Distribution and intrinsic membrane properties of basal forebrain GABAergic and parvalbumin neurons in the mouse

The basal forebrain (BF) strongly regulates cortical activation, sleep homeostasis, and attention. Many BF neurons involved in these processes are GABAergic, including a subpopulation of projection neurons containing the calcium‐binding protein, parvalbumin (PV). However, technical difficulties in identification have prevented a precise mapping of the distribution of GABAergic and GABA/PV+ neurons in the mouse or a determination of their intrinsic membrane properties. Here we used mice expressing fluorescent proteins in GABAergic (GAD67‐GFP knock‐in mice) or PV+ neurons (PV‐Tomato mice) to study these neurons. Immunohistochemical staining for GABA in GAD67‐GFP mice confirmed that GFP selectively labeled BF GABAergic neurons. GFP+ neurons and fibers were distributed throughout the BF, with the highest density in the magnocellular preoptic area (MCPO). Immunohistochemistry for PV indicated that the majority of PV+ neurons in the BF were large (>20 μm) or medium‐sized (15–20 μm) GFP+ neurons. Most medium and large‐sized BF GFP+ neurons, including those retrogradely labeled from the neocortex, were fast‐firing and spontaneously active in vitro. They exhibited prominent hyperpolarization‐activated inward currents and subthreshold “spikelets,” suggestive of electrical coupling. PV+ neurons recorded in PV‐Tomato mice had similar properties but had significantly narrower action potentials and a higher maximal firing frequency. Another population of smaller GFP+ neurons had properties similar to striatal projection neurons. The fast firing and electrical coupling of BF GABA/PV+ neurons, together with their projections to cortical interneurons and the thalamic reticular nucleus, suggest a strong and synchronous control of the neocortical fast rhythms typical of wakefulness and REM sleep. J. Comp. Neurol., 521:1225–1250, 2013. © 2012 Wiley Periodicals, Inc.     

Somatostatin- and Neuropeptide Y-immunoreactive neurons in the neocortex in senile dementia of alzheimer's type

Morphological changes in neocortical somatostatin- and neuropeptide-Y-immunoreactive cells in senile dementia of the Alzheimer type (SDAT) were studied using light-microscopic immunohistochemical methods. The density of somatostatin-immunoreactive cells in the neocortex did not decrease in cases of SDAT compared with aged normal subjects. However, many somatostatin-positive fibers were abnormally swollen and bulbous in shape and they were often observed within senile plaques. The morphology of these swollen and bulbous fibers was similar to that of the swollen neurites present in senile plaques demonstrated by the silver-impregnation method. Similar fiber abnormalities were observed in sections stained with antibodies to neuropeptide Y. Somatostatin-positive cells in aged normal subjects were distributed from layer II through to the subcortical white matter. These cells were multipolar, bitufted, or pyramidal in shape, with the majority of cells being of the multipolar type. Neuropeptide Y-positive cells also were distributed from layer II through to the subcortical white matter. Most of these cells were multipolar, but a few bipolar cells were also observed. We suggest that a primary degenerative process might begin at the fiber terminals of the somatostatin neuronal system in the neocortex in SDAT.     

Preprodynorphin‐expressing neurons constitute a large subgroup of somatostatin‐expressing GABAergic interneurons in the mouse neocortex

Dynorphins, leumorphin, and neoendorphins are preprodynorphin (PPD)‐derived peptides and ligands for κ‐opioid receptors. Using an antibody to PPD C‐terminal, we investigated the chemical and molecular characteristics of PPD‐expressing neurons in mouse neocortex. PPD‐immunopositive neuronal somata were distributed most frequently in layer 5 and less frequently in layers 2–4 and 6 throughout neocortical regions. Combined labeling of immunofluorescence and fluorescent mRNA signals revealed that almost all PPD‐immunopositive neurons expressed glutamic acid decarboxylase but not vesicular glutamate transporter, indicating their γ‐aminobutyric acid (GABA)ergic characteristics, and that PPD‐immunopositive neurons accounted for 15% of GABAergic interneurons in the primary somatosensory area. As GABAergic interneurons were divided into several groups by specific markers, we further examined the chemical characteristics of PPD‐expressing neurons by the double immunofluorescence labeling method. More than 95% of PPD‐immunopositive neurons were also somatostatin (SOM)‐immunopositive in the primary somatosensory, primary motor, orbitofrontal, and primary visual areas, but only 24% were SOM‐immunopositive in the medial prefrontal cortex. In the primary somatosensory area, PPD‐immunopositive neurons constituted 50%, 79%, 55%, and 17% of SOM‐immunopositive neurons in layers 2–3, 4, 5, and 6, respectively. Although SOM‐expressing neurons contained calretinin‐, neuropeptide Y‐, nitric oxide synthase‐, and reelin‐expressing neurons as subgroups, only reelin immunoreactivity was detected in many PPD‐immunopositive neurons. These results indicate that PPD‐expressing neurons constitute a large subgroup of SOM‐expressing cortical interneurons, and the PPD/SOM‐expressing GABAergic neurons might serve not only as inhibitory elements in the local cortical circuit, but also as modulators for cortical neurons expressing κ‐opioid and/or SOM receptors. J. Comp. Neurol. 522:1506–1526, 2014. © 2013 Wiley Periodicals, Inc.     

