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,2
Departments of
* Physiology,
Paediatrics and Medicine, Queen’s University, Kingston, Ontario, Canada; and Departments of
Medicine and
Cell Biology, Duke University, Durham, North Carolina, USA
2Correspondence: Department of Physiology, 4th floor Botterell Hall, Queen’s University, Kingston, ON K7L 3N6, Canada. E-mail: fisherjt{at}post.queensu.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: DAT • hypoxia • ADHD • chemoreception • respiration • circadian rhythm • body temperature
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, whereas exogenously applied DA inhibits the tonic discharge of the carotid sinus nerve (2)
. In vivo studies using a canine model showed inhibition of chemoreceptor afferent activity at low doses of dopamine but excitation followed by inhibition at higher doses (3)
.
The presence of both presynaptic dopamine DA-2 autoreceptors on type I cells and postsynaptic DA-2 receptors on sensory nerve endings (1)
suggests a complex modulatory role for DA. The net effect of DA-2 receptor blockade is to increase basal arterial chemoreceptor activity (4)
and block the tonic inhibition seen in exogenously applied DA (2)
. Similarly, ventilation and respiratory frequency are increased after carotid body DA-2 receptor blockade (5)
during normoxia and remain unaltered in response to hypoxia. Thus, overall DA appears to play an inhibitory role in peripheral chemoreception.
The exact role of dopamine in the central transmission of the hypoxic ventilatory response remains elusive. Afferent fibers from the carotid body provide feedback to central dopaminergic systems located in the nucleus tractus solitarius (NTS; refs 6
, 7
). Extracellular dopamine is released in the NTS of the rabbit in response to severe hypoxia; however, this release relies on the intact peripheral chemoreceptor input from the carotid sinus nerve (8)
. A study done in mutant mice lacking both central and peripheral DA-2 receptors has shown that DA-2 (–/–) mice show a significant increase in ventilation in response to 5 min of hypoxia characterized by an increase in respiratory frequency and tidal volume, when compared with DA-2 (+/+) mice (9)
. However, other studies in rats show that blocking the central DA-2 receptors by haloperidol does not affect the hypoxia mediated hyperventilation (5
, 10)
and that blocking both central and peripheral DA-2 receptors results in a decrease in respiratory frequency in response to hypoxia (5)
.
Central DA receptors also influence the hypoxia-induced decrease in body temperature (Tb) and metabolic thermogenesis, due to hypothalamic mechanisms (11)
. In the rat, stimulation of DA receptors in the preoptic area of the hypothalamus by DA or the dopamine agonist apomorphine causes hypothermia and a decreased metabolic rate (12; 3). Further, injection of the DA-2 antagonist haloperidol into the preoptic anterior hypothalamus and the striatal nuclei causes hyperthermia associated with vasoconstriction and increased metabolism, suggesting that central activation of dopaminergic pathways inhibits metabolic heat production (13)
. Indeed, blockade of central DA-2 receptors by haloperidol attenuated a hypoxia-induced drop in Tb (10)
, suggesting, at least in part, a role for DA in hypoxia-induced hypothermia.
The dopamine transporter (DAT) protein, a high affinity monoamine transporter protein located on the presynaptic dopaminergic neuron, plays a critical role in the termination of dopamine signaling (14)
. The development of a mutant mouse lacking functional DA transporter (DAT–/–) protein provides a powerful tool to alter the inherent DA concentration without the complications of exogenous agents. The DAT–/– mouse displays environment-dependent hyperactivity, cognitive impairment in maze trials, and a calming response to methylphenidate that is strikingly similar to the response seen in attention deficit hyperactivity disorder (ADHD) patients (15
, 16)
. Although this mouse model has been used to examine behavioral responses to psychostimulants such as amphetamine and methylphenidate (14
15
16)
, the respiratory phenotype remains uncharacterized and may yield insight into the potential for a cardiopulmonary phenotype in behavioral disorders.
