The basolateral nuclear complex (BNC) of the amygdala plays an important role in the generation of emotional/motivational behavior and the consolidation of emotional memories. Activation of M1 cholinergic receptors (M1Rs) in the BNC is critical for memory consolidation. Previous receptor binding studies in the monkey amygdala demonstrated that the BNC has a high density of M1Rs, but did not have sufficient resolution to identify which neurons in the BNC expressed them. This was accomplished in the present immunohistochemical investigation using an antibody for the m1 receptor (m1R). Analysis of m1Rs in the monkey BNC using immunoperoxidase techniques revealed that their expression was very dense in the BNC, and suggested that virtually all of the pyramidal projection neurons (PNs) in all of the BNC nuclei were m1R-immunoreactive (m1R+). This was confirmed with dual-labeling immunofluorescence using staining for calcium/calmodulin-dependent protein kinase II (CaMK) as a marker for BNC PNs. However, additional dual-labeling studies indicated that one-third of inhibitory interneurons (INs) expressing glutamic acid decarboxylase (GAD) were also m1R+. Moreover, the finding that 60% of parvalbumin (PV) immunoreactive neurons were m1R+ indicated that this IN subpopulation was the main GAD+ subpopulation exhibiting m1R expression. The cholinergic innervation of the amygdala is greatly reduced in Alzheimer's disease and there is currently considerable interest in developing selective M1R positive allosteric modulators (PAMs) to treat the symptoms. The results of the present study indicate that M1Rs in both PNs and INs in the primate BNC would be targeted by M1R PAMs.

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 ( approximately 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.

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 (CCKL ) and small (CCKS ). 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.

The basolateral nuclear complex of the amygdala (BLC) receives a dense serotonergic innervation that appears to play a critical role in the regulation of mood and anxiety. However, little is known about how serotonergic inputs interface with different neuronal subpopulations in this region. To address this question, dual-labeling immunohistochemical techniques were used at the light and electron microscopic levels to examine inputs from serotonin-immunoreactive (5-HT+) terminals to different neuronal subpopulations in the rat BLC. Pyramidal cells were labeled by using antibodies to calcium/calmodulin-dependent protein kinase II, whereas different interneuronal subpopulations were labeled by using antibodies to a variety of interneuronal markers including parvalbumin (PV), vasoactive intestinal peptide (VIP), calretinin, calbindin, cholecystokinin, and somatostatin. The BLC exhibited a dense innervation by thin 5-HT+ axons. Electron microscopic examination of the anterior basolateral nucleus (BLa) revealed that 5-HT+ axon terminals contained clusters of small synaptic vesicles and a smaller number of larger dense-core vesicles. Serial section reconstruction of 5-HT+ terminals demonstrated that 76% of these terminals formed synaptic junctions. The great majority of these synapses were symmetrical. The main targets of 5-HT+ terminals were spines and distal dendrites of pyramidal cells. However, in light microscopic preparations it was common to observe apparent contacts between 5-HT+ terminals and all subpopulations of BLC interneurons. Electron microscopic analysis of the BLa in sections dual-labeled for 5-HT/PV and 5-HT/VIP revealed that many of these contacts were synapses. These findings suggest that serotonergic axon terminals differentially innervate several neuronal subpopulations in the BLC.

Cortical pyramidal neurons (PNs) containing nonphosphorylated neurofilaments (NNFs) localized with the SMI-32 monoclonal antibody have been shown to be especially vulnerable to degeneration in Alzheimer's disease (AD). The present investigation is the first to study the expression of SMI-32+ NNFs in neurons of the basolateral nuclear complex of the amygdala (BNC), which contains cortex-like PNs and nonpyramidal neurons (NPNs). We observed that PNs in the rat basolateral nucleus (BL), but not in the lateral (LAT) or basomedial (BM) nuclei, have significant levels of SMI-32-ir in their somata with antibody diluents that did not contain Triton X-100, but staining in these cells was greatly attenuated when the antibody diluent contained 0.3% Triton. Using Triton-containing diluents, we found that all SMI-32+ neurons in all three of the BNC nuclei were NPNs. Using a dual-labeling immunoperoxidase technique, we demonstrated that most of these SMI-32+ NPNs were parvalbumin-positive (PV+) or somatostatin-positive NPNs but not vasoactive intestinal peptide-positive or neuropeptide Y-positive NPNs. Using a technique that combines retrograde tracing with SMI-32 immunohistochemistry using intermediate levels of Triton in the diluent, we found that all BNC neurons projecting to the mediodorsal thalamic nucleus (MD) were large NPNs, and most were SMI-32+. In contrast, BNC neurons projecting to the ventral striatum or cerebral cortex were PNs that expressed low levels of SMI-32 immunoreactivity (SMI-32-ir) in the BL, and no SMI-32-ir in the LAT or BM. These data suggest that the main neuronal subpopulations in the BNC that degenerate in AD may be PV+ and MD-projecting NPNs.

As is well recognized, serotonergic (5-HT) fibers distribute widely throughout the forebrain, including the amygdala. Although a few reports have examined the 5-HT innervation of select nuclei of the amygdala in the rat, no previous report has described overall 5-HT projections to the amygdala in the rat. Using immunostaining for the serotonin transporter, SERT, we describe the complete pattern of distribution of 5-HT fibers to the amygdala (proper) and to the extended amygdala in the rat. Based on its ontogenetic origins, the amygdala was subdivided into two major parts, pallial and subpallial components, with the pallial component further divided into superficial and deep nuclei (Olucha-Bordonau et al. 2015). SERT+ fibers were shown to distributed moderately to densely to the deep and cortical pallial nuclei, but, by contrast, lightly to the subpallial nuclei. Specifically, 1) of the deep pallial nuclei, the lateral, basolateral, and basomedial nuclei contained a very dense concentration of 5-HT fibers; 2) of the cortical pallial nuclei, the anterior cortical and amygdala-cortical transition zone rostrally and the posteromedial and posterolateral nuclei caudally contained a moderate concentration of 5-HT fibers; and 3) of the subpallial nuclei, the anterior nuclei and the rostral part of the medial (Me) nuclei contained a moderate concentration of 5-HT fibers, whereas caudal regions of Me as well as the central nuclei and the intercalated nuclei contained a sparse/light concentration of 5-HT fibers. With regard to the extended amygdala (primarily the bed nucleus of stria terminalis; BST), on the whole, the BST contained moderate numbers of 5-HT fibers, spread fairly uniformly throughout BST. The findings are discussed with respect to a critical serotonergic influence on the amygdala, particularly on the basal complex, and on the extended amygdala in the control of states of fear and anxiety. J. Comp. Neurol. 525:116-139, 2017. © 2016 Wiley Periodicals, Inc.