Journal of Applied Physiology
Vol. 81, No. 5, pp. 1987-1995, November 1996
CONTROL OF BREATHING, CIRCULATION, AND TEMPERATURE

Central chemoreception in the region of the ventral respiratory group in the rat

Eugene E. Nattie and Aihua Li

Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Nattie, Eugene E., and Aihua Li. Central chemoreception in the region of the ventral respiratory group in the rat. J. Appl. Physiol. 81(5): 1987-1995, 1996.We injected acetazolamide (AZ; 5 × 10⁶ M, 1 nl) into the region of the ventral respiratory group (VRG) of anesthetized paralyzed ventilated rats. Control injections (mock cerebrospinal fluid, n = 6, or the inactive AZ analogue 2-acetylamino-1,3,4-thiadiazole-5-sulfon-t-butylamide, n = 6) did not increase the integrated phrenic neurogram [phrenic nerve amplitude (PNA)]. The AZ injections produced a focal region of tissue acidosis with a radius < 300-400 µm and are used as a probe for sites of central chemosensitivity. Injection location is determined by anatomic analysis. Of 22 VRG injections of AZ, 14 increased the amplitude of the PNA over 15-90 min; 8 had no effect. In 17 cases, we measured medullary tissue pH at the injection center and/or at a distant site and reaffirmed the size of the acidotic region produced by such small AZ injections. Of injections with pH electrodes within 300-400 µm of the injection center, all responders showed an acid pH; three nonresponders showed an acid pH, and one an alkaline pH. In a subgroup of five rats, at VRG sites with known respiratory effects identified by prior glutamate injection (10 nl, 100 mM), all subsequent AZ injections produced a PNA response. Simultaneous measurement of PNA and tissue pH responses at the injection center of eight rats did not show a uniform correlation in time; initially, both changed with a similar time course, but PNA recovered more quickly. We conclude that 1) the region of the VRG contains sites of ventilatory chemoreception, 2) ineffective AZ injections do produce a tissue acidosis but at sites with minimal impact on breathing, and 3) tissue pH does not uniquely represent the chemoreceptor stimulus.

ventral medulla; control of breathing; carbon dioxide sensitivity; central chemoreceptors

INTRODUCTION

BERNARD ET AL. (1) and Coates et al. (6) have reported that there are widespread sites of brain stem ventilatory chemoreception. Their approach has been to produce a tissue acidosis in various medullary regions by a 1-nl microinjection of the carbonic anhydrase inhibitor acetazolamide (AZ). Such an injection results in a focal circumscribed region of decreased pH. Coates et al. have previously evaluated the size of this low pH region by using glass micro pH electrodes with their tips placed at varying distances from the center of an AZ injection. At the center, the decrease in pH was approximately equivalent to that produced by a 36-Torr increase in end-tidal PCO₂ (from 28 to 64 Torr). At a distance of 100 µm from the center, the decrease in pH was equivalent to an increase in end-tidal PCO₂ of ~9 Torr (from 28 to 37 Torr), and at >350 µm from the injection center, there was no detectable change in tissue pH (6). With systemic PCO₂ maintained at eucapnic levels, an increase in phrenic nerve discharge that occurs after such an AZ injection is attributed to a central chemoreceptor response occurring within this focal acidotic region because the remainder of the brain stem is eucapnic.

In both anesthetized cats and rats, this approach has identified the presence of central chemoreception in regions near the ventral medullary surface (6), in keeping with traditional ideas of ventral medullary surface central chemoreceptor locations (17, 25). Responsive injection sites were also observed more dorsally at the region of the locus ceruleus and in the region of the nucleus tractus solitarius (NTS) (6). Recent work in brain slice preparations has shown the presence of neurons that are excited by local acidosis in both the locus ceruleus (21) and NTS (7, 26), supporting the interpretation that central chemoreception is present in these regions. In anesthetized rats, using the same 1-nl AZ injection approach, Bernard et al. (1) have also shown that central chemoreception is present in the midline caudal medullary raphé. Again, work in brain slice preparations has shown the presence of chemosensitive neurons in the raphé (22). Not all injections of AZ at each chemoreceptor region increased phrenic nerve amplitude (PNA); some AZ injections had no effect on phrenic output. Whether this reflects less tissue acidosis with those injections or fewer respiratory-related neurons and less impact on PNA at these sites is not known. Control injections at these sites of an inactive AZ analogue, 2-acetylamino-1,3,4-thiadiazole-5-sulfon-t-butylamide (AN), had no effect (1, 6).

