Microinjections of glycine into the pre-Bötzinger complex inhibit phrenic nerve activity in the rat

Microinjections of L-glutamate were used to identify the pre-Bötzinger complex in urethane-anesthetized, immobilized, bilaterally vagotomized, artificially ventilated, adult male Wistar rats. Unilateral microinjections (20-30 nl) of L-glutamate into the pre-Bötzinger complex on either side elicited a bilateral continuous phrenic nerve discharge superimposed on which was an increase in burst-frequency. Neurokinin-1 receptor immunoreactivity in the semi-compact region of the nucleus ambiguus and the area immediately ventral to it indicated that the site of microinjections was in the general region of pre-Bötzinger complex. Unilateral microinjections of glycine into the pre-Bötzinger complex caused an inhibition of phrenic nerve activity bilaterally in a concentration-dependent manner. At lower concentrations (1 and 3 mM) phrenic nerve burst-frequency as well as burst-amplitude were decreased. At higher concentrations (6 mM), complete bilateral cessation of phrenic nerve activity was observed. The effects of glycine were prevented by a prior microinjection of strychnine (0.5 mM) into the pre-Bötzinger complex. The specificity of strychnine as an antagonist for glycine receptors was established by its lack effect on GABA(A) receptors; muscimol was used as a GABA(A) receptor agonist. Unilateral microinjections of muscimol (0.01 and 0.1 mM) into previously identified pre-Bötzinger complex also caused a bilateral decrease in phrenic nerve burst-frequency and burst-amplitude. At higher concentrations (0.3 and 1 mM) muscimol microinjections into the pre-Bötzinger elicited a complete bilateral cessation of phrenic nerve activity. The effects of muscimol were not altered by prior microinjections of strychnine (0.5 mM) at the same site. These results demonstrate pharmacologically the presence of glycine receptors in the pre-Bötzinger complex. The role of these receptors in the regulation of respiration remains to be elucidated.