Research reportImmunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala
Introduction
The basolateral nuclear complex of the amygdala (ABL) is critical for the generation of emotional responses and the formation of emotional memories [1], [2]. Understanding the manner in which the ABL processes emotionally relevant information will require identification of the major cell types in this region and an analysis of their interconnections. Previous studies have shown that there are two major cell classes in the ABL, pyramidal neurons and nonpyramidal neurons. Although these cells do not exhibit a laminar organization, their morphology, synaptology, electrophysiology, and pharmacology are remarkably similar to their namesakes in the cerebral cortex [3], [4], [12], [24], [30], [45], [47], [56]. The principal neurons in the ABL are large pyramidal-like projection neurons with spiny dendrites that utilize glutamate as an excitatory neurotransmitter [9], [25], [29], [30], [32], [41]. In contrast, most nonpyramidal neurons in the ABL are spine-sparse interneurons that utilize GABA as an inhibitory neurotransmitter [3], [25], [26], [41]. As in the cerebral cortex, subpopulations of nonpyramidal cells in the ABL can be distinguished on the basis of their content of calcium-binding proteins (parvalbumin [PV], calbindin [CB], and calretinin [CR]) and neuropeptides (somatostatin [SOM], neuropeptide Y [NPY], vasoactive intestinal polypeptide [VIP], and cholecystokinin [CCK]) [19], [27], [28], [36]. The results of previous studies indicate that there are at least three distinct subpopulations of interneurons in the ABL: (1) PV+ neurons (most of which also contain CB); (2) SOM+ neurons (most of which also contain CB), and (3) CR+ neurons that are morphologically similar to VIP+ neurons [35], [36], [38].
Immunohistochemical studies have demonstrated that the neuropeptide cholecystokinin (CCK) may be found in two types of ABL interneurons [27], [34]. One subpopulation consists of small neurons whose morphology resembles that of CR+ and VIP+ neurons, whereas the other subpopulation is comprised of larger neurons. Recent studies indicate that the large CCK+ neurons, but not the small CCK+ neurons, express significant amounts of the CB1 cannabinoid receptor [15], [37]. Although it is known that CCK+ neurons do not contain SOM [28], there have been no colocalization studies of CCK with other peptides or calcium-binding proteins. It is also not known if there is colocalization of VIP with CR in the population of small ABL interneurons. These questions are addressed in the present study using double-labeling immunohistochemical techniques.
Section snippets
Tissue preparation
A total of 26 male Sprague–Dawley rats (250–350 g; Harlan) 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. All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to produce reliable scientific data. Rats were anesthetized with chloral hydrate (580 mg/kg) and perfused intracardially with phosphate-buffered saline (PBS; pH 7.4) containing
Single-label immunoperoxidase experiments
In agreement with previous studies, all nuclei of the basolateral amygdalar complex contained a subpopulation of CCK+ nonpyramidal neurons [27], [34], [37], [40]. The great majority of these neurons could easily be classified as large or small (Fig. 1) [27], [34], [37]. The large neurons (type L) were multipolar, bipolar, or bitufted neurons with somata that averaged 15–20 μm in diameter (Fig. 2, Fig. 3). Most small neurons (type S) were bipolar or bitufted neurons with somata that averaged 10
Discussion
This investigation is the first comprehensive analysis of the relationship of CCK+ neurons to previously described interneuronal populations in the ABL recognized on the basis of their content of other neuropeptides or calcium-binding proteins. The results confirm the notion that there are two subpopulations of CCK+ interneurons in the ABL. Small type S CCK+ neurons exhibited extensive colocalization of CR and VIP, whereas the larger type L CCK+ neurons often exhibited colocalization of CB.
Acknowledgments
The donation of the mouse monoclonal antibody to gastrin/CCK (#9303) by Dr John H. Walsh and Helen Wong (CURE/Gastroenteric Biology Center, Los Angeles, CA, USA) and the rabbit anti-calbindin and anti-parvalbumin antisera by Kenneth G. Baimbridge (University of British Columbia) is greatly appreciated. This work was supported by NIH Grant NS38998.
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