In this paper, we look for central chemoreception in the region of the ventral respiratory group (VRG) in the anesthetized rat. This is a major site of respiratory-related neurons in the rat brain stem (9, 30), it would likely be a reasonable site for chemoreceptor location, and existing data on the possible presence of chemoreception within the VRG are in conflict (7, 11-13, 20, 23, 24, 26, 27, 29). Given the widespread nature of central chemoreception and the importance of the VRG, it seems reasonable to expect its presence within the VRG.

We ask the following questions. 1) Are central chemoreceptors present in the VRG? 2) Do ineffective AZ injections produce a decrease in tissue pH? 3) Are all VRG sites with known effects on respiration as predetermined by glutamate injection responsive to AZ? 4) What are the time courses of the PNA and tissue pH responses measured simultaneously after an AZ injection? Our expectations are that the VRG will contain central chemoreception; ineffective AZ sites will be acidotic, suggesting a nonhomogeneous distribution of chemoreceptors or of sites that can affect respiratory system output; and the PNA response will have a time course similar to that of tissue pH.

METHODS

General preparation. Adult male Sprague-Dawley rats (290-410 g) were used in all experiments. With the rat in an enclosed box, 2.0% halothane in O₂ was administered to initially anesthetize the animal. The trachea was cannulated, and catheters were placed in the femoral artery and vein. Urethan (550 mg/kg) and -chloralose (60 mg/kg) were injected into the femoral vein over several minutes while at the same time the inspired halothane concentration was decreased. Gallamine triethiodide (3 mg/kg) was used to paralyze the rats, with supplemental doses administered as needed. The rats were artificially ventilated (rodent ventilator model 683, Harvard Instruments) with 100% O₂. Arterial blood pressure was monitored with a strain gauge connected to the femoral arterial catheter, rectal temperature was maintained at 37°C with a feedback system, and end-tidal PCO₂ was monitored with a CO₂ analyzer (Biochem, ETCO₂ monitor, Lifespan 100) and maintained at any set value by manual control of the ventilator rate and/or tidal volume. Bilateral thoracotomies were performed, and a positive end-expiratory pressure of 3-5 cmH₂O was maintained during the experiment.

Bilateral vagotomy was performed before the ventral medullary surface was exposed. The phrenic nerve was isolated and cut, and the central end was placed on bipolar electrodes and covered with Wacker sil-gel (an insulating medium). Phrenic nerve activity was amplified (BMA 831 amplifiers, Charles Ward), integrated (Payntar filter in an MA-821 moving averager, Charles Ward), and displayed on a storage oscilloscope. Integrated PNA, arterial blood pressure, and end-tidal PCO₂ were recorded on a strip-chart recorder (MFE 1400, Gould). An on-line program calculated, for experimenter-determined sequences of breaths, the amplitude of the integrated phrenic activity, phrenic burst frequency, blood pressure, and end-tidal PCO₂.

Blood pressure and the frequency and integrated amplitude of phrenic discharge were continuously monitored during the experiment to monitor the depth of anesthesia. An increase in frequency or blood pressure that could not be attributed to a microinjection or that occurred in response to a noxious stimulus, the pinch of a hind paw, was viewed as a sign that the animal needed additional anesthesia, which was given in the form of one-fourth to one-third of the initial dose.

Microinjections. Microinjections (1 nl) of mock cerebrospinal fluid (mCSF), AZ (5 × 10⁶ M), or AN (5 × 10 ⁵ M; CL 13850, Lederle Laboratories Division) were made with a Picospritzer connected to a glass micropipette with a tip diameter of 10 µm. The system was calibrated by observing, under the microscope, the diameter of the fluid droplet ejected with given pressure injection parameters and calculating the volume. Each injection was monitored by observation under a microscope of the movement of the air-liquid interface in tubing of 500 µm ID. In this size tubing, a 1-nl volume change is associated with a 5-µm linear displacement of the meniscus. To enhance detection of this small linear movement, we stretched a section of the tubing to narrow the cross-sectional area. AZ and AN were prepared in mCSF of normal ionic and acid-base composition (6). The solution was kept at 37°C and bubbled with 5% CO₂ to maintain a pH of 7.4 before the addition of calcium. Fluorescent microbeads (Polysciences) were then added to the test solutions for subsequent microscopic identification of the injection sites. We also estimated the injection volume by anatomic analysis. Typically, in the tissue, each injection formed not a sphere but an ellipsoid shape with its long axis in the rostral-to-caudal dimension. We counted each 50-µm-thick medullary section containing fluorescent beads. The section with the largest area of beads was used to estimate the area of the base of two adjoining cones. The number of sections containing beads was used to determine the height of each cone.

