A model of dynamic exercise: the decerebrate rat locomotor preparation TOBY G. BEDFORD, P. K. LOI, AND CRAIG C. CRANDALL Department of Exercise and Movement Science, University of Oregon, Eugene, Oregon 97403 BEDFORD, TOBY G., P. K. LOI, AND CRAIG C. CRANDALL.A model of dynamic exercise: the decerebrate rat locomotor preparation. J. Appl. Physiol. 72(l): 121-127, 1992.-The purpose of
this study was to develop a dynamic exercise model in the rat that could be usedto study central nervous system control of the cardiovascular system. Rats of both sexes were decerebrated under halothane anesthesia and prepared for induced locomotion on a freely turning wheel. Electrical stimulation of the mesencephalic locomotor region (MLR) elicited locomotion at different speedsand gait patterns and increased heart rate and blood pressure. Two maneuvers were performed to illustrate the potential useof the preparation. The first maneuver consistedof muscular paralysis, which prevents excitation of muscle mechanoreceptors and chemoreceptors resulting from exercise.MLR stimulation still increasedblood pressure. The secondmaneuver was performed to determine whether the blood pressureresponseobtained during paralysis was an artifact of electrical stimulation of the MLR. After microinjection of y-aminobutyric acid into the MLR, electrical current thresholds for blood pressure and locomotion increased in parallel. y-Aminobutyric acid injection also reduced the pressor responseto suprathreshold electrical stimulation by 76%. The injection results suggestthat electrical stimulation of the MLR activates cellsrather than fibers of passage. The blood pressure responseof the exercise model is probably not an artifact of stimulation. The decerebraterat locomotor preparation should offer another approach to investigate difficult problemsin exercisephysiology. brain; mesencephaliclocomotor region; feedforward control; central command; pedunculopontine tegmental nucleus; gammaaminobutyric acid
CONSIDERINGTHE WIDESPREAD USE and availability of the rat in exercise physiology, it is important to develop a model of dynamic exercise in the rat that would enable difficult problems to be addressed. For example, the problem of nerve recording cannot be addressed adequately in conscious exercising rats because of technical difficulties. A model of dynamic exercise is needed in which technical difficulties are reduced. Such a model is the locomotor preparation (20). Locomotor preparations are decerebrate yet can run spontaneously or with chemical/electrical stimulation of locomotor regions. Certain technical difficulties are considerably reduced, because extensive surgery for experiments can be performed owing to the decerebrate condition. The advantage of electrical stimulation is that the onset of locomotion is rapid and repeatable. Furthermore the 0161-7567/92
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speed/gait of locomotion can be changed by changing stimulus current (20). With chemical stimulation, the onset of locomotion is delayed and gradual and cannot be rapidly terminated (8), whereas with spontaneous locomotion the onset, speed, or duration cannot be predicted (2). Although spontaneous and chemically induced locomotion preparations are useful for study of brain problems, electrical stimulation should provide a much wider use of the preparation to examine not only nervous system mechanisms but also cardiovascular hemodynamics, respiratory, endocrine, and skeletal motor issues in nontrained and trained animals. In addition to these areas of study, a rat preparation would allow specialty rat strains, such as hypertensives (SHR, Dahl), stroke prone, and obese, to be studied. Commonly, two regions of the brain can be stimulated to induce locomotion, the mesencephalic locomotor region (MLR) (6,9,20,21) and the subthalamic locomotor region (SLR) (9, 20, 21). We have focused on the MLR because more is known about its neuroanatomy, efferent projections, and pharmacology (4) than the SLR and there is considerable question as to the neural anatomic basis of the SLR (1). In the cat the MLR is essential to spontaneous locomotion in the decerebrate preparation inasmuch as microinjection of the inhibitory amino acid y-aminobutyric acid (GABA) into the MLR arrests (8) ongoing spontaneous locomotion; furthermore this effect is reversed by picrotoxin (8). The purpose of this study was to establish a decerebrate rat MLR preparation and use it to describe blood pressure-locomotor responses during electrical stimulation of the MLR. Besides describing the basic locomotor preparation, two other maneuvers were performed. To demonstrate a potential use of this preparation in conjunction with our focus on cardiovascular control, a paralyzed preparation was used. After completion of a stimulus-locomotor response regimen animals were paralyzed and MLR stimulation was repeated. Paralysis eliminates the mechanoreceptor and chemoreceptor feedback from muscle (14) so that central nervous system (CNS) mechanisms can be investigated. The second maneuver consisted of microinjecting GABA into the locomotor region after locomotion had been obtained with electrical stimulation. This was done to demonstrate that the blood pressure response during locomotion was not an artifact of electrical stimulation but rather an integral part of the total exercise response to locomotion.
