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Hypothalamic GABAergic influences on treadmill exercise responses in rats
Brain Research Bulletin, Vol. 33, No. 5, pp. 517-522, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + .OO
Pergamon 0361.9230(93)EOOO7-9
Hypothalamic GABAergic Influences on Treadmill Exercise Responses in Rats J. MICHAEL OVERTON,*’ MICHAEL W. REDDING,? SUSAN L. YANCEYf AND RICHARD W. STREMELS *Department of Nutrition, Food, and Movement Sciences, College of Human Sciences, Florida State University, 203 Montgomery, Tallahassee, FL 32306-4079 fExercise Physiology Program and the SDepartment of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY 40292 Received 16 March 1993; Accepted 4 October 1993 OVERTON, .I. M., M. W. REDDING, S. L. YANCEY AND R. W. STREMEL. Hypothalamic GABAergic influences on treadmill exercise responses in ruts. BRAIN RES BULL 33(5) 517-522,1994.-Microinjection of GABAergic antagonists in the posterior
hypothalamus (PH) produces exercise-like adjustments in cardiovascular function. To test the hypothesis that a hypothalamic GABAergic mechanism within the PH modulates the cardiovascular adjustments to dynamic exercise in conscious animals, Sprague-Dawley rats (n = 10) were instrumented with bilateral guide cannula directed at the PH, an arterial cammla, and Doppler flow probes on the iliac and mesenteric arteries. Saline (100 nl) or the GABA, receptor agonist muscimol(125 ng. 100 nl-‘) was bilaterally injected into the PH during treadmill exercise (20 rn’rnin-I). Microinjection of saline had no effect on mean arterial pressure (MAP), heart rate (HR), mesenteric vascular resistance (MR), or iliac vascular resistance (IR) during exercise. Microinjection of muscimol during exercise produced no significant changes in MAP (mean change ? SE, +0 2 1 mmHg), HR (+ 17 2 12 b.min-‘), or MR (+7 t 13%). However, microinjection of muscimol produced a significant increase in IR during exercise (16 + 6%). In addition, muscimol significantly decreased treadmill run time (saline = 19.6 5 0.4 min; muscimol = 17.8 2 0.6 min) and produced behavioral effects (including mild sedation) that were most evident after exercise. The results of these experiments suggest that while the posterior hypothalamic GABAergic system may modulate iliac blood flow during exercise in rats, this system does not modulate HR and MR responses to dynamic exercise. Muscimol
Posterior hypothalamus
Blood pressure
Regional blood flow
LARGE muscle dynamic exercise requires an increase in cardiac output and a redistribution of blood flow to provide enhanced substrate delivery to contracting muscle. These responses are produced to a large extent by the autonomic nervous system (ANS). The mechanisms by which the ANS is recruited during exercise have been under intensive study for decades. Two primary mechanisms have emerged to explain the regulation of the ANS during exercise (8,12,13). One well-established mechanism is the exercise pressor reflex, which involves activation of nerve endings in muscle that are sensitive to either muscle contraction per se or the metabolic products of contraction. Increased afferent nerve activity is transmitted to the central nervous system (CNS), producing an activation of the ANS. The other primary mechanism thought to produce the ANS adjustments to exercise is central command. Central command has been described as irradiation of impulses from motor centers to cardiovascular centers (5). This would represent a mechanism for coactivation of motor and cardiovascular systems in a manner proportional to muscular activity. While the concept of central command is well accepted, a more precise physiological definition of central command has not been generated.
Central command
The viability of the central command hypothesis would be enhanced by the determination of the precise neuroanatomical substrates responsible for producing adjustments in ANS activity with the initiation of exercise. There are multiple central nervous sites that produce exercise-like cardiovascular and sympathoadrenal responses upon activation (7). Of these, the posterior hypothalamus (PH) has been frequently identified as a potential neuroanatomical substrate of central command (1,2,20,21). This postulate is based on experiments demonstrating that both electrical and pharmacological stimulation of this region produce motor and cardiorespiratory activation (1,2,20). Furthermore, GABAergic stimulation in this region has been shown to elicit exercise-like redistribution of blood flow independent of muscle contraction (21). This point requires some clarification because it is the administration of a GABA receptor antagonist that produces the motor and cardiovascular responses (2,18,20,21). Taken together, these studies suggest that a posterior hypothalamic GABAergic mechanism could be involved in the CNS regulation on the cardiorespiratory responses to exercise. However, direct evidence that this region is actively involved in cardiovascular regulation during exercise is lacking. Therefore, the pur-
’ To whom requests for reprints should be addressed.
517
518
OVE%‘TON ET AL..
pose of this study was to examine whether the GABAergic system in the PH is involved in mediating the hemodynamic adjustments to treadmill exercise in the conscious rat. METHOD
Male Sprague-Dawley rats (Harlan, Indiana~lis, IN, n = 10) weighing 269 + 4 g, were gradually introduced to running on a multilane rodent treadmill with minimal use of negative reinforcement (i.e., electrical shock grid located at the back of the treadmill). Treadmill familiarization consisted of 7- 10 bouts of exercise during a 7 day period. The duration and intensity of exercise were increased until rats were running 30 m.min-’ for the last 5 min of a 30 min session. During this period they were maintained on a standard 1ight:dark cycle (lights on from 0700 to 1900 h) and provided food and water ad lib. Chronic Instrumentation
After an overnight fast, rats were anesthetized with an acepromazineflretamine mixture (1.1/l 10 mg . kg-’ IP). Miniaturized pulsed Doppler crystals (DBF-120A-XS, Crystal-Biotech, Holliston, MA) configured into silastic cuffs were secured around the superior mesenteric and left iliac arteries. The wire leads from each probe were anchored to the psoas muscle with suture and tunneled subcutaneously to the nape of the neck. After closing the midline incision, the flow probe leads were soldered to a miniaturized receptacle (GF-6, Microtech, Boothwyn, PA). A polyethylene catheter (PE-50, Clay Adams, Parsippany, NJ) filled with heparinized saline was inserted into the right carotid artery, secured in position, externalized at the nape of the neck, and sealed. The rats were then mounted in a stereotaxic unit (Kopf Instruments, Tujunga, CA) for bilateral placement of 26 gauge stainless steel guide cannula (C315G, Plastics One, Roanoke, VA) 1.0 mm above the PH through small holes drilled in the skull. The stereotaxic coordinates relative to bregma (caudal -3.8 mm; medial + 0.7 mm; and ventral -7.7 mm) were derived from the atlas of Paxinos and Watson (10). Because of the medial location of the PH, guide cannulae were inserted at angles which were 220” from the perpendicular and parallel to the sagittal sinus. The guide cannulae and the receptacle housing the flow probe leads were secured to the cranium with jeweler’s screws and cranioplastic cement. The guide cannulae were closed via insertion of dummy cannulae. Finally, animals received saline (5 ml SC), topical antibiotics (0.2% nitrofurazone) and intramuscular antibiotics (sulfadimethoxine, 12.5 mg kg-’ IP). The animals were given 5-7 days of recovery during which they performed additional treadmill familiarization. Submaximal Exercise Testing
On the day of an experiment, extension tubing was attached to the arterial catheter for measurement of AP (Gold-Stath~ P23ID transducer). A light, flexible cable was plugged into the receptacle housing the flow probe leads. Thirty-two gauge injection stylets, which extended 1.0 mm beyond the tip of the guide cannulae, were connected to PE-20 tubing and filled with distilled water. The tubing was then backfilled with about 3-5 ~1 of mineral oil and with muscimol or saline. This procedure creates a fluid-filled system, which is essential for accuracy when injecting small volumes. At the same time that extension lines and cables were attached to the rat for me~urement of cardiov~cul~ variables, the injection stylets were inserted through the guide cannulae and secured by screw down collars (C35lc; Plastics One; Roanoke, VA).
