AMPA GluR2 subunit is differentially distributed on GABAergic neurons and pyramidal cells in the macaque monkey visual cortex

The cellular and synaptic distribution of the AMPA receptor subunit GluR2 was analyzed in the monkey primary visual cortex (area V1), by immunocytochemistry and postembedding immunogold methods. GluR2 immunoreactivity was widely distributed in all of the layers of area V1. A quantitative double labeling analysis in layers II and III revealed that the vast majority of GABAergic interneurons in this area also contained GluR2. Postembedding immunogold analysis revealed that GluR2 immunoreactivity was present at asymmetric synapses on both GABAergic interneurons and pyramidal cells. A quantitative study indicated that the number of GluR2 immunogold particles at asymmetric synapses on pyramidal cells was significantly higher than that on GABAergic interneurons. These results from the primate neocortex are in agreement with and extend our previous studies on the rat hippocampus and amygdala. In view of the dominant role of the GluR2 subunit in regulating calcium flux through AMPA receptors, the differential synaptic distribution of GluR2 on different neuronal types might provide a mechanism for cell-specific response properties to glutamate as well as clues to selective neuronal vulnerability and cell death mediated by calcium-dependent excitotoxic mechanisms.     

Synaptic coexistence of AMPA and NMDA receptors in the rat hippocampus: A postembedding immunogold study

Synaptic distributions of N-methyl-d -aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor subunits, NMDAR1 and GluR2, respectively, were examined by electron microscopy with the high spatial resolution of postembedding immunogold localization. We provide direct evidence for colocalization at individual axodendritic asymmetric synapses within the CA1 subfield of rat hippocampus. AMPA/ NMDA receptor colocalization was found both in γ-aminobutyric acid (GABA)ergic dendrites and non-GABAergic dendritic shafts, as well as dendritic spines. Some asymmetric synapses were found to contain only NMDAR1 or GluR2; however, most immunopositive synapses contained both subunits. Many NMDAR1 and/ or GluR2 immunopositive profiles received GABAergic innervation at an adjacent synapse, providing a substrate for GABAergic modulation of both GluR classes. These data suggest that excitatory neuronal transmission in CA1 neurons may generally involve activation of both NMDA and AMPA receptor subunits at a single synapse, however, they also offer ultrastructural evidence for NMDAR1-only synapses that might represent silent synapses. J. Neurosci. Res. 54:444–449, 1998. © 1998 Wiley-Liss, Inc.     

Pyramidal cells of the rat basolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons

The generation of emotional responses by the basolateral amygdala is determined largely by the balance of excitatory and inhibitory inputs to its principal neurons, the pyramidal cells. The activity of these neurons is tightly controlled by gamma-aminobutyric acid (GABA)-ergic interneurons, especially a parvalbumin-positive (PV(+)) subpopulation that constitutes almost half of all interneurons in the basolateral amygdala. In the present semiquantitative investigation, we studied the incidence of synaptic inputs of PV(+) axon terminals onto pyramidal neurons in the rat basolateral nucleus (BLa). Pyramidal cells were identified by using calcium/calmodulin-dependent protein kinase II (CaMK) immunoreactivity as a marker. To appreciate the relative abundance of PV(+) inputs compared with excitatory inputs and other non-PV(+) inhibitory inputs, we also analyzed the proportions of asymmetrical (presumed excitatory) synapses and symmetrical (presumed inhibitory) synapses formed by unlabeled axon terminals targeting pyramidal neurons. The results indicate that the perisomatic region of pyramidal cells is innervated almost entirely by symmetrical synapses, whereas the density of asymmetrical synapses increases as one proceeds from thicker proximal dendritic shafts to thinner distal dendritic shafts. The great majority of synapses with dendritic spines are asymmetrical. PV(+) axon terminals form mainly symmetrical synapses. These PV(+) synapses constitute slightly more than half of the symmetrical synapses formed with each postsynaptic compartment of BLa pyramidal cells. These data indicate that the synaptology of basolateral amygdalar pyramidal cells is remarkably similar to that of cortical pyramidal cells and that PV(+) interneurons provide a robust inhibition of both the perisomatic and the distal dendritic domains of these principal neurons.     

