Cell-type-specific responses to associative learning in the primary motor cortex.

The primary motor cortex (M1) is known to be a critical site for movement initiation and motor learning. Surprisingly, it has also been shown to possess reward-related activity, presumably to facilitate reward-based learning of new movements. However, whether reward-related signals are represented among different cell types in M1, and whether their response properties change after cue-reward conditioning remains unclear. Here, we performed longitudinal in vivo two-photon Ca2+ imaging to monitor the activity of different neuronal cell types in M1 while mice engaged in a classical conditioning task. Our results demonstrate that most of the major neuronal cell types in M1 showed robust but differential responses to both the conditioned cue stimulus (CS) and reward, and their response properties undergo cell-type-specific modifications after associative learning. PV-INs' responses became more reliable to the CS, while VIP-INs' responses became more reliable to reward. Pyramidal neurons only showed robust responses to novel reward, and they habituated to it after associative learning. Lastly, SOM-INs' responses emerged and became more reliable to both the CS and reward after conditioning. These observations suggest that cue- and reward-related signals are preferentially represented among different neuronal cell types in M1, and the distinct modifications they undergo during associative learning could be essential in triggering different aspects of local circuit reorganization in M1 during reward-based motor skill learning.

Untangling the cortico-thalamo-cortical loop: cellular pieces of a knotty circuit puzzle.

Functions of the neocortex depend on its bidirectional communication with the thalamus, via cortico-thalamo-cortical (CTC) loops. Recent work dissecting the synaptic connectivity in these loops is generating a clearer picture of their cellular organization. Here, we review findings across sensory, motor and cognitive areas, focusing on patterns of cell type-specific synaptic connections between the major types of cortical and thalamic neurons. We outline simple and complex CTC loops, and note features of these loops that appear to be general versus specialized. CTC loops are tightly interlinked with local cortical and corticocortical (CC) circuits, forming extended chains of loops that are probably critical for communication across hierarchically organized cerebral networks. Such CTC-CC loop chains appear to constitute a modular unit of organization, serving as scaffolding for area-specific structural and functional modifications. Inhibitory neurons and circuits are embedded throughout CTC loops, shaping the flow of excitation. We consider recent findings in the context of established CTC and CC circuit models, and highlight current efforts to pinpoint cell type-specific mechanisms in CTC loops involved in consciousness and perception. As pieces of the connectivity puzzle fall increasingly into place, this knowledge can guide further efforts to understand structure-function relationships in CTC loops.

The localization and expression of gonadotropin inhibitory hormone in the hypothalamus of turkey hens during the prepubertal, pubertal and postpubertal phases.

Gonadotropin inhibitory hormone (GnIH), initially discovered in birds as a hypothalamic neuropeptide, inhibits the synthesis and release of gonadotropins by affecting GnRH neurons and gonadotropes. Therefore, it may be a key neuropeptide in reproduction in birds. The aim of the present study was to investigate the prepubertal, pubertal, and postpubertal localization of GnIH and changes in hypothalamic GnIH expression in British United Turkey hens. In prepubertal, pubertal, and postpubertal periods, the brains of turkey hens (n = 15) were removed after fixation. Sections (30 µm) were prepared from the entire hypothalamus and stained immunohistochemically against GnIH antibody. Gonadotropin inhibitory hormone-immunoreactive neurons were observed in the paraventricular nucleus. These neurons were significantly more abundant in the prepubertal turkeys than pubertal and postpubertal turkeys (P < 0.05). The results suggested that GnIH neurons have an important role in regulating the pubertal events in British United Turkey hens.

Phenotypic variation of transcriptomic cell types in mouse motor cortex.

