Elsevier

Neuroscience

Volume 357, 15 August 2017, Pages 349-362
Neuroscience

Evidence for M2 muscarinic receptor modulation of axon terminals and dendrites in the rodent basolateral amygdala: An ultrastructural and electrophysiological analysis

https://doi.org/10.1016/j.neuroscience.2017.06.019Get rights and content

Highlights

  • Electrophysiological studies show suppression of IPSCs in the basolateral amygdala (BLa) by M2 muscarinic receptors (M2Rs).

  • Dual cell recording indicated that M2Rs suppressed IPSCs generated by only a subpopulation of BLa interneurons.

  • M2R immunoreactivity was found in 23% of axon terminals of parvalbumin (PV) interneurons targeting somata and dendrites.

  • Many PV-negative inhibitory axon terminals, excitatory axon terminals, dendrites, and spines are also M2R+ in BLa.

  • One-quarter to two-thirds of M2Rs were plasma membrane-associated in M2R+ structures in BLa, depending on the structure.

Abstract

The basolateral amygdala receives a very dense cholinergic innervation from the basal forebrain that is important for memory consolidation. Although behavioral studies have shown that both M1 and M2 muscarinic receptors are critical for these mnemonic functions, there have been very few neuroanatomical and electrophysiological investigations of the localization and function of different types of muscarinic receptors in the amygdala. In the present study we investigated the subcellular localization of M2 muscarinic receptors (M2Rs) in the anterior basolateral nucleus (BLa) of the mouse, including the localization of M2Rs in parvalbumin (PV) immunoreactive interneurons, using double-labeling immunoelectron microscopy. Little if any M2R-immunoreactivity (M2R-ir) was observed in neuronal somata, but the neuropil was densely labeled. Ultrastructural analysis using a pre-embedding immunogold-silver technique (IGS) demonstrated M2R-ir in dendritic shafts, spines, and axon terminals forming asymmetrical (excitatory) or symmetrical (mostly inhibitory) synapses. In addition, about one-quarter of PV+ axon terminals and half of PV+ dendrites, localized using immunoperoxidase, were M2R+ when observed in single thin sections. In all M2R+ neuropilar structures, including those that were PV+, about one-quarter to two-thirds of M2R+ immunoparticles were plasma-membrane-associated, depending on the structure. The expression of M2Rs in PV+ and PV-negative terminals forming symmetrical synapses indicates M2R modulation of inhibitory transmission. Electrophysiological studies in mouse and rat brain slices, including paired recordings from interneurons and pyramidal projection neurons, demonstrated M2R-mediated suppression of GABA release. These findings suggest cell-type-specific functions of M2Rs and shed light on organizing principles of cholinergic modulation in the BLa.

Introduction

Cholinergic neurons in the basal forebrain innervate several major structures in the forebrain including the neocortex, hippocampus, and amygdala. Although the neocortex and hippocampus have been the focus of most anatomical and electrophysiological studies of the basal forebrain cholinergic system, the anterior basolateral amygdalar nucleus (BLa) is actually the region that receives the densest cholinergic innervation from the basal forebrain (Mesulam et al., 1983a, Mesulam et al., 1983b, Carlsen et al., 1985, Rao et al., 1987, Kordower et al., 1989). Acetylcholine is important for memory consolidation by the basolateral amygdala (McGaugh, 2004). Injections of muscarinic cholinergic antagonists into the basolateral amygdala, or lesions of the basal forebrain cholinergic neurons projecting to the amygdala, produce impairments in several kinds of learning related to emotion and motivation including fear conditioning and inhibitory avoidance (Power et al., 2003a, Wilson and Fadel, 2016).

