Postnatal plasticity in the paralaminar nucleus of the pallial amygdala in juvenile swine brain

Postnatal plasticity in the paralaminar nucleus of the pallial amygdala in juvenile swine brain | 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 Postnatal plasticity in the paralaminar nucleus of the pallial amygdala in juvenile swine brain Júlia Freixes, Ester Desfilis, Loreta Medina This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8544141/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Brain Structure and Function → Version 1 posted 9 You are reading this latest preprint version Abstract The aim of this study was to investigate the presence of immature cells and their phenotype in the pallial amygdala of juvenile swine brains by way of immunoreactivity for the microtubule-associated protein doublecortin (DCX), combined with the cell proliferation marker Ki-67 and different neuronal markers, including NeuN and the transcription factor COUP-TFII (NR2F2, critical for amygdalar development and adult phenotype maintenance). Our results showed the existence of numerous DCX + cells along the external border of the basal amygdalar complex, adjacent to the amygdalar capsule, in an area identified as the swine paralaminar nucleus. DCX + cells in this nucleus showed a patchy distribution, with shell-like clusters of DCX + cells partially surrounding islands of non-stained large cells of the basal amygdalar complex. Both, the paralaminar nucleus and the other nuclei of the basal amygdalar complex also contained abundant neurons expressing COUP-TFII (NR2F2), but not the transcription factor FOXP2, which defines the clusters of intercalated amygdalar cells. Paralaminar patches of DCX + cells were more abundant at posterior levels, where they were continuous with chains of DCX + cells with migratory-like morphology and other immature DCX + cells of the subventricular zone surrounding the temporal horn of the lateral ventricle (tlv). This part of the ventricular/subventricular zone (vz/svz) also expressed COUP-TFII / NR2F2, suggesting that this might be the source of the immature cells found in the paralaminar nucleus and other parts of the pallial amygdala. A major difference between this part of the vz/svz and the Arc (giving rise to DCX + cells of the rostral migratory stream and, apparently, the migratory cell chains seen in the external capsule and piriform cortex) is that the latter does not express COUP-TFII / NR2F2. Overall, these results show that the swine pallial amygdala shows a prolonged postnatal plasticity and contains a reservoir of immature neurons mainly located in the paralaminar nucleus and related vz/svz, resembling the situation seen in humans. These findings also point to the swine as an excellent model to study mechanisms behind postnatal plasticity in the amygdala of gyrencephalic animals, and the role of this protracted plasticity in amygdala function and dysfunction. postnatal neurogenesis gyrencephalic brain migratory streams neuroplasticity amygdalar pallium paralaminar nucleus intercalated amygdalar cells pig Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 INTRODUCTION In humans and non-human primates, the amygdala shows a remarkably prolonged maturation, extending through adolescence and beyond, which may be related to the increasing involvement of this structure over time in social cognition and adaptation to changing social contexts (Chareyron et al. 2012 ; Avino et al. 2018 ). The protracted postnatal development of the amygdala involves the presence of a large number of immature neurons, especially concentrated in the paralaminar amygdalar nucleus (PL) (De Campo and Fudge, 2012 ), which appears as an immature cell reservoir from where new neurons migrate, mature and incorporate into the adjacent basal and accessory amygdalar nuclei (Chareyron et al. 2012 ; Avino et al. 2018 ; Sorrells et al. 2019 ). Since the PL is a major target of hippocampal projections (Fudge et al. 2012 ), it has been proposed to be an interface linking autobiographical memories with emotional valence (Jin et al. 2015 ; Kim et al. 2017 ; discussed by Sorrell et al. 2019). In humans and monkeys, the PL increases in size and neuron number during juvenile ages, but this fails to occur in autism spectrum disorder (Avino et al. 2018 ). In humans and other primates, immature neurons are abundant in PL of children and express microtubule-associated protein doublecortin (DCX), polysialylated neural cell adhesion molecule (PSA-NCAM) and B cell lymphoma 2 protein (BCL2) (Bernier et al. 2002 ; De Campo and Fudge, 2012 ; Sorrells et al. 2019 ; Ghibaudi et al. 2025 ). Many of these immature neurons transition during adolescence into mature glutamatergic neurons that contain the vesicular glutamate transporter VGLUT2 (SCL17A6) and the transcription factors TBR1 and COUP-TFII (NR2F2), but not other transcription factors typical of the caudal ganglionic eminence such as SP8 (Sorrells et al. 2019 ). This suggests that these immature neurons originate in the pallium, but their exact spatial and temporal origin, either prenatally and/or postnatally, remain unknown or controversial (Bernier et al. 2002 ; Sorrell et al. 2019; Ghibaudi et al. 2025 ). Independent of their origin, the existence of large numbers of excitatory neurons in primate PL that remain immature for relatively long periods (decades in humans) indicates an extraordinary plasticity of the pallial amygdala (Sorrells et al. 2019 ; Ghibaudi et al. 2025 ), which function remains to be explored. Immature neurons are more abundant and persist during a longer postnatal period in the basal complex of the amygdala in large-brained, mostly gyrencephalic mammals (especially primates, but also carnivores as the cat and artiodactyls as the sheep), but they are scarce and decline rapidly in small-brained mammalian species (including rodents) (Piumatti et al. 2018 ; Alderman et al. 2024 ; Ghibaudi et al. 2025 ). In primates, immature neurons continue to be observed in the basal complex of the amygdala even in aged adults, while in rodents (like mouse) their density is much lower and there is a sharp decline in juveniles (Alderman et al. 2024 ; Ghibaudi et al. 2025 ). This is likely related to the very small PL found in rodents, difficult to distinguish from the intercalated cells (De Campo and Fudge, 2012 ). Only recently, with the use of multiple fluorescent labeling and single cell transcriptomics, it was possible to reliably distinguish in mouse between PL (expressing DCX, COUP-TFII / NR2F2, and several glutamatergic markers) from the intercalated amygdalar cells (expressing FOXP2 and GABAergic markers) (Alderman et al. 2024 ). In spite of the detailed knowledge on the distribution, phenotype and changes throughout ontogeny of amygdalar immature neurons in some primates and rodents, data in other species is still scarce and a putative paralaminar nucleus remains poorly defined. In recent years, the swine has emerged as a large-brained gyrencephalic species, which brain development and organization resemble more closely that of humans, compared to the murine brain (Lind et al. 2007 ; Kinder et al. 2019 ; Liu et al. 2021 ; Ghibaudi et al. 2025 ). Moreover, it shows abundant levels of DCX + immature neurons throughout all olfactory areas in piglets and juveniles, which appear to migrate from different rostrocaudal levels of the subventricular zone of the lateral ventricle (SVZ) and/or the adjacent white matter (Costine et al. 2015 ; Torrijos-Saiz et al. 2025 ; Freixes et al. 2025 ). While the SVZ is usually known as a source of GABAergic interneurons for the olfactory bulb (Alvarez-Buylla and García-Verdugo 2002; Kohwi et al. 2005 , 2007 ; Young et al. 2007 ), studies in different mammalian species including swine show that it also gives rise to glutamatergic neurons that migrate through the rostral migratory stream and the external capsule to different olfactory structures of the piriform lobe (Freixes et al. 2025 ). DCX + immature neurons were also found in the olfacto-recipient cortical area of the amygdala in juvenile swine, but their presence in the basal amygdalar complex and the existence of a PL is unexplored in swine. An excellent approach to help with the identification of homologue brain structures is by studying their developmental origin, as this is a good reference for understanding their topological position, which remains identical throughout ontogeny and in evolution (Nieuwenhuys and Puelles, 2016 ). In primates, the basal amygdalar complex, including the PL, was proposed to derive from the so-called “inferior ganglionic eminence”, adjacent to the temporal horn of the lateral ventricle (tlv), and postnatally the PL resides in the same location adjacent to the former inferior ganglionic eminence (reviewed by De Campo and Fudge, 2012 ). The developmental origin of the basal complex and cortical areas of the amygdala has recently been reformulated in mouse, and they appear to derive from a distinct posteroventral ventricular sector of the telencephalon, named the amygdalar pallium (García-Calero et al. 2020), which appears comparable to the primate inferior ganglionic eminence. Thus, in spite of its name suggesting a subpallial location, the inferior ganglionic eminence would be the progenitor zone of the amygdalar pallium. According to García-Calero et al. (2020), this amygdalar pallial sector contains at least four radial subunits giving rise to different neuron subgroups of the basal complex and cortical amygdala. However, the PL was not considered in this study, and its exact origin remains elusive. This information could help to better understand the topological location and possible migratory routes of immature cells found postnatally in the PL of different mammals. The aim of this study was to investigate the distribution of immature cells in the basolateral amygdalar complex of juvenile swine brains, trying to identify the PL as well as the phenotype and possible migratory routes of PL immature cells. To that aim, we performed immunolabeling for DCX, combined with the cell proliferation marker Ki-67 and different neuronal markers, including NeuN (neuronal nuclear protein, also known as FOX3), the transcription factors BRN2 (POU3F2, present in subsets of glutamatergic neurons) and COUP-TFII / NR2F2 (critical for amygdalar development and adult phenotype maintenance, and present in PL cells in both human and mouse). To distinguish PL from intercalated amygdalar cell clusters, we combined DCX with the transcription factor FOXP2. To better understand PL cytoarchitecture with respect to that of other amygdalar nuclei, we also performed Nissl staining in complete series of sections adjacent to those processed for immunohistochemistry of immunofluorescence. Our results demonstrated that the swine pallial amygdala shows a prolonged postnatal plasticity similar to that of humans and contains a reservoir of immature neurons coexpressing DCX and COUP-TFII / NR2F2 mainly located in the PL. We also identified the possible origin of these cells in a posteroventral ventricular/subventricular pallial sector adjacent to the temporal horn of the lateral ventricle expressing COUP-TFII / NR2F2. MATERIAL AND METHODS In the present study, we used brains from swine (Sus scrofa domesticus ) provided by the Applied Biomedical Research Centre (CREBA) of the Institute of Biomedical Research of Lleida- Dr. Pifarré Foundation (IRBLleida, Spain), located in Torrelameu (Lleida, Spain) (registered as a user center of experimental animals: L9900008; REGA: ES252310036907). In total, we used 4 juvenile swine of both sexes (1 male and 3 females), ranging in age from 2.5 to 3.5 months, and weighted between 30 to 40kg. At this age, pigs are considered juvenile, as they have not yet reached sexual or full neurodevelopmental maturity. Animals were housed and handled in the pig CREBA facilities, according to the protocol approved by the Animal Experimentation Ethics Committee of the CREBA and following the regulations and laws of the European Union (Directive 2010/63/EU) and the Spanish Government (Royal Decrees 53/2013) for the care and handling of animals in research. These animals were previously used for practicing and improving surgical procedures by M.D. surgeons. After, animals were euthanized, and their brains were extracted and processed as explained below. Tissue collection and fixation For cerebral tissue collection, the animals previously employed for surgery research were sacrificed, just before brain extraction, with a lethal dose of sodium pentobarbital (200mg/kg; IV). After extracting the brain, the tissue was washed with abundant water to remove blood and hemispheres were separated and fixed by immersion in a solution of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), for 2–4 days. Sample preparation and sectioning Some hemispheres were cut into four blocks (frontal, temporoparietal, occipital, and brainstem with cerebellum), while others were conserved in a single piece. Following fixation, the tissue was cryoprotected in a solution of increasing glycerol concentrations (10% and 20%) and 2% DMSO in 0.1M PB for 6–10 days. The tissue was frozen by immersion in -65/-70°C isopentane (2-methyl butane, Sigma-Aldrich, Germany) for 1-2min, following the protocol of Rosene et al. ( 1986 ). Frozen brains were wrapped in aluminum foil and stored in a container at -80°C until further use. For sectioning, samples were cut using a freezing sliding microtome (Microm HM 450; Thermo Fisher Scientific), equipped with a large size Freezing Stage (BFS-40MPA, Physitemp Instruments, LLC, USA). Coronal, sagittal and horizontal sections of 100µm thick were collected at 4°C in 0.1M PB, into twelve parallel series with about 20 sections per each block for coronal sections and each sagittal- or horizontally sectioned hemisphere. For each series, selected sections of the appropriate levels including the areas of interest were processed for studying. The sections not used immediately were stored in a 30% sucrose PB solution and frozen at -20°C. In one animal (male), the right hemisphere was sectioned in the sagittal plane, and two parallel series were stained for Nissl and immunohistochemistry for DCX. Similarly, in a female, the right hemisphere was sectioned in the horizontal plane, and two parallel series were processed for Nissl and DCX-immunohistochemistry staining. Horizontal sections from another female were processed, but in this case the occipital lobe of the right hemisphere was separated and stored for further analysis. Regarding frontal sections, the amygdala was studied from anterior to posterior levels (from 10.375 mm to 6.125 mm antero-posterior stereotaxic coordinates from the MRI Atlas, Saikali et al. 2010 ) by using Nissl and immunohistochemistry for DCX staining. Following the initial analysis of Nissl and DCX chromogenic immunostained sections, selected frontal, sagittal and horizontal sections were processed for double and triple immunofluorescence, combining DCX with other markers of interest (see Table 1 ). Overall, 55 sections were Nissl-stained (10 frontal sections, 25 horizontal sections and 20 sagittal sections), about 100 sections were processed for single immunohistochemistry (including DCX, COUP-TFII, FOXP2 and Ki-67 staining), and 50 sections were processed for double or triple immunofluorescence. Nissl staining To study the tissue organization and to help with the identification of the areas, complete series of sections of each plane were processed for Nissl staining as follows. After being mounted with a solution of gelatine from porcine skin at 0.5% in Tris buffer and dried, slides with sections were sequentially immersed in 100% ethanol and xylene to dissolve out the lipids and allow a better staining. After, sections were dried and incubated for 1 minute with filtered 1% Toluidine blue in 0.1M acetate buffer at pH 4.6. Sections were then rinsed with abundant water, and differentiated with acid alcohol (0.01% acetic acid solution in 70% ethanol) for 90 seconds. Subsequently, the slides were dehydrated with ascending ethanol concentrations (70%, 96%, and 100%), followed by a brief xylene wash, and finally coverslipped with Permount mounting medium (Thermo Fischer Scientific). Immunohistochemistry Free-floating brain sections were processed for immunohistochemistry to detect DCX, COUP-TFII, FOXP2, or Ki-67, using specific primary antibodies (Table 1 ). Sections were washed with 0.1M PB and then processed for antigen retrieval by incubation in a sodium citrate buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for 1h at 60ºC. After rinsing, sections were permeabilized with PB containing 0.3% Triton-X 100 (PB-Tx; 0.1 M), and incubated for 2h at room temperature in a blocking solution containing 2% of bovine serum albumin (BSA) and 20% of normal serum (according to the species in which the secondary antibody was raised) in 0.1M PB. Next, sections were incubated in primary antibody solution (for dilutions see Table 1 ) in PB-Tx for 72h at 4°C with gentle shaking. After rinsing, sections were incubated in a biotinylated secondary antibody (Table 2 ) in PB-Tx overnight, at 4°C with gentle shaking. Successively, sections were washed, and endogen peroxidase was blocked with a solution of PB with 20% methanol and 2% H 2 O 2 for 20min. After, sections were washed and incubated in the avidin–biotin complex (AB Complex, Vector Laboratories Ltd.) for 1 h at room temperature. After incubation in the ABC solution, the sections were first washed in PB, followed by Tris buffer (0.05M, pH 7.6). To reveal the staining, the sections were incubated with diaminobenzidine (DAB), prepared according to the manufacturer’s instructions (SIGMAFAST™ tablets, Sigma-Aldrich Co. LLC; or DAB Chromogen/Substrate Bulk Pack, ScyTek Laboratories INC.). The reaction was stopped by rinsing the sections with Tris buffer. Sections were then mounted, dehydrated and covered with Permount™ as described before. As negative control, some sections were processed following the immunohistochemistry procedure but omitting primary antibodies. No specific cell labeling was observed (Freixes et al. 2025 ). Table 1 Primary antibodies Type Antibody Antigen recognized Dilution Manufacturer and Reference RRID Polyclonal Goat anti-doublecortin Doublecortin C-18 1:1000–1:2000 for IHC/IF 1:1000 for WB Santa Cruz Biotechnologies Ref. sc-8066 AB_2088494 Rabbit anti-doublecortin Doublecortin 1:3000 for IHC/IF 1:1000 for WB Abcam Ref. ab18723 AB_732011 Rabbit anti-FOXP2 Forkhead box P2 1:2000 for IHC/IF Abcam Ref. ab16046 AB_2107107 Rabbit anti-actin N-terminal actin peptide attached to a multiple antigen peptide backbone. 