Cortical neurons exhibit extreme diversity in gene expression as well as in morphological and electrophysiological properties1,2. Most existing neural taxonomies are based on either transcriptomic3,4 or morpho-electric5,6 criteria, as it has been technically challenging to study both aspects of neuronal diversity in the same set of cells7. Here we used Patch-seq8 to combine patch-clamp recording, biocytin staining, and single-cell RNA sequencing of more than 1,300 neurons in adult mouse primary motor cortex, providing a morpho-electric annotation of almost all transcriptomically defined neural cell types. We found that, although broad families of transcriptomic types (those expressing Vip, Pvalb, Sst and so on) had distinct and essentially non-overlapping morpho-electric phenotypes, individual transcriptomic types within the same family were not well separated in the morpho-electric space. Instead, there was a continuum of variability in morphology and electrophysiology, with neighbouring transcriptomic cell types showing similar morpho-electric features, often without clear boundaries between them. Our results suggest that neuronal types in the neocortex do not always form discrete entities. Instead, neurons form a hierarchy that consists of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior.

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms-SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)-and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Specific subpopulations of hypothalamic leptin receptor-expressing neurons mediate the effects of early developmental leptin receptor deletion on energy balance.

OBJECTIVE: To date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance. METHODS: We generated new mouse lines deleted for LepRb in ARC GhrhCre neurons or in Htr2cCre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells. RESULTS: The early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance. CONCLUSIONS: Although early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH.

Thalamo-cortical projections to the macaque superior parietal lobule areas PEc and PE.

The exposed surface of the superior parietal lobule in macaque brain contains two architectonically defined areas named PEc and PE. The aim of the present study is the characterization of thalamic afferents of these two areas. For this purpose, retrograde neuronal tracers were injected, or placed in crystal form, in areas PEc and PE. We found that the two areas show a similar pattern of thalamic inputs, mainly originating from Lateral Posterior (LP), Pulvinar (Pul), Ventral Posterior Lateral (VPL), and Ventral Lateral (VL) nuclei, all structures known to be involved in visual, somatosensory, and/or sensorimotor processing. Minor afferents were observed from the Centromedian/Parafascicular complex (CM/PF), Central Lateral (CL), Ventral Anterior (VA), and Medial Dorsal (MD) nuclei. LP and VL were more strongly connected to PEc than to PE, while the other main thalamic inputs to the two areas showed slight differences in strength. The part of the Pul mostly connected with areas PEc and PE was the Medial Pul. No labeled cells were found in the retinotopically organized Lateral and Inferior Pul. In the somatotopically organized VPL and VL nuclei, labeled neurons were mainly found in regions likely to correspond to the trunk and limb representations (in particular the legs). These findings are in line with the sensory-motor nature of areas PEc and PE, and with their putative functional roles, being them suggested to be involved in the preparation and control of limb interaction with the environment, and in locomotion.

Infrabarrels Are Layer 6 Circuit Modules in the Barrel Cortex that Link Long-Range Inputs and Outputs.

The rodent somatosensory cortex includes well-defined examples of cortical columns-the barrel columns-that extend throughout the cortical depth and are defined by discrete clusters of neurons in layer 4 (L4) called barrels. Using the cell-type-specific Ntsr1-Cre mouse line, we found that L6 contains infrabarrels, readily identifiable units that align with the L4 barrels. Corticothalamic (CT) neurons and their local axons cluster within the infrabarrels, whereas corticocortical (CC) neurons are densest between infrabarrels. Optogenetic experiments showed that CC cells received robust input from somatosensory thalamic nuclei, whereas CT cells received much weaker thalamic inputs. We also found that CT neurons are intrinsically less excitable, revealing that both synaptic and intrinsic mechanisms contribute to the low firing rates of CT neurons often reported in vivo. In summary, infrabarrels are discrete cortical circuit modules containing two partially separated excitatory networks that link long-distance thalamic inputs with specific outputs.

Trace Conditioning in Drosophila Induces Associative Plasticity in Mushroom Body Kenyon Cells and Dopaminergic Neurons.