Mossy Cells of the Rat Dentate Gyrus are lmmunoreactive for Calcitonin Gene-related Peptide (CGRP)

The neuropeptide calcitonin gene-related peptide (CGRP) was localized in the hippocampus and dentate gyrus of the rat by immunocytochemistry at the light and electron microscopic levels. Without colchicine treatment only faint neuropil labelling was found in the inner molecular layer of the dentate gyrus. Following colchicine treatment, a large number of neurons with numerous complex spines along the proximal dendrites were visualized in the hilus of the dentate gyrus, particularly in the ventral areas, and, in addition, staining of the inner molecular layer became stronger. Several CA3c pyramidal cells located adjacent to the hilar region in the ventral hippocampus also appeared to be faintly positive, although in most cases only their axon initial segments were labelled. Outside this region, the subicular end of the CA1 subfield contained occasional CGRP-positive non-pyramidal cells. The hilar CGRP-positive neurons were negative for parvalbumin, calretinin, cholecystokinin and somatostatin, whereas most of them were immunoreactive for GluR2/3 (the AMPA-type glutamate receptor known to be expressed largely by principal cells). Correlated electron microscopy showed that the spines along the proximal dendritic shafts indeed correspond to thorny excrescences engulfed by large complex mossy terminals forming asymmetrical synapses. Pre-embedding immunogold staining demonstrated that CGRP immunoreactivity in the inner molecular layer was confined to axon terminals that form asymmetrical synapses, and the labelling was associated with large dense-core vesicles. The present data provide direct evidence that CGRP is present in mossy cells of the dentate gyrus and to a lesser degree in CA3c pyramidal cells of the ventral hippocampus. These CGRP-containing principal cells terminate largely in the inner molecular layer of the dentate gyrus, and may release the neuropeptide in conjunction with their 'classical' neurotransmitter, glutamate.     

Synaptic Distribution of the AMPA-GluR2 Subunit and Its Colocalization with Calcium-Binding Proteins in Rat Cerebral Cortex: An Immunohistochemical Study Using a GluR2-Specific Monoclonal Antibody

Due to its role as the dominant AMPA receptor subunit in respect to regulation of calcium permeability, information on the neuronal localization of GluR2 is of particular importance, yet has been hampered by the lack of a GluR2-specific antibody. Monoclonal antibodies were raised against the putative N-terminal portion (amino acids 175–430) of GluR2, using the fusion protein linked to trpE as an antigen. Western blot analysis and immunocytochemistry of transiently transfected human embryonic kidney 293 cells unambiguously confirmed the specificity of monoclonal antibody 6C4 for GluR2, which did not recognize or cross-react with any other AMPA/Kainate GluR subunits expressed. 6C4 was used in immunohistochemical studies to characterize the regional, cellular, and subcellular distribution of the GluR2 subunit at the light and electron microscopic levels in rat hippocampus and somatosensory cortex and in colocalization studies with the three calcium-binding proteins: parvalbumin, calbindin, and calretinin. GluR2 was widely distributed in both pyramidal cells and interneurons. Asymmetric synapses were labeled on both spines and small dendritic shafts. In contrast to previous reports, our double labeling studies using monoclonal antibody 6C4 with polyclonal antisera against calcium-binding proteins demonstrated that 84–97% of parvalbumin and calbindin-immunoreactive and 45–66% of the calretinin-immunoreactive interneurons in CA1 and somatosensory cortex also contain GluR2. These data have important implications regarding heterogeneity in calcium permeability of AMPA receptors across cell types in neocortex and hippocampus, as well as for differential vulnerability to excitotoxic injury.     

