Elsevier

Brain Research

Volume 976, Issue 2, 27 June 2003, Pages 171-184
Brain Research

Research report
Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala

https://doi.org/10.1016/S0006-8993(03)02625-8Get rights and content

Abstract

Specific neuronal populations in the basolateral amygdala (ABL) exhibit immunoreactivity for distinct neuropeptides and calcium-binding proteins. In the present study, immunohistochemical techniques were used to analyze neurons in the rat ABL that contain cholecystokinin (CCK). Some pyramidal projection neurons in the anterior subdivision of the basolateral nucleus exhibited low levels of CCK immunoreactivity in rats that received injections of colchicine to interrupt axonal transport; staining was concentrated in the axon initial segments of these cells. High levels of CCK immunoreactivity were observed in two subpopulations of nonpyramidal interneurons in all nuclei of the ABL: (1) type L neurons (characterized by large somata and thick dendrites), and (2) type S neurons (characterized by small somata and thin dendrites). Dual-labeling immunofluorescence studies using confocal laser scanning microscopy revealed that many (30–40%) type L CCK+ interneurons exhibited immunoreactivity for calbindin (CB), but not for parvalbumin (PV), calretinin (CR), or vasoactive intestinal polypeptide (VIP). In contrast, there was extensive colocalization of CR and VIP with CCK in type S neurons, but no significant colocalization with CB or PV. In addition, the majority of CR and VIP interneurons exhibited colocalization of both neurochemicals. Collectively, the results of this and previous studies indicate that there are at least four distinct interneuronal subpopulations in the ABL: (1) PV+ neurons (the great majority of which are CB+); (2) SOM+ neurons (many of which are CB+ and NPY+); (3) large CCK+ neurons (some of which are CB+); and (4) small bipolar/bitufted neurons that exhibit various amounts of colocalization of CCK, VIP, and CR.

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.

References (58)

  • A.J. McDonald

    Calretinin immunoreactive neurons in the basolateral amygdala of the rat and monkey

    Brain Res.

    (1994)
  • A.J. McDonald et al.

    Coexistence of GABA and peptide immunoreactivity in non-pyramidal neurons of the basolateral amygdala

    Neurosci. Lett.

    (1989)
  • A.J. McDonald et al.

    Parvalbumin containing neurons in the rat basolateral amygdala: morphology and colocalization of calbindin D-28k

    Neuroscience

    (2001)
  • A.J. McDonald et al.

    Colocalization of calcium-binding proteins and gamma-aminobutyric acid in neurons of the rat basolateral amygdala

    Neuroscience

    (2001)
  • A.J. McDonald et al.

    Localization of the CB1 type cannabinoid receptor in the rat basolateral amygdala: High concentrations in a subpopulation of cholecystokinin containing interneurons

    Neuroscience

    (2001)
  • A.J. McDonald et al.

    Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala

    Brain Res.

    (2002)
  • O.E. Millhouse et al.

    Neuronal configurations in lateral and basolateral amygdala

    Neuroscience

    (1983)
  • M. Savasta et al.

    Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization

    Neurosci. Lett.

    (1988)
  • H. Sorvari et al.

    Parvalbumin-immunoreactive neurons make inhibitory synapses on pyramidal cells in the human amygdala: a light and electron microscopic study

    Neurosci. Lett.

    (1996)
  • H. Sorvari et al.

    Calretinin-immunoreactive terminals make synapses on calbindin D28k-immunoreactive neurons in the lateral nucleus of the human amygdala

    Brain Res.

    (1998)
  • K. Tsou et al.

    Cannabinoid CB1 receptors are localized primarily on cholecystokinin-containing Gabaergic interneurons in the rat hippocampal formation

    Neuroscience

    (1999)
  • M.M. Voigt et al.

    Preprocholecystokinin mRNA in rat brain: regional expression includes thalamus

    Brain Res.

    (1988)
  • F.G. Wouterlood et al.

    Two-laser dual-immunofluorescence confocal laser scanning microscopy using Cy2- and Cy5-conjugated secondary antibodies: unequivocal detection of co-localization of neuronal markers

    Brain Res. Protoc.

    (1998)
  • L. Zaborszky et al.

    Cholecystokinin innervation of the ventral striatum: a morphological and radioimmunological study

    Neuroscience

    (1985)
  • J. Carlsen

    Immunocytochemical localization of glutamate decarboxylase in the rat basolateral amygdaloid nucleus, with special reference to GABAergic innervation of amygdalostriatal projection neurons

    J. Comp. Neurol.

    (1988)
  • F. Conde et al.

    Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: distribution and morphology

    J. Comp. Neurol.

    (1994)
  • T.F. Freund et al.

    Interneurons of the hippocampus

    Hippocampus

    (1996)
  • Cited by (0)

    View full text