Biochemical differences in the modes of synaptic vesicle release between control and streptozotocin-induced diabetic rats and possible relationship to changes in behaviour

Abstract

In order to understand the huge complexity of brain function, and to determine the mechanisms underlying various psychiatric (e.g. schizophrenia) and neuronal disorders (Alzheimer’s disease), it is imperative that the basic machinery involved in neuronal transmission is fully elucidated. This involves exocytosis of synaptic vesicles (SVs) and subsequent release of neurotransmitter. SVs can exocytose by two different modes: full fusion (FF) and kiss-and-run (K&R). There is much debate as to whether SV fusion in the nerve terminal can occur via K&R mode of exocytosis and this has been studied herein, using cerebrocortical synaptosomes from adult rats. Switching between the two modes depends upon the secondary messenger calcium and protein phosphorylation reactions. Dr. Ashton has previously demonstrated that an increase in intracellular calcium levels regulates the switch between these modes of exocytosis, and thus the role of voltage-gated calcium channels (VGCCs) was studied. Blockade of L-type (but not N-and P-type) VGCCs switch K&R exocytosis to FF mode in control terminals, indicating that such channels contribute to the calcium increase that induces K&R mode. These results (for the first time) demonstrate that distinct VGCC subtypes contribute to the specific mode of exocytosis. Very surprisingly, it has been discovered that in diabetic terminals (prepared from streptozotocin treated rats: a model for type 1 diabetes), higher amount of K&R exocytosis occurs relative to non-diabetic terminals due to a higher stimulus evoked change in intracellular calcium. Whether this was due to an over-activation of certain VGCCs was studied. Fascinatingly, L-type channels did not regulate the mode of exocytosis but diabetic terminals displayed a higher dependence on N-type channels. Blockade of calcium/calmodulin dependent kinase II (CaMKII) was found to inhibit completely the release of reserve pool (RP) of vesicles, with no effect on readily releasable pool (RRP) of vesicles in control terminals. However, by studying the release of just the RRP of SV it was discovered that, inhibition of CaMKII leads to a switch from K&R to FF, suggesting this enzyme when activated can phosphorylate substrate proteins that induces K&R mode of exocytosis. Inhibition of myosin II induces a switch from K&R to FF in both control and diabetic terminals; although results suggest that myosin II may only regulate the fusion mode of the RRP. In control terminals, blockade of calcineurin induces more K&R. By blocking both myosin II and calcineurin in control synaptosomes, more K&R was apparent. This indicates that RP of vesicles switch to K&R mode of exocytosis independently of the role of myosin II, and that calcineurin exclusively works on RP of vesicles. The inhibition of dynamins switched the mode of exocytosis of the RP of SVs from K&R to FF in diabetic terminals, whilst failing to regulate the mode of exocytosis of the RRP for both control and diabetic terminals when a strong stimulus was applied. It has been established that inhibition of protein phosphatase 2A and activation of protein kinase C induces RRPs that undergo K&R in control terminals to FF mode of exocytosis. Similar experiments were performed on diabetic terminals. Each drug treatment alone switched the RRP vesicles in diabetic terminals to undergo FF, but the dual treatment switched all vesicles that previously underwent K&R (i.e. the RRPs and some RPs) to a FF mode of exocytosis. The results obtained should help future research to understand precisely the molecular mechanisms that occur in the switching of the mode of different pools of SVs.
The diabetic terminals respond differently to various drugs that perturb protein phosphorylation, [Ca2+]i and specific phospho-proteins. We hypothesised that these characterized biochemical changes may affect synaptic plasticity and could result in some behavioural changes. With the long-term goal of establishing a link between the biochemical and behavioural findings, which may represent subtle changes in synaptic plasticity, difference in the behaviours of STZ-induced diabetic rats in comparison to the age-matched control rats were measured. A newly developed behaviour registration system, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS) was utilized. The diabetic animals showed significantly decreased locomotive and rearing behaviour whilst the grooming, drinking and eating behaviour was substantially increased over this period. Intriguingly, whilst the incidence of locomotive behaviour was decreased in diabetic animals, the average speed and distance covered over a period of 24hrs was significantly more in such rats than the control rats. These initial observations established behavioural differences that could be related to the biochemical changes seen, and future experiments will attempt to find a correlation between these.