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J Appl Physiol 89: 1142-1150, 2000;
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Vol. 89, Issue 3, 1142-1150, September 2000

Ventilatory responses to acute and chronic hypoxia in mice: effects of dopamine D2 receptors

K. A. Huey1, M. J. Low3, M. A. Kelly3, R. Juarez3, J. M. Szewczak2, and F. L. Powell1,2

1 Department of Medicine and 2 White Mountain Research Station, University of California, San Diego, La Jolla, California 92093-0623; and 3 Vollum Institute, Oregon Health Sciences Center, Portland, Oregon 97201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We used genetically engineered D2 receptor-deficient [D2-(-/-)] and wild-type [D2-(+/+)] mice to test the hypothesis that dopamine D2 receptors modulate the ventilatory response to acute hypoxia [hypoxic ventilatory response (HVR)] and hypercapnia [hypercapnic ventilatory response (HCVR)] and time-dependent changes in ventilation during chronic hypoxia. HVR was independent of gender in D2-(+/+) mice and significantly greater in D2-(-/-) than in D2-(+/+) female mice. HCVR was significantly greater in female D2-(+/+) mice than in male D2-(+/+) and was greater in D2-(-/-) male mice than in D2-(+/+) male mice. Exposure to hypoxia for 2-8 days was studied in male mice only. D2-(+/+) mice showed time-dependent increases in "baseline" ventilation (inspired PO2 = 214 Torr) and hypoxic stimulated ventilation (inspired PO2 = 70 Torr) after 8 days of acclimatization to hypoxia, but D2-(-/-) mice did not. Hence, dopamine D2 receptors modulate the acute HVR and HCVR in mice in a gender-specific manner and contribute to time-dependent changes in ventilation and the acute HVR during acclimatization to hypoxia.

hypoxic ventilatory response; hypercapnic ventilatory response; acclimatization to hypoxia; carotid body; transgenic mice


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VENTILATORY ACCLIMATIZATION to hypoxia produces a time-dependent increase in ventilation, a decrease in arterial PCO2 (PaCO2), and an increase in the hypoxic ventilatory response (HVR). This process has been well characterized in humans (10, 30, 39) and several animal models, such as goats (7, 32), ponies (8), cats (36, 38), and rats (23). Although all species studied exhibit ventilatory acclimatization to hypoxia, differences exist in the time course and mechanisms of acclimatization (4). The time course of acclimatization to hypoxia can range from only 6 h in the goat to weeks in the rat, which exhibits the most humanlike ventilatory acclimatization to hypoxia (23). However, the time course of acclimatization in the mouse has not been fully characterized. This is important, because recent advances in molecular biology have produced many genetically manipulated mice that can be used to test physiological mechanisms of ventilatory acclimatization to hypoxia. Knowledge about the time course of ventilatory acclimatization to hypoxia in mice will provide a framework for interpreting results in genetically manipulated mice.

Changes in dopaminergic pathways are hypothesized to contribute to ventilatory acclimatization to hypoxia. Dopamine D2 receptors modulate the HVR peripherally in the carotid body and in the central nervous system (CNS). During exposure to hypoxia, dopamine (DA) is released from carotid body glomus cells (13) and chemoafferent fibers in the nucleus tractus solitarius (NTS) (12), which is the primary synapse for afferent fibers from the carotid body (15, 37). At the carotid body, DA activates pre- and postsynaptic D2 receptors (5, 22) and tonically inhibits carotid body neural output (16, 18, 36) and ventilation (2). Chronic hypoxia modifies the levels of carotid body DA and reduces D2 receptor inhibition of carotid body neural output in rats and cats (2, 17). In contrast to the carotid body, D2 receptors in the CNS tonically stimulate ventilation in rats (14), mice (25), and cats (16, 31). Chronic hypoxia also increases DA levels in the CNS (24). It is unclear whether D2 receptors are involved in the increased CNS gain of the HVR with chronic hypoxia, but increased dopaminergic transmission in the NTS could increase D2 receptor facilitation of the HVR.