Activation of both M1 and M2 muscarinic receptors (M1Rs and M2Rs) in the BLa is required for memory consolidation by the amygdala (Power et al., 2003b). However, almost nothing is known about muscarinic mechanisms and their role in mnemonic function in the amygdala. It is clear that basic immunohistochemical and electrophysiological studies of muscarinic receptor subtypes in the BLa will be an important first step in clarifying their involvement in amygdalar memory circuits. In general, M1Rs are excitatory and M2Rs are inhibitory (Richelson, 1995, Ehlert et al., 1995). Although it is well established that excitatory inputs to pyramidal projection neurons (PNs) of the BLa are important for associative learning, including fear conditioning, recent studies indicate that inhibition of GABAergic interneurons (INs) may also play a key role by providing disinhibition of PNs (Wolff et al., 2014, Letzkus et al., 2015, Tovote et al., 2015). The initial hypothesis of the present study, which was confirmed in our electrophysiological studies in brain slices in both rat and mouse, was that inhibitory M2Rs in the axon terminals of GABAergic INs of the BLa disinhibit PNs by suppressing GABA release, as they do in hippocampus (Szabó et al., 2010).

However, like the hippocampus and neocortex, the BLa contains several distinct GABAergic IN subpopulations that can be identified on the basis of their expression of calcium binding proteins and neuropeptides (Spampanato et al., 2011). INs expressing the calcium binding protein parvalbumin (PV) are the predominant IN subpopulation in the BLa, constituting about 40% of the total IN population (McDonald and Betette, 2001, Mascagni and McDonald, 2003). As in the hippocampus and neocortex, many of the PV+ INs in the BLa are basket cells or axo-axonic chandelier cells (Muller et al., 2006, Rainnie et al., 2006, Vereczki et al., 2016, Veres et al., 2017). Since PV+ INs in the hippocampus express M2Rs in their axon terminals that suppress GABA release (Hájos et al., 1998, Szabó et al., 2010), we hypothesized that the terminals of BLa PV+ INs would also express M2Rs. To test this second hypothesis we used dual-labeling immunoelectron microscopy in mouse brain. PV was localized using an immunoperoxidase (IP) technique whereas M2Rs were localized using a high-resolution pre-embedding immunogold-silver (IGS) technique. The use of the IGS technique for M2R permitted differentiation of membrane-associated versus cytoplasmic M2Rs. In addition, since this is the first study of M2Rs in the BLa using the high-resolution IGS method, we also analyzed the extent of membrane-associated M2Rs in PV-negative structures in the BLa, including dendritic shafts, spines, axon terminals forming asymmetrical (excitatory) synapses, and axon terminals forming symmetrical (inhibitory/neuromodulatory) synapses.

Section snippets

Animals

Experiments were performed using either male B6129SF2/J mice (Jackson Laboratory, Bar Harbor, Me) or male Sprague–Dawley rats (Envigo; Indianapolis, IN). All animals were housed on a 12-h light/dark cycle with ad libitum access to food and water. 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

Brain slice electrophysiology: M2Rs inhibit IPSCs in BLa pyramidal neurons

To investigate the functional effect of M2Rs on inhibitory transmission we recorded monosynaptic IPSCs from BLa PNs in mouse brain slice preparations. In the presence of CNQX and D-APV to block AMPA/kainate and NMDA receptors, electrical stimulation near the recorded PN evoked a monosynaptic GABAA IPSC. This IPSC was significantly inhibited (p < 0.05, n = 5) by bath application of a saturating concentration of muscarine (10 µM, Fig. 1A). The selective M2R antagonist, AF-DX 116 (1 µM) reversed this

Discussion

This investigation includes the first light and electron microscopic studies of M2Rs in the mouse basolateral amygdala. These studies demonstrated that the neuropil of the mouse BLa, like that of the rat (Muller et al., 2016), has a very high density of M2R+ structures, including dendritic shafts, dendritic spines, axon terminals forming asymmetrical (glutamatergic) synapses, and axon terminals forming symmetrical (GABAergic or neuromodulatory) synapses. In all of these structures approximately

Acknowledgments

We thank James Warren for technical assistance with this study. This work was supported by NIH grant R01MH104638.

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