DOI: 10.5061/dryad.69p8cz8xp
title: Identification of an inhibitory neuron subtype, the L-stellate cells of the cochlear nucleus

description: Auditory processing depends upon inhibitory signaling by interneurons,
 even at its earliest stages in the ventral cochlear nucleus (VCN).
 Remarkably, to date only a single subtype of inhibitory neuron has been
 documented in the VCN, a projection neuron termed the D-stellate cell.
 With the use of a transgenic mouse line, optical clearing and imaging
 techniques, combined with electrophysiological tools, we revealed a
 population of glycinergic cells in the VCN distinct from the D-stellate
 cell. These novel multipolar glycinergic cells were smaller in soma size
 and dendritic area, but over 10-fold more numerous than D-stellate cells.
 They were activated by auditory nerve fibers 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.
description: All procedures were approved by the Oregon Health and Science
 University’s Institutional Animal Care and Use Committee. GlyT2-GFP mice
 (Zeilhofer et al., 2005) and Somatostatin(Sst)-Cre tdTomato::GlyT2-GFP of
 either sex, postnatal days(P) 17-30 were used in this study. Mice were
 deeply anesthetized with isoflurane, then perfused transcardially with
 0.9% warm saline followed by 4% paraformaldehyde buffered in PBS. Brains
 were removed and post-fixed overnight at 4 °C. The entire cochlear nucleus
 (450-500 mm) was cut from the brainstem using a vibratome (Leica VT1000S).
 Because the cochlear nucleus sits at the lateral edge of the brainstem,
 only a single cut was required to prepare the specimen. Samples were
 washed in 0.1 M PBS before clearing for 72 h at room temperature on a
 shaker table with an accelerated ‘clear unobstructed brain imaging
 cocktail’ (CUBIC)-mount solution (Lee et al., 2016) containing sucrose (50
 %, w/v), urea (25%, w/v), and
 N,N,N′,N′-tetrakis (2- hydroxypropyl)ethylenediamine (25%, w/v) dissolved
 in 30 ml of dH<sub>2</sub>O. Once
 each sample was cleared and almost transparent, it was then mounted on a
 glass slide with 0.5 mm deep silicone spacers (EMS) in CUBIC-mount. The
 images were acquired within 24 h to reduce volume changes in CUBIC-mount
 (10-20% increase in volume after incubation). The samples were imaged
 using a fast Airyscan LSM880 microscope with a 25X objective. Tiles and
 z-stack of whole cochlear nucleus were imaged at 5-mm step size with the
 exception of one sample where images were obtained at 2-mm step size.
 Images were post-processed and individual tiles were stitched together
 using Zeiss Zen Black software. Analysis of cell count and soma volume
 were quantified using Imaris software (Bitplane 12.1). VCN was selected
 for quantification by drawing regions of interest around the VCN followed
 by applying a mask outside the region of interest. The dorsal border of
 the VCN was defined by the granule cell lamina, and the medial border by
 the absence of glycinergic somata (Muniak et al., 2013).  For cell count
 quantification, we used the built-in “spot detection” algorithm in Imaris
 in which the program places a “spot” on the soma of each cell. The spots
 are used for counting of GFP positive cells in the VCN. Automatically
 detected spots were verified and corrected manually. Thus, if the computer
 failed to detect a cell or erroneously placed a spot on a background
 bright spot, we manually added spots and deleted spots respectively. To
 test the validity of automated spot function, certain regions of VCN were
 manually counted and compared against the spot function. The spot function
 detected more than 95% of all cells present in that area. For volume measurements, we
 used the in-built “surface” rendering function in Imaris. The program
 rendered 3D surfaces on the soma of GFP positive cells. Rendered surfaces
 were used to extract the volume statistics. Surfaces were also verified
 and corrected manually. Spike
 waveforms were analyzed using IgorPro8. Threshold was defined as the
 voltage at 5% of the peak of the spike’s first derivative, measured after
 subtracting the baseline dV/dt generated by the neuron’s electronic
 charging. This method was found empirically to identify the visually
 identified spike ‘onset’ much more reliably than the oft-used peak of the
 3<sup>rd</sup> derivative. The latter method, when applied to
 diverse neurons in our dataset, often failed when the spike had a
 prominent initial segment ‘hump’ on the rising phase, or when the
 3<sup>rd</sup> derivative was noisy. Spike overshoot was
 calculated as spike peak voltage relative to zero. Absolute amplitude of
 the spike was calculated from threshold to peak. Undershoot was the
 negative-most excursion of voltage, relative to threshold, and AHP latency
 was the time delay from spike peak to undershoot peak. Spike AHP was an
 arbitrary definition that measured the difference between voltage at the
 peak of the undershoot to the voltage one ms later. This time point
 captured well differences among neurons, since the D-stellate cells, and
 not the smaller cells, had extremely fast decaying AHPs. Membrane input
 resistance (R<sub>in</sub>) was measured by injecting a small
 hyperpolarizing current in voltage clamp mode, R<sub>in</sub>
 was calculated offline using Ohm’s law. Average spike rate was calculated
 in response to depolarizing 500 pA current injections.