Ontogeny of CL- homeostasis in spherical bushy cells (SBC) on the anteroventral cochlear nucleus (AVCN): impact on GABAergic and glycinergic neurotransmission

• Applicant: Professor Dr. Ivan Milenkovic
• Subject Area: Cognitive, Systems and Behavioural Neurobiology
• Term: from 2005 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5452597

Spherical bushy cells (SBC) of the mammalian cochlear nucleus transmit phaselocked activity of the auditory nerve in pathways that contribute to sound localization based on interaural time differences. However, the firing of SBC is determined by an interaction between excitatory (glutamatergic) and inhibitory inputs (GABAergic and glycinergic). Depending on the Cl- gradient across the membrane, activation of GABAA and glycine receptors can either depolarize neurons or, like in most mature neurons, hyperpolarize them. The aim of this project is to investigate the ontogeny of the Cl- regulation mechanisms and their outcome on the inhibitory neurotransmission in SBC. We will conduct three series of experiments: First, we intent to investigate the developmental pattern of the KCC2, a neuronal specific isoform of the K+-Cl- cotransporter which is, by extruding internal Cl-, critical for inhibitory GABA responses in mature neurons. Immunofluorescence and Western blot studies, during early postnatal development (before and after hearing onset), will examine the expression pattern of the KCC2 in SBC. Using brain slice preparations, gramicidin-perforated patch clamp recordings of SBC will scrutinize the driving force for Cl- by determining EGABA, EGly, and Vrest. Second, we will address the basis for excitatory GABAergic activity in SBC which we observed even after the hearing onset (P12). These experiments will explore the activity of the Cl¿accumulating Na+-K+-2Cl- cotransporter (NKCC1) which is responsible for excitatory GABAergic activity in diverse immature brain structures and in mature sensory neurons. Using loop diuretic drugs and assuming the differential activity pattern of both Clcotransporters throughout development, we intent to challenge Cl--accumulation early in development and Cl--extrusion in later stages which is, most likely, correlated with KCC2 activity. Third, Ca2+-imaging measurements around the hearing onset will address the GABA- and glycine-evoked [Ca2+]i responses since they, indirectly, provide evidence about the nature (depolarizing or hyperpolarizing) of the Cl- gradient and, directly, about the effects of inhibitory transmission on the Ca2+ signalling in SBC. If NKCC1 does not accompany the depolarizing action of GABA and glycine in SBC, our research will focus on another potential candidate which was recently postulated to render depolarizing Cl- gradient in auditory brainstem neurons, HCO3 -/Cl- exchanger AE3. Better understanding of the chloride regulation mechanisms in SBC will provide new insight into roles of GABA and glycine in coding acoustic signals at the first central synapse of the auditory pathway.

• DFG Programme: Research Grants