Chemosensory responses to CO 2 in multiple brainstem nuclei determined using a voltage - 1 sensitive dye in brain slices from rats

293 words). 23 We used epifluorescence microscopy and a voltage sensitive dye, di-8-ANEPPS, to study 24 changes in membrane potential during hypercapnia with or without synaptic blockade in 25 chemosensory brainstem nuclei: the locus coeruleus (LC), the nucleus of the solitary tract (NTS), 26 lateral paragigantocellularis nucleus (PGCL), raphé pallidus and raphé obscurus, and in putative 27 non-chemosensitive nuclei, the gigantocellularis reticular nucleus (GCR) and the spinotrigeminal 28 nucleus (STG). We studied the response to hypercapnia in LC cells to evaluate the performance 29 characteristics of the voltage-sensitive dye. Hypercapnia depolarized many LC cells, and the 30 voltage responses to hypercapnia were diminished, but not eradicated by synaptic blockade 31 (there were intrinsically CO2 sensitive cells in the LC). The voltage response to hypercapnia was 32 substantially diminished after inhibiting fast Na channels with tetrodotoxin (TTX). Thus, action 33 potential related activity was responsible for most of the optical signal that we detected. We 34 systematically examined CO2 sensitivity among cells in brainstem nuclei to test the hypothesis 35 that CO2 sensitivity is a ubiquitous phenomenon, not restricted to nominally CO2 chemosensory 36 nuclei. We found intrinsically CO2 sensitive neurons in all the nuclei that we examined; even the 37 non-chemosensory nuclei had small numbers of intrinsically CO2 sensitive neurons. However, 38 synaptic blockade significantly altered the distribution of CO2 sensitive cells in all of the nuclei 39 so that the cellular response to CO2 in more intact preparations may be difficult to predict based 40 on studies of intrinsic neuronal activity. Thus, CO2 sensitive neurons are widely distributed in 41 chemosensory and non-chemosensory nuclei, and CO2 sensitivity is dependent on inhibitory and 42 excitatory synaptic activity even within brain slices. Neuronal CO2 sensitivity important for the 43 behavioral response to CO2 in intact animals will, therefore, be determined as much by synaptic 44 mechanisms and patterns of connectivity throughout the brain as by intrinsic CO2 sensitivity. 45 Voltage-sensitive dyes to assess CO2 chemosensitivity 3 46