pH microelectrode. A double-barreled borosilicate glass pipette (1.5 mm OD; Frederick Haer) was vertically pulled, then broken, so that the tip diameter of each barrel was ~20 µm. One barrel was silanized in an oven with 5% dimethyldichlorosaline (Fluka 40136) in xylene (80-100°C) overnight. The pipette was dipped in hydrogen ionophore II-cocktail A (Fluka 95297) until the H⁺ exchanger was drawn up into the barrel a distance of 500-1,000 µm. Then the barrel was backfilled with 100 mM NaCl (pH 6.1). The pH barrel was coupled to a potentiometer (EA 920, Orion Research) with a platinum-iridium wire. Single-barrel pH pipettes were constructed the same way. All electrodes were calibrated in vitro before and after the experiment with precision buffer solutions with pH values of 4, 6, 7, and 10 at 24°C. In addition, the electrode responses to the initial and final whole animal CO₂-response tests were compared as an in vivo calibration. To allow comparison of tissue pH data among animals and groups, the responses in each animal were normalized to the response of that electrode in vivo to changes in end-tidal CO₂ from 4 to 9%.

Experimental protocol. All animals were first tested for their responsiveness to inspired CO₂. Baseline end-tidal PCO₂ was set just above the apneic threshold (5 Torr) while the animal was ventilated with 100% O₂. To determine the ventilatory response to CO₂, the end-tidal CO₂ was increased in ~7-Torr steps by introducing controlled amounts of 100% CO₂ into the inspiratory circuit, keeping ventilator frequency and tidal volume constant. Responses were measured when the PNA had stabilized (3-5 min at any CO₂ level). When the animal had completely recovered from the CO₂ challenge and showed stable baseline phrenic activity, microinjections of mCSF, AZ, or AN were made in the VRG, and the effects on blood pressure, phrenic frequency, and PNA were observed. The mean ± SE period of stable baseline PNA before the first injection was 15 ± 4 min. When the response was completed, another CO₂ test was performed. CO₂ responses were normalized to a percentage of the maximum; injection responses were normalized to a percentage of the baseline. Normalization of injection responses to percent maximum does not alter the conclusions.

At the end of each experiment, the rat was killed and the brain stem was quickly and carefully removed. It was placed rostral side up in dry ice and stored at 24°C. Sections (50 µm) were cut in a cryostat (Cryocut 1800, Reíchert-Jung) and examined for the location of the fluorescent microbeads. All sections with beads were counted, and the section with the largest area of beads was identified as the center of the injection. An adjacent slide was stained with cresyl violet for identification of anatomic landmarks.

Criteria for inclusion in the study. All data reported in RESULTS were obtained in experiments that fulfilled the following criteria. 1) The injection site was in the region of the VRG as shown by anatomic evaluation of the fluorescent microbeads mixed in with each injection. 2) The initial preinjection amplitude of the integrated phrenic neurogram obtained under baseline conditions was, at most, 65% of the maximum obtained by systemic CO₂ stimulation during the control CO₂ response. This allowed for a measurable PNA response to the focal acidosis produced by the AZ injection. 3) There was a final response to systemic CO₂ stimulation after the injection protocol, indicating that CO₂ sensitivity was maintained during the protocol. 4) Mean arterial blood pressure was at least 80 mmHg during the protocol.

Acceptance criteria for AZ injection being positive. Data from control (mCSF or AN) and AZ injections were expressed as a percent change from the preinjection baseline, with mean values for integrated PNA determined from 10 to 25 respiratory cycles at the end of the baseline period just before the injection. An increase of 10% or greater measured at 15 and 30 min was the criterion for inclusion as a responder. This criterion was chosen based on the absence of any PNA response to control injections of mCSF or AN that was 10% or greater.