In these experiments we measured acute HVR, hypercapnic ventilatory response (HCVR), and ventilatory acclimatization to hypoxia in D2 receptor-deficient [D2-(-/-)] and wild-type [D2-(+/+)] mice. Using the D2-(-/-) mice allowed us to characterize acute ventilatory responses and ventilatory acclimatization to hypoxia in the absence of D2 receptors and without the complications of pharmacological blockade. This study was designed to test two hypotheses: 1) acute HVR and HCVR in D2-(-/-) mice are different from those in D2-(+/+) mice because of the involvement of D2 receptors in the arterial chemoreflex pathway, and 2) the time course of ventilatory acclimatization in D2-(-/-) mice is different from that in D2-(+/+) mice because of time-dependent changes in D2 receptor modulation of the HVR with chronic hypoxia.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ventilatory Responses to Acute Hypoxia and/or Hypercapnia

Ventilatory responses were quantified in restrained, awake, adult (16- to 26-wk-old) male and female D2-(+/+) (8 males and 8 females) and D2-(-/-) mice (7 males and 8 females, with 7 females exposed to hypoxic hypercapnia) with use of a head-out, dual-chamber plethysmograph (Buxco). The D2-(+/+) and D2-(-/-) mice were age-matched siblings derived from the breeding of D2-(+/-) parents and have been described previously (20). The mutant allele had been backcrossed onto the C57BL/65 genetic background for five successive generations.

Protocol. Male and female D2-(+/+) and D2-(-/-) mice were tested under the following conditions: 1) hypoxia (8% O2), 2) hypercapnia (10% CO2 and 21% O2), and 3) hypoxic hypercapnia (10% CO2 and 8% O2). At the start of an experiment, a mouse was removed from its cage, weighed, and then placed into the plethysmograph. The mouse was allowed to acclimatize to the plethysmograph for 30 min while it breathed room air before exposure to each experimental gas mixture in a randomized order. Between experimental conditions, ventilation was allowed to return to baseline values during a 15- to 30-min washout period. BioSystem XA software (Buxco) was used to acquire tidal volume (VT), respiratory frequency (f), and minute ventilation (VI) every 30 s during the 5-min exposure to hypoxia or hypercapnia and the 10-min exposure to hypoxic hypercapnia.

Data analysis. To determine the effects of gender, genotype, and time-dependent ventilatory responses, a two-between-factor (gender and genotype), one-within-factor (time) ANOVA (Statview version 5.0.1) was used to determine significant differences in ventilatory responses to hypoxia, hypercapnia, or hypoxic hypercapnia. A two-way ANOVA was also used to determine significant effects of gender and genotype on steady-state ventilatory responses after 5 min of exposure to a given gas mixture. Significance was set at P < 0.05. Values are means ± SE.

Ventilatory Responses to Chronic Hypoxia

Acute ventilatory responses were measured in awake, unrestrained, adult (36- to 40-wk-old) male mice. D2-(+/+) (n = 10) and D2-(-/-) (n = 10) mice were studied before (day 0) and after 2 and 8 days of exposure to normobaric hypoxia [inspiratory PO2 (PIO2) = 70 Torr]. The acclimatization chamber consisted of a clear plastic box that continuously received a hypoxic gas mixture at flow rates sufficient to prevent CO2 buildup (fraction of inspired CO2 < 0.003). Food and water were provided ad libitum.

A small-volume (5-cm-diameter, 10-cm-long) flow-through plethysmograph was used for simultaneous measurements of O2 uptake (VO2) and ventilation. Side ports of the metabolic and reference chambers were connected to opposite sides of a differential pressure transducer (model DP103-18, Validyne). The reference chamber isolated the ventilation signal from ambient thermal and pressure noise. Total resistance in each circuit and between the chambers was balanced with flow resistors. The pressure transducer signal was demodulated (model CD-15, Validyne), sent to a chart recorder (model 2400S, Gould) for amplification and analog low-pass filtering, and then sampled at 250 Hz for real-time monitoring and data storage by computer (Apple Quadra 700 with PowerPC 601 CPU; National Instruments Lab-NB A/D board and Labview software).

VT values were determined from the differential pressure data according to the principles described by Drorbaugh and Fenn (6) and adapted to a flow-through system by Jacky (19). Humidity data were acquired with a humidity sensor (model IH-3602, Hy-Cal Engineering, El Monte, CA) fitted just downstream of the chamber. To calibrate VT, 0.1-ml pulses from a gastight syringe attached to a port on top of the chamber were injected into the plethysmograph at a rate similar to f. The chamber-ambient pressure differential was measured with a water manometer and ranged from 6 to 18 cmH2O.

