A novel subpopulation of 5-HT type 3A receptor subunit immunoreactive interneurons in the rat basolateral amygdala

Abstract

The amygdalar basolateral nuclear complex (BLC) has very high levels of the 5-HT type 3 receptor (5-HT(3)R). Previous studies have reported that 5-HT(3)R protein in the BLC is expressed in interneurons and that 5-HT(3)R mRNA is coexpressed with GABA and certain neuropeptides or calcium-binding proteins in these cells. However, there have been no detailed descriptions of the distribution of 5-HT(3)R+ neurons in the rat amygdala, and no quantitative studies of overlap of neurons expressing 5-HT(3)R protein with distinct interneuronal subpopulations in the BLC. The present investigation employed dual-labeling immunohistochemistry using antibodies to the 5-HT-3A receptor subunit (5-HT(3A)R) and specific interneuronal markers to address these questions. These studies revealed that there was a moderate density of nonpyramidal 5-HT(3A)R+ neurons in the BLC at all levels of the amygdala. In addition, immunostained cells were also seen in anterior portions of the cortical and medial nuclei. Although virtually all 5-HT(3A)R+ neurons in the BLC were GABA+, very few expressed neuropeptide or calcium-binding protein markers for individual subpopulations. The main interneuronal marker expressed by 5-HT(3A)R+ neurons was cholecystokinin (CCK), but only 8-16% of 5-HT(3)R+ neurons in the BLC, depending on the nucleus, were CCK+. Most of these CCK+/5-HT(3A)R+ double-labeled neurons appeared to belong to the subpopulation of large type L CCK+ interneurons. Very few 5-HT(3A)R+ neurons expressed calretinin, vasoactive intestinal peptide, or parvalbumin, and none expressed somatostatin or calbindin. Thus, the great majority of neurons expressing 5-HT(3A)R protein appear to constitute a previously unrecognized subpopulation of GABAergic interneurons in the BLC.

Fig. 1

Distribution of 5-HT_3AR+ neurons in the rat amygdala. Neurons were plotted from two 50 μm thick sections at each level of the amygdala (Bregma −2.3, −3.2, and −4.0 of Paxinos and Watson, 1997). A third section located between the two sections was stained with cresyl violet to identify nuclear borders. Abbreviations: AHA, amygdalohippocampal area; BLa, anterior subdivision of the basolateral nucleus; BLp, posterior subdivision of the basolateral nucleus; BMA, anterior basomedial nucleus; BMP, posterior basomedial nucleus; CL, lateral subdivision of the central nucleus; Coa, anterior cortical nucleus; Copl, posterolateral cortical nucleus; Copm, posteromedial cortical nucleus; Ldl, dorsolateral subdivision of the lateral nucleus; Lvm, ventromedial subdivision of the lateral nucleus; M, medial nucleus; ST, stria terminalis.

Fig. 2

5-HT_3AR immunoreactivity in the BLC. Arrows indicate immunostained cells, all of which appear to be nonpyramidal. A) Low power photomicrograph of the anterior subdivision of the basolateral nucleus (BLa) and the ventromedial subdivision of the lateral nucleus (Lvm) at the bregma −2.6 level (Paxinos and Watson, 1997). Asterisk is located between two immunostained neurons shown at higher magnification in C. ec, external capsule. Scale bar = 50 μm. B) Higher power photomicrograph of the Lvm at the bregma −3.3 level. Scale bar = 50 μm. C) High power photomicrograph of the two immunostained neurons indicated by an asterisk in A, but mediolateral orientation is flipped. Scale bar = 25 μm.

Fig. 3

Dual localization of 5-HT_3AR immunoreactivity (green) and interneuronal marker immunoreactivity (red) in the BLC using immunofluorescence confocal laser scanning microscopy. All images are merged images. A–C) Dual localization of 5-HT_3AR (green) and GABA (red) immunoreactivity in the Lvm (A, C) and BLa (B). Note that all 5-HT_3AR+ neurons in these fields are double-labeled (yellow). D–G) Dual localization of 5-HT_3AR (green) and CCK (red) immunoreactivity in the Lvm (D, E) and BLa (F, G). Note that most CCK+ neurons are either large (type L neurons) or small (type S neurons). Most single-labeled 5-HT_3AR+ neurons (green) are medium-sized or small, whereas double-labeled 5-HT_3AR+/CCK+ neurons (yellow) are large. (H–I) Dual localization of 5-HT_3AR (green) and CR (red) immunoreactivity in the BLa (H) and Lvm (I). Although both 5-HT_3AR+ neurons and CR+ neurons are robustly labeled, there are no double-labeled 5-HT_3AR+/CR+ neurons in these fields. All scale bars = 25 μm.

Fig. 4

Diagram showing the overlap and relative proportions of neuronal populations containing calcium-binding proteins, neuropeptides, and 5-HT_3AR in the anterior subdivision of the basolateral nucleus (BLa). The relationship of the 5-HT_3AR+ subpopulation to the other interneuronal subpopulations is similar in the lateral nucleus. Thick lines indicate the 5-HT_3AR + subpopulation. Lines of intermediate thickness indicate two populations of calcium-binding proteins (CB+ and CR+ neurons), each of which is comprised of several subpopulations. Data on the relationships among SOM+, PV+, CB+, CR+, VIP, and CCK+ neurons is taken from previous quantitative studies (for a review see Mascagni and McDonald, 2003); the relative sizes of the rectangles representing these subpopulations are depicted in relation to the total population of GABA+ neurons in each nucleus (see Fig. 3 of McDonald and Mascagni, 2002). Note that the 5-HT_3AR+ subpopulation is relatively small and slightly overlaps the subpopulation of large type L CCK+ neurons (CCK_L) that do not contain CB. In the present study it appeared that none of the 5-HT_3AR+ neurons corresponded to the subpopulation of small type S CCK+ neurons (CCK_S).