Experiments were conducted on a single lane, Plexiglas cnclosed treadmill modified to allow continuous determination of cardiovascular variables during exercise. After attachment of extension lines and cables, the rat was placed on the treadmill for at least 1 h prior to testing to establish resting cardiovascular conditions (i.e., resting heart rate zz 37.5 beats.min- ‘). Graded submaximal exercise was initiated at 10 m .min ’ (0% grade) for 5 min. The speed was then increased to 20 m min ’ and kept there for 10 min. After 4 min of exercise, saline (100 nl) or muscimol (125 ng in 100 nl) was injected bilaterally using a 1 ~1 syringe. The injections were completed within l-2 min. After 10 min of exercise at 20 m’min--‘, the treadmill speed was increased to 30 rn. min ’ for an additional 5 min or until the rat was unable to continue running. Measurement of CardiovascuLar Variables
Changes in regional blood flow distribution were determined using a Doppler flowmeter (545C-4, University of Iowa Bioengineering, Iowa City, IA). The Doppler blood flow measurement system determines flow velocity in kHz of Doppler shift on a continuous basis. Zero flow is determined electronically, and pulsatile flow curves are examined to ensure appropriate signal quality. Changes in Doppler shift have been shown to be linearly related to changes in volume flow measured using electromagnetic flow probes (3). Because blood pressure is obtained concomitantly, an index of regional vascular resistance (mmHgikHz Doppler shift) can be calculated. Therefore, relative changes in regional blood Aows and resistances during exercise can be determined. Blood pressure and flow velocity from the mesenteric and iliac arteries were recorded simultaneously on a chart recorder (Narcotrace SO, Houston, TX). Heart rate was also obtained and recorded continuously via a cardiotach coupler triggered by input from one of the pulsed Doppler signals. Histology
At the conclusion of experiments, the animals were anesthetized (sodium pentobarbital, 50 mg. kg-’ IP) and 100 nl of fast green dye (5% solution) was microinjected to allow determination of the injection sites. Brains were removed, fixed in 10% formalin for 5-7 days, blocked, and sliced in 50 pm coronal sections. Alternate serial sections were stained with cresyl violet for determination of the injection sites by comparison of a magnified image of the section to the atlas of Paxinos and Watson (10). Data Analysis and Statistics
All variables were sampled at 200 Hz by a computer based data acquisition system (DAS-16, Metrabyte, Taunton, MA) for 20 s each minute. The average value for each variable was calculated by the computer and saved for subsequent statistical analysis. The effects of saline and muscimol on MAP, HR, mesenteric blood flow (MBF), mesenteric vascular resistance (MR), iliac blood flow (IBF), and iliac vascular resistance (IR) during exercise at 20 m/min were evaluated using Student’s t-tests. Student’s t-tests were also used to determine if there was a statistically significant difference between the effect on each variable produced by saline and the effect produced by muscimol. The significance level for statistical analyses wasp < 0.05. RESULTS Body weight decreased slightly, but significantly after surgery from 269 rt-.4 g to 260 4 5 g. In spite of this modest reduction in body weight, all animals appeared healthy after surgery and
HYPOTHALAMIC
REGULATION
OF EXERCISE
creased run time on the treadmill (saline = 19.6 + 0.4 min; muscimol = 17.8 + 0.6 min). Bilateral microinjection of muscimol during exercise produced behavioral effects that were generally evident during and after exercise. After muscimol injection, some rats ran lower to the ground and with a slight head tilt. We did not observe any change in breathing patterns during exercise. After exercise, most rats remained motionless on the treadmill. Upon removal from the treadmill, they were sedate and occasionally did not demonstrate a righting reflex. The behavior of rats treated with muscimol returned to normal after 2-3 h. Microinjection sites marked by dye injection are illustrated in Fig. 2. Injections were located just lateral to the third ventricle and medial to the mammillothalamic tract in an area between -3.8 mm and -4.3 mm rostra1 to bregma.