Metabolic measurements. We also measured VO2 by using dried and CO2-scrubbed gas (Drierite and soda lime, respectively) to provide a determination of VO2 independent of the respiratory quotient (11). A differential O2 analyzer (models S-A3II and N-37M sensor, Applied Electrochemistry) determined the change in O2 fraction by comparing the excurrent metabolic chamber O2 fraction with that measured in the reference chamber. Metabolic chamber flow (600-650 ml/min) was measured with a Fleisch pneumotachograph calibrated against a glass float flowmeter (Cole-Parmer Instrument, ±2% accuracy). The signal output from the O2 analyzer was acquired simultaneously with the ventilation signal onto a computer. After analog low-pass filtration to eliminate noise spikes, the signal was sampled at 250 Hz.

Protocols. Ventilatory responses and VO2 were measured before (day 0) and after 2 and 8 days of exposure to chronic hypoxia. At the start of an experiment, a mouse was removed from its cage, weighed, and then placed into the plethysmograph. The chamber setup was then immersed in a water bath controlled at 30°C. If a mouse became noticeably agitated in the chamber, it was removed and the experiment was ended.

The mouse was allowed to acclimatize to the chamber at its chronic PIO2 for 1 h before data collection. The poikilocapnic HVR was determined using three to four different PIO2 levels (57-214 Torr). We used 214 Torr PIO2 (sea level fraction of inspiratory O2 = 0.30) as our normoxic level to minimize afferent input from arterial chemoreceptors. Pilot measurements of VI in three mice showed that VI decreased from 1,758 ± 85 to 1,472 ± 33 ml · min-1 · kg-1 when PIO2 increased from 150 to 214 Torr. This is similar to results reported for rats (1) and suggests that 30% O2 breathing at sea level can minimize ventilatory drive from O2-sensitive arterial chemoreceptors. VI was measured 2 min after a change in PIO2 and again 10-15 min after a change, before the animal was returned to its chronic PIO2 level for 15 min between tests. VO2 was measured continuously during the protocol, but the values reported here correspond to the ventilatory periods selected for analysis.

Data analysis. Data were analyzed for VI, VT, and f with Labview software. VO2 values were measured during the ventilatory periods selected for analysis. A two-way ANOVA was done at each PIO2 to determine significant ventilatory responses and interactions between the different durations of acclimatization to hypoxia (0, 2, and 8 days) and genotype [D2-(+/+) and D2-(-/-)]. F tests for simple effects were used when the ANOVA yielded a significant interaction of day and genotype. Paired t-tests were used to compare VI, VT, f, and VO2 after 2 and 15 min of hypoxia to test for hypoxic "roll-off."


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ventilatory Responses to Acute Hypoxia and Hypercapnia

The average weight for the male D2-(-/-) mice was significantly lower than that for male D2-(+/+) mice (29.5 ± 0.7 and 35.5 ± 0.8 g, respectively). In contrast, the average weight was not significantly different between female D2-(-/-) and D2-(+/+) mice (22.3 ± 1.4 and 24.0 ± 1.1 g, respectively).

Effects of gender. The effects of gender, independent of genotype, were studied by comparing ventilatory responses in male and female D2-(+/+) mice. There was no significant effect of gender on weight-normalized VI in the mice breathing room air. VI in room air for D2-(+/+) mice was 2,629 ± 135 and 2,549 ± 59 ml · min-1 · kg-1 for male and female mice, respectively. Similarly, ventilatory responses to acute hypoxia were not affected by gender (Fig. 1). In addition, the time-dependent decline in VI during sustained hypoxia was similar between genders in D2-(+/+) mice.


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  		Fig. 1.   Time course of ventilation (VI), frequency (f), and tidal volume (VT) responses to 5 min of hypoxia (8% O2) in male and female D2-(+/+) () and D2-(-/-) (open circle ) mice. Hypoxic responses were not affected by gender alone but were significantly greater in female D2-(-/-) than in female D2-(+/+) mice. All groups exhibited ventilatory decline with sustained hypoxia. * Significant difference between genotypes at 5 min.

In contrast to room air breathing and hypoxia, HCVR were significantly affected by gender in the D2-(+/+) mice (Fig. 2). HCVR were significantly greater in female than in male D2-(+/+) mice; VI after 5 min of 10% CO2 inhalation was 4,843 ± 374 and 3,583 ± 225 ml · min-1 · kg-1, respectively. This greater HCVR was primarily due to a larger VT in the female D2-(+/+) mice (15.4 ± 1.4 vs. 11.6 ± 0.9 ml/kg at 5 min for females and males, respectively), inasmuch as f was similar (322 ± 10 and 313 ± 10 min-1 at 5 min for females and males, respectively). The significant gender effect on HCVR was not evident when the D2-(+/+) mice were exposed to hypoxia and hypercapnia (Fig. 3).


