Localization of the M2 muscarinic cholinergic receptor in dendrites, cholinergic terminals, and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis

Abstract

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. In the present investigation immunocytochemistry at the electron microscopic level was used to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine if M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R-immunoreactivity (M2R-ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R-negative. About 95% of dendritic shafts and 60% of dendritic spines were M2-immunoreactive (M2R+). Some M2R+ dendrites had spines suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R-ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R+ terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R+. The main targets of M2R+ terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R+ dendritic shafts. M2R-ir was also seen in VAChT+ cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R-mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites, as well as regulating release of glutamate, GABA and acetylcholine from presynaptic axon terminals.

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

“…2A). M 2 R-ir in the BLa was very dense in the neuropil, but virtually all cell bodies appeared unstained as in previous studies in the rat (McDonald and Mascagni 2011, Muller et al 2016) (Fig. 2B).…”

“…In fact, about three-quarters of the total M 2 R+ immunoparticles in the neuropil of the BLa were contained in dendritic shafts and spines. The prominent expression of M 2 Rs in these dendritic structures was also seen in the rat BLa using immunoperoxidase (IP) techniques, where it was found that over 90% of dendritic shafts and over 50% of dendritic spines were M 2 R+ (Muller et al, 2016). In the rat BLa using the IP technique it was observed that label for M 2 R was sometimes found adjacent to or contacting the plasma membranes of dendrites and spines, including at the edges of cholinergic synapses, suggesting that the excitability of dendrites could be modulated via M 2 Rs (Muller et al, 2016).…”

“…The prominent expression of M 2 Rs in these dendritic structures was also seen in the rat BLa using immunoperoxidase (IP) techniques, where it was found that over 90% of dendritic shafts and over 50% of dendritic spines were M 2 R+ (Muller et al, 2016). In the rat BLa using the IP technique it was observed that label for M 2 R was sometimes found adjacent to or contacting the plasma membranes of dendrites and spines, including at the edges of cholinergic synapses, suggesting that the excitability of dendrites could be modulated via M 2 Rs (Muller et al, 2016). However, since the extent to which the IP reaction product could diffuse from the antigenic binding site was not clear, the precise localization of M 2 R-ir to the plasma membrane of dendrites could not be determined with certainty.…”

“…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 M 2 R+ 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 20–70% of the M 2 R+ immunoparticles were associated with the plasma membrane.…”

“…Likewise, the great majority of all dendritic shafts in the rat BLa appeared to be M 2 R+ (Muller et al, 2016). Since most dendritic spines in the BLa arise from PN distal dendrites (Muller et al, 2006), the great majority of M 2 R+ spines observed in this study probably belong to PN dendrites.…”