Synaptic protein profiles and neurotransmitter release in relation to alcoholism

Kuo, S. (2005). Synaptic protein profiles and neurotransmitter release in relation to alcoholism PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Kuo, S.
Thesis Title Synaptic protein profiles and neurotransmitter release in relation to alcoholism
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2005
Thesis type PhD Thesis
Supervisor Dodd, P.
Total pages 211
Collection year 2005
Language eng
Subjects L
270101 Analytical Biochemistry
780105 Biological sciences
Formatted abstract

Alcoholism is a major health, social and economical problem in most countries. Severe chronic alcohol misuse leads to localized damage in human cerebral cortex, particularly in prefrontal areas, that is accompanied by differences in factors mediating amino acid neurotransmission. Selective differences between GABA and glutamate receptors suggest there may be a shift in the excitation: inhibition balance in pathologically susceptible cortical regions, provided that the quantities of the relevant transmitters released synaptically are not selectively altered in a compensatory manner. Microarray analyses have found differences in the frontal cortex between alcoholics and controls in the expression of several transcripts coding for synaptic proteins involved in exocytosis. The present work aimed to characterize the release process at both molecular and dynamic levels in chronic severe alcoholics and matched controls. 


Protein was extracted from superior frontal cortex and motor cortex of human brain samples obtained at autopsy. These represent pathologically damaged and spared regions respectively. Cases were selected based on alcohol consumption and matched for post-mortem delay, cause of death and age at death. lmmunoblotting was carried out on the protein extract from each brain region from 28 controls, 25 uncomplicated alcoholics and 12 cirrhotic alcoholics using antibodies directed against synapsin lla, synaptophysin, α-SNAP, ϒ-SNAP, α-synuclein, β-synuclein, and ϒ-synuclein. A common protein standard was used in all experiments to eliminate inter-assay variables such as differences in transfer efficiency, the parameters of chemiluminescence and film exposure, and detection and quantitation by densitometry. The results showed that these presynaptic proteins were differentially expressed among groups. For the vesicle-associated proteins synapsin IIa, synaptophysin, and α-SNAP, there was a trend of increasing expression with increasing severity of alcoholism, as indexed by the absence or presence of cirrhosis. The synucleins exhibited a different pattern, with the greatest expression in non-cirrhotic alcoholics. These differing patterns may underlie alterations in the neurotransmitter release process in chronic alcoholics. The expression profile of ϒ-SNAP was significantly different from that of other synaptic proteins, suggesting a distinct role for this protein in exocytosis. All proteins measured showed significantly higher expression in female subjects than in male subjects overall, although in the main this was not further influenced by alcohol abuse. 


A paradigm was developed to investigate the dynamics of synaptosomal amino acid transmitter release in tissue from chronic alcoholics and controls obtained at autopsy. Subjects were divided on the basis of alcohol consumption and matched for post-mortem delay, cause of death and age. Tissue was processed and synaptosomes were prepared and pre-incubated with radio labeled transmitters. 


In rat cerebral cortex synaptosomes preloaded with L-[3H]-glutamate and [14C]-GABA, vesicular release could be successfully differentiated from cytoplasmic release when the depolarizing stimulus was raised K+ ion concentration. However, human synaptosomes did not give clear-cut results. Experiments were carried out to optimize the release paradigm as well as to improve the vesicular uptake of labeled transmitters. A "pulsechase" protocol was developed and performed in human tissue samples in order to enhance the release signal derived from exocytosis, by reducing the cytoplasmic pool of L-[3H]-glutamate. However, the two release profiles remained very close to one another; total release was not enhanced effectively relative to the non-pulse-chase protocol. It was concluded that the pulse-chase protocol did not build up the vesicular pool of L-[3H]-glutamate, though the cytoplasmic L-[3H]-glutamate was effectively depressed by D-aspartate chasing. 


Further studies applied 4-AP to trigger release of amino acid transmitters, to prevent the problem of the reversal of plasma membrane transporters caused by raised K+ ion concentration. In addition, 1 mM ATP was added to the incubation medium to enhance the vesicular uptake of labeled transmitters. The results showed that the application of 4-AP caused the amino acid transmitters to be released from the rat synaptosomes, but failed to produce successful release signals in the human tissue experiments. 


The findings suggest that vesicular exocytosis may be impaired by freezing and affected by post-mortem delay (PMD). Model studies showed that the freezing step had a major effect on Ca2+-dependent release, as less L-[3H]-glutamate and [14C]-GABA were released from synaptosomes prepared from frozen rat tissue. There was an indication of a decline in L-[3H]-glutamate release with increasing PMD, though the regression test failed to achieve significance because of the small sample size. 


Finally, electrical stimulation was used to distinguish the vesicular from the cytoplasmic component of transmitter release in human synaptosomes. The application of electrical pulses causes rapid changes in neural activity that mimics the physiological input to nerve-endings, and can elicit successive release peaks from synaptosomes. After incubation with radiolabeled transmitters and washout, synaptosomes were stimulated with electrical pulses (10 V bipolar square waves, 0.4 msec pulse width, 100/s) for 1.5 min epochs in the presence and absence of Ca2+ ions (these stimulation parameters were found to be optimal). Fractions were collected and analyzed for released radioactivity. Both L-[3H]-glutamate and [14C]-GABA showed a clear enhancement of release in the presence of Ca2+ ions (P < 0.001 in both instances); the increment is most likely due to release from synaptic vesicles. L-[3H]-glutamate (P < 0.05), but not [14C]-GABA (NS), release was significantly higher in synaptosomes from alcoholics. The excitation: inhibition balance may be altered in chronic alcoholics in a way that would tend to promote the loss of neurons by excitotoxicity. 

Keyword Alcohol -- Physiological effect
Neurotransmitter receptors

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