posterior. \u003cstrong\u003eB.\u003c/strong\u003e Retrograde labeling of thalamocortical (TC) neurons in dLGN (outlined by dotted lines). Some TC neurons in pulvinar (medial of dLGN) were labeled but the ventrobasal complex (VB) and medial geniculate nucleus (MGN, not shown) were devoid of CTB. \u003cstrong\u003eC.\u003c/strong\u003e Coronal section through TRN (outlined by dotted lines) shows CTB-labeled TC projections restricted to the head of TRN. There was an absence of CTB labeling below the apex. \u003cstrong\u003eD.\u003c/strong\u003e The cortical surface of a P2 brain illustrates the location of a CTB injection into somatosensory cortex (yellow). Coronal sections from this case are shown in E and F. \u003cstrong\u003eE.\u003c/strong\u003e Retrograde labeling of TC neurons was seen throughout much of VB, but no CTB was observed in dLGN. \u003cstrong\u003eF.\u003c/strong\u003e CTB-labeled TC projections from VB were detected at and below the apex of TRN. \u003cstrong\u003eG.\u003c/strong\u003e The cortical surface of a P2 Math 5\u003csup\u003e-/-\u003c/sup\u003e brain showing a CTB (blue) injection into the visual cortex. Coronal sections from this case are shown in H and I. \u003cstrong\u003eH.\u003c/strong\u003e Retrograde labeling of TC neurons was prevalent in dLGN and adjacent pulvinar nucleus but absent in VB (ventral to dLGN) or vMGN (not shown). \u003cstrong\u003eI.\u003c/strong\u003e TC axons passing through TRN were restricted to the head of TRN, dorsal to the apex. \u003cstrong\u003eJ. \u003c/strong\u003eThe cortical surface of a P2 Math 5\u003csup\u003e-/-\u003c/sup\u003e brain illustrating a CTB injection (yellow) into the somatosensory cortex. Coronal sections from this case are shown in K and L.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eK.\u003c/strong\u003e TC neurons in VB were retrogradely labeled by CTB but none were found in dLGN. \u003cstrong\u003eL.\u003c/strong\u003e In TRN, TC labeled axons were confined to regions ventral to the apex.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/8af3f79078a58cef770db9a2.jpg"},{"id":52139733,"identity":"2d0bbafb-d8af-4190-99c6-f76015dece5e","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50743,"visible":true,"origin":"","legend":"\u003cp\u003eThalamocortical (TC) projections coursing through TRN in CRH-Cre x Ai9 (tdtomato) WT and CRH-Cre x Ai9 mice crossed onto a Math 5\u003csup\u003e-/-\u003c/sup\u003e background. Coronal sections from CRH-Cre x Ai9 WT (top panels) and Math 5\u003csup\u003e-/-\u003c/sup\u003e mice (bottom panels) at postnatal day (P) 0, P7, P14, P21 and P28. At all ages and for both groups, robust td-tomato was evident in TC neurons located in VB as well as TC axons coursing through the TRN in route to neocortex. Scale bar=100um.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/eb0ee201775c1b5f63f2ef38.jpg"},{"id":52139736,"identity":"57034f92-0bf1-4e45-85ec-c833b6c4a824","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116951,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of TC axon terminals in TRN using vesicular glutamate transporter 2 (vGluT2) immunohistochemistry. \u003cstrong\u003eA.\u003c/strong\u003e Coronal sections through TRN (outlined by dotted lines) from a P28 CRH-Cre x Ai9 mouse with vGluT2 immunohistochemistry. Cre-dependent tdTomato (purple) labeled TC neurons and their axons coursing through TRN. vGluT2 immunohistochemistry (blue) labeled TC terminals in TRN. NeuN (yellow) is a marker of somatic nuclei and used to delineate the borders of TRN. \u003cstrong\u003eB.\u003c/strong\u003e Coronal sections depict a progressive increase in vGluT2-positive TC terminals within TRN for WT (top) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (bottom) mice at P7, P10, P14 and P21. In both WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e, vGluT2 labeling was nearly absent at P7 (left) with labeled puncta emerging by P10 and increasing in density with age. \u003cstrong\u003eC.\u003c/strong\u003e Summary plot depicting the spatial extent of TC axon collateral terminals in visual TRN (regions above the apex) as a function of postnatal age. Each data point represents the median percent of visual TRN area covered by vGluT2 labeling (± 95% confidence interval) in both WT (solid symbols, n=104 sections) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (open symbols, n=72 sections) mice. Values were derived from \u003cu\u003e\u0026gt;\u003c/u\u003e8 hemispheres from \u003cu\u003e\u0026gt;\u003c/u\u003e2 animals using 2-3 successive sections of visual TRN. The area covered by vGluT2 labeling increased with age for both WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e mice.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/ad9975bc6b79765217e799f1.jpg"},{"id":52139742,"identity":"95e66754-19b2-4a2f-926e-4fe84fae9389","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":130882,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment\u003cstrong\u003e \u003c/strong\u003eof TRN projections to dLGN in the presence and absence of retinal input. GAD65-EGFP mice were used to visualize TRN projections in WT. Math 5\u003csup\u003e-/-\u003c/sup\u003e mutants crossed with GAD65-EGFP mice were used to examine projections in the absence of retinal signaling. \u003cstrong\u003eA.\u003c/strong\u003e Examples of confocal images of TRN and dLGN at P0 (left) and P28 (right) from WT GAD65 EGFP mice depict robust labeling of TRN neurons. Note the lack of EGFP expression in dLGN interneurons. \u003cstrong\u003eB.\u003c/strong\u003e Coronal sections from early postnatal GAD65 mice containing the middle region of dLGN. Examples show the developmental time course of TRN innervation of dLGN both in the presence (WT, top) and absence (Math 5\u003csup\u003e-/-\u003c/sup\u003e, bottom) of retinal input. \u003cstrong\u003eC.\u003c/strong\u003e Summary plot showing the percentage of dLGN area covered in fluorescent fibers as a function of postnatal age for both WT (solid symbols) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (open symbols). Each data point (green) is the median value of 47 WT and 59 Math 5-/- sections obtained from \u0026nbsp;\u003cu\u003e\u0026gt;\u003c/u\u003e3 hemispheres of \u003cu\u003e\u0026gt;\u003c/u\u003e2 mice with error bars representing 95% confidence intervals.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/6bf780442312c8986513f2fc.jpg"},{"id":52139738,"identity":"75944afe-10d6-4197-9a60-23b4fe1f67e7","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61068,"visible":true,"origin":"","legend":"\u003cp\u003eLight evoked synaptic responses of TRN neurons from WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e CRH-Cre x ChR2 mice. Examples of excitatory synaptic responses recorded in voltage clamp mode to trains of blue light (20, 1ms pulses delivered at 1, 5 and 10Hz) are depicted by postnatal week for WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e mice. Trains of blue light are shown in blue beneath each corresponding response. The inset depicts feedforward (purple) and feedback green circuits between TRN and dLGN and the blue circle indicates the location of blue light stimulation. All neurons held at -70mV.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/d2fb7c5f576559ed7cbacf2b.jpg"},{"id":52140926,"identity":"acb96c01-66d6-4fb4-b1ca-36b5c9eaffa2","added_by":"auto","created_at":"2024-03-07 10:58:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":79567,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the light evoked synaptic responses of TRN neurons from WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e CRH-Cre x ChR2 mice. \u0026nbsp;\u003cstrong\u003eA.\u003c/strong\u003e Plot of the percent of responsive cells by age for WT (solid symbols, n=186) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (open symbols, n=114) mice. Each point represents data from 4-39 neurons (median = 15). For both groups, incidence increased with age, nearing 100% by P20. \u003cstrong\u003eB.\u003c/strong\u003e The amplitude of the initial (maximal) EPSC is plotted by postnatal week in WT (solid symbols, n=96) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (open symbols, n=50). Bars represent the mean amplitude (± SEM) at each postnatal week. EPSC amplitude increased with age. \u003cstrong\u003eC.\u003c/strong\u003e A plot of the paired pulse ratios as a function of stimulus number for a 1Hz stimulus train. Ratios of EPSC amplitudes were calculated by dividing the n\u003csup\u003eth\u003c/sup\u003e response by the initial response. Each point represents the mean (± SEM) of at least 14-43 neurons. PPRs \u0026lt; 1 reflect synaptic depression. Symbols depict different weeks with solid ones representing WT and open ones Math 5\u003csup\u003e-/-\u003c/sup\u003e groups. Both groups showed comparable degrees of depression with the largest attenuation noted during week 2. \u003cstrong\u003eD. \u003c/strong\u003eThe paired pulse ratios of EPSC amplitudes during a 0.25, 0.50, 1, 5, 10 and 50Hz Hz stimulus train was calculated by dividing the 10\u003csup\u003eth\u003c/sup\u003e response by the initial response. Each point represents the mean (± SEM) of at least 6-43 (median = 14) neurons. Temporal frequency of stimulation (0.25Hz – 50Hz) is organized by color. Both age and stimulus frequency led to lower PPRs, which indicates a greater degree of synaptic depression.