Whole-cell patch clamp recording

For electrophysiological recording, acute brain slices (350 or 225 μm in thickness) were incubated in gassed ACSF at 34 °C for ~45 minutes to allow recovery from the slicing, then transferred to a submersion chamber on a fixed-stage Axio Examiner microscope (Carl Zeiss Microscopy, LLC) and bathed in continuous flow of ACSF. Whole-cell patch clamp recording was performed using Multiclamp 700B amplifier, Axon Digidata 1550B digitizer, and pClamp 10 software (Molecular Devices). The recording pipette was pulled from KG-33 borosilicate glass (King Precision Glass) using a Sutter P-2000 puller (Sutter Instruments). Electrode solution contained (in mM): 105 K-gluconate, 36 KCl, 2 NaCl, 10 HEPES, 0.2 EGTA, 4 MgATP, 0.3 GTP, and 10 Tris-phosphocreatine, with pH adjusted to 7.2. All reported voltages were adjusted with a junction potential of −12 mV. To visualize cellular morphology, Alexa Fluor 488 (final concentration of 0.01% by weight) was added to the electrode solution for online fluorescent imaging as well as subsequent processing in immunohistochemistry. Five μM CNQX was added to the ACSF to block glutamatergic excitatory synaptic transmission. All recordings were made at 34 °C.

Under current clamp mode, membrane responses to depolarizing and hyperpolarizing step current injections were obtained from all three major cell types of the AVCN, including bushy, T-stellate and D-stellate neurons. Cell type was identified based on both electrophysiological and morphological features as described in previous studies (Brawer et al., 1974;Cant and Morest, 1979;Wu and Oertel, 1984;Xie and Manis, 2013;2017). Specifically, bushy neurons were characterized by having short primary dendrites and heavily-branched tufts in resemble of a “bush” (Brawer, et al., 1974;Cant and Morest, 1979;Lauer et al., 2013;Webster and Trune, 1982), as well as firing only one or a few small spikes to prolonged suprathreshold current injections (Wu and Oertel, 1984). In contrast, both T- and D-stellate neurons had thin and long dendrites without heavily-branched tufts, and electrically fired tonic spikes to prolonged current injections (Fujino and Oertel, 2001;Wu and Oertel, 1984;Xie and Manis, 2017). T- and D-stellates were further distinguished by the orientation of their primary dendrites, in which T-stellate neurons have dendrites that ran parallel to the fascicles of auditory nerve fibers, while D-stellate neurons have dendrites spanned multiple fascicles (Doucet and Ryugo, 1997;Smith and Rhode, 1989;Xie and Manis, 2017). They also responded differently to large hyperpolarizing current injections, in which T-stellate neurons show slow and small depolarization sags, whereas D-stellate neurons show large and fast depolarization sags (Fujino and Oertel, 2001;Rodrigues and Oertel, 2006;Xie and Manis, 2017). Glycinergic IPSCs were obtained under voltage clamp mode in response to electrical stimulation at the DCN, which presumably activated the DCN tuberculoventral neurons that provide glycinergic inhibitory inputs to AVCN neurons (Muniak and Ryugo, 2014;Wickesberg and Oertel, 1990;Xie and Manis, 2013;2013;Zhang and Oertel, 1993). Stimulus pulse had a duration of 100 μs and was delivered via a 75 μm diameter concentric stimulating electrode (Frederick Haer Company). Stimulus amplitude was determined by gradually increasing the stimulus strength from zero until stimuli evoked reliable IPSCs in the target neuron.