Copyright 0 1992 the American Physiological Society
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Sprague-Dawley rats (n = 19) of both sexes (250-450 g) were used in these studies. Rats were housed in animal quarters that were temperature (22 t 2OC) and light controlled (12 h on-off). Rat chow and water were freely available. Animals were anesthetized with halothane (3.5%) in oxygen through a vaporizer (Vapor 19, Drager). A tracheotomy was performed, after which artificial ventilation (Harvard Apparatus) was used to maintain the anesthetic mixture in the animal. A pressure relief hole allowed room air to dilute the pressurized oxygen-anesthetic mixture; thus the inspired oxygen was ~100%. Tidal volumes (1.8-2.0 ml) were adjusted according to the size of the animal and dead space while maintaining a frequency of 100Imin. Blood gas and pH values (0.25 ml blood) were measured just before electrical stimulation in this preparation and were 7.386 t 0.022 pH units for 40.5 t 2.4 Torr PCO, and 158.9 t 8.7 Torr PO,. The right carotid artery was ligated to reduce brain bleeding. The left carotid artery was cannulated for measurement of arterial pressure (Statham P23 ID). The right external jugular vein was cannulated for drug injection. Body temperature was maintained at 37 t 1°C with a temperature controller unit and infrared bulb. The animal’s head was centered in a stereotaxic head frame (Kopf), and an opening was made in the parietal bone on the right side. The sagittal and transverse sinuses remained intact. Cortical tissue was aspirated until the midbrain and thalamus were visible. A vertical decerebration was performed ~2 mm rostra1 to the superior colliculi by means of aspiration. Completeness of the decerebration was confirmed postmortem. The decerebration removed the forebrain, thalamus, and hypothalamus. Multistranded bipolar stainless steel electromyogram (EMG) electrodes were placed into the triceps surae muscle group (12). The EMG signals were routed through EMG-averaging couplers (Sensormedics, type 9852A) that have a direct mode bandwidth of 5.3-1,000 Hz. The EMG signal was full-wave rectified and passed through an integrating circuit with a time constant of 100 ms. Amplification was x1,000-5,000. The area of the rectified smoothed curve was integrated for total integrated EMG activity (IEMG). After surgery, halothane was discontinued but artificial ventilation with oxygen continued. Lidocaine HCl (2%) was liberally applied on the exposed head and neck wounds to eliminate unwanted nociceptor input that might change background cardiovascular activity. The animal was placed on a freely moving wheel (27 in. diam) that was turned by the animal during locomotion. The head was kept in the head frame to provide lateral stability and accurate electrical stimulation. Between periods of electrical stimulation, all four limbs and the abdomen rested on the surface of the wheel. After the withdrawal of halothane (40 min) the junction of the superior and inferior colliculi in the midbrain was searched unilaterally for the MLR (20) with a semimicro monopolar stainless steel electrode (Rhodes, SNE-300, (0.1 mm diam) with 0.25 mm of the tip exposed. The electrode was designated the cathode. The
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anode was placed on exposed muscle and skin tissue in the head wound. Rectangular current pulses were monitored on an oscilloscope (Tektronix 502A) as the voltage drop across a 2-kQ resistor in series with the circuit. A 1-MQ resistor was also placed in the circuit to give virtual constant-current stimulation (range lo-60 PA) as provided by a Grass SIU-4B stimulator and S4G stimulus isolation unit. Effective stimulus parameters for inducing locomotion were 60 Hz and 1-ms duration and were utilized for all studies. Stimulation periods were short (~30 s) because this was all that was needed to establish the gait and blood pressure changes. It is important to determine the location of the MLR by physiological criteria (6) rather than just anatomic coordinates. The area is circumscribed, and it is the unique physiological response to stimulation that defines the area. Criteria for accepting a point as the MLR were (6) 1) locomotion thresholds < 60 PA, 2) stimulus-bound locomotion, and 3) graded speed of locomotion and gait changes with increased stimulation current. Once a locomotor point was located, the threshold current for a 10.mmHg rise in blood pressure was determined to the nearest 5 PA. A stimulation-response regimen was initiated with ~-PA increases in current until it appeared that the animals’ blood pressure or speed of running was maximal. The time between stimulations varied because blood pressure was allowed to return to control levels before the next stimulation (30 s-3 min). For skeletal muscle paralysis, a single dose of decamethonium bromide (20 mg/kg, Sigma Chemical) was slowly injected intravenously. This dose was found to completely eliminate observable movement during strong stimulation. Decamethonium has little effect on histamine release and blockade of autonomic ganglia, both of which would lower blood pressure (23). Paralysis was induced to eliminate possible mechanoreceptor and chemoreceptor activation in the muscle, which could contribute