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/5749265ea9bf044baf4988db.jpg"},{"id":52139741,"identity":"d8889044-01e7-4c77-a4e2-1758a7d494ad","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":76263,"visible":true,"origin":"","legend":"\u003cp\u003eLight evoked synaptic responses of dLGN neurons from WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e CRH-Cre x ChR2 mice. Examples of inhibitory synaptic responses recorded in voltage clamp mode to trains of blue light (twenty 1ms pulses delivered at 1, 5 and 10Hz) are depicted by postnatal week for WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e mice. Trains of blue light are shown in blue beneath each corresponding response. Inset depicts feedforward (purple) and feedback green circuits between TRN and dLGN and the blue circle indicates the location of blue light stimulation. All neurons held at 0mV.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/51d3d795c78c4960d1eb7421.jpg"},{"id":52139743,"identity":"972d3434-e382-4d7d-b06a-7f38a5d74a58","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":84385,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the light evoked synaptic responses of dLGN neurons from WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e CRH-Cre x ChR2 mice. \u0026nbsp;\u003cstrong\u003eA.\u003c/strong\u003e A plot of the percent of responsive cells by age for WT (solid symbols, n=352) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (open symbols, n=195) mice. Each point represents data from 7-66 neurons (median=25). For both groups incidence increased with age, nearing 100% by P8. \u003cstrong\u003eB.\u003c/strong\u003e The amplitude of the initial (maximal) EPSC is plotted by postnatal week in WT (solid symbols, n=64) and Math 5\u003csup\u003e-/-\u003c/sup\u003e (open symbols, n=102). \u003cstrong\u003eC.\u003c/strong\u003e A plot of the paired pulse ratios as a function of stimulus number for a 1Hz stimulus train. Ratios of EPSC amplitudes were calculated by dividing the n\u003csup\u003eth\u003c/sup\u003e response by the initial response. Each point represents the mean (± SEM) of 7-15 neurons. PPRs \u0026lt; 1 reflect synaptic depression. Symbols depict different weeks with solid ones representing WT and open ones Math 5\u003csup\u003e-/-\u003c/sup\u003e groups. Both groups showed comparable degrees of depression with the largest attenuation noted during week 2. \u003cstrong\u003eD. \u003c/strong\u003eA plot of the paired pulse ratios as a function of age for 0.25, 0.50, 1, 5, 10 and 50Hz stimulus trains. Ratios of EPSC amplitudes were calculated by dividing the 10\u003csup\u003eth\u003c/sup\u003e response by the initial response. Each point represents the mean (± SEM) of at least 4-40 neurons (median=13). Temporal frequency of stimulation (0.25Hz – 50Hz) is organized by color. Stimulus frequency led to low PPRs, which indicates a greater degree of synaptic depression.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/43d409c64f6002870649374b.jpg"},{"id":52139739,"identity":"81d2124a-ff8e-4f32-9aea-49b285d2a363","added_by":"auto","created_at":"2024-03-07 10:50:08","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":102708,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of TRN-mediated inhibition of dLGN activity.\u003cstrong\u003e A.\u003c/strong\u003e Examples of voltage responses obtained from P14 (week 2, top) and P28 (week 4, bottom) SST-Cre x ChR2 WT and SST-Cre x ChR2 mice crossed on a Math 5\u003csup\u003e-/-\u003c/sup\u003e background. Each group shows spiking evoked by a square wave current pulse (1500ms) injection (gray trace beneath response). Responses in the left column show spiking in the absence of TRN stimulation (control), while those on the right depict responses during blue light stimulation at 10Hz and 50Hz. The rate of firing was measured during the middle 500ms of the depolarization (black bar, top) in the absence (control, left) and presence (stimulation, right) of blue light pulses (blue). \u003cstrong\u003eB.\u003c/strong\u003e Summary plots showing the changes in spike firing brought about by TRN stimulation. Left: Summary of firing rates measured as a function of postnatal week during a 500ms (see above) epoch in the absence (control) and presence of TRN terminal stimulation.\u0026nbsp; Each point represents the mean ± SEM for WT (solid symbols, n=73) and Math 5\u003csup\u003e-/-\u003c/sup\u003e, (open symbols, n=81) neurons. Firing rates during control recordings remained relatively stable. TRN activation had little impact on firing during the first two postnatal weeks but suppressed firing during weeks 4 and 6. Right: Plot showing firing rate as a percent change from a matching period of control. Each point represents the mean ± SEM for WT and Math 5\u003csup\u003e-/-\u003c/sup\u003e groups. There was a progressive decline after week 2.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/6fae53925304d0d6ae3c313c.jpg"},{"id":52140925,"identity":"72c02a3b-7315-41fa-8b0b-01d9904b0c52","added_by":"auto","created_at":"2024-03-07 10:58:08","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":74658,"visible":true,"origin":"","legend":"\u003cp\u003eTimeline illustrating the development of the connections between TRN and dLGN. \u003cstrong\u003eA.\u003c/strong\u003e Development of feedforward input from dLGN to TRN. Top: Thalamocortical projections (purple) are present in TRN at birth but terminals on axon collaterals (blue) do not emerge until postnatal week 2. The density of axon collateral terminals gradually increases up until postnatal week 4. Bottom: Excitatory responses are absent during the first postnatal week and highly immature during week 2. EPSCs are weak and exhibit fatigue in the face of repetitive stimulation. EPSC activity gradually strengthens so that by week 3, evoked activity is of large amplitude and responds faithfully to repetitive stimulation. \u003cstrong\u003eB.\u003c/strong\u003e Development of TRN feedback projections to dLGN. Top: TRN fibers (green) are present at the ventral-medial edge of dLGN at birth. During week 1, fibers innervate dLGN and cover much of the nucleus. Middle: Inhibitory responses arise during postnatal week 1 but are of low amplitude and cannot follow trains of stimuli \u0026gt;0.5Hz. Responses continue to mature during postnatal weeks 2 and 3 so that by week 4, evoked activity is large and able to follow high temporal frequency stimulation. Bottom: TRN inhibition has little if any impact on dLGN spiking during the first two postnatal weeks. During week 3, activation of TRN terminals decreases TC firing, and by week 4 can fully suppress transmission.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/afdc3d086abf2e23e2dbcb3e.jpg"},{"id":52141176,"identity":"eb82ebac-2e40-489a-b415-cc962f5c4f72","added_by":"auto","created_at":"2024-03-07 11:06:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1054774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/17645dde-1534-4a98-86e8-d62cf606b828.pdf"},{"id":52139745,"identity":"3ce502db-b4be-4b77-a74e-354ad610f392","added_by":"auto","created_at":"2024-03-07 10:50:09","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":689337,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigurescampbelletal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4014221/v1/15da1285af5337d626a54f0b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of feedforward and feedback connections between the dorsal lateral geniculate nucleus and the thalamic reticular nucleus","fulltext":[{"header":"Background","content":"\u003cp\u003eThe thalamic reticular nucleus (TRN) is a shell-like structure that surrounds the dorsal and lateral aspects of the thalamus [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Comprised entirely of GABAergic neurons, the TRN serves as an important nexus for thalamocortical (TC) and corticothalamic (CT) communication. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This nucleus receives excitatory input from thalamocortical and corticothalamic axon collaterals, which in turn serve to activate GABAergic inhibitory feedback to many thalamic nuclei. This pattern of connectivity is arranged in a sectorial manner that allows for both modality-specific and network-wide interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe TRN plays a key role in TC function by modulating sensory signaling during different behavioral states, participating in the generation and propagation of thalamocortical rhythms during sleep and wakefulness [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, a disruption in connectivity or in the intrinsic membrane properties of TRN neurons has been implicated in a number of neurodevelopmental diseases including epilepsy, autism spectrum disorder and attention hyperactivity disorder [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although the organization of TRN and its impact on state dependent behavior has been studied extensively, we still lack a fundamental understanding of how and when these intrathalamic connections develop and become operational. Studies in rodent have begun to characterize early postnatal TC network activity [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], however, the underlying circuitry linking the TRN to first-order thalamic nuclei remain unexplored.