|Bushy cells (BCs) of the anteroventral cochlear nucleus (aVCN) are the first relay station of ascending auditory information. Sound is transformed from mechanical to electrical stimuli by the inner hair cells of the cochlea and transmitted to the auditory brainstem by spiral ganglion neurons (SGNs). To enable consistent firing of Bushy cells, auditory nerve fibers, arising from SGNs terminate on BCs with large calyceal axosomatic endings, called the endbulbs of Held. Since BCs are tuned to respond with short action potentials, phase lock and entrain to the sound stimulus, endbulbs display pronounced short term depression and fast recovery which allow fast sound-onset coding and consistent synaptic transmission even at high frequencies, produced by high sound intensities. These properties are important for the localization of sound in the azimuth plane and BCs are connected with other nuclei of the auditory brainstem to enable these computations, which crucially depend on precise timing. This thesis sheds new light on the molecular composition of pre-synaptic endbulb active zones and how it is connected with their unique physiology. It also investigates how the auditory system can be modulated by hormone-like neurotransmitters, that are released in a brain-wide fashion and, importantly, are able to tweak the molecular properties of calyceal synapses and active zones of the auditory pathway, affecting physiology.
First, collaborators and I studied synaptic transmission and the cytoarchitecture of mouse endbulb terminals from RIM-BP2 constitutive knockout (KO) mice (See Chapter 1 and Appendix). RIM-BP2 is a protein that belongs to the pre-synaptic cytomatrix of the active (CAZ) and interacts with other members of the CAZ forming a network of interactions that are important for the function of active zones. In its absence, endbulbs of Held elicit smaller evoked post-synaptic currents unto BCs, their synaptic vesicle (SV) release probability (Pvr) was reduced and the replenishment rate of new SVs was slower. These effects altered the characteristic endbulb short-term plasticity (STP), especially at higher stimulation frequencies. The effect of RIM-BP2 deficiency on STP was rescued by applying high extracellular Ca2+ concentrations. Recovery from SV pool depletion was slower in the KO, which was corroborated by the presence of less SVs close to the pre-synaptic plasma membrane, observed with electron tomography. Immunofluorescence imaging experiments showed that the levels of other CAZ proteins were unaltered at KO endbulbs, but the distance between Ca2+ channels and core CAZ components was increased, disrupting the pre-synaptic terminal’s cytoarchitecture. We postulate a role of RIM-BP2 in controlling the distance between the SV release machinery and Ca2+ and additionally promoting SV tethering and priming, increasing endbulb Pvr, possibly through interactions with Munc13-1.
Second, the effects of monoamines on the mouse endbulb of Held synapse were studied, with a focus on mechanisms of pre-synaptic neuromodulation. Immunofluorescence imaging of aVCN slices revealed pronounced innervation of the lower auditory pathway by noradrenergic and serotonergic fibers, while the presence of dopamine was not as prominent, consistent with existing literature on other rodents and cats. Preliminary experiments with monoamine receptor blockers show that baseline transmission is important for setting up synaptic properties by regulating the amplitude, frequency and kinetics of spontaneous neurotransmitter release events. Evoked release was not similarly affected by the blockers. Application of monoamine receptor agonists, especially norepinephrine (NE), altered the short-term plasticity of endbulbs, reducing Pvr and variability of evoked release, while it slowed down the recovery from SV pool depletion. We cautiously interpret these preliminary results, and consider a role of NE in reducing STD and post-synaptic receptor desensitization at endbulbs, promoting onset coding through convergence of subthreshold inputs on BCs.