to the blood pressure response (14). The same series of stimulation currents was then applied after paralysis. The animals were ventilated at all times. Microinjections of freshly made 0.5 M GABA (Sigma Chemical) in 0.9% saline (pH 7.2-7.6) were made in a group of five animals. The MLR was located by electrical stimulation, resulting in locomotion, which was followed by injection of GABA in 0.5 ~1 saline. The injection cannula (0.15 mm OD) was attached to the side of the stimulating electrode and recessed 0.25 mm from the tip of the electrode. Volumes were injected over 30 s. Thresholds for pressor responses and locomotion were determined ~5 min after GABA injection and every 10 min thereafter until thresholds appeared to return to control levels. Controls with saline in 0.5-~1 volumes following the same protocol were also run. Injection spread of GABA was estimated in an additional four animals by injecting 0.5 ~1 India ink-l% Alcian blue (22) into a confirmed MLR location. Brain tissue was processed in the same manner as all other brain tissues. At the conclusion of the electrical stimulation experiments and the GABA injection experiments, a lesion was placed at the stimulation point in the brain by use of 1.5-mA direct current for 3 s. All animals were killed with
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an overdose of halothane or pentobarbital sodium. The brains were placed in 10% Formalin-saline solution and allowed to fix for 3-7 days, after which the solution was replaced with 20-40% sucrose in water. Frozen tissue sections 20-40 pm thick were cut. Sections were stained with thionin to highlight cell bodies and coverslipped. Lesioned areas were located, enlarged, and traced onto a representative tissue section. Data processing and calculations. Almost all recordings were made on a Beckman R-511 dynograph. Mean arterial pressure was calculated from planimetry of selected portions of the pressure tracing during control conditions and in the steady state during stimulation (5-10 s after start of stimulation). Heart rate was calculated from measurements of systolic pressure intervals corresponding to the section chosen for planimetry. For each animal, changes in blood pressure were calculated as the difference between the prestimulation values for each level of stimulation and the steady state during stimulations. Blood pressure and heart rate consistently reached steady state during the stimulation. The threshold stimulation current for blood pressure changes was considered stimulation level 1 regardless of the absolute amount of current. Results from all animals were grouped according to the stimulation level. IEMG was averaged over time for each stimulation and was expressed as a percentage of the maximal IEMG for a given series of stimulations. Locomotion rates were calculated from the number of peaks of integrated activity. Analysis of variance procedures (Crunch Software, Oakland, CA) were used to compare 1) percent changes of blood pressure with IEMG at each level of stimulation and 2) blood pressure changes after muscular paralysis with those before paralysis. Repeated measures was used to determine significant increases in blood pressure and heart rate from prestimulation values and compare step frequency of stimulation Zeuel 1 with other stimulation levels. A paired t test (Crunch Software) was run to determine whether the blood pressure and locomotor thresholds changed significantly from each other during the initial time period after microinjection of GABA. An alpha of 0.05 was chosen as the level of significance. Results are expressed as means t SE. RESULTS
Locomotion was obtained consistently by stimulation at the border of the inferior and superior colliculus, 0.30.7 mm anterior, 1.9 mm lateral, and 3.5-4.5 mm deep from the surface of the colliculi, as identified from the atlas of Paxinos and Watson (17). The location is in agreement with Skinner and Garcia-Rill (21). The typical sequence of events during MLR stimulation at lowlevel currents consisted of flexion of the hindlimbs until the feet were solidly placed on the wheel surface. All limbs then extended, and the locomotor gait appropriate to the level of stimulation was initiated. Gait patterns ranged in sequence as the stimulus level increased from fast walking, to running, to galloping (Fig. 1). Unlike the dog (unpublished observations) and cat (2, 4), the rat easily supports its body weight during stimulation of the MLR locomotor region. Locomotion was stimulus bound