\u003c/p\u003e \u003cp\u003eThe dLGN of the mouse has emerged as a model system to study thalamic circuit development [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Much of our present understanding is based on the study of the retinogeniculate pathway [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies have also delineated when and how input from nonretinal sources, such as those arising from layer VI of visual cortex, as well as those originating from cholinergic nuclei of the brainstem, innervate dLGN and form functional connections with thalamocortical relay neurons [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Taken together, these studies reveal that circuit assembly is dLGN is a highly orchestrated event, with retinogeniculate connections emerging first, followed by CT innervation during the second postnatal week, and then finally by input from cholinergic brainstem areas which proceeds slowly through the end of the first postnatal month. Interestingly, the arrival of retinal afferents plays a key role in the timing of nonretinal innervation. For example, the absence or elimination of retinal signaling accelerates the arrival of CT input by disrupting the expression of aggrecan, a repulsive CPSG that normally inhibits cortical axons from entering dLGN prematurely [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the emergence of connections between dLGN and TRN have yet to explored in this context. More specifically, what is lacking is an understanding about how and when the connections linking dLGN and TRN occur, and whether the absence of retinal input affects the development of these feedforward and feedback loops. To accomplish this, we made use of specific Cre-driver, reporter, and transgenic lines that allow for the visualization and interrogation of intrathalamic feedforward and feedback circuits. To study the impact of retinal signaling, we took a loss of function approach and employed a novel form of genetic deafferentation by utilizing a Math5 null (Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mouse. This mutant lacks the transcription factor Math5, which is essential for the differentiation of retinal progenitors into retinal ganglion cells. As a result, Math5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibit a wholesale loss (\u0026gt;\u0026thinsp;95%) of retinal ganglion cells, a failure to develop an optic nerve, and a brain that is devoid of retinofugal projections [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Finally, because our focus is on visual intrathalamic circuits, we also assessed whether the modality-specific sectors of TRN were present at birth and if such an arrangement was altered by visual deafferentation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eSubjects\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExperiments were conducted in mice P0-P46 of either sex. We used the GAD65 EGFP transgenic strain that expresses enhanced green fluorescent protein (EGFP) in TRN [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We also utilized two Cre-driver lines, somatostatin-Cre (SST-Cre; Jax stock no. 013044, RRID: IMSR_JAX:013044) and corticotropin releasing hormone-Cre (CRH-Cre, MMRRC no. 030850-UCD, RRID: MMRRC_030850-UCD), to target reporter expression within TRN (SST-Cre) or thalamocortical neurons (CRH-Cre). The Cre-driver lines were crossed with reporters to enable Cre-dependent expression of either tdTomato (Ai9; Jax stock no. 007909; RRID: IMSR_JAX:007909) or channelrhodopsin 2 \u0026ndash; EYFP (ChR2; Ai32; Jax stock no. 012569; RRID: IMSR_JAX:012569). All lines were crossed onto a Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e background in order to study TRN and dLGN circuit assembly in the absence of retinal input [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. All breeding and experimental procedures were approved by the University of Louisville Institutional Animal Care and Use Committee.\u003c/p\u003e\n\u003ch3\u003eCholera toxin subunit B injections\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eCholera toxin subunit B injections\u003c/div\u003e \u003cp\u003eTo retrogradely label TC projections in TRN, P1 CRH-Cre or CRH-Cre x Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were deeply anesthetized using isoflurane vapors. The skull was pierced with a sterile needle and then a glass pipette (10-20um tip diameter) filled with a 1% solution of Cholera toxin subunit B (CTB) conjugated to different Alexa Fluors (488, 546 or 647; Invitrogen) dissolved in distilled water was lowered into the targeted region. The pipette was attached to a picospritzer and ~\u0026thinsp;2uL of CTB was injected into visual cortex or somatosensory cortex. After a 24-hr survival period, animals were deeply anesthetized by isoflurane vapors and transcardially perfused with PBS followed by 4% paraformaldehyde in 0.1M phosphate buffer (4% PFA). Brains were removed and post-fixed for 24hr in 4% PFA and then transferred to phosphate buffered saline [PBS: 0.01 M phosphate buffer (PB) with 0.9% NaCl]. To verify the injection site, the cortical surface of excised brains was imaged using a stereomicroscope (Olympus SZX2-ILLB) with fluorescence illumination (Prior Scientific Lumen 200).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTo visualize thalamocortical axon collateral terminals in TRN we used vesicular glutamate transporter 2 (vGluT2) labeling [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. At least three coronal sections (35um) containing the visual sector of TRN from at least two different mice were collected from CRH-Cre and CRH-Cre x Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e at ages that ranged from P5-P28. Sections were blocked (10% normal goat serum [NGS; Vector Labs, S-1000, RRID: AB_2336615] and 0.3% Triton X-100 in PBS) for 1hr and then incubated overnight in rabbit anti-vGluT2 (SysSys, AB135403 1:500, RRID: AB_887883; diluted 1:100 with 10% NGS in PBS). After a wash in PBS, a secondary antibody, goat anti-rabbit 488 (ThermoFisher, A11034, RRID: AB_2576217) was applied for 1hr. Sections were washed, then immunolabeled to visualize neurons, and incubated overnight in mouse NeuN (Milipore, MAB377, RRID: AB_2298772). After a wash, sections were incubated for 1hr with biotinylated goat anti-mouse (Vector Labs, BA9200, RRID: AB_2336171) and then labeled with streptavidin-AF647 (ThermoFisher, S21374, RRID: AB_2336066, 1:100 diluted in PBS). Sections were mounted with Prolong Gold (Invitrogen, P36931), coverslipped, imaged using confocal microscopy, and analyzed using methods described below.\u003c/p\u003e \u003cp\u003eFor all in vitro recordings, biocytin (0.5%, Sigma) was included in the internal pipette solution for intracellular filling and 3D neuron reconstruction using confocal microscopy [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Following the completion of the recording session, slices were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), then washed with PBS and incubated overnight with AlexaFluor 647-conjugated streptavidin (Invitrogen, S21374) in a PBS solution containing Triton X-100 (0.1%).\u003c/p\u003e\n\u003ch3\u003eImage acquisition and analysis\u003c/h3\u003e\n\u003cp\u003eTo visualize TRN terminals in dLGN, and TC axons in TRN, fixed brains from GAD-65 EGFP and CRH-Cre XAi9 mice were cut in the coronal plane (70uM) and mounted on slides using ProLong mounting medium containing DAPI (Life Technologies P36931). Sections containing TRN or dLGN were imaged using a multiphoton laser scanning confocal microscope (Olympus, model no. FV1200BX61) equipped with a 20x (0.75 NA) objective. Fluorophores were excited using Ar (488nm) and HeNe (635nm) lasers, and Z-stacked images (1.26um optical sections) were acquired using Fluoview software at a scanning resolution of 1600 x 1600 pixels.\u003c/p\u003e \u003cp\u003eTo quantify the spatial extent of innervation or the density of VGlut2 staining at different postnatal ages, we analyzed at least three coronal sections through the middle of TRN or dLGN [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Z-stacks of each image were generated and imported into Photoshop (Adobe). Images were binarized using a threshold that clearly distinguished signal and background fluorescence. Binarized images were imported into ImageJ (NIH, RRID: SCR_003070) in order to count the number of fluorescent pixels as well as the total number of pixels within a nucleus. These values were used to calculate the percent area that contained fluorescent pixels. At each postnatal age, values were obtained from 3\u0026ndash;28 hemispheres taken from \u0026gt;\u0026thinsp;2 mice. For summary statistics, each hemisphere served as a unit of observation and the median value with a 95% confidence interval were plotted.