Spontaneous Ca2+ signals in the developing mammalian cochlea of live mice under different anaesthetic regimes

The pre-hearing mouse cochlea undergoes critical periods of spontaneous Ca2+-dependent activity that spreads across non-sensory supporting cells and inner hair cells (IHCs). These signals have been shown to regulate not only the refinement of neural circuits along the auditory pathway towards functional maturity, but also the maturation of the hair cells into sensory receptors. Although the origin and interplay of these Ca2+ signals during cochlear development have recently been investigated in live mice, the impact of anaesthesia on in vivo functional measurements was not explored. Here, we investigate the effects of different anaesthetic regimes (ketamine and xylazine; 2.5% isoflurane; and 1.0%–1.5% isoflurane with the sedative acepromazine) that provided an effective unconsciousness to perform the surgery and Ca2+-imaging recordings from the intact cochlea of live mice. The IHCs, supporting cells and spiral ganglion neuron terminals onto the IHCs showed spontaneous Ca2+-dependent activity under all anaesthetic regimes, with a few significant differences observed between conditions. Calcium waves from supporting cells synchronized the activity of IHCs. Moreover, we found that the endocochlear potential, which is crucial for cochlear function, was unaffected by the different anaesthetics. However, low concentrations of isoflurane produced the most stable recordings of vital physiological signs in mice, including heart rate and breathing rate. Although all anaesthetic regimes tested appeared to be suitable for performing Ca2+ imaging from the cochlea of pre-hearing live mice, a low concentration of isoflurane (1.0%–1.5%), combined with the pre-anaesthetic sedative acepromazine and oxygenation, represents the most suitable approach to maintain a stable and long-lasting depth of anaesthesia.