ATP Hydrolysis Is Critically Required for Function of Caᵥ1.3 Channels in Cochlear Inner Hair Cells via Fueling Ca²⁺ Clearance

Sound encoding is mediated by Ca²⁺ influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca²⁺ current through Caᵥ1.3 channels and Ca²⁺ homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca²⁺ imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca²⁺ influx in inner hair cells via fueling Ca²⁺-ATPases to avoid an increase in cytosolic [Ca²⁺] and subsequent Ca²⁺/calmodulin-dependent inactivation of Caᵥ1.3 channels

EF-hand protein Ca²⁺ buffers regulate Ca²⁺ influx and exocytosis in sensory hair cells

EF-hand Ca²⁺-binding proteins are thought to shape the spatiotemporal properties of cellular Ca²⁺ signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca²⁺ signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca²⁺-dependent inactivation of their Ca²⁺ current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca²⁺ channels and release sites (effective “coupling distance” of 17 nm). Substitution experiments with synthetic Ca²⁺ chelators indicated the presence of endogenous Ca²⁺ buffers equivalent to 1 mM synthetic Ca²⁺-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca²⁺ buffers regulate presynaptic IHC function for metabolically efficient sound coding

Exocytotic release of ATP from cultured astrocytes.

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared

Macromolecular and electrical coupling between inner hair cells in the rodent cochlea

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in ‘mini-syncytia’. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding

Astrocytic Ion Dynamics: Implications for Potassium Buffering and Liquid Flow

We review modeling of astrocyte ion dynamics with a specific focus on the implications of so-called spatial potassium buffering, where excess potassium in the extracellular space (ECS) is transported away to prevent pathological neural spiking. The recently introduced Kirchoff-Nernst-Planck (KNP) scheme for modeling ion dynamics in astrocytes (and brain tissue in general) is outlined and used to study such spatial buffering. We next describe how the ion dynamics of astrocytes may regulate microscopic liquid flow by osmotic effects and how such microscopic flow can be linked to whole-brain macroscopic flow. We thus include the key elements in a putative multiscale theory with astrocytes linking neural activity on a microscopic scale to macroscopic fluid flow.Comment: 27 pages, 7 figure

Neuronal energy consumption: biophysics, efficiency and evolution

Electrical and chemical signaling within and between neurons consumes energy. Recent studies have sought to refine our understanding of the processes that consume energy and their relationship to information processing by coupling experiments with computational models and energy budgets. These studies have produced insights into both how neurons and neural circuits function, and why they evolved to function in the way they do