were able to perform well on the treadmill during the exercise tests. Due to probe malfunction, one iliac flow signal was not available for measurement and analysis. Baseline Data and Response to Exercise (Table 1, Fig. 1) There were no significant differences in baseline MAP, HR, MBF, MR, IBF, and IR between the 2 days of treadmill testing (Table 1). The response to exercise and the protocol for this study are illustrated in Fig. 1. The initiation of mild exercise (10 rn. mini’) produced an immediate increase in IBF, MAP, and HR and an immediate decrease in MBF. The initial changes in HR, MBF, and IBF represent somewhat of an overshoot response because after 5 min of exercise at 10 m.min-’ these variables have reached a steady-state level closer to preexercise values. After 4 min of exercise at 20 m min-‘, the microinjections were given and the cardiovascular values for exercise at 20 rn. min-’ and when possible, at 30 m.min-’ were observed. Many of the rats that received muscimol (n = 6) could not complete 5 min of exercise at 30 m.min-‘. Therefore, data for exercise at 30 m’min-’ was not analyzed. The absolute values for MAP, HR, MBF, MR, IBF, and IR during steady-state exercise at 20 m.min-’ prior to microinjection for the two exercise trials are given in Table 1. There were no significant differences in these exercise values between the two trials prior to administration of saline or muscimol. Effect of Treatments on Exercise Hemodynamics
DISCUSSION
There are two primary findings from this study. First, microinjection of the GABA, receptor agonist muscimol into the region of the PH of the rat during exercise had no significant effect on blood pressure, heart rate, or mesenteric vascular resistance. Secondly, administration of muscimol into the PH during exercise produced significant decreases in iliac blood flow and significant increases in iliac vascular resistance. Taken together, the findings suggest that posterior hypothalamic GABAergic receptors are not involved in the elevation of cardiac output and redistribution of blood flow during steady-state exercise. However, the results do suggest that posterior hypothalamic GABAergic receptors may modulate blood flow to active skeletal muscle during steady-state exercise. Several indirect lines of evidence support the possibility that the hypothalamus is involved in mediating the ANS adjustments to exercise. Electrical stimulation of the PH produces running activity, increases in blood pressure, and increases in respiratory activity in the unanesthetized, decorticate cat (1). Studies with paralyzed cats demonstrated that the cardiovascular and respiratory activation were not dependent on feedback from exercising muscles. Thus, this region represents a possible neural substrate for central command. Several studies have since demonstrated that specific activation of cell bodies within this region by administration of GABAergic antagonists (picrotoxin or bicuculline methiodide) produce increases in motor activity, HR, blood pressure, and respiratory activity (2,18,20,21). Furthermore, GABAergic activation in this region produces exercise-like redistri-
(Table 2)
During exercise at 20 m *mini’, bilateral administration of saline (100 nl) produced no significant change in MAP, HR, MBF, MR, IBF, or IR measured during the last minute of exercise at 20 m/min. Compared to the effect of saline, bilateral administration of muscimol(125 ng) produced no significant effect on MAP, HR, MBF, and MR during exercise at 20 m.min~‘. However, muscimol treatment did produce a significant decrease (-12 ? 4%) in IBF, and a significant increase (16 ? 6%) in IR during exercise. Behavioral
Observations
and Histology (Fig. 2)
Bilateral microinjection of saline during exercise produced no observable behavioral effects. As mentioned previously, animals receiving bilateral administration of muscimol frequently did not complete the exercise protocol. Thus, muscimol significantly de-
TABLE CARDIOVASCULAR
519
RESPONSES
1
VALUES DURING REST AND EXERCISE FOR SALINE AND MUSCIMOL TRIALS Baseline Saline
MAP (mmHg) HR
@.min-‘)
MBF &Hz) MR (mmHg kH..‘) IBF @Hz) IR (mmHg.IrHz-‘)
115 331 5.3 24.6 2.6 47.7
23 k8 + 0.7 -t 2.5 2 0.2 2 4.4
Exercise Muscimol
116 337 5.1 26.3 2.6 47.4
+ 2 A 10 k 0.6 2 3.3 2 0.2 + 4.3
Saline
135 460 3.6 44.8 9.1 15.3
k 4 k 11 + 0.5 + 6.0 2 0.6 k 0.9
Muscimol
132 462 3.3 45.3 8.8 15.8
2 2 t 13 2 0.3 * 5.4 + 0.6 2 1.2
Values are mean ? SE for 10 rats (n = 9 for IBF and IR). Baseline values were obtained while animals were resting quietly on the treadmill prior to exercise. Exercise values were obtained after 4 mm of exercise at 20 m’rnin-’ (prior to microinjection). MAP, mean arterial pressure; HR, heart rate; MBF, mesenteric blood flow; MR, mesenteric resistance; IBF, iliac blood flow; IR, iliac resistance. There were no significant differences between the saline and muscimol trials for any variable at baseline or during exercise prior to microinjection.
520
OVERTON ET AL.
bution of blood flow without concordant muscle contraction (20). Because antagonist administration produces the excitatory response (disinhibition), it was necessary to use the GABA agonist muscimol in these experiments to determine if this GAEJAergic mechanism controlled cardiovascular adjustments during dynamic exercise. Direct examination of central neural regulation of cardiovascular function during exercise requires the use of experimental approaches such as lesion or microinjection in discrete CNS sites in animals that have been chronically instrumented for assessment of ANS function. Few studies have utilized these experimental paradigms. In experiments with small sample sizes, hypothalamic lesions have been shown to attenuate the cardiovascular responses to treadmill exercise in beagles (14) and to static exercise in baboons (4). However, it has recently been reported that bilateral electrolytic lesions of PH do not significantly alter the blood pressure, heart rate, or regional blood flow responses to treadmill exercise in a group of nine beagles (9). This provides compelling evidence that neural pathways passing through or originating in the area of the PH are not requisite for generation of the hemodynamic responses to exercise in the beagle. Furthermore, the primary finding from the present study, i.e., that muscimol administration in the PH does not influence the blood pressure, heart rate, and mesenteric blood flow responses to exercise, supports this contention.
5
TABLE 2 EFFECT OF BILATERAL POSTERIOR HYPOTHALAMIC ADMINISTRATION OF SALINE (100 nl) AND MUSCIMOL (125 “g IN 100 nl SALINE) ON CARDIOVASCULAR VALUES DURING EXERCISE
Saline
MAP (mmHg) HR (beats’min-‘) MBF (%) MR (%) IBF (%) IR (%)
-22
Muscimol 1
ui
127
17-c
13 2 7
1%
7
--9 -t 6 0+4
7-t -122
13
o?z4
16 t_
I-
10 min -1
7
Iliac Blood Flow (kHz)
1
80
500 Heart Rate (blmin) 3ooJ----yJl
1
6’
Hypothalamic activation has frequently been shown to produce increases in blood flow to skeletal muscle (20,24,25). The increased blood flow is due to an initial withdrawal of sympa-
0 I--
180
4*
Values (mean + SE) represent changes from exercise values shown in Table 1 for 10 rats (n = 9 for IBF and IR). The exercise values were obtained during the 10th min of exercise at 20 m. min-‘, MAP, mean arterial pressure; HR, heart rate; MBF, mesenteric blood flow; MR, mesenteric resistance; IBF, iliac blood flow;IR, iliac resistance. * Indicates significant difference at p < 0.05.