1:5000 for WB Sigma-Aldrich Ref. A5060 AB_476738 Monoclonal Rat anti-Ki-67 Ki-67 SolA15 1:100 for IHC/IF Invitrogen Ref. 14-5698-82 AB_10854564 Mouse anti-NeuN Neuronal Nuclei (NeuN); clone A60 1:1000 for IHC/IF Sigma-Aldrich Ref. MAB377 AB_2298772 Mouse anti-COUP-TF2/NR2F2 COUP-TF II/NR2F2 clone H7147 1:500 for IHC/IF 1:1000 for WB Bio-Techne R&D Systems Ref. PP-H7147-00 AB_1964214 Mouse anti-Brn2 IgG1 POU class 3 homeobox 2 1:200 for IF Santa Cruz Biotechnologies Ref. sc-393324 AB_2737347 Mouse anti-actin Slightly modified β-cytoplasmic actin N-terminal peptide. 1:5000 for WB Sigma-Aldrich Ref. A5441 AB_476744 Double and triple immunofluorescence Multiple labeling immunofluorescence was performed to detect cells coexpressing different markers. Following tissue permeabilization and blocking of non-specific binding, sections were incubated at 4°C for 72h with a cocktail of primary antibodies (Table 1 ). After rinsing, sections were incubated overnight at 4°C in PB-Tx with the corresponding cocktail of fluorescent secondary antibodies (Table 2 ). Finally, the sections were mounted with a solution of gelatine at 0.5% and covered with antifading mounting medium (Vectashield Hardset Antifade mounting medium, Vector Laboratories Ltd.). As done for immunohistochemistry, some sections were incubated omitting the cocktail of primary antibodies to ensure their specificity. No labelling was detected. Table 2 Secondary antibodies Type Antibody Dilution Manufacturer and Reference RRID Biotinylated for IHC Rabbit anti-goat IgG (H + L) 1:200 Vector Laboratories Ref. BA-5000 AB_2336126 Goat anti-mouse IgG (H + L) 1:200 Vector Laboratories Ref. BA-9200 AB_2336171 Goat anti-rabbit IgG (H + L) 1:200 Vector Laboratories Ref. BA-1000 AB_2313606 Goat Anti-Rat IgG (H + L) 1:200 Vector Laboratories Ref. BA-9400 AB_2336202 HRP-linked for WB Anti-rabbit IgG 1:20000 Cell Signaling Ref. 7074 AB_2099233 Anti-mouse IgG 1:20000 Cell Signaling Ref. 7076 AB_330924 Anti-goat IgG 1:5000 Vector Laboratories Ref. PI-9500 AB_2336124 Fluorescent for IF Donkey anti-goat IgG (H + L), coupled to Alexa 488 1:500 Jackson ImmunoResearch Ref. 705-546-147 AB_2340430 Donkey anti-mouse IgG (H + L), coupled to Alexa 488 1:500 Invitrogen Ref. A21202 AB_141607 Donkey anti-goat IgG (H + L), coupled to Alexa 568 1:500 Invitrogen Ref. A11057 AB_2534104 Donkey anti-rabbit IgG (H + L), coupled to Alexa 568 1:500 Invitrogen Ref. A10042 AB_2534017 Goat anti-rabbit IgG (H + L), coupled to Alexa Plus 405 1:250 Invitrogen Ref. A48254 AB_2890548 Goat anti-mouse IgG (H + L), coupled to Alexa Plus 405 1:250 Invitrogen Ref. A48255 AB_2890536 Goat anti-rat IgG (H + L), coupled to Alexa Plus 405 1:250 Invitrogen Ref. A48261 AB_2890550 Western blotting for validation of primary antibodies Since the employed antibodies were not validated by the respective manufacturers for swine tissue, we employed the Western blot technique for validation. Samples consisted of brain tissue, obtained from juvenile and embryos at 50 days of gestation (E50). One hemisphere of a juvenile swine was dissected to obtain samples from the caudate nucleus (Cn), the piriform cortex (Pir) and the neocortex (NCx), while for the embryonic sample, one whole hemisphere was used. All the samples were fragmented and homogenized using an electric homogenizer (Tissue Grinder) in ice-cold radioimmunoprecipitation assay (RIPA) containing lysis buffer (150 mM NaCl; 1% NP-40; 0.5% Na-deoxycholate; 0.1% SDS; 50 mM Tris-HCl [pH 7.4]), protease inhibitor (Sigma-Aldrich, cat # P8340) and PhosSTOP (Roche). Homogenized samples were centrifuged at 12000 rpm for 15 min at 4°C and then protein concentrations of the supernatants were determined by BIO-RAD Micro DC protein assay (BIO-RAD, Laboratories, Inc.). Samples containing 20–40 µg of protein were mixed with an equivalent volume of sample buffer containing 8% of SDS and 2% mecaptoethanol and heated at 100°C for 5 min. Following, samples were first loaded onto a denaturing 10% sodium dodecyl sulphate-polyacrylamide gel, together with chemiluminescent (MagicMark™ XP, Invitrogen) and multicolor prestained (PageRuler™ Plus, Thermo Fisher) ladders. Proteins were separated by electrophoresis for 90 to 120 minutes at an intensity of 25 mA per gel and then were electrotransferred to a nitrocellulose membrane in Tris-glycine-methanol buffer, using semidry transfer. The membrane was blocked for 1 h at room temperature in a blocking solution mixture of 5% nonfat dry milk in 0.1% Tween 20 and Tris-buffered saline pH 8 (TBST). After washing with TBST, membranes were incubated overnight at 4°C with anti-DCX and anti-COUP-TFII antibodies (Table 1 ) for immunodetection. Next day, membranes were washed and incubated with the appropriate peroxidase-conjugated secondary antibodies (Table 2 ) for 60 min at room temperature, washed in TBST, and visualized using the ECL Prime Western blotting Detection Reagent detection kit (GE Healthcare), as described by the manufacturer. Chemi-Doc MP Imaging System (BIO-RAD Laboratories, Inc.) was used for the visualization of bands. Following, the membrane was incubated with anti-actin antibody as loading controls. Doublecortin antibodies showed a band of 40kDa, as described by the manufacturer (Supplementary Fig. 1). Similarly, brain sample reacted with COUP-TFII antibody giving a band of a similar weight as the one reported by the manufacturer (~ 55kDa). Identification of regions of interest To identify the regions of interest, we used our own Nissl-stained sections and those of the wild boar brain from the University of Wisconsin-Madison, as well as the MRI 3D atlas from Saikali et al. ( 2010 ). Our identification was based on both the topological position and cytoarchitecture of the structures. Digital photographs and figures Digital microphotographs of the sections processed for immunohistochemistry were taken on a Leica microscope (DM2500 LED, Leica Microsystems GmbH) equipped with a digital camera (Zeiss Axiovision Digital Camera -Carl Zeiss- and Flexacam C5 LSR Camera -Leica Microsystems GmbH-). Serial images from fluorescent material were taken under confocal microscopes (Olympus FV1000 -Olympus Corporation- and Nikon AX NSPAR -Nikon). Selected digital fluorescent images were adjusted and extracted using Olympus FV10-ASW 4.2 Viewer (Olympus Corporation) and Image J (Fiji). Finally, the figures were mounted using Affinity Designer 2 (version 2.6.5, Serif Corporation). RESULTS We analyzed coronal, sagittal, and horizontal sections from juvenile swine brains of both sexes, at different levels, to carry out a systematic description of the distribution of DCX + cells throughout the amygdala. The expression pattern of DCX was similar across animals and in both sexes, even if the limited brain number did not allow a reliable comparison between sexes or ages. We found DCX + cells in the basal complex of the amygdala, mainly localized in clusters or patches along the external border of the complex, resembling in topological position the PL. To know more about the phenotype of the DCX + cells of the clusters and distinguish whether they truly represent the swine PL or belong to clusters of intercalated amygdalar cells (ITC), we compared the results between adjacent sections single or double/triple-labeled for DCX, NeuN (that is expressed in neurons since they start to differentiate), and/or the transcription factors BRN2 (POU3F2, which is expressed in subpopulations of immature glutamatergic cortical neurons), COUP-TFII (NR2F2, which is crucial for amygdala patterning and is expressed in PL neurons), and FOXP2 (that is expressed in intercalated cells of the amygdala). As many of the DCX + cells in the clusters had a migratory cell morphology and some of them formed chains (in agreement with Torrijos- Saiz et al. 2025; Freixes et al. 2025 ), we tried to identify possible progenitor areas and explore the possible migratory pathways that immature cells might follow from these progenitor areas to their final position in PL. To help in this analysis, we also studied the coexpression of Ki-67, a mitotic marker, with other markers. Distribution of DCX + cells in the pallial amygdala and identification of swine PL The pallial amygdala consists of the basal complex of the amygdala (BCA) and several cortical amygdalar areas near the surface. Like in other gyrencephalic mammals, the swine pallial amygdala has a relatively large size, as compared to the central and medial amygdalar nuclei (Fig. 1 A and Supplementary Fig. 2). We found numerous DCX + cells in both the BCA and the cortical areas of juvenile swine, indicating high levels of plasticity. The majority of the DCX + cells were observed near the external border of BCA, close to the amygdalar capsule, and in its posteroventral continuation towards the subventricular zone adjacent to the temporal horn of the lateral ventricle (Figs. 1 , 2 ). These cells resembled the primate PL in topological position and postnatal DCX expression. Like primate PL, DCX + cells of swine PL were often broken into clusters which overlapped with COUP-TFII expressing cells (Fig. 2 C,D), but not with FOXP2, which identified the swine intercalated amygdalar cells (Figs. 2 F-H; 3 B,D,H). In contrast to PL clusters, the intercalated cell clusters showed negligible levels of DCX immunoreaction (Figs. 2 G; 3 E,I) and did not express COUP-TFII (Fig. 3 F,J). The intercalated cell clusters and those of PL also differed in location: while the first were mainly found anterior to BCA (the main intercalated nucleus or IM) or along its medial border with the central or medial amygdala (Fig. 3 ), the PL clusters were mainly observed laterally and became more abundant at posterior levels (Figs. 2 – 3 and Supplementary Fig. 2). Like in primates, the swine PL also displayed lateral and medial subdivisions that surrounded laterally and ventromedially the BCA (Fig. 1 B; details of lateral and medial subdivisions are shown in Fig. 1 D and 1 F, respectively). DCX + cells of the lateral subdivision were organized in patches or clusters, which were more abundant ventrally, adjacent to the basolateral (BL) and basomedial (BM) nuclei (Fig. 1 B, Supplementary Fig. 2). In frontal sections, clusters of DCX + cells of the lateral PL displayed a shell-like organization, surrounding islands of non-stained large cells of the BCA (Fig. 1 C,D). As seen in Nissl staining, the PL cells of the shell-like clusters showed a predominant fusiform shape, but these were mixed with cells of round or multipolar morphology of different sizes (Fig. 1 C). These different cell morphologies were also seen with DCX immunohistochemical labeling, with a predominance of small, round cells (Fig. 1 D). In the medial PL, DCX + cells were more dispersed and rarely formed clusters, although these did not display a shell-like organization (Fig. 1 E,F). Different morphologies and soma sizes were observed in medial PL but most DCX + cells were round and small (Fig. 1 F). Phenotype of DCX + cells in PL To better understand the phenotype of the immature DCX + cells of PL, we carried out double and triple immunofluorescence (Figs. 4 , 5 , 6 , 7 ). This combination also allowed a better correlation between distinct morphologies and soma sizes of DCX + cells and specific phenotypes. The majority of the DCX + cells of PL expressed the neuronal marker NeuN (Fig. 4 A-D’’), and cells coexpressing DCX and NeuN (arrowheads in Fig. 4 B’-B’’, D’-D’’) were generally smaller than those expressing only NeuN. Among the cells coexpressing DCX and NeuN we could distinguish at least two types, one with a larger (medium-sized) soma and dendrites, and another one with very small and round or fusiform soma (Fig. 4 A-D’’). Double labeling with Ki-67 showed that a few of the small and round DCX + cells coexpressed Ki-67 (Figs. 4 E-H’’), indicating that at least a few of these immature cells were proliferating in both lateral (Fig. 4 E-F’’) and medial (Fig. 4 G-H’’) parts of PL. In addition to NeuN, most DCX + cells in PL showed COUP-TFII immunofluorescence (Figs. 5 A-D’’). As typical of transcription factors, COUP-TFII was mainly located in the cellular nucleus. However, we found COUP-TFII + cell nuclei of two sizes, large and small. Almost all DCX + cells showed a small COUP-TFII + nucleus. Most DCX + cells had a round or fusiform soma, some with a migratory-like morphology. Large COUP-TFII non-DCX cells were abundant in both PL and the rest of the BCA, and resembled mature neurons. We only found very few DCX + cells coexpressing BRN2 in PL (Fig. 5 E-F’’), which is a major difference with our previous findings in the olfactory areas, where many cells coexpressed DCX and BRN2 (Freixes et al. 2025 ). Possible progenitor areas of DCX + cells and migratory routes to PL Many DCX + cells in PL had an elongated morphology, and some displayed a leading process resembling migratory cells (Figs. 5 A,B’,C,D’). Since the vast majority of the DCX + cells of PL coexpressed COUP-TFII, we analyzed expression of this transcription factor in the ventricular/subventricular zones (vz/svz) of the temporal horn of the lateral ventricle (tlv) in order to understand the origin of PL immature cells. We identified a posterolateral inferior sector of the vz/svz rich in radially oriented DCX + cells and small round COUP-TFII cells (Fig. 6 ). Remarkably, many cells in this vz/svz sector coexpressed COUP-TFII and Ki-67 and DCX and Ki-67 (Fig. 6 C-C’’’, 7A-A’’), resembling a hotspot for production of new cells. We also found coexpression of DCX and COUP-TFII (Fig. 6 D-D’’). A major difference between this vz/svz sector and the Arc (giving rise to immature neurons of the rostral migratory stream and those found in the external capsule and many olfactory areas) is that the Arc did not express COUP-TFII (Supplementary Fig. 3). These observations discarded the Arc as a possible origin of immature cells found in PL and pointed to the posterolateral and inferior vz/svz sector adjacent to the tlv as the most likely origin. Moreover, from this hotspot of the vz/svz, we found chains of radially oriented migratory-like DCX + cells, coexpressing COUP-TFII, that were in continuity with the cell clusters of lateral PL (Figs. 6 , 8 ). Thus, this particular posterolateral and inferior sector of vz/svz might be the source of the DCX + immature neurons found in lateral PL. The horizontal plane was the best to follow this continuity from the vz/svz to the PL clusters (Fig. 6 , 8 ), and showed that the chains of migratory-like DCX + cells in continuity with the DCX+/COUP-TFII + cell clusters of PL occupied an inner position in the amygdalar capsule (Figs. 6 , 8 ), while other DCX + cell chains located more superficially did not overlapped nor coexpressed COUP-TFII and appeared to be related to the endopiriform/piriform region. The continuity of DCX + cell chains from the posterolateral inferior vz/svz to the PL cell clusters could not be observed in the frontal (Fig. 9 ) of sagittal planes (Fig. 10 ), although in these section planes it was possible to observe short chains of DCX + cells in the inner part of the amygdalar capsule, which appeared to reach the lateral PL clusters (Figs. 9 A,B,F; filled arrowheads in F; 10E,H,K; empty arrowheads in K). In contrast, DCX + cell chains of the external part of the amygdalar capsule were oriented toward the endopiriform/piriform region (empty arrowheads in Figs. 9 F empty arrowheads). Finally, we observed tangentially oriented chains of DCX + cells, aligned parallel to the ventricle, that appear to separate from the same posterolateral and inferior vz/svz rich in small cells immunoreactive for DCX and COUP-TFII mentioned above (Fig. 6 E-F’’). These tangentially oriented cell chains might contribute to at least part of the DCX + cells observed in medial PL, but we cannot discard that other immature cells might be produced in the vz/svz sector adjacent to medial PL, since we also observed a few cases of DCX/Ki-67 coexpression there (Fig. 7 B-B’’). DISCUSSION Our results on DCX expression showed prolonged postnatal plasticity in the swine pallial amygdala that extends at least until juvenile ages, which is comparable to the situation described in primates (De Campo and Fudge, 2012 ; Sorrells et al. 2019 ; Ghibaudi et al. 2025 ). As previously shown in primates (De Campo and Fudge, 2012 ; Sorrells et al. 2019 ; Ghibaudi et al. 2025 ), in swine this protracted plasticity of the amygdala is associated to the presence of immature cells in the PL, which we identified as a subdivision located along the external border of BCA, rich in immature DCX + cells, most of which express NeuN (indicating a neuronal phenotype) and the transcription factor COUP-TFII / NR2F2, but not FOXP2 (which identified the intercalated amygdalar cells). In primates, it has been proposed that the PL could serve as a reservoir to provide new neurons for the basal (basolateral) and accessory (basomedial) nuclei of BCA (Chareyron et al. 2012 ; Avino et al. 2018 ; Sorrells et al. 2019 ). As in primates, the swine PL could serve as a reservoir of immature neurons for the pallial amygdala. However, the PL seems to be a different nucleus of BCA, containing neurons with different maturation degree as suggested by the shell-like organization of DCX + cell clusters around non-DCX cells with mature morphology (Fig. 1 ). Therefore, most immature cells of PL might continue residing within this nucleus upon maturation, although we cannot discard that a few migrate to other BCA nuclei. Previous studies indicated that the size of the PL reservoir correlates with the degree of cortical expansion, being larger in gyrencephalic than in lissencephalic brains (Ghibaudi et al. 2025 ). Species with a gyrencephalic brain also have a relatively large pallial amygdala, which is particularly evident in the BCA (De Campo and Fudge, 2012 ; Piumatti et al. 2018 ; Ghibaudi et al. 2025 ). Our results agree with this proposal, as the swine has a gyrencephalic brain, with large cortices and BCA relative to subpallial structures such as the striatum and central amygdala, and we also observed a