Many auditory brainstem neurons are able to maintain high-bandwidth, high-frequency firing over prolonged periods of time. For example, excitatory (glutamatergic) and inhibitory (glycinergic) neurons projecting to the lateral superior olive (LSO), which form the basis of sound localization via interaural intensity detection, drive their postsynaptic target neurons up to ca. 600 times per second. We have shown that fast synaptic transmission at these synapses is remarkably robust during sustained stimulation as evidenced by resistance to fatigue and low failure rates. These features set them apart from ‘conventional’ synapses, which depress more profoundly and at significantly lower frequency, and recover much less efficiently from their depressed state. Apparently, auditory synapses employ very efficient vesicle replenishment to indefatigably encode sound. The underlying molecular mechanisms for the differences in synaptic performance are unknown.
We want to analyze the molecular regulation of high-frequency synaptic transmission to LSO neurons by focusing on four key proteins in the presynaptic release machinery which appear to be determinants of the speed and fidelity of transmitter release (Bassoon, Munc13-1, Synaptotagmin2, CAPS1). Causal relationship will be demonstrated by specific gene deletion. To circumvent problems immanent to systemic gene deletion, we will perform spatially restricted gene ablation, which became available only very recently through the generation of transgenic mice with floxed genes for the candidate molecules. Temporally and spatially restricted gene silencing will be pursued via stereotaxic gene delivery (Cre recombinase) through viral transfection. As a benchmark, we will also analyze two synapse types in the hippocampal formation, namely the glutamatergic CA3-CA1 connection and the GABAergic connection from the entorhinal cortex to the dentate gyrus. We will quantify basic synaptic transmission as well as synaptic plasticity with a battery of established stimulus protocols. We hypothesize that robust high-frequency transmission and resistance to synaptic fatigue are manifested by synapse-specific characteristics in the presynaptic release machinery. We also suggest that the four candidate proteins contribute in a manner that overlaps only partially with their peer proteins, thereby achieving a moderate degree of redundancy. Histological investigations comprising light and electron microscopic immunohistochemistry will complement our physiological analyses.
In total, we expect to better understand the underlying mechanisms for high frequency neurotransmission and to elucidate the role of some key players in synapse function. Our project shall also contribute to comprehending general aspects of synaptic information transfer in various neuronal systems.