Opera Medica et Physiologica


Effects of Endocannabinoid-Related Compounds on the Activity of Septal and Hippocampal Neurons in a Model of Kainic Neurotoxicity: Study Ex Vivo

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The goal of the work was to study the effect of the cannabinoid receptor agonist WIN55,212-2 and the cannabinoid type 1 receptor antagonist AM251 on electrophysiological changes in the hippocampus and the medial septal region (MS) induced by the intracerebral administration of excitotoxin kainic acid. Kainate injected into the right brain ventricle provoked persistent seizures (status epilepticus, SE) in all rats. A morphological analysis of the right hippocampus performed one month after the SE revealed the death of neurons, which was most pronounced in the hilus of the dentate gyrus and in the CA3a field of the dorsal hippocampus. In brain slices taken one month after the SE, the spontaneous activity of MS neurons and population EPSP (pEPSP) in the CA1 field of the hippocampus evoked by the stimulation of Shaffer collaterals (SC) was recorded; the changes in the activity were compared with the activity in slices of healthy animals injected with normal saline (“control slices”). It was found that the activity in MS slices from the brain of animals injected with kainic acid (“kainate slices”) was almost twice higher than in the control. After the application of WIN55,212-2, the frequency of discharges in the control did not change, whereas in kainate slices, the level of neuronal activity decreased to the control value. The application of AM251 led to an increase in the frequency of discharges in the control and its decrease in kainate slices. The registration of pEPSPs in the hippocampal slices revealed a twofold increase in the responses to SC stimulation in kainate slices compared with those in the control, i.e., an abrupt increase in neuronal excitability. A tendency for a decrease in excitability after the application of WIN55,212-2 and, conversely, for its increase by the action of AM251 was noted in evoked responses in the hippocampal kainate slices. Our results allow to assume the protective impact of cannabinoid agonist WIN55,212-2 on neuronal activity in the medial septum and hippocampus that disturbed by neurotoxic kainate influence. 


Long Term Potentiation (LTP) and Long Term Depression (LTD) Cause Differential Spatial Redistribution of the Synaptic Vesicle Protein Synaptophysin in the Middle Molecular Layer of the Dentate Gyrus in Rat Hippocampus

Synaptophysin is a synaptic vesicle glycoprotein with four transmembrane domains and has a molecular weight of 38 kD. It accounts for 7% of the total vesicle membrane proteins (Nadol et al., 1993) and is considered a reliable marker of nerve terminal differentiation due to its differential expression in terms of level and localization of immunoreactivity (Lupa and Hall, 1989). It has been identified as a calcium-binding protein (Shin, 2014) and hence may be a major component of the mechanism of neurotransmitter release (Reichardt et al., 1983).

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The presynaptic modifications that accompany long-term changes in synaptic plasticity are still not fully understood. Synaptophysin is a major synaptic vesicle protein involved in neurotransmitter release. We have used quantitative electron microscopy to study synaptophysin (Syn) immunolabelling in the hippocampus of adult rats 24h after induction in vivo of long term potentiation (LTP), and long term depression (LTD). Electrodes were implanted chronically in hippocampus with stimulation at either the medial (MPP) or lateral perforant path (LPP). 24h following induction of LTP or LTD rats were rapidly perfusion fixed and hippocampal tissue processed to electron microscopy via freeze substitution method. Anti-synaptophysin post-embedding immunolabelling was performed and tissue was imaged in the middle molecular layer (MML) of the dentate gyrus. There was a significant decrease in number of Syn labelled vesicles per unit area of bouton after LTP, but not LTD. An analysis of the spatial distribution of Syn labelled synaptic vesicles showed an increase in nearest neighbour distances, more so in the LTP than the LTD group, which is consistent with the overall decrease of Syn after LTP. These data are in agreement with the suggestion that Syn is involved in clathrin-dependent and “kiss and run” endocytosis which occurs perisynaptically. Thus, an increase in release of neurotransmitter and in consequence endocytosis would be consistent with an increased active zone distance for vesicles containing Syn.

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