Dopaminergic neurons (DANs) signal punishment and reward during associative learning. In mammals, DANs show associative plasticity that correlates with the discrepancy between predicted and actual reinforcement (prediction error) during classical conditioning. Also in insects, such as Drosophila, DANs show associative plasticity that is, however, less understood. Here, we study associative plasticity in DANs and their synaptic partners, the Kenyon cells (KCs) in the mushroom bodies (MBs), while training Drosophila to associate an odorant with a temporally separated electric shock (trace conditioning). In most MB compartments DANs strengthened their responses to the conditioned odorant relative to untrained animals. This response plasticity preserved the initial degree of similarity between the odorant- and the shock-induced spatial response patterns, which decreased in untrained animals. Contrary to DANs, KCs (α'/ß'-type) decreased their responses to the conditioned odorant relative to untrained animals. We found no evidence for prediction error coding by DANs during conditioning. Rather, our data supports the hypothesis that DAN plasticity encodes conditioning-induced changes in the odorant's predictive power.

Heterogeneity of hypothalamic pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing.

OBJECTIVE: Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight. Often characterized simply as direct targets for leptin, recent data suggest a more complex architecture. METHODS: Using single cell RNA sequencing, we have generated an atlas of gene expression in murine POMC neurons. RESULTS: Of 163 neurons, 118 expressed high levels of Pomc with little/no Agrp expression and were considered "canonical" POMC neurons (P+). The other 45/163 expressed low levels of Pomc and high levels of Agrp (A+P+). Unbiased clustering analysis of P+ neurons revealed four different classes, each with distinct cell surface receptor gene expression profiles. Further, only 12% (14/118) of P+ neurons expressed the leptin receptor (Lepr) compared with 58% (26/45) of A+P+ neurons. In contrast, the insulin receptor (Insr) was expressed at similar frequency on P+ and A+P+ neurons (64% and 55%, respectively). CONCLUSION: These data reveal arcuate POMC neurons to be a highly heterogeneous population. Accession Numbers: GSE92707.

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations.

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population. The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing "Golgi-like" complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills. Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

Distribution of hypothalamic vasoactive intestinal peptide immunoreactive neurons in the male native Thai chicken.

Avian prolactin (PRL) secretion is under stimulatory control by the PRL-releasing factor (PRF), vasoactive intestinal peptide (VIP). The neuroendocrine regulation of the avian reproductive system has been extensively studied in females. However, there are limited data in males. The aim of this study was to elucidate the VIPergic system and its relationship to PRL and testosterone (T) in the male native Thai chicken. The distributions of VIP-immunoreactive (-ir) neurons and fibers were determined by immunohistochemistry. Changes in VIP-ir neurons within the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) areas were compared across the reproductive stages. Plasma levels of PRL and T were determined by enzyme-linked immunosorbent assay and then compared across the reproductive stages. The results revealed that the highest accumulations of VIP-ir neurons were concentrated only within the IH-IN, and VIP-ir neurons were not detected within other hypothalamic nuclei. Within the IH-IN, VIP-ir neurons were low in premature and aging males and markedly increased in mature males. Changes in VIP-ir neurons within the IH-IN were directly mirrored with changes in PRL and T levels across the reproductive stages. These results suggested that VIP neurons in the IH-IN play a regulatory role in year-round reproductive activity in males. The present study also provides additional evidence that VIP is the PRF in non-seasonal, continuously breeding equatorial species.

Awake dynamics and brain-wide direct inputs of hypothalamic MCH and orexin networks.

The lateral hypothalamus (LH) controls energy balance. LH melanin-concentrating-hormone (MCH) and orexin/hypocretin (OH) neurons mediate energy accumulation and expenditure, respectively. MCH cells promote memory and appropriate stimulus-reward associations; their inactivation disrupts energy-optimal behaviour and causes weight loss. However, MCH cell dynamics during wakefulness are unknown, leaving it unclear if they differentially participate in brain activity during sensory processing. By fiberoptic recordings from molecularly defined populations of LH neurons in awake freely moving mice, we show that MCH neurons generate conditional population bursts. This MCH cell activity correlates with novelty exploration, is inhibited by stress and is inversely predicted by OH cell activity. Furthermore, we obtain brain-wide maps of monosynaptic inputs to MCH and OH cells, and demonstrate optogenetically that VGAT neurons in the amygdala and bed nucleus of stria terminalis inhibit MCH cells. These data reveal cell-type-specific LH dynamics during sensory integration, and identify direct neural controllers of MCH neurons.

