Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influence dendritic spine morphology and long-term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4-labeled dendritic spines and axon terminals corresponded to 9-19% and 25-40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre- or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP.     

Immunogold localization of phocein in dendritic spines

Phocein, a widely expressed intracellular protein involved in clathrin- and dynamin-dependent membrane dynamics, has been shown to interact with members of the striatin family of proteins, striatin, SG2NA, and zinedin. Immunogold labeling was performed to assess subcellular localization of phocein in neurons of the rodent cerebellar cortex and hippocampal Ammon's horn. Most of the phocein-bound gold particles were located within dendritic thorns and spines of the cerebellar Purkinje cells and hippocampal pyramidal neurons, as observed previously for striatin in striatal neurons. The postsynaptic profiles containing phocein were engaged in asymmetric synapses with the main types of afferents in the cerebellum and in the hippocampus. In the cerebellum, phocein-bound immunogold particle numbers ranged from 1-20 in approximately 50% of the Purkinje cell spines. In these spines most of the immunogold particles were found in the neuroplasm ( approximately 70%) and on nonsynaptic plasma membrane domains and related structures such as endocytic-like profiles ( approximately 18%). As soon as the first postnatal week, phocein was detected in the Purkinje cell somatic and dendritic thorns making asymmetric synapses with climbing fibers. During the following weeks the protein was located in the dendritic spines, as observed in the adult molecular layer. Finally, double immunogold labeling revealed a distribution of phocein and SG2NA suggesting that the two proteins could interact in the Purkinje cell spines. The early postnatal expression of phocein, a protein involved in membrane dynamics, suggests that it may have functional relevance in dendritic remodeling during development and potentially in spine plasticity during adulthood.     

Cholinergic innervation of the rat hippocampus as revealed by choline acetyltransferase immunocytochemistry: a combined light and electron microscopic study

The cholinergic innervation of the rat hippocampus proper and fascia dentata was investigated by using a monoclonal antibody against choline acetyltransferase (ChAT). At the light microscopic level, thin varicose ChAT-immunoreactive fibers were observed mainly in the vicinity of the pyramidal and granular layers where they formed a fine network around proximal dendrites of pyramidal and granule cells. In addition, many ChAT-immuno-reactive fibers were found in the hilar region and in stratum oriens, radiatum, and lacunosum-moleculare of all hippocampal sectors. Electron microscopic analysis revealed ChAT immunoreactivity in thin unmyelinated varicose axons and terminals which established synaptic contacts. Asymmetric contacts of ChAT-immunoreactive terminals were found on small spines in the dendritic layers of the hippocampus proper and in the molecular layer of the fascia dentata. Symmetric synaptic contacts were formed on the cell bodies of pyramidal and granule cells. Both symmetric and asymmetric synaptic contacts occurred on dendritic shafts. The analysis of serial thin sections, which allows identification of postsynaptic elements, suggests that pyramidal cells, granule cells, and nonpyramidal neurons of the hippocampus receive a cholinergic input.     

Role of Ca2+/calmodulin‐dependent protein kinase II in dendritic spine remodeling during epileptiform activity in vitro

Epileptiform activity (EA) in vivo and in vitro induces a loss of dendritic spines and synapses. Because CaMKII has been implicated in synaptogenesis and synaptic plasticity, we investigated the role of CaMKII in the effects of EA on spines, using rat hippocampal slice cultures. To visualize dendrites and postsynaptic densities (PSDs) in pyramidal neurons in the slices, we used biolistic transfection to express either free GFP or a PSD95‐YFP construct that specifically labels PSDs. This allowed us to distinguish two classes of dendritic protrusions: spines that contain PSDs, and filopodia that lack PSDs and that are, on average, longer than spines. By these criteria, 48 hr of EA caused a decrease specifically in the number of spines. Immunoblots showed that EA increased CaMKII activity in the slices. Inhibition of CaMKII by expression of AIP, a specific peptide inhibitor of CaMKII, reduced spine number under basal conditions and failed to prevent EA‐induced spine loss. However, under EA conditions, AIP increased the number of filopodia and the number of PSDs on the dendritic shaft. These data show at least two roles for CaMKII activity in maintenance and remodeling of dendritic spines under basal or EA conditions. First, CaMKII activity promotes the maintenance of spines and spine PSDs. Second, CaMKII activity suppresses EA‐induced formation of filopodia and suppresses an increase in shaft PSDs, apparently by promoting translocation of PSDs from dendritic shafts to spines and/or selectively stabilizing spine rather than shaft PSDs. © 2009 Wiley‐Liss, Inc.     