Ultrastructure and aspects of functional organization of pyramidal and nonpyramidal entorhinal projection neurons contributing to the perforant path

Identified entorhino-hippocampal projection neurons were investigated for their ultrastructure. Spinous projection neurons (pyramidal and spiny stellate cells) display common features such as symmetric axosomatic terminals on their somata, asymmetric synapses on the spines, and both types of synapses on the dendritic shafts. Their axons descend towards the white matter, branching occasionally via collaterals which establish contact with local spines and rarely on dendritic shafts and somata. The sparsely spinous projection neurons (multipolar and horizontal-bipolar) typically show deep nuclear infolds and symmetric and asymmetric synapses on their somata and dendritic shafts. Axons also collateralize in the soma vicinity and form local synapses. It is concluded that the entorhino-hippocampal projection neurons (both spiny and sparsely spinous) act locally and distally thus performing simultaneously as local-circuit and as projection neurons. In accordance with other morphological and electrophysiological reports it appears likely that the generation, modulation, and suppression of entorhinal excitation waves is mediated by these neurons through direct excitation, feed-forward and feed-back inhibition, and disinhibition.     

Ultrastructural localization of neurotransmitter immunoreactivity in mossy cell axons and their synaptic targets in the rat dentate gyrus

Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter γ-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements. Hippocampus 1997;7:559–570. © 1997 Wiley-Liss, Inc.     

Ultrastructural relationship between the AMPA-GluR2 receptor subunit and the mu-opioid receptor in the mouse central nucleus of the amygdala

Activation of GluR2-expressing non-calcium-permeable AMPA-type glutamate receptors in the central nucleus of the amygdala (CeA) may play an important role in integrating emotion and memory with goal-directed behaviors involved in opioid addiction. The location of non-calcium-permeable AMPA receptors within distinct neuronal compartments (i.e., soma, dendrite, or axon) is an important functional feature of these proteins; however, their ultrastructural location and subcellular relationship with mu-opioid receptors (μOR) in the CeA are unknown. Immunocytochemical electron microscopy was used to characterize the ultrastructural distribution of GluR2 and its association with μOR in the mouse CeA. A single-labeling analysis of GluR2 distribution employing immunoperoxidase or immunogold markers revealed that this protein was frequently affiliated with intracellular vesicular organelles, as well as the plasma membrane of CeA neuronal profiles. Among all GluR2-labeled neuronal structures, over 85% were dendrites or somata. Unlabeled axon terminals frequently formed asymmetric excitatory-type synaptic junctions with GluR2-labeled dendritic profiles. Dual-labeling immunocytochemical analysis showed that GluR2 and μOR were co-localized in neuronal compartments. Among all dual-labeled structures, approximately 80% were dendritic. Synaptic inputs to these dual-labeled dendrites were frequently from unlabeled axon terminals forming asymmetric excitatory-type synapses. The presence of GluR2 in dendritic profiles receiving asymmetric synapses suggests that activation of the non-calcium-permeable AMPA receptor plays a role in the postsynaptic modulation of excitatory signaling involving CeA neuronal circuits that coordinate sensory, affective, and behavioral processes involved in drug addiction. Given the critical role of non-calcium-permeable AMPA receptor function in neural and behavioral adaptability, their dendritic association with μOR in CeA dendrites provides a neuronal substrate for opioid-mediated plasticity.     

Glutamate receptor subunit 3 (GluR3) immunoreactivity delineates a subpopulation of parvalbumin-containing interneurons in the rat hippocampus