\u003c/p\u003e\n\u003ch3\u003eIn vitro slice preparation and whole cell recording\u003c/h3\u003e\n\u003cp\u003eAcutely prepared, thalamic brain slices were made from mice that were deeply anesthetized and rapidly decapitated. The brain was excised and placed into 4\u0026deg;C oxygenated cutting solution (in mM): Sucrose 234, glucose 11, NaHCO3 26, MgSO4 10, KCl 2.5, CaCl2 0.5, NaH2PO4 1.25. Bran slices containing thalamus were cut in the coronal plane (270 \u0026micro;m thick) on a vibratome (Leica), placed in a chamber and bathed for 30 min in warm (32\u0026deg;C) oxygenated artificial cerebrospinal fluid (aCSF) (in mM: 126 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4). Recordings were conducted in a chamber mounted on a perfused continuously with 32\u0026deg;C aCSF at a rate of 2\u0026ndash;3 mL/min. Thalamic nuclei were visualized on an upright microscope (Olympus BX51WI) with DIC optics and fluorescent filters (GFP: Chroma 49002; tdTomato: Chroma 49005) using a 10x or 60x water immersion objective. A vertical puller (Narashige PC-10) was used to pull patch electrodes from borosilicate glass. For voltage clamp recordings, the electrode solution contained (in mM) 117 Cs gluconate, 11.0 CsCl, 1.0 MgCl2, 1.0 CaCl2, 0.1 EGTA, 10.0 HEPES, 2 Na2-ATP, 0.4 Na2-GTP. For current clamp recordings, the electrode internal solution contained (in mM) 117 K-gluconate, 13 KCl, 1.0 MgCl2 1, 0.07 CaCl2, 0.1 EGTA, 10 HEPES. A 0.5% The final electrode tip resistance was 4\u0026ndash;7 M Ohms. Whole cell recordings were made in current or voltage clamp mode using an amplifier (Multiclamp 700B, Molecular Devices), filtered at 3\u0026ndash;10 kHz, and digitized (Digidata 1440A) at 20 kHz. Pipette capacitance, series resistance, input resistance, and whole-cell capacitance were monitored throughout the recording session. Inhibitory postsynaptic currents were measured by holding the neuron at 0mV using a cesium-containing electrode while excitatory currents were measured at -70mV using a potassium-containing electrode.\u003c/p\u003e\n\u003ch3\u003eOptogenetic stimulation and analysis\u003c/h3\u003e\n\u003cp\u003eWe took an optogenetic approach to assess functional connections between TRN and dLGN. By crossing CRH-Cre [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] or SST-Cre [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] with an Ai32 mouse [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], we were able to selectively stimulate feedforward or feedback projections in thalamic slices in WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e backgrounds.\u003c/p\u003e \u003cp\u003eLight-gated postsynaptic responses were evoked using a light emitting diode (LED, Prizmatix) that delivered blue light through a 60x objective. Blue light pulses were 0.3mm in diameter with a light power of 525mW/mm2 and with a pulse width of 1ms. Trains of 10 blue light pulses were presented at different temporal frequencies (0.25, 0.5, 1, 5, 10 and 50 Hz). The incidence of optically evoked postsynaptic responses was assessed by determining if the amplitude was \u0026gt;\u0026thinsp;2 x RMS (root mean squared) of baseline [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The peak amplitude was measured relative to baseline levels obtained for 1s just prior to photostimulation. To examine the changes in the synaptic response to repetitive stimulation, paired pulse ratios were calculated by comparing the amplitude of the initial response to that evoked by the nth pulse (EPSCn/EPSC1) as well as the 10th pulse (EPSC10/ EPSC). To examine how TRN stimulation affects spiking activity, dLGN neurons were held at -70 mV and injected with a 1.5-s square wave depolarizing current pulse of sufficient strength to evoke a steady train of spike firing 5 Hz [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Changes in spike firing were then calculated by comparing equivalent periods (0.5 s) of activity in the presence or absence of blue light stimulation (2, 5, 10, 20, and 50 Hz).\u003c/p\u003e \u003cp\u003eTypically, the measurements described above were based on the average of four stimulus presentations. All traces reflect the averaged responses of individual trials. All statistical tests were performed using Prism 10.1.1 (Graphpad Software, La Jolla, CA) and reported in the \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDevelopment of the Sectorial Arrangement of TRN\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe TRN is comprised of nonoverlapping, modality-specific sectors that are defined by the source of their ascending thalamocortical (TC) and descending corticothalamic (CT) collateral inputs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. While TC and CT projections pass through TRN at perinatal ages, [\u003cspan additionalcitationids=\"CR53 CR54 CR55 CR56\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], it remains unclear whether they innervate the TRN in a segregated, modality-specific manner. To address this, we made injections of a retrograde tracer (CTB conjugated to either 488 or 647 Alexa dyes) into visual (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, blue) or somatosensory cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, yellow) in WT mice at postnatal day 1 (n\u0026thinsp;=\u0026thinsp;6). As early as P2, CTB injections into visual cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) resulted in retrograde labeling of TC neurons in dLGN as well as the pulvinar, but not in ventrobasal complex (VB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In all six cases, the labeled axons within TRN were restricted to the head (i.e., dorsal-caudal region) of the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, we failed to detect any axonal labeling ventral to the apex of TRN, a boundary that delineates visual and nonvisual sectors of the TRN in the adult mouse [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A similar modality specific patterning was evident following CTB injections into somatosensory cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These injections labeled TC neurons that were restricted to somatosensory thalamic nuclei such as the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei (i.e., ventrobasal complex (VB), Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In TRN, the axons from somatosensory nuclei were restricted to the ventro-medial region at or below the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) adjacent to VB.\u003c/p\u003e \u003cp\u003eWhile TRN sectors are established at perinatal ages, it is unclear whether this arrangement is preserved in the absence of peripheral sensory input. To test for this, we made similar CTB injections in Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, a mutant mouse that lacks\u0026thinsp;\u0026gt;\u0026thinsp;95% of retinal ganglion cells and that is completely devoid of retinal input to the brain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Similar to WT, injections into visual cortex of Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H) led to retrograde labeling in the head of TRN above the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, blue, n\u0026thinsp;=\u0026thinsp;3), while injections into somatosensory cortex (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and K) labeled axons in the ventromedial region of TRN at or below the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, yellow, n\u0026thinsp;=\u0026thinsp;2).\u003c/p\u003e \u003cp\u003eTaken together, these results reveal that the sensory specific organization of TRN is established by perinatal ages and that such organization is retained in the absence of sensory stimulation. These results also allowed for us to examine the formation of visual feedforward and feedback connections by targeting the dorsal caudal aspect (head) of TRN (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003edLGN innervation of TRN\u003c/h3\u003e\n\u003cp\u003eTo visualize excitatory feedforward projections from dLGN to TRN, we crossed corticotropin releasing hormone-Cre (CRH-Cre) mice with an Ai9 reporter line [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] in order to express tdTomato in TC neurons of 1st order thalamic nuclei such as the dLGN and VB complex [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e provides examples of coronal sections of labeled TC axons at different postnatal ages in both WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) mice. For both groups, as early as P0, TC axons coursed through TRN on a trajectory from the medial aspect of the nucleus to the lateral edge where they aggregate to form the internal capsule. At later ages, axons within TRN were arranged into fascicles forming a reticular configuration. This arrangement emerged as early as P7 and was readily apparent within the head of vTRN by P14.\u003c/p\u003e \u003cp\u003eIt is important to note that the projections from dLGN to TRN involve fine caliber axon collaterals while thalamocortical axons are much larger and bundled into fascicles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Indeed, these processes are difficult to resolve at the light microscopic level. Thus, to visualize terminal boutons of thalamocortical axon collaterals in TRN we used an antibody against a vesicular glutamate transporter 2 (vGluT2) [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA illustrates the punctate vGluT2 labeling of TC axon collaterals (blue) in relation to TC tdTomato axonal labeling (purple) and TC somatic NeuN labeling (yellow) in the same coronal section through the TRN of an adult mouse.