Elsevier

Neuroscience

Volume 144, Issue 3, 9 February 2007, Pages 1015-1024
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2006.10.044Get rights and content

Abstract

The amygdalar basolateral nuclear complex (BLC) has very high levels of the 5-HT type 3 receptor (5-HT3R). Previous studies have reported that 5-HT3R protein in the BLC is expressed in interneurons and that 5-HT3R 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-HT3R+ neurons in the rat amygdala, and no quantitative studies of overlap of neurons expressing 5-HT3R 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-HT3AR) and specific interneuronal markers to address these questions. These studies revealed that there was a moderate density of nonpyramidal 5-HT3AR+ 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-HT3AR+ 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-HT3AR+ neurons was cholecystokinin (CCK), but only 8–16% of 5-HT3R+ neurons in the BLC, depending on the nucleus, were CCK+. Most of these CCK+/5-HT3AR+ double-labeled neurons appeared to belong to the subpopulation of large type L CCK+ interneurons. Very few 5-HT3AR+ neurons expressed calretinin, vasoactive intestinal peptide, or parvalbumin, and none expressed somatostatin or calbindin. Thus, the great majority of neurons expressing 5-HT3AR protein appear to constitute a previously unrecognized subpopulation of GABAergic interneurons in the BLC.

Section snippets

Tissue preparation

A total of 14 male Sprague–Dawley rats (250–350 g; Harlan, Indianapolis, IN, USA) were used in this study. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Use and Care Committee (IACUC) of the University of South Carolina. All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to produce reliable scientific data.

Rats

Results

5-HT3AR staining in the forebrain at the level of the amygdala appeared to be identical to that obtained in previous studies using this antibody (Morales et al., 1998). Thus, there was a moderate density of nonpyramidal 5-HT3AR+ neurons in the neocortex, hippocampus, and striatum, as well as in the BLC of the amygdala (Fig. 1). In addition, immunostained cells were also seen in anterior portions of the cortical and medial amygdalar nuclei. 5-HT3AR+ neurons were of similar morphology in all of

Discussion

This is the first study to use double-labeling immunohistochemistry to study the phenotypes of 5-HT3AR+ neurons in the BLC. As suggested in a previous immunohistochemical study (Morales et al., 1998), 5-HT3AR+ neurons in the amygdala were nonpyramidal interneurons that were mainly located in the BLC. The present investigation demonstrated that virtually all neurons in the BLC that expressed 5-HT3AR protein were GABA-immunoreactive, consistent with a previous report that 70–95% of telencephalic

Conclusion

In summary, some of the neurons expressing 5-HT3AR subunit protein in the BLC belong to a subpopulation of large GABAergic interneurons that are CCK+ (type L CCK+ interneurons), but most appear to constitute a previously unrecognized subpopulation of medium-sized GABAergic interneurons. In the awake state typical serotonergic neurons in the raphe fire in a clock-like manner at about 1–2 Hz, depending on the state of behavioral arousal (Jacobs and Azmitia, 1992). Thus, 5-HT3AR+ interneurons in

Acknowledgment

We are very grateful for the generous donation of the antibody to the 5-HT3AR subunit (antiserum 0165) by Dr. Marisela Morales (National Institute on Drug Abuse, Baltimore, MD, USA), the mouse anti-CCK antibody (antibody #9303) and mouse anti-VIP antibody (antibody CURE-V55) by Dr. John Walsh (CURE/Digestive Diseases Research Center, Antibody/RIA Core, NIH Grant #DK41301, Los Angeles, CA, USA), and the mouse anti-GABA antibody (antibody 115-AD5-A9) by Dr. Ismo Virtanen (University of Helsinki,

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