A light and electron microscopic study of the metabotropic glutamate receptor mGluR1a in the normal and kainate-lesioned rat hippocampus

The distribution of the metabotropic glutamate receptor mGluR1a was studied in the normal and kainate-lesioned rat hippocampus using a monoclonal (MAb) and a polyclonal antibody to mGluR1a. Many labeled nonpyramidal neurons were observed in the stratum oriens of CA1 in sections incubated with MAb. In comparison, fewer labeled neurons were observed in this layer in sections incubated with polyclonal antibody. Many nonpyramidal neurons were observed in the stratum lucidum of CA3 and the hilus of the dentate gyrus, with both antibodies. The cell bodies of pyramidal neurons were unlabeled. A dense network of labeled processes was observed in the neuropil of the CA fields at electron microscopy. Some dendrites were very densely labeled and did not contain dendritic spines. These were identified as dendrites of nonpyramidal neurons. Other dendrites contained lightly labeled dendritic shafts, but densely labeled dendritic spines, and were identified as dendrites of pyramidal neurons. Intravenous kainate injections resulted in destruction of pyramidal neurons and a massive decrease in mGluR1a immunoreactivity in the CA fields. This decrease was obvious even at 1–5 d postinjection, when the nonpyramidal neurons in the stratum oriens remained densely labeled, suggesting that pyramidal neurons contributed significantly to mGluR1a staining in the CA fields. We conclude that the dendritic spines of hippocampal pyramidal neurons contain mGluR1a, even though little staining is observed in their parent dendritic shafts or cell bodies.     

Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain

We recently showed by electron microscopy that the postsynaptic density (PSD) from hippocampal cultures undergoes rapid structural changes after ischemia-like conditions. Here we report that similar structural changes occur after delay in transcardial perfusion fixation of the mouse brain. Delay in perfusion fixation, a condition that mimics ischemic stress, resulted in 70%, 90%, and 23% increases in the thickness of PSDs from the hippocampus (CA1), cerebral cortex (layer III), and cerebellar cortex (Purkinje spines), respectively. In step with PSD thickening, the amount of PSD-associated alpha-calcium calmodulin-dependent protein kinase II (alpha- CaMKII) label increased more in cerebral cortical spines than in Purkinje spines. Although the Purkinje PSDs thickened only slightly after delayed fixation, they became highly curved, and many formed sub-PSD spheres approximately 80 nm in diameter that labeled for CaMKII. Delayed perfusion fixation also produced more cytoplamic CaMKII clusters ( approximately 110 nm in diameter) in the somas of pyramidal cells (from hippocampus and cerebral cortex) than in Purkinje cells. Thus a short delay in perfusion fixation produces cell-specific structural changes at PSDs and neuronal somas. Purkinje cells respond somewhat differently to delayed perfusion fixation, perhaps owing to their lower levels of CaMKII, and CaMKII binding proteins at PSDs. We present here a catalogue of structural changes that signal a perfusion fixation delay, thereby providing criteria by which to assess perfusion fixation quality in experimental structural studies of brain and to shed light on the subtle changes that occur in intact brain following metabolic stress.     

