Subtypes of substance P receptor immunoreactive interneurons in the rat basolateral amygdala

Local injections of the neurotoxin SP-saporin into the basolateral amygdala (BLA) are reported to specifically lesion substance P receptor immunoreactive (SPR-IR) interneurons, and to reduce anxiety related behavior. Hence, this technique might provide a means to study how defined interneuron populations regulate neuronal activity in the BLA. However, what interneuron subgroups in the BLA might be targeted by SP-saporin lesions has not been established. This study has used dual-labeling immunofluorescence in the rat BLA to examine SPR-IR neurons for their colocalization with the calcium-binding proteins; calbindin-D28k (CB), parvalbumin (PV), and calretinin (CR); and the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY). We found that all NPY-IR neurons and 45% of SOM-IR interneurons expressed SPR-IR, and that 50% and 51% of the SPR-IR interneuron population expressed NPY- and SOM-IR, respectively. Previous studies have reported that approximately a third of SOM-IR interneurons also express NPY, which suggests a large degree of overlap between the NPY, SOM and SPR expressing neurons in the BLA. We also found that the majority of SPR-IR cells were CB-IR (62%), but that these interneurons represented only 2.8% of the total CB-IR population. Moreover, SPR-IR interneurons did not express either PV-or CR- IR. Hence, SP-saporin lesions would ablate all interneurons in the BLA that contain NPY, but leave the majority of the CB-IR cells intact, and have no effect on the CR- and PV-IR populations. Consequently, these results support the use of SP-saporin lesions as a useful technique to study the role of NPY-IR interneurons in the BLA.     

The Roles of Basolateral Amygdala Parvalbumin Neurons in Fear Learning

The basolateral amygdala (BLA) is obligatory for fear learning. This learning is linked to BLA excitatory projection neurons whose activity is regulated by complex networks of inhibitory interneurons, dominated by parvalbumin (PV)-expressing GABAergic neurons. The roles of these GABAergic interneurons in learning to fear and learning not to fear, activity profiles of these interneurons across the course of fear learning, and whether or how these change across the course of learning all remain poorly understood. Here, we used PV cell-type-specific recording and manipulation approaches in male transgenic PV-Cre rats during pavlovian fear conditioning to address these issues. We show that activity of BLA PV neurons during the moments of aversive reinforcement controls fear learning about aversive events, but activity during moments of nonreinforcement does not control fear extinction learning. Furthermore, we show expectation-modulation of BLA PV neurons during fear learning, with greater activity to an unexpected than expected aversive unconditioned stimulus (US). This expectation-modulation was specifically because of BLA PV neuron sensitivity to aversive prediction error. Finally, we show that BLA PV neuron function in fear learning is conserved across these variations in prediction error. We suggest that aversive prediction-error modulation of PV neurons could enable BLA fear-learning circuits to retain selectivity for specific sensory features of aversive USs despite variations in the strength of US inputs, thereby permitting the rapid updating of fear associations when these sensory features change.SIGNIFICANCE STATEMENT The capacity to learn about sources of danger in the environment is essential for survival. This learning depends on complex microcircuitries of inhibitory interneurons in the basolateral amygdala. Here, we show that parvalbumin-positive GABAergic interneurons in the rat basolateral amygdala are important for fear learning during moments of danger, but not for extinction learning during moments of safety, and that the activity of these neurons is modulated by expectation of danger. This may enable fear-learning circuits to retain selectivity for specific aversive events across variations in expectation, permitting the rapid updating of learning when aversive events change.     

Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala

Specific neuronal populations in the basolateral amygdala (ABL) exhibit immunoreactivity for distinct neuropeptides and calcium-binding proteins. In the present study, immunohistochemical techniques were used to analyze neurons in the rat ABL that contain cholecystokinin (CCK). Some pyramidal projection neurons in the anterior subdivision of the basolateral nucleus exhibited low levels of CCK immunoreactivity in rats that received injections of colchicine to interrupt axonal transport; staining was concentrated in the axon initial segments of these cells. High levels of CCK immunoreactivity were observed in two subpopulations of nonpyramidal interneurons in all nuclei of the ABL: (1) type L neurons (characterized by large somata and thick dendrites), and (2) type S neurons (characterized by small somata and thin dendrites). Dual-labeling immunofluorescence studies using confocal laser scanning microscopy revealed that many (30-40%) type L CCK+ interneurons exhibited immunoreactivity for calbindin (CB), but not for parvalbumin (PV), calretinin (CR), or vasoactive intestinal polypeptide (VIP). In contrast, there was extensive colocalization of CR and VIP with CCK in type S neurons, but no significant colocalization with CB or PV. In addition, the majority of CR and VIP interneurons exhibited colocalization of both neurochemicals. Collectively, the results of this and previous studies indicate that there are at least four distinct interneuronal subpopulations in the ABL: (1) PV+ neurons (the great majority of which are CB+); (2) SOM+ neurons (many of which are CB+ and NPY+); (3) large CCK+ neurons (some of which are CB+); and (4) small bipolar/bitufted neurons that exhibit various amounts of colocalization of CCK, VIP, and CR.     

