Rapid, activity-dependent co-release of GABA and glycine in the mouse brainstem

Abstract

Presympathetic neurons in the ventrolateral and ventromedial medulla (VLM/VMM) control the sympathetic tone, and GABA was identified as a primary mechanism for sympathoinhibition. Intriguingly, there is evidence that both GABA and glycine efficiently inhibit sympathetic tone; however, the mechanism of glycine release is unknown. In this study, we tested the hypothesis that GABA and glycine are co-released in the VLM/VMM. Glycine is recycled by glycine transporter 2 (GlyT2), which is a reliable marker of glycinergic neurons. Therefore, we crossed heterozygous GlyT2Cre mice with floxed channelrhodopsin 2 (ChR2-EYFP) mice to generate GlyT2 reporter mice (GlyT2ChR2/EYFP), which were used to determine the mechanism of glycine release and reveal the location of glycinergic neurons. Whole-cell patch-clamp recordings from presympathetic VLM/VMM neurons revealed that GABA mediates most spontaneous inhibitory postsynaptic currents (sIPSCs); whereas increased activity of inhibitory inputs promotes the release of glycine. Light stimulation of glycinergic fibers expressing ChR2 in GlyT2ChR2/EYFP mice evoked IPSCs (eIPSCs) with glycinergic and GABAergic components. Bath application of GABAA receptors antagonist (bicuculline, 10 μM) decreased the amplitude of eIPSCs, suggesting that approximately 70% of the eIPSCs are generated by glycine. Moreover, blockade of GlyT2 partially diminished the glycinergic component of the light-evoked IPSCs. Using GlyT2ChR2/EYFP mice, we found that glycinergic fibers are abundant throughout the rostral brainstem, and GlyT2-expressing neurons were distributed within the ventral gigantocellular nucleus and medial reticular formation. Immunofluorescence labeling of glycine receptor subunits in the VLM/VMM revealed the expression of two prevalent forms of glycine receptors formed by α1 and α3. In summary, we found that GABA and glycine are co-released in the VLM/VMM, and blockage of GlyT2 decreased the amplitude of light-evoked IPSCs, suggesting that GABA and glycine are likely located in separate vesicles. Our data provide evidence for the co-inhibition of presympathetic neurons in the VLM/VMM by GABA and glycine and suggest that GABA controls the threshold excitability, while glycine is released on demand to increase the strength of inhibition. Supported by: NIDDK122842 and Tulane Brain Institute Marko Spark Innovation Research Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.