Fine structure of identified neurons in the primate hippocampus: a combined Golgi/EM study in the baboon

The hippocampi of two 1-year-old female baboons (Papio anubis) were used for a combined Golgi/electron microscope (EM) study of characteristic cell types in the hippocampus proper and fascia dentata. Results were compared with previous Golgi/EM studies of hippocampal neurons in small laboratory animals. Cell bodies of pyramidal neurons in CA1 were more loosely distributed than known from studies on the rat or guinea pig. Numerous basal and horizontal dendrites originating from the perikaryon filled in the space between neighboring cell bodies. Apical stem dendrites were varying in length, depending on the position of the parent cell body in outer or inner portions of the pyramidal layer. Dendrites were densely covered with spines which in the EM showed very complex synaptic contacts. In contrast to our observations in rats and guinea pigs, CA3 pyramidal cells in the monkey hippocampus exhibited numerous large spines or excrescences not only on apical dendrites but also on basal dendrites running through stratum oriens. These excrescences appeared to be more complex than in small rodents. They often branched, protruding deeply into presynaptic mossy fiber boutons, and formed multiple asymmetric synaptic contacts. Granule cells of the monkey fascia dentata, in contrast to those of the rodent, occasionally had basal dendrites extending into the hilar region. In the EM, granule cells either with or without basal dendrites exhibited fine structural characteristics that were very similar to those described in Golgi/EM studies of granule cells in the rat fascia dentata. Of the various types of nonpyramidal neurons the horizontal cells in stratum oriens with dendrites parallel to the alveus were analyzed. As seen in rats, these cells exhibited large amounts of rough endoplasmic reticulum, indentations of the nuclear membrane, and nuclear inclusions. Numerous terminals formed synaptic contacts on dendritic shafts. In contrast to rodents, numerous spines arose from dendrites and cell bodies of these neurons. In the EM, often single spines were found to establish synaptic contacts with several presynaptic boutons. In summary, our correlated light and EM study of four characteristic cell types, which are present in both nonprimates and primates, demonstrates a much more complex dendritic pattern and synaptic organization of these neurons in primates than in commonly studied small laboratory animals.     

Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus

From embryonic development to adulthood, the EphA4 receptor and several of its ephrin‐A or ‐B ligands are expressed in the hippocampus, where they presumably play distinct roles at different developmental stages. To help clarify these diverse roles in the assembly and function of the hippocampus, we examined the cellular and subcellular localization of EphA4 in postnatal rat hippocampus by light and electron microscopic immunocytochemistry. On postnatal day (P) 1, the EphA4 immunostaining was robust in most layers of CA1, CA3, and dentate gyrus and then decreased gradually, until P21, especially in the cell body layers. At the ultrastructural level, focal spots of EphA4 immunoreactivity were detected all over the plasma membrane of pyramidal and granule cells, between P1 and P14, from the perikarya to the dendritic and axonal extremities, including growth cones and filopodia. This cell surface immunoreactivity then became restricted to the synapse‐associated dendritic spines and axon terminals by P21. In astrocytes, the EphA4 immunolabeling showed a similar cell surface redistribution, from the perikarya and large processes at P1–P7, to small perisynaptic processes at P14–P21. In both cell types, spots of EphA4 immunoreactivity were also detected, with an incidence decreasing with maturation, on the endoplasmic reticulum, Golgi apparatus, and vesicles, organelles involved in protein synthesis, posttranslational modifications, and transport. The cell surface evolution of EphA4 localization in neuronal and glial cells is consistent with successive involvements in the developmental movements of cell bodies first, followed by process outgrowth and guidance, synaptogenesis, and finally synaptic maintenance and plasticity. J. Comp. Neurol. 512:798–813, 2009. © 2008 Wiley‐Liss, Inc.     

Distribution,morphological features,and synaptic connections of parvalbumin- and calbindin D28k-immunoreactive neurons in the human hippocampal formation