Cell‐specific expression of neuropeptide Y Y1 receptor immunoreactivity in the rat basolateral amygdala

Activation of neuropeptide Y (NPY) Y1 receptors (Y1r) in the rat basolateral nuclear complex of the amygdala (BLA) produces anxiolysis and interferes with the generation of conditioned fear. NPY is important in regulating the output of the BLA, yet the cell types involved in mediating this response are currently unknown. The current studies employed multiple label immunocytochemistry to determine the distribution of Y1r‐immunoreactivity (‐ir) in glutamatergic pyramidal and GABAergic cell populations in the BLA using scanning laser confocal stereology. Pyramidal neurons were identified by expression of calcium‐calmodulin dependent kinase II (CaMKII‐ir) and functionally distinct interneuron subpopulations were distinguished by peptide (cholecystokinin, somatostatin) or calcium‐binding protein (parvalbumin, calretinin) content. Throughout the BLA, Y1r‐ir was predominately on soma with negligible fiber staining. The high degree of coexpression of Y1r‐ir (99.9%) in CaMKII‐ir cells suggests that these receptors colocalize on pyramidal cells and that NPY could influence BLA output by directly regulating the activity of these projection neurons. Additionally, Y1r‐ir was also colocalized with the interneuronal markers studied. Parvalbumin‐ir interneurons, which participate in feedforward inhibition of BLA pyramidal cells, represented the largest number of Y1r expressing interneurons in the BLA (≈4% of the total neuronal population). The anatomical localization of NPY receptors on different cell populations within the BLA provides a testable circuit whereby NPY could modulate the activity of the BLA via actions on both projection cells and interneuronal cell populations. J. Comp. Neurol. 517:166–176, 2009. © 2009 Wiley‐Liss, Inc.     

The effects of acute stress on the regulation of central and basolateral amygdala CRF-binding protein gene expression

Corticotropin-releasing factor (CRF) is a key mediator of the behavioral, autonomic, and endocrine responses to stress. CRF binds two receptors and a CRF-binding protein (CRF-BP), which may inactivate or modulate the actions of CRF at its receptors. The amygdala is an important anatomical substrate for CRF and contains CRF, its receptors, and CRF-BP. Few studies have examined the effects of acute stress on the regulation of amygdala CRF-BP with other CRF system genes. Therefore, we examined the time course of the effects of acute restraint stress on central (CeA) and basolateral (BLA) amygdala CRF system genes. Consistent with our previous study, acute stress increased BLA CRF-BP mRNA shortly after stress offset. Surprisingly, BLA CRF-BP mRNA remained elevated up to 21 h after the stressor. This effect was selective in the BLA as stress did not alter CeA CRF-BP mRNA, and there were no changes in CRF or CRF receptor mRNAs in either amygdala nucleus. These results suggest that alterations in BLA CRF-BP gene expression are a primary response of the BLA/CeA CRF system to acute stress. Because CRF-BP can modulate CRF action, changes in amygdala CRF-BP levels after stress exposure may affect the ability of an organism to adapt to future stressors.     