Experimental design. In the VRG AZ group, injections of AZ were made into the region of the VRG of 22 rats, with tissue pH measured at the injection center in eight and with pH measured at a distant site in these eight and in another nine. In control group I, six animals received injections of mCSF with no tissue pH measurements. In control group II, six animals received VRG-region injections of the AZ analogue AN, with tissue pH measured at the injection center. In the VRG glutamate and AZ group, five animals received a VRG-region injection of glutamate at different sites until the PNA was stimulated. At that site, AZ was then injected.

RESULTS

Injection locations. Figure 1 shows the location of the center of each injection included in this report. All injections were in the region of the rat VRG (9, 30) extending, relative to the bregma, from 12.78 mm caudally to 14.30 mm, based on the atlas of Paxinos and Watson (19). Our anatomic estimate of injection volume, based on the measured parameters of the fluorescent-bead distribution, was 1.8 ± 0.7 (SE) nl. This may be a slight underestimate if the beads concentrate within the central portion of the injected volume. The mean calculated volumes of AZ injections did not differ from those of control injections; the volumes of responsive and nonresponsive injections did not differ.

Table 1. Integrated PNA and frequency values during systemic CO2 responses onset and completion of experiment Initial Final PNA Frequency PNA Frequency 4% 9% 4% 9% 4% 9% 4% 9% Responders (n = 14) 52 ± 2  100 44 ± 2  44 ± 1  50 ± 2  71 ± 4  48 ± 2  52 ± 2  Nonresponders (n = 8) 61 ± 2  100 38 ± 2  42 ± 1  56 ± 4  78 ± 8  47 ± 2  51 ± 1  Control (n = 12) 61 ± 4  100 40 ± 2  45 ± 2  57 ± 5  72 ± 6  53 ± 2  56 ± 3  Glu/AZ (n = 6) 54 ± 3  100 46 ± 3  43 ± 2  49 ± 8  70 ± 11  47 ± 4  45 ± 4 Values are means ± SE; n, no. of subjects. PNA, phrenic nerve amplitude; initial, onset of experiment; final, completion of experiment; 4%, baseline end-tidal CO2 values; 9%, maximum end-tidal CO2 value; Glu, glutamate; AZ, acetazolamide.

1

1

DISCUSSION

Major findings. This study has three new findings: 1) the region of the VRG contains central chemoreceptors, 2) AZ injections that do not stimulate PNA do produce an acidosis, and 3) the time course of the respiratory response to focal acidification by AZ microinjection differs from that of tissue pH measured simultaneously at the injection site.

The region of the rat VRG contains central chemoreception. One-nanoliter injections of AZ (5 × 10⁶ M) into the VRG region of anesthetized rats significantly increased integrated PNA, showing the presence of central chemoreception. In the VRG region, 14 of 22 injections increased integrated PNA by 23 ± 3% (SE) above baseline at the time of maximum response (32 ± 5 min). Expressed as percent maximum (the value achieved with end-tidal PCO₂ = 9%), this was a change from 55 to 67% maximum. In previous studies, 12 of 17 AZ injections increased PNA from 49 to 69% maximum (6) and 8 of 14 raphé injections increased PNA from 50 to 66% maximum (1). Control injections into the VRG region with mCSF or an inactive AZ analogue had no effect on PNA, and not all AZ injections were effective even when located in anatomic proximity to those that produce a response.

The VRG is a major site of respiratory-related neurons in the rat brain stem (9, 30); it seems likely that it would be a reasonable site for chemoreceptor location. However, existing data are in conflict on the presence of chemoreception within the VRG. In brain slices, neurons depolarized by CO₂ seem abundant in the NTS and ventrolateral medulla (VLM) (7, 26) but not in the nucleus ambiguus (VRG) (7, 26). Some experiments identifying brain sites that stain for c-Fos protein after stimulation by systemic hypercapnia have found positive staining along the VLM surface (24, 27), in the NTS (24), and in the locus ceruleus (11) but have not found prominent involvement in the VRG (24, 27).