Calcium binding proteins calbindin D_28k (CaBP) and parvalbumin (PV) are known to form distinct subpopulations of gamma-aminobutyric acid (GABA)ergic neurons in the rodent hippocampal formation. Light and electron microscopic morphology and connections of these protein-containing neurons are only partly known in the primate hippocampus. In this study, CaBP and PV were localized in neurons of the human hippocampal formation including the subicular complex (prosubiculum, subiculum, and presubiculum) in order to explore to what extent these subpopulations of hippocampal neurons differ in phylogenetically distant species. CaBP immunoreactivity was present in virtually all granule cells of the dentate gyrus and in a proportion of pyramidal neurons in the CA1 and CA2 regions. A distinct population of CaBP-positive local circuit neurons was found in all layers of the dentate gyrus and Ammon's horn. Most frequently they were located in the molecular layer of the dentate gyrus and the pyramidal layer of Ammon's horn. In the subicular complex pyramidal neurons were not immunoreactive for CaBP. In the prosubiculum and subiculum immunoreactive nonpyramidal neurons were equally distributed in all layers, whereas in the presubiculum they occurred mainly in the superficial layers. Electron microscopy showed typical somatic and dendritic features of the granule, pyramidal, and local circuit neurons. CaBP-positive mossy fiber terminals in the hilus of the dentate gyrus and terminals of presumed pyramidal neurons of Ammon's horn formed asymmetric synapses with dendrites and spines. CaBP-positive terminals of nonprincipal neurons formed symmetric synapses with dendrites and dendritic spines, but never with somata or axon initial segments. PV was exclusively present in local circuit neurons in both the hippocampal formation and subicular complex. Most of the PV-positive cell bodies were located among or close to the principal cell layers. However, large numbers of immunoreactive neurons were also found in the molecular layer of the dentate gyrus and in strata oriens of Ammon's horn. PV-positive cells were equally distributed in all layers of the subicular complex. Electron microscopy showed the characteristic somatic and dendritic features of local circuit neurons. PV-positive axon terminals formed exclusively symmetric synapses with somata, axon initial segments and dendritic shafts, and in a few cases with dendritic spines. The CaBP- and PV-containing neurons formed similar subpopulations in rodents, monkeys, and humans, although the human hippocampus displayed the largest variability of these immunoreactive neurons in their morphology and location. Calcium binding protein-containing neurons frequently occurred in the molecular layer of the human dentate gyrus and in the stratum lacunosum-moleculare of Ammon's horn. The corresponding areas of the rat or monkey hippocampus were devoid of such neurons. In both rodents and primates similar populations of principal neurons contained CaBP. In addition, CaBP and PV were localized in distinct and nonoverlapping populations of nonprincipal cells. Their target selectivity did not change during phylogeny (e.g., PV-positive cells mainly innervate the perisomatic region and CaBP-positive cells the distal dendritic region of principal cells). © 1993 Wiley-Liss,Inc.     

Intraventricular administration of substance p increases the dendritic arborisation and the synaptic surfaces of Purkinje cells in rat's cerebellum

Substance P was infused in the lateral ventricles of twenty Lewis rats for twenty days. On the twentieth day the animals were sacrificed and the cerebellar cortex was processed for electron microscopy. The ultrastructural morphometric analysis revealed that the Purkinje cell dendritic arborisation and the number of the synapses between the parallel fibres and the Purkinje cell dendritic spines were much higher than in control animals. Numerous unattached spines of the secondary and tertiary dendritic branches of the Purkinje cells were also seen in the molecular layer either free or surrounded by astrocytic sheath. The increased number of synapses between the Purkinje cell dendrites and the parallel fibres in the animals, which received substance P intraventricularly, in correlation to control animals, supports a neurotrophine-like activity of the substance P in the mammalian cerebellum, enforcing the pre-programmed capability of the Purkinje cells to develop new synaptic surfaces.     

Specific expression of inositol 1,4,5-trisphosphate 3-kinase in dendritic spines

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase (IP3K) in the rat cerebral cortex and hippocampus was studied immunohistochemically. In both regions, the major structure expressing a high level of IP3K was the dendritic spines of pyramidal neurons, where immunoreactivity was associated with the spine apparatuses and plasmalemma. The postsynaptic densities showed the most intense labelling. Taking into account the results of our previous observations, which demonstrated the restricted localization of the enzyme in the dendritic spines of Purkinje and basket cells in cerebellum, IP3K may be localized specifically in dendritic spines in various regions of the central nervous system, and involved in synaptic signal transduction at the spines.     