Thalamocortical Connections Drive Intracortical Activation of Functional Columns in the Mislaminated Reeler Somatosensory Cortex.

Neuronal wiring is key to proper neural information processing. Tactile information from the rodent's whiskers reaches the cortex via distinct anatomical pathways. The lemniscal pathway relays whisking and touch information from the ventral posteromedial thalamic nucleus to layer IV of the primary somatosensory "barrel" cortex. The disorganized neocortex of the reeler mouse is a model system that should severely compromise the ingrowth of thalamocortical axons (TCAs) into the cortex. Moreover, it could disrupt intracortical wiring. We found that neuronal intermingling within the reeler barrel cortex substantially exceeded previous descriptions, leading to the loss of layers. However, viral tracing revealed that TCAs still specifically targeted transgenically labeled spiny layer IV neurons. Slice electrophysiology and optogenetics proved that these connections represent functional synapses. In addition, we assessed intracortical activation via immediate-early-gene expression resulting from a behavioral exploration task. The cellular composition of activated neuronal ensembles suggests extensive similarities in intracolumnar information processing in the wild-type and reeler brains. We conclude that extensive ectopic positioning of neuronal partners can be compensated for by cell-autonomous mechanisms that allow for the establishment of proper connectivity. Thus, genetic neuronal fate seems to be of greater importance for correct cortical wiring than radial neuronal position.

Distribution of histaminergic neuronal cluster in the rat and mouse hypothalamus.

Histidine decarboxylase (HDC) catalyzes the biosynthesis of histamine from L-histidine and is expressed throughout the mammalian nervous system by histaminergic neurons. Histaminergic neurons arise in the posterior mesencephalon during the early embryonic period and gradually develop into two histaminergic substreams around the lateral area of the posterior hypothalamus and the more anterior peri-cerebral aqueduct area before finally forming an adult-like pattern comprising five neuronal clusters, E1, E2, E3, E4, and E5, at the postnatal stage. This distribution of histaminergic neuronal clusters in the rat hypothalamus appears to be a consequence of neuronal development and reflects the functional differentiation within each neuronal cluster. However, the close linkage between the locations of histaminergic neuronal clusters and their physiological functions has yet to be fully elucidated because of the sparse information regarding the location and orientation of each histaminergic neuronal clusters in the hypothalamus of rats and mice. To clarify the distribution of the five-histaminergic neuronal clusters more clearly, we performed an immunohistochemical study using the anti-HDC antibody on serial sections of the rat hypothalamus according to the brain maps of rat and mouse. Our results confirmed that the HDC-immunoreactive (HDCi) neuronal clusters in the hypothalamus of rats and mice are observed in the ventrolateral part of the most posterior hypothalamus (E1), ventrolateral part of the posterior hypothalamus (E2), ventromedial part from the medial to the posterior hypothalamus (E3), periventricular part from the anterior to the medial hypothalamus (E4), and diffusely extended part of the more dorsal and almost entire hypothalamus (E5). The stereological estimation of the total number of HDCi neurons of each clusters revealed the larger amount of the rat than the mouse. The characterization of histaminergic neuronal clusters in the hypothalamus of rats and mice may provide useful information for further investigations.

[Neurotology and cochlear implants]. / Neurotologia e implantacion coclear.

