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.

Chemogenetically decreasing activity of the dorsal cochlear nucleus can ameliorate noise-induced tinnitus in mice

“…This would explain the fact that mice that recovered after CNO injection in the first set of experiments presented only a partial recovery (Figure 2B), meaning the startle suppression was not restored to pre-noise exposure values, but significantly improved compared to post-noise exposure after NaCl injection. It is also important to point out that we are only targeting a small area of the DCN using our electrode shank, as well as the virus injections being local and might only affect neurons in the vicinity of the injection sites (Malfatti et al, 2021). Here we could not confirm hM4Di spread of infection due to the weak expression of the mCitrine fluorescent protein.…”

“…To investigate the cellular contribution to noise-induced tinnitus mice were initially screened for capability to carry out the gap prepulse inhibition of acoustic startle (GPIAS) test developed for evaluating tinnitus in rodents (Turner et al, 2006). Mice were acclimatized and habituated to the test equipment before subjected to GPIAS (Figure 1A) testing the capability of detecting a short (40ms) silence in background noise (60dBSPL) 100ms prior to a loud startle pulse (105dBSPL, 50ms duration), thereby suppressing the acoustic startle reflex by at least 30% (Li et al, 2013 1A) for adequate hM4Di expression in CaMKIIα expressing cells, comprising both excitatory and some inhibitory cell populations (Malfatti et al, 2021). Hearing threshold was evaluated by recording auditory brainstem responses (ABRs, Figure 1B-C) three days prior to noise exposure (1h, 90dBSPL, 9-11kHz filtered uniform white noise, followed by 2h in silence) under anesthesia in order to induce tinnitus-like behavior (Winne et al, 2020).…”

“…and sound-evoked activity was recorded using a 16-channel single-shank silicon probe lowered into the left DCN (Malfatti et al, 2021) in response to auditory stimuli following NaCl and CNO i.p. injections (30 min prior to recordings, Figure 3A).…”

“…Finally, we did not record from units of either experimental group (noise-exposed or noise-exposed + CNO) at any particular depth or layer, as we did not want to bias data to any particular region of the DCN (Figure 8). Unit recordings describes units responding to sound when CaMKIIα+ neurons of the DCN circuit had the firing frequency in response to sound chemogenetically lowered, and does thus not reflect recordings from CaMKIIα+ units only (Malfatti et al, 2021).…”

“…Here we behaviorally examine if tinnitus perception can be reduced by lowering the activity of CaMKII α positive DCN neurons using chemogenetics. We have recently shown this promoter to be expressed by both excitatory and inhibitory DCN neurons, but with a preference for slow-firing units (Malfatti et al, 2021), presumable excitatory fusiform cells (Ochiishi et al, 1998; Oh et al, 2014). We specifically investigated if noise-induced tinnitus, without hearing loss, can be ameliorated by lowering DCN neuronal activity.…”