We tested the hypothesis that loss of DAT function would result in a significant cardiorespiratory phenotype, which would reflect a significant role of dopamine in the chemical control of breathing, metabolism, and thermoregulation. To test our hypothesis we measured the ventilatory, metabolic, and thermoregulatory responses to reduced oxygen concentrations (hypoxia) and/or increased CO2 (hypercapnia). We also examined the circadian variation in body temperature, heart rate, and motor activity. We report herein that the DAT–/– mouse displays a shift in core body temperature and a blunted ventilatory response to hypoxia. Our results should be of significance to those interested in the diagnosis of pathological conditions involving hyperdopaminergia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Mice used in this study were obtained from the mutant mouse colony at Duke University (Durham, NC). The DAT –/– mouse is characterized by a 5-fold increase in extracellular DA concentration (14
, 17)
, a reduction in the overall amount of releasable DA (17)
, as well as a prolonged synaptic cleft dopamine clearance rate (at least 300 times longer than the DAT +/+ mouse) (17)
. The overall condition of the DAT–/– mouse is one of heightened extracellular dopaminergic tone. The behavioral phenotype associated with this hyperdopaminergia is one of enhanced locomotor activity in novel environments (17)
. Experiments were performed on conscious, unrestrained DAT–/– knockout mice (n=12, 26.1±2.0 g) and on age-matched DAT+/+ wild-type (WT) littermate controls (n=14, 36.3±2.3 g). Mice were acclimated to a standard 12:12 h light-dark cycle under standard animal care facilities at 24 ± 1°C. Mouse chow and water were provided ad libitum.
Respiratory measurements
Mice were placed in a whole body plethysmograph and allowed to acclimate for at least 45 min before measurements. Mice were exposed to either a hypoxia challenge (FIO2=0.10) consisting of a 1 h exposure to normoxic air followed by a 4 h exposure to hypoxia (DAT–/–: n=5; WT: n=6) or a hypercapnia challenge (FICO2=0.05) consisting of a 15 min exposure to normoxic air, a 40 min exposure to hypercapnia, and a 40 min recovery exposure to normoxic air (DAT–/–: n=6; WT: n=8). All air sources were humidified before passing through the chamber. Ventilation was measured by the barometric method, and a breath-by-breath analysis of tidal volume (VT), respiratory frequency (f), and inspiratory (TI), and expiratory times (TE) was performed.
Metabolic and temperature measurements
To measure core body temperature, sterile temperature transmitters (Data Sciences International, St. Paul, MN, USA) were surgically implanted into the abdominal cavity. Mice were anesthetized with an intraperitoneally injection of Ketamine-Xylazine (80:4 mg/kg) mixture; transmitters were implanted under aseptic conditions. Two weeks postsurgery mice (DAT–/–: n=4; WT: n=4) were exposed to the 4 h hypoxia challenge during which their core body temperature was recorded by a telemetry receiver (Data Science International). Metabolic responses were measured by passing a continuous flow of air through the plethysmograph chamber into a CO2 analyzer for the measurement of carbon dioxide production (QUBIT systems). In a similarly instrumented group of mice (DAT–/–: n=6; WT: n=6), continuous biotelemetry (Data Sciences International) recorded circadian variation of core body temperature, heart rate and activity levels over a 10 day period. Body temperature was averaged over a ten second period and recorded every 5 min. Heart rate was calculated from ECG waves and reported as a single value every 5 min. Activity count was tallied by the receiver over a 10 s sample period and reported as a relative measure of locomotor activity every 5 min.
Analysis
Data were converted to numeric files for graphical and numerical analysis using Excel (Microsoft, Redmond, WA, USA). Data are mean ± SE. The effects of time course of hypoxia or hypercapnia and strain were evaluated by statistical analysis using a two way repeated measures ANOVA on a one factor general linear model with a Student-Newman Keuls method for multiple comparisons [Statistical Packages for the Social Sciences (SPSS) version 10.0, SPSS Inc., Chicago, IL, USA]. Circadian rhythm data were time-synchronized and averaged for each genotype. Maximum and minimum values were calculated as the top 10 percentile and bottom 10 percentile, respectively (11)
. Fourier analysis was conducted on circadian body temperature data using Sigma Plot (SPSS Inc.). A significant difference was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E; E=VT*f) between the two genotypes (P>0.05). The lower respiratory frequency in the DAT–/– mice was characterized by a significantly longer TI than the WT mice (0.177 vs. 0.112 s, respectively, P<0.05). TE was not statistically different between the two groups. Average VT/TI ratio was significantly lower in the DAT–/– mice compared to the WT mice (1.35±0.15 vs. 3.10±0.23 ml/s, respectively; P<0.05).
Hypoxic respiratory phenotype
In response to the hypoxic challenge (Fig. 1
), the WT mice showed a significant increase in minute ventilation (55% increase from control) at 10 min of hypoxia (P<0.05) with a roll off to control values for the remainder of the hypoxic challenge. In contrast, DAT–/– mice showed a smaller, more variable increase in minute ventilation, which was not significantly different from control values (P>0.05). The smaller minute ventilation response of the DAT–/– mice at 10 min of hypoxia was due to the lack of a tidal volume response (average increase in tidal volume of 1.6±8.9% from control compared to 12.8±2.6% in the WT mice). In fact, four out of five DAT–/– mice displayed a reduction in tidal volume from control (–6.5±4.8%).
