We produced local tissue acidosis in various brain stem regions with 1-nl injections of acetazolamide (AZ) to locate the sites of central chemoreception. To determine whether the local acidosis resulted in a stimulation of breathing, we performed the experiment in chloralose-urethan anesthetized vagotomized carotid-denervated (cats) paralyzed servo-ventilated cats and rats and measured phrenic nerve activity (PNA) as the response index. Measurements of extracellular brain tissue pH by glass microelectrodes showed that AZ injections induced a change in pH at the injection center equivalent to that produced by an increase in end-tidal PCO2 of approximately 36 Torr and that the change in brain pH was limited to a tissue volume with a radius of < 350 microns. We found AZ injections sites that caused a significant increase in PNA to be located 1) within 800 microns of the ventrolateral medullary surface at locations within traditional rostral and caudal chemosensitive areas and the intermediate area, 2) within the vicinity of the nucleus tractus solitarii, and 3) within the vicinity of the locus coeruleus. Single AZ injections produced increases in PNA that were < or = 69% of the maximum value observed with an increase in end-tidal PCO2. We conclude that central chemoreceptors are distributed at many locations within the brain stem, all within 1.5 mm of the surface, and that stimulation of a small fraction of all central chemoreceptors can result in a large ventilatory response.

SUMMARY1. Acetazolamide (0 1 mM) applied to the surface of the rostral ventrolateral medulla or microinjected beneath the medullary surface in chloralose-urethaneanaesthetized, vagotomized, carotid-denervated, paralysed, servo-ventilated cats produced a long-lasting increase in integrated phrenic nerve activity.2. Extracellular pH measured beneath the rostral ventrolateral medulla exhibited a long-lasting decrease after surface acetazolamide but was not a good predictor, in each individual animal, of changes in phrenic activity.3. Medullary carbonic anhydrase inhibition reduced the slope and the half-time of the phrenic response to rapid step CO2 increases. Conversely, acetazolamide did not affect the phrenic response to steady-state CO2 increases.4. These data indicate that localized inhibition of medullary carbonic anhydrase causes a centrally mediated increase in ventilation that we attribute to medullary tissue hypercapnia and acidosis. In addition, these data indicate that medullary carbonic anhydrase may play a role in central CO2 chemotransduction.

Central CO(2) chemoreception and the role of carbonic anhydrase were assessed in brain stems from Rana catesbeiana tadpoles and frogs. Buccal and lung rhythms were recorded from cranial nerve VII and spinal nerve II during normocapnia and hypercapnia before and after treatment with 25 microM acetazolamide. The lung response to acetazolamide mimicked the hypercapnic response in early-stage and midstage metamorphic tadpoles and frogs. In late-stage tadpoles, acetazolamide actually inhibited hypercapnic responses. Acetazolamide and hypercapnia decreased the buccal frequency but had no effect on the buccal duty cycle. Carbonic anhydrase activity was present in the brain stem in every developmental stage. Thus more frequent lung ventilation and concomitantly less frequent buccal ventilation comprised the hypercapnic response, but the response to acetazolamide was not consistent during metamorphosis. Therefore, acetazolamide is not a useful tool for central CO(2) chemoreceptor studies in this species. The reversal of the effect of acetazolamide in late-stage metamorphosis may reflect reorganization of central chemosensory processes during the final transition from aquatic to aerial respiration.

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Previous studies indicate that Long-Evans rats can be operantly trained to discriminate inspired CO(2) concentrations as low as 0.5%. This ability has been proposed to be due to the presence of CO(2)-sensitive olfactory receptors that contain the enzyme carbonic anhydrase (CA). The objectives of the present study were as follows: 1) to determine whether Zucker rats could be operantly conditioned to discriminate low concentrations of CO(2) from control air and 2) to determine the rats' CO(2) detection thresholds before and after nasal perfusion of mammalian Ringers or methazolamide, a CA inhibitor. Rats were operantly trained to discriminate between 25% CO(2) and control air (0% CO(2)) and were then subjected to various CO(2) concentrations (0.5-12.5%) to determine their CO(2) detection thresholds. The average (+/-standard error of mean) baseline CO(2) detection threshold of 7 Zucker rats was 0.48 +/- 0.07% CO(2), whereas the average CO(2) detection thresholds after nasal perfusion of either mammalian Ringers or 10(-2) M methazolamide were 1.41 +/- 0.30% and 5.92 +/- 0.70% CO(2), respectively. The average CO(2) detection threshold after methazolamide was significantly greater (P<0.0001) than the baseline detection threshold. These findings demonstrate that like Long-Evans rats, Zucker rats can be trained to discriminate low concentrations of CO(2) and that inhibition of nasal CA reduces the ability of the rats to detect low concentrations (3.5% and below) but not higher concentrations of CO(2) (12.5%). These results add to the growing evidence that olfactory neurons exhibiting CA activity are CO(2) chemoreceptors sensitive to physiological concentrations of CO(2).

The objectives of this study were to determine: (1) the frequency and distribution of carbonic anhydrase (CA) activity in the bullfrog nasal cavities, and (2) whether inhibition of nasal CA affects the olfactory receptor response to CO2 or other odorants. It was found, using Hansson's staining technique, that some olfactory receptor neurons exhibited CA activity and that these CA-positive receptors were distributed throughout the nasal cavity with peak densities in the dorsal and ventral sensory epithelial regions. To test for the role of CA in olfactory transduction, electro-olfacto-grams (EOGs) were recorded from the surface of the ventral sensory epithelium in response to 2-s pulses of 5% CO2 and amyl acetate before and after topical CA inhibition with acetazolamide (10(-3) mol.l-1). In 52 bullfrogs, 1222 sites on the ventral epithelium were tested resulting in 23 locations that exhibited a response to 5% CO2. Inhibition of CA caused an immediate 65% reduction in the EOG response to CO2 while the response to amyl acetate was not affected. These results, along with the histochemical localization of CA in some olfactory receptor neurons, indicate that CA plays a role in the detection of CO2 in frog olfactory neurons and that only a small population of olfactory receptor neurons are CO2 sensitive.

The main olfactory system of mice contains a small subset of olfactory sensory neurons (OSNs) that are stimulated by CO₂. The objective of this study was to record olfactory receptor responses to a range of CO₂ concentrations to further elucidate steps in the proposed CO₂ transduction pathway in mice. Electro-olfactograms (EOGs) were recorded before and after inhibiting specific steps in the CO₂ transduction pathway with topically applied inhibitors. Inhibition of extracellular carbonic anhydrase (CA) did not significantly affect EOG responses to CO₂ but did decrease EOG responses to several control odorants. Inhibition of intracellular CA or cyclic nucleotide-gated channels attenuated EOG responses to CO₂, confirming the role of these components in CO₂ sensing in mice. We also show that, like canonical OSNs, CO₂-sensitive OSNs depend on Ca²⁺-activated Cl⁻ channels for depolarization of receptor neurons. Lastly, we found that guanylyl cyclase-D knockout mice were still able to respond to CO₂, indicating that other pathways may exist for the detection of low concentrations of nasal CO₂. We discuss these findings as they relate to previous studies on CO₂-sensitive OSNs in mice and other animals.