well developed PL, including lateral and medial subdivisions (schemes in Figs. 11 and 12 ), as described in primates (De Campo and Fudge, 2012 ). In contrast, lissencephalic rodents as the mouse and rat, with smaller cortices and BCA, only have a rudimentary PL, which remained unnoticed or poorly described until recently (De Campo and Fudge, 2012 ; Alderman et al. 2024 ). A major distinctive feature of the immature DCX + cells of PL is that they express COUP-TFII and several glutamatergic markers such as TBR1 and VGLUT2 (SCL17A6), but not GABAergic markers as SP8, GAD65 and GAD67 (Sorrells et al. 2019 ; Alderman et al. 2024 ). Moreover, deletion of COUP-TFII had a significant impact on excitatory populations (TBR1 and GLU2R) of the rodent BCA, while few effects were reported in inhibitory GAD67 + interneurons (Tang et al. 2012 ). In our material in swine, we found that a few DCX + cells of PL express the transcription factor BRN2 (POU3F2), present in some subsets of immature glutamatergic cells (Dominguez et al. 2012; Brunjes and Osterberg, 2015 ), such as many of those of the swine postnatal olfactory structures (Freixes et al. 2025 ). However, most DCX + cells of swine PL did not express BRN2 (POU3F2), at least during juvenile postnatal ages, suggesting that at this age they express glutamatergic markers different to those found in immature neurons of olfactory structures. We observed that many DCX+/COUP-TFII + cells found in juvenile swine PL had an elongated, migratory-like morphology, which prompted us to investigate possible origin and migration routes by analyzing sections in different planes. We could follow the immature cells posteroventrally, until the vicinity of the temporal horn of the lateral ventricle, where we found a reservoir of DCX+/COUP-TFII + immature cells in a posterolateral inferior sector of the vz/svz, from where cells appeared to migrate radially to lateral PL and tangentially to medial PL (schemes in Figs. 11 , 12 ). However, this proposal would require confirmation using cell migration assays. The vz/svz associated to PL appears to relate to the lateralmost progenitor domain of the pallial amygdala, and it has been identified previously in the lateral part of the “inferior ganglionic eminence” (De Campo and Fudge, 2012 ), which is not a subpallial domain as the name suggests, but it really represents the progenitor zone of the pallial amygdala (García-Calero et al. 2020). Notably, many DCX + and COUP-TFII + cells in this vz/svz sector expressed Ki-67 in juvenile swine, indicating that new neurons for the PL could be produced postnatally, at least until juvenile stages. This would agree with previous findings in primates (Bernier et al. 2002 ), including humans (Sorrells et al. 2019 ; Roeder et al., 2022 ), and with our results of the swine olfactory areas (Freixes et al., 2025 ). In addition, other DCX+/COUP-TFII + cells found in this vz/svz sector and the associated PL might represent quiescent immature neurons that were generated previously, as suggested in adult animals of different mammalian species (Ghibaudi et al. 2025 ). In contrast to glutamatergic COUP-TFII neurons of PL that appear to be produced in the amygdalar pallium (as discussed above), GABAergic COUP-TFII cells are produced during development in the caudal ganglionic and part of the medial ganglionic eminences of the subpallium, and they constitute a source of interneurons that migrate tangentially to the cortex (Tang et al., 2012 ; Alderman et al. 2024 ; Kim et al. 2025 ). Remnants of GABAergic immature neurons continue to be produced postnatally from the vz/svz of the Arc, at least in human and non-human primate infants and in two-days-old piglets (Kim et al. 2025 ). This could be the source of the COUP-TFII+/SP8 + cells found in the cerebral cortex and the piriform cortex. However, by using single-cell transcriptome of different rostrocaudal parts of the Arc, Kim et al. ( 2025 ) also identified that the vz/svz of the Arc gives rise to a temporo-ventral migratory stream of DCX + cells that express both COUP-TFII and TBR1, suggesting that cells of this stream are glutamatergic. Many of these cells appear to migrate to the temporal cortex, but some were also found in the piriform cortex (Kim et al. 2025 ). It is unclear whether the latter temporo-ventral stream of DCX+/COUP-TFII+/TBR1 + cells includes part of the PL-associated vz/svz cells. In our juvenile swine data, we found that the Arc does not express COUP-TFII, but we did not carry out a systematic analysis of all levels of Arc. More studies will be needed to clarify this issue. Compared to that in other mammals, the density of DCX + immature cells in the pallial amygdala, concentrated in PL, is not only high in gyrencephalic human and non-human primates, but also in small lissencephalic primates as the marmoset (Ghibaudi et al. 2025 ). Thus, primates stand above other mammals regarding pallial amygdala plasticity, independent of the secondary loss of gyri as it appears to occur in marmosets (Kelava et al. 2013 ). In other mammalian radiations, gyrencephalic brains have often been associated with high levels of non-neurogenic postnatal plasticity (Piumatti et al. 2018 ; Freixes et al. 2025 ; Ghibaudi et al. 2025 ). However, this prolonged plasticity and the location of the immature cells in the brain need to be understood in the context of the ecological adaptations of each species: for example, the high postnatal plasticity in the olfactory areas of swine could be related to the relevance of olfactory cues for feeding and social interactions in these animals (Brunjes et al. 2016 ; also discussed by Freixes et al. 2025 ). Regarding the high postnatal plasticity found in the pallial amygdala, what could be the advantage of having a prolonged production of new neurons in this brain structure? In primates, the BCA is the part of the amygdala more broadly connected to the neocortex (Ghashghaei and Barbas, 2002 ; Ghashghaei et al. 2007 ; Pessoa, 2008 ). Having a large reservoir of immature neurons in this nuclear complex may be required for achieving a more sophisticated regulation of emotional learning (Jin et al. 2015 ; Kim et al. 2017 ; discussed by Sorrell et al. 2019), as well as a more plastic adaptation to ever-changing complex social contexts (Chareyron et al. 2012 ; Avino et al. 2018 ; Ghibaudi et al. 2025 ). Overall, our data, together with other studies (Kostović et al., 2019 ; Liu et al., 2021 ), highlight the similarities between swine and humans in terms of brain anatomy and ontogeny, including a protracted development of the pallial amygdala. Since the incorporation of immature neurons in the human PL at juvenile ages seems to be dysfunctional in autism spectrum disorder (Avino et al. 2018 ), the swine emerges as a good animal model to further study postnatal plasticity in functional and dysfunctional contexts​. Abbreviations A: anterior AA: anterior amygdalar area ABC: avidin–biotin complex ac: anterior commissure ACo: anterior cortical amygdalar area AHi: amygdalohippocampal area amc: amygdalar capsule AO: anterior olfactory area BCA: basal complex of the amygdala BCL2: B cell lymphoma 2 protein BL: basolateral nucleus of the basal complex of the amygdala BLA: basolateral nucleus of the basal complex of the amygdala, anterior part BLP: basolateral nucleus of the basal complex of the amygdala, posterior part BM: basomedial nucleus of the basal complex of the amygdala Brn2 (Pou3f2): POU class 3 homeobox 2 BSA: bovine serum albumin CA: cornu ammonis Ce: central nucleus of the amygdala Cl: claustrum Cn: caudate nucleus Co: cortical amygdalar area COUP-TFII (NR2F2): Chicken Ovalbumin Upstream Promoter Transcription Factor II CxA: cortex-amygdala transition zone D: dorsal DCX: doublecortin DG: dentate gyrus EC: entohrinal cortex ec: external capsule En: endopiriform nucleus fi: fimbria FOXP2: Forkhead box P2 fx: fornix GP: globus pallidus Gt: goat HF: hippocampal formation HRP: Horseradish peroxidase conjugated secondary antibodies ic: internal capsule ICx: insular cortex IF: immunofluorescence IHC: immunohistochemistry IM: intercalated amygdalar nucleus, main part ITC: intercalated cells of the amygdala IV: intravenous L: lateral La: lateral nucleus of the basal complex of the amygdala lot: lateral olfactory tract LOT: nucleus of the lateral olfactory tract lv: lateral ventricle Me: medial nucleus of the amygdala MeA: medial nucleus of the amygdala, anterior part MePD: medial nucleus of the amygdala, posterodorsal part Ms: mouse NeuN: Neuronal nuclear protein (also knows as FOX3) ot: optic tract P: posterior PB: phosphate buffer PB-Tx: phosphate buffer containing Triton-X 100 PFA paraformaldehyde PH: parahippocampal area Pir: piriform nucleus PL: paralaminar nucleus PL lat: paralaminar nucleus, lateral subdivision PL med: paralaminar nucleus, medial subdivision PLCo: posterolateral cortical amygdalar area PMCo: posteromedial cortical amygdalar area PSA-NCAM: polysialylated neural cell adhesion molecule PTh: prethalamus Pu: putamen Rb: rabbit RIPA: radioimmunoprecipitation assay RMS: rostral migratory stream Rt: reticular nucleus of the prethalamus S: subiculum SP8: Specificity protein 8 (SP8 transcription factor) st: stria terminalis SVZ/svz: subventricular zone Tbr1: T-box brain transcription factor 1 TBST: Tris-buffered saline containing Tween 20 Th: Thalamus tlv: temporal horn of the lateral ventricle Tu: olfactory tubercle VGLUT2: vesicular glutamate transporter 2 VZ/vz: ventricular zone WB : Western blot Declarations Funding JF has a predoctoral FPU contract by the Spanish Council for Education, Professional Education and Sports (FPU22/03133). This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades and Agencia Estatal de Investigación, MICIU/AEI/10.13039/501100011033 and FEDER-EU (Grant no. PID2023-151927OB-I00 to LM and ED), and by the AGAUR/Generalitat de Catalunya (2021 SGR 01359 to LM). Competing Interests All the authors declare that they have no financial interests to disclose. Author Contributions This work is part of the Doctoral Thesis of JF, supervised by ED and LM. JF contributed to the experimental design, conducted immunohistochemical and immunofluorescence experiments, analyzed data, prepared the figures and wrote the first draft of the manuscript. LM and ED conceived and designed the study, supervised the project, secured funding, contributed to samples characterization, interpretation of results, and improving the manuscript. All authors approved the final manuscript and agreed with the results presented. Data Availability The most relevant data are included in the Figures of this article. Additional data is available upon request and agreement. Ethics Approval This study was performed in line with the 3Rs principles (Russell and Burch, 1959). Brain samples were obtained from animals that were previously used for practicing and improving surgical procedures by M.D. surgeons of the Arnau de Vilanova University Hospital of Lleida. Since brains were extracted once animals were euthanized, Animal Research Ethics Committee of the IRBLleida confirmed that no ethical approval is required. Acknowledgement We deeply thank all Agencies that funded our research: the Spanish Ministerio de Ciencia, Innovación y Universidades and Agencia Estatal de Investigación, MICIU/AEI/10.13039/501100011033 and FEDER-EU (Grant no. PID2023-151927OB-I00 to LM and ED), and the AGAUR/Generalitat de Catalunya (2021 SGR 01359 to LM). 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(B) Western blot of rabbit anti-doublecortin antibody (Abcam, ab18723) with a sample from a homogenized whole-hemisphere of an E50 embryo, also showing a band of ~40kDa. (C) Western blot of mouse anti-COUP-TFII antibody (Bio-Techne R&D Systems, PP-H7147-00) with two samples of 20µg and 40 µg of protein from a homogenized whole-hemisphere of an E50 embryo, showing a band of ~55kDa, consistent with COUP-TFII. SupplFig2.jpg Supplementary Figure 2. Distribution of DCX+ clusters in the paralaminar nucleus (PL) of juvenile swine pallial amygdala. (A, B) Frontal sections at two different levels of the amygdala and (C) horizontal section at the level of the amygdala, immunohistochemically stained for DCX. (A) Anterior and (B) posterior frontal sections allow the visualization of DCX+ cell clusters in the PL, with and increasing gradient along the anteroposterior axis. DCX+ cells are found in lateral and medial subdivisions of PL (PL lat and PL med), but those in PL lat show a shell-like organization, around islands of non-stained cells (asterisks). (C) Horizontal section also shows DCX+ cells in the posterolateral inferior ventricular/subventricular zones (vz/svz), adjacent to the temporal horn of the lateral ventricle, as well as in the PL lat, which decrease as we move toward anterior parts of the pallial amygdala. Asterisks point to DCX+ cells with shell-like distribution in PL lat. Schematic representation of coronal sections and mediolateral and dorsoventral axes are shown in A and B for orientation. Schematic representation of the horizontal section and mediolateral and anteroposterior axes are shown in C for orientation. For abbreviations, see list. Scales: A, B, C = 2 mm. SupplFig3.jpg Supplementary Figure 3. Distribution of DCX+ cells in the Arc and through the rostral migratory stream, and their relation to COUP-TFII. (A-C, E-F, H-I) Sagittal sections at similar level (same level to that in Fig. 11), stained for Nissl (A) or immunohistochemistry for DCX (C, F, I). Note the presence of DCX+ cells in the ventricular/subventricular zones (vz/svz) of the Arc, adjacent to the dorsal part of the lateral ventricle, and in the rostral migratory stream (RMS) or in the external capsule (ec) adjacent to the putamen. Squared areas in A are shown at higher magnification in B, E and H. (B, C) Detail of the Arc, where DCX+ cells are grouped in clusters and form migratory-like chains entering the RMS. (D-D’’) Confocal images of a sagittal section (similar level to that in A) processed for double immunofluorescence for DCX (green) and COUP-TFII (magenta). Note the abundant DCX+ cells and their processes, but the lack of coexpression with COUP-TFII. (E, F) Detail of the DCX+ cell patches along the ec, adjacent to the putamen (Pu), with processes oriented along the anteroposterior axis (F). (G-G’’) Confocal images of a sagittal section (similar level to that in A) processed for double immunofluorescence for DCX (green) and COUP-TFII (magenta), showing DCX+ cells in ec (G’), but none of them coexpresses COUP-TFII (G’’). (H, I) Detail of the rostral part of the ec, adjacent to the RMS, where DCX+ elongated cells align along the anteroposterior axis. (J-J’’) Confocal images of a sagittal section (similar level to that in A) processed for double immunofluorescence for DCX (green) and COUP-TFII (magenta), showing no coexpression of DCX and COUP-TFII in migratory-like chains of the RMS. Schematic representation of the sagittal section and the anteroposterior and dorsoventral axes are shown in A for orientation. For other abbreviations, see list. Scales: A = 2 mm; B, E, H = 250 μm (also applies to C, F and I); D, G, J = 100 μm (also applies to D’-D’’, G’-G’’ and J’-J’’). Cite Share Download PDF Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Brain Structure and Function → Version 1 posted Editorial decision: Revision requested 30 Jan, 2026 Reviews received at journal 30 Jan, 2026 Reviews received at journal 21 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers invited by journal 09 Jan, 2026 Editor assigned by journal 08 Jan, 2026 Submission checks completed at journal 08 Jan, 2026 First submitted to journal 07 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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07:57:28","extension":"html","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185522,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/5d8c0dace54e8b637ab3a5f0.html"},{"id":100170205,"identity":"1b3b45e6-b682-4d5e-a4bb-21a679f85a75","added_by":"auto","created_at":"2026-01-13 16:30:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":530507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCX expression in the paralaminar nucleus of the juvenile swine pallial amygdala.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Parallel frontal sections at a similar level of the juvenile swine amygdala stained for Nissl (A) and immunohistochemistry for DCX (B). Note the DCX+ cell clusters near the external and ventromedial borders of the basal amygdalar complex, within the paralaminar nucleus (PL). Squared areas in A are shown at higher magnification in C and E, while squared areas in B are shown at higher magnification in D and F. (C-D) Details of lateral subdivision of PL showing shell-like clusters of DCX+ cells with small, round or fusiform somas, that surround islands of non-immunoreactive, large cells (islands are pointed with asterisks). (E-F) Details of medial subdivision of PL containing groups of small DCX+ cells near the ventricular (vz) and subventricular (svz) zones, adjacent to the temporal horn of the lateral ventricle (tlv). In contrast to those of lateral PL, DCX+ cells of medial PL do not display a shell-like organization. A schematic representation of the frontal section, and the mediolateral and dorsoventral axes are shown in A for orientation. For other abbreviations, see list. Scales: A, B = 2 mm; C, D, E, F = 200 μm.\u003c/p\u003e","description":"","filename":"Figure01.