In contrast, a recent report (29) did show c-Fos activation by hypercapnia of neurons nearby to the rat VRG. And neurons harvested from the VRG region (nucleus ambiguus) of neonatal rat brain stem and grown in culture do show chemosensitivity, although the stimulus intensity in this experiment was severe (23). Intracellular injection of neurobiotin into VRG neurons has shown long projections that extend to the VLM surface in the adult cat (12, 20). On the basis of the projections, these VRG neurons have been proposed as possible chemoreceptors. Our observations support this proposal. Cells in the VRG region have been shown responsive to local acidification in the adult cat (13) and, recently, in the isolated neonatal rat brain stem preparation (12). Given the widespread nature of central chemoreception, it seems reasonable to expect its presence within the VRG, and we conclude that the VRG region does contain chemoreception. It is unclear why brain slice preparations do not demonstrate CO₂-sensitive neurons in the VRG.

In all cases (1, 6; present study), the response to a single AZ injection is, on average, a surprisingly large fraction of that observed with stimulation of all chemoreceptors by increased end-tidal PCO₂. We propose the following interpretations for this observation. 1) At the AZ injection center, the focal acidosis is moderately severe (approximately equivalent to that produced by a change in end-tidal PCO₂ from 28 to 75 Torr). It is possible that central chemoreceptor sites have different stimulus-intensity thresholds and the AZ injection stimulus is above threshold at all sites. We envision here "physiological" vs. "emergency" central chemoreceptor sites depending on the stimulus threshold (this hypothesis arises from discussions with P. Scheid). 2) All sites are operative in the physiological stimulus-intensity range and provide a redundant system able to sense pH abnormalities at many locations and to produce a respiratory system response after an abnormality at any site. 3) There may be an unexplained effect of AZ either within or, possibly, outside the region of decreased tissue pH. This could reflect AZ diffusion to sites outside the region of tissue acidosis. 4) Axon terminals from more than one chemoreceptor neuron could be the source of the large response. Chemoreceptor stimuli could be integrated at the axonal level, with stimulation of a pool of chemoreceptor neurons via gap junctions or collateral synaptic connections.

Time course of tissue pH and PNA after VRG AZ injection. Measurement of medullary tissue pH in this study confirms that AZ injections result in a focal region of tissue acidosis. At the center of the injection, the tissue pH decreased, on average, an amount equivalent to that produced by an increase in end-tidal PCO₂ from 28 to 75 Torr. In a previous study by Coates et al. (6), the tissue pH change at the injection center was equivalent to that produced by a change in end-tidal PCO₂ from 28 to 64 Torr. With increasing distance from the injection center, the tissue pH change diminished, such that at ~300-400 µm pH was unchanged. At distances beyond 400 µm, tissue pH appears to become alkaline, although the number of observations is limited. The cause of this alkalosis, if real, is unknown. We can speculate that if cerebral blood flow is increased at the AZ-induced region of acidosis, this could promote CO₂ loss from surrounding tissue that is unaffected by the AZ.

In this study, tissue pH measurements were made at the injection center simultaneously with PNA. We expected that, on average, the tissue pH and PNA responses would have similar time courses (Fig. 7) and that tissue pH was an approximate estimate of the chemoreceptor stimulus. The initial increase in PNA does correlate well with the initial decrease in tissue pH. But, PNA plateaus and begins its decline back to baseline at 60 min, whereas the tissue acidosis peaks later, at 90 min, before beginning its decline. Control experiments with inactive AZ analogue injections appear to rule out electrode drift as the cause of the sustained pH change. Teppema et al. (28) pointed out the absence of a tight correlation between medullary tissue pH and the ventilatory response in experiments with systemic carbonic anhydrase (CA) inhibition. Here the CA inhibition is focal and the pH measurement is at the center of the region of inhibition.

There are a number of possible interpretations of these observations. 1) The chemoreceptor stimulus is intracellular not extracellular (tissue) pH. Data supporting an intracellular pH-sensing site have been reported for the carotid body type I cells thought responsible for sensing CO₂ and/or H⁺ (3, 14, 18) and for central chemoreception in an invertebrate model (8). The slower PNA response to an abrupt increase in CO₂ in animals with brain stem CA inhibition also supports the view that intracellular pH is sensed; with rapid changes in CO₂, CA speeds the conversion of CO₂ to H⁺ (5). And Lassen (15) has suggested that intracellular pH is the central chemoreceptor stimulus based on an analysis of intra- and extracellular pH measurements after hypercapnia or systemic AZ administration. 2) The pH electrode at the center of the AZ injection does not reflect the tissue pH at the chemoreceptor site, i.e., the site is not at the injection center. 3) The chemoreceptor mechanism might show accommodation of some type during the period of tissue acidosis. When central chemoreceptors are identified, this hypothesis can be tested. 4) The respiratory system response might accommodate, decreasing its output as it continues to receive stimulatory information only from this single site. We can test this idea by evaluation of the effects on PNA of stimulating more than one central chemoreceptor site at different times.