Selective populations of hippocampal interneurons express ErbB4 and their number and distribution is altered in ErbB4 knockout mice

Neuregulins (NRGs) are ligands of ErbB receptor tyrosine kinases. The NRG1‐ErbB4 pathway has been shown to modulate hippocampal synaptic plasticity and network oscillations in the adult rodent brain. To identify cells that mediate these effects, here we determine the expression pattern of ErbB4 in four functionally distinct classes of interneurons that represent the majority of all inhibitory neurons in the adult hippocampus. On the basis of data from nine mice and 25,000 cells, we show that ErbB4 is expressed in cells that are positive for cholecystokinin (CCK, 54%), parvalbumin (PV, 42%), or neuronal nitric oxide synthase (nNOS, 39%) in a layer‐specific and region‐specific manner, whereas cells expressing somatostatin (SOM) are rarely immunoreactive for ErbB4 (1%). We next compared the numerical density (cells/mm³) and the distribution of interneurons between ErbB4−/− mice and wildtype controls. Based on data from 25 mice and 56,000 cells, we detected reductions of PV‐positive and nNOS‐positive cells in knockouts (−24% and −27%, respectively) but only a minor reduction of CCK‐positive cells; no changes in SOM‐positive cells were observed. The overall reduction of interneurons was verified by quantification of GAD67‐immunoreactive cells (−24% in ErbB4−/− mice). The reduction of interneurons along the dorsoventral axis was more severe in intermediate and ventral portions than in the dorsal hippocampus, and regional reductions occurred in the CA1–3 regions and subiculum, whereas we found no significant changes in the dentate gyrus (DG). The expression by different populations of interneurons suggests that ErbB4 can modulate several microcircuits within the hippocampus and mediate the previously reported effects of NRG1 on network oscillations and synaptic plasticity. The selective reduction of GABAergic cells in ErbB4−/− mice is consistent with the role of NRG‐ErbB4 signaling in the generation and migration of interneurons during development, and with neuronal and behavioral functional deficits in adult ErbB4 knockouts. © 2009 Wiley‐Liss, Inc.     

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.     

Cholecystokinin immunoreactive neurons in the basolateral amygdala of the rhesus monkey (Macaca mulatta)

Several distinct subpopulations of interneurons (INs) in the amygdalar basolateral nuclear complex (BNC) of the rat can be recognized on the basis of their expression of calcium-binding proteins and neuropeptides, including parvalbumin (PV), somatostatin (SOM), calretinin (CR), and cholecystokinin (CCK). In the rat BNC CCK is expressed in two separate IN subpopulations, termed large (CCK_L) and small (CCK_S). These subpopulations exhibit distinct connections indicative of discrete functional roles in the circuitry of the BNC. Although there have been several studies of PV+, SOM+, and CR+ INs in the primate BNC, there is almost no information regarding CCK+ INs in these species. Therefore, in the present study the distribution and morphology of CCK+ INs and their axon terminals in the BNC of the monkey was investigated. CCK immunoreactivity in the BNC was observed in somata and proximal dendrites of nonpyramidal neurons, as well as in axon terminals. A moderate density of CCK+ INs was found in all nuclei of the BNC. CCK+ INs in the BNC were morphologically heterogeneous, with both small and large varieties observed. All CCK+ somata gave rise to 2–4 dendrites that branched sparingly and were aspiny. CCK+ axon terminals in the BNC were found both in the neuropil and forming pericellular baskets contacting somata of pyramidal cells. In addition, many CCK+ neurons were contacted by multiple CCK+ terminals, indicative of the existence of a CCK interneuronal network. These data indicate that the morphology of CCK+ INs in the monkey is very similar to that of the rat.     

Multiple anxiogenic drugs recruit a parvalbumin-containing subpopulation of GABAergic interneurons in the basolateral amygdala

The basolateral amygdala is a nodal structure within a distributed and interconnected network that regulates anxiety states and anxiety-related behavior. Administration of multiple anxiogenic drugs increases cellular responses (i.e., increases c-Fos expression) in a subregion of the basolateral amygdala, but the neurochemical phenotypes of these cells are not known. The basolateral amygdala contains glutamatergic projection neurons and several populations of γ-aminobutyric acid-synthesizing (GABAergic) interneurons, including a population of parvalbumin (PV)-expressing GABAergic interneurons that co-express the excitatory 5-HT_2A receptor. The role for these PV-expressing GABAergic interneurons in anxiety-states is unclear. In this experiment we examined the effects of multiple anxiogenic drugs including the 5-HT_2C/2A receptor agonist m-chlorophenyl piperazine (mCPP), the adenosine receptor antagonist caffeine, the α₂-adrenoreceptor antagonist yohimbine and the partial inverse agonist at the benzodiazepine allosteric site on the GABA_A receptor, N-methyl-beta-carboline-3-carboxamide (FG-7142), on c-Fos expression in PV-immunoreactive (PV-ir) interneurons in subdivisions of the basolateral amygdala. All drugs with the exception of mCPP increased c-Fos expression in PV-ir neurons in the basolateral amygdaloid nucleus, anterior part (BLA). The numbers of c-Fos-immunoreactive (c-Fos-ir)/PV-ir GABAergic interneurons in the BLA were positively correlated with the numbers of c-Fos-ir serotonergic neurons in the mid-rostrocaudal dorsal raphe nucleus (DR) and with a measure of anxiety-related behavior. All four drugs increased c-Fos expression in non-PV-ir cells in most of the subdivisions of the basolateral amygdala that were sampled, compared with vehicle-injected controls. Together, these data suggest that the PV/5-HT_2A receptor expressing GABAergic interneurons in the basolateral amygdala are part of a DR-basolateral amygdala neuronal circuit modulating anxiety-states and anxiety-related behavior.     

