Elsevier

Neuroscience

Volume 196, 24 November 2011, Pages 49-65
Neuroscience

Cellular and Molecular Neuroscience
Research Paper
Neuronal localization of M2 muscarinic receptor immunoreactivity in the rat amygdala

https://doi.org/10.1016/j.neuroscience.2011.08.032Get rights and content

Abstract

Muscarinic cholinergic neurotransmission in the amygdala is critical for memory consolidation in emotional/motivational learning tasks, but little is known about the neuronal distribution of different receptor subtypes. Immunohistochemistry was used in the present investigation to localize the m2 receptor (M2R). Differential patterns of M2R-immunoreactivity (M2R-ir) were observed in the somata and neuropil of the various amygdalar nuclei. Neuropilar M2R-ir was strongest in rostral portions of the basolateral nuclear complex (BLC). M2R-positive (M2R+) somata were seen in low numbers in all nuclei of the amygdala. Most M2R+ neurons associated with the BLC were in the lateral nucleus and external capsule. These cells were nonpyramidal neurons that contained glutamatic acid decarboxylase (GAD), somatostatin (SOM), and neuropeptide Y (NPY), but not parvalbumin (PV), calretinin (CR), or cholecystokinin (CCK). Little or no M2R-ir was observed in GAD+, PV+, CR+, or CCK+ axons in the BLC, but it was seen in some SOM+ axons and many NPY+ axons. M2R-ir was found in a small number of spiny and aspiny neurons of the central nucleus that were mainly located along the lateral and ventral borders of its lateral subdivision. Many of these cells contained SOM and NPY. M2R+ neurons were also seen in the medial nucleus, including a distinct subpopulation of neurons that surrounded its anteroventral subdivision. The latter neurons were negative for all neuronal markers analyzed. The intercalated nuclei (INs) were associated with two types of large M2R+ neurons, spiny and aspiny. The small principal neurons of the INs were M2R-negative. The somata and dendrites of the large spiny neurons, which were actually found in a zone located just outside of the rostral INs, expressed SOM and NPY, but not GAD. These findings indicate that acetylcholine can modulate a variety of discrete neuronal subpopulations in various amygdalar nuclei via M2Rs, especially neurons that express SOM and NPY.

Highlights

▶There is a high density of neuropilar M2 immunoreactivity in the amygdala, especially in rostral portions of the basolateral amygdala. ▶A subset of M2+ nonpyramidal neurons that express GABA, somatostatin, and neuropeptide Y were observed in the lateral nucleus and adjacent external capsule. ▶Large spiny and large aspiny neurons associated with the intercalated nuclei were M2R+. ▶Many neuropeptide Y containing axons in the basolateral amygdala were M2R+. ▶Discrete subpopulations of M2R+ neurons were also seen in the central and medial nuclei.

Section snippets

Tissue preparation

A total of 16 adult 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.

Immunoperoxidase studies

All of the immunoperoxidase studies were performed using the rabbit M2R antiserum. The pattern of M2R immunoreactivity (M2R-ir) in the forebrain at the level of the amygdala appeared to be identical to that obtained in previous studies using this antiserum (Levey et al., 1991, Levey et al., 1995b). Thus, staining in the cerebral cortex was predominately neuropilar and exhibited a bistratified configuration. M2R-ir in the striatum was mainly found in a population of large multipolar neurons. The

Discussion

This is the first detailed immunohistochemical investigation of M2R localization in the amygdala. Earlier immunohistochemical and in situ hydridization studies of muscarinic receptor protein and mRNA localization in the rat brain reported that M2R+ neurons were seen in low numbers in the amygdala, but they provided no details regarding their location within the nuclear complex (Levey et al., 1991). The present investigation found low numbers of M2R+ neurons in all nuclei of the amygdala,

Acknowledgments

The authors would like to thank Dr. John H. Walsh (CURE/Digestive Diseases Research Center, Antibody/RIA Core, NIH grant number DK41301, Los Angeles, CA) for the donation of the mouse anti-CCK antibody and Dr. Alison Buchan (University of British Columbia) for the donation of the mouse anti-somatostatin antibody. The authors would like to thank Dr. Jay F. Muller for his comments on an earlier version of this manuscript. This work was supported by NIH grant R01-DA027305.

References (84)

  • A.J. McDonald et al.

    Localization of GABA-like immunoreactivity in the monkey amygdala

    Neuroscience

    (1993)
  • 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.

    Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala

    Brain Res

    (2002)
  • A.J. McDonald et al.