In this review we analyse cochlear implantation in terms of the fundamental aspects of the functioning of the auditory system. Concepts concerning neuronal plasticity applied to electrical stimulation in perinatal and adult deep hypoacusis are reviewed, and the latest scientific bases that justify early implantation following screening for congenital deafness are discussed. Finally, this review aims to serve as an example of the importance of fostering the sub-specialty of neurotology in our milieu, with the aim of bridging some of the gaps between specialties and thus improving both the knowledge in the field of research on auditory pathologies and in the screening of patients. The objectives of this review, targeted above all towards specialists in the field of otorhinolaryngology, are to analyse some significant neurological foundations in order to reach a better understanding of the clinical events that condition the indications and the rehabilitation of patients with cochlear implants, as well as to use this means to foster the growth of the sub-specialty of neurotology.

TITLE: Neurotologia e implantacion coclear.En esta revision se analiza la implantacion coclear en relacion con los fundamentos del funcionamiento del sistema auditivo. Se revisan los conceptos sobre plasticidad neuronal aplicados a la estimulacion electrica en la hipoacusia profunda perinatal y del adulto, y se comentan las bases cientificas actuales que justifican la implantacion precoz tras el cribado de la sordera congenita. Finalmente, se propone esta revision como un ejemplo de la importancia de fomentar la subespecialidad de neurotologia en nuestro medio, a fin de tender puentes entre especialidades que mejoren tanto el conocimiento en el campo de la investigacion de la patologia auditiva como en el cuidado de los pacientes. Los objetivos de esta revision, dirigida sobre todo a especialistas del campo de la otorrinolaringologia, son los de analizar algunos fundamentos neurologicos de relevancia para comprender mejor los eventos clinicos que condicionan las indicaciones y la rehabilitacion de los pacientes con implantes cocleares, asi como estimular por este medio la potenciacion de la subespecialidad en neurotologia.

Hypothalamic Agrp neurons drive stereotypic behaviors beyond feeding.

The nervous system evolved to coordinate flexible goal-directed behaviors by integrating interoceptive and sensory information. Hypothalamic Agrp neurons are known to be crucial for feeding behavior. Here, however, we show that these neurons also orchestrate other complex behaviors in adult mice. Activation of Agrp neurons in the absence of food triggers foraging and repetitive behaviors, which are reverted by food consumption. These stereotypic behaviors that are triggered by Agrp neurons are coupled with decreased anxiety. NPY5 receptor signaling is necessary to mediate the repetitive behaviors after Agrp neuron activation while having minor effects on feeding. Thus, we have unmasked a functional role for Agrp neurons in controlling repetitive behaviors mediated, at least in part, by neuropeptidergic signaling. The findings reveal a new set of behaviors coupled to the energy homeostasis circuit and suggest potential therapeutic avenues for diseases with stereotypic behaviors.

Immunohistochemical study on the neuronal diversity and three-dimensional organization of the mouse entopeduncular nucleus.

The entopeduncular nucleus (EPN) is one of the major output nuclei of the basal ganglia in rodents. Previous studies have divided it into rostral and caudal halves, with the former containing somatostatin (SOM)-immunoreactive neurons and the latter dominated by parvalbumin (PV)-containing neurons, respectively. However, it is unclear whether this simple rostrocaudal segmentation is appropriate, and the possibility of the existence of other neuronal populations remains to be investigated. In this study the cytoarchitecture of the mouse EPN was analyzed immunohistochemically. Substance P (SP)-immunoreactivity determined the extent of the EPN, which was 800 µm-long along the rostrocaudal axis. PV-positive neurons were concentrated in the caudal two-thirds of this range. PV-negative neurons were abundant in the rostral half but were further located caudally around the PV neuron-rich core. PV(+)/SOM(-) and PV(-)/SOM(+) neurons constituted 28.6% and 45.7% of EPN neurons, respectively, whereas the remaining population (25.7%) exhibited neither immunoreactivity. Eleven percent of EPN neurons lacked immunoreactivity for glutamic acid decarboxylase, indicating their non-GABAergic nature. Three-dimensional reconstruction revealed that PV-rich/SP-poor core was surrounded by PV-poor/SP-rich shell region. Therefore, presumptive thalamus-targeting PV neurons are outnumbered by other populations, and the regional heterogeneity shown here might be related to functionally distinct pathways through the basal ganglia.