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The respiratory frequency of the WT mouse increased significantly from control and remained significantly elevated for most of the hypoxic challenge (Fig. 1)
. In contrast, DAT–/– mice had a transient increase in respiratory frequency (at 10 min), followed by a return to control values until 3 h of hypoxia, at which time frequency increased slightly. Overall, the respiratory frequency of the DAT–/– mice remained significantly lower than the WT genotype for over 3.5 h of the 4 h hypoxic challenge.
During the hypoxic challenge, the increase in respiratory frequency from control was accounted for by a significant decrease in TE (P<0.05) in the WT mice (Fig. 2
). In contrast, TE transiently decreased in the DAT–/– mice at 10 min of hypoxia and then returned to values not significantly different from control until over 3.5 h of hypoxia. TI of the DAT–/– mice (Fig. 2)
remained significantly longer during the hypoxia challenge compared to the WT genotype (P<0.05). The smaller increase in tidal volume and longer TI of the DAT–/– mice at 10 min of hypoxia resulted in a lower VT/TI ratio compared to the WT group (1.75±0.32 vs. 3.90±0.26 ml/s, respectively; P<0.05).
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Hypercapnic respiratory phenotype
Both groups showed a robust ventilatory response (Fig. 3
) to hypercapnia that was characterized by a significant increase in minute ventilation and respiratory frequency (P<0.05) from control. Minute ventilation was not statistically different between the two groups for either the control, hypercapnia, or recovery periods of the challenges. Tidal volume although slightly elevated in response to hypercapnia was not significantly different from control for either genotype nor was there a statistically significant difference between the groups during any period. The increase in respiratory frequency in response to hypercapnia in the WT mouse was marked by a significant reduction from control in TE alone (Fig. 4
), whereas the DAT–/– mice responded with a significant reduction in both TE and TI.
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As with the normoxia and hypoxia data, the respiratory frequency of the DAT–/– mice remained significantly lower than that of the WT mice for both control and hypercapnic periods. Similarly, the TI of the DAT–/– mice was significantly longer than the WT mice throughout the entire protocol (Fig. 4)
.
Body temperature and metabolic phenotype during hypoxia
To examine hypoxia induced hypothermia and the inhibition of metabolic thermogenesis, measurements of CO2 production (Fig. 5
), as an index of metabolism, and core body temperature (Fig. 6
) were taken. Taken as an average over the 60 min control period, the WT and DAT–/– mice showed no difference in metabolic rates, although during normoxia the CO2 production of the DAT–/– mice was more variable (Fig. 5)
. Both groups showed a decrease in metabolism in response to hypoxia, although the DAT–/– mice had a larger, although not statistically significant, relative decrease (58±5%) during hypoxia when compared to WT mice (45±6%). During the normoxic control period, DAT–/– mice exhibited a significantly lower core body temperature (35.8±0.2°C) when compared to the WT mice (37±0.2°C, P<0.05). Both groups showed a drop in core body temperature in response to hypoxia. The DAT–/– mice had a lower core body temperature than the WT mice at 2 h of hypoxia, although the percent change from control was not significantly different between the two groups.
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To examine whether the lower body temperature seen in the DAT–/– mice during normoxia was a constant phenotypic observation, biotelemetry was performed on a separate group of mice. Figure 7
illustrates the mean core body temperature responses of separate groups of WT (top panel, n=6) and DAT–/– mice (bottom panel, n=6) recorded over 5 days. The nocturnal activity/arousal pattern of both groups of mice is illustrated as an increase in body temperature during lights off (indicated by dark bars). Both strains of mice show normal photoentrainment patterns with a dominant circadian rhythm period of 23 h, as analyzed by Fourier analysis. The average body temperature and 10th percentile maximum body temperature for the DAT–/– mice were not significantly different from those of the WT mice. However, DAT–/– mice did have a significantly lower 10th percentile minimum body temperature at 34.50 ± 0.27°C than the WT mice at 35.71 ± 0.1°C. The swing in body temperature (maximum–minimum) for the WT mice was 2.3°C, while the DAT–/– mice had a significantly larger swing in body temperature of 3.8°C (P<0.001).
