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bioRxiv · 10.1101/2020.09.19.304550

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

Malfatti, T.·Ciralli, B.·Hilscher, M. M.·Edwards, S. J.·Kullander, K.·Leao, R. N.·Leao, K. E.
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Abstract

The dorsal cochlear nucleus (DCN) is the first auditory region that integrates somatosensory and auditory inputs. The region is of particular interest for auditory research due to the large incidence of somatic tinnitus and increased aberrant activity in other forms of tinnitus. Yet, the lack of useful genetic markers for in vivo manipulations hinders the elucidation of the DCN contribution to tinnitus pathophysiology. In this work, we assessed whether adeno-associated viral vectors (AAV) containing the calcium/calmodulin-dependent protein kinase 2 alpha (CaMKII) promoter and our mouse line of nicotinic acetylcholine receptor alpha 2 subunit (Chrna2)-Cre can be used to target specific DCN populations. The CaMKII promoter is usually applied in studies of principal neurons of neo and paleocortex while Chrna2-cre mice express Cre recombinase in cortical dendrite inhibiting interneurons. We found that CaMKII cannot be used to specifically target excitatory fusiform DCN neurons. EYFP expression driven by the CaMKII promoter was stronger in the fusiform layer but labelled cells showed a diverse morphology indicating that they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 (ChR2) by the CaMKII promoter generated spikes in some units but firing rate decreased when light stimulation coincide with sound presentation. Expression and activation of eArch3.0 (CaMKII driven) in the DCN produced spike inhibition in some units but, most importantly, 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 observed profuse projections arising from the ventral cochlear nucleus (VCN). Anterograde labeling Cre dependent AAV injected in the VCN revealed two main projections: one arising in the ipsilateral superior olive and the 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). Stimulating ChR2 expressing terminals (light applied on the DCN) of VCN Chrna2+ cells increased firing of sound responding and nonresponding DCN units. This work shows that molecular tools intensively used in cortical studies may be useful for manipulating the DCN especially in tinnitus studies.

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Malfatti, T., Ciralli, B., Hilscher, M. M., Edwards, S. J., Kullander, K., Leao, R. N., Leao, K. E.. 2020-09-19. Using cortical neuron markers to target cells in the dorsal cochlear nucleus. https://doi.org/10.1101/2020.09.19.304550

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