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  		Fig. 2.   Time course of VI, f, and VT responses to 5 min of hypercapnia (10% CO2 and 21% O2) in male and female D2-(+/+) () and D2-(-/-) (open circle ) mice. Hypercapnic responses were significantly greater in female than in male mice. However, hypercapnic responses were significantly greater in male D2-(-/-) than in male D2-(+/+) mice because of a significantly greater VT response. * Significant difference between genotypes at 5 min.



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  		Fig. 3.   Time course of VI, f, and VT responses to 5 min of hypoxic hypercapnia (8% O2 and 10% CO2) in male and female D2-(+/+) () and D2-(-/-) (open circle ) mice. Hypoxic-hypercapnic responses were not affected by gender alone but were significantly greater in female D2-(-/-) than in female D2-(+/+) mice. * Significant difference between genotypes at 5 min.

Effects of D2 receptor genotype and gender. There were no significant effects of genotype on VI in mice breathing room air. VI in the male and female D2-(-/-) mice were similar to VI reported above for the D2-(+/+) mice [2,896 ± 83 and 2,674 ± 59 ml · min-1 · kg-1 for male and female D2-(-/-) mice, respectively].

In male and female mice there was a significant HVR (8% O2) that was independent of gender but significantly affected by genotype (Fig. 1). The significant VI response to hypoxia was the result of significant VT and f responses; however, only f was significantly affected by gender and genotype. Although the acute HVR was not significantly affected by gender alone, there was a significant interaction of gender, genotype, and the time-dependent ventilatory response to hypoxia. For example, in the female mice, peak VI in hypoxia was different between genotypes, occurring after 30 and 60 s for the D2-(+/+) and D2-(-/-) mice, respectively (3,344 ± 118 and 3,603 ± 203 ml · min-1 · kg-1, respectively). In contrast, in the male mice, peak VI in hypoxia occurred after 30 s for both genotypes [3,144 ± 259 and 3,532 ± 224 ml · min-1 · kg-1 for D2-(+/+) and D2-(-/-) mice, respectively]. After peak responses to hypoxia, all groups of mice exhibited ventilatory decline during sustained hypoxia or roll-off (Fig. 1). However, at the end of the 5-min exposure to hypoxia, VI was significantly different between genotypes in the female mice only.

Exposure to 5 min of hypercapnia significantly increased VI in male and female mice, and the responses were significantly affected by genotype and gender (Fig. 2). The significant VI responses to hypercapnia were the result of significant increases in f and VT. Gender and genotype significantly affected VT responses, whereas f was significantly affected by genotype only. There were also significant interactions between the time-dependent VI responses to hypercapnia and both genotype and gender. The time-dependent change in VI with hypercapnia was a slow "on" response, making ventilation stable after 60 s, in contrast to roll-off with hypoxia. Steady-state VI at the end of the 5-min hypercapnic exposure was significantly greater in both groups of female mice than in the male mice. However, genotype significantly affected hypercapnic VI in male mice only, inasmuch as it was significantly greater in D2-(-/-) than in D2-(+/+) mice. This was the result of a significantly greater VT in male D2-(-/-) mice coupled with a nonsignificantly higher f.

The significant VI response to hypoxic hypercapnia (10% CO2 and 8% O2) in male and female mice was also dependent on gender and genotype (Fig. 3). Hypoxic hypercapnia significantly increased VT and f. Similar to hypercapnia alone, VT responses were significantly affected by gender and genotype, whereas f responses were significantly affected by genotype only. In addition, there were significant interactions between time-dependent changes in VI during exposure to hypoxic hypercapnia and both gender and genotype. The time-dependent changes with hypoxic hypercapnia were similar to the changes with hypercapnia alone, i.e., "roll-on," not roll-off. Steady-state VI responses after 5 min of hypoxic hypercapnia were greater in D2-(-/-) male and female mice than in D2-(+/+) mice; however, the difference was only significant in female mice.