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The circadian pattern of heart rate for both genotypes closely followed that of body temperature. Although the DAT–/– mice exhibited a slightly lower average heart rate for both lights on and lights off periods, there was no statistical difference between the two groups (data not shown). There was also no significant difference between the two groups for either the 10th percentile minimum heart rate or the 10th percentile maximum heart rate. DAT–/– mice had a slightly higher average activity rate compared to the WT mice, although again this was not statistically significant. However, the 10th percentile maximum activity in the DAT–/– mice, which represents peak activity values, was significantly higher when compared with values from the WT mice (80.4±8.9 vs. 56.7±4.4, P=0.039).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normoxic respiratory phenotype
Normoxic resting ventilation was not significantly different between genotypes, although DAT–/– mice displayed a significantly lower respiratory frequency. This observation is consistent with data for human subjects, which show a reduction in resting minute ventilation and respiratory frequency in response to an intravenously infusion of dopamine (18)
. The lower respiratory frequency in the DAT–/– mice was further characterized by a significantly prolonged TI compared to WT mice, which is consistent with the effects of activation of central DA receptors by DA agonists where modifications of phrenic nerve timing are present (19
, 20)
.
Loss of the DAT function in the DAT–/– mutant and the corresponding increase in extracellular dopamine could result in a sensitization of carotid body DA-2 receptors and a reduction in the tonic discharge of sensory neurons. DA-2 receptor mRNA is expressed in the carotid body in type 1 glomus cells as well as in sensory neurons of the petrosal ganglion that innervate the carotid body (21, 22). Whether the predominant action of dopamine on DA-2 receptors occurs presynaptically or postsynaptically is unclear; however, the net effect of activation of DA-2 receptors at the carotid body is to cause an inhibition of the tonic discharge of the sensory nerves and a reduction in respiratory frequency (10)
. DAT–/– mice are under the influence of high levels of dopamine throughout their development, and although mRNA and protein levels of postsynaptic DA-1 and DA-2 receptors are down-regulated by 50% (14)
, specific populations of these receptors are supersensitive (14
, 17)
, resulting in an overall effect of hyperdopaminergic tone. Whether the hyperdopaminergic tone principally acts centrally to influence phrenic output or at the level of the carotid body to inhibit tonic sensory discharge remains to be seen; however, the net effect on respiratory timing during normoxia is clearly consistent with such actions.
Hypoxic respiratory phenotype
In response to hypoxia, the hyperdopaminergic DAT–/– mice showed a blunted acute ventilatory response compared to the WT mice. Release of DA from the carotid body is thought to be in direct proportion to the intensity of the hypoxic stimulus (1)
, which confers a major role for DA in the chemoreception of hypoxia. Studies using exogenous DA or DA antagonists suggest that DA plays an inhibitory rather than a stimulatory role at the level of the carotid body and may modulate chemoreceptor activity by tonically inhibiting activity from sensory afferent nerves (2
3
4)
. Our results are consistent with those in human subjects, where high altitude or administration of dopamine both show a negative correlation between blood DA concentration and the ventilatory response to hypoxia (23
, 24)
. Indeed, our findings are consistent with the hypothesis that hyperdopaminergia at the level of the carotid body modulates both the respiratory frequency and tidal volume response to hypoxia.
Since the hypoxic ventilatory response is characterized by an acute increase in ventilation followed by a "roll off" response, it might be argued that our sampling design may have missed the peak ventilatory response in the DAT–/– mice. This is unlikely based on preliminary studies in WT mice in which we measured the hypoxic ventilatory response every 5 min and observed a greater ventilatory response at 10 min of hypoxia. Indeed, this observation formed the basis for the timing of our first measurement period. An increase in sample size would also be unlikely to alter our results, since not only was the average increase in tidal volume during hypoxia extremely modest in the DAT–/– mice (<2%), but four out of five DAT–/– mice displayed a decrease in tidal volume.
Hypoxic body temperature and metabolic phenotypes
The DAT–/– mice had a similar metabolic rate that was accompanied by a significantly lower core body temperature than the WT mice during normoxia (15)
. Our data clearly support a role for DAT-induced changes in DA in the regulation of basal thermoregulation, which is independent of metabolism. In response to hypoxia, both genotypes decreased metabolism and body temperature to a similar extent. Our data are consistent with the behavior of several adult mammalian species, in which both ventilation and metabolism change in response to hypoxia (25)
. We suspect that the slightly greater hypoxia-induced fall in metabolic rate and body temperature in the DAT–/– mice may be physiologically relevant. Our data contrast that reported for rats in which haloperidol injected into the third ventricle caused an attenuated decrease in body temperature and O2 during hypoxia and no difference in normoxic body temperature or O2 (10)
. Although it is possible that alterations in thermoregulation and metabolism between species explain these differences, surgical procedures or the influence of the injection volume in the rat third ventricle may be a more likely explanation.
