Abstract:
The role of P2X4, one of the most abundant ionotropic purinergic receptors in the central nervous system, is explored in the context of auditory function. We observed, by using constitutive and conditional P2X4mCherryIN knock-in adult mouse models of either sex, a specific high expression of mCherry-tagged P2X4 in living cochlear outer hair cells (OHCs), from immature postnatal stages to adulthood. This P2X4-mCherry expression, confirmed by confocal immunofluorescence microscopy in wild-type mice, was mainly concentrated in the intracellular apical region of the OHCs, in the area of the Hensen's body, a lysosomal rich region, specifically labeled with the fluorescent dye lysotracker. In addition, the basal cholinergic efferent synaptic region of the OHCs was found to express P2X4 at the cell membrane. Surprisingly, the assessment of the hearing function in constitutive P2X4 knock-out (P2X4KO) mice showed improved auditory brainstem responses (ABRs) with smaller latencies and lower thresholds. These P2X4KO mice, as well as conditional Myo15-Cre:P2X4KO mice, displayed enhanced distortion product otoacoustic emissions (DPOAEs), suggesting an improved electromechanical 'amplification' activity by OHCs. These mutant animals showed reduced inhibition of DPOAEs by contralateral noise, consistent with a weaker inhibitory effect of the medial cholinergic olivocochlear efferent circuit (MOC) on OHCs. When P2X4KO mice were exposed to noise (white-noise 95dB-SPL, 12h) ABRs decreased and partially recovered much like wild-type mice, but DPOAEs showed faster recovery. We concluded that the MOC negative feedback modulation of cochlear micromechanics, in addition to involve Ca(2+) permeable alpha9/alpha10 nicotinic receptors, also requires the activation of postsynaptic P2X4 receptors in OHCs.Significance statement Our study reveals a specific strong expression of the purinergic P2X4 receptor, the main ATP-gated-cation channels in the central nervous system, in mouse cochlear outer hair cells. These cells are essential for generating the distortion products otoacoustic emissions (DPOAES) and tuning the sensitivity and frequency selectivity of the hearing organ, the cochlea. Mice lacking P2X4 showed a deficient inhibitory control of their cochlear DPOAEs when activating the medial olivocochlear efferent pathway innervating the OHCs. We propose P2X4 receptors as an important Ca(2+) regulatory component of the micromechanics of OHCs and that genetic defects in these purinergic receptors may potentially lead to hearing disorders.

Abstract:
Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains limited. To address this question, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof(+/GFP) mice exhibited a normal hearing function, homozygote Otof(GFP/GFP) mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof (GFP/GFP) IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.

Abstract:
It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions. Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest. This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting(1). By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Abstract:
Stimuli that induce rhythmic firing in trigeminal neurons also increase astrocytic coupling and reveal networks that define the boundaries of this particular population. Rhythmic firing depends on astrocytic coupling which in turn depends on S100beta. In many nervous functions that rely on the ability of neuronal networks to generate a rhythmic pattern of activity, coordination of firing is an essential feature. Astrocytes play an important role in some of these networks, but the contribution of astrocytic coupling remains poorly defined. Here we investigate the modulation and organization of astrocytic networks in the dorsal part of the trigeminal main sensory nucleus (NVsnpr), which forms part of the network generating chewing movements. Using whole-cell recordings and the dye coupling approach by filling a single astrocyte with biocytin to reveal astrocytic networks, we showed that coupling is limited under resting conditions, but increases importantly under conditions that induce rhythmic firing in NVsnpr neurons. These are: repetitive electrical stimulation of the sensory inputs to the nucleus, local application of NMDA and decrease of extracellular Ca(2+) . We have previously shown that rhythmic firing induced in NVsnpr neurons by these stimuli depends on astrocytes and their Ca(2+) -binding protein S100beta. Here we show that extracellular blockade of S100beta also prevents the increase in astrocytic coupling induced by local application of NMDA. Most of the networks were small and remained confined to the functionally distinct area of dorsal NVsnpr. Disrupting coupling by perfusion with the nonspecific gap junction blocker, carbenoxolone or with GAP26, a selective inhibitor of connexin 43, mostly expressed in astrocytes, abolished NMDA-induced rhythmic firing in NVsnpr neurons. These results suggest that astrocytic coupling is regulated by sensory inputs, necessary for neuronal bursting, and organized in a region specific manner.