Chronic stress reduces the number of GABAergic interneurons in the adult rat hippocampus,dorsal‐ventral and region‐specific differences

Major depressive disorder is a common and complex mental disorder with unknown etiology. GABAergic dysfunction is likely to contribute to the pathophysiology since disrupted GABAergic systems are well documented in depressed patients. Here we studied structural changes in the hippocampal GABAergic network using the chronic mild stress (CMS) model, as one of the best validated animal models for depression. Rats were subjected to 9 weeks of daily stress and behaviorally characterized using the sucrose consumption test into anhedonic and resilient animals based on their response to stress. Different subtypes of GABAergic interneurons were visualized by immunohistochemistry using antibodies for parvalbumin (PV), calretinin (CR), calbindin (CB), cholecystokinin (CCK), somatostatin (SOM), and neuropeptide Y (NPY). We used an unbiased quantification method to systematically count labeled cells in different subareas of the dorsal and ventral hippocampus. Chronic stress reduced the number of specific interneurons in distinct hippocampal subregions significantly. PV+ and CR+ neurons were reduced in all dorsal subareas, whereas in the ventral part only the CA1 was affected. Stress had the most pronounced effect on the NPY+ and SOM+ cells and reduced their number in almost all dorsal and ventral subareas. Stress had no effect on the CCK+ and CB+ interneurons. In most cases the effect of stress was irrespective to the behavioral phenotype. However, in a few specific areas the number of SOM+, NPY+, and CR+ neurons were significantly reduced in anhedonic animals compared to the resilient group. Overall, these data clearly demonstrate that chronic stress affects the structural integrity of specific GABAergic neuronal subpopulations and this should also affect the functioning of these hippocampal GABAergic networks. © 2014 Wiley Periodicals, Inc.     

Innervation of the serotonin-immunoreactive cells distributed in the wall of the common carotid artery and its branches in the chicken

In the chicken, serotonin-immunoreactive cells were widely distributed not only in the carotid body but also in the wall of the common carotid artery and around each artery arising from the common carotid artery. Almost all of the serotonin cells in the wall of the common carotid artery were intensely immunoreactive to the neuropeptide Y, met- and leu-enkephalin antisera, whereas in the carotid body only a few cells were immunoreactive to these antisera. Innervation of the serotonin cells in and around arteries of chickens was investigated by immunohistochemistry and electron microscopy, in comparison with that of the carotid body. The serotonin cell groups in and around arteries, as well as the carotid body, received numerous peptidergic nerve fibers. Calcitonin gene-related peptide (CGRP)- and substance P-immunoreactive varicose nerve fibers were densely distributed, and somatostatin-immunoreactive fibers were moderately distributed in the serotonin cell groups. Galanin- and vasoactive intestinal peptide (VIP)-immunoreactive fibers were sparsely distributed in the cell groups. By electron microscopy, the serotonin cells in and around arteries were characterized by the presence of numerous dense-cored vesicles, 70-220 nm in diameter. The granule-containing cells were in close association with numerous axons. Naked axons regarded axon terminals were frequently apposed on the granular cells. The axon terminals were usually long and often partly invested the granular cells. Numerous synaptic junctions were detected along the contact between the granular cells and axon terminals. Most of the synaptic junctions showed afferent morphology; the secretory granules were accumulated near and attached to the asymmetrical membrane thickenings. Thus, the serotonin cells in and around arteries, like the carotid body, constitute chemoreceptive tissue.     

