Using Cortical Neuron Markers to Target Cells in the Dorsal Cochlear Nucleus.

Thawann Malfatti,Markus M Hilscher,Klas Kullander,Katarina E Leao,Steven J Edwards,Barbara Ciralli,Richardson N Leao

doi:10.1523/eneuro.0413-20.2020

Thawann Malfatti, Markus M Hilscher + Show 5 more

Open Access

https://doi.org/10.1523/eneuro.0413-20.2020

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Abstract

The dorsal cochlear nucleus (DCN) is a region of particular interest for auditory and tinnitus research. However, 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 2α (CaMKIIα) promoter and a mouse line of nicotinic acetylcholine receptor α2 subunit (Chrna2)-Cre can target specific DCN populations. We found that CaMKIIα cannot be used to target excitatory fusiform DCN neurons as labeled cells showed diverse morphology indicating they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 (ChR2) 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 (MNTB; 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.

Highlights

The dorsal cochlear nucleus (DCN) of the auditory brainstem is the first integrator of auditory and multisensory signals and has been pointed as a key structure in tinnitus physiopathology (Kaltenbach et al, 2005; Tzounopoulos, 2008; Baizer et al, 2012)
Auditory brainstem responses (ABRs) can be extracted during optogenetic excitation of CaMKIIa-ChR2-positive DCN neurons
Smaller neuronal somas were labeled with enhanced yellow fluorescent protein (eYFP) in the fusiform and deeper layers, as well as several large neuronal somas of the deep layer of the DCN with dendrites stretching along the internal edge of the DCN

Summary

Introduction

The dorsal cochlear nucleus (DCN) of the auditory brainstem is the first integrator of auditory and multisensory signals and has been pointed as a key structure in tinnitus physiopathology (Kaltenbach et al, 2005; Tzounopoulos, 2008; Baizer et al, 2012). Cells in the DCN receive direct or indirect [e.g., relayed by the ventral cochlear nucleus (VCN)] sound input onto different cell populations in a layer arrangement. The most cell-populated DCN field is the fusiform cell layer formed by excitatory fusiform cells intercalated with interneurons (Oertel and Young, 2004). An interesting aspect of the DCN is its architectural similarity to the cerebellum (Devor, 2000) that is thought to be responsible for integrative processing (e.g., sound/somatosensory; Oertel and Young, 2004). A large number of studies have shown altered synaptic and intrinsic cellular properties within the DCN circuit relating

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