\u003c/p\u003e \u003cp\u003eTo determine when TC axon collateral terminals appear in vTRN and whether they are affected by the removal of retinal signaling, we examined vGluT2 labeling in coronal sections from age-matched WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, top) and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, bottom) mice. To analyze the rate of TRN innervation by TC axon collaterals, we quantified the percentage of vTRN area covered by vGluT2 puncta across different postnatal ages and plotted the median values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]). Measurements were confined to the head of TRN above the apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.) In WT, the area covered by vGluT2 puncta increased with age (filled circles, n\u0026thinsp;=\u0026thinsp;104 sections, Kruskal-Wallis: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). During the first postnatal week (P5-P7) vGluT2 labeling was sparse but showed a progressive increase, eventually covering nearly half of vTRN by P10 (P5 and P7 vs P10, Dunn\u0026rsquo;s multiple comparison test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and then all of it by P21 (all comparisons between P10, P14 and P21, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). At P21, thalamic innervation of visual TRN plateaued and did not differ from P28 (p\u0026thinsp;=\u0026thinsp;0.3063). Thus, innervation of vTRN by TC axon collaterals occurred largely during the second postnatal week (P7-P14) and encompassed the entire visual sector by the end of the third postnatal week (P21). Similar to WT, there was an age-related increase in vGluT2 puncta in the vTRN of Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, open circles, n\u0026thinsp;=\u0026thinsp;72 sections, Kruskal-Wallis: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Moreover, the age-related increase in vGluT2 labeling had a similar trajectory in WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (two-way ANOVA, F\u003csub\u003egenotype (1,164)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5503, p\u0026thinsp;=\u0026thinsp;0.4593). Together, these data demonstrate that while TC axons course through TRN at birth, the appearance of axon collaterals increases during the second postnatal week and reaches adult-like levels by the end of the third postnatal week (P21). Furthermore, the absence of retinal inputs does not alter the time course of TC innervation of visual TRN.\u003c/p\u003e\n\u003ch3\u003eTRN axon innervation of dLGN\u003c/h3\u003e\n\u003cp\u003eTo visualize inhibitory feedback projections from TRN to dLGN we used GAD65-EGFP mice [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which labels TRN neurons with EGFP as early as P0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We also crossed Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants with GAD65-EGFP mice to assess whether the absence of retinal signaling disrupted the timing of feedback innervation.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e provides coronal sections of dLGN from early postnatal GAD65 and GAD65 x Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. In WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, top row), TRN fibers were absent from dLGN at birth, but began to innervate the nucleus at P2 from the medial-ventral border. By the end of the first postnatal week TRN input encompassed all of dLGN in a dense plexus of terminals. We analyzed the rate of TRN innervation by quantifying the spatial extent of terminals expressed as a percentage of dLGN area [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and plotted the median values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) for WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e groups. In WT (filled circles, n\u0026thinsp;=\u0026thinsp;47 sections), TRN terminals arrived in dLGN at P2 and showed a progressive increase with age (Kruskal-Wallis, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Most of the projections arrived between P2-4 and showed nearly a 3-fold increase over this period (median values, P2\u0026thinsp;=\u0026thinsp;22.40 vs P4\u0026thinsp;=\u0026thinsp;61.34). By P6, TRN innervation was complete, forming a dense but diffuse network that spanned the entire nucleus (median values, P6\u0026thinsp;=\u0026thinsp;83.87, P9\u0026thinsp;=\u0026thinsp;82.67, P18\u0026thinsp;=\u0026thinsp;86.08). In Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;59 sections). The absence of retinal signaling appeared to accelerate TRN innervation of dLGN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB bottom row). At P0, TRN axons could be seen innervating the ventral medial border, and by P2 they nearly encompassed all of dLGN with some axons extending close to the dorsal lateral border just beneath the optic tract. TRN terminals showed a two-fold increase in innervation between P0 and P2, (open circles n\u0026thinsp;=\u0026thinsp;59) which rose steadily between P4-6 (median values: P0\u0026thinsp;=\u0026thinsp;26.18, P2\u0026thinsp;=\u0026thinsp;51.02, P4\u0026thinsp;=\u0026thinsp;61.28). This pattern of innervation was significantly different from WT mice showing greater innervation between P0-3 (multiple K-S tests, for P0, P2 and P3, D\u0026thinsp;=\u0026thinsp;1.000, adjusted p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Together, these data demonstrate that TRN projections arrive in dLGN during the first postnatal week and that loss of retinal signaling accelerates innervation but does not seem to alter the overall density or pattern of innervation.\u003c/p\u003e\n\u003ch3\u003eDevelopment of feedforward synaptic connections between dLGN and TRN\u003c/h3\u003e\n\u003cp\u003eWe adopted an optogenetic approach to assess when functional feedforward connections between dLGN and TRN neurons emerge and to test whether this time course was influenced by the loss of retinal signaling. To photoactivate TC axon collateral terminals and record postsynaptic responses in TRN neurons, we used CRH-Cre x Ai32 (ChR2-EYFP) mice and crossed these onto a Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e background. To determine if ChR2 expression in TC neurons was sufficient to drive postsynaptic activity in TRN at early postnatal ages, we recorded light evoked activity directly from TC neurons. At P4, ChR2-EYFP was evident in TC neurons, and blue light stimulation with brief pulses (1ms) led to light evoked depolarizations and spike firing (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003eTo examine feedforward excitatory postsynaptic activity in TRN, we presented repetitive trains of blue light at different temporal frequencies (0.25Hz, 0.5Hz, 1Hz, 5Hz, 10Hz, 50Hz) and conducted voltage clamp recordings using a potassium-based internal solution while holding vTRN neurons at -70mV. We recorded from 186 WT and 114 Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e neurons between P5-28 that were verified to be in vTRN based on their biocytin filled reconstructions.\u003c/p\u003e \u003cp\u003eExamples of the light evoked responses to blue light pulse trains (1ms, 20 pulses) trains presented at 0.5Hz, 5Hz, and 10Hz are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. During the first postnatal week (P5-7), photoactivation of TC axon collaterals in visual TRN evoked little postsynaptic activity. EPSC activity was rare, of low amplitude and could only follow low rates of stimulation (e.g., 0.5Hz). Between the second and third postnatal weeks in both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the incidence of light evoked EPSC activity was more prevalent, greater in amplitude and capable of following higher rates of stimulation (e.g., 10 and 50Hz by week 3). A similar profile was observed during the fourth postnatal week with robust excitatory responses recorded throughout the stimulus train. Of notable significance was the emergence of synaptic depression when higher rates of stimulation were used. For example, by week 3, at 5 Hz and 50 Hz the amplitude of EPSCs began to attenuate after the initial pulse but then stabilized to values that were about half the amplitude of the first response. This form of synaptic depression can be quantified by generating paired pulse ratios where the amplitude of the N\u003csup\u003eth\u003c/sup\u003e response within a stimulus train can be is divided by the initial response (EPSCn/EPSC1; see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003eA summary of these age-related changes for WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, where we plot the incidence of responsive neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), the amplitude of the initial (and maximal) response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), and the paired pulse ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC EPSCn/EPSC1; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD EPSC10/ESPC1). For both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, during the first postnatal week, only a few neurons showed weak light evoked responses (P5-7, WT: 3/18, 17%; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 0/8, 0%). However, between P11-13, the incidence of light evoked EPSC activity showed about a three-fold increase (P11-13, WT: 25/38, 66%; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 20/26, 77%). The incidence of light evoked activity continued to rise so that by P21 nearly all neurons tested were responsive (P19-21, WT: 28/31, 90%; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 16/18, 89%). This progression was unaffected by the loss of retinal signaling with Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showing a similar trajectory as WT (Fisher\u0026rsquo;s exact test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Consistent with the increase in incidence, both groups showed an age-related increase in peak amplitude. Overall, amplitude increased with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, two-way ANOVA, F\u003csub\u003eage(2,138)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.743, p\u0026thinsp;=\u0026thinsp;0.0040) and was unaffected by the loss of retinal signaling (F\u003csub\u003egenotype(1,138)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2572, p\u0026thinsp;=\u0026thinsp;0.6128). In WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, peak amplitude increased during the second postnatal week but was stable between weeks 3 and 4 (Tukey\u0026rsquo;s multiple comparison test, week 2 vs week 3: p\u0026thinsp;=\u0026thinsp;0.0174; week 2 vs week 4: p\u0026thinsp;=\u0026thinsp;0.0096; week 3 vs week 4: p\u0026thinsp;=\u0026thinsp;0.7982).