Ventilatory Responses to Chronic Hypoxia

Similar to the younger male mice used to study acute ventilatory responses, the average weight was significantly lower for male D2-(-/-) mice than for D2-(+/+) mice (31.2 ± 1.1 and 38.1 ± 1.4 g, respectively, n = 10/group). However, VO2 normalized to body weight was not significantly different between the groups in normoxia or hypoxia (Table 1). Acute hypoxia produced a significant hypometabolism, independent of genotype, on all days.

                              
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  		Table 1.   Effects of acute hypoxia on VO2 in D2-(+/+) and D2-(-/-) mice after 0, 2, and 8 days of continuous hypoxia

Similar to the restrained mice studied during acute hypoxia, ventilatory decline was observed during sustained acute hypoxia in these unrestrained male mice. Between 2 and 15 min of exposure to hypoxia, there was a significant decrease in VI from 3,120 ± 280 to 2,741 ± 203 ml · min-1 · kg-1 in the D2-(+/+) mice. This ventilatory decline was due to a significant decrease in VT and was accompanied by a significant decrease in VO2. There was also a nonsignificant decrease in hypoxic VI in the D2-(-/-) mice, from 3,427 ± 163 to 3,121 ± 195 ml · min-1 · kg-1 (P = 0.06). In contrast to the D2-(+/+) mice, ventilatory decline was due to a significant decrease in f between 2 and 15 min in the D2-(-/-) mice. Similar to the D2-(+/+) mice, the D2-(-/-) mice also significantly decreased their VO2 between 2 and 15 min of hypoxia. All data reported below are measured at 10-15 min of acute hypoxia, i.e., after the decline has occurred.

On all days, VI significantly increased with decreasing PIO2 in both groups (Fig. 4). However, the HVR were significantly different between D2-(+/+) and D2-(-/-) mice. Under control conditions (day 0), VI was similar in both genotypes, except at the lowest level of hypoxia, where VI was significantly greater in the D2-(-/-) than in the D2-(+/+) mice. With longer hypoxic exposures, VI became more different between D2-(-/-) and D2-(+/+) mice, as VI increased more in the D2-(+/+) mice. The differences were most pronounced on day 8, such that VI was significantly lower in the D2-(-/-) mice at all PIO2 levels. Differences in VI between D2-(-/-) and D2-(+/+) mice on days 0 and 2 were due to significant differences in f, whereas the differences on day 8 were due to significant differences in f and VT.


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  		Fig. 4.   VI, f, and VT responses to poikilocapnic hypoxia in D2-(+/+) () and D2-(-/-) (open circle ) mice after 0, 2, and 8 days of hypoxia. There was a significant effect of inspiratory PO2 (PIO2) on all variables on all days. There was a significant effect of genotype on VI on all days, on f on days 0 and 2, and on VT on day 8. * Significant difference between D2-(+/+) and D2-(-/-) at a given PIO2. An additional, lower level of hypoxia (PIO2 = 57 Torr) was studied at days 2 and 8.

With chronic hypoxia, D2-(+/+) mice exhibited a progressive and significant increase in VI at high PO2 (PIO2 = 214 Torr) from days 0 to 2 and from days 2 to 8, whereas it was unchanged in D2-(-/-) mice (Fig. 4). This indicates that "baseline" ventilatory drive (i.e., without significant hypoxic ventilatory drive) is changing differently between genotypes. The time-dependent change in baseline VI from 0 to 8 days of hypoxia was significantly different between genotypes. The differences in baseline VI from days 0 to 2 between genotypes were due to significant differences in f, whereas differences from days 2 to 8 were the result of significant differences in VT.

There were also significant differences in hypoxic (PIO2 = 70 Torr) ventilatory drive during acclimatization between D2-(-/-) and D2-(+/+) mice (Fig. 4). Hypoxic VI decreased in both groups from 0 to 2 days; however, the decrease was significant only in the D2-(-/-) group because of a significant decrease in f. Both genotypes showed a similar increase in hypoxic VI between days 2 and 8. In the D2-(+/+) mice, the increase in hypoxic VI from 2 to 8 days resulted in a significantly greater hypoxic VI after 8 days than in control. However, hypoxic VI after 8 days in D2-(-/-) mice remained below control levels, because hypoxic VI after 2 days was so low compared with control. The higher hypoxic VI on day 8 in the D2-(+/+) mice was due to a greater VT than in D2-(-/-) mice with a similar f.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These findings suggest that D2 receptors are involved in acute ventilatory responses to hypoxia and/or hypercapnia and ventilatory acclimatization to hypoxia. The contribution of D2 receptors to acute hypoxic, hypercapnic, and hypoxic-hypercapnic ventilatory responses is dependent on gender. Furthermore, data from male and female mice strongly support a net inhibitory effect of D2 receptors on acute HVR and HCVR. Although not always significant, ventilation was higher with all acute ventilatory challenges in the D2-(-/-) mice. However, chronic hypoxia removed this net inhibitory effect of D2 receptors on ventilation, and the HVR were lower in D2-(-/-) mice after 2 and 8 days of exposure to hypoxia.

