Using cortical neuron markers to target cells in the dorsal cochlear nucleus

Abstract

The dorsal cochlear nucleus (DCN) is a region of particular interest for auditory and tinnitus research. Yet, lack of useful genetic markers for in vivo manipulations hinders elucidation of the DCN contribution to tinnitus pathophysiology. This work assesses whether adeno-associated viral vectors (AAV) containing the calcium/calmodulin-dependent protein kinase 2 alpha (CaMKIIα) promoter and a mouse line of nicotinic acetylcholine receptor alpha 2 subunit (Chrna2)-Cre can target specific DCN populations. We found that CaMKIIα cannot be used to target excitatory fusiform DCN neurons as labelled cells showed diverse morphology indicating they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 by the CaMKIIα promoter generated spikes in some units but firing rate decreased when light stimulation coincided with sound. Expression and activation of CaMKIIα-eArchaerhodopsin3.0 in the DCN produced inhibition in some units but sound-driven spikes were delayed by concomitant light stimulation. We explored the existence of Cre+ cells in the DCN of Chrna2-Cre mice by hydrogel embedding technique (CLARITY). There were almost no Cre+ cell bodies in the DCN; however, we identified profuse projections arising from the ventral cochlear nucleus (VCN). Anterograde labeling in the VCN revealed projections to the ipsilateral superior olive and contralateral medial nucleus of the trapezoid body (bushy cells); and a second bundle terminating in the DCN, suggesting the latter to be excitatory Chrna2+ T-stellate cells. Exciting Chrna2+ cells increased DCN firing. This work shows that cortical molecular tools may be useful for manipulating the DCN especially for tinnitus studies.

Significance statement Cortical neuron markers could be used to label subcortical regions such as the dorsal cochlear nucleus (DCN) that integrates sound and somatosensory input. Here we examine if excitatory (CaMKIIα) or inhibitory (Chrna2) neuron markers used in neo/paleo-cortex studies label unique DCN populations. We found CaMKIIα to be expressed by different DCN neuron populations (affecting sound sensitive and nonsensitive neurons). The Chrna2 promoter was specifically expressed in excitatory cells of the ventral cochlear nucleus and could drive indirect activity in the DCN. This study highlights novel ways of regulating DCN neuron activity, which can provide new means for treatment of bothersome tinnitus.