Vesicular glutamate transporter 2 is required for the respiratory and parasympathetic activation produced by optogenetic stimulation of catecholaminergic neurons in the rostral ventrolateral medulla of mice in vivo

Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM-CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM-CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine-beta-hydroxylase-expressing neurons in mice [D beta H-Cre/0;VGLUT2(flox/flox)) mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM-CA neurons in cKO vs. control mice (D beta H-Cre/0, using channelrhodopsin-2 (ChR2-mCherry) optogenetics. ChR2-mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (similar to 400 neurons), with identical selectivity for catecholaminergic neurons (90-99% colocalisation with tyrosine hydroxylase). RVLM-CA neurons had similar morphology and axonal projections in D beta H-Cre/0 and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2-positive RVLM-CA neurons in D beta H-Cre/0 and cKO mice. Photostimulation in conscious mice produced frequency-dependent respiratory activation in D beta H-Cre/0 mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in D beta H-Cre/0 mice only. Vagal responses were unaffected by alpha(1)-adrenoreceptor blockade. In conclusion, two responses evoked by RVLM-CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM-CA neurons are mediated by glutamate.