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.     

Physiological and morphological characterization of parvalbumin-containing interneurons of the rat basolateral amygdala

The basolateral amygdala (BLA) is critical for the generation of emotional behavior and the formation of emotional memory. Understanding the neuronal mechanisms that contribute to emotional information processing in the BLA will ultimately require knowledge of the anatomy and physiology of its constituent neurons. Two major cell classes exist in the BLA, pyramidal projection neurons and nonpyramidal interneurons. Although the properties of projection neurons have been studied in detail, little is known about the properties of BLA interneurons. We have used whole-cell patch clamp recording techniques to examine the physiological properties of 48 visually identified putative interneurons from the rat anterior basolateral amygdalar nucleus. Here, we report that BLA interneurons can be differentiated into four electrophysiologically distinct subtypes based on their intrinsic membrane properties and their response to afferent synaptic input. Interneuron subtypes were named according to their characteristic firing pattern generated in response to transient depolarizing current injection and were grouped as follows: 1) burst-firing interneurons (n = 13), 2) regular-firing interneurons (n = 11), 3) fast-firing interneurons (n = 10), and 4) stutter-firing interneurons (n = 14). Post hoc histochemical visualization confirmed that all 48 recorded neurons had morphological properties consistent with their being local circuit interneurons. Moreover, by using triple immunofluorescence (for biocytin, calcium-binding proteins, and neuropeptides) in conjunction with patch clamp recording, we further demonstrated that over 60% of burst-firing and stutter-firing interneurons also expressed the calcium-binding protein parvalbumin (PV(+)). These data demonstrate that interneurons of the BLA show both physiological and neurochemical diversity. Moreover, we demonstrate that the burst- and stutter-firing patterns positively correlate with PV(+) immunoreactivity, suggesting that these neurons may represent functionally distinct subpopulations.     

Laminar Distribution of Neurochemically-Identified Interneurons and Cellular Co-expression of Molecular Markers in Epileptic Human Cortex

Inhibitory GABAergic interneurons are fundamental elements of cortical circuits and play critical roles in shaping network activity. Dysfunction of interneurons can lead to various brain disorders, including epilepsy, schizophrenia, and anxiety. Based on the electrophysiological properties, cell morphology, and molecular identity, interneurons could be classified into various subgroups. In this study, we investigated the density and laminar distribution of different interneuron types and the co-expression of molecular markers in epileptic human cortex. We found that parvalbumin (PV) and somatostatin (SST) neurons were distributed in all cortical layers except layer I, while tyrosine hydroxylase (TH) and neuropeptide Y (NPY) were abundant in the deep layers and white matter. Cholecystokinin (CCK) neurons showed a high density in layers IV and VI. Neurons with these markers constituted ~7.2% (PV), 2.6% (SST), 0.5% (TH), 0.5% (NPY), and 4.4% (CCK) of the gray-matter neuron population. Double- and triple-labeling revealed that NPY neurons were also SST-immunoreactive (97.7%), and TH neurons were more likely to express SST (34.2%) than PV (14.6%). A subpopulation of CCK neurons (28.0%) also expressed PV, but none contained SST. Together, these results revealed the density and distribution patterns of different interneuron populations and the overlap between molecular markers in epileptic human cortex.     

Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala

There are discrete subpopulations of GABAergic interneurons in the basolateral amygdala (ABL) that contain particular neuropeptides or calcium-binding proteins (calbindin-D28k, parvalbumin (PV), or calretinin). The present study employed a dual-labeling immunofluorescence technique combined with confocal laser scanning microscopy to investigate the neurochemical characteristics of the interneuronal subpopulation containing somatostatin (SOM). The great majority of SOM+ neurons in the ABL exhibited GABA immunoreactivity (66-82% depending on the nucleus). These SOM+ neurons constituted 11-18% of the GABA+ population. There was also extensive colocalization of SOM with calbindin (CB) in all nuclei of the ABL, but no colocalization of SOM with parvalbumin, calretinin, or vasoactive intestinal polypeptide. In the basolateral nucleus more than 90% of SOM+ neurons also exhibited CB immunoreactivity, whereas in the lateral nucleus about two-thirds of SOM+ neurons contained significant levels of CB. These SOM/CB neurons constituted about one quarter of the CB+ population in the basolateral nucleus and about one third of the CB+ population in the lateral nucleus. These results, in conjunction with the findings of previous studies, indicate that there are at least three major subpopulations of GABAergic interneurons in the ABL: (i) SOM+ neurons (most of which also contain CB and/or neuropeptide Y); (ii) PV+ neurons (most of which also contain CB); and (iii) CR+ neurons (most of which also contain vasoactive intestinal polypeptide).     

Postsynaptic targets of somatostatin-containing interneurons in the rat basolateral amygdala

The basolateral amygdala contains several subpopulations of inhibitory interneurons that can be distinguished on the basis of their content of calcium-binding proteins or peptides. Although previous studies have shown that interneuronal subpopulations containing parvalbumin (PV) or vasoactive intestinal peptide (VIP) innervate distinct postsynaptic domains of pyramidal cells as well as other interneurons, very little is known about the synaptic outputs of the interneuronal subpopulation that expresses somatostatin (SOM). The present study utilized dual-labeling immunocytochemical techniques at the light and electron microscopic levels to analyze the innervation of pyramidal cells, PV+ interneurons, and VIP+ interneurons in the anterior basolateral amygdalar nucleus (BLa) by SOM+ axon terminals. Pyramidal cell somata and dendrites were selectively labeled with antibodies to calcium/calmodulin-dependent protein kinase II (CaMK); previous studies have shown that the vast majority of dendritic spines, whether CAMK+ or not, arise from pyramidal cells. Almost all SOM+ axon terminals formed symmetrical synapses. The main postsynaptic targets of SOM+ terminals were small-caliber CaMK+ dendrites and dendritic spines, some of which were CaMK+. These SOM+ synapses with dendrites were often in close proximity to asymmetrical (excitatory) synapses to these same structures formed by unlabeled terminals. Few SOM+ terminals formed synapses with CaMK+ pyramidal cell somata or large-caliber (proximal) dendrites. Likewise, only 15% of SOM+ terminals formed synapses with PV+, VIP+, or SOM+ interneurons. These findings suggest that inhibitory inputs from SOM+ interneurons may interact with excitatory inputs to pyramidal cell distal dendrites in the BLa. These interactions might affect synaptic plasticity related to emotional learning.     

Immunochemical characterization of inhibitory mouse cortical neurons: Three chemically distinct classes of inhibitory cells

The cerebral cortex has diverse types of inhibitory neurons. In rat cortex, past research has shown that parvalbumin (PV), somatostatin (SOM), calretinin (CR), and cholecystokinin (CCK) label four distinct chemical classes of GABAergic interneurons. However, in contrast to rat cortex, previous studies indicate that there is significant colocalization of SOM and CR in mouse cortical inhibitory neurons. In the present study we further characterized immunochemical distinctions among mouse inhibitory cortical neurons by double immunochemical labeling with chemical markers. We found that, PV, SOM, and vasointenstinal peptide (VIP) reliably identify three nonoverlapping distinct subpopulations, as there was no overlap of immunoreactivity between PV and all the other chemical markers tested, and SOM and VIP did not show any overlap in labeled neurons in all the cortical areas. In comparison, there was significant overlap in combinations of other chemical markers. With some laminar and regional variations, the average overlap of SOM/CR (percentage of SOM+ cells expressing CR) and SOM/neuropeptide tyrosine (NPY) across all examined layers and cortical regions was 21.6% and 7.1%, respectively. The average overlap of VIP/CR, VIP/NPY, and CR/NPY was 34.2%, 9.5%, and 10%, respectively. We quantified and assessed the percentages of marker‐positive GABAergic cells, and showed that the nonoverlapping subpopulations (i.e., PV+, SOM+ and VIP+ cells) accounted for about 60% of the GABAergic cell population. Taken together, our data reveal important chemical distinctions between mouse inhibitory cortical neurons and indicate that PV, SOM, and VIP can differentially label a majority of mouse inhibitory cortical neurons. J. Comp. Neurol. 518:389–404, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Adult microbiota‐deficient mice have distinct dendritic morphological changes: differential effects in the amygdala and hippocampus