Fetal cell microchimerism in the maternal mouse spinal cord

Fetal cell microchimerism refers to the persistence of fetal cells in the maternal tissues following pregnancy. It has been detected in peripheral organs and the brain, but its existence in the spinal cord has not been reported. Our aim was to detect fetal cell microchimerism in the spinal cord of maternal mice. C57BL/6 female mice were crossed with GFP transgenic male mice and sacrificed after their first or third delivery. GFP-positive cells, which were presumably from fetuses whose fathers were GFP transgenic, were detected in the spinal cord by fluorescence microscopy and immunohistochemistry. PCR was also performed to detect GFP DNA, which must come from GFP hemizygous fetuses. We found GFP-positive cells and detectable GFP DNA in most of the maternal spinal cords. Twenty percent (1/5) of the mice that were only pregnant once had detectable fetal cells, while 80% (4/5) of those that were pregnant three times had detectable fetal cells. Some fetal cells, which not only emitted green fluorescence but also expressed NeuN, were detected in the spinal cords from maternal mice. These results indicate that fetal cells migrate into the spinal cord of a maternal mouse during and/or after the gestational period, and the fetal cells may differentiate into neurons in the spinal cord.     

Neuronal expression of the proteolipid protein gene in the medulla of the mouse

The proteolipid protein (PLP) gene (Plp) encodes the major myelin proteins, PLP and DM20. Expression of Plp occurs predominantly in oligodendrocytes, but evidence is accumulating that this gene is also expressed in neurons. In earlier studies, we demonstrated that myelin‐deficient (MD) rats, which carry a mutation in the Plp gene, exhibit lethal hypoxic ventilatory depression. Furthermore, we found that, in the MD rat, PLP accumulated in neuronal cell bodies in the medulla oblongata. In the current study, we sought to determine which neurons expressed the Plp gene in the medulla oblongata and whether Plp gene expression changed in neurons with maturation. A transgenic mouse expressing the Plp promoter driving expression of enhanced green fluorescent protein (Plp‐EGFP) was used to identify neurons expressing this gene. Plp expression in neurons was confirmed by immunostaining EGFP‐positive cells for NeuN and by in situ hybridization for PLP mRNA. The numbers of neurons expressing Plp‐EGFP and their distribution increased between P5 and P10 in the medulla. Immunostaining for surface receptors and classes of neurons expressing Plp‐EGFP revealed that Plp gene expression in brainstem neurons was restricted to neurons expressing specific ligand‐gated channels and biosynthetic enzymes, including glutamatergic NMDA receptors, GABA_A receptors, and ChAT in defined areas of the medulla. Plp gene expression was rarely found in interneurons expressing GABA and was never found in AMPA receptor‐ or tyrosine hydroxylase‐expressing neurons. Thus, Plp expression in the mouse caudal medulla was found to be developmentally regulated and restricted to specific groups of neurons. © 2009 Wiley‐Liss, Inc.     

神经元型一氧化氮合酶在血管性痴呆大鼠海马中的表达

目的 探讨神经元型一氧化氮合酶(nNOS)在血管性痴呆(VD)大鼠海马中的表达。方法 将60只大鼠随机分为：对照组、VD12h组、VD1d组、VD3d组、VD7d组。采用反复夹闭双侧颈总动脉方法建立VD大鼠模型，用HE染色观察各组大鼠海马CA1区神经元的数目；应用免疫组化染色和Western印迹方法检测nNOS在大鼠海马中的表达。结果 VD12h组、VD1d组、VD3d组、VD7d组大鼠海马CA1区神经元数均明显下降。nNOS在对照组大鼠海马CA1区中弱表达．在VD12h组表达增强．VD1d组进一步增强，VD3d和7d组表达逐渐减弱。结论 nNOS可能参与缺血早期海马神经元的损害，是VD的发病机制之一。     

GABAergic nonpyramidal neurons in intracerebral transplants of the rat hippocampus and fascia dentata: a combined light and electron microscopic immunocytochemical study