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/984a629423345d4e568f9dfa.jpg"},{"id":100368562,"identity":"c733054e-28f8-411a-ad1d-cfb99abd992c","added_by":"auto","created_at":"2026-01-16 07:58:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":587149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCX and COUP-TFII expression in the paralaminar nucleus (PL), and distinction from the intercalated amygdalar cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) Parallel sagittal sections or details at similar level of the amygdala stained for Nissl (A, detail in B) and immunohistochemistry for DCX (C) or COUP-TFII (D). Squared area in A is shown at higher magnification in B. (B-D) Details of a DCX+ cell cluster in the lateral subdivision of PL, showing a high density of small cells, adjacent to the amygdalar capsule (amc) (B, C). Abundant COUP-TFII immunoreactive cells with small, round or fusiform somas are also observed in lateral PL, overlapping the DCX+ cells (D). In addition, many COUP-TFII+ large cells are found in PL as well as in other nuclei of the basal amygdalar complex (BCA). (E-H) Parallel sagittal sections or details at a similar level of the amygdala, medial to the sections shown in (A-D), stained for Nissl (E, detail in F), or processed for immunohistochemistry for DCX (G) or FOXP2 (H). Squared area in E is shown at higher magnification in F. (F-H) Details of a cluster of intercalated amygdalar cells (ITC) rich in FOXP2 immunoreactivity (H). In contrast to PL cell clusters, ITC clusters do not express or only show negligible immunoreactivity for DCX (G). Schematic representations of the sagittal sections, and the anteroposterior and dorsoventral axes are shown in A and E for orientation. For other abbreviations, see list. Scales: A, E = 2 mm; B-D, F-H = 200 μm.\u003c/p\u003e","description":"","filename":"Figure02.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/a2926c73346bf3c988fe48b5.jpg"},{"id":100367645,"identity":"aab47f78-9898-4683-a8e4-30a0f2f12080","added_by":"auto","created_at":"2026-01-16 07:57:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":524447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the intercalated amygdalar cells (ITC) of juvenile swine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-J) Parallel horizontal sections at a similar level of the amygdala stained for Nissl (A, details in C and G) and immunohistochemically stained for FOXP2 (B, details in D and H), DCX (E, I) or COUP-TFII (F, J). Dashed square in A is shown at higher magnification and immunohistochemically stained for FOXP2 in B. Squared areas in A are shown at higher magnification in C and G. Squared areas in B are shown at higher magnification in D and H. Note the abundance of FOXP2 immunoreactive cells in ITC, including its main nucleus (IM). (C-F) Details of IM showing a high density of cells, immunoreactive for FOXP2 (D), but with negligible expression of DCX (E) and no expression of COUP-TFII (F). Similarly, (G-J) show an ITC cluster with high density of FOXP2+ cells (H), but with negligible expression of DCX (I) and no expression of COUP-TFII (J). This is a major difference of the ITC clusters with those of the paralaminar nucleus (seen in previous figures). In addition, ITC clusters are often located anterior or medial to the basal amygdalar complex (BCA), while the PL is mainly located along the external and ventromedial margins on BCA. A schematic representation of the horizontal section, and the anteroposterior and mediolateral axes are shown in A for orientation. For other abbreviations, see list. Scales: A, B = 1 mm; C-J = 200 μm.\u003c/p\u003e","description":"","filename":"Figure03.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/71b96edcf27b1e7114398212.jpg"},{"id":100368764,"identity":"da22c501-20ea-4f2b-9471-acc7872a6593","added_by":"auto","created_at":"2026-01-16 07:58:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":551484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuN and Ki-67 expression in DCX+ cells of the swine paralaminar nucleus (PL).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D’’) Confocal images of a frontal section at the level of the lateral paralaminar nucleus (PL) (similar level to that Fig. 1), processed for immunofluorescence for DCX (green) and NeuN (blue). (B’-B’’, D’-D’’) Details of the squared areas in A and C, with separate confocal channels. These details show numerous coexpressing cells (arrowheads) in the lateral PL. Note that coexpressing cells are small and often have a round soma, while large NeuN-expressing cells do not coexpress DCX. (E-F’’) Confocal images of a frontal section (similar level to that Fig. 1) at the level of lateral PL (E-F’’) and medial PL (G-H’’), processed for immunofluorescence for DCX (green) and Ki-67 (blue). (F-F’’, H-H’’) Details of the squared areas in F and H, showing separate confocal channels. Note the scarce double labeled small cells in lateral and medial PL (arrowheads). Anteroposterior and mediolateral axes are indicated in A for orientation (applies to C, E and G). For other abbreviations, see list. Scales: A, C, E, G = 50 μm; B’,D’,F’,H’ = 25 μm (applies to B-B’’, D’-D’’, F’-F’’, H’-H’’).\u003c/p\u003e","description":"","filename":"Figure04.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/2a2463d3529b9b28ad369b01.jpg"},{"id":100369660,"identity":"5510464a-5666-46b7-84b9-4f50e0f6075c","added_by":"auto","created_at":"2026-01-16 07:59:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":672114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCOUP-TFII and BRN2 expression in DCX+ cells of the swine paralaminar nucleus (PL).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D’’) Confocal images of a horizontal section (similar level to that in Fig. 3), at the level of the lateral PL (A-B’’) or medial PL (C-D’’), processed for immunofluorescence for DCX (green) and COUP-TFII (magenta). (B’-B’’, D’-D’’) Details of the squared areas in A and C, showing separate confocal channels. Arrowheads point to numerous double labeled cells, which usually display small, round or fusiform somas. Note that the large COUP-TFII immunoreactive cells do not coexpress DCX. (E-F’’) Confocal images of a frontal section (similar level to that in Fig. 1) processed for immunofluorescence for DCX (green) and BRN2 (blue). (F’, F’’) Details of the squared area in E, showing separate confocal channels. Note the presence of a few double-labeled cells (arrowheads). Anteroposterior and mediolateral axes are indicated in A for orientation (also applies to C). Dorsoventral and mediolateral axes are indicated in E for orientation. For other abbreviations, see list. Scales: A, C, E = 50 μm; B’-B’’ = 25 μm (also applies to D’-D’’, F’-F’’).\u003c/p\u003e","description":"","filename":"Figure05.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/4e8e2ea0993c06c30f7bee2b.jpg"},{"id":100368856,"identity":"d1c066e3-1152-4451-a115-082d9374ed24","added_by":"auto","created_at":"2026-01-16 07:58:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":814418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDCX+, COUP-TFII+ and Ki-67+ cells in the posterolateral inferior ventricular and subventricular zones (vz/svz), adjacent to the temporal horn of the lateral ventricle (tlv).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D’’) Confocal images of a horizontal section processed for immunofluorescence for DCX (green), COUP-TFII (magenta) and Ki-67 (blue). (B’-B’’) Details at higher magnification of the squared area in A, with separate green and magenta confocal channels, showing abundant DCX immunoreactive cells and their radial processes (B’) and abundant COUP-TFII+ cells with small, round somas (B’’) in the posterolateral inferior part of the svz and vz. Note their continuity with labeled migratory-like cells in the inner part of the amygdalar capsule (amc) and a cluster of cells in the adjacent lateral paralaminar nucleus (PL). (C-C’’’) Detail of the vz/svz, showing DCX+ radial processes and some somas (C’; most cell somas are found in a different z plane), COUP-TFII+ small cells (C’’; immunoreaction is mainly found in the cellular nucleus) and abundant Ki-67+ cells (C’’’). Arrowheads point to COUP-TFII and Ki-67 double labeled cells. (D-D’’) Another detail of the vz/svz, showing the presence of some somas coexpressing DCX (D’) and COUP-TFII (D’’). (E-F’’) Confocal images of a horizontal section (similar level to that in Fig. 3) processed for immunofluorescence for DCX (green) and COUP-TFII (magenta). (F’-F’’) Details at higher magnification of the squared area in E, with separate confocal channels, showing tangentially-oriented DCX immunoreactive cells and their processes, forming chains parallel or oblique to the ventricle. These cellular chains seem to separate from the posterolateral svz and extend toward the medial subdivision of PL (F’). These tangentially-oriented migratory-like cells also coexpress COUP-TFII (F’’). Schematic representation of the horizontal sections and anteroposterior and mediolateral axes are shown in A and E for orientation. Asterisks (\u003csub\u003e*\u003c/sub\u003e) in A and E indicate blood vessels. For other abbreviations, see list. Scales: A, B’, E, F’ = 250 μm (also applies to B’’, F’’); C, D = 50 μm (also applies to C’-C’’’, D’-D’’).\u003c/p\u003e","description":"","filename":"Figure06.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/d8bb4dc7c3cfa66e6e49acfa.jpg"},{"id":100368598,"identity":"8cb9706f-a517-4c9f-8c27-7c85272796a5","added_by":"auto","created_at":"2026-01-16 07:58:09","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":573515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKi-67+/COUP-TFII+ and Ki-67+/DCX+ proliferating cells in the ventricular/subventricular zones (vz/svz).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-A’’) Confocal images of a horizontal section (similar level to that in Fig. 3), at the level of the posterolateral inferior vz/svz, processed for immunofluorescence for COUP-TFII (magenta) and Ki-67 (blue). Detail of the vz/svz, with some cells coexpressing COUP-TFII (A’) and Ki-67 (A’’) (arrowheads point to double labeled cells). (B-B’’) Confocal images of a frontal section (similar level to that in Fig. 1), at the level of the medial PL showing immunofluorescence for DCX (green) and Ki-67 (blue) in the vz/svz. Some cells are coexpressing DCX (B’) and Ki-67 (B’’) (arrowheads). Schematic representations of the horizontal (A) and frontal (B) sections and the corresponding axes are indicated for orientation. For other abbreviations, see list. Scales: A, B = 50 μm (also applies to A’-A’’, B’-B’’).\u003c/p\u003e","description":"","filename":"Figure07.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/24dee4c39899a329eb38151c.jpg"},{"id":100170220,"identity":"520c9346-2ce2-4ad4-8d01-f5f3da9eec81","added_by":"auto","created_at":"2026-01-13 16:30:57","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":641927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHorizontal sections showing migratory-like DCX+ and COUP-TFII+ cells extending rostrally from the vz/svz along the amygdalar capsule (amc)\u003c/strong\u003e \u003cstrong\u003eand adjacent paralaminar nucleus (PL).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-G) Parallel horizontal sections at a similar level of the pallial amygdala and its related posterior vz/svz, adjacent to the temporal horn of the lateral ventricle (tlv), stained for Nissl (A) or immunohistochemistry for DCX (C) or COUP-TFII (D). Squared area in A is shown at higher magnification in B. Squared areas in B, C, and D are shown at higher magnification in E, F, and G respectively. (B, C, D) Detail of the vz/svz and its continuation through the inner part of the amc, adjacent to the paralaminar nucleus (PL) and other parts of the basal amygdalar complex (BCA, including BLA and BLP). Clusters of small cells immunoreactive for DCX (C) and COUP-TFII (D) can be detected along the inner part of amc and adjacent PL. An increasing gradient of these clusters can be detected throughout the anteroposterior axis, being more abundant in the posterior part of the amc (near the svz) and scarcer in its anterior parts. (E-G) Detail of a cluster with some migratory-like cells with small, round somas expressing DCX (F) and COUP-TFII (G). A schematic representation of the horizontal section, and the anteroposterior and lateromedial axes are shown in A for orientation. For other abbreviations, see list. Scales: A = 2 mm; B, C, D = 500 μm; E, F, G = 50 μm.\u003c/p\u003e","description":"","filename":"Figure08.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/7b9d3677f8eda0ca328d376d.jpg"},{"id":100369534,"identity":"4e8327d7-52f1-4822-90f2-49c237edd2d8","added_by":"auto","created_at":"2026-01-16 07:59:07","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":544118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFrontal sections showing migratory-like DCX+ cells through the amygdalar capsule (amc) and adjacent paralaminar nucleus (PL).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-G) Parallel frontal sections (similar level to that in Fig. 1) stained for immunohistochemistry for DCX (A, details in B, D, F) or Nissl (C, E, G), showing DCX+ cells in the inner part of amc and adjacent PL. Squared areas in A are shown at higher magnification in B, D, and F. (B) Detail of dorsal part of the amc, adjacent to the lateral nucleus (L) of the basal amygdalar complex (BCA), showing small DCX+ cells with a migratory-like morphology. (D, F) Detail of DCX immunoreactive cells in more ventral parts of the amc, some having a migratory-like morphology with the leading process oriented toward the PL clusters. Other DCX+ cells are organized in chains oriented toward PL (filled arrowheads in F). In the external part of the amc (F), DCX+ cells show the opposite orientation, toward the endopiriform nuclei and the piriform cortex (empty arrowheads in F). Schematic representation of the frontal section, and the mediolateral and dorsoventral axes are shown in A for orientation. For other abbreviations, see list. Scales: A = 500 μm; B, C, D, E, F, G = 100 μm.\u003c/p\u003e","description":"","filename":"Figure09.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/1dd1798fd7bfbf7120934368.jpg"},{"id":100369752,"identity":"a46b8c28-80c6-4417-b09c-aed999455aa6","added_by":"auto","created_at":"2026-01-16 07:59:23","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":639280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSagittal sections showing DCX+ and COUP-TFII+ cells through the amygdalar capsule (amc) and adjacent paralaminar nucleus (PL).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-L) Parallel sagittal sections or their details at similar lateral level stained for Nissl (A, details in B, D, G, J) or immunohistochemistry for DCX (C, details in E, H, K) or COUP-TFII (F, I, L), showing labeled cells in the amygdalar capsule (amc) and adjacent paralaminar nucleus. Note the small size of the cells and the overlapping position of DCX+ and COUP-TFII+ labeling. Squared area in A is shown at higher magnification in B. Squared areas in B are shown at higher magnification in D, G, and J. Squared areas in C are shown at higher magnification in E, H, and K. (B, C) Detail of the amc laterally and ventrally surrounding the basal magnocellular complex (BCA), stained for Nissl (B) and immunohistochemically processed for DCX (C). (D-F, G-I, and J-L) Details showing groups of DCX+ and COUP-TFII+ cells with small, round or fusiform somas in the inner border of amc (empty arrowheads in K), some of which are oriented toward the clusters of labeled cells in the adjacent PL (pointed with filled arrowheads in K). Schematic representation of the sagittal section, and the anteroposterior and dorsoventral axes are shown in A for orientation. For other abbreviations, see list. Scales: A, B, C = 500 μm; D, E, F, G, H, I, J, K, L = 200 μm.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/450b69ae2e429616678aa1c1.jpg"},{"id":100170226,"identity":"4fb30c83-239f-4d5d-990c-0ef0ed8ef764","added_by":"auto","created_at":"2026-01-13 16:30:57","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":260874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic drawing of a frontal section showing the possible origin, migratory pathways and distribution of immature DCX+ and DCX+/COUP-TFII+ neurons in the pallial amygdala of juvenile swine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of a frontal section of juvenile swine brain at the level of the amygdala, showing DCX+ and DCX+/COUP-TFII+ cells. Round cells in the ventricular/subventricular zones (vz/svz) represent possible sources of immature cells, while elongated somas represent the migratory-like cells found along the external (ec) and amygdalar capsules (amc), plus the adjacent areas of the endopiriform/piriform region and the basal complex of the amygdala (BCA), including the paralaminar nucleus (PL). Single labeled DCX+ cells are drawn as empty circles (such as those in the vz/svz of the Arc) or empty fusiform cells (as those in ec, the external part of amc, and those found in the endopiriform/piriform region). In contrast, double labeled cells immunoreactive for DCX and COUP-TFII are represented with a black dot in the center, indicating the COUP-TFII immunoreactive cell nucleus, and are found in the inner part of amc and lateral and medial subdivisions of PL (PL lat, PL med). Since DCX+ cells of the Arc do not express COUP-TFII, it is unlikely they produce the DCX+/COUP-TFII+ immature cells of the pallial amygdala. Thus, as previously shown, the Arc might mainly produce immature cells for the olfactory bulb and anterior olfactory areas (through the rostral migratory stream) and the olfactory structures of the piriform lobe (through ec and external parts of amc). Dorsoventral and mediolateral axes are indicated for orientation. For other abbreviations, see list. Scales: A = 1 mm.\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/12364edbf6ede56b8d6298e5.jpg"},{"id":100170228,"identity":"2133d3d4-8d28-4dc2-b2db-2c46fd49ed8e","added_by":"auto","created_at":"2026-01-13 16:30:57","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":358836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic drawings of sagittal and horizontal sections showing the possible origin, migratory pathways and distribution of immature DCX+ and DCX+/COUP-TFII+ neurons in the pallial amygdala of juvenile swine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Schematic representation of sagittal (A) and horizontal (B) sections from juvenile swine brain, at the level of the pallial amygdala, showing DCX+ and DCX+/COUP-TFII+ cells. Round cells in the ventricular/subventricular zones (vz/svz) represent possible sources of immature cells, while elongated somas represent the migratory-like cells found along the external (ec) and amygdalar capsules (amc), plus the adjacent areas of the endopiriform/piriform region and the basal complex of the amygdala (BCA), including the paralaminar nucleus (PL). Single labeled DCX+ cells are drawn as empty circles (such as those in the vz/svz of the Arc) or empty fusiform cells (as those in ec, the external part of amc, and those found in the endopiriform/piriform region). In contrast, double labeled cells immunoreactive for DCX and COUP-TFII are drawn with a black dot in the center, representing the COUP-TFII immunoreactive cell nucleus. Double labeled cells are found in the posterolateral inferior vz/svz, as well as in the inner part of amc and adjacent PL. This posterolateral and inferior vz/svz sector rich in double-labeled cells is likely the origin of the double-labeled immature cells found in the inner part of amc, in PL and other parts of the pallial amygdala. Some cells might also migrate from this vz/svz sector to adjacent parahippocampal areas (PH). In contrast, the Arc only contains single-labeled DCX+ cells, and thus it is unlikely that it is a source of immature cells for the pallial amygdala. Anteroposterior and mediolateral axes are indicated in A for orientation. Dorsoventral and anteroposterior axes are indicated in B for orientation. For other abbreviations, see list. Scales: A = 1 mm.