Molecular Anatomy of a Trafficking Organelle

Microglia Promote Learning-Dependent Synapse Formation through Brain-Derived Neurotrophic Factor

This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.

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Physiology and Pathophysiology of Purinergic Neurotransmission

We used an antiserum against purified cholinergic synaptic vesicles from Torpedo and expression screening to isolate a cDNA clone encoding synuclein, a 143 amino acid neuron-specific protein. A cDNA clone was also isolated from a rat brain cDNA library that encodes a highly homologous 140 amino acid protein. The amino terminal 100 amino acids of both proteins are comprised of an 11 amino acid repeating unit that contains a conserved core of 6 residues. The synuclein gene is expressed only in nervous system tissue, not in electric organ, muscle, liver, spleen, heart, or kidney. In the electric organ synapse Torpedo synuclein-immunoreactive proteins are found in 3 major molecular-weight classes of 17.5, 18.5, and 20.0 kDa. In the neuronal cell soma the 17.5 kDa species is predominant and immunoreactivity is localized to a portion of the nuclear envelope.

https://www.jneurosci.org/content/jneuro/8/8/2804.full.pdf

Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.

/pdf/synaptic-vesicle-exocytosis-captured-by-quick-freezing-and-1qgnb5cggj.pdf

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.

The density and free water of cholinergic synaptic vesicles as a function of osmotic pressure.

The Na+ and K+ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israel et al. have high internal Na+ (290 MM) and low internal K+ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na+, 96 mM; K+, 81 mM); 0.1 mM-ouabain abolished these gradients. The trans-membrane Na+ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na+ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain-sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.

The Na+ and K+ content of isolated Torpedo synaptosomes and its effect on choline uptake.

— Rabbit antisera have been prepared against whole cholinergic vesicles purified from the electric organ of Torpedo marmorata. The sera contain two major and two minor precipitating systems against membranous proteins, as revealed by Ouchterlony diffusion. No immunoprecipitation could be detected against the soluble vesicle protein constituent ‘vesiculin’. Fractions from cephalopod, amphibian and mammalian neural tissue were shown to exhibit no immunochemical homology with Torpedo cholinergic vesicle proteins.

Immunological approach to the characterization of cholinergic vesicular protein

Cholinergic nerve terminals utilize glycoconjugates in several ways, as surface markers and as structural components of the synaptic vesicles present within them. The surface markers have been discovered immunochemically: antibodies raised against them are able specifically to sensitize the cholinergic subpopulation of mammalian brain synaptosomes to complement-mediated lysis. One such group of antigens (Chol-1) have been identified as a novel series of minor gangliosides having in common a sialylatedN-acetylgalactosamine residue. These gangliosides may constitute the major gangliosides at cholinergic terminals. A second surface antigen (Chol-2) is thought to be a protein with an epitope in common with aTorpedo electric organ ganglioside. Cholinergic synaptic vesicles are rich in a proteoglycan which appears to assist in the sequestration of acetylcholine within the vesicle and to stabilize the vesicle membrane during cycles of exocytosis and recovery. It may be the cholinergic equivalent of the chromogranins.

Cholinergic-specific glycoconjugates

Preparation of ultra pure synaptic vesicles from the electric organ of torpedo marmorata by porous glass bead chromatography and estimation of their acetyl choline content

Victor P. Whittaker

Papers

The density and free water of cholinergic synaptic vesicles as a function of osmotic pressure.

The Na+ and K+ content of isolated Torpedo synaptosomes and its effect on choline uptake.

Immunological approach to the characterization of cholinergic vesicular protein

Cholinergic-specific glycoconjugates

Preparation of ultra pure synaptic vesicles from the electric organ of torpedo marmorata by porous glass bead chromatography and estimation of their acetyl choline content