Glutamate decarboxylase (GAD) immunocytochemistry was used to study GABAergic neurons and synapses in intracerebral allografts of the rat hippocampus and fascia dentata. Tissue blocks of regio inferior of Ammon's horn (hippocampal field CA3) or of the fascia dentata were taken from newborn rats and transplanted to the hippocampal region of young adult rats. After 6 1/2 months' survival the recipient brains were fixed by perfusion and serially sectioned on a Vibratome. Sections containing the transplant and/or the host hippocampal region were immunostained for GAD and flat-embedded in Araldite for a correlated light and electron microscopic analysis. Immunostained neurons and terminals in the transplants were compared to immunoreactive elements in the hippocampus and fascia dentata of the hosts and other, normal rats. As in the hippocampal formation in situ, GAD-immunoreactive neurons and terminals in the transplants were observed in all layers. In dentate transplants a preponderance of immunostained cells was found just beneath the granule cell layer. In both hippocampal and dentate transplants, immunoreactive terminals were most abundant in the cell layers where they formed characteristic pericellular baskets around the pyramidal and granule cell bodies. In the electron microscope, the transplant GAD-immunoreactive neurons exhibited numerous cytoplasmic organelles, deeply infolded nuclei, and nuclear rods. Immunoreactive terminals formed symmetric synaptic contacts on the cell bodies, dendritic shafts, and spines of transplant pyramidal cells, granule cells, and hilar neurons. These are normal characteristics of GAD-immunoreactive neurons and terminals as also observed in the hippocampus of the host rats and the normal controls. Our results demonstrate that GABAergic neurons survive transplantation and develop a cell-specific morphology that includes the axonal projections.     

大鼠脊髓背角浅层神经元中神经肽Y Y1受体与生长抑素共存

用间接免疫荧光双标记方法,研究大鼠脊髓Ⅱ层神经元中神经肽Ｙ Ｙ１受体与生长抑素共存关系。结果显示在Ⅱ层深部许多神经元含Ｙ１受体－免疫反应性物质,只观察到少量的生长抑素免疫反应阳性神经元胞体,其中个别Ｙ１受体阳性神经元也含生长抑素免疫反应性物质;在局部使用秋水仙素后,大多数Ｙ１受体免疫反应阳性神经元含有生长抑素免疫反应性物质。推论脊髓背角Ⅱ层神经元中的Ｙ１受体可能与生长抑素在脊髓水平的功能有关。     

Morphometric multivariate analysis of GABAergic neurons containing calretinin and neuronal nitric oxide synthase in the mouse hippocampus

Several studies reported the morphology of calretinin-positive (CR+) neurons and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeled or neuronal nitric oxide synthase-positive (nNOS+) neurons in the rodent hippocampus, where these neurons showed similar morphological features. In addition, a previous study reported the frequent colocalization of CR and NADPH-d in the rat hippocampus. In this study, we aimed to examine whether CR+ neurons and nNOS+ neurons belong to a same morphological subpopulation of GABAergic neurons in the mouse hippocampus. Neurons were immunocytochemically classified into three groups, i.e., CR+/nNOS−, CR−/nNOS+ and CR+/nNOS+ groups. The present morphometric analysis was performed in the mouse Ammon’s horn, because CR+/nNOS+ neurons were rarely found in the mouse dentate gyrus. We selected three morphometric parameters, i.e., soma area, soma form factor (FF) and number of primary dendrites. Dunnett’s post-hoc analysis revealed that soma area, soma FF and number of primary dendrites were significantly larger in CR−/nNOS+ group than in CR+/nNOS− and CR+/nNOS+ groups. The morphometric data of CR+/nNOS+ group were quite similar to those of CR+/nNOS− group. The morphometric multivariate logistic regression analysis revealed that these three morphometric parameters were independent significant variables to discriminate between CR+/nNOS− and CR−/nNOS+ groups, and the majority of CR+/nNOS− and CR−/nNOS+ groups were correctly classified from the morphometric features. The present results clearly indicate that CR+/nNOS− neurons and CR−/nNOS+ neurons belong to different morphological subpopulations, and lead us to speculate that they might play different functional roles in the hippocampal circuit. The further application of morphometric multivariate analysis would be valuable to understand the functional roles of chemically defined neurons in the various brain regions.     