\u003c/p\u003e","description":"","filename":"Figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/859e79d9bcbef3e90a75c050.jpg"},{"id":106809742,"identity":"cf07838f-21da-4ca6-9aea-a2734288d9eb","added_by":"auto","created_at":"2026-04-13 16:12:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8237447,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/61f7cba0-db64-4448-aeed-65e41e92cf30.pdf"},{"id":100368797,"identity":"1ea41373-0d43-4a0a-a6cf-9bdfc2f0196d","added_by":"auto","created_at":"2026-01-16 07:58:22","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":201300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Validation of primary antibodies by western blotting.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis of DCX and COUP-TFII antibodies in swine brain tissue. (A) Western blot of goat anti-doublecortin antibody (Santa Cruz Biotechnologies, sc-8066) with samples of juvenile brain, taken from the caudate nucleus (Cn), the neocortex (NCx), the piriform cortex (Pir) and a homogenized whole-hemisphere of an E50 embryo. Results show a band of \u003csub\u003e~\u003c/sub\u003e40kDa in the embryo brain tissue, consistent with that of DCX. (B) Western blot of rabbit anti-doublecortin antibody (Abcam, ab18723) with a sample from a homogenized whole-hemisphere of an E50 embryo, also showing a band of ~40kDa. (C) Western blot of mouse anti-COUP-TFII antibody (Bio-Techne R\u0026amp;D Systems, PP-H7147-00) with two samples of 20µg and 40 µg of protein from a homogenized whole-hemisphere of an E50 embryo, showing a band of ~55kDa, consistent with COUP-TFII.\u003c/p\u003e","description":"","filename":"SupplFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/1aa23d07246ca7863c09d784.jpg"},{"id":100369308,"identity":"6e6fe56d-e599-4951-bf5e-7b5892602248","added_by":"auto","created_at":"2026-01-16 07:58:53","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":388074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Distribution of DCX+ clusters in the paralaminar nucleus (PL) of juvenile swine pallial amygdala.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Frontal sections at two different levels of the amygdala and (C) horizontal section at the level of the amygdala, immunohistochemically stained for DCX. (A) Anterior and (B) posterior frontal sections allow the visualization of DCX+ cell clusters in the PL, with and increasing gradient along the anteroposterior axis. DCX+ cells are found in lateral and medial subdivisions of PL (PL lat and PL med), but those in PL lat show a shell-like organization, around islands of non-stained cells (asterisks). (C) Horizontal section also shows DCX+ cells in the posterolateral inferior ventricular/subventricular zones (vz/svz), adjacent to the temporal horn of the lateral ventricle, as well as in the PL lat, which decrease as we move toward anterior parts of the pallial amygdala. Asterisks point to DCX+ cells with shell-like distribution in PL lat. Schematic representation of coronal sections and mediolateral and dorsoventral axes are shown in A and B for orientation. Schematic representation of the horizontal section and mediolateral and anteroposterior axes are shown in C for orientation. For abbreviations, see list. Scales: A, B, C = 2 mm.\u003c/p\u003e","description":"","filename":"SupplFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/54b2403bef551871847b5477.jpg"},{"id":100368229,"identity":"61be51d7-f816-494b-89b5-ef72dc7d009b","added_by":"auto","created_at":"2026-01-16 07:57:44","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":664848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Distribution of DCX+ cells in the Arc and through the rostral migratory stream, and their relation to COUP-TFII.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C, E-F, H-I) Sagittal sections at similar level (same level to that in Fig. 11), stained for Nissl (A) or immunohistochemistry for DCX (C, F, I). Note the presence of DCX+ cells in the ventricular/subventricular zones (vz/svz) of the Arc, adjacent to the dorsal part of the lateral ventricle, and in the rostral migratory stream (RMS) or in the external capsule (ec) adjacent to the putamen. Squared areas in A are shown at higher magnification in B, E and H. (B, C) Detail of the Arc, where DCX+ cells are grouped in clusters and form migratory-like chains entering the RMS. (D-D’’) Confocal images of a sagittal section (similar level to that in A) processed for double immunofluorescence for DCX (green) and COUP-TFII (magenta). Note the abundant DCX+ cells and their processes, but the lack of coexpression with COUP-TFII. (E, F) Detail of the DCX+ cell patches along the ec, adjacent to the putamen (Pu), with processes oriented along the anteroposterior axis (F). (G-G’’) Confocal images of a sagittal section (similar level to that in A) processed for double immunofluorescence for DCX (green) and COUP-TFII (magenta), showing DCX+ cells in ec (G’), but none of them coexpresses COUP-TFII (G’’). (H, I) Detail of the rostral part of the ec, adjacent to the RMS, where DCX+ elongated cells align along the anteroposterior axis. (J-J’’) Confocal images of a sagittal section (similar level to that in A) processed for double immunofluorescence for DCX (green) and COUP-TFII (magenta), showing no coexpression of DCX and COUP-TFII in migratory-like chains of the RMS. Schematic representation of the sagittal section and the anteroposterior and dorsoventral axes are shown in A for orientation. For other abbreviations, see list. Scales: A = 2 mm; B, E, H = 250 μm (also applies to C, F and I); D, G, J = 100 μm (also applies to D’-D’’, G’-G’’ and J’-J’’).\u003c/p\u003e","description":"","filename":"SupplFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8544141/v1/e5e336ba78b0dd9043719101.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Postnatal plasticity in the paralaminar nucleus of the pallial amygdala in juvenile swine brain","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn humans and non-human primates, the amygdala shows a remarkably prolonged maturation, extending through adolescence and beyond, which may be related to the increasing involvement of this structure over time in social cognition and adaptation to changing social contexts (Chareyron et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Avino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The protracted postnatal development of the amygdala involves the presence of a large number of immature neurons, especially concentrated in the paralaminar amygdalar nucleus (PL) (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which appears as an immature cell reservoir from where new neurons migrate, mature and incorporate into the adjacent basal and accessory amygdalar nuclei (Chareyron et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Avino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since the PL is a major target of hippocampal projections (Fudge et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), it has been proposed to be an interface linking autobiographical memories with emotional valence (Jin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; discussed by Sorrell et al. 2019). In humans and monkeys, the PL increases in size and neuron number during juvenile ages, but this fails to occur in autism spectrum disorder (Avino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn humans and other primates, immature neurons are abundant in PL of children and express microtubule-associated protein doublecortin (DCX), polysialylated neural cell adhesion molecule (PSA-NCAM) and B cell lymphoma 2 protein (BCL2) (Bernier et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Many of these immature neurons transition during adolescence into mature glutamatergic neurons that contain the vesicular glutamate transporter VGLUT2 (SCL17A6) and the transcription factors TBR1 and COUP-TFII (NR2F2), but not other transcription factors typical of the caudal ganglionic eminence such as SP8 (Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This suggests that these immature neurons originate in the pallium, but their exact spatial and temporal origin, either prenatally and/or postnatally, remain unknown or controversial (Bernier et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Sorrell et al. 2019; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Independent of their origin, the existence of large numbers of excitatory neurons in primate PL that remain immature for relatively long periods (decades in humans) indicates an extraordinary plasticity of the pallial amygdala (Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which function remains to be explored. Immature neurons are more abundant and persist during a longer postnatal period in the basal complex of the amygdala in large-brained, mostly gyrencephalic mammals (especially primates, but also carnivores as the cat and artiodactyls as the sheep), but they are scarce and decline rapidly in small-brained mammalian species (including rodents) (Piumatti et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Alderman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In primates, immature neurons continue to be observed in the basal complex of the amygdala even in aged adults, while in rodents (like mouse) their density is much lower and there is a sharp decline in juveniles (Alderman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This is likely related to the very small PL found in rodents, difficult to distinguish from the intercalated cells (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Only recently, with the use of multiple fluorescent labeling and single cell transcriptomics, it was possible to reliably distinguish in mouse between PL (expressing DCX, COUP-TFII / NR2F2, and several glutamatergic markers) from the intercalated amygdalar cells (expressing FOXP2 and GABAergic markers) (Alderman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn spite of the detailed knowledge on the distribution, phenotype and changes throughout ontogeny of amygdalar immature neurons in some primates and rodents, data in other species is still scarce and a putative paralaminar nucleus remains poorly defined. In recent years, the swine has emerged as a large-brained gyrencephalic species, which brain development and organization resemble more closely that of humans, compared to the murine brain (Lind et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kinder et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, it shows abundant levels of DCX\u0026thinsp;+\u0026thinsp;immature neurons throughout all olfactory areas in piglets and juveniles, which appear to migrate from different rostrocaudal levels of the subventricular zone of the lateral ventricle (SVZ) and/or the adjacent white matter (Costine et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Torrijos-Saiz et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While the SVZ is usually known as a source of GABAergic interneurons for the olfactory bulb (Alvarez-Buylla and Garc\u0026iacute;a-Verdugo 2002; Kohwi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Young et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), studies in different mammalian species including swine show that it also gives rise to glutamatergic neurons that migrate through the rostral migratory stream and the external capsule to different olfactory structures of the piriform lobe (Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). DCX\u0026thinsp;+\u0026thinsp;immature neurons were also found in the olfacto-recipient cortical area of the amygdala in juvenile swine, but their presence in the basal amygdalar complex and the existence of a PL is unexplored in swine. An excellent approach to help with the identification of homologue brain structures is by studying their developmental origin, as this is a good reference for understanding their topological position, which remains identical throughout ontogeny and in evolution (Nieuwenhuys and Puelles, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In primates, the basal amygdalar complex, including the PL, was proposed to derive from the so-called \u0026ldquo;inferior ganglionic eminence\u0026rdquo;, adjacent to the temporal horn of the lateral ventricle (tlv), and postnatally the PL resides in the same location adjacent to the former inferior ganglionic eminence (reviewed by De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The developmental origin of the basal complex and cortical areas of the amygdala has recently been reformulated in mouse, and they appear to derive from a distinct posteroventral ventricular sector of the telencephalon, named the amygdalar pallium (Garc\u0026iacute;a-Calero et al. 2020), which appears comparable to the primate inferior ganglionic eminence. Thus, in spite of its name suggesting a subpallial location, the inferior ganglionic eminence would be the progenitor zone of the amygdalar pallium. According to Garc\u0026iacute;a-Calero et al. (2020), this amygdalar pallial sector contains at least four radial subunits giving rise to different neuron subgroups of the basal complex and cortical amygdala. However, the PL was not considered in this study, and its exact origin remains elusive. This information could help to better understand the topological location and possible migratory routes of immature cells found postnatally in the PL of different mammals. The aim of this study was to investigate the distribution of immature cells in the basolateral amygdalar complex of juvenile swine brains, trying to identify the PL as well as the phenotype and possible migratory routes of PL immature cells. To that aim, we performed immunolabeling for DCX, combined with the cell proliferation marker Ki-67 and different neuronal markers, including NeuN (neuronal nuclear protein, also known as FOX3), the transcription factors BRN2 (POU3F2, present in subsets of glutamatergic neurons) and COUP-TFII / NR2F2 (critical for amygdalar development and adult phenotype maintenance, and present in PL cells in both human and mouse). To distinguish PL from intercalated amygdalar cell clusters, we combined DCX with the transcription factor FOXP2. To better understand PL cytoarchitecture with respect to that of other amygdalar nuclei, we also performed Nissl staining in complete series of sections adjacent to those processed for immunohistochemistry of immunofluorescence. Our results demonstrated that the swine pallial amygdala shows a prolonged postnatal plasticity similar to that of humans and contains a reservoir of immature neurons coexpressing DCX and COUP-TFII / NR2F2 mainly located in the PL. We also identified the possible origin of these cells in a posteroventral ventricular/subventricular pallial sector adjacent to the temporal horn of the lateral ventricle expressing COUP-TFII / NR2F2.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eIn the present study, we used brains from swine \u003cem\u003e(Sus scrofa domesticus\u003c/em\u003e) provided by the Applied Biomedical Research Centre (CREBA) of the Institute of Biomedical Research of Lleida- Dr. Pifarr\u0026eacute; Foundation (IRBLleida, Spain), located in Torrelameu (Lleida, Spain) (registered as a user center of experimental animals: L9900008; REGA: ES252310036907). In total, we used 4 juvenile swine of both sexes (1 male and 3 females), ranging in age from 2.5 to 3.5 months, and weighted between 30 to 40kg. At this age, pigs are considered juvenile, as they have not yet reached sexual or full neurodevelopmental maturity. Animals were housed and handled in the pig CREBA facilities, according to the protocol approved by the Animal Experimentation Ethics Committee of the CREBA and following the regulations and laws of the European Union (Directive 2010/63/EU) and the Spanish Government (Royal Decrees 53/2013) for the care and handling of animals in research. These animals were previously used for practicing and improving surgical procedures by M.D. surgeons. After, animals were euthanized, and their brains were extracted and processed as explained below.