Mesenteric Blood Flow IkHz)
Arterial Pressure (mm Hg)
1 12
2
3
4
5
8
FIG. 1. Example of the effects of exercise and posterior hypothalamic muscimol administration on mesenteric blood flow, iliac blood flow, arterial blood pressure, and heart rate. At event label 1, exercise was initiated at 10 m’min-‘, at 2 the at 3 and 4 muscimol was injected into the PH, at 5 the speed was increased to 30 speed was increased to 20 m.miC’, m.min-‘. and at 6 the exercise bout was ended.
HYPOTHALAMIC
REGULATION
521
OF EXERCISE RESPONSES
3.80 mm
mm
mm
FIG. 2. Diagrammatic representation of the diencephalic sites of microinjection (0) are unilaterally represented for labeling. Unilateral sites for all 10 rats studied are depicted. 3V = 3rd ventricle, PH = posterior hypothalamus, mt = mammillothalamic tract, f = fornix, and ARH = arcuate nucleus of the hypothalamus. Projections are referenced from bregma.
thetic tone and to increases in plasma epinephrine levels (25). Interestingly, microinjection of the GABA antagonist bicuculline methio~de into the PH produces micro~irculatory vasodilation mediated by sympathetic withdrawal in striated muscle (18). In this study, hypothalamic microinjection of the GABA agonist muscimol was associated with a small (-12 rtr 4%), but significant decrease in iliac blood flow. We do not know the mechanism responsible for this effect of muscimol. Tbe reduction in iliac blood flow may be explained by either a reduction in the degree of sympathetic withdrawal to the vasculature, or by a reduction in epinephrine release after muscimol injection. Another potential explanation for the change in iliac blood flow is related to the observation of subtle effects of muscimol on running behavior. Metabolic rate was not measured during these studies. All the rats ran very well at 20 ml’min; however, it is conceivable that the changes in iliac blood flow were related to changes in metabolic rate or motor unit recruitment patterns. It should be recognized that the increases in iliac vascular resistance could represent a reduced blood flow to tissues other than muscle. The methodolo~ used for these studies does not allow a differentiation between blood flow going to muscle and Aow to skin or other tissues. We recognize that there are some potential limitations of this study that preclude a definitive conclusion regarding the role of the
PH in regulating the cardiovascular responses to exercise. One of the most obvious is that our muscimol injections may have been generally ineffective because an iMpprop~ate concentration of drug was used or because the appropriate receptor population was not affected. Regarding the former, the dose used was about 10 times higher than that which has been shown to greatly reduce HR response to air jet stress (6). We, therefore, believe that this dose should have been adequate to identify an effect during exercise. As indicated in Fig. 2, the injection sites were spread fairly widely through the caudal hypothalamus. While we did not routinely determine that each site was involved in cardiovascular regulation, we did administer bicuculline (125 @side) in one conscious rat and observed vigorous cardiovascular and behavioral responses. In addition, we gave fairly si~ifi~t volumes (100 nl) in an attempt to have a substantial spread of the injection. Yet, we did not observe marked alterations in exercise MAP, HR, or MBF in any of the animals studied. Because of the possibility of muscimol diffusion to receptor populations outside of the PH due to the use of 100 nl injection volumes (19), it is possible that the observed reduction in iliac blood flow was not due to a localized action in the PH. The neural substrates responsible for central command remain unidentified. It should be noted that the findings from this study do not necessarily suggest that the GABAergic hypothalamic system is not involved in producing exercise-induced changes in
522
OVERTON E’l’ Al.
ANS activity. Rather, the results indicate that this system is not requisite for the maintenance of cardiovascular responses during exercise. In other words, there may be multiple central neural pathways involved in producing the autonomic adjustments to exercise so that it could be very difficult to isolate and identify these pathways (8,9). During steady-state submaximal exercise, it is likely that both feedback from exercising muscle and central feed-forward mechanisms are operative. Furthermore, the neural substrates that produce central command may not be independent of those that produce the autonomic adjustments associated with feedback from contracting muscle. For example, hindlimb contraction in the anesthetized animal has been shown to produce increases in extracellular single-unit activity in the PH (22). In the conscious animal, one approach to study responses that might be primarily mediated by central command is to examine cardiovascular control at the onset of exercise. In the rat, exercise produces an immediate and pronounced increase in blood pressure, heart rate, mesenteric vascular resistance, and iliac blood flow (see Fig. 1). Therefore, we also performed preliminary experiments involving microinjection of muscimol prior to exercise to examine if GABAergic receptors in the PH influenced the marked cardiovascular responses observed at the onset of treadmill exercise performed by rats. However, bilateral muscimol injection prior to exercise rendered some animals unable to perform exercise adequately to study these responses. There has been some confusion in the literature regarding the precise neuroanatomical location of the PH due to major differences in the atlases of Pelligrino et al. (11) and Paxinos and
Watson (10). This issue has recently been discussed in detail (1.5,16). The atlas of Pelligrino et al. incorrectly extends the anterior border of the posterior hypothalamic nucleus well into the tuberal hypothalamus. Thus, some reports that have attributed cardioexcitatory responses to the PH actually represent actions of the dorsomedial nucleus of the hypothalamus (6,23,24). Furthermore, it has been suggested that some earlier studies examining the PH as a potential region of central command were also in the area of the dorsomedial nucleus of the hypothalamus (17). The injection sites in the present study were compared to the atlas of Paxinos and Watson (10) and were clearly in the region of the PH. Additional studies in other hypothalamic sites, including the dorsomedial nucleus, may lead to a more precise understanding of the central neural substrates responsible for producing the ANS adjustments during dynamic exercise. In conclusion, this study has demonstrated that microinjection of muscimol in the PH during exercise increases iliac vascular resistance and impairs exercise performance. However, the findings indicate that hypothalamic microinjection of the GABAergic receptor agonist muscimol does not significantly influence blood pressure, heart rate, or mesenteric vascular resistance during treadmill exercise in the rat. The precise neural substrates responsible for eliciting the Ah3 adjustments to exercise remain undetermined. ACKNOWLEDGEMENTS
These studies were funded by grants from the American sociation, KY Affiliate, and the University of Louisville School.