\u003c/p\u003e \u003cp\u003eTo estimate the degree of synaptic depression, we computed paired pulse ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC EPSCn/EPSC1; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD EPSC10/ESPC1). A summary of these ratios for each stimulus pulse (EPSCn/EPSC1) for a 1 Hz train for both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC. For both groups, paired pulse ratios exhibited a progressive decline after the initial response that stabilized by the 5th pulse (two-way ANOVAs with repeated measures, F\u003csub\u003estimulus (1.415, 167.0)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;637.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). However, the magnitude of depression was significantly greater during the second postnatal week (red circles) than either the third (green squares) or fourth (blue triangles) weeks (two-way ANOVAs, WT: F\u003csub\u003eage(2,79)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.63, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: F\u003csub\u003eage(2,39)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.98, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Tukey\u0026rsquo;s multiple comparison tests: week 2 vs week 3 or 4: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 in both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eTo quantify the degree of synaptic depression at different temporal frequencies (0.25, 0.50, 1, 5, 10, and 50Hz) we calculated the paired pulse ratios between the 10th and 1st response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). For both groups, there was an age dependent depression with higher temporal frequencies exhibiting the greatest decrements (two-way ANOVAs with Tukey\u0026rsquo;s multiple comparison testing, WT: F\u003csub\u003eweek(2,410)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.131 p\u0026thinsp;=\u0026thinsp;0.0024, F\u003csub\u003efrequency(5,410)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;92.47, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: F\u003csub\u003eweek(2,208)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21.26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, F\u003csub\u003efrequency(5,208)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;115.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, for both genotypes, 0.25Hz or 0.5Hz vs all other stimulation rates: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, 10Hz vs 50Hz WT: p\u0026thinsp;=\u0026thinsp;0.1822, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: p\u0026thinsp;=\u0026thinsp;0.2735). Additionally, we noted that the degree of synaptic depression weakened with age with weeks 3 and 4 exhibiting larger paired pulse ratios than week 2 (WT: week 2 vs week 3: p\u0026thinsp;=\u0026thinsp;0.0015, week 2 vs week 4: p\u0026thinsp;=\u0026thinsp;0.0449, week 3 vs week 4: p\u0026thinsp;=\u0026thinsp;0.8169; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: week 2 vs week 3 or 4: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, week 3 vs week 4: p\u0026thinsp;=\u0026thinsp;0.4586)\u003c/p\u003e\n\u003ch3\u003eDevelopment of feedback synaptic connections between TRN and dLGN\u003c/h3\u003e\n\u003cp\u003eTo examine the emergence of feedback inhibitory activity from TRN to dLGN, we conducted whole cell recordings from SST-Cre x ChR2 mice and ones crossed on a Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e background. To determine if ChR2 expression in TRN neurons was sufficient to drive postsynaptic activity in dLGN at early postnatal ages, we recorded light evoked activity from vTRN neurons (supplemental Fig.\u0026nbsp;1C). As early as P2, ChR2-EYFP was evident in TRN and photoactivation with brief pulses (1ms) of blue light led to reliable depolarizations and spike firing (supplemental Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eTo investigate TRN-mediated inhibition of TC neurons in dLGN, we conducted voltage clamp recordings using a cesium-based internal solution while holding neurons at 0 mV [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. We recorded 352 WT and 195 Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e neurons in dLGN between postnatal ages P2-P35.\u003c/p\u003e \u003cp\u003eExamples of the light evoked responses at different postnatal weeks to trains of blue light pulses (1ms) presented at different temporal frequencies (0.5Hz, 5Hz, and 10Hz) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. In both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e dLGN, during the first postnatal week, light evoked inhibitory activity was weak and infrequent. When present, it was typically limited to the first pulse of a stimulus train. By the second postnatal week, inhibitory activity was stronger and more prevalent, but responses to repetitive stimulus trains were limited to low rates of stimulation (e.g., 0.5Hz, 1Hz and 5Hz). By the start of the third postnatal week, all TC neurons exhibited light evoked inhibition that was also accompanied by a form of synaptic depression. At temporal frequencies\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;5Hz, IPSC activity began to diminish after the initial pulse but stabilized midway through the stimulus train to values that were about half the amplitude of the first response.\u003c/p\u003e \u003cp\u003eA summary of these age-related inhibitory responses for WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e where we plot the incidence of light evoked responses, (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA), the amplitude of the initial evoked (and maximal) IPSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), and paired pulse ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC EPSCn/EPSC1; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD EPSC10/ESPC1). For both groups, the incidence of light evoked responses followed a similar time course (Fisher\u0026rsquo;s exact test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 at all ages) with IPSC activity emerging as early as P4 (P4 WT: 5/14, 36%, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 33%) and increasing rapidly thereafter so that by the end of the first postnatal week virtually all TC neurons exhibited responses (P7 WT: 18/21, 86%; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 8/9, 89%, P10-12 WT: 18/18, 100%, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 11/11, 100%; \u0026gt;P12 WT: 198/198, 100%, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 116/121, 96%). Both groups showed a comparable age-related increase in IPSC amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB; two-way ANOVA with Tukey multiple comparison testing, F\u003csub\u003eage(4,156)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;30.26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F\u003csub\u003egenotype(1,156)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1095, p\u0026thinsp;=\u0026thinsp;0.7412; ). Between weeks 1\u0026ndash;3, IPSC amplitude exhibited a progressive increase (WT or Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: week 1 vs week 3, 4 or 5: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, week 2 vs week 4 or 5: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) that stabilized after week 3 (WT or Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: week 3 vs week 4 or 5: p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFor both groups, measurements of paired pulse ratios taken for a 1Hz stimulus train (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC EPSC\u003csub\u003en\u003c/sub\u003e/EPSC\u003csub\u003e1\u003c/sub\u003e) or across a range of temporal frequencies (0.25, 0.50, 1, 5, 10 and 50Hz; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD) reflected a sustained synaptic depression. At 1 Hz, ratios computed for each pulse within the stimulus train show an initial reduction that remained constant throughout stimulation with the greatest attenuation seen prior to the third postnatal week (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC; two-way ANOVA with repeated measures, F\u003csub\u003eage(7,115)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.31, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). However there was no difference between WT (solid symbols) and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (open symbols; two-way ANOVAs, week 2: F\u003csub\u003egenotype (1,41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0003, p\u0026thinsp;=\u0026thinsp;0.9866; week3: F\u003csub\u003egenotype (1,32)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0620, p\u0026thinsp;=\u0026thinsp;0.8049; week 4: F\u003csub\u003egenotype (1,26)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.911, p\u0026thinsp;=\u0026thinsp;0.0999; week 5: F\u003csub\u003egenotype (1,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0048, p\u0026thinsp;=\u0026thinsp;0.9455). When examined across a range of temporal frequencies, paired pulse ratios based on the 10th pulse revealed a sustained depression, with higher temporal frequencies exhibiting the largest decline (two-way ANOVAs WT: F\u003csub\u003efrequency(5,231)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;60.54, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: F\u003csub\u003efrequency(5,385)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;187.