Rasmussen's encephalitis is a childhood disease resulting in intractable seizures associated with hippocampal and neocortical inflammation. An autoantibody against the GluR3 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors is implicated in the pathophysiology of Rasmussen's encephalitis. AMPA receptors mediate excitatory neurotransmission in the brain and contain combinations of four subunits (GluR1-4). Although the distributions of GluR1, GluR2, and GluR4 are known in some detail, the cellular distribution of GluR3 in the mammalian brain remains to be described. We developed and characterized a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in CA1 of the rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and astrocytes and in most interneurons. We quantified the intensity of GluR3 immunoreactivity in interneuron subtypes defined by their calcium-binding protein content. GluR3 immunofluorescence, but not GluR1 or GluR2 immunofluorescence, was significantly elevated in somata of parvalbumin-containing interneurons compared to pyramidal somata. Strikingly, increased GluR3 immunofluorescence was not observed in calbindin- and calretinin-containing interneurons. Furthermore, 24% of parvalbumin-containing interneurons could be distinguished from surrounding neurons based on their intense GluR3 immunoreactivity. This subpopulation had significantly elevated GluR3 immunoreactivity compared to the rest of parvalbumin-containing interneurons. Electron microscopy revealed enriched GluR3 immunoreactivity in parvalbumin-containing perikarya at cytoplasmic and postsynaptic sites. Parvalbumin-containing interneurons, potent inhibitors of cortical pyramidal neurons, are vulnerable in the brains of epileptic patients. Our findings suggest that the somata of these interneurons are enriched in GluR3, which may render them vulnerable to pathological states such as epilepsy and Rasmussen's encephalitis.     

Effects of context‐drug learning on synaptic connectivity in the basolateral nucleus of the amygdala in rats

Context‐drug learning produces structural and functional synaptic changes in the circuitry of the basolateral nucleus of the amygdala (BLA). However, how the synaptic changes translated to the neuronal targets was not established. Thus, in the present study, immunohistochemistry with a cell‐specific marker and the stereological quantification of synapses was used to determine if context‐drug learning increases the number of excitatory and inhibitory/modulatory synapses contacting the gamma‐aminobutyric acid (GABA) interneurons and/or the pyramidal neurons in the BLA circuitry. Amphetamine‐conditioned place preference increased the number of asymmetric (excitatory) synapses contacting the spines and dendrites of pyramidal neurons and the number of multisynaptic boutons contacting pyramidal neurons and GABA interneurons. Context‐drug learning increased asymmetric (excitatory) synapses onto dendrites of GABA interneurons and increased symmetric (inhibitory or modulatory) synapses onto dendrites but not perikarya of these same interneurons. The formation of context–drug associations alters the synaptic connectivity in the BLA circuitry, findings that have important implications for drug‐seeking behavior.     

Differential postsynaptic compartments in the laterocapsular division of the central nucleus of amygdala for afferents from the parabrachial nucleus and the basolateral nucleus in the rat

Neurons in the laterocapsular division of the central nucleus of the amygdala (CeC), which is known as the "nociceptive amygdala," receive glutamatergic inputs from the parabrachial nucleus (PB) and the basolateral nucleus of amygdala (BLA), which convey nociceptive information from the dorsal horn of the spinal cord and polymodal information from the thalamus and cortex, respectively. Here, we examined the ultrastructural properties of PB- and BLA-CeC synapses identified with EGFP-expressing lentivirus in rats. In addition, the density of synaptic AMPA receptors (AMPARs) on CeC neurons was studied by using highly sensitive SDS-digested freeze-fracture replica labeling (SDS-FRL). Afferents from the PB made asymmetrical synapses mainly on dendritic shafts (88%), whereas those from the BLA were on dendritic spines (81%). PB-CeC synapses in dendritic shafts were significantly larger (median 0.072 μm(2)) than BLA-CeC synapses in spines (median 0.058 μm(2); P = 0.02). The dendritic shafts that made synapses with PB fibers were also significantly larger than those that made synapses with BLA fibers, indicating that the PB fibers make synapses on more proximal parts of dendrites than the BLA fibers. SDS-FRL revealed that almost all excitatory postsynaptic sites have AMPARs in the CeC. The density of AMPAR-specific gold particles in individual synapses was significantly higher in spine synapses (median 510 particles/μm(2)) than in shaft synapses (median 427 particles/μm(2); P = 0.01). These results suggest that distinct synaptic impacts from PB- and BLA-CeC pathways contribute to the integration of nociceptive and polymodal information in the CeC.     

