Evidence for glycinergic respiratory neurons: Botzinger neurons express mRNA for glycinergic transporter 2

Botzinger (BOTZ) neurons in the rostral ventrolateral medulla fire during t he late expiratory phase of the respiratory cycle. These cells inhibit phre nic motor neurons and several types of respiratory neurons in the medulla o blongata. BOTZ cells produce a fast, chloride-mediated inhibition of their target neurons, but the neurotransmitter used by these cells has not been d etermined. In the present study, we examine whether gamma-aminobutyric acid (GABA) or glycine could be the inhibitory neurotransmitter of BOTZ cells. In chloralose-anesthetized rats, we individually filled 20 physiologically characterized BOTZ neurons with biotinamide by using a juxtacellular labeli ng method. Medullary sections containing the labeled BOTZ neurons were proc essed for in situ hybridization by using digoxigenin-labeled riboprobes for glutamic acid decarboxylase isoform 67 (GAD67), a marker for GABAergic neu rons, or for glycine transporter 2 (GLYT2), a marker for glycinergic neuron s. All BOTZ cells examined contained GLYT2 mRNA (n = 10), whereas none had detectable levels of GAD67 mRNA(n = 10). For a positive control, 12 GABAerg ic neurons in the substantia nigra pars reticulata also were recorded and f illed with biotinamide in vivo. Most of these cells, as expected, had detec table levels of GAD67 mRNA (11 out of 12). These results demonstrate that t he juxtacellular labeling method can be combined with in situ hybridization to identify physiologically characterized cells with probable GABAergic or glycinergic phenotypes. Furthermore, these data suggest that BOTZ neurons use the neurotransmitter glycine and not GABA to provide widespread inhibit ion of respiratory-related neurons. J. Comp. Neurol. 407:583-597, 1999. (C) 1999 Wiley-Liss, Inc.