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in the majority of cases, whereas at some currents, locomotion spontaneously ended even though stimulation continued. Animals sometimes showed poststimulus locomotion for a few cycles. This phenomenon has also been noted in the cat (2, 4) and the dog (unpublished observations). EMG activity increased with increased levels of stimulation as shown in Figs. 1 and 2. The threshold for locomotion was 20 t 2.2 (SE) PA (n = 10). Stepping frequency ranged from 2.9 to 5.0 Hz over the range of stimulation levels as shown in Fig. 3. If stepping frequencies were grouped according to the gait, then the frequency for fast walking was 3.1 t 0.25 Hz, 4.6 t 0.21 Hz for running, and 5.3 t 0.30 Hz for galloping. It is likely that the criterion of self-supported hindlimb locomotion to indicate the threshold skewed the stepping frequency toward faster gait patterns in which the animal was able to self-support. Stepping movements without self-support could be elicited by turning the wheel during subthreshold stimulation. Resting blood pressure was 124 t 6 mmHg, which is similar to values in intact conscious rats of 107-119 mmHg (3, 16). Resting heart rate was 450 t 18 beats/ min, which was elevated compared with 389 t 15 beats/ min in conscious resting rats (16). Blood pressure increased with increased levels of stimulation and concomitantly with locomotion as shown in Figs. 1, 2, and 4. Blood pressure increased 47 mmHg above the control pressure at the highest level of MLR stimulation. The blood pressure rise from stimulation level 1 to 6 (from ~20 to 45 PA stimulation current) was only 25 mmHg. This latter pressure increase is similar to that observed in conscious rats running on a treadmill in which the increase in blood pressure is ~27 mmHg over the lowest work load (11). The electrical threshold for blood pressure increases of 210 mmHg was 18.5 t 2.5 PA. In Fig. 2, blood pressure and EMG values are normalized to the percentage of the maximal response for each measurement. This provides a comparison between blood pressure and EMG changes at all levels of stimulation. Blood pressure increased relatively more than EMG at the two lowest stimulation levels. Heart rate increased with stimulation and running on the wheel to 503 t 29 beats/min (Fig. 4). Resting control blood pressure after paralysis was 136 t 13 mmHg and heart rate was 5Olk 19 beats/min. Blood pressure increased during stimulation of the physiologically identified MLR after muscular paralysis as shown in Fig. 5, At lower levels of stimulation, blood pressure changes were nearly the same as before paralysis. At stimulation EeueZs5 and 6, blood pressure increased significantly more after paralysis. At the highest level of stimulation, blood pressure increased 120 t 16 mmHg. Heart rate did not change during stimulation, but considering the high resting heart rate this is not surprising. Microinjection of 0.5 M GABA (0.5 ~1) into the MLR (as verified by electrically induced locomotion) was followed by elevated electrical thresholds for both locomotion and pressor responses. The peak change in thresholds occurred during the first time period of 5-14 min postinjection. Thereafter the thresholds gradually re-
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1. Digitized (1,000 Hz) record of decerebrate rat stimulated (60 Hz, 1 ms) in mesencephalic locomotor region to evoke locomotion on a freely turning wheel. Time lines at bottom represent 1 s. Animal rested between each stimulation. EMG is from each hindlimb. SAP, systemic arterial pressure. Right: brain section from the same animal; dark region represents electrolytic lesion. FIG.
(MLR)
turned to control values by 35-50 min. These changes are shown in Fig. 6. The increase in thresholds for the first time period was 15 t 3 PA for blood pressure and 22 t 5 PA for locomotion. These values were not significantly different from each other. There were no significant changes from control in threshold for locomotion or pressor responses when saline (0.5 ~1) was injected (locomotion 20.5 t 3.8 vs. 22.0 t 3.3 PA, pressor response 17.3 t 2.2 vs. 18.5 t 1.8 PA). When the blood pressure response to 30% suprathreshold stimulation was compared with control values, there was no change in resting blood pressure after injection of GABA [87.4 t 9.2 mmHg
(control) vs. 83.4 t 11.3 mmHg (GABA)], but the pressor response to stimulation was significantly reduced by 76% [39.4 k 11.9 mmHg (control) vs. 9.6 t 6.9 mmHg (GABA)]. The brain sites of GABA injection are illustrated in Fig. 7. The lesion areas correspond to the pedunculopontine tegmentum nucleus, which is a portion of the MLR (21). Estimation of spread of injectate (0.5 ~1) was made by successively sectioning those areas of brain tissue that demonstrated stain (Fig. 8). The size and location of the stained areas agree with Garcia-Rill et al. (6) for MLR
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localization. There was no stain in the inferior colliculus, an area that also elicits blood pressure-locomotion responses (5, 13). Furthermore there was no stain in the parabrachial nucleus, which has been shown to elevate blood pressure when stimulated (24). DISCUSSION
The rat has served as a useful model for investigation of the cardiovascular response to dynamic exercise and the effects of training (10,19). The decerebrate rat locomotor preparation offers another investigative approach to study physiological responses to exercise and exercise training. The preparation exhibited several characteristics of the dynamic exercise response. Muscle recruitment is very similar to that in normal rats running at
15-24 25-34 35-50 Minutes post-injection FIG. 6. Change in threshold current after injection of GABA into M&R, which was confirmed physiologically by locomotion and anatomically by location of electrolytic lesions (n = 5). Increases in threshold current for blood pressure and locomotion during first time period were not significantly different from each other.