GABAergic innervation of alpha type II calcium/calmodulin-dependent protein kinase immunoreactive pyramidal neurons in the rat basolateral amygdala

Although calcium/calmodulin-dependent protein kinase II (CaMK) has been shown to play a critical role in long-term potentiation (LTP) and emotional learning mediated by the basolateral amygdala, little is known about its cellular localization in this region. We have utilized immunohistochemical methods to study the neuronal localization of CaMK, and its relationship to gamma-aminobutyric acid (GABA)-ergic structures, in the rat basolateral amygdala (ABL). Light microscopic observations revealed dense CaMK staining in the ABL. Although the cell bodies and proximal dendrites of virtually every pyramidal cell appeared to be CaMK(+), the cell bodies of small nonpyramidal neurons were always unstained. Dual localization of CaMK and GABA immunoreactivity with confocal immunofluorescence microscopy revealed that CaMK and GABA were found in different neuronal populations in the ABL. CaMK was contained only in pyramidal neurons; GABA was contained only in nonpyramidal cells. At the ultrastructural level, it was found that CaMK was localized to pyramidal cell bodies, thick proximal dendrites, thin distal dendrites, most dendritic spines, axon initial segments, and axon terminals forming asymmetrical synapses. These findings suggest that all portions of labeled pyramidal cells, with the exception of some dendritic spines, can exhibit CaMK immunoreactivity. By using a dual immunoperoxidase/immunogold-silver procedure at the ultrastructural level, GABA(+) axon terminals were seen to innervate all CaMK(+) postsynaptic domains, including cell bodies (22%), thick (>1 microm) dendrites (34%), thin (<1 microm) dendrites (22%), dendritic spines (17%), and axon initial segments (5%). These findings indicate that CaMK is a useful marker for pyramidal neurons in ultrastructural studies of ABL synaptology and that the activity of pyramidal neurons in the ABL is tightly controlled by a high density of GABAergic terminals that target all postsynaptic domains of pyramidal neurons.     

Subcellular and subsynaptic distribution of the NR1 subunit of the NMDA receptor in the neostriatum and globus pallidus of the rat: co-localization at synapses with the GluR2/3 subunit of the AMPA receptor

Glutamatergic neurotransmission in the neostriatum and the globus pallidus is mediated through NMDA-type as well as other glutamate receptors and is critical in the expression of basal ganglia function. In order to characterize the cellular, subcellular and subsynaptic localization of NMDA receptors in the neostriatum and globus pallidus, multiple immunocytochemical techniques were applied using antibodies that recognize the NR1 subunit of the NMDA receptor. In order to determine the spatial relationship between NMDA receptors and AMPA receptors, double labelling was performed with the NR1 antibodies and an antibody that recognizes the GluR2 and 3 subunits of the AMPA receptor. In the neostriatum all neurons with characteristics of spiny projection neurons, some interneurons and many dendrites and spines were immunoreactive for NR1. In the globus pallidus most perikarya and many dendritic processes were immunopositive. Immunogold methods revealed that most NR1 labelling is associated with asymmetrical synapses and, like the labelling for GluR2/3, is evenly spread across the synapse. Double immunolabelling revealed that in neostriatum, over 80% of NR1-positive axospinous synapses are also positive for GluR2/3. In the globus pallidus most NR1-positive synapses are positive for GluR2/3. In both regions many synapses labelled only for GluR2/3 were also detected. These results, together with previous data, suggest that NMDA and AMPA receptor subunits are expressed by the same neurons in the neostriatum and globus pallidus and that NMDA and AMPA receptors are, at least in part, colocalized at individual asymmetrical synapses. The synaptic responses to glutamate in these regions are thus likely be mediated by both AMPA and NMDA receptors at the level of individual synapses.     