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection and fixation\u003c/h2\u003e \u003cp\u003eFor cerebral tissue collection, the animals previously employed for surgery research were sacrificed, just before brain extraction, with a lethal dose of sodium pentobarbital (200mg/kg; IV). After extracting the brain, the tissue was washed with abundant water to remove blood and hemispheres were separated and fixed by immersion in a solution of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), for 2\u0026ndash;4 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample preparation and sectioning\u003c/h3\u003e\n\u003cp\u003eSome hemispheres were cut into four blocks (frontal, temporoparietal, occipital, and brainstem with cerebellum), while others were conserved in a single piece. Following fixation, the tissue was cryoprotected in a solution of increasing glycerol concentrations (10% and 20%) and 2% DMSO in 0.1M PB for 6\u0026ndash;10 days. The tissue was frozen by immersion in -65/-70\u0026deg;C isopentane (2-methyl butane, Sigma-Aldrich, Germany) for 1-2min, following the protocol of Rosene et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Frozen brains were wrapped in aluminum foil and stored in a container at -80\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003eFor sectioning, samples were cut using a freezing sliding microtome (Microm HM 450; Thermo Fisher Scientific), equipped with a large size Freezing Stage (BFS-40MPA, Physitemp Instruments, LLC, USA). Coronal, sagittal and horizontal sections of 100\u0026micro;m thick were collected at 4\u0026deg;C in 0.1M PB, into twelve parallel series with about 20 sections per each block for coronal sections and each sagittal- or horizontally sectioned hemisphere. For each series, selected sections of the appropriate levels including the areas of interest were processed for studying. The sections not used immediately were stored in a 30% sucrose PB solution and frozen at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003eIn one animal (male), the right hemisphere was sectioned in the sagittal plane, and two parallel series were stained for Nissl and immunohistochemistry for DCX. Similarly, in a female, the right hemisphere was sectioned in the horizontal plane, and two parallel series were processed for Nissl and DCX-immunohistochemistry staining. Horizontal sections from another female were processed, but in this case the occipital lobe of the right hemisphere was separated and stored for further analysis. Regarding frontal sections, the amygdala was studied from anterior to posterior levels (from 10.375 mm to 6.125 mm antero-posterior stereotaxic coordinates from the MRI Atlas, Saikali et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) by using Nissl and immunohistochemistry for DCX staining.\u003c/p\u003e \u003cp\u003eFollowing the initial analysis of Nissl and DCX chromogenic immunostained sections, selected frontal, sagittal and horizontal sections were processed for double and triple immunofluorescence, combining DCX with other markers of interest (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, 55 sections were Nissl-stained (10 frontal sections, 25 horizontal sections and 20 sagittal sections), about 100 sections were processed for single immunohistochemistry (including DCX, COUP-TFII, FOXP2 and Ki-67 staining), and 50 sections were processed for double or triple immunofluorescence.\u003c/p\u003e\n\u003ch3\u003eNissl staining\u003c/h3\u003e\n\u003cp\u003eTo study the tissue organization and to help with the identification of the areas, complete series of sections of each plane were processed for Nissl staining as follows. After being mounted with a solution of gelatine from porcine skin at 0.5% in Tris buffer and dried, slides with sections were sequentially immersed in 100% ethanol and xylene to dissolve out the lipids and allow a better staining. After, sections were dried and incubated for 1 minute with filtered 1% Toluidine blue in 0.1M acetate buffer at pH 4.6. Sections were then rinsed with abundant water, and differentiated with acid alcohol (0.01% acetic acid solution in 70% ethanol) for 90 seconds. Subsequently, the slides were dehydrated with ascending ethanol concentrations (70%, 96%, and 100%), followed by a brief xylene wash, and finally coverslipped with Permount mounting medium (Thermo Fischer Scientific).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFree-floating brain sections were processed for immunohistochemistry to detect DCX, COUP-TFII, FOXP2, or Ki-67, using specific primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sections were washed with 0.1M PB and then processed for antigen retrieval by incubation in a sodium citrate buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for 1h at 60\u0026ordm;C. After rinsing, sections were permeabilized with PB containing 0.3% Triton-X 100 (PB-Tx; 0.1 M), and incubated for 2h at room temperature in a blocking solution containing 2% of bovine serum albumin (BSA) and 20% of normal serum (according to the species in which the secondary antibody was raised) in 0.1M PB. Next, sections were incubated in primary antibody solution (for dilutions see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in PB-Tx for 72h at 4\u0026deg;C with gentle shaking. After rinsing, sections were incubated in a biotinylated secondary antibody (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in PB-Tx overnight, at 4\u0026deg;C with gentle shaking. Successively, sections were washed, and endogen peroxidase was blocked with a solution of PB with 20% methanol and 2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 20min. After, sections were washed and incubated in the avidin\u0026ndash;biotin complex (AB Complex, Vector Laboratories Ltd.) for 1 h at room temperature. After incubation in the ABC solution, the sections were first washed in PB, followed by Tris buffer (0.05M, pH 7.6).\u003c/p\u003e \u003cp\u003eTo reveal the staining, the sections were incubated with diaminobenzidine (DAB), prepared according to the manufacturer\u0026rsquo;s instructions (SIGMAFAST\u0026trade; tablets, Sigma-Aldrich Co. LLC; or DAB Chromogen/Substrate Bulk Pack, ScyTek Laboratories INC.). The reaction was stopped by rinsing the sections with Tris buffer. Sections were then mounted, dehydrated and covered with Permount\u0026trade; as described before.\u003c/p\u003e \u003cp\u003eAs negative control, some sections were processed following the immunohistochemistry procedure but omitting primary antibodies. No specific cell labeling was observed (Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary antibodies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAntigen recognized\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eManufacturer and Reference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRRID\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003ePolyclonal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat anti-doublecortin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDoublecortin C-18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u0026ndash;1:2000 for IHC/IF\u003c/p\u003e \u003cp\u003e1:1000 for WB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta Cruz Biotechnologies\u003c/p\u003e \u003cp\u003eRef. sc-8066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_2088494\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit anti-doublecortin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDoublecortin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:3000 for IHC/IF\u003c/p\u003e \u003cp\u003e1:1000 for WB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003cp\u003eRef. ab18723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_732011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit anti-FOXP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eForkhead box P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:2000 for IHC/IF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003cp\u003eRef. ab16046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_2107107\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit anti-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN-terminal actin peptide attached to a multiple antigen peptide backbone.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000 for WB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003cp\u003eRef. A5060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_476738\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eMonoclonal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRat anti-Ki-67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKi-67 SolA15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:100\u0026nbsp;for IHC/IF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. 14-5698-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_10854564\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse anti-NeuN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeuronal Nuclei (NeuN); clone A60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u0026nbsp;for IHC/IF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003cp\u003eRef. MAB377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_2298772\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse anti-COUP-TF2/NR2F2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCOUP-TF II/NR2F2 clone H7147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:500\u0026nbsp;for IHC/IF\u003c/p\u003e \u003cp\u003e1:1000 for WB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBio-Techne R\u0026amp;D Systems\u003c/p\u003e \u003cp\u003eRef. PP-H7147-00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_1964214\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse anti-Brn2 IgG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePOU class 3 homeobox 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:200\u0026nbsp;for IF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta Cruz Biotechnologies\u003c/p\u003e \u003cp\u003eRef. sc-393324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_2737347\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse anti-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlightly modified β-cytoplasmic actin N-terminal peptide.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000 for WB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003cp\u003eRef. A5441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_476744\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eDouble and triple immunofluorescence\u003c/h3\u003e\n\u003cp\u003eMultiple labeling immunofluorescence was performed to detect cells coexpressing different markers. Following tissue permeabilization and blocking of non-specific binding, sections were incubated at 4\u0026deg;C for 72h with a cocktail of primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After rinsing, sections were incubated overnight at 4\u0026deg;C in PB-Tx with the corresponding cocktail of fluorescent secondary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Finally, the sections were mounted with a solution of gelatine at 0.5% and covered with antifading mounting medium (Vectashield Hardset Antifade mounting medium, Vector Laboratories Ltd.).\u003c/p\u003e \u003cp\u003eAs done for immunohistochemistry, some sections were incubated omitting the cocktail of primary antibodies to ensure their specificity. No labelling was detected.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSecondary antibodies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eManufacturer and Reference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRRID\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBiotinylated for IHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit anti-goat IgG (H\u0026thinsp;+\u0026thinsp;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVector Laboratories\u003c/p\u003e \u003cp\u003eRef. BA-5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2336126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVector Laboratories\u003c/p\u003e \u003cp\u003eRef. BA-9200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2336171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVector Laboratories\u003c/p\u003e \u003cp\u003eRef. BA-1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2313606\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat Anti-Rat IgG (H\u0026thinsp;+\u0026thinsp;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVector Laboratories\u003c/p\u003e \u003cp\u003eRef. BA-9400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2336202\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHRP-linked for WB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-rabbit IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:20000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling\u003c/p\u003e \u003cp\u003eRef. 7074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2099233\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-mouse IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:20000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling\u003c/p\u003e \u003cp\u003eRef. 7076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_330924\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-goat IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVector Laboratories\u003c/p\u003e \u003cp\u003eRef. PI-9500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2336124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e\u003cb\u003eFluorescent for IF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey anti-goat IgG (H\u0026thinsp;+\u0026thinsp;L),\u003c/p\u003e \u003cp\u003ecoupled to Alexa 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e \u003cp\u003eRef. 705-546-147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2340430\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L),\u003c/p\u003e \u003cp\u003ecoupled to Alexa 488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. A21202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_141607\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey anti-goat IgG (H\u0026thinsp;+\u0026thinsp;L),\u003c/p\u003e \u003cp\u003ecoupled to Alexa 568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. A11057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2534104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonkey anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L),\u003c/p\u003e \u003cp\u003ecoupled to Alexa 568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. A10042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2534017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), coupled to Alexa Plus 405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. A48254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2890548\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L), coupled to Alexa Plus 405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. A48255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2890536\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat anti-rat IgG (H\u0026thinsp;+\u0026thinsp;L),\u003c/p\u003e \u003cp\u003ecoupled to Alexa Plus 405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003eRef. A48261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAB_2890550\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting for validation of primary antibodies\u003c/h2\u003e \u003cp\u003eSince the employed antibodies were not validated by the respective manufacturers for swine tissue, we employed the Western blot technique for validation. Samples consisted of brain tissue, obtained from juvenile and embryos at 50 days of gestation (E50).\u003c/p\u003e \u003cp\u003eOne hemisphere of a juvenile swine was dissected to obtain samples from the caudate nucleus (Cn), the piriform cortex (Pir) and the neocortex (NCx), while for the embryonic sample, one whole hemisphere was used. All the samples were fragmented and homogenized using an electric homogenizer (Tissue Grinder) in ice-cold radioimmunoprecipitation assay (RIPA) containing lysis buffer (150 mM NaCl; 1% NP-40; 0.5% Na-deoxycholate; 0.1% SDS; 50 mM Tris-HCl [pH 7.4]), protease inhibitor (Sigma-Aldrich, cat # P8340) and PhosSTOP (Roche). Homogenized samples were centrifuged at 12000 rpm for 15 min at 4\u0026deg;C and then protein concentrations of the supernatants were determined by BIO-RAD Micro DC protein assay (BIO-RAD, Laboratories, Inc.). Samples containing 20\u0026ndash;40 \u0026micro;g of protein were mixed with an equivalent volume of sample buffer containing 8% of SDS and 2% mecaptoethanol and heated at 100\u0026deg;C for 5 min. Following, samples were first loaded onto a denaturing 10% sodium dodecyl sulphate-polyacrylamide gel, together with chemiluminescent (MagicMark\u0026trade; XP, Invitrogen) and multicolor prestained (PageRuler\u0026trade; Plus, Thermo Fisher) ladders. Proteins were separated by electrophoresis for 90 to 120 minutes at an intensity of 25 mA per gel and then were electrotransferred to a nitrocellulose membrane in Tris-glycine-methanol buffer, using semidry transfer. The membrane was blocked for 1 h at room temperature in a blocking solution mixture of 5% nonfat dry milk in 0.1% Tween 20 and Tris-buffered saline pH 8 (TBST). After washing with TBST, membranes were incubated overnight at 4\u0026deg;C with anti-DCX and anti-COUP-TFII antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for immunodetection. Next day, membranes were washed and incubated with the appropriate peroxidase-conjugated secondary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) for 60 min at room temperature, washed in TBST, and visualized using the ECL Prime Western blotting Detection Reagent detection kit (GE Healthcare), as described by the manufacturer. Chemi-Doc MP Imaging System (BIO-RAD Laboratories, Inc.) was used for the visualization of bands. Following, the membrane was incubated with anti-actin antibody as loading controls.\u003c/p\u003e \u003cp\u003eDoublecortin antibodies showed a band of 40kDa, as described by the manufacturer (Supplementary Fig.\u0026nbsp;1). Similarly, brain sample reacted with COUP-TFII antibody giving a band of a similar weight as the one reported by the manufacturer (~\u0026thinsp;55kDa).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification of regions of interest\u003c/h3\u003e\n\u003cp\u003eTo identify the regions of interest, we used our own Nissl-stained sections and those of the wild boar brain from the University of Wisconsin-Madison, as well as the MRI 3D atlas from Saikali et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our identification was based on both the topological position and cytoarchitecture of the structures.\u003c/p\u003e\n\u003ch3\u003eDigital photographs and figures\u003c/h3\u003e\n\u003cp\u003eDigital microphotographs of the sections processed for immunohistochemistry were taken on a Leica microscope (DM2500 LED, Leica Microsystems GmbH) equipped with a digital camera (Zeiss Axiovision Digital Camera -Carl Zeiss- and Flexacam C5 LSR Camera -Leica Microsystems GmbH-). Serial images from fluorescent material were taken under confocal microscopes (Olympus FV1000 -Olympus Corporation- and Nikon AX NSPAR -Nikon). Selected digital fluorescent images were adjusted and extracted using Olympus FV10-ASW 4.2 Viewer (Olympus Corporation) and Image J (Fiji). Finally, the figures were mounted using Affinity Designer 2 (version 2.6.5, Serif Corporation).