Synaptic localization of receptor-type protein tyrosine phosphatase zeta/beta in the cerebral and hippocampal neurons of adult rats

Receptor-type protein tyrosine phosphatase (RPTP) zeta/beta is a nervous tissue-specific chondroitin sulfate proteoglycan. In this study, we investigated the immunohistochemical localization of RPTPzeta/beta in adult rat cerebral cortex and hippocampus at light and electron microscopic levels. Double labeling immunofluorescence microscopy revealed that the immunoreactivity of RPTPzeta/beta was observed at MAP2-positive dendrites and PSD-95-positive spines of pyramidal neurons in the cerebral cortex and hippocampus. Electron microscopic observation demonstrated a strong immunoreactivity of RPTPzeta/beta at the postsynaptic membrane of dendritic spines and shafts, and its moderate immunoreactivity at the dendritic membrane. In cultured cortical neurons, the immunoreactivity of RPTPzeta/beta was observed at some of PSD-95-positive spines. These results demonstrate that RPTPzeta/beta is localized mainly at the postsynaptic membrane of pyramidal neurons in adult cerebral cortex and hippocampus.     

Estradiol induces a phasic Fos response in the hippocampal CA1 and CA3 regions of adult female rats

Previous studies have shown that estradiol induces structural and functional changes in hippocampal CA1 pyramidal cells of the adult female rat. Estradiol increases the density of dendritic spines and axospinous synapses on CA1 pyramidal cells, and increases these cells' sensitivity to NMDA receptor-mediated synaptic input. Curiously, while estradiol effects are observed in CA1 pyramidal cells, the majority of the evidence indicates that these cells lack genomic estradiol receptors. In contrast, genomic estradiol receptors are expressed in at least some hippocampal interneurons in CA1. The goal of the present study was to determine which hippocampal neuronal populations are activated by estradiol, as determined by induction of c-Fos immunoreactivity, as well as the time-course of this activation. We quantified c-Fos expression in each of the major subdivisions of the hippocampus in adult female rats at various time points during the same estradiol treatment regimen known to regulate dendritic spines and synapses on CA1 pyramidal cells. Our results show a phasic estradiol-induced c-Fos response in the pyramidal cell layers of both CA1 and CA3. c-Fos was induced within 2 h of treatment, decreased at 6 and 12 h, and subsequently increased again at 24 h after treatment with estradiol. Double labeling for c-Fos and GAD 65 or GAD 67 suggests that c-Fos is induced primarily in principal cells, though a small proportion of GABAergic cells is also labeled. These estradiol-induced changes in c-Fos expression may reflect phasic neuronal activation and coupling to gene expression, which could be involved in estradiol's effects on excitatory synaptic connectivity in the hippocampus.     

Polarized distribution of alpha5 integrin in dendrites of hippocampal and cortical neurons

The distribution of immunoreactivity for the alpha5 subunit of the fibronectin receptor was evaluated in adult rat brain with particular interest in the cellular localization of immunostaining in the hippocampal formation and neocortex. Beyond localization to neuronal perikarya and short dendritic fragments within most brain areas, alpha5 immunoreactivity (-ir) was particularly dense within primary apical dendrites of pyramidal cells in both hippocampus and neocortex and within the dendritic arbors of cerebellar Purkinje cells. In hippocampal and cortical pyramidal cells, immunostaining was clearly polarized: alpha5-ir was not detectable in basal dendrites in hippocampal neurons and was limited to proximal arbors or absent from basal dendrites in pyramidal cells in superficial and deep layers of neocortex. Beyond this, alpha5-ir was distributed within the dendritic ramifications of the dentate gyrus granule cells and within perikarya and dendrites of occasional nonpyramidal neurons. Developmental studies demonstrated that, in both hippocampus and neocortex, alpha5-ir appears first within perikarya and is distributed to dendrites during the second postnatal week. These results are in accord with the broad hypothesis that integrins contribute to apical-basal differences in dendrites and that the integrin fibronectin (alpha5beta1) receptor, in particular, contributes to some late developing features of dendritic structure or function.     