Compartmentation of NADPH-diaphorase activity in the mouse cerebellar cortex

The mammalian cerebellum is built around an array of parasagittal bands of Purkinje cells that can be demonstrated by immunocytochemical staining for the differentiation antigen zebrin II. Climbing and Mossy fiber afferents also terminate in bands, and the afferent terminal fields and the Purkinje cell bands are aligned. The convergence of mossy and climbing fiber pathways onto the Purkinje cells, which are the sole output of the cerebellar cortex, is a characteristic feature of cerebellar circuitry. Previous studies showed that when both afferent pathways are activated synchronously there develops a long-term depression of synaptic efficacy at the parallel fiber-Purkinje cell synapse. Two second messenger pathways mediate long-term depression: one involves diacylglyceroland protein kinase C, and the other involves nitric oxide that is generated by a nitric oxide synthase. We have studied the distribution of nitric oxide synthase in the adult Mouse cerebellum by using nicatinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. NADPH-diaphorase activity is found mainly in the granule and basket cells. Within the granular layer NADPH-diaphorase activity is expressed nonuniformly patches of granule cells and synaptic glomeruli. The patches are yseen in all lobules, are reproducible from individual to individual, and are topographically ordered with respect to the Purkinje cell compartments as revealed by using anti-zebrin II immunocytochemistry. These data imply that nitric oxide-dependent, long-term depression may only involve a subset of mossy fiber/granule cell projections, and that one role for nitric oxide may be to refine cerebellar receptive fields. © 1994 Wiley-Liss, Inc.     

Subclass-specific formation of perineuronal nets around parvalbumin-expressing GABAergic neurons in Ammon's horn of the mouse hippocampus

Perineuronal nets (PNNs) are closely associated with parvalbumin-positive (PV+) neurons, and play a major role in controlling developmental neural plasticity. Considering the recent advances in classification of PV+ neurons, here we aimed to clarify whether PNNs might be associated with specific subclasses of PV+ neurons in the hippocampus. In this study, we labeled PNNs by Wisteria floribunda agglutinin (WFA), and classified PV+ neurons based on the combination of cellular location, molecular expression (neuropeptide Y [NPY], somatostatin [SOM], special AT-rich sequence-binding protein-1 [SATB1]), and retrograde tracing through stereotaxic injection of Fluoro-Gold (FG) into the medial septum. The criteria of each subclass can be summarized as follows: axo-axonic cells, PV+/SATB1–/NPY– cells in the stratum pyramidale; basket cells, PV+/SATB1+/NPY– cells in the stratum pyramidale; bistratified cells, PV+/SATB1+/NPY+ cells in the stratum pyramidale; oriens-lacunosum-moleculare (O-LM) cells, PV+/SOM+/FG– cells in the stratum oriens; hippocampo-septal projection (H-S) cells, and PV+/SOM+/FG+ cells in the stratum oriens. The ratios of formation of WFA+ PNNs around each subclass of PV+ neurons were estimated according to the optical disector principle. The vast majority (over 90%) of putative PV+ basket cells were surrounded by PNNs, while only a minor population (less than 10%) of putative PV+ axo-axonic, O-LM, and H-S cells were enwrapped with PNNs. The ratios of formation of PNNs around putative PV+ bistratified cells were intermediate (25–50%). These findings indicate that PNNs may be specifically associated with PV+ basket cells, and also provide a key to understand the functional significance of PNNs and PV+ neurons in the hippocampus. J. Comp. Neurol. 523:790–804, 2015. © 2015 Wiley Periodicals, Inc.     

Melanocortin-4 receptor expression in different classes of spinal and vagal primary afferent neurons in the mouse

Melanocortin‐4 receptor (MC4R) ligands are known to modulate nociception, but the site of action of MC4R signaling on nociception remains to be elucidated. The current study investigated MC4R expression in dorsal root ganglia (DRG) of the MC4R‐GFP reporter mouse. Because MC4R is known to be expressed in vagal afferent neurons in the nodose ganglion (NG), we also systematically compared MC4R‐expressing vagal and spinal afferent neurons. Abundant green fluorescent protein (GFP) immunoreactivity was found in about 45% of DRG neuronal profiles (at the mid‐thoracic level), the majority being small‐sized profiles. Immunohistochemistry combined with in situ hybridization confirmed that GFP was genuinely produced in MC4R‐expressing neurons in the DRG. While a large number of GFP profiles in the DRG coexpressed Nav1.8 mRNA (84%) and bound isolectin B4 (72%), relatively few GFP profiles were positive for NF200 (16%) or CGRP (13%), suggesting preferential MC4R expression in C‐fiber nonpeptidergic neurons. By contrast, GFP in the NG frequently colocalized with Nav1.8 mRNA (64%) and NF200 (29%), but only to a moderate extent with isolectin B4 (16%). Lastly, very few GFP profiles in the NG expressed CGRP (5%) or CART (4%). Together, our findings demonstrate variegated MC4R expression in different classes of vagal and spinal primary afferent neurons, and underscore the role of the melanocortin system in modulating nociceptive and nonnociceptive peripheral sensory modalities. J. Comp. Neurol. 520:3933–3948, 2012. © 2012 Wiley Periodicals, Inc.     

