["The Journal of Physiology, Volume 604, Issue 7, Page 2939-2957, 1 April 2026. ", "\nAbstract figure legend Our study demonstrates that both GABA and glycine mediate inhibitory postsynaptic currents in the ventrolateral/ventromedial medulla (VLM/VMM) and increasing the activity of inhibitory synapses facilitates glycine release. We found that presympathetic VLM/VMM neurons receive glycinergic inputs, which release both glycine and GABA, and inhibition of glycine transporter 2 (GlyT2) decreased glycine release. In summary, our study suggests that in the mouse brainstem, glycinergic neurons release both GABA and glycine, and these inhibitory mechanisms may be equally important for the regulation of presympathetic neurons.\n\n\n\n\n\n\n\n\n\nAbstract\nPresympathetic neurons in the ventrolateral and ventromedial medulla (VLM/VMM) control sympathetic tone, and GABA and glycine were identified as inhibitory neurotransmitters. Although glycinergic inhibition of presympathetic neurons in the VLM/VMM has been shown, the sources of glycinergic inputs and the mechanism of glycine release are ill‐defined. Here, we tested whether presympathetic neurons in the VLM/VMM receive glycinergic inputs. Glycine is recycled by glycine transporter 2 (GlyT2), which is a reliable marker of glycinergic neurons. GlyT2Cre mice were cross bred with channelrhodopsin (ChR2) mice to generate GlyT2ChR2/EYFP mice to be able to selectively stimulate GlyT2‐expressing fibres. Presympathetic neurons in the VLM/VMM were retrogradely labelled with pseudorabies virus and whole‐cell, patch clamp recordings were conducted in brainstem slices. Our study demonstrates that inhibitory synaptic currents recorded from presympathetic VLM/VMM neurons are mediated by GABA and glycine. We found that increasing the activity of inhibitory synapses facilitates glycine release. Light stimulation of GlyT2ChR2/EYFP fibres triggered monosynaptic evoked currents composed of GABA and glycine and we found that sustained glycine release depends on glycine uptake mediated by GlyT2. In addition, we used a combination of monosynaptic and transsynaptic viral tracings to identify the location of presympathetic glycinergic neurons in the ventral brainstem, and immunostaining to reveal the expression of GlyR α1, α3 and β subunits in VLM/VMM neurons. Our study identified that GlyT2‐expressing neurons rely on release of both GABA and glycine to generate inhibitory currents. However, the potential role and the necessity for both neurotransmitters in the control of presympathetic VLM/VMM neurons require additional studies.\n\n\n\n\n\n\n\n\n\nKey points\n\nPresympathetic neurons in the mouse ventrolateral/ventromedial medulla (VLM/VMM) receive monosynaptic glycinergic inputs that release both GABA and glycine.\nGlycinergic neurons were identified in the lateral paragigantocellular nucleus and Bötzinger complex.\nIncreasing the activity of inhibitory synapses facilitates glycine release and sustained glycine release depends on glycine uptake mediated by glycine transporter 2 (GlyT2).\nThree prevalent forms of glycine receptors (α1, α3 and β) are expressed in the VLM/VMM.\nOur study demonstrates the necessity of GABA and glycine for the regulation of presympathetic neurons and thus highlights the need for a better understanding of glycinergic circuits.\n\n\n"]