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Tukey multiple comparison testing in both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: 0.25Hz, 0.5Hz or 1Hz vs 5Hz, 10Hz or 50Hz: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005). Such depression weakened with age (two-way ANOVAs WT: F\u003csub\u003eweek(4,231)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;24.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e: F\u003csub\u003eweek(4, 385)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;61.23, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; within each stimulus and genotype, week 1 or week 2 vs weeks 3\u0026ndash;5: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, week 4 vs week 5: p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) but still remained robust for both groups.\u003c/p\u003e \u003cp\u003eFinally, we assessed how the emergence of feedback inhibition from TRN influenced spike activity of dLGN TC neurons. We conducted current clamp recordings (WT n\u0026thinsp;=\u0026thinsp;52, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e n\u0026thinsp;=\u0026thinsp;47 cells) and photoactivated TRN terminals while injecting a depolarizing current pulse to trigger a steady train spiking in TC neurons. Changes in spike firing were then calculated by comparing equivalent periods (0.5 s) of activity in the presence or absence of blue light stimulation (10 or 50 Hz).\u003c/p\u003e \u003cp\u003eExamples for WT (top) and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (bottom) neurons recorded at 2 and 4 weeks are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA in which a square current pulse (gray, 1500ms) evoked tonic firing before (left) and during blue light stimulation (500ms, blue) at 10Hz (middle) and 50Hz (right; see also supplemental Fig.\u0026nbsp;2 for recordings at week 1\u0026ndash;4, and 6). For both groups, during postnatal weeks 1 and 2, activation of TRN terminals at 10Hz or 50Hz had little impact on spike firing. However, by week 3, TRN activation began to reduce TC spiking, and by week 4, it led to an almost complete suppression of activity. These data are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB which plots the average firing rate (left) observed during control and photostimulation epochs. In the absence of photostimulation, both WT and Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e groups showed comparable firing rates (three-way ANOVA, mixed effects analysis of control epoch: F\u003csub\u003eweek(4,90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.626, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, F\u003csub\u003egenotype(1,90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.06348, p\u0026thinsp;=\u0026thinsp;0.8017). During photostimulation, there was an age-related suppression of TC spiking that emerged at week 3, continued to decline through week 4 and then led to a nearly complete suppression by week 6 (three-way ANOVA, mixed effects analysis: F\u003csub\u003eweek(4,90)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.73, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Tukey multiple comparison testing: week 1 or 2 vs week 4 or 6: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005). A similar pattern emerged when firing rates are converted to percent change (stim-control/control) in activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB, right) with values approaching 100% reduction at 6 weeks of age (mean +/- SEM: WT 10Hz: 90.107 +/- 4.893, 50Hz: 92.755 +/- 3.916, Math 5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e 10Hz: 87.517 +/- 5.035, 50Hz: 88.483 +/- 8.670).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThese studies provide new information about the postnatal development of the feedforward and feedback projections between dLGN and TRN. Our retrograde tracing experiments reveal that thalamocortical axons passed through TRN in an orderly, modality-specific manner that resembles the arrangement noted in adult mice. As early as P2 there was a well-defined and non-overlapping organization of sensory afferents, with visual TC axons restricted to a region that is dorsal to the apex and in the head of TRN while nonvisual ones pass through the \u0026ldquo;tail\u0026rdquo;, ventral to the apex [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Moreover this sectorial arrangement persists even in the absence of retinal signaling (see also [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]), suggesting that the organization of TRN is determined largely by intrinsic guidance cues and seems impervious to alterations in signaling from the periphery [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur anatomical and electrophysiological experiments in strains that isolate the reciprocal connections between TRN and dLGN revealed that feedback connections from TRN to dLGN were established before feedforward TC collateral input onto TRN neurons. (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). TRN axons passed through the ventral-medial border of dLGN at birth and began to innervate dLGN at P2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, green), fully encompassing the nucleus by P7. At this time, optogenetic stimulation of TRN terminals in dLGN led to weak, light-evoked inhibitory postsynaptic activity among TC neurons. Over the course of the second and third postnatal weeks, responses increased in amplitude and showed some form of synaptic suppression during repetitive stimulation. During weeks 3\u0026ndash;5 this depression remained relatively strong, especially during high rates of repetitive stimulation. Interestingly, TRN mediated inhibition had little impact on TC firing during weeks 1\u0026ndash;2, perhaps because synaptic currents remained relatively weak. However, the continued age-related increase in current strength between weeks 3\u0026ndash;5 led to a concomitant reduction in TC spiking. Indeed, by week 4 TRN stimulation led to an almost complete suppression of TC spiking, especially at high rates of stimulation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The synaptic depression noted during weeks 1\u0026ndash;2 may in part reflect the immature state of a developing synapse since even low rates of stimulation evoked responses that showed substantial fatigue. However, the depression noted in subsequent weeks is likely to reflect the emergence of an adult-like property that is consistent with their ultrastructural synaptic profile [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy contrast, excitatory feedforward connections between TC collaterals and TRN neurons appear later. Although TC axons passed through visual TRN at birth, the labeling of axon collaterals with vGluT2 was not detected until the second week (P10) and did not fully encompass visual TRN until the third week. The onset of synaptic activity followed a similar trajectory. Optogenetic stimulation of TC collaterals in TRN evoked weak excitatory responses during the second week which peaked in amplitude by the end of the third week. Accompanying this increase in synaptic strength was a frequency dependent depression, suggesting that excitatory TC input to TRN is driver-like in nature [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile retinal afferents are the first to arrive to dLGN and make functional connections with TC neurons [\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], TRN feedback connections seem to precede the arrival and establishment of other nonretinal inputs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For example, descending CT projections from layer 6 begin to arrive in force and make connections with TC neurons during the second postnatal week [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], while those from brainstem cholinergic nuclei take weeks to arrive and fully innervate dLGN [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Indeed, feedback inhibition from TRN to dLGN also occurs prior to the onset of feedforward inhibition from GABAergic intrinsic interneurons [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which form connections with TC neurons near the end of the second week [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The latter sequence has important implications for understanding the source of thalamic inhibition as a contributing factor underlying the maturation of TC network dynamics. The emergence of thalamic inhibition has been postulated to account for the transformation of network activity from an immature state dominated by weak intrinsic oscillatory activity to a more mature one that is comprised of stimulus evoked events and sleep/wake EEG patterns [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The early postnatal arrival of TRN feedback inhibition appears to roughly coincide with the maturation of TC network dynamics and thus makes for a suitable candidate mechanism.