The non-ionic role of Kv3.3 on the distribution of vesicles in hippocampal synapses and the calyx of Held

Changes in postsynaptic neuronal excitability have been well characterised in epilepsy, but presynaptic contributions are less understood. Hyperexcitability and seizures in a mouse model of spinocerebellar ataxia type 13 (SCA13) where the function of Kv3.3 potassium channels are impaired, causing increased neurotransmitter release at the presynaptic neuron. My project studied the organisation of synaptic vesicles (SV) near to transmitter release sites. I measured the proximity of SVs to the release site using electron microscopy in osmium-stained tissue from wild-type (WT) and SCA13 transgenic mice.

I tested the following hypothesis: Kv3.3 influences SV density at the release site of hippocampal synapses. I measured hippocampal synapses across 4 genotypes: WT, Kv3.3 knockout (KO), 2 SCA13 point mutations (R420H and G592R). I compared SV density at synapses from CA1 (little Kv3.3) and CA3 (high Kv3.3). Mice were killed and perfused with low calcium aCSF and then fixed. Osmium-thiocarbohydrazide was used as a staining and contrast agent, and sectioned tissue was imaged on a JEOL JEM-1400 electron microscope.

SCA13-causing mutations of Kv3.3 increase vesicle density in excitatory CA1 hippocampal synapses, but only a Kv3.3 knockout (KO) and the G592R mutant increased vesicle density in the CA3. However, at the calyx of Held, only the R420H and G592R mutation showed increased vesicle density. I conclude that Kv3.3 controls vesicle recycling at hippocampal synapses through a direct or indirect ionic mechanism, causing increased proximity of SV to the active zones of synapses when this channel is mutated. The mechanism is either attributed to Kv3.3 interaction with the actin cytoskeleton, or an indirect ionic mechanism controlling intracellular calcium. This would increase neurotransmission when the channel is mutated, and contribute to seizure activity and enhance epileptic susceptibility. I also conclude that the same mechanism operates at the calyx of Held is the same mechanism, but the Kv3.3-KO is compensated by an over-expression of Kv3.1 channels, or that Kv3.3 has an alternative role, depending on the cell type. Further research is needed to comprehensively understand the role of Kv3-channels in different cell types.