LRRC52 regulates BK channel function and localization in mouse cochlear inner hair cells.

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+ channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (-60 mV) even in the absence of intracellular Ca2+ elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+ channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat-containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs from Lrrc26 knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs from Lrrc52 KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about -90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.

Preparation of the intact rodent organ of Corti for RNAscope and immunolabeling, confocal microscopy, and quantitative analysis.

This protocol describes the preparation of the mouse organ of Corti for RNAscope, immunolabeling, confocal microscopy, and quantitative image analysis to examine transcript and protein localization, sensory hair cells, and synapses. This protocol can be applied to mice and other rodents (juvenile and adult) and can be adapted for other techniques, including electrophysiology and RNA sequencing. This protocol features minimal tissue processing to preserve viability for downstream assays, while isolating the organ of Corti is the most challenging step. For additional details on the use and execution of this protocol, please refer to McLean et al. (2009); Schuth et al. (2014); Lingle et al. (2019); Pyott et al. (2020).

Regulation of auditory plasticity during critical periods and following hearing loss.

Sensory input has profound effects on neuronal organization and sensory maps in the brain. The mechanisms regulating plasticity of the auditory pathway have been revealed by examining the consequences of altered auditory input during both developmental critical periods-when plasticity facilitates the optimization of neural circuits in concert with the external environment-and in adulthood-when hearing loss is linked to the generation of tinnitus. In this review, we summarize research identifying the molecular, cellular, and circuit-level mechanisms regulating neuronal organization and tonotopic map plasticity during developmental critical periods and in adulthood. These mechanisms are shared in both the juvenile and adult brain and along the length of the auditory pathway, where they serve to regulate disinhibitory networks, synaptic structure and function, as well as structural barriers to plasticity. Regulation of plasticity also involves both neuromodulatory circuits, which link plasticity with learning and attention, as well as ascending and descending auditory circuits, which link the auditory cortex and lower structures. Further work identifying the interplay of molecular and cellular mechanisms associating hearing loss-induced plasticity with tinnitus will continue to advance our understanding of this disorder and lead to new approaches to its treatment.

Chronic postnatal monoamine oxidase inhibition affects affiliative behavior in rat pupso.

Monoamine neurotransmitters serotonin (5-HT), dopamine (DA), and noradrenaline (NA) act as important modulators of mammalian brain development and represent neurobiological substrates of affiliative behavior reflected in rat pups as a tendency to huddle or produce ultrasonic vocalizations (USV) when separated from the nest. Monoamines are metabolized through oxidative deamination catalyzed by the mitochondrial enzyme monoamine oxidase (MAO). In this study, we examined the consequences of postnatal MAO inhibition on affiliative behavior in rat pups. Pups received daily injections of either an irreversible non-selective MAO inhibitor tranylcypromine (TCP) or saline, from post-natal day (PND) 1 to PND 22. Quantitative and qualitative components of USV were analyzed on PNDs 10, 13 and 16 in order to determine the level of separation-induced anxiety and the modality of vocal communication. In comparison to control pups, TCP-treated pups displayed higher cortical 5-HT, DA and NA levels, higher peripheral 5-HT concentration, lower body mass throughout the pre-weaning period, higher isolation-induced drop in body temperature, and reduced total number of calls. Furthermore, they produced lower pitched calls of longer average duration without a preferable waveform. Our results demonstrate that chronic MAO inhibition by TCP primarily affects 5-HT concentrations, but also raises central catecholamine levels. They further indicate that disturbed monoaminergic homeostasis during early postnatal development leads to decreased weight-gain, compromised thermoregulation, and altered affiliative behavior in pre-weaning pups as reflected in reduced separation anxiety and inadequate vocal communication. Finally, they suggest a need for thorough examination of the potential effects of TCP and other monoamine inhibitors on the developing human brain.