Activity of CaMKIIa+ dorsal cochlear nucleus neurons are crucial for tinnitus perception but not for tinnitus induction

1 The dorsal cochlear nucleus (DCN) is a region known to integrate somatosensory and auditory inputs 2 and is identified as a potential key structure in the generation of phantom sound perception, especially 3 noise-induced tinnitus. Yet, how altered homeostatic plasticity of the DCN induces and maintains the 4 sensation of tinnitus is not clear. Here, we chemogenetically decrease activity of a subgroup of DCN 5 neurons, Ca/Calmodulin kinase 2α (CaMKIIα) positive DCN neurons, using Gi-coupled human M4 6 Designer Receptors Exclusively Activated by Designer Drugs (hM4Di DREADDs), to investigate their 7 role in noise-induced tinnitus. Mice were exposed to loud noise (9-11kHz, 90dBSPL, 1h, followed by 2h of 8 silence) and auditory brainstem responses (ABRs) and gap prepulse inhibition of acoustic startle (GPIAS) 9 were recorded two days before and two weeks after noise exposure to identify animals with a significantly 10 decreased inhibition of startle, indicating tinnitus but without permanent hearing loss. Neuronal activity of 11 CaMKIIα+ neurons expressing hM4Di in the DCN was lowered by administration of clozapine-N-oxide 12 (CNO). We found that acutely decreasing firing rate of CaMKIIα+ DCN units decrease tinnitus-like 13 responses (p = 0.038, n = 11 mice), compared to the control group that showed no improvement in GPIAS 14 (control virus; CaMKIIα-YFP + CNO, p = 0.696, n = 7 mice). Extracellular recordings confirmed CNO to 15 decrease unit firing frequency of CaMKIIα-hM4Di+ mice and alter best frequency and tuning width of 16 response to sound. However, these effects were not seen if CNO had been previously administered during 17 the noise exposure (n = 6 experimental and 6 control mice). Our results suggest that CaMKIIα-hM4Di 18 positive cells in the DCN are not crucial for tinnitus induction but play a significant role in maintaining 19 tinnitus perception in mice. 20