different speeds (15). Because the animals were able to support themselves over most levels of stimulation and achieve high stepping frequencies, muscle metabolism probably increased. Arterial blood pressure and heart rate increased and respiration was also noted to increase during stimulation. These observations and fklings suggest that the decerebrate rat locomotor preparation offers another method to study many integrative aspects of dynamic exercise. Two maneuvers were performed with the preparation to illustrate its potential use for cardiovascular studies, which is our focus. Paralysis was used to separate the contribution of muscle chemo- and mechanoreceptors and muscle vasodilation from CNS mechanisms to the pressor response during locomotion. A pressor response
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was obtained after paralysis during MLR stimulation and suggests a CNS contribution. This is a new finding concerning the MLR. Blood pressure increased significantly more at the highest levels of stimulation compared with that obtained during locomotion. The exaggerated pressor response during paralysis may reflect the absence of massive muscle vasodilation, which would oppose vasoconstriction. The second maneuver was performed to determine whether the pressor response observed during paralysis was an artifact of electrical stimulation. If the model is to be realistic, blood pressure responses should not be an artifact resulting from stimulation of unrelated neuronal elements in the CNS. Electrical stimulation activates both axons and cells (18). Thus it is impossible to state whether only cell bodies and/or axons of passage were activated, which resulted in pressor responses. There is strong evidence that the locomoti .on component of MLR electrical/chemical stimulation is due to activation of cell bodies and not axonal fibers of passage (47). The blood pressure component of the response to electrical stimulation was tested by microinjection of GABA into the MLR. After injection, electrical stimulation thresholds for blood pressure and locomotion increased in parallel. In addition, there was a 76% reduction of the pressor response to a standard suprathreshold stimulation. These new findings strongly suggest that the blood pressure response to electrical stimulation of the MLR during paralysis was not an artifact. Microinjection of dye enabled an estimate of the spread of GABA to various surrounding neuronal groups (Fig. 8). Spread in the caudal direction did not reach the
ANT, 0.5 FIG. 7, Brain section taken through MLR with extent of electrolytic lesions outlined on right. Each lesion represents a GABA injection site. Lesions are in pedunculopontine tegmental nucleus, which is part of MLR. Brain section is 0.5 anterior, and lesions are ~1.9 mm lateral. Abbreviations from Paxinos and Watson (17); Aq, aqueduct; CG, central gray; CNF, cuneiform nucleus; IC, inferior colliculus; PPTg, pedunculopontine tegmental nucleus.
MODEL
FIG. 8. Extent of India ink-Alcian blue stain after 0.5~~1 injection into MLR. MLR was confirmed by inducing locomotion. Sagittal sections (1.9 mm lateral) indicate that stain did not enter inferior colliculus or parabrachial nucleus. Stain overlaps pedunculopontine tegmental nucleus (hatched area), which is part of MLR. Identification of nuclei from Paxinos and Watson (17): BC, brachium conjunctivum; CB, cerebellum; IC, inferior colliculus; PB, parabrachial nucleus; SC, superior colliculus; SN, substantia nigra.
parabrachial nucleus. This is important inasmuch as electrical and chemical stimulation of the parabrachial nucleus with glutamate (24) causes pressor responses. In the dorsal direction, stain did not reach the inferior colliculus, another region that causes locomotion and pressor responses (13 l). The bulk of the stain overlapped the pedunculopontine tegmental nucleus, which has been shown to be part of the MLR (4,6). The dye experiments corroborate the suggestion that GABA was acting on cells within the MLR and that electrical stimulation does not create pressor artifacts. In summary, a decerebrate locomotor preparation has been described in the rat that enables an alternate aPpreach to the study of exercise physiology. Electrical stimulation of the MLR in the absence of peripheral feedback elevated blood pressure. Parallel blockade of locomotion and pressor responses by microinjection of GABA suggests that cells within MLR are mediating the combined responses and that blood pressure changes are not an artifact of electrical stimulation. The authors acknowledge Susan Grieve and A. M. Sidek who participated in the experiments and R. Bremiller for assistance and use of the histology laboratory. Support for this study was provided by National Heart, Lung, and Blood Institute Grant R23-HL-39664. Address reprint requests to T. G. Bedford. Received 15 March 1991; accepted in final form 3 September 1991. REFERENCES 1. ARMSTRONG, D. M. Supraspinal contributions to the initiation and control of locomotion in the cat. Prog. Neurobt’ol. 26: 273-361,1986. 2. ELDRIDGE, F. L., D. E. MILLHORN, J. P. KILEY, AND T. G. WAL-