Glutamate identification of VRG sites that effect respiration; effective vs. ineffective AZ injections. Some AZ microinjections have no effect on PNA even though they appear to be located in anatomic proximity to effective injection sites (1, 6; present study). In another study, we (16) have identified respiratory-responsive sites by glutamate microinjections (10 nl, 100 mM) before injection of the neuroactive agent under study. By using this approach, it took from two to six injections of glutamate at different VRG sites in each of five animals to obtain a PNA response. Subsequent injection of AZ at these responsive sites increased PNA in all cases. Even within a region with potent respiratory effects, some pressure injections produce no response, presumably because an insufficient number neurons with respiratory system effects have been affected by the injection. The radius of the glutamate injection volume of 10 nl is ~134 µm, assuming no diffusion. This compares qualitatively to the known region of tissue acidosis after a 1-nl AZ injection that has a radius of <300-400 µm. Anatomic comparisons of injection locations among animals is imprecise. Plots like Fig. 1 compile data from adjacent sections in different animals and do not represent exact relationships among the injection sites. It is clear from functional data that not all glutamate or AZ injections produce a respiratory effect even though they are located anatomically within the same general region of interest. We know in each case that an injection was made into the tissue because of the presence of the fluorescent beads and, in the case of AZ, the change in tissue pH.

What is the function of brain CA? The function of brain CA, present in glia, neurons, neuronal membranes, and extracellular fluid (ECF) and at the blood-brain barrier, is not well understood (4). We mention three possibilities here and evaluate them for possible involvement in the tissue acidosis produced by AZ injections.

1) CA minimizes changes in intra- and extracellular pH that occur with neuronal activation via ligand-gated ion channels (4). These pH changes are small and transient and unlikely to be involved in the sustained increase in respiratory activity caused by the focal AZ injection. Furthermore, acidosis inhibits glutamate-induced cell excitation (4) and enhances -aminobutyric acid-induced cell inhibition (4), responses that diminish synaptic excitability.

2) CA-facilitated diffusion of CO₂ can be important in skeletal muscle (10); it has not been demonstrated in neurons. AZ interference with facilitated diffusion could acidify tissue.

3) CA helps to minimize changes in H⁺ produced by cell metabolism or other sources. Rapid conversion of H⁺ to CO₂ inside or outside the cell allows the CO₂ to leave the system by diffusion. AZ can slow these processes, promoting an intra- and extracellular acidosis. Cell buffer power exceeds that of ECF, so with AZ changes in ECF pH should be greater. Bickler et al. (2) have introduced the idea of a carbonic acidosis with CA inhibition in the brain, which, in essence, reflects a loss of this CA-H⁺ "buffer" function. Measurements of brain cortical surface pH show an acidosis even with control of tissue surface PCO₂. This acidosis has been measured only in the ECF; it is unclear what happens to intracellular pH.

We suggest that the mechanism producing the decrease in tissue pH in focal AZ injections involves diminished ability to excrete H⁺ via CO₂, i.e., by decreased CA-related buffering. The change in ECF pH should be greater than that of intracellular pH. An AZ-related decrease in intracellular pH has not been demonstrated, but Lassen (15) has argued that such an intracellular acidosis, although small, is present based on his analysis of brain intra- and extracellular pH responses to systemic hypercapnia and to systemic AZ administration.

ACKNOWLEDGEMENTS

In respect to possible functions for brain carbonic anhydrase, the authors acknowledge many discussions with S. M. Tenney, James C. Leiter, Donald Bartlett, Jr., Peter Scheid, and Joseph Erlichman. We thank Lederle Laboratory Division for the inactive acetazolamide analogue.

FOOTNOTES

Address for reprint requests: E. E. Nattie, Dept. of Physiology, 706E Borwell, Dartmouth Medical School, Lebanon, NH 03756-0001 (E-mail: Eugene.Nattie{at}Dartmouth.EDU).

Received 14 March 1996; accepted in final form 11 June 1996.

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