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe analyzed coronal, sagittal, and horizontal sections from juvenile swine brains of both sexes, at different levels, to carry out a systematic description of the distribution of DCX\u0026thinsp;+\u0026thinsp;cells throughout the amygdala. The expression pattern of DCX was similar across animals and in both sexes, even if the limited brain number did not allow a reliable comparison between sexes or ages. We found DCX\u0026thinsp;+\u0026thinsp;cells in the basal complex of the amygdala, mainly localized in clusters or patches along the external border of the complex, resembling in topological position the PL. To know more about the phenotype of the DCX\u0026thinsp;+\u0026thinsp;cells of the clusters and distinguish whether they truly represent the swine PL or belong to clusters of intercalated amygdalar cells (ITC), we compared the results between adjacent sections single or double/triple-labeled for DCX, NeuN (that is expressed in neurons since they start to differentiate), and/or the transcription factors BRN2 (POU3F2, which is expressed in subpopulations of immature glutamatergic cortical neurons), COUP-TFII (NR2F2, which is crucial for amygdala patterning and is expressed in PL neurons), and FOXP2 (that is expressed in intercalated cells of the amygdala). As many of the DCX\u0026thinsp;+\u0026thinsp;cells in the clusters had a migratory cell morphology and some of them formed chains (in agreement with Torrijos- Saiz et al. 2025; Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we tried to identify possible progenitor areas and explore the possible migratory pathways that immature cells might follow from these progenitor areas to their final position in PL. To help in this analysis, we also studied the coexpression of Ki-67, a mitotic marker, with other markers.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDistribution of DCX\u0026thinsp;+\u0026thinsp;cells in the pallial amygdala and identification of swine PL\u003c/h2\u003e \u003cp\u003eThe pallial amygdala consists of the basal complex of the amygdala (BCA) and several cortical amygdalar areas near the surface. Like in other gyrencephalic mammals, the swine pallial amygdala has a relatively large size, as compared to the central and medial amygdalar nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;2). We found numerous DCX\u0026thinsp;+\u0026thinsp;cells in both the BCA and the cortical areas of juvenile swine, indicating high levels of plasticity. The majority of the DCX\u0026thinsp;+\u0026thinsp;cells were observed near the external border of BCA, close to the amygdalar capsule, and in its posteroventral continuation towards the subventricular zone adjacent to the temporal horn of the lateral ventricle (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These cells resembled the primate PL in topological position and postnatal DCX expression. Like primate PL, DCX\u0026thinsp;+\u0026thinsp;cells of swine PL were often broken into clusters which overlapped with COUP-TFII expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D), but not with FOXP2, which identified the swine intercalated amygdalar cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB,D,H). In contrast to PL clusters, the intercalated cell clusters showed negligible levels of DCX immunoreaction (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE,I) and did not express COUP-TFII (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF,J). The intercalated cell clusters and those of PL also differed in location: while the first were mainly found anterior to BCA (the main intercalated nucleus or IM) or along its medial border with the central or medial amygdala (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the PL clusters were mainly observed laterally and became more abundant at posterior levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLike in primates, the swine PL also displayed lateral and medial subdivisions that surrounded laterally and ventromedially the BCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; details of lateral and medial subdivisions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, respectively). DCX\u0026thinsp;+\u0026thinsp;cells of the lateral subdivision were organized in patches or clusters, which were more abundant ventrally, adjacent to the basolateral (BL) and basomedial (BM) nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;2). In frontal sections, clusters of DCX\u0026thinsp;+\u0026thinsp;cells of the lateral PL displayed a shell-like organization, surrounding islands of non-stained large cells of the BCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D). As seen in Nissl staining, the PL cells of the shell-like clusters showed a predominant fusiform shape, but these were mixed with cells of round or multipolar morphology of different sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These different cell morphologies were also seen with DCX immunohistochemical labeling, with a predominance of small, round cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In the medial PL, DCX\u0026thinsp;+\u0026thinsp;cells were more dispersed and rarely formed clusters, although these did not display a shell-like organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE,F). Different morphologies and soma sizes were observed in medial PL but most DCX\u0026thinsp;+\u0026thinsp;cells were round and small (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhenotype of DCX\u0026thinsp;+\u0026thinsp;cells in PL\u003c/h2\u003e \u003cp\u003eTo better understand the phenotype of the immature DCX\u0026thinsp;+\u0026thinsp;cells of PL, we carried out double and triple immunofluorescence (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This combination also allowed a better correlation between distinct morphologies and soma sizes of DCX\u0026thinsp;+\u0026thinsp;cells and specific phenotypes. The majority of the DCX\u0026thinsp;+\u0026thinsp;cells of PL expressed the neuronal marker NeuN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D\u0026rsquo;\u0026rsquo;), and cells coexpressing DCX and NeuN (arrowheads in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026rsquo;-B\u0026rsquo;\u0026rsquo;, D\u0026rsquo;-D\u0026rsquo;\u0026rsquo;) were generally smaller than those expressing only NeuN. Among the cells coexpressing DCX and NeuN we could distinguish at least two types, one with a larger (medium-sized) soma and dendrites, and another one with very small and round or fusiform soma (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D\u0026rsquo;\u0026rsquo;). Double labeling with Ki-67 showed that a few of the small and round DCX\u0026thinsp;+\u0026thinsp;cells coexpressed Ki-67 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H\u0026rsquo;\u0026rsquo;), indicating that at least a few of these immature cells were proliferating in both lateral (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F\u0026rsquo;\u0026rsquo;) and medial (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H\u0026rsquo;\u0026rsquo;) parts of PL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to NeuN, most DCX\u0026thinsp;+\u0026thinsp;cells in PL showed COUP-TFII immunofluorescence (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D\u0026rsquo;\u0026rsquo;). As typical of transcription factors, COUP-TFII was mainly located in the cellular nucleus. However, we found COUP-TFII\u0026thinsp;+\u0026thinsp;cell nuclei of two sizes, large and small. Almost all DCX\u0026thinsp;+\u0026thinsp;cells showed a small COUP-TFII\u0026thinsp;+\u0026thinsp;nucleus. Most DCX\u0026thinsp;+\u0026thinsp;cells had a round or fusiform soma, some with a migratory-like morphology. Large COUP-TFII non-DCX cells were abundant in both PL and the rest of the BCA, and resembled mature neurons.\u003c/p\u003e \u003cp\u003eWe only found very few DCX\u0026thinsp;+\u0026thinsp;cells coexpressing BRN2 in PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F\u0026rsquo;\u0026rsquo;), which is a major difference with our previous findings in the olfactory areas, where many cells coexpressed DCX and BRN2 (Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePossible progenitor areas of DCX\u0026thinsp;+\u0026thinsp;cells and migratory routes to PL\u003c/h2\u003e \u003cp\u003eMany DCX\u0026thinsp;+\u0026thinsp;cells in PL had an elongated morphology, and some displayed a leading process resembling migratory cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B\u0026rsquo;,C,D\u0026rsquo;). Since the vast majority of the DCX\u0026thinsp;+\u0026thinsp;cells of PL coexpressed COUP-TFII, we analyzed expression of this transcription factor in the ventricular/subventricular zones (vz/svz) of the temporal horn of the lateral ventricle (tlv) in order to understand the origin of PL immature cells. We identified a posterolateral inferior sector of the vz/svz rich in radially oriented DCX\u0026thinsp;+\u0026thinsp;cells and small round COUP-TFII cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Remarkably, many cells in this vz/svz sector coexpressed COUP-TFII and Ki-67 and DCX and Ki-67 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-C\u0026rsquo;\u0026rsquo;\u0026rsquo;, 7A-A\u0026rsquo;\u0026rsquo;), resembling a hotspot for production of new cells. We also found coexpression of DCX and COUP-TFII (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-D\u0026rsquo;\u0026rsquo;). A major difference between this vz/svz sector and the Arc (giving rise to immature neurons of the rostral migratory stream and those found in the external capsule and many olfactory areas) is that the Arc did not express COUP-TFII (Supplementary Fig.\u0026nbsp;3). These observations discarded the Arc as a possible origin of immature cells found in PL and pointed to the posterolateral and inferior vz/svz sector adjacent to the tlv as the most likely origin.\u003c/p\u003e \u003cp\u003eMoreover, from this hotspot of the vz/svz, we found chains of radially oriented migratory-like DCX\u0026thinsp;+\u0026thinsp;cells, coexpressing COUP-TFII, that were in continuity with the cell clusters of lateral PL (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Thus, this particular posterolateral and inferior sector of vz/svz might be the source of the DCX\u0026thinsp;+\u0026thinsp;immature neurons found in lateral PL. The horizontal plane was the best to follow this continuity from the vz/svz to the PL clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), and showed that the chains of migratory-like DCX\u0026thinsp;+\u0026thinsp;cells in continuity with the DCX+/COUP-TFII\u0026thinsp;+\u0026thinsp;cell clusters of PL occupied an inner position in the amygdalar capsule (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), while other DCX\u0026thinsp;+\u0026thinsp;cell chains located more superficially did not overlapped nor coexpressed COUP-TFII and appeared to be related to the endopiriform/piriform region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe continuity of DCX\u0026thinsp;+\u0026thinsp;cell chains from the posterolateral inferior vz/svz to the PL cell clusters could not be observed in the frontal (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) of sagittal planes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), although in these section planes it was possible to observe short chains of DCX\u0026thinsp;+\u0026thinsp;cells in the inner part of the amygdalar capsule, which appeared to reach the lateral PL clusters (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA,B,F; filled arrowheads in F; 10E,H,K; empty arrowheads in K). In contrast, DCX\u0026thinsp;+\u0026thinsp;cell chains of the external part of the amygdalar capsule were oriented toward the endopiriform/piriform region (empty arrowheads in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF empty arrowheads).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we observed tangentially oriented chains of DCX\u0026thinsp;+\u0026thinsp;cells, aligned parallel to the ventricle, that appear to separate from the same posterolateral and inferior vz/svz rich in small cells immunoreactive for DCX and COUP-TFII mentioned above (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F\u0026rsquo;\u0026rsquo;). These tangentially oriented cell chains might contribute to at least part of the DCX\u0026thinsp;+\u0026thinsp;cells observed in medial PL, but we cannot discard that other immature cells might be produced in the vz/svz sector adjacent to medial PL, since we also observed a few cases of DCX/Ki-67 coexpression there (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-B\u0026rsquo;\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur results on DCX expression showed prolonged postnatal plasticity in the swine pallial amygdala that extends at least until juvenile ages, which is comparable to the situation described in primates (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As previously shown in primates (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), in swine this protracted plasticity of the amygdala is associated to the presence of immature cells in the PL, which we identified as a subdivision located along the external border of BCA, rich in immature DCX\u0026thinsp;+\u0026thinsp;cells, most of which express NeuN (indicating a neuronal phenotype) and the transcription factor COUP-TFII / NR2F2, but not FOXP2 (which identified the intercalated amygdalar cells). In primates, it has been proposed that the PL could serve as a reservoir to provide new neurons for the basal (basolateral) and accessory (basomedial) nuclei of BCA (Chareyron et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Avino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As in primates, the swine PL could serve as a reservoir of immature neurons for the pallial amygdala. However, the PL seems to be a different nucleus of BCA, containing neurons with different maturation degree as suggested by the shell-like organization of DCX\u0026thinsp;+\u0026thinsp;cell clusters around non-DCX cells with mature morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, most immature cells of PL might continue residing within this nucleus upon maturation, although we cannot discard that a few migrate to other BCA nuclei.\u003c/p\u003e \u003cp\u003ePrevious studies indicated that the size of the PL reservoir correlates with the degree of cortical expansion, being larger in gyrencephalic than in lissencephalic brains (Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Species with a gyrencephalic brain also have a relatively large pallial amygdala, which is particularly evident in the BCA (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Piumatti et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Our results agree with this proposal, as the swine has a gyrencephalic brain, with large cortices and BCA relative to subpallial structures such as the striatum and central amygdala, and we also observed a well developed PL, including lateral and medial subdivisions (schemes in Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e), as described in primates (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In contrast, lissencephalic rodents as the mouse and rat, with smaller cortices and BCA, only have a rudimentary PL, which remained unnoticed or poorly described until recently (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Alderman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA major distinctive feature of the immature DCX\u0026thinsp;+\u0026thinsp;cells of PL is that they express COUP-TFII and several glutamatergic markers such as TBR1 and VGLUT2 (SCL17A6), but not GABAergic markers as SP8, GAD65 and GAD67 (Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Alderman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, deletion of COUP-TFII had a significant impact on excitatory populations (TBR1 and GLU2R) of the rodent BCA, while few effects were reported in inhibitory GAD67\u0026thinsp;+\u0026thinsp;interneurons (Tang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In our material in swine, we found that a few DCX\u0026thinsp;+\u0026thinsp;cells of PL express the transcription factor BRN2 (POU3F2), present in some subsets of immature glutamatergic cells (Dominguez et al. 2012; Brunjes and Osterberg, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), such as many of those of the swine postnatal olfactory structures (Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, most DCX\u0026thinsp;+\u0026thinsp;cells of swine PL did not express BRN2 (POU3F2), at least during juvenile postnatal ages, suggesting that at this age they express glutamatergic markers different to those found in immature neurons of olfactory structures.