Heart AsGraduate
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Pergamon 0361.9230(93)EOOO7-9
Hypothalamic GABAergic Influences on Treadmill Exercise Responses in Rats J. MICHAEL OVERTON,*’ MICHAEL W. REDDING,? SUSAN L. YANCEYf AND RICHARD W. STREMELS *Department of Nutrition, Food, and Movement Sciences, College of Human Sciences, Florida State University, 203 Montgomery, Tallahassee, FL 32306-4079 fExercise Physiology Program and the SDepartment of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY 40292 Received 16 March 1993; Accepted 4 October 1993 OVERTON, .I. M., M. W. REDDING, S. L. YANCEY AND R. W. STREMEL. Hypothalamic GABAergic influences on treadmill exercise responses in ruts. BRAIN RES BULL 33(5) 517-522,1994.-Microinjection of GABAergic antagonists in the posterior
hypothalamus (PH) produces exercise-like adjustments in cardiovascular function. To test the hypothesis that a hypothalamic GABAergic mechanism within the PH modulates the cardiovascular adjustments to dynamic exercise in conscious animals, Sprague-Dawley rats (n = 10) were instrumented with bilateral guide cannula directed at the PH, an arterial cammla, and Doppler flow probes on the iliac and mesenteric arteries. Saline (100 nl) or the GABA, receptor agonist muscimol(125 ng. 100 nl-‘) was bilaterally injected into the PH during treadmill exercise (20 rn’rnin-I). Microinjection of saline had no effect on mean arterial pressure (MAP), heart rate (HR), mesenteric vascular resistance (MR), or iliac vascular resistance (IR) during exercise. Microinjection of muscimol during exercise produced no significant changes in MAP (mean change ? SE, +0 2 1 mmHg), HR (+ 17 2 12 b.min-‘), or MR (+7 t 13%). However, microinjection of muscimol produced a significant increase in IR during exercise (16 + 6%). In addition, muscimol significantly decreased treadmill run time (saline = 19.6 5 0.4 min; muscimol = 17.8 2 0.6 min) and produced behavioral effects (including mild sedation) that were most evident after exercise. The results of these experiments suggest that while the posterior hypothalamic GABAergic system may modulate iliac blood flow during exercise in rats, this system does not modulate HR and MR responses to dynamic exercise. Muscimol
Posterior hypothalamus
Blood pressure
Regional blood flow
LARGE muscle dynamic exercise requires an increase in cardiac output and a redistribution of blood flow to provide enhanced substrate delivery to contracting muscle. These responses are produced to a large extent by the autonomic nervous system (ANS). The mechanisms by which the ANS is recruited during exercise have been under intensive study for decades. Two primary mechanisms have emerged to explain the regulation of the ANS during exercise (8,12,13). One well-established mechanism is the exercise pressor reflex, which involves activation of nerve endings in muscle that are sensitive to either muscle contraction per se or the metabolic products of contraction. Increased afferent nerve activity is transmitted to the central nervous system (CNS), producing an activation of the ANS. The other primary mechanism thought to produce the ANS adjustments to exercise is central command. Central command has been described as irradiation of impulses from motor centers to cardiovascular centers (5). This would represent a mechanism for coactivation of motor and cardiovascular systems in a manner proportional to muscular activity. While the concept of central command is well accepted, a more precise physiological definition of central command has not been generated.
Central command
The viability of the central command hypothesis would be enhanced by the determination of the precise neuroanatomical substrates responsible for producing adjustments in ANS activity with the initiation of exercise. There are multiple central nervous sites that produce exercise-like cardiovascular and sympathoadrenal responses upon activation (7). Of these, the posterior hypothalamus (PH) has been frequently identified as a potential neuroanatomical substrate of central command (1,2,20,21). This postulate is based on experiments demonstrating that both electrical and pharmacological stimulation of this region produce motor and cardiorespiratory activation (1,2,20). Furthermore, GABAergic stimulation in this region has been shown to elicit exercise-like redistribution of blood flow independent of muscle contraction (21). This point requires some clarification because it is the administration of a GABA receptor antagonist that produces the motor and cardiovascular responses (2,18,20,21). Taken together, these studies suggest that a posterior hypothalamic GABAergic mechanism could be involved in the CNS regulation on the cardiorespiratory responses to exercise. However, direct evidence that this region is actively involved in cardiovascular regulation during exercise is lacking. Therefore, the pur-
’ To whom requests for reprints should be addressed.
517
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OVE%‘TON ET AL..
pose of this study was to examine whether the GABAergic system in the PH is involved in mediating the hemodynamic adjustments to treadmill exercise in the conscious rat. METHOD
Male Sprague-Dawley rats (Harlan, Indiana~lis, IN, n = 10) weighing 269 + 4 g, were gradually introduced to running on a multilane rodent treadmill with minimal use of negative reinforcement (i.e., electrical shock grid located at the back of the treadmill). Treadmill familiarization consisted of 7- 10 bouts of exercise during a 7 day period. The duration and intensity of exercise were increased until rats were running 30 m.min-’ for the last 5 min of a 30 min session. During this period they were maintained on a standard 1ight:dark cycle (lights on from 0700 to 1900 h) and provided food and water ad lib. Chronic Instrumentation
After an overnight fast, rats were anesthetized with an acepromazineflretamine mixture (1.1/l 10 mg . kg-’ IP). Miniaturized pulsed Doppler crystals (DBF-120A-XS, Crystal-Biotech, Holliston, MA) configured into silastic cuffs were secured around the superior mesenteric and left iliac arteries. The wire leads from each probe were anchored to the psoas muscle with suture and tunneled subcutaneously to the nape of the neck. After closing the midline incision, the flow probe leads were soldered to a miniaturized receptacle (GF-6, Microtech, Boothwyn, PA). A polyethylene catheter (PE-50, Clay Adams, Parsippany, NJ) filled with heparinized saline was inserted into the right carotid artery, secured in position, externalized at the nape of the neck, and sealed. The rats were then mounted in a stereotaxic unit (Kopf Instruments, Tujunga, CA) for bilateral placement of 26 gauge stainless steel guide cannula (C315G, Plastics One, Roanoke, VA) 1.0 mm above the PH through small holes drilled in the skull. The stereotaxic coordinates relative to bregma (caudal -3.8 mm; medial + 0.7 mm; and ventral -7.7 mm) were derived from the atlas of Paxinos and Watson (10). Because of the medial location of the PH, guide cannulae were inserted at angles which were 220” from the perpendicular and parallel to the sagittal sinus. The guide cannulae and the receptacle housing the flow probe leads were secured to the cranium with jeweler’s screws and cranioplastic cement. The guide cannulae were closed via insertion of dummy cannulae. Finally, animals received saline (5 ml SC), topical antibiotics (0.2% nitrofurazone) and intramuscular antibiotics (sulfadimethoxine, 12.5 mg kg-’ IP). The animals were given 5-7 days of recovery during which they performed additional treadmill familiarization. Submaximal Exercise Testing
On the day of an experiment, extension tubing was attached to the arterial catheter for measurement of AP (Gold-Stath~ P23ID transducer). A light, flexible cable was plugged into the receptacle housing the flow probe leads. Thirty-two gauge injection stylets, which extended 1.0 mm beyond the tip of the guide cannulae, were connected to PE-20 tubing and filled with distilled water. The tubing was then backfilled with about 3-5 ~1 of mineral oil and with muscimol or saline. This procedure creates a fluid-filled system, which is essential for accuracy when injecting small volumes. At the same time that extension lines and cables were attached to the rat for me~urement of cardiov~cul~ variables, the injection stylets were inserted through the guide cannulae and secured by screw down collars (C35lc; Plastics One; Roanoke, VA).