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DROP. Stimulation by central command of locomotion, respiration and circulation during exercise. Respir. Physiol. 59: 313-337, 1985, 3. FINK, G. D., W. J. BRYAN, M. PV3[ANN, J. OSBORN, AND A. WERBER. Continuous blood pressure measurement in rats with aortic baroreceptor deafferentation. Am. J. Physiol. 241 (Heart Circ. Physiol. 10): H268-H272,1981. 4. GARCIA-RILL, E. The basal ganglia and the locomotor regions. Brain Res. Rev. 11: 47-63, 1986. 5. GARCIA-RILL, E., C. R. HOUSER, R. D. SKINNER, W. SMITH, AND D. J. WOODWARD. Locomotion-inducing sites in the vicinity of the pedunculopontine nucleus. Brain Res. BuZZ. 18: 731-738, 1987. 6. GARCIA-RILL, E., N. KINJQ Y. ATSUTA, Y. ISHIKAWA, M. WEBBER, AND R. D. SKINNER, Posterior midbrain-induced locomotion. Brain Res. Bull. 24: 499-508, 1990. 7. GARCIA-RILL, E., AND R. D. SKINNER. Modulation of rhythmic function in the posterior midbrain. Neuroscience 27: 639-654,1988. 8. GARCIA-RILL, E., R. D. SKINNER, AND J. A. FITZGERALD. Chemical activation of the mesencephalic locomotor region. Brain Res. 330: 43-54,1985. 9. GRILLNER, S. Control of locomotion in bipeds, tetrapods, and fish. In: Handbook of Physiology. The Nervous System. Motor Control. Bethesda, MD: Am. Physiol. Sot., 1981, sect. 1, vol. II, pt. 2, chapt. 26. 10. LAUGHLIN, M. H. Skeletal muscle blood flow capacity: role of muscle pump in exercise hyperemia. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H993-H1004, 1987. 11. LAUGHLIN, M. H., AND R. B. ARMSTRONG. Muscular blood flow distribution patterns as a function of running speed in rats. Am. J. Physiol. 243 (Heart Circ. PhysioZ. 12): H296-H306, 1982. 12. LOEB, G. E., AND C. GANS. Electromyography for Experimental&s. Chicago, IL: University of Chicago Press, 1986, p. 115-117. 13. LOI, P. K., AND T. BEDFORD. GABA injections into the mesence-
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Physiology. The Cardiovascular Sys tern. Peripheral CircuZa tion and Organ Blood FZow, Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. III, pt. 2, chapt. 17, p. 623-658. 15. NICOLOPOULOS-STOURNAFUS, S., AND J. F. ILES. Hindlimb muscle activity during locomotion in the rat (Rattus norvegicus) (Rodentia:Muridae). J. Physiol. Land. 203: 427-440, 1984. 16. NORMAN, R. A., T. G. COLEMAN, AND A. C. DENT. Continuous
monitoring of arterial pressure indicates sinoaortic denervated rats are not hypertensive. Hypertension 3: 119-125, 1981. 17. PAXINOS, G., AND C. WATSON. The Rat Brain in Stereotaxic Coordinates. Orlando, FL: Academic, 1986. 18. RANCK, J. B. Which elements are excited in electrical stimulation of mammalian central nervous system: a review. Brain Res. 98: 417-440,1975. 19. SCHEUER, 20. 21. 22.
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J., AND C. M. TIPTON. Cardiovascular adaptations to physical training. Annu. Rev. Physiol. 39: 221-251, 1977. SHIK, M. L., AND G, N. ORLOVSKY. Neurophysiology of locomotor automatism. Physiol. Rev. 56: 465-500, 1976. SKINNER, R. J., AND E. GARCIA-RILL. The mesencephalic locomotor region (MLR) in the rat. Bruin Res. 323: 385-389,1984, SUNDARAM, K., J. MURAGAIAN, A. KRIEGER, AND H. SAPRU. Microinjections of cholinergic agonists into the intermediolateral cell column of the spinal cord at T,-T, increase heart rate and contractility. Brain Res. 503: 22-31, 1989. TAYLOR, I? Neuromuscular blocking agents. In: The Pharmacological Basis of Therapeutics, edited by A. G. Gillman, L. S. Goodman, T. W. Rall, and F. Murad. New York: Macmillan, 1985, chapt. 11, p.
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D. G. Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure. Brain Res. 462: 383390,1988,
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this study was to develop a dynamic exercise model in the rat that could be usedto study central nervous system control of the cardiovascular system. Rats of both sexes were decerebrated under halothane anesthesia and prepared for induced locomotion on a freely turning wheel. Electrical stimulation of the mesencephalic locomotor region (MLR) elicited locomotion at different speedsand gait patterns and increased heart rate and blood pressure. Two maneuvers were performed to illustrate the potential useof the preparation. The first maneuver consistedof muscular paralysis, which prevents excitation of muscle mechanoreceptors and chemoreceptors resulting from exercise.MLR stimulation still increasedblood pressure. The secondmaneuver was performed to determine whether the blood pressureresponseobtained during paralysis was an artifact of electrical stimulation of the MLR. After microinjection of y-aminobutyric acid into the MLR, electrical current thresholds for blood pressure and locomotion increased in parallel. y-Aminobutyric acid injection also reduced the pressor responseto suprathreshold electrical stimulation by 76%. The injection results suggestthat electrical stimulation of the MLR activates cellsrather than fibers of passage. The blood pressure responseof the exercise model is probably not an artifact of stimulation. The decerebraterat locomotor preparation should offer another approach to investigate difficult problemsin exercisephysiology. brain; mesencephaliclocomotor region; feedforward control; central command; pedunculopontine tegmental nucleus; gammaaminobutyric acid
CONSIDERINGTHE WIDESPREAD USE and availability of the rat in exercise physiology, it is important to develop a model of dynamic exercise in the rat that would enable difficult problems to be addressed. For example, the problem of nerve recording cannot be addressed adequately in conscious exercising rats because of technical difficulties. A model of dynamic exercise is needed in which technical difficulties are reduced. Such a model is the locomotor preparation (20). Locomotor preparations are decerebrate yet can run spontaneously or with chemical/electrical stimulation of locomotor regions. Certain technical difficulties are considerably reduced, because extensive surgery for experiments can be performed owing to the decerebrate condition. The advantage of electrical stimulation is that the onset of locomotion is rapid and repeatable. Furthermore the 0161-7567/92