Synaptic relationships between axon terminals from the mediodorsal thalamic nucleus and gamma-aminobutyric acidergic cortical cells in the prelimbic cortex of the rat

Although the reciprocal interconnections between the prefrontal cortex and the mediodorsal nucleus of the thalamus (MD) are well known, the involvement of inhibitory cortical interneurons in the neural circuit has not been fully defined. To address this issue, we conducted three combined neuroanatomical studies on the rat brain. First, the frequency and the spatial distribution of synapses made by reconstructed dendrites of nonpyramidal neurons were identified by impregnation of cortical cells with the Golgi method and identification of thalamocortical terminals by degeneration following thalamic lesions. Terminals from MD were found to make synaptic contacts with small dendritic shafts or spines of Golgi-impregnated nonpyramidal cells with very sparse dendritic spines. Second, a combined study that used anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and postembedding gamma-aminobutyric acid (GABA) immunocytochemistry indicated that PHA-L-labeled terminals from MD made synaptic junctions with GABA-immunoreactive dendritic shafts and spines. Nonlabeled dendritic spines were found to receive both axonal inputs from MD with PHA-L labelings and from GABAergic cells. In addition, synapses were found between dendritic shafts and axon terminals that were both immunoreactive for GABA. Third, synaptic connections between corticothalamic neurons that project to MD and GABAergic terminals were investigated by using wheat germ agglutinin conjugated to horseradish peroxidase and postembedding GABA immunocytochemistry. GABAergic terminals in the prelimbic cortex made symmetrical synaptic contacts with retrogradely labeled corticothalamic neurons to MD. All of the synapses were found on cell somata and thick dendritic trunks. These results provide the first demonstration of synaptic contacts in the prelimbic cortex not only between thalamocortical terminals from MD and GABAergic interneurons but also between GABAergic terminals and corticothalamic neurons that project to MD. The anatomical findings indicate that GABAergic interneurons have a modulatory influence on excitatory reverberation between MD and the prefrontal cortex.     

Excitatory synapses on dendritic shafts of the caudal basal amygdala exhibit elevated levels of GABAA receptor α4 subunits following the induction of activity‐based anorexia

Anorexia nervosa (AN) is an eating disorder characterized by self‐imposed severe starvation, excessive exercise, and anxiety. The onset of AN is most often at puberty, suggesting that gonadal hormonal fluctuations may contribute to AN vulnerability. Activity‐based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction. The basal amygdala as well as the GABAergic system regulate trait anxiety. We therefore examined the subcellular distribution of GABA receptors (GABARs) in the basal amygdala of female pubertal rats and specifically of their α4 subunits, because expression of α4‐containing GABARs is regulated by gonadal hormone fluctuations. Moreover, because these GABARs reduce neuronal excitability through shunting of EPSPs, we quantified the frequency of occurrence of these GABARs adjacent to excitatory synapses. Electron microscopic immunoctychemistry revealed no change in the frequency of association of α4 subunits with excitatory synapses on dendritic spines, whether in the anterior (Bregma ?2.8 mm) or caudal (Bregma ?3.8 mm) portion of the basal amygdala. Sholl analysis of golgi‐stained neurons also revealed no change in the extent of dendritic branching by these densely spiny, pyramidal‐like neurons. However, there was an increase of membranous α4 subunits near excitatory synapses on dendritic shafts, specifically in the caudal basal amygdala, and this was accompanied by a rise of α4 subunits intracellularly. Because most dendritic shafts exhibiting excitatory synapses are GABAergic interneurons, the results predict disinhibition, which would increase excitability of the amygdaloid network, in turn augmenting ABA animals' anxiety. Synapse, 68:1–15, 2014 . © 2013 Wiley Periodicals, Inc.     

