SUMMARY Spatial patterns of functional organization, resolved by microelectrode mapping, comprise a core principle of sensory cortices. In auditory cortex, however, recent two-photon Ca2+ imaging challenges this precept, as the traditional tonotopic arrangement appears weakly organized at the level of individual neurons. To resolve this fundamental ambiguity about the organization of auditory cortex, we developed multiscale optical Ca2+ imaging of unanesthetized GCaMP transgenic mice. Single-neuron activity monitored by two-photon imaging was precisely registered to large-scale cortical maps provided by transcranial wide field imaging. Neurons in the primary field responded well to tones, neighboring neurons were appreciably co-tuned, and preferred frequencies adhered tightly to a tonotopic axis. By contrast, nearby secondary-field neurons exhibited heterogeneous tuning. The multiscale imaging approach also readily localized vocalization regions and neurons. Altogether, these findings cohere electrode and two-photon perspectives, resolve new features of auditory cortex, and offer a promising approach generalizable to any cortical area.

Neurons in the developing auditory system exhibit spontaneous bursts of activity before hearing onset. How this intrinsically generated activity influences development remains uncertain, because few mechanistic studies have been performed in vivo. We show using macroscopic calcium imaging in unanesthetized mice that neurons responsible for processing similar frequencies of sound exhibit highly synchronized activity throughout the auditory system during this critical phase of development. Spontaneous activity normally requires synaptic excitation of spiral ganglion neurons (SGNs). Unexpectedly, tonotopic spontaneous activity was preserved in a mouse model of deafness in which glutamate release from hair cells is abolished. SGNs in these mice exhibited enhanced excitability, enabling direct neuronal excitation by supporting cell-induced potassium transients. These results indicate that homeostatic mechanisms maintain spontaneous activity in the pre-hearing period, with significant implications for both circuit development and therapeutic approaches aimed at treating congenital forms of deafness arising through mutations in key sensory transduction components.

The regulation of CaV2.1 (P/Q-type) channels by calmodulin (CaM) showcases the powerful Ca2+ decoding capabilities of CaM in complex with the family of CaV1-2 Ca2+ channels. Throughout this family, CaM does not simply exert a binary on/off regulatory effect; rather, Ca2+ binding to either the C- or N-terminal lobe of CaM alone can selectively trigger a distinct form of channel modulation. Additionally, Ca2+ binding to the C-terminal lobe triggers regulation that appears preferentially responsive to local Ca2+ influx through the channel to which CaM is attached (local Ca2+ preference), whereas Ca2+ binding to the N-terminal lobe triggers modulation that favors activation via Ca2+ entry through channels at a distance (global Ca2+ preference). CaV2.1 channels fully exemplify these features; Ca2+ binding to the C-terminal lobe induces Ca2+-dependent facilitation of opening (CDF), whereas the N-terminal lobe yields Ca2+-dependent inactivation of opening (CDI). In mitigation of these interesting indications, support for this local/global Ca2+ selectivity has been based upon indirect inferences from macroscopic recordings of numerous channels. Nagging uncertainty has also remained as to whether CDF represents a relief of basal inhibition of channel open probability (P o) in the presence of external Ca2+, or an actual enhancement of P o over a normal baseline seen with Ba2+ as the charge carrier. To address these issues, we undertake the first extensive single-channel analysis of CaV2.1 channels with Ca2+ as charge carrier. A key outcome is that CDF persists at this level, while CDI is entirely lacking. This result directly upholds the local/global Ca2+ preference of the lobes of CaM, because only a local (but not global) Ca2+ signal is here present. Furthermore, direct single-channel determinations of P o and kinetic simulations demonstrate that CDF represents a genuine enhancement of open probability, without appreciable change of activation kinetics. This enhanced-opening mechanism suggests that the CDF evoked during action-potential trains would produce not only larger, but longer-lasting Ca2+ responses, an outcome with potential ramifications for short-term synaptic plasticity.