\u003c/p\u003e \u003cp\u003eFinally, it is important to note the role of retinal signaling in intrathalamic circuit formation. Our results in Math5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, a mutant that is devoid of retinofugal projections [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], indicate that the loss of retinal signaling accelerates TRN innervation of dLGN, but has little or no impact on the development of dLGN input to TRN. The premature innervation of dLGN by TRN afferents is consistent with several other studies that underscore the role of retinal signals in regulating the timing of non-retinal innervation of dLGN [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Indeed, in the absence of retinal signaling, intrinsic interneurons fail to target dLGN appropriately, leading to a loss of their input onto TC neurons [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. It also results in a delay and misrouting of brainstem cholinergic input to dLGN [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], as well as an acceleration of descending layer VI innervation of dLGN [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The latter is mediated by the early degradation of aggrecan an extracellular matrix molecule that serves as a repellent for CT innervation of dLGN [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, in the absence of retinal input this repulsive molecule is nearly absent at perinatal ages due to abnormally high levels of degradative proteases that cleave it. Whether aggrecan also regulates the timing of TRN innervation remains untested. Interestingly, the absence of retinal input to dLGN did not appear to alter the timing or nature of TRN synapse formation. The incidence, strength, and degree of synaptic suppression were unaffected. Therefore, the presence of retinal inputs at early ages, while altering the rate of TRN innervation, does not appear to have an impact on TRN synapse formation or maturation. This would suggest that these developmental steps in circuit assembly (innervation and synapse formation) are separable and regulated by independent mechanisms.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe TRN is organized into modality specific sectors at or near birth. The feedback connections from TRN to dLGN are established before feedforward TC collateral input onto TRN neurons. While TC axons pass through TRN at birth, their collateral sprouting and synaptic innervation of TRN neurons occurs largely during weeks 2-3. Feedback projections from TRN to dLGN begins at P2, with terminals encompassing most of dLGN by the end of the first week and functional inhibitory responses emerging soon after, reaching an adult-like state by 3 weeks of age. There is an age-related increase in TRN mediated inhibition which is accompanied by a progressive increase in the suppression of TC spiking. The absence of retinal signaling, leads largely to an acceleration of TRN innervation of dLGN but has little impact on the development of feedforward projections from dLGN to TRN. Together, these studies provide a foundation to investigate the development of TC network dynamics as well as a reference to understand neurodevelopmental diseases that implicate TRN circuitry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate: \u003c/strong\u003eN/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e N/A\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding: \u003c/strong\u003eThis work was supported by EY12716 (WG) and EY026792 (PC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions: \u003c/strong\u003eP.W.C., G.G., and W.G. conceived and designed research;\u003c/p\u003e\n\u003cp\u003eP.W.C., G.G., and W.G. performed experiments; P.W.C. and W.G. analyzed data;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP.W.C., and W.G. prepared figures; P.W.C. and W.G. drafted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u003c/strong\u003eWe thank B. 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Proc Natl Acad Sci. 2020;201913053.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"[email protected]","identity":"discover-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ndev","sideBox":"Learn more about [Neural Development](http://neuraldevelopment.biomedcentral.com/)","snPcode":"13064","submissionUrl":"https://submission.nature.com/new-submission/13064/3","title":"Discover Neuroscience","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"dorsal lateral geniculate nucleus, thalamic reticular nucleus, development, mouse","lastPublishedDoi":"10.21203/rs.3.rs-4014221/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4014221/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe thalamic reticular nucleus (TRN) serves as an important node between the thalamus and neocortex, regulating thalamocortical rhythms and sensory processing in a state dependent manner. Disruptions in TRN circuitry also figures prominently in several neurodevelopmental disorders including epilepsy, autism, and attentional defects. An understanding of how and when connections between TRN and 1\u003csup\u003est\u003c/sup\u003e order thalamic nuclei, such as the dorsal lateral geniculate nucleus (dLGN), develop is lacking. We used the mouse visual thalamus as a model system to study the organization, pattern of innervation and functional responses between TRN and the dLGN. Genetically modified mouse lines were used to visualize and target the feedforward and feedback components of these intra-thalamic circuits and to understand how peripheral input from the retina impacts their development.\u003c/p\u003e\n\u003cp\u003eRetrograde tracing of thalamocortical (TC) afferents through TRN revealed that the modality-specific organization seen in the adult, is present at perinatal ages and seems impervious to the loss of peripheral input. To examine the formation and functional maturation of intrathalamic circuits between the visual sector of TRN and dLGN, we examined when projections from each nuclei arrive, and used an acute thalamic slice preparation along with optogenetic stimulation to assess the maturation of functional synaptic responses. Although thalamocortical projections passed through TRN at birth, feedforward axon collaterals determined by vGluT2 labeling, emerged during the second postnatal week, increasing in density through the third week. Optogenetic stimulation of TC axon collaterals in TRN showed infrequent, weak excitatory responses near the end of week 1. During weeks 2-4, responses became more prevalent, grew larger in amplitude and exhibited synaptic depression during repetitive stimulation. Feedback projections from visual TRN to dLGN began to innervate dLGN as early as postnatal day 2 with weak inhibitory responses emerging during week 1. During week 2-4, inhibitory responses continued to grow larger, showing synaptic depression during repetitive stimulation. During this time TRN inhibition started to suppress TC spiking, having its greatest impact by week 4-6. Using a mutant mouse that lacks retinofugal projections revealed that the absence of retinal signaling led to an acceleration of TRN innervation of dLGN but had little impact on the development of feedforward projections from dLGN to TRN. Together, these experiments reveal how and when intrathalamic connections emerge during early postnatal ages and provide foundational knowledge to understand the development of thalamocortical network dynamics as well as neurodevelopmental diseases that involve TRN circuitry.\u003c/p\u003e","manuscriptTitle":"Development of feedforward and feedback connections between the dorsal lateral geniculate nucleus and the thalamic reticular nucleus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 10:50:03","doi":"10.21203/rs.3.rs-4014221/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-27T16:55:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-27T02:33:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"fd214d81-a87c-4325-957d-6c57d82b8382","date":"2024-03-08T01:26:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1dfbbacf-706b-425c-a60a-f36a047d7ba1","date":"2024-03-06T18:06:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-06T01:10:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-06T00:52:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-05T04:13:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neural Development","date":"2024-03-04T19:31:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ndev","sideBox":"Learn more about [Neural Development](http://neuraldevelopment.biomedcentral.com/)","snPcode":"13064","submissionUrl":"https://submission.nature.com/new-submission/13064/3","title":"Discover Neuroscience","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e47e0647-25d0-4edb-a9e0-00a362c22176","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-22T15:18:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 10:50:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4014221","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4014221","identity":"rs-4014221","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}