\u003c/p\u003e \u003cp\u003eWe observed that many DCX+/COUP-TFII\u0026thinsp;+\u0026thinsp;cells found in juvenile swine PL had an elongated, migratory-like morphology, which prompted us to investigate possible origin and migration routes by analyzing sections in different planes. We could follow the immature cells posteroventrally, until the vicinity of the temporal horn of the lateral ventricle, where we found a reservoir of DCX+/COUP-TFII\u0026thinsp;+\u0026thinsp;immature cells in a posterolateral inferior sector of the vz/svz, from where cells appeared to migrate radially to lateral PL and tangentially to medial PL (schemes in Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e,\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). However, this proposal would require confirmation using cell migration assays. The vz/svz associated to PL appears to relate to the lateralmost progenitor domain of the pallial amygdala, and it has been identified previously in the lateral part of the \u0026ldquo;inferior ganglionic eminence\u0026rdquo; (De Campo and Fudge, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which is not a subpallial domain as the name suggests, but it really represents the progenitor zone of the pallial amygdala (Garc\u0026iacute;a-Calero et al. 2020). Notably, many DCX\u0026thinsp;+\u0026thinsp;and COUP-TFII\u0026thinsp;+\u0026thinsp;cells in this vz/svz sector expressed Ki-67 in juvenile swine, indicating that new neurons for the PL could be produced postnatally, at least until juvenile stages. This would agree with previous findings in primates (Bernier et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), including humans (Sorrells et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Roeder et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and with our results of the swine olfactory areas (Freixes et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition, other DCX+/COUP-TFII\u0026thinsp;+\u0026thinsp;cells found in this vz/svz sector and the associated PL might represent quiescent immature neurons that were generated previously, as suggested in adult animals of different mammalian species (Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast to glutamatergic COUP-TFII neurons of PL that appear to be produced in the amygdalar pallium (as discussed above), GABAergic COUP-TFII cells are produced during development in the caudal ganglionic and part of the medial ganglionic eminences of the subpallium, and they constitute a source of interneurons that migrate tangentially to the cortex (Tang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Alderman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Remnants of GABAergic immature neurons continue to be produced postnatally from the vz/svz of the Arc, at least in human and non-human primate infants and in two-days-old piglets (Kim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This could be the source of the COUP-TFII+/SP8\u0026thinsp;+\u0026thinsp;cells found in the cerebral cortex and the piriform cortex. However, by using single-cell transcriptome of different rostrocaudal parts of the Arc, Kim et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) also identified that the vz/svz of the Arc gives rise to a temporo-ventral migratory stream of DCX\u0026thinsp;+\u0026thinsp;cells that express both COUP-TFII and TBR1, suggesting that cells of this stream are glutamatergic. Many of these cells appear to migrate to the temporal cortex, but some were also found in the piriform cortex (Kim et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It is unclear whether the latter temporo-ventral stream of DCX+/COUP-TFII+/TBR1\u0026thinsp;+\u0026thinsp;cells includes part of the PL-associated vz/svz cells. In our juvenile swine data, we found that the Arc does not express COUP-TFII, but we did not carry out a systematic analysis of all levels of Arc. More studies will be needed to clarify this issue.\u003c/p\u003e \u003cp\u003eCompared to that in other mammals, the density of DCX\u0026thinsp;+\u0026thinsp;immature cells in the pallial amygdala, concentrated in PL, is not only high in gyrencephalic human and non-human primates, but also in small lissencephalic primates as the marmoset (Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Thus, primates stand above other mammals regarding pallial amygdala plasticity, independent of the secondary loss of gyri as it appears to occur in marmosets (Kelava et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In other mammalian radiations, gyrencephalic brains have often been associated with high levels of non-neurogenic postnatal plasticity (Piumatti et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, this prolonged plasticity and the location of the immature cells in the brain need to be understood in the context of the ecological adaptations of each species: for example, the high postnatal plasticity in the olfactory areas of swine could be related to the relevance of olfactory cues for feeding and social interactions in these animals (Brunjes et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; also discussed by Freixes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Regarding the high postnatal plasticity found in the pallial amygdala, what could be the advantage of having a prolonged production of new neurons in this brain structure? In primates, the BCA is the part of the amygdala more broadly connected to the neocortex (Ghashghaei and Barbas, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ghashghaei et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pessoa, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Having a large reservoir of immature neurons in this nuclear complex may be required for achieving a more sophisticated regulation of emotional learning (Jin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; discussed by Sorrell et al. 2019), as well as a more plastic adaptation to ever-changing complex social contexts (Chareyron et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Avino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ghibaudi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, our data, together with other studies (Kostović et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), highlight the similarities between swine and humans in terms of brain anatomy and ontogeny, including a protracted development of the pallial amygdala. Since the incorporation of immature neurons in the human PL at juvenile ages seems to be dysfunctional in autism spectrum disorder (Avino et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), the swine emerges as a good animal model to further study postnatal plasticity in functional and dysfunctional contexts​.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eA: anterior\u003c/p\u003e\n\u003cp\u003eAA: anterior amygdalar area\u003c/p\u003e\n\u003cp\u003eABC:\u0026nbsp;avidin\u0026ndash;biotin complex\u003c/p\u003e\n\u003cp\u003eac: anterior commissure\u003c/p\u003e\n\u003cp\u003eACo: anterior cortical amygdalar area\u003c/p\u003e\n\u003cp\u003eAHi: amygdalohippocampal area\u003c/p\u003e\n\u003cp\u003eamc: amygdalar capsule\u003c/p\u003e\n\u003cp\u003eAO: anterior olfactory area\u003c/p\u003e\n\u003cp\u003eBCA: basal complex of the amygdala\u003c/p\u003e\n\u003cp\u003eBCL2: B cell lymphoma 2 protein\u003c/p\u003e\n\u003cp\u003eBL: basolateral nucleus of the basal complex of the amygdala\u003c/p\u003e\n\u003cp\u003eBLA: basolateral nucleus of the basal complex of the amygdala, anterior part\u003c/p\u003e\n\u003cp\u003eBLP: basolateral nucleus of the basal complex of the amygdala, posterior part\u003c/p\u003e\n\u003cp\u003eBM: basomedial nucleus of the basal complex of the amygdala\u003c/p\u003e\n\u003cp\u003eBrn2 (Pou3f2): POU class 3 homeobox 2\u003c/p\u003e\n\u003cp\u003eBSA:\u0026nbsp;bovine serum albumin\u003c/p\u003e\n\u003cp\u003eCA: cornu ammonis\u003c/p\u003e\n\u003cp\u003eCe: central nucleus of the amygdala\u003c/p\u003e\n\u003cp\u003eCl: claustrum\u003c/p\u003e\n\u003cp\u003eCn: caudate nucleus\u003c/p\u003e\n\u003cp\u003eCo: cortical amygdalar area\u003c/p\u003e\n\u003cp\u003eCOUP-TFII (NR2F2): Chicken Ovalbumin Upstream Promoter Transcription Factor II\u003c/p\u003e\n\u003cp\u003eCxA: cortex-amygdala transition zone\u003c/p\u003e\n\u003cp\u003eD: dorsal\u003c/p\u003e\n\u003cp\u003eDCX: doublecortin\u003c/p\u003e\n\u003cp\u003eDG: dentate gyrus\u003c/p\u003e\n\u003cp\u003eEC: entohrinal cortex\u003c/p\u003e\n\u003cp\u003eec: external capsule\u003c/p\u003e\n\u003cp\u003eEn: endopiriform nucleus\u003c/p\u003e\n\u003cp\u003efi: fimbria\u003c/p\u003e\n\u003cp\u003eFOXP2: Forkhead box P2\u003c/p\u003e\n\u003cp\u003efx: fornix\u003c/p\u003e\n\u003cp\u003eGP: globus pallidus\u003c/p\u003e\n\u003cp\u003eGt: goat\u003c/p\u003e\n\u003cp\u003eHF: hippocampal formation\u003c/p\u003e\n\u003cp\u003eHRP: Horseradish peroxidase conjugated secondary antibodies\u003c/p\u003e\n\u003cp\u003eic: internal capsule\u003c/p\u003e\n\u003cp\u003eICx: insular cortex\u003c/p\u003e\n\u003cp\u003eIF: immunofluorescence\u003c/p\u003e\n\u003cp\u003eIHC: immunohistochemistry\u003c/p\u003e\n\u003cp\u003eIM: intercalated amygdalar nucleus, main part\u003c/p\u003e\n\u003cp\u003eITC: intercalated cells of the amygdala\u003c/p\u003e\n\u003cp\u003eIV: intravenous\u003c/p\u003e\n\u003cp\u003eL: lateral\u003c/p\u003e\n\u003cp\u003eLa: lateral nucleus of the basal complex of the amygdala\u003c/p\u003e\n\u003cp\u003elot: lateral olfactory tract\u003c/p\u003e\n\u003cp\u003eLOT: nucleus of the lateral olfactory tract\u003c/p\u003e\n\u003cp\u003elv: lateral ventricle\u003c/p\u003e\n\u003cp\u003eMe: medial nucleus of the amygdala\u003c/p\u003e\n\u003cp\u003eMeA: medial nucleus of the amygdala, anterior part\u003c/p\u003e\n\u003cp\u003eMePD: medial nucleus of the amygdala, posterodorsal part\u003c/p\u003e\n\u003cp\u003eMs: mouse\u003c/p\u003e\n\u003cp\u003eNeuN: Neuronal nuclear protein (also knows as FOX3)\u003c/p\u003e\n\u003cp\u003eot: optic tract\u003c/p\u003e\n\u003cp\u003eP: posterior\u003c/p\u003e\n\u003cp\u003ePB: phosphate buffer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePB-Tx: phosphate buffer containing Triton-X 100\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePFA paraformaldehyde\u003c/p\u003e\n\u003cp\u003ePH: parahippocampal area\u003c/p\u003e\n\u003cp\u003ePir: piriform nucleus\u003c/p\u003e\n\u003cp\u003ePL: paralaminar nucleus\u003c/p\u003e\n\u003cp\u003ePL lat: paralaminar nucleus, lateral subdivision\u003c/p\u003e\n\u003cp\u003ePL med: paralaminar nucleus, medial subdivision\u003c/p\u003e\n\u003cp\u003ePLCo: posterolateral cortical amygdalar area\u003c/p\u003e\n\u003cp\u003ePMCo: posteromedial cortical amygdalar area\u003c/p\u003e\n\u003cp\u003ePSA-NCAM: polysialylated neural cell adhesion molecule\u003c/p\u003e\n\u003cp\u003ePTh: prethalamus\u003c/p\u003e\n\u003cp\u003ePu: putamen\u003c/p\u003e\n\u003cp\u003eRb: rabbit\u003c/p\u003e\n\u003cp\u003eRIPA:\u0026nbsp;radioimmunoprecipitation assay\u003c/p\u003e\n\u003cp\u003eRMS: rostral migratory stream\u003c/p\u003e\n\u003cp\u003eRt: reticular nucleus of the prethalamus\u003c/p\u003e\n\u003cp\u003eS: subiculum\u003c/p\u003e\n\u003cp\u003eSP8: Specificity protein 8 (SP8 transcription factor)\u003c/p\u003e\n\u003cp\u003est: stria terminalis\u003c/p\u003e\n\u003cp\u003eSVZ/svz: subventricular zone\u003c/p\u003e\n\u003cp\u003eTbr1: T-box brain transcription factor 1\u003c/p\u003e\n\u003cp\u003eTBST:\u0026nbsp;Tris-buffered saline containing Tween 20\u003c/p\u003e\n\u003cp\u003eTh: Thalamus\u003c/p\u003e\n\u003cp\u003etlv: temporal horn of the lateral ventricle\u003c/p\u003e\n\u003cp\u003eTu: olfactory tubercle\u003c/p\u003e\n\u003cp\u003eVGLUT2: vesicular glutamate transporter 2\u003c/p\u003e\n\u003cp\u003eVZ/vz: ventricular zone\u003c/p\u003e\n\u003cp\u003eWB : Western blot\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJF has a predoctoral FPU contract by the Spanish Council for Education, Professional Education and Sports (FPU22/03133). This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades and Agencia Estatal de Investigación, MICIU/AEI/10.13039/501100011033 and FEDER-EU (Grant no. PID2023-151927OB-I00 to LM and ED), and by the AGAUR/Generalitat de Catalunya (2021 SGR 01359 to LM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare that they have no financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is part of the Doctoral Thesis of JF, supervised by ED and LM. JF contributed to the experimental design, conducted immunohistochemical and immunofluorescence experiments, analyzed data, prepared the figures and wrote the first draft of the manuscript. LM and ED conceived and designed the study, supervised the project, secured funding, contributed to samples characterization, interpretation of results, and improving the manuscript. All authors approved the final manuscript and agreed with the results presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe most relevant data are included in the Figures of this article. Additional data is available upon request and agreement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the 3Rs principles (Russell and Burch, 1959). Brain samples were obtained from animals that were previously used for practicing and improving surgical procedures by M.D. surgeons of the Arnau de Vilanova University Hospital of Lleida. Since brains were extracted once animals were euthanized, Animal Research Ethics Committee of the IRBLleida confirmed that no ethical approval is required.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe deeply thank all Agencies that funded our research: the Spanish Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades and Agencia Estatal de Investigaci\u0026oacute;n, MICIU/AEI/10.13039/501100011033 and FEDER-EU (Grant no. PID2023-151927OB-I00 to LM and ED), and the AGAUR/Generalitat de Catalunya (2021 SGR 01359 to LM). J.F. has a predoctoral FPU (Formaci\u0026oacute;n de Profesorado Universitario) contract from the Spanish Council for Education, Professional Education and Sports (FPU22/03133). We thank Dr. Sara Hern\u0026aacute;ndez and Ms. Lidia Piedrafita for their help and advice with the Western blots. We also thank the technicians and other staff of the Department of Experimental Medicine, the Service of Microscopy of the University of Lleida and IRBLleida, as well as the veterinarians, technicians and other staff of the Center for Experimental Research in Applied Biomedicine (CREBA) of the Institute for Biomedical Research of Lleida (IRBLleida).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlderman PJ, Saxon D, Torrijos-Saiz LI, Sharief M, Page CE, Baroudi JK, Sorrells SF (2024) Delayed maturation and migration of excitatory neurons in the juvenile mouse paralaminar amygdala. 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J Neurosci 27(31):8286\u0026ndash;8296. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.0476-07.2007\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.0476-07.2007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"brain-structure-and-function","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bsaf","sideBox":"Learn more about [Brain Structure and Function](https://www.springer.com/journal/429)","snPcode":"429","submissionUrl":"https://submission.nature.com/new-submission/429/3","title":"Brain Structure and Function","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"postnatal neurogenesis, gyrencephalic brain, migratory streams, neuroplasticity, amygdalar pallium, paralaminar nucleus, intercalated amygdalar cells, pig","lastPublishedDoi":"10.21203/rs.3.rs-8544141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8544141/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to investigate the presence of immature cells and their phenotype in the pallial amygdala of juvenile swine brains by way of immunoreactivity for the microtubule-associated protein doublecortin (DCX), combined with the cell proliferation marker Ki-67 and different neuronal markers, including NeuN and the transcription factor COUP-TFII (NR2F2, critical for amygdalar development and adult phenotype maintenance). Our results showed the existence of numerous DCX\u0026thinsp;+\u0026thinsp;cells along the external border of the basal amygdalar complex, adjacent to the amygdalar capsule, in an area identified as the swine paralaminar nucleus. DCX\u0026thinsp;+\u0026thinsp;cells in this nucleus showed a patchy distribution, with shell-like clusters of DCX\u0026thinsp;+\u0026thinsp;cells partially surrounding islands of non-stained large cells of the basal amygdalar complex. Both, the paralaminar nucleus and the other nuclei of the basal amygdalar complex also contained abundant neurons expressing COUP-TFII (NR2F2), but not the transcription factor FOXP2, which defines the clusters of intercalated amygdalar cells. Paralaminar patches of DCX\u0026thinsp;+\u0026thinsp;cells were more abundant at posterior levels, where they were continuous with chains of DCX\u0026thinsp;+\u0026thinsp;cells with migratory-like morphology and other immature DCX\u0026thinsp;+\u0026thinsp;cells of the subventricular zone surrounding the temporal horn of the lateral ventricle (tlv). This part of the ventricular/subventricular zone (vz/svz) also expressed COUP-TFII / NR2F2, suggesting that this might be the source of the immature cells found in the paralaminar nucleus and other parts of the pallial amygdala. A major difference between this part of the vz/svz and the Arc (giving rise to DCX\u0026thinsp;+\u0026thinsp;cells of the rostral migratory stream and, apparently, the migratory cell chains seen in the external capsule and piriform cortex) is that the latter does not express COUP-TFII / NR2F2. Overall, these results show that the swine pallial amygdala shows a prolonged postnatal plasticity and contains a reservoir of immature neurons mainly located in the paralaminar nucleus and related vz/svz, resembling the situation seen in humans. These findings also point to the swine as an excellent model to study mechanisms behind postnatal plasticity in the amygdala of gyrencephalic animals, and the role of this protracted plasticity in amygdala function and dysfunction.\u003c/p\u003e","manuscriptTitle":"Postnatal plasticity in the paralaminar nucleus of the pallial amygdala in juvenile swine brain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 16:30:52","doi":"10.21203/rs.3.rs-8544141/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-30T17:14:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T14:28:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-21T10:54:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270046634782930194440874756955358350185","date":"2026-01-12T06:29:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12184720634669536592672237843439592158","date":"2026-01-09T14:57:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T14:09:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-08T12:23:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-08T08:09:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Brain Structure and Function","date":"2026-01-07T16:55:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"brain-structure-and-function","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bsaf","sideBox":"Learn more about [Brain Structure and Function](https://www.springer.com/journal/429)","snPcode":"429","submissionUrl":"https://submission.nature.com/new-submission/429/3","title":"Brain Structure and Function","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6e901268-8a92-4fac-9446-3367b01eb4f9","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:07:11+00:00","versionOfRecord":{"articleIdentity":"rs-8544141","link":"https://doi.org/10.1007/s00429-026-03106-8","journal":{"identity":"brain-structure-and-function","isVorOnly":false,"title":"Brain Structure and Function"},"publishedOn":"2026-04-06 15:57:21","publishedOnDateReadable":"April 6th, 2026"},"versionCreatedAt":"2026-01-13 16:30:52","video":"","vorDoi":"10.1007/s00429-026-03106-8","vorDoiUrl":"https://doi.org/10.1007/s00429-026-03106-8","workflowStages":[]},"version":"v1","identity":"rs-8544141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8544141","identity":"rs-8544141","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}