Increasing evidence implicates the microbiota in the regulation of brain and behaviour. Germ‐free mice (GF; microbiota deficient from birth) exhibit altered stress hormone signalling and anxiety‐like behaviours as well as deficits in social cognition. Although the mechanisms underlying the ability of the gut microbiota to influence stress responsivity and behaviour remain unknown, many lines of evidence point to the amygdala and hippocampus as likely targets. Thus, the aim of this study was to determine if the volume and dendritic morphology of the amygdala and hippocampus differ in GF versus conventionally colonized (CC) mice. Volumetric estimates revealed significant amygdalar and hippocampal expansion in GF compared to CC mice. We also studied the effect of GF status on the level of single neurons in the basolateral amygdala (BLA) and ventral hippocampus. In the BLA, the aspiny interneurons and pyramidal neurons of GF mice exhibited dendritic hypertrophy. The BLA pyramidal neurons of GF mice had more thin, stubby and mushroom spines. In contrast, the ventral hippocampal pyramidal neurons of GF mice were shorter, less branched and had less stubby and mushroom spines. When compared to controls, dentate granule cells of GF mice were less branched but did not differ in spine density. These findings suggest that the microbiota is required for the normal gross morphology and ultrastructure of the amygdala and hippocampus and that this neural remodelling may contribute to the maladaptive stress responsivity and behavioural profile observed in GF mice.     

Corticotropin-releasing factor (CRF), but not corticosterone, increases basolateral amygdala CRF-binding protein

Corticotropin-releasing factor (CRF) is a key mediator of the behavioral, autonomic, and endocrine responses to stress. CRF binds two receptors and a CRF-binding protein (CRF-BP), which may inactivate or modulate the actions of CRF at its receptors. The amygdala is an important anatomical substrate for CRF and contains CRF, its receptors, and CRF-BP. Our previous studies demonstrated that acute stress increases basolateral amygdala (BLA) CRF-BP mRNA. However, factors that may be responsible for this increase remain unclear. Both CRF and corticosterone are released during stress and are known to increase CRF-BP in vitro. However, the effects of these agents in vivo on brain CRF-BP have not been studied. Therefore, we examined the effects of CRF and corticosterone administration on BLA CRF-BP mRNA in rats. The findings demonstrate that intracerebroventricular CRF (5 microg) significantly increases BLA CRF-BP mRNA 9 h post-infusion, a time point consistent with that observed for the effects of acute stress-induced increases in CRF-BP. In contrast, injection of corticosterone at a dose mimicking acute stress (6.5 mg/kg sc) failed to increase BLA CRF-BP mRNA 9 h post-injection. Surprisingly, two different CRF antagonists failed to block CRF-induced increases in CRF-BP mRNA. These results suggest that CRF, but not corticosterone, may be responsible for stress-induced increases in BLA CRF-BP gene expression. Furthermore, this effect appears to be mediated by mechanisms other than the identified CRF receptors.     

Cholecystokinin enhances GABAergic inhibitory transmission in basolateral amygdala

The neuropeptide cholecystokinin (CCK) is anxiogenic in studies of human and animal behavior. As the amygdala formation has been implicated in generation of emotional states such as anxiety, we tested the effect of CCK on spontaneous synaptic events in the basolateral amygdala (BLA) using whole cell patch recordings in rat brain slice preparation. We found that CCK increased the frequency of spontaneous inhibitory postsynaptic potentials (sIPSPs) and currents (sIPSCs). This effect was blocked by the fast sodium channel blocker tetrodotoxin (TTX), indicating that the CCK effect is likely mediated by direct excitation of GABAergic interneurons. The CCK(B) receptor subtype antagonist, CR2945, blocked the CCK effect, while CCK4, a specific CCK(B) agonist, increased sIPSC frequency. We hypothesize that these actions may underlie the anxiogenic effects of CCK observed in behavioral studies.     

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.