Experiments were conducted on a single lane, Plexiglas cnclosed treadmill modified to allow continuous determination of cardiovascular variables during exercise. After attachment of extension lines and cables, the rat was placed on the treadmill for at least 1 h prior to testing to establish resting cardiovascular conditions (i.e., resting heart rate zz 37.5 beats.min- ‘). Graded submaximal exercise was initiated at 10 m .min ’ (0% grade) for 5 min. The speed was then increased to 20 m min ’ and kept there for 10 min. After 4 min of exercise, saline (100 nl) or muscimol (125 ng in 100 nl) was injected bilaterally using a 1 ~1 syringe. The injections were completed within l-2 min. After 10 min of exercise at 20 m’min--‘, the treadmill speed was increased to 30 rn. min ’ for an additional 5 min or until the rat was unable to continue running. Measurement of CardiovascuLar Variables
Changes in regional blood flow distribution were determined using a Doppler flowmeter (545C-4, University of Iowa Bioengineering, Iowa City, IA). The Doppler blood flow measurement system determines flow velocity in kHz of Doppler shift on a continuous basis. Zero flow is determined electronically, and pulsatile flow curves are examined to ensure appropriate signal quality. Changes in Doppler shift have been shown to be linearly related to changes in volume flow measured using electromagnetic flow probes (3). Because blood pressure is obtained concomitantly, an index of regional vascular resistance (mmHgikHz Doppler shift) can be calculated. Therefore, relative changes in regional blood Aows and resistances during exercise can be determined. Blood pressure and flow velocity from the mesenteric and iliac arteries were recorded simultaneously on a chart recorder (Narcotrace SO, Houston, TX). Heart rate was also obtained and recorded continuously via a cardiotach coupler triggered by input from one of the pulsed Doppler signals. Histology
At the conclusion of experiments, the animals were anesthetized (sodium pentobarbital, 50 mg. kg-’ IP) and 100 nl of fast green dye (5% solution) was microinjected to allow determination of the injection sites. Brains were removed, fixed in 10% formalin for 5-7 days, blocked, and sliced in 50 pm coronal sections. Alternate serial sections were stained with cresyl violet for determination of the injection sites by comparison of a magnified image of the section to the atlas of Paxinos and Watson (10). Data Analysis and Statistics
All variables were sampled at 200 Hz by a computer based data acquisition system (DAS-16, Metrabyte, Taunton, MA) for 20 s each minute. The average value for each variable was calculated by the computer and saved for subsequent statistical analysis. The effects of saline and muscimol on MAP, HR, mesenteric blood flow (MBF), mesenteric vascular resistance (MR), iliac blood flow (IBF), and iliac vascular resistance (IR) during exercise at 20 m/min were evaluated using Student’s t-tests. Student’s t-tests were also used to determine if there was a statistically significant difference between the effect on each variable produced by saline and the effect produced by muscimol. The significance level for statistical analyses wasp < 0.05. RESULTS Body weight decreased slightly, but significantly after surgery from 269 rt-.4 g to 260 4 5 g. In spite of this modest reduction in body weight, all animals appeared healthy after surgery and
HYPOTHALAMIC
REGULATION
OF EXERCISE
creased run time on the treadmill (saline = 19.6 + 0.4 min; muscimol = 17.8 + 0.6 min). Bilateral microinjection of muscimol during exercise produced behavioral effects that were generally evident during and after exercise. After muscimol injection, some rats ran lower to the ground and with a slight head tilt. We did not observe any change in breathing patterns during exercise. After exercise, most rats remained motionless on the treadmill. Upon removal from the treadmill, they were sedate and occasionally did not demonstrate a righting reflex. The behavior of rats treated with muscimol returned to normal after 2-3 h. Microinjection sites marked by dye injection are illustrated in Fig. 2. Injections were located just lateral to the third ventricle and medial to the mammillothalamic tract in an area between -3.8 mm and -4.3 mm rostra1 to bregma.
