Elsevier

Neuroscience

Volume 146, Issue 1, 25 April 2007, Pages 306-320
Neuroscience

Neuroanatomy
Neuronal localization of 5-HT type 2A receptor immunoreactivity in the rat basolateral amygdala

https://doi.org/10.1016/j.neuroscience.2007.01.047Get rights and content

Abstract

Although it is well established that there are alterations in type 2A 5-HT receptors (5-HT2ARs) in the basolateral nuclear complex of the amygdala (BLC) in several neuropsychiatric disorders, very little is known about the neuronal localization of these receptors in this brain region. Single-labeling and dual-labeling immunohistochemical techniques were utilized in the rat to address this question. Three different 5-HT2AR antibodies were used, each producing distinct but overlapping patterns of immunostaining. Two of three 5-HT2AR antibodies mainly stained pyramidal projection neurons in the BLC. The third antibody only stained pyramidal cells in the dorsolateral subdivision of the lateral amygdalar nucleus. With one of the antibodies, the most intensely stained neurons were a population of large nonpyramidal neurons whose morphology and distribution closely resembled those shown in previous studies to project to the mediodorsal thalamic nucleus (MD). This was confirmed in the present study using a technique that combined 5-HT2AR immunohistochemistry with fluorogold retrograde tract-tracing. Two of three 5-HT2AR antibodies stained large numbers of parvalbumin-containing interneurons in the BLC. One of these two antibodies also stained a subpopulation of somatostatin-containing neurons. None of the 5-HT2AR antibodies stained significant numbers of the other two main interneuronal subpopulations, the large cholecystokinin-positive neurons or the small interneurons that exhibit extensive colocalization of calretinin and cholecystokinin. Since each of the three antibodies was raised against a distinct immunizing antigen, they may recognize different conformations of 5-HT2AR in different neuronal domains. The expression of 5-HT2ARs in pyramidal cells and parvalbumin-positive interneurons in the BLC is consistent with the results of previous electrophysiological studies, and suggests that 5-HT may produce excitation of several neuronal populations in the BLC via 5-HT2ARs.

Section snippets

Tissue preparation

A total of 28 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

The pattern of immunostaining obtained in the amygdala with the three 5-HT2AR antibodies was different (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7), and each will be described separately.

Discussion

Although there is evidence that the monoclonal and polyclonal antibodies used in this study are specific for 5-HT2AR (see above), their staining patterns in the BLC were different. The OC antibody mainly stained nonpyramidal interneurons that expressed PV or SOM, although numerous dendrite-like processes were stained in the Ldl. The density of the latter processes indicates that they probably arise from pyramidal cells, the principal neurons of the BLC. In contrast, the BD antibody appeared to

Acknowledgments

The authors would like to thank the following investigators for their donations of the antibodies used in this study: Dr. Kenneth Baimbridge (University of British Columbia) for the rabbit polyclonal antibody to parvalbumin, Dr. John H. Walsh (CURE/Digestive Diseases Research Center, Antibody/RIA Core, NIH grant #DK41301, Los Angeles, CA, USA) for the mouse anti-CCK antibody, Dr. Bryan Roth (University of North Carolina School of Medicine) for the Ab51 polyclonal 5-HT2AR antibody, and Dr.

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