Acute HVR and HCVR in Mice

Effects of experimental methods to measure VI. Differences in the methods used to measure ventilation may explain differences in the magnitude of the HVR between the two groups of transgenic mice we studied. Restraint has been shown to significantly increase absolute values of f and VI, but not VT, in adult female outbred mice, although increases in VI with hypoxia or hypercapnia were not affected (3). The first set of transgenic mice we used to measure acute ventilatory responses were restrained in a "head-out" plethysmograph, and this potential stress may explain why f and VI in normoxia were greater in this group than in the chronically hypoxic group studied with whole body plethysmography. The maximum levels of ventilation we observed were similar in both groups of animals we studied, so the change in ventilation with chemoreceptor stimulation (i.e., the HVR and HCVR) was larger in our unrestrained mice. This contrasts with results showing no significant difference between the HVR and HCVR in restrained and unrestrained mice (3). We speculate that the effect of restraint may have been larger on our first group of mice in baseline conditions, thereby decreasing the absolute HVR.

Male and female restrained mice exhibited ventilatory decline during the 5-min exposure to 8% O2 independent of genotype (Fig. 1). The experimental system used to measure ventilation in unrestrained male mice was not suitable to measure the time course of ventilatory changes with the same resolution obtained for the restrained mice because of the time necessary to change gas mixtures in the whole body chamber. However, in the unrestrained male mice, we observed a significant decrease in VI and VO2 between 2 and 15 min of exposure to 10% O2. Because VO2 was not measured in the restrained mice, it is unknown whether the ventilatory decline observed in only 5 min of hypoxia was also accompanied by a fall in metabolic rate. Also, because isocapnia was not maintained in the restrained or unrestrained mice, the ventilatory decline may have resulted from decreased CO2 stimulation as PaCO2 was decreased during hypoxic hyperventilation. Thus we cannot use these data to determine whether mice exhibit hypoxic ventilatory decline comparable to that described in humans and other mammals (29). Hypoxic ventilatory decline has been defined as a decline in ventilation during the first several minutes of hypoxia that can occur independent of decreases in metabolism or PaCO2.

Effects of gender. In this study, gender did not significantly affect ventilation in our transgenic mice breathing room air or the peak acute HVR (Fig. 1). In contrast, acute HCVR were significantly greater in female than in male D2-(+/+) mice (Fig. 2). These data agree, in part, with a previous study measuring ventilation in aged (32- to 44-wk-old) male and female 129/Sv mice (27). In this study, ventilation in room air and hypoxia was similar in male and female mice. In the 129/Sv mice, VI in hypoxia was not greater than VI in room air. However, this may be due to the fact that hypoxic VI was reported at 5 min of hypoxia, after roll-off may have occurred. In this previous study, HCVR were greater in female mice; however, the difference was not statistically significant. Our hypercapnic results, taken together with the previous results showing a trend for greater responses to hypercapnia in female mice, agree with studies in humans and rats showing that female hormones (progestin and estrogen) increase ventilatory responses to CO2 (21, 35).

Effects of D2 receptor genotype. D2 receptor genotype significantly affected acute hypoxic, hypercapnic, and hypoxic-hypercapnic responses, as well as ventilatory acclimatization to hypoxia (Figs. 1-4). The acute ventilatory responses strongly suggest a net inhibitory effect of D2 receptors on acute HVR or HCVR, inasmuch as ventilation was consistently higher in D2-(-/-) than in D2-(+/+) mice under these conditions. Furthermore, the effects of D2 receptor genotype on acute ventilatory responses were gender dependent. Genotype significantly affected HVR in female mice only. In contrast, HCVR were significantly affected by genotype in male mice only. This suggests that the lower HCVR may be due to a D2 receptor-dependent effect in males instead of (or in addition to) a lack of facilitation by female hormones. The significant interaction between genotype and ventilation during exposure to hypoxic hypercapnia in male and female mice may reflect the independent effects of D2 receptor involvement in HVR and HCVR in female and male mice, respectively. The differences in HVR and HCVR seen between male and female mice may be due to hormonal differences (34).