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speed/gait of locomotion can be changed by changing stimulus current (20). With chemical stimulation, the onset of locomotion is delayed and gradual and cannot be rapidly terminated (8), whereas with spontaneous locomotion the onset, speed, or duration cannot be predicted (2). Although spontaneous and chemically induced locomotion preparations are useful for study of brain problems, electrical stimulation should provide a much wider use of the preparation to examine not only nervous system mechanisms but also cardiovascular hemodynamics, respiratory, endocrine, and skeletal motor issues in nontrained and trained animals. In addition to these areas of study, a rat preparation would allow specialty rat strains, such as hypertensives (SHR, Dahl), stroke prone, and obese, to be studied. Commonly, two regions of the brain can be stimulated to induce locomotion, the mesencephalic locomotor region (MLR) (6,9,20,21) and the subthalamic locomotor region (SLR) (9, 20, 21). We have focused on the MLR because more is known about its neuroanatomy, efferent projections, and pharmacology (4) than the SLR and there is considerable question as to the neural anatomic basis of the SLR (1). In the cat the MLR is essential to spontaneous locomotion in the decerebrate preparation inasmuch as microinjection of the inhibitory amino acid y-aminobutyric acid (GABA) into the MLR arrests (8) ongoing spontaneous locomotion; furthermore this effect is reversed by picrotoxin (8). The purpose of this study was to establish a decerebrate rat MLR preparation and use it to describe blood pressure-locomotor responses during electrical stimulation of the MLR. Besides describing the basic locomotor preparation, two other maneuvers were performed. To demonstrate a potential use of this preparation in conjunction with our focus on cardiovascular control, a paralyzed preparation was used. After completion of a stimulus-locomotor response regimen animals were paralyzed and MLR stimulation was repeated. Paralysis eliminates the mechanoreceptor and chemoreceptor feedback from muscle (14) so that central nervous system (CNS) mechanisms can be investigated. The second maneuver consisted of microinjecting GABA into the locomotor region after locomotion had been obtained with electrical stimulation. This was done to demonstrate that the blood pressure response during locomotion was not an artifact of electrical stimulation but rather an integral part of the total exercise response to locomotion.
Copyright 0 1992 the American Physiological Society
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Sprague-Dawley rats (n = 19) of both sexes (250-450 g) were used in these studies. Rats were housed in animal quarters that were temperature (22 t 2OC) and light controlled (12 h on-off). Rat chow and water were freely available. Animals were anesthetized with halothane (3.5%) in oxygen through a vaporizer (Vapor 19, Drager). A tracheotomy was performed, after which artificial ventilation (Harvard Apparatus) was used to maintain the anesthetic mixture in the animal. A pressure relief hole allowed room air to dilute the pressurized oxygen-anesthetic mixture; thus the inspired oxygen was ~100%. Tidal volumes (1.8-2.0 ml) were adjusted according to the size of the animal and dead space while maintaining a frequency of 100Imin. Blood gas and pH values (0.25 ml blood) were measured just before electrical stimulation in this preparation and were 7.386 t 0.022 pH units for 40.5 t 2.4 Torr PCO, and 158.9 t 8.7 Torr PO,. The right carotid artery was ligated to reduce brain bleeding. The left carotid artery was cannulated for measurement of arterial pressure (Statham P23 ID). The right external jugular vein was cannulated for drug injection. Body temperature was maintained at 37 t 1°C with a temperature controller unit and infrared bulb. The animal’s head was centered in a stereotaxic head frame (Kopf), and an opening was made in the parietal bone on the right side. The sagittal and transverse sinuses remained intact. Cortical tissue was aspirated until the midbrain and thalamus were visible. A vertical decerebration was performed ~2 mm rostra1 to the superior colliculi by means of aspiration. Completeness of the decerebration was confirmed postmortem. The decerebration removed the forebrain, thalamus, and hypothalamus. Multistranded bipolar stainless steel electromyogram (EMG) electrodes were placed into the triceps surae muscle group (12). The EMG signals were routed through EMG-averaging couplers (Sensormedics, type 9852A) that have a direct mode bandwidth of 5.3-1,000 Hz. The EMG signal was full-wave rectified and passed through an integrating circuit with a time constant of 100 ms. Amplification was x1,000-5,000. The area of the rectified smoothed curve was integrated for total integrated EMG activity (IEMG). After surgery, halothane was discontinued but artificial ventilation with oxygen continued. Lidocaine HCl (2%) was liberally applied on the exposed head and neck wounds to eliminate unwanted nociceptor input that might change background cardiovascular activity. The animal was placed on a freely moving wheel (27 in. diam) that was turned by the animal during locomotion. The head was kept in the head frame to provide lateral stability and accurate electrical stimulation. Between periods of electrical stimulation, all four limbs and the abdomen rested on the surface of the wheel. After the withdrawal of halothane (40 min) the junction of the superior and inferior colliculi in the midbrain was searched unilaterally for the MLR (20) with a semimicro monopolar stainless steel electrode (Rhodes, SNE-300, (0.1 mm diam) with 0.25 mm of the tip exposed. The electrode was designated the cathode. The
MODEL