    Neuropeptide Y and somatostatin-like immunoreactivity in the monkey amygdala: distribution, morphology, and differential coexistence

    Neuroscience

    (1995)
  • A.J. McDonald et al.

    Identification of putative nitric oxide producing neurons in the rat amygdala using NADPH-diaphorase histochemistry

    Neuroscience

    (1993)
  • M.M. Mesulam et al.

    Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1-Ch6)

    Neuroscience

    (1983)
  • A.E. Power et al.

    Muscarinic cholinergic influences in memory consolidation

    Neurobiol Learn Mem

    (2003)
  • S.T. Rouse et al.

    Localization of M(2) muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus

    Neurosci Lett

    (2000)
  • S.T. Rouse et al.

    Muscarinic receptor subtypes involved in hippocampal circuits

    Life Sci

    (1999)
  • J.F. Smiley et al.

    Infracortical interstitial cells concurrently expressing m2-muscarinic receptors, acetylcholinesterase and nicotinamide adenine dinucleotide phosphate-diaphorase in the human and monkey cerebral cortex

    Neuroscience

    (1998)
  • D.G. Spencer et al.

    Direct autoradiographic determination of M1 and M2 muscarinic acetylcholine receptor distribution in the rat brain: relation to cholinergic nuclei and projections

    Brain Res

    (1986)
  • M.J. Stillman et al.

    Elevation of hippocampal extracellular acetylcholine levels by methoctramine

    Brain Res Bull

    (1993)
  • C.N. Svendsen et al.

    Acetylcholinesterase staining of the human amygdala

    Neurosci Lett

    (1985)
  • W.A. Truitt et al.

    Anxiety-like behavior is modulated by a discrete subpopulation of interneurons in the basolateral amygdala

    Neuroscience

    (2009)
  • P. Turrini et al.

    Cholinergic nerve terminals establish classical synapses in the rat cerebral cortex: synaptic pattern and age-related atrophy

    Neuroscience

    (2001)
  • N.J. Woolf et al.

    Cholinergic systems in the rat brain: IProjections to the limbic telencephalon

    Brain Res Bull

    (1984)
  • 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 Brain Res Protoc

    (1998)
  • G.F. Alheid et al.

    Amygdala and extended amygdala

  • D.G. Amaral et al.

    Cholinergic innervation of the monkey amygdala: an immunohistochemical analysis with antisera to choline acetyltransferase

    J Comp Neurol

    (1989)
  • J. Apergis-Schoute et al.

    Muscarinic control of long-range GABAergic inhibition within the rhinal cortices

    J Neurosci

    (2007)
  • Y. Ben-Ari et al.

    Regional distribution of choline acetyltransferase and acetylcholinesterase within the amygdaloid complex and stria terminalis system

    Brain Res

    (1977)
  • D. Busti et al.

    Different fear states engage distinct networks within the intercalated cell clusters of the amygdala

    J Neurosci

    (2011)
  • N.S. Canteras et al.

    Organization of projections from the medial nucleus of the amygdala: a PHAL study in the rat

    J Comp Neurol

    (1995)
  • J. Carlsen et al.

    A correlated light and electron microscopic immunocytochemical study of cholinergic terminals and neurons in the rat amygdaloid body with special emphasis on the basolateral amygdaloid nucleus

    J Comp Neurol

    (1986)
  • J. Carlsen et al.

    Cholinergic projections from the basal forebrain to the basolateral amygdaloid complex: a combined retrograde fluorescent and immunohistochemical study

    J Comp Neurol

    (1985)
  • J.S. De Olmos et al.

    The projection field of the stria terminalis in the rat brain

    J Comp Neurol

    (1972)
  • A.A. Disney et al.

    Muscarinic acetylcholine receptors in macaque V1 are most frequently expressed by parvalbumin-immunoreactive neurons

    J Comp Neurol

    (2008)
  • F.J. Ehlert et al.

    Molecular biology, pharmacology, and brain distribution of subtypes of the muscarinic receptor

  • A. Erisir et al.

    Muscarinic receptor M(2) in cat visual cortex: laminar distribution, relationship to gamma-aminobutyric acidergic neurons, and effect of cingulate lesions

    J Comp Neurol

    (2001)
  • T.F. Freund et al.

    Interneurons of the hippocampus

    Hippocampus

    (1996)
  • M. Girgis

    Acetylcholinesterase enzyme localization in the amygdala: a comparative histochemical and ultrastructural study

    Acta Anat (Basel)

    (1980)
  • Cited by (0)

    View full text