were able to perform well on the treadmill during the exercise tests. Due to probe malfunction, one iliac flow signal was not available for measurement and analysis. Baseline Data and Response to Exercise (Table 1, Fig. 1) There were no significant differences in baseline MAP, HR, MBF, MR, IBF, and IR between the 2 days of treadmill testing (Table 1). The response to exercise and the protocol for this study are illustrated in Fig. 1. The initiation of mild exercise (10 rn. mini’) produced an immediate increase in IBF, MAP, and HR and an immediate decrease in MBF. The initial changes in HR, MBF, and IBF represent somewhat of an overshoot response because after 5 min of exercise at 10 m.min-’ these variables have reached a steady-state level closer to preexercise values. After 4 min of exercise at 20 m min-‘, the microinjections were given and the cardiovascular values for exercise at 20 rn. min-’ and when possible, at 30 m.min-’ were observed. Many of the rats that received muscimol (n = 6) could not complete 5 min of exercise at 30 m.min-‘. Therefore, data for exercise at 30 m’min-’ was not analyzed. The absolute values for MAP, HR, MBF, MR, IBF, and IR during steady-state exercise at 20 m.min-’ prior to microinjection for the two exercise trials are given in Table 1. There were no significant differences in these exercise values between the two trials prior to administration of saline or muscimol. Effect of Treatments on Exercise Hemodynamics
DISCUSSION
There are two primary findings from this study. First, microinjection of the GABA, receptor agonist muscimol into the region of the PH of the rat during exercise had no significant effect on blood pressure, heart rate, or mesenteric vascular resistance. Secondly, administration of muscimol into the PH during exercise produced significant decreases in iliac blood flow and significant increases in iliac vascular resistance. Taken together, the findings suggest that posterior hypothalamic GABAergic receptors are not involved in the elevation of cardiac output and redistribution of blood flow during steady-state exercise. However, the results do suggest that posterior hypothalamic GABAergic receptors may modulate blood flow to active skeletal muscle during steady-state exercise. Several indirect lines of evidence support the possibility that the hypothalamus is involved in mediating the ANS adjustments to exercise. Electrical stimulation of the PH produces running activity, increases in blood pressure, and increases in respiratory activity in the unanesthetized, decorticate cat (1). Studies with paralyzed cats demonstrated that the cardiovascular and respiratory activation were not dependent on feedback from exercising muscles. Thus, this region represents a possible neural substrate for central command. Several studies have since demonstrated that specific activation of cell bodies within this region by administration of GABAergic antagonists (picrotoxin or bicuculline methiodide) produce increases in motor activity, HR, blood pressure, and respiratory activity (2,18,20,21). Furthermore, GABAergic activation in this region produces exercise-like redistri-
(Table 2)
During exercise at 20 m *mini’, bilateral administration of saline (100 nl) produced no significant change in MAP, HR, MBF, MR, IBF, or IR measured during the last minute of exercise at 20 m/min. Compared to the effect of saline, bilateral administration of muscimol(125 ng) produced no significant effect on MAP, HR, MBF, and MR during exercise at 20 m.min~‘. However, muscimol treatment did produce a significant decrease (-12 ? 4%) in IBF, and a significant increase (16 ? 6%) in IR during exercise. Behavioral
Observations
and Histology (Fig. 2)
Bilateral microinjection of saline during exercise produced no observable behavioral effects. As mentioned previously, animals receiving bilateral administration of muscimol frequently did not complete the exercise protocol. Thus, muscimol significantly de-
TABLE CARDIOVASCULAR
519
RESPONSES
1
VALUES DURING REST AND EXERCISE FOR SALINE AND MUSCIMOL TRIALS Baseline Saline
MAP (mmHg) HR
@.min-‘)
MBF &Hz) MR (mmHg kH..‘) IBF @Hz) IR (mmHg.IrHz-‘)
115 331 5.3 24.6 2.6 47.7
23 k8 + 0.7 -t 2.5 2 0.2 2 4.4
Exercise Muscimol
116 337 5.1 26.3 2.6 47.4
+ 2 A 10 k 0.6 2 3.3 2 0.2 + 4.3
Saline
135 460 3.6 44.8 9.1 15.3
k 4 k 11 + 0.5 + 6.0 2 0.6 k 0.9
Muscimol
132 462 3.3 45.3 8.8 15.8
2 2 t 13 2 0.3 * 5.4 + 0.6 2 1.2
Values are mean ? SE for 10 rats (n = 9 for IBF and IR). Baseline values were obtained while animals were resting quietly on the treadmill prior to exercise. Exercise values were obtained after 4 mm of exercise at 20 m’rnin-’ (prior to microinjection). MAP, mean arterial pressure; HR, heart rate; MBF, mesenteric blood flow; MR, mesenteric resistance; IBF, iliac blood flow; IR, iliac resistance. There were no significant differences between the saline and muscimol trials for any variable at baseline or during exercise prior to microinjection.
520
OVERTON ET AL.
bution of blood flow without concordant muscle contraction (20). Because antagonist administration produces the excitatory response (disinhibition), it was necessary to use the GABA agonist muscimol in these experiments to determine if this GAEJAergic mechanism controlled cardiovascular adjustments during dynamic exercise. Direct examination of central neural regulation of cardiovascular function during exercise requires the use of experimental approaches such as lesion or microinjection in discrete CNS sites in animals that have been chronically instrumented for assessment of ANS function. Few studies have utilized these experimental paradigms. In experiments with small sample sizes, hypothalamic lesions have been shown to attenuate the cardiovascular responses to treadmill exercise in beagles (14) and to static exercise in baboons (4). However, it has recently been reported that bilateral electrolytic lesions of PH do not significantly alter the blood pressure, heart rate, or regional blood flow responses to treadmill exercise in a group of nine beagles (9). This provides compelling evidence that neural pathways passing through or originating in the area of the PH are not requisite for generation of the hemodynamic responses to exercise in the beagle. Furthermore, the primary finding from the present study, i.e., that muscimol administration in the PH does not influence the blood pressure, heart rate, and mesenteric blood flow responses to exercise, supports this contention.
5
TABLE 2 EFFECT OF BILATERAL POSTERIOR HYPOTHALAMIC ADMINISTRATION OF SALINE (100 nl) AND MUSCIMOL (125 “g IN 100 nl SALINE) ON CARDIOVASCULAR VALUES DURING EXERCISE
Saline
MAP (mmHg) HR (beats’min-‘) MBF (%) MR (%) IBF (%) IR (%)
-22
Muscimol 1
ui
127
17-c
13 2 7
1%
7
--9 -t 6 0+4
7-t -122
13
o?z4
16 t_
I-
10 min -1
7
Iliac Blood Flow (kHz)
1
80
500 Heart Rate (blmin) 3ooJ----yJl
1
6’
Hypothalamic activation has frequently been shown to produce increases in blood flow to skeletal muscle (20,24,25). The increased blood flow is due to an initial withdrawal of sympa-
0 I--
180
4*
Values (mean + SE) represent changes from exercise values shown in Table 1 for 10 rats (n = 9 for IBF and IR). The exercise values were obtained during the 10th min of exercise at 20 m. min-‘, MAP, mean arterial pressure; HR, heart rate; MBF, mesenteric blood flow; MR, mesenteric resistance; IBF, iliac blood flow;IR, iliac resistance. * Indicates significant difference at p < 0.05.
