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This article is from Physiological Reports , volume 2 . Abstract The type of neurotransmitter secreted by a neuron is a product of the vesicular transporters present on its synaptic vesicle membranes and the available transmitters in the local cytosolic environment where the synaptic vesicles reside. Synaptic vesicles isolated from electroplaques of the marine ray, Torpedo californica, have served as model vesicles for cholinergic neurotransmission. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. We identified the types of vesicular neurotransmitter transporters present at the electroplaque using immunoblot and immunofluoresence techniques with antibodies against the vesicle acetylcholine transporter (VAChT), the vesicular glutamate transporters (VGLUT1, 2, and 3), and the vesicular nucleotide transporter (VNUT). We found that VAChT, VNUT, VGLUT 1 and 2, but not 3 were present by immunoblot, and confirmed that the antibodies were specific to proteins of the axons and terminals of the electroplaque. We used a single‐vesicle imaging technique to determine whether these neurotransmitter transporters were present on the same or different populations of synaptic vesicles. We found that greater than 85% of vesicles that labeled for VAChT colabeled with VGLUT1 or VGLUT2, and approximately 70% colabeled with VNUT. Based upon confidence intervals, at least 52% of cholinergic vesicles isolated are likely to contain all four transporters. The presence of multiple types of neurotransmitter transporters – and potentially neurotransmitters – in individual synaptic vesicles raises fundamental questions about the role of cotransmitter release and neurotransmitter synergy at cholinergic synapses.

From morphological and electrophysiological data, performed proteinaceous channels have been implicated in the release of neurotransmitters from their vesicular stores, synaptic vesicles. We have characterized different synaptic proteins which qualify as potential components of such hypothesized 'synaptopore' or 'fusion pore' structures: (1) Synaptophysin is a major integral membrane protein of synaptic vesicles that shares its transmembrane topolgy and structural organization with connexins, the subunits of gap junction proteins. By crosslinking and reconstitution techniques we have demonstrated that native synaptophysin contains six identical subunits and forms channels upon reconstitution in planar lipid bilayers. This protein therefore may correspond to the to the vesicular portion of a putative fusion pore structure. (2) Synaptoporin is another member of the synaptophysin/connexin channel protein family that is localized in small synaptic vesicles. In situ hybridization shows that this protein is selectively expressed in a subset of central neurons. (JS)

Neuromuscular junctions (NMJs) from diaphragm, soleus, and extensor digitorum longus muscles of male albino rats were assessed for morphological alterations following acute exposure by single subcutaneous injections of pyridostigmine bromide and following subacute exposure by subcutaneously implanted osmotic minipumps that contained 10 milligrams/milliliters of pyridostigmine. Recovery processes were monitored up to 90 days. Drug exposure resulted in pre- and postsynaptic alterations. These included partial withdrawal of the nerve terminals from junctional folds, disruption of the myofibrillar organization, and damage to membrane-bound organelles. These lesions were observed as independent occurrences in some NMJs but were present concurrently in other fibers. The extent of the pathology was dose-dependent. Keywords: Pyridostigmine, Carbamate, Acute toxicity, Chronic toxicity, Neuromuscular, NMJ, ChE, Cholinesterase, Anticholinesterase drug, EDL.

The workshop addressed the following topics: Synaptogenesis and Regulation; Acetylcholinesterase; Presynaptic Mechanisms; Signal Transduction and Modulation; Acetylcholine Receptor.

Diaphragms from male albino, Charles River or Edgewood rates were assessed for morphological alterations following acute or subacute exposures to pyridostigmine bromide. Low acute does (threshold 0.001 Lethal dose 50) seemed to affect the presynaptic area with no apparent effects of the muscle cell. At higher doses (0.01 - 1.0 LD50) both pre- and postsynaptic elements became involved. A 14-day subacute exposure to 20 milligrams of pyridostigmine produced pre- and post-synaptic alterations which were more extensive than those seen following exposure to a 3 mg 14 day exposure. However, no subacute dose created damage equivalent to that seen 30 minutes after a single LD50 dose. Presynaptic alterations included withdrawal of the terminal from the junctional folds, invasion of the synaptic cleft with Schwann cell processes, and disruption oa axon terminal organelles. Keywords: Pyridostigmine, Carbamate, Acute toxicity, Chronic toxicity, Neuromuscular, Anticholinesterase drugs, Che, NMJ, ACHE.

Neuromuscular junctions (NMJs) from diaphragm, soleus and extensor digitorum longus muscles of male albino rats were assessed for morphological alterations following acute exposure (30 minutes) by single subcutaneous injections of pyridostigmine bromide (0.36 to 1.0 milligrams/kilogram) and following subacute exposure (2-14 days) by subcutaneously implanted osmotic minipumps that contained 10 milligrams/millimeters of pyridostigmine. Recovery processes were monitored up to 60 days. Drug exposure resulted in pre-and postsynaptic alterations. These included partial withdrawal of the nerve terminals from junctional folds, disruption of the myofibrillar organization and damage to membrane-bound organelles. These lesions were observed as independent occurrences in some NMJs but were present concurrently in other fibers. The extent of the pathology was dose-dependent. Keywords: Pyridostigmine, Carbamate, Acute toxicity, Chronic toxicity, Neuromuscular, ChE, Anticholinesterase drug.

Neuromuscular junctions (NMJs) from diaphragm, soleus and extensor digitorum longus muscles of male albino rats were assessed for morphological alterations following acute exposure (30 minutes) by single subcutaneous injections of pyridostigmine bromide (0.36 to 1.0 milligrams/kilogram) and following subacute exposure (2-14 days) by subcutaneously implanted osmotic minipumps that contained 10 milligrams/millimeters of pyridostigmine. Recovery processes were monitored up to 60 days. Drug exposure resulted in pre-and postsynaptic alterations. These included partial withdrawal of the nerve terminals from junctional folds, disruption of the myofibrillar organization and damage to membrane-bound organelles. These lesions were observed as independent occurrences in some NMJs but were present concurrently in other fibers. The extent of the pathology was dose-dependent. Keywords: Pyridostigmine, Carbamate, Acute toxicity, Chronic toxicity, Neuromuscular, ChE, Anticholinesterase drug.

The conference dealt with recent developments in the structure and function of the nicotinic acetylcholine receptor and acetylcholinesterase. During the four day period, over 50 talks and 31 poster sessions were presented. The high caliber of the presentations combined with the limited number of attendees, and secluded site, allowed for much fruitful interaction an in-depth discussions. The sessions were divided into five major categories:'Synaptogenesis and Regulation.' 'Acetycholinesterase,' 'Presynaptic Mechanisms,'Signal Transduction and Modulation,' and 'Acetylcholine Receptor.' Keywords: Cholinergic synapses, Cholinergic receptors; Ion channels; Cholinergic neurotransmitters, Gene cloning.