Acetylcholine (ACh) is an important neurotransmitter in the mammalian brain; it is implicated in arousal, learning, and other cognitive functions. Recent studies indicate that nicotinic receptors contribute to these cholinergic effects, in addition to the established role of muscarinic receptors. In the hippocampus, where cholinergic involvement in learning and memory is particularly well documented, alpha7 nicotinic acetylcholine receptor subunits (alpha7 nAChRs) are highly expressed, but their precise ultrastructural localization has not been determined. Here, we describe the results of immunogold labeling of serial ultrathin sections through stratum radiatum of area CA1 in the rat. Using both anti-alpha7 nAChR immunolabeling and alpha-bungarotoxin binding, we find that alpha7 nAChRs are present at nearly all synapses in CA1 stratum radiatum, with immunolabeling present at both presynaptic and postsynaptic elements. Morphological considerations and double immunolabeling indicate that GABAergic as well as glutamatergic synapses bear alpha7 nAChRs, at densities approaching those observed for glutamate receptors in CA1 stratum radiatum. Postsynaptically, alpha7 nAChRs often are distributed at dendritic spines in a perisynaptic annulus. In the postsynaptic cytoplasm, immunolabeling is associated with spine apparatus and other membranous structures, suggesting that alpha7 nAChRs may undergo dynamic regulation, with insertion into the synapse and subsequent internalization. The widespread and substantial expression of alpha7 nAChRs at synapses in the hippocampus is consistent with an important role in mediating and/or modulating synaptic transmission, plasticity, and neurodegeneration.
Aplysia VAP-33 (VAMP-associated protein) has been previously proposed to be involved in the control of neurotransmitter release. Here, we show that a Drosophila homolog of VAP-33, DVAP-33A, is localized to neuromuscular junctions. Loss of DVAP-33A causes a severe decrease in the number of boutons and a corresponding increase in bouton size. Conversely, presynaptic overexpression of DVAP-33A induces an increase in the number of boutons and a decrease in their size. Gain-of-function experiments show that the presynaptic dose of DVAP-33A tightly modulates the number of synaptic boutons. Our data also indicate that the presynaptic microtubule architecture is severely compromised in DVAP-33A mutants. We propose that a DVAP-33A-mediated interaction between microtubules and presynaptic membrane plays a pivotal role during bouton budding.
Presynaptic GABAA receptors modulate synaptic transmission in several areas of the CNS but are not known to have this action in the cerebral cortex. We report that GABAA receptor activation reduces hippocampal mossy fibers excitability but has the opposite effect when intracellular Cl- is experimentally elevated. Synaptically released GABA mimics the effect of exogenous agonists. GABAA receptors modulating axonal excitability are tonically active in the absence of evoked GABA release or exogenous agonist application. Presynaptic action potential-dependent Ca2+ transients in individual mossy fiber varicosities exhibit a biphasic dependence on membrane potential and are altered by GABAA receptors. Antibodies against the alpha2 subunit of GABAA receptors stain mossy fibers. Axonal GABAA receptors thus play a potentially important role in tonic and activity-dependent heterosynaptic modulation of information flow to the hippocampus.
VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum-resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis.
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