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 water spaces in cholinergic synaptic vesicles from Torpedo measured by changes in density induced by permeating substances

The esterases of horse blood. 2. The specificity of horse erythrocyte cholinesterase

Acetylcholine is synthesized in the terminal cytoplasm of neurones and released from storage sites in synaptic vesicles on stimulation. V. P. Whittaker describes recent work on the molecular heterogeneity of synaptic vesicles and vesicular recycling. Although the process of endocytosis and exocytosis can be depicted, the molecular mechanisms remain a challenge to cell biologists.

/pdf/the-storage-and-release-of-acetylcholine-3iyri0qqwm.pdf

The storage and release of acetylcholine.

Synexin, a protein from the cytosol of the adrenal medulla, selectively increases the ability of Ca2+ to aggregate chromaffin granules and other membrane-bound particles. The ability of synexin to self-aggregate in the presence of Ca2+ can be employed in the purification of the protein by monitoring purification with parallel assays that utilize the aggregation of both chromaffin granule membranes and phosphatidylserine liposomes. It is shown that the enhancement of the Ca2+-induced aggregation of both liposomes and chromaffin granule membranes is a property associated with a 47,000 Mr protein. Trypsin inactivated synexin. We found that if granule membranes were well washed after trypsin treatment, they were still excellent substrates for synexin aggregation. This finding cannot be explained by extinction changes owing to synexin self-aggregation. The 47,000 Mr protein enhancement Ca2+ aggregation of phosphatidylserine liposomes containing up to 40% phosphatidylcholine, liposomes made from lipids extracted from chromaffin granule membranes, and trypsin-treated chromaffin granule membranes, thus suggesting that synexin activity in vivo may be independent of specific membrane proteins but dependent on the presence of acidic phospholipids in the membrane.

Victor P. Whittaker

Papers

The water spaces in cholinergic synaptic vesicles from Torpedo measured by changes in density induced by permeating substances

The esterases of horse blood. 2. The specificity of horse erythrocyte cholinesterase

The storage and release of acetylcholine.

Purification and Mode of Action of Synexin: A Protein Enhancing Calcium‐Induced Membrane Aggregation

The characterization of the esterases of human plasma.