Despite differences and age and experimental techniques, the effects of D2 receptor genotype on normoxic and hypoxic ventilation are similar in the two groups of male mice studied. The acute responses to 8% O2 in restrained male mice and the acute responses to 10% O2 (day 0) in unrestrained male mice before acclimatization to hypoxia are in agreement, in that D2 receptors do not have large effects on acute HVR in male mice. However, in both groups studied, VI was higher in D2-(-/-) than in D2-(+/+) mice at all O2 levels before exposure to chronic hypoxia (Figs. 1 and 4).

Ventilation in 10% inspired CO2 was higher in male and female D2-(-/-) mice than in the corresponding D2-(+/+) mice (Fig. 2). The significantly greater HCVR in the male D2-(-/-) mice contrasts with a previous study in which D2 receptor antagonists were used (26). In male QS mice, total body D2 receptor blockade with droperidol significantly decreased ventilation in 7.5% inspired CO2. In addition, stimulation of CNS dopamine receptors by simultaneous administration of L-dopa and carbidopa significantly increased ventilation in 7.5% inspired CO2 compared with administration of carbidopa alone (25). The differences between these previous studies and the present results may be due to side effects of droperidol, which could depress HCVR, independent of D2 receptors. Additional possibilities are differences in HCVR among different strains of mice that have been previously documented (33): similar responses to hypercapnia (8% CO2 and 21% O2) and hypoxic hypercapnia (8% CO2 and 10% O2) were reported in C57 and 129 mouse strains; however, QS mice were not studied.

Ventilatory Acclimatization to Chronic Hypoxia in Mice

This is the first study to quantify the time course of changes in the HVR during acclimatization to hypoxia in mice. We measured poikilocapnic HVR in D2-(+/+) and D2-(-/-) mice after 0, 2, and 8 days of exposure to 70 Torr PIO2 (Fig. 4). Olson and Saunders (26) investigated acclimatization to hypoxia in male QS mice, but normoxic VI, f, and VT were not reported. Because hyperventilation in normoxia, in addition to increased ventilation during hypoxic stimulation, is considered an important component of ventilatory acclimatization to hypoxia (9), changes in normoxic ventilation are integral in characterizing the time course of acclimatization in mice. Our results demonstrate that the time course of ventilatory acclimatization to hypoxia in transgenic wild-type mice is similar to that previously reported in rats (23). In rats exposed to 14 days of hypobaric hypoxia, significant ventilatory acclimatization occurred within 4 days but was not complete until 1 wk (23). In another study on rats, normoxic VI increased up to 2 days of hypoxia and remained at that level through 8 days of hypoxia (28). In contrast, other animal species acclimatize to hypoxia more rapidly than rats or mice. Specifically, goats acclimatize to 4,300 m in 4-6 h (7, 32), whereas significant acclimatization to hypoxia is apparent in cats after 2 days (38).

Effects of D2 receptors on ventilatory acclimatization to hypoxia. Our results in the D2-(-/-) mice are consistent with a previous study investigating the acute effects of whole body D2 receptor blockade with droperidol during acclimatization to hypoxia in QS mice (26). Systemic D2 receptor blockade demonstrates the net effects of D2 receptors on the HVR. The ventilatory responses of D2-(-/-) mice compared with those of D2-(+/+) mice are similar to the effects of acute droperidol administration during chronic hypoxia. Before exposure to chronic hypoxia, droperidol significantly increased VI in mice acutely breathing 70 Torr PIO2, suggesting that the carotid body D2 receptor effect predominates in control mice because D2 receptors in the carotid body depress O2 sensitivity and the HVR (13, 16, 18, 28, 36). Similarly, VI was higher in unacclimatized D2-(-/-) mice than in unacclimatized D2-(+/+) mice at all PIO2 levels, with the largest difference at 70 Torr PIO2 (Fig. 4). After 2, 4, and 8 days of hypoxia, acute droperidol significantly decreased VI in the QS mice (26). Similarly, VI was greater in the D2-(+/+) than in the D2-(-/-) mice at all PIO2 levels after 2 and 8 days of hypoxia (Fig. 4). This suggests a predominantly CNS effect in mice after exposure to chronic hypoxia, because D2 receptors facilitate the HVR in rats and cats (14, 16, 25, 28, 31). The lack of significant increase in baseline ventilation (PIO2 = 214 Torr) in D2-(-/-) mice over 8 days of hypoxia suggests that D2 receptors are critical for components of ventilatory acclimatization other than changes in the HVR. The difference in baseline ventilation after chronic hypoxia between genotypes may involve the effects of D2 receptors on ventilatory sensitivity to CO2, as suggested by differences in the acute HCVR (Fig. 2).

