Mapping and Identification of GABAergic Neurons in Transgenic Mice Projecting to Cardiac Vagal Neurons in the Nucleus Ambiguus Using Photo-Uncaging

The neural control of heart rate is determined primarily by the activity of preganglionic parasympathetic cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA) in the brain stem. GABAergic inputs to CVNs play an essential role in determining the activity of these neurons including a robust inhibition during each inspiratory burst. The origin of GABAergic innervation has yet to be determined however. A transgenic mouse line expressing green florescent protein (GFP) in GABAergic cells was used in conjunction with caged glutamate to identify both clusters and individual GABAergic neurons that evoke inhibitory GABAergic synaptic responses in CVNs. Transverse slices were taken with CVNs patch-clamped in the whole cell configuration. Sections containing both the pre-Botzinger complex as well as the calamus scriptorius were divided into approximately 90 quadrants, each 200 x 200 microm and were sequentially photostimulated. Inhibitory post synaptic currents (IPSCs) were recorded in CVNs after a 5-ms photostimulation of 50 microM caged glutamate. The four areas that contained GABAergic cells projecting to CVNs were 200 microm medial, 400 microm medial, 200 microm ventral, and 1,200 microm dorsal and 1,000 microm medial to patched CVNs. Once foci of GABAergic cells projecting to CVNs were determined, photostimulation of individual GABAergic neurons was conducted. The results from this study suggest that GABAergic cells located in four specific areas project to CVNs, and that these cells can be individually identified and stimulated using photouncaging to recruit GABAergic neurotransmission to CVNs.