We examined the effects of focal tissue acidosis in the pre-Bötzinger complex (pre-BötC; the proposed locus of respiratory rhythm generation) on phrenic nerve discharge in chloralose-anesthetized, vagotomized, paralyzed, mechanically ventilated cats. Focal tissue acidosis was produced by unilateral microinjection of 10-20 nl of the carbonic anhydrase inhibitors acetazolamide (AZ; 50 microM) or methazolamide (MZ; 50 microM). Microinjection of AZ and MZ into 14 sites in the pre-BötC reversibly increased the peak amplitude of integrated phrenic nerve discharge and, in some sites, produced augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst). Microinjection of AZ and MZ into this region also reversibly increased the frequency of eupneic phrenic bursts in seven sites and produced premature bursts (i.e., doublets) in five sites. Phrenic nerve discharge increased within 5-15 min of microinjection of either agent; however, the time to the peak increase and the time to recovery were less with AZ than with MZ, consistent with the different pharmacological properties of AZ and MZ. In contrast to other CO(2)/H(+) brain stem respiratory chemosensitive sites demonstrated in vivo, which have only shown increases in amplitude of integrated phrenic nerve activity, focal tissue acidosis in the pre-BötC increases frequency of phrenic bursts and produces premature (i.e., doublet) bursts. These data indicate that the pre-BötC has the potential to play a role in the modulation of respiratory rhythm and pattern elicited by increased CO(2)/H(+) and lend additional support to the concept that the proposed locus for respiratory rhythm generation has intrinsic chemosensitivity.

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The pre-Bötzinger complex (pre-BötC) is hypothesized to be the site for respiratory rhythm generation in mammals. Studies examining the cellular mechanisms mediating rhythm generation have focused on the role of chemically mediated synaptic interactions; however, electrotonic synaptic interactions (i.e., electrotonic coupling), which occur by means of gap junctions, may also play a role. Here, we used immunoblot and immunohistochemical analyses to determine whether the pre-BötC contains the gap junction proteins necessary for electrotonic communication and whether the presence and distribution of these gap junction proteins show a developmental change in expression. We found that both connexin26 (Cx26) and connexin32 (Cx32) were expressed in pre-BötC neurons of neonatal and adult rats; however, the relative amounts and their distribution varied by age. Cx26 labeling was seen in a high proportion of pre-BötC neurons in neonatal rats < or = 7 days postnatal (P7) but declined with increasing age. In contrast, Cx32 labeling was sparse in pre-BötC neurons of neonatal rats < or = P7, but increased with increasing age; the highest proportion was seen in adult rats. These data suggest the potential for gap junctional communication in the pre-BötC of both neonatal and adult rats, and we propose that the gap junction proteins Cx26 and Cx32 form the neuroanatomic substrate for this gap junctional communication, which may be important in the synchronization of neural activity generating respiratory rhythm.