Distribution and origin of vesicular glutamate transporter 2-immunoreactive fibers in the rat hippocampus

This study examined the distribution of vesicular glutamate transporter 2 (VGLUT2)-immunoreactive neuronal structures in the ipsilateral and contralateral hippocampi of unilateral fimbria/fornix transected, unilateral entorhinal cortex ablated, and intact female and male rats. In the hippocampi of intact animals, the highest density of VGLUT2-positive boutons was observed in the supragranular layer of the dentate gyrus, followed by the CA2 pyramidal and oriens layers, and the stratum lacunosum-moleculare of the CA1 field. This staining pattern was identical both in males and in females. Electron microscopic examination revealed that the immunolabeling was confined to axon terminals forming exclusively asymmetric synaptic contacts. The quantitative analysis of the synaptic targets of VGLUT2-positive terminals showed that in the dentate gyrus, 59% of the synaptic targets were dendritic spines, followed by dendritic shafts (22%) and granule cell somata (19%). In the pyramidal layer of the CA2 field, VGLUT2-immunoreactive boutons contacted mostly dendritic shafts (85%), only some of which (15%) synapsed with spines. The synaptic targets of VGLUT2-positive varicosities were dendritic spines (71%) and shafts (29%) in the stratum lacunosum-moleculare of the CA1 field. The fimbria/fornix transection caused a significant reduction in the density of VGLUT2-positive boutons only in the CA2 field, while entorhinal cortex ablation elicited no change in fiber density in any of the areas analyzed. Furthermore, our latest experiments on colchicine-treated animals revealed a large population of VGLUT2-positive neurons in the hippocampus that may be a possible intrinsic source of hippocampal VGLUT2 boutons. Our results suggest that the most likely sources of VGLUT2-positive boutons in the dentate supragranular layer, the CA2 area, as well as in the stratum lacunosum-moleculare of the CA1 field, might be the mossy cells, the supramammillary area, and the nucleus reuniens thalami, respectively.     

Dendritic spine density of adult hippocampal pyramidal cells is sensitive to thyroid hormone

In order to determine whether pyramidal cells of the adult hippocampus are morphologically sensitive to thyroid hormone, we performed single-section Golgi impregnation analyses on brains from hyperthyroid and control rats. Quantitative analyses of Golgi-impregnated pyramidal cells from the CA1 region showed a significant decrease in the density of apical dendritic spines with hyperthyroidism. In contrast, no changes were observed in spine density of basal dendrites or in cross-sectional cell body area of CA1 pyramidal cells. No changes in any of these morphological variables were detected in pyramidal cells of the CA3 region with hyperthyroidism. These results suggest that spine density of the apical dendrites of CA1 pyramidal cells is specifically affected by thyroid hormone in adulthood. Since dendritic spines are thought to represent postsynaptic sites it is likely that this morphological change results in altered hippocampal function.     

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.     

Differential synaptic distribution of the AMPA-GluR2 subunit on GABAergic and non-GABAergic neurons in the basolateral amygdala

The cellular and ultrastructural distribution patterns of the AMPA glutamate receptor subunit, GluR2, were determined in the rat basolateral amygdala. GluR2 immunoreactivity was widely and uniformly distributed in the basolateral nucleus, with both pyramidal and non-pyramidal neurons labelled. In fact, double label immunohistochemical analyses demonstrated that over 90% of the GABAergic interneurons were labelled for GluR2. Electron microscopic analyses further confirmed the presence of GluR2 in the soma and dendrites of GABAergic interneurons as well as in the soma, spines and dendritic shafts of pyramidal cells. As in our parallel study in the rat hippocampus, immunogold analyses revealed that GluR2 immunoreactivity was frequently preferentially located at asymmetric synapses on both pyramidal cell spines and shafts, as well as the dendritic processes and soma of GABAergic interneurons. However, the number of immunogold particles per labelled synapse on GABAergic neurons was significantly lower than at similar labelled asymmetric synapses on spines of presumed pyramidal cells. Given that the presence of GluR2 within the AMPA receptor complex decreases calcium flux, these data indicate that GABAergic local circuit neurons might possess AMPA receptors with higher calcium permeability on average than pyramidal cells, as has been suggested for hippocampus. Such cell class-specific differences in the subunit representation and resultant channel properties of AMPA receptors have implications for response properties as well as selective vulnerability of neurons within the basolateral nucleus of the amygdala.