anode was placed on exposed muscle and skin tissue in the head wound. Rectangular current pulses were monitored on an oscilloscope (Tektronix 502A) as the voltage drop across a 2-kQ resistor in series with the circuit. A 1-MQ resistor was also placed in the circuit to give virtual constant-current stimulation (range lo-60 PA) as provided by a Grass SIU-4B stimulator and S4G stimulus isolation unit. Effective stimulus parameters for inducing locomotion were 60 Hz and 1-ms duration and were utilized for all studies. Stimulation periods were short (~30 s) because this was all that was needed to establish the gait and blood pressure changes. It is important to determine the location of the MLR by physiological criteria (6) rather than just anatomic coordinates. The area is circumscribed, and it is the unique physiological response to stimulation that defines the area. Criteria for accepting a point as the MLR were (6) 1) locomotion thresholds < 60 PA, 2) stimulus-bound locomotion, and 3) graded speed of locomotion and gait changes with increased stimulation current. Once a locomotor point was located, the threshold current for a 10.mmHg rise in blood pressure was determined to the nearest 5 PA. A stimulation-response regimen was initiated with ~-PA increases in current until it appeared that the animals’ blood pressure or speed of running was maximal. The time between stimulations varied because blood pressure was allowed to return to control levels before the next stimulation (30 s-3 min). For skeletal muscle paralysis, a single dose of decamethonium bromide (20 mg/kg, Sigma Chemical) was slowly injected intravenously. This dose was found to completely eliminate observable movement during strong stimulation. Decamethonium has little effect on histamine release and blockade of autonomic ganglia, both of which would lower blood pressure (23). Paralysis was induced to eliminate possible mechanoreceptor and chemoreceptor activation in the muscle, which could contribute to the blood pressure response (14). The same series of stimulation currents was then applied after paralysis. The animals were ventilated at all times. Microinjections of freshly made 0.5 M GABA (Sigma Chemical) in 0.9% saline (pH 7.2-7.6) were made in a group of five animals. The MLR was located by electrical stimulation, resulting in locomotion, which was followed by injection of GABA in 0.5 ~1 saline. The injection cannula (0.15 mm OD) was attached to the side of the stimulating electrode and recessed 0.25 mm from the tip of the electrode. Volumes were injected over 30 s. Thresholds for pressor responses and locomotion were determined ~5 min after GABA injection and every 10 min thereafter until thresholds appeared to return to control levels. Controls with saline in 0.5-~1 volumes following the same protocol were also run. Injection spread of GABA was estimated in an additional four animals by injecting 0.5 ~1 India ink-l% Alcian blue (22) into a confirmed MLR location. Brain tissue was processed in the same manner as all other brain tissues. At the conclusion of the electrical stimulation experiments and the GABA injection experiments, a lesion was placed at the stimulation point in the brain by use of 1.5-mA direct current for 3 s. All animals were killed with
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an overdose of halothane or pentobarbital sodium. The brains were placed in 10% Formalin-saline solution and allowed to fix for 3-7 days, after which the solution was replaced with 20-40% sucrose in water. Frozen tissue sections 20-40 pm thick were cut. Sections were stained with thionin to highlight cell bodies and coverslipped. Lesioned areas were located, enlarged, and traced onto a representative tissue section. Data processing and calculations. Almost all recordings were made on a Beckman R-511 dynograph. Mean arterial pressure was calculated from planimetry of selected portions of the pressure tracing during control conditions and in the steady state during stimulation (5-10 s after start of stimulation). Heart rate was calculated from measurements of systolic pressure intervals corresponding to the section chosen for planimetry. For each animal, changes in blood pressure were calculated as the difference between the prestimulation values for each level of stimulation and the steady state during stimulations. Blood pressure and heart rate consistently reached steady state during the stimulation. The threshold stimulation current for blood pressure changes was considered stimulation level 1 regardless of the absolute amount of current. Results from all animals were grouped according to the stimulation level. IEMG was averaged over time for each stimulation and was expressed as a percentage of the maximal IEMG for a given series of stimulations. Locomotion rates were calculated from the number of peaks of integrated activity. Analysis of variance procedures (Crunch Software, Oakland, CA) were used to compare 1) percent changes of blood pressure with IEMG at each level of stimulation and 2) blood pressure changes after muscular paralysis with those before paralysis. Repeated measures was used to determine significant increases in blood pressure and heart rate from prestimulation values and compare step frequency of stimulation Zeuel 1 with other stimulation levels. A paired t test (Crunch Software) was run to determine whether the blood pressure and locomotor thresholds changed significantly from each other during the initial time period after microinjection of GABA. An alpha of 0.05 was chosen as the level of significance. Results are expressed as means t SE. RESULTS
Locomotion was obtained consistently by stimulation at the border of the inferior and superior colliculus, 0.30.7 mm anterior, 1.9 mm lateral, and 3.5-4.5 mm deep from the surface of the colliculi, as identