Muscarinic cholinergic receptor M1 in the rat basolateral amygdala: ultrastructural localization and synaptic relationships to cholinergic axons.

Muscarinic neurotransmission in the anterior basolateral amygdalar nucleus (BLa) mediated by the M1 receptor (M1R) is critical for memory consolidation. Although knowledge of the subcellular localization of M1R in the BLa would contribute to an understanding of cholinergic mechanisms involved in mnemonic function, there have been no ultrastructural studies of this receptor in the BLa. In the present investigation, immunocytochemistry at the electron microscopic level was used to determine which structures in the BLa express M1R. The innervation of these structures by cholinergic axons expressing the vesicular acetylcholine transporter (VAChT) was also studied. All perikarya of pyramidal neurons were labeled, and about 90% of dendritic shafts and 60% of dendritic spines were M1R+. Some dendrites had spines suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of interneurons. M1R immunoreactivity (M1R-ir) was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M1R+ terminals forming asymmetrical synapses were dendritic spines, most of which were M1R+. The main targets of M1R+ terminals forming symmetrical synapses were M1R+ perikarya and dendritic shafts. About three-quarters of VAChT+ cholinergic terminals formed synapses; the main postsynaptic targets were M1R+ dendritic shafts and spines. In some cases M1R-ir was seen near the postsynaptic membrane of these processes, but in other cases it was found outside of the active zone of VAChT+ synapses. These findings suggest that M1R mechanisms in the BLa are complex, involving postsynaptic effects as well as regulating release of neurotransmitters from presynaptic terminals.

Postsynaptic muscarinic m2 receptors at cholinergic and glutamatergic synapses of mouse brainstem motoneurons.

In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons; therefore, we analyzed the localization of the m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial, and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses in both motoneuronal perikarya and dendrites. A relatively large proportion (20-30%) of plasma membrane-associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through a synaptic as well as diffuse type of transmission.

Imaging the post-fusion release and capture of a vesicle membrane protein.

The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter's capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.

Vesicular acetylcholine transporter-immunoreactive axon terminals enriched in the pontine nuclei of the mouse.

Information to the cerebellum enters via many afferent sources collectively known as precerebellar nuclei. We investigated the distribution of cholinergic terminal-like structures in the mouse precerebellar nuclei by immunohistochemistry for vesicular acetylcholine transporter (VAChT). VAChT is involved in acetylcholine transport into synaptic vesicles and is regarded as a reliable marker for cholinergic terminals and preterminal axons. In adult male mice, brains were perfusion-fixed. Polyclonal antibodies for VAChT, immunoglobulin G-peroxidase and diaminobenzidine were used for immunostaining. In the mouse brain, immunoreactivity was seen in almost all major cholinergic cell groups including brainstem motoneurons. In precerebellar nuclei, the signal could be detected as diffusely beaded terminal-like structures. It was seen heaviest in the pontine nuclei and moderate in the pontine reticulotegmental nucleus; however, it was seen less in the medial solitary nucleus, red nucleus, lateral reticular nucleus, inferior olivary nucleus, external cuneate nucleus and vestibular nuclear complex. In particular, VAChT-immunoreactive varicose fibers were so dense in the pontine nuclei that detailed distribution was studied using three-dimensional reconstruction of the pontine nuclei. VAChT-like immunoreactivity clustered predominantly in the medial and ventral regions suggesting a unique regional difference of the cholinergic input. Electron microscopic observation in the pontine nuclei disclosed ultrastructural features of VAChT-immunoreactive varicosities. The labeled bouton makes a symmetrical synapse with unlabeled dendrites and contains pleomorphic synaptic vesicles. To clarify the neurons of origin of VAChT-immunoreactive terminals, VAChT immunostaining combined with wheat germ agglutinin-conjugated horseradish peroxidase retrograde labeling was conducted by injecting a retrograde tracer into the right pontine nuclei. Double-labeled neurons were seen bilaterally in the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus. It is assumed that mesopontine cholinergic neurons negatively regulate neocortico-ponto-cerebellar projections at the level of pontine nuclei.