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.

When small blocks comprising four columns of electrocytes were excised from electric organs of Torpedo marmorata after stimulation in vivo via the electric lobe at 1 Hz for 1 h and allowed to recover at 20-22 degrees C for several hours in medium containing 100 microM d4 choline and 500 microM propionate, small quantities of propionylcholine amounting to no more than 1% of the endogenous acetylcholine of the tissue could be detected in tissue extracts by gas chromatography-mass spectrometry (GCMS). Kinetic studies demonstrated that there was no nonexchangeable propionylcholine in the tissue and in the absence of added propionate, propionylcholine levels were less than 0.2% of tissue acetylcholine. Vesicular propionylcholine amounted to less than 0.5% of vesicular acetylcholine and the distribution of d0 and d4 propionylcholine suggested that an appreciable proportion (up to one-third) of this could be an artifact of preparation for GCMS determinations. Propionylcholine formation during extraction and demethylation of an artificial mixture of acetylcholine, choline, and propionate was indeed detected. It is concluded that propionylcholine has no significance as an endogenous or as a false transmitter at this terminal, in conformity with the work of Sheridan et al. [Z. Zellforsch. 74, 281-307 (1966)] but in contrast to the report of O'Regan [J. Neurochem. 39, 764-772 (1982)].

Is Propionylcholine Present in or Synthesized by Electric Organ

The historical significance of work with electric organs for the study of cholinergic transmission.

Cocultures of septal and hippocampal tissues taken from 6- to 7-day-old rats were maintained in culture for up to 30 days in the presence and absence of nerve growth factor (NGF), and their Chol-1 contents determined at varying time intervals by a modified enzyme-linked immunosorption assay (ELISA). The major brain gangliosides were determined densitometrically after spraying chromatograms with resorcinol reagent. There was little change in the contribution of the major gangliosides to the total ganglioside content of the explants either with time or the presence or absence of NGF, the only exception being an NGF-insensitive fall in the contribution of GM1 to about 60% of its initial value at 20 and 30 days. By contrast, the concentration of Chol-1 expressed either per unit weight of ganglioside sialic acid or protein increased considerably in culture and this increase was enhanced by NGF. The effect of NGF resembles that on other cholinergic markers, choline acetyltransferase and acetylcholinesterase, and may be attributed to an NGF-stimulated hippocampal ingrowth of cholinergic fibres and enhanced survival of cholinergic septal neurons. The Chol-1 concentration finally attained in the presence of NGF and the time course of its increase parallel those previously found in vivo and indicate the potential usefulness of septal-hippocampal cocultures for investigating the function of Chol-1.

/pdf/nerve-growth-factor-enhances-the-expression-of-the-4l80zygj78.pdf

Nerve growth factor enhances the expression of the cholinergic-specific ganglioside Chol-1 in cocultures of rat septum and hippocampus.

Monoclonal antibodies (MABs) have been raised against acidic glycolipids extracted from the electric organ of Torpedo marmorata. One of these, designated L9, appears to recognize acidic glycolipids in adult T. marmorata electric organ, electromotor nerves and brain, adult rat sciatic nerve, and in embryonic and neonatal rat brain, starting at embryonic day (ED) 15 and disappearing by the 20th day of post-natal life. The epitope is present in growth cones isolated from 4-day-old rats; its proportion relative to total gangliosides is, however, no higher than that found in whole neonatal brain membranes. Desialidation of the acidic glycolipid fraction modifies neither the immunoreactivity nor the RF value following thin-layer chromatography (TLC) of the antigen; it is concluded that the antigen is not a ganglioside. The MAB, HNK-1, recognizes the L9 antigen. Both HNK-1 and L9 recognize a sulphoglycolipid of the same RF in TLC. The function of the L9 antigen is not known but its evolutionary conservation, presence in growth cones and its developmental regulation in the mammalian central nervous system indicate that it plays an important role in nervous system maturation.

/pdf/characterization-of-a-monoclonal-antibody-directed-against-a-46w6ex15l5.pdf

Characterization of a monoclonal antibody directed against a sulphoglycolipid that is evolutionarily conserved and developmentally regulated in rat brain

The subcellular distribution of the cholinergic-specific gangliosidic Chol-1 antigens in rat brain has been compared with that of total ganglioside as a marker for neural plasma membranes, particle-bound choline acetyltransferase (ChAT) as a synaptosomal marker, and supernatant ChAT as a marker for neuronal perikaryal cytoplasm. Over 50% of brain Chol-1 was recovered in a synaptosome fraction prepared by a standard density-gradient method. The specific concentration of Chol-1 relative to protein or major gangliosides was also highest in the synaptosome fraction and low in a supernatant fraction which contained most of the neuronal plasma membrane fragments. ChAT was likewise mainly recovered in the synaptosome fraction but its relative concentration there was significantly less than that of Chol-1 (p < 0.01).

Victor P. Whittaker

Papers

Is Propionylcholine Present in or Synthesized by Electric Organ

The historical significance of work with electric organs for the study of cholinergic transmission.

Nerve growth factor enhances the expression of the cholinergic-specific ganglioside Chol-1 in cocultures of rat septum and hippocampus.

Characterization of a monoclonal antibody directed against a sulphoglycolipid that is evolutionarily conserved and developmentally regulated in rat brain

The cholinergic marker Chol-1 is selectively localized in nerve terminals.