Distribution of AMPA receptor subunit glur1 in the bed nucleus of the stria terminalis and effect of stress

The brain circuitry thought to be involved in stress responses includes several nuclei of the extended amygdala. The bed nucleus of the stria terminalis (BNST) is thought to be involved in the generation of sustained, nonspecific anxiety. Previous behavioral and electrophysiological experiments demonstrate that glutamate systems are involved in anxiety‐like behaviors in the BNST. Antagonists for AMPA receptors injected into the BNST decrease anxiety‐like behaviors. However, little is known about the role of AMPA receptors and the mechanism by which they act in the establishment of anxiety‐like behavior in response to a stressor. We hypothesized that the distribution of AMPA receptors is changed following a paradigm of unpredictable footshock as has been seen in the basolateral amygdala (BLA). We examined the subcellular localization of the GluR1 subunits of the AMPA receptor. We found that the neuropil of the BNST had a lower density of dendritic spines compared to dendritic shafts in the BLA. The majority of elements immunolabeled for GluR1 were dendritic shafts and spines with axonal and glial elements rarely labeled. Compared with controls, no significant effect was observed on days 1, 6, or 14 poststress. However, there was a trend for an increase at 6 and 14 days poststress. These data demonstrate that GluR1 subunits are primarily located on postsynaptic elements in the BNST. Moreover, it was shown that the response of the AMPA GluR1 subunit does not undergo a significant migration into spines from dendrites in response to a stressor as has been demonstrated in the BLA. Synapse 68:194–201, 2014 . Published 2014     

Immunocytochemical localization of GABABR1 receptor subunits in the basolateral amygdala

Gamma-aminobutyric acid B (GABAB) receptors (GBRs) are G-protein-coupled receptors that mediate a slow, prolonged form of inhibition in the basolateral amygdala (ABL) and other brain areas. Recent studies indicate that this receptor is a heterodimer consisting of GABABR1 (GBR1) and GABABR2 subunits. In the present investigation, antibodies to the GABABR1 subunit were used to study the neuronal localization of GBRs in the rat ABL. GBR immunoreactivity was mainly found in spine-sparse interneurons and astrocytes at the light microscopic level. Very few pyramidal neurons exhibited perikaryal staining. Dual-labeling immunofluorescence analysis indicated that each of the four main subpopulations of interneurons exhibited GBR immunoreactivity. Virtually 100% of large CCK+ neurons in the basolateral and lateral nuclei were GBR+. In the basolateral nucleus 72% of somatostatin (SOM), 73% of parvalbumin (PV) and 25% of VIP positive interneurons were GBR+. In the lateral nucleus 50% of somatostatin, 30% of parvalbumin and 27% of VIP positive interneurons were GBR+. Electron microscopic (EM) analysis revealed that most of the light neuropil staining seen at the light microscopic level was due to the staining of dendritic shafts and spines, most of which probably belonged to spiny pyramidal cells. Very few axon terminals (Ats) were GBR+. In summary, this investigation demonstrates that the distal dendrites of pyramidal cells, and varying percentages of each of the four main subpopulations of interneurons in the ABL, express GBRs. Because previous studies suggest that GBR-mediated inhibition modulates NMDA-dependent EPSPs in the ABL, these receptors may play an important role in neuronal plasticity related to emotional learning.     

Cortical inputs innervate calbindin‐immunoreactive interneurons of the rat basolateral amygdaloid complex

The present study was undertaken to shed light on the synaptic organization of the rat basolateral amygdala (BLA). The BLA contains multiple types of GABAergic interneurons that are differentially connected with extrinsic afferents and other BLA cells. Previously, it was reported that parvalbumin immunoreactive (PV⁺) interneurons receive strong excitatory inputs from principal BLA cells but very few cortical inputs, implying a prevalent role in feedback inhibition. However, because prior physiological studies indicate that cortical afferents do trigger feedforward inhibition in principal cells, the present study aimed to determine whether a numerically important subtype of interneurons, expressing calbindin (CB⁺), receives cortical inputs. Rats received injections of the anterograde tracer Phaseolus vulgaris‐leucoagglutinin (PHAL) in the perirhinal cortex or adjacent temporal neocortex. Light and electron microscopic observations of the relations between cortical inputs and BLA neurons were performed in the lateral (LA) and basolateral (BL) nuclei. Irrespective of the injection site (perirhinal or temporal neocortex) and target nucleus (LA or BL), ～90% of cortical axon terminals formed asymmetric synapses with dendritic spines of principal BLA neurons, while 10% contacted the dendritic shafts of presumed interneurons, half of which were CB⁺. Given the previously reported pattern of CB coexpression among GABAergic interneurons of the BLA, these results suggest that a subset of PV‐immunonegative cells that express CB, most likely the somatostatin‐positive interneurons, are important mediators of cortically evoked feedforward inhibition in the BLA. J. Comp. Neurol. 522:1915–1928, 2014. © 2013 Wiley Periodicals, Inc.     