The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice

To obtain viable GABAergic interneurons in cerebral cortical slices of adult mice, we investigated the effects of slice cutting solutions on the viability of green fluorescent protein (GFP)-expressing cortical neurons in GAD67-GFP knock-in mice. Almost no nuclei of GFP-positive neurons were labeled with propidium iodide in incubated slices, suggesting that GFP fluorescence was a useful indicator for the viability of GABAergic interneurons. When several cutting solutions were compared with saline-based solution, N-methyl-d-glucamine-based sodium-free solution was most effective to keep the number of GFP-positive neurons near the level of perfusion-fixed brain. GFP-positive neurons in slices cut with sodium-free solution were more numerous in cortical layers V–VI, at 30–60 μm depth from the cut surface and 1–6 h after cutting than those with saline-based solution. Furthermore, the number of GFP-positive neurons decreased in the cutting condition of high calcium concentration (5 mM) or high temperature (37 °C), and GFP fluorescence decreased when cut at 0 °C. The present results indicate that cutting the brain at 20 °C in sodium-free solution is a method for preparing cortical slices with GABAergic interneurons viable. This method would thus be useful for electrophysiological and morphological studies of cortical interneurons in slice preparations of the adult brain.     

Selective protection against oxidative damage in brain of mice with a targeted disruption of the neuronal nitric oxide synthase gene

Nitric oxide (NO) is an essential messenger molecule in brain, where it is produced in neurons mostly by the activity of the neuronal isoform of nitric oxide synthase (nNOS). To understand the participation of the different isoforms of NOS in physiological functioning and in pathological processes, mice with null mutations for each of the NOS isoforms have been generated. In the present paper, we report that there is a selective protection from oxidative damage in the brain of mice with a targeted disruption of the nNOS gene. The cerebellum of these mice shows reduced levels of lipid peroxidation (LP) at the different ages tested, compared with wild-type mice, and also a reduction in the formation of reactive oxygen species (ROS). We observed a decrease of LP in cortex, and no effect on either LP or ROS formation was observed in striatum of knockout mice compared with wild type. We also report increased spontaneous motor activity of knockout mice. The expression and activity of nNOS are crucial to maintain redox status in brain, and we consider that the alteration in oxidative damage may help us to explain the phenotypical characteristics of nNOS knockout mice and their differential susceptibility to brain insults.     

Distinct immunohistochemically defined areas in the medial amygdala in the developing and adult mouse

The medial amygdala has been considered a subpallial structure, but various studies have shown that is a somewhat complex structure expressing both pallial and subpallial gene markers. In this regard, we analyzed the immunohistochemical expression of the neurotransmitter GABA, the vesicular glutamate transporter type 2 (VGLUT2), the neuronal nitric oxide synthase (nNOS), and the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the developing and adult mouse medial amygdala. From intermediate embryonic stages on, neurochemical data show a distinctive superficial region forming a band all along the medial amygdalar surface. This superficial band displays a strong VGLUT2-immunoreactive neuropil and numerous CR-immunoreactive fibers, as well as some nNOS-, CR- or CB-positive cells. In contrast, the superficial region of the posterior medial amygdala appears to be non-GABA immunoreactive. This band in the posterior medial amygdala matches a Tbr1-expressing territory. Our results also show differences between dorsal and ventral parts of the postnatal and adult posterior medial amygdala. Especially, a compact cell aggregate of nNOS immunoreactive cells was found in the ventral portion of the medial amygdala, whereas the dorsal part is occupied by scattered weakly stained cells. Comparison of our results with gene expression patterns and fiber-tracing studies, let us to propose that the superficial band is a pallial derivative, whereas the dorsal and ventral nuclei of the posterior medial amygdala receive each neurons from different subpallial compartments, and the latter one a subset of excitatory, pallial projection neurons.