Data CitationsTrussell L. area, but over ten-fold more numerous than D-stellate cells. They were activated by auditory nerve and T-stellate cells, and made local inhibitory synaptic contacts on principal cells of the VCN. Given their abundance, combined with their narrow dendritic fields and axonal projections, it is likely that these neurons, here termed L-stellate cells, play a significant role in frequency-specific processing of acoustic signals. (Keine and Rbsamen, 2015) and (Campagnola and Manis, 2014). Indeed, no local inhibitory interneuron has ever been described for VCN, which is unlike most known brain regions. Early anatomical studies suggested Desmopressin Acetate the presence of short axon cells in the VCN (Lorente de N, 1981). Furthermore, previous histological studies also reported the presence of small inhibitory neurons (glycinergic or possibly GABAergic) that are different from the D-stellate cells, but the identity of such neurons is unknown (Wenthold, 1987; Kolston et al., 1992; Doucet and Ryugo, 1997; Gleich and Vater, 1998; Doucet et al., 1999). Here we comprehensively examined the diversity of inhibitory neurons in the VCN using a well-characterized transgenic mouse line, GlyT2-GFP, (Zeilhofer et al., 2005; Kuo et al., 2012; Moore and Trussell, 2017) which labels virtually all glycinergic neurons in the CN. With the use of this transgenic mouse, as well as optical tissue clearing, whole CN super-resolution microscopy, electrophysiological, and morphological tools, we discovered a large population of inhibitory glycinergic cell types distinct RaLP from the D-stellate cell. The novel glycinergic neurons, which have a narrower dendritic field than D-stellate cells, form the vast majority of inhibitory neurons in the VCN. We show that these cells, which we have termed L-stellate cells, receive monosynaptic ANF inputs and polysynaptic inputs from axon collaterals of T-stellate cells, and in turn locally inhibit bushy and T-stellate cells in the VCN. Thus, the VCN has a rich diversity of glycinergic interneurons to provide maximum flexibility to control the excitability of projection neurons in the VCN. Results Cell counts and soma size We used a well-characterized GlyT2-GFP transgenic mouse (Zeilhofer et al., 2005; Albrecht et al., 2014; Moore and Trussell, 2017) in order to study the prevalence of glycinergic cells in the VCN. The neuronal glycine transporter, GlyT2, is a reliable marker of glycinergic cells, and GFP is selectively expressed in? 90% of glycinergic cells in the CN. To visualize all the glycinergic cells, we optically cleared whole CN (450C500 m) using CUBIC-mount (Lee et al., 2016). Next, we imaged the entire CN, lateral to medial (Figure 1). Figure 1A shows a series of 50 m thick image stacks of CN, Desmopressin Acetate lateral to medial. Not surprisingly, we observed a dense population of glycinergic cells in the DCN as described in previous studies (Oertel and Wu, 1989; Zhang and Oertel, 1993b; Zhang and Oertel, 1993a; Kuo et al., 2012; Apostolides and Trussell, 2014). Also apparent were thick tracts of glycinergic fibers that entered the dorsal part of VCN, Desmopressin Acetate presumably projections of the glycinergic tuberculoventral neurons (vertical cells) in the DCN (Figure 1A). There was an obvious lack of glycinergic cells in the octopus cell region of the Desmopressin Acetate posterior VCN, consistent with previous studies (Wickesberg and Oertel, 1988; Wickesberg et al., 1991). However, we found a large population of glycinergic cells distributed across the rest of the VCN. To obtain a global view of their distribution, the images were stitched and combined to create a 3D image of the entire CN (Figure 1Bi, ii). The high density of glycinergic cells throughout VCN was surprising, because D-stellate cells are thought to be sparse, and the.