Hypercapnic respiratory phenotype
The exact role of DA in the chemoreception of hypercapnia is complex. DA is thought to act both at the level of the carotid body (26)
and centrally to modify chemoreceptor activity and alter inspiratory motor output (8)
. We found no significant difference between the two groups in their ventilatory or respiratory frequency response to hypercapnia. This is consistent with a study in cats, which showed that blockade of DA-2 receptors by haloperidol does not change the ventilatory response to hypercapnia (27)
. However, the increase in ventilation in the DAT–/– mice was associated with a significant reduction in both TE and TI from control compared to a response that was restricted to TE for the WT genotype. Activation of central DA receptors has been shown to alter respiratory timing (20)
, and exogenous DA has been shown to significantly inhibit the discharge frequency of lung rapidly adapting receptors (28)
. Thus, afferent input from the carotid body and airway mechanoreceptors may be modified by the hyperdopaminergic tone of the DAT–/– mice consistent with our findings.
Body temperature, heart rate, and activity phenotype
DAT–/– mice exhibited a significantly lower daytime (inactive period) body temperature that was accompanied by significantly greater swings in body temperature compared to the WT mice. In the rat, stimulation of DA receptors in the preoptic area of the hypothalamus causes hypothermia and a decrease in metabolic rate (12
, 13)
. The similar resting metabolic rates in the DAT–/– and WT mice during normoxia and the lack of a difference in heart rate between the two strains suggest the hypothermia experienced by the DAT–/– mice can not be explained easily by a change in heart rate or metabolism. However, thermoregulatory mechanisms include not only those under hypothalamic influence but also behavioral modifications such as increased activity.
Consistent with previous studies characterizing the DAT–/– model of hyperdopaminergia, our mice displayed a significantly larger maximum motor activity level than the WT mice. The activity data record for the DAT–/– mice is consistent with the enhanced locomotor activity and hyperactivity previously reported for this genotype (17)
and is one of the primary reasons for using this model in pharmacological studies related to the study of ADHD. Since hypothermia exhibited in the DAT–/– mouse was limited to the inactive (lights on) periods, we examined the length and frequency of the inactive periods over a single 24 h period (data not shown). DAT–/– mice showed fewer inactive periods during the day (waking more often) and, despite having a more intense activity period, had longer inactive periods during the active (lights off) period than the WT mice. Our observations of increased wakefulness are consistent with those made by Wisor et al. (29)
in the same murine model and are also consistent with observations of daytime sleepiness and nocturnal wakefulness seen in ADHD patients (30
, 31)
. The hypothermia exhibited during inactivity and normal body temperature exhibited during intense hyperactivity suggest a greater sensitivity for behavioral modification of body temperature than the WT mice. It is not clear how DA affects thermal inertia, and it would be interesting to know whether similar behaviors exist for patients displaying hyperdopaminergia.
Loss of the DAT protein affects the homeostasis of the neurotransmitter DA by removing the primary uptake mechanism of DA from the synaptic cleft (see Fig. 8
). These alterations in DA transmission and signaling result in a state of global hyperdopaminergia (17)
. Our study is the first to examine the effect of disruption of the DAT protein on the chemical control of breathing in conscious, unrestrained mutant mice and to characterize the respiratory phenotype of an animal model of hyperdopaminergia. We found that loss of the DAT protein was characterized by a respiratory phenotype that was present during normoxia and hypoxia consistent with the proposed inhibitory role for DA (2
, 3
, 5)
.
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Implications for ADHD
ADHD is a heterogeneous disorder involving both genetic and environmental components (32)
. Although ADHD is likely the result of aberrant interactions between several neurological systems, rather than a single neurological dysfunction, the principal target for the most widely used anti-ADHD medications is DAT, which presents as a unique molecular target for the investigation of the pathophysiology of ADHD (for review see refs 33
34
35
). The DAT–/– mouse is but one of several animal models developed to assist in the investigation of ADHD (14
15
16
, 36)
. The clinical diagnosis of ADHD may be enhanced by a reliable physiologically based diagnostic tool. Our findings of a unique respiratory and thermoregulatory phenotype in the DAT–/– model suggest that such measurements represent a promising target that could aid in the development of noninvasive diagnostic tools for ADHD. Further, our data on the respiratory role of the DAT protein are consistent with a highly complex and integrated system, which is of primary interest to the pathophysiology of ADHD. Our results should be of broad clinical relevance to those interested in both the physiology and diagnosis of ADHD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 31, 2006. Accepted for publication December 14, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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