Mesenteric Blood Flow IkHz)
Arterial Pressure (mm Hg)
1 12
2
3
4
5
8
FIG. 1. Example of the effects of exercise and posterior hypothalamic muscimol administration on mesenteric blood flow, iliac blood flow, arterial blood pressure, and heart rate. At event label 1, exercise was initiated at 10 m’min-‘, at 2 the at 3 and 4 muscimol was injected into the PH, at 5 the speed was increased to 30 speed was increased to 20 m.miC’, m.min-‘. and at 6 the exercise bout was ended.
HYPOTHALAMIC
REGULATION
521
OF EXERCISE RESPONSES
3.80 mm
mm
mm
FIG. 2. Diagrammatic representation of the diencephalic sites of microinjection (0) are unilaterally represented for labeling. Unilateral sites for all 10 rats studied are depicted. 3V = 3rd ventricle, PH = posterior hypothalamus, mt = mammillothalamic tract, f = fornix, and ARH = arcuate nucleus of the hypothalamus. Projections are referenced from bregma.
thetic tone and to increases in plasma epinephrine levels (25). Interestingly, microinjection of the GABA antagonist bicuculline methio~de into the PH produces micro~irculatory vasodilation mediated by sympathetic withdrawal in striated muscle (18). In this study, hypothalamic microinjection of the GABA agonist muscimol was associated with a small (-12 rtr 4%), but significant decrease in iliac blood flow. We do not know the mechanism responsible for this effect of muscimol. Tbe reduction in iliac blood flow may be explained by either a reduction in the degree of sympathetic withdrawal to the vasculature, or by a reduction in epinephrine release after muscimol injection. Another potential explanation for the change in iliac blood flow is related to the observation of subtle effects of muscimol on running behavior. Metabolic rate was not measured during these studies. All the rats ran very well at 20 ml’min; however, it is conceivable that the changes in iliac blood flow were related to changes in metabolic rate or motor unit recruitment patterns. It should be recognized that the increases in iliac vascular resistance could represent a reduced blood flow to tissues other than muscle. The methodolo~ used for these studies does not allow a differentiation between blood flow going to muscle and Aow to skin or other tissues. We recognize that there are some potential limitations of this study that preclude a definitive conclusion regarding the role of the
PH in regulating the cardiovascular responses to exercise. One of the most obvious is that our muscimol injections may have been generally ineffective because an iMpprop~ate concentration of drug was used or because the appropriate receptor population was not affected. Regarding the former, the dose used was about 10 times higher than that which has been shown to greatly reduce HR response to air jet stress (6). We, therefore, believe that this dose should have been adequate to identify an effect during exercise. As indicated in Fig. 2, the injection sites were spread fairly widely through the caudal hypothalamus. While we did not routinely determine that each site was involved in cardiovascular regulation, we did administer bicuculline (125 @side) in one conscious rat and observed vigorous cardiovascular and behavioral responses. In addition, we gave fairly si~ifi~t volumes (100 nl) in an attempt to have a substantial spread of the injection. Yet, we did not observe marked alterations in exercise MAP, HR, or MBF in any of the animals studied. Because of the possibility of muscimol diffusion to receptor populations outside of the PH due to the use of 100 nl injection volumes (19), it is possible that the observed reduction in iliac blood flow was not due to a localized action in the PH. The neural substrates responsible for central command remain unidentified. It should be noted that the findings from this study do not necessarily suggest that the GABAergic hypothalamic system is not involved in producing exercise-induced changes in
522
OVERTON E’l’ Al.
ANS activity. Rather, the results indicate that this system is not requisite for the maintenance of cardiovascular responses during exercise. In other words, there may be multiple central neural pathways involved in producing the autonomic adjustments to exercise so that it could be very difficult to isolate and identify these pathways (8,9). During steady-state submaximal exercise, it is likely that both feedback from exercising muscle and central feed-forward mechanisms are operative. Furthermore, the neural substrates that produce central command may not be independent of those that produce the autonomic adjustments associated with feedback from contracting muscle. For example, hindlimb contraction in the anesthetized animal has been shown to produce increases in extracellular single-unit activity in the PH (22). In the conscious animal, one approach to study responses that might be primarily mediated by central command is to examine cardiovascular control at the onset of exercise. In the rat, exercise produces an immediate and pronounced increase in blood pressure, heart rate, mesenteric vascular resistance, and iliac blood flow (see Fig. 1). Therefore, we also performed preliminary experiments involving microinjection of muscimol prior to exercise to examine if GABAergic receptors in the PH influenced the marked cardiovascular responses observed at the onset of treadmill exercise performed by rats. However, bilateral muscimol injection prior to exercise rendered some animals unable to perform exercise adequately to study these responses. There has been some confusion in the literature regarding the precise neuroanatomical location of the PH due to major differences in the atlases of Pelligrino et al. (11) and Paxinos and
Watson (10). This issue has recently been discussed in detail (1.5,16). The atlas of Pelligrino et al. incorrectly extends the anterior border of the posterior hypothalamic nucleus well into the tuberal hypothalamus. Thus, some reports that have attributed cardioexcitatory responses to the PH actually represent actions of the dorsomedial nucleus of the hypothalamus (6,23,24). Furthermore, it has been suggested that some earlier studies examining the PH as a potential region of central command were also in the area of the dorsomedial nucleus of the hypothalamus (17). The injection sites in the present study were compared to the atlas of Paxinos and Watson (10) and were clearly in the region of the PH. Additional studies in other hypothalamic sites, including the dorsomedial nucleus, may lead to a more precise understanding of the central neural substrates responsible for producing the ANS adjustments during dynamic exercise. In conclusion, this study has demonstrated that microinjection of muscimol in the PH during exercise increases iliac vascular resistance and impairs exercise performance. However, the findings indicate that hypothalamic microinjection of the GABAergic receptor agonist muscimol does not significantly influence blood pressure, heart rate, or mesenteric vascular resistance during treadmill exercise in the rat. The precise neural substrates responsible for eliciting the Ah3 adjustments to exercise remain undetermined. ACKNOWLEDGEMENTS
These studies were funded by grants from the American sociation, KY Affiliate, and the University of Louisville School.
Heart AsGraduate
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