Ultrastructural evidence for presynaptic mu opioid receptor modulation of synaptic plasticity in NMDA-receptor-containing dendrites in the dentate gyrus

Physiological studies have demonstrated that long-term potentiation (LTP) induction in N-methyl-D-aspartate (NMDA) receptor containing dentate granule cells following lateral perforant path stimulation is opioid dependent, involving mu-opioid receptors (MORs) on gamma-aminobutyric acid (GABA)-ergic neurons. To determine the cellular relationships of MORs to postsynaptic NMDA receptor-containing dendrites, immunoreactivity (-I) against MOR and the NMDA receptor subunit 1 (NMDAR1) was examined in the outer molecular layer of the dentate gyrus using electron microscopy. MOR-I was predominantly in axons and axon terminals. NMDAR1-I was almost exclusively in spiny dendrites, but was also in a few terminals. Using immunogold particles to localize precisely NMDAR1, one-third of the NMDAR1-I was detected on the dendritic plasmalemma; in dendritic spines plasmalemmal immunogold particles were near synaptic densities. Many MOR-labeled axons and terminals contacted NMDAR1-labeled dendrites. MOR-labeled terminals formed symmetric (inhibitory-type) synapses on NMDAR1-labeled dendritic shafts or nonsynaptically contacted NMDAR1-labeled shafts and spines. MOR-labeled axons often abutted NMDAR1-containing dendritic spines which received asymmetric (excitatory-type) synapses from unlabeled terminals. Occasionally, MOR-labeled terminals and dendrites were apposed to NMDAR1-containing terminals. These results provide anatomical evidence that endogenous enkephalins or exogenous opioid agonists could inhibit GABAergic terminals that modulate granule cell dendrites, thus boosting depolarizing events in granule cells and facilitating the activation of NMDA receptors located on their dendrites.     

Demonstration of competition between endogenous dopamine and [11C]raclopride binding in in vitro brain slices using a dynamic autoradiography technique

Although the basolateral nucleus (BL) of the amygdala is known to contain an abundance of gamma-aminobutyric acid (GABA)ergic neurons that regulate the amygdaloid projection neurons and influence storage and consolidation of memory, it remains to be determined what type of neuronal input controls GABAergic neurons in the BL. We examined the synapses that GABAergic neurons form with GABAergic and noradrenergic neurons and terminals with unknown transmitters by double-labeling immunoelectron microscopy using anti-GABA and dopamine-beta-hydroxylase (DBH) antisera. The medium and small dendrites of the GABAergic neurons were shown to receive symmetric, inhibitory-type synapses from GABAergic axon terminals and asymmetric, excitatory-type synapses from noradrenergic axon terminals. Each segment of the GABAergic neurons from perikarya to dendritic spines received both symmetric and asymmetric synapses from unlabeled axon terminals of various forms and sizes. The incidence rates of the two types of synapses were almost identical. Our results suggest that GABAergic neurons in the BL of the rat amygdala might be affected by the excitatory influence of the noradrenergic system and the inhibitory influence of the GABAergic system. Furthermore, these neurons are also strongly influenced by both excitatory and inhibitory-type synapses from neuronal systems other than the GABAergic and noradrenergic systems.