Critique of Experimental Design and Methods

Arterial chemoreceptor stimuli. One limitation of the present study is that we were unable to obtain arterial blood gases and, therefore, measure the true arterial chemoreceptor stimuli. However, after 2 and 8 days of hypoxia, VI was lower in the D2-(-/-) than in the D2-(+/+) mice, and this would decrease arterial PO2 (PaO2) at a given PIO2, so the HVR would be even lower in the D2-(-/-) mice if calculated as a function of PaO2. Furthermore, VT was lower at 8 days in the D2-(-/-) mice, so dead space would be greater, which would increase PaCO2, with the assumption of similar CO2 production. VO2 was similar (Table 1), and we would not expect respiratory exchange ratios to be different between groups. Consequently, the predicted changes in PaO2 and PaCO2 in D2-(-/-) mice would stimulate VI and cannot account for the lower VI at a given PIO2 with acclimatization compared with the D2-(+/+) mice.

Transgenic model of D2 receptor deficiency. Although genetically manipulated mice provide the opportunity to study physiological adaptations in the absence of the gene of interest, several limitations in this model must be considered. One limitation of the knockout methodology is that the genetic mutation is generally on a hybrid background. The genetic and behavioral differences between inbred mouse strains present some problems in the interpretation of mutant phenotypes as well as in the choice of appropriate control mice. The mice in this study are of a mixed 129 and C57BL/6 background (20). Further studies are necessary to determine whether the wild-type mice must be used for control studies or whether the phenotype is the same as one of the original strains.

In addition, results may be complicated by compensatory mechanisms during development, for example, in other dopamine receptor subtypes. However, the results show that even if these secondary effects occur, they are insufficient to completely overcome the direct effects of gene ablation. D2-(-/-) mice lacked a normal ventilatory response to chronic hypoxia, despite a normal control HVR. It is also possible that the difference in ventilatory acclimatization in D2-(-/-) mice does not involve D2 receptors during exposure to chronic hypoxia but, rather, results from another D2 receptor-dependent mechanism during development. However, our results are consistent with previous results with acute administration of a dopamine antagonist during acclimatization (26).

Conclusions

These results demonstrate gender-specific involvement of D2 receptors in acute HVR and/or HCVR. Specifically, these results suggest a net inhibitory effect of D2 receptors on the acute HVR and HCVR. In addition, the absence of the D2 receptor gene prevented normal ventilatory acclimatization to hypoxia in male mice, despite a normal control HVR and metabolic rate compared with D2-(+/+) mice. Results with knockouts are most clearly interpreted in experimental paradigms that take place over days to weeks, since the results can be interpreted within the context of complete and continuous absence of receptor function (e.g., acclimatization, addiction, and sensitization). Consequently, D2-(-/-) mice provide a good model to study ventilatory acclimatization to hypoxia, which occurs over days to weeks, when continuous (rather than acute) D2 receptor inactivation may be advantageous. Future studies with inducible or region-specific knockouts will further our understanding of the role of D2 receptors in ventilatory acclimatization to hypoxia.


    ACKNOWLEDGEMENTS

We thank Charlene Bryan for excellent technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-17331 (F. L. Powell, K. A. Huey, and J. M. Szewczak), HL-07212 (F. L. Powell and K. A. Huey), RO1 DA-12062 (M. J. Low), and T32 DA-07262 (M. A. Kelly); the University of California White Mountain Research Station (F. L. Powell and J. M. Szewczak); and the Minority Medical Faculty Development Award, Robert Wood Johnson Foundation (R. Juarez).

Address for reprint requests and other correspondence: K. A. Huey, Physiology and Biophysics, University of California, Irvine, 346-D Med Sci I, Irvine, CA 92697-4560 (E-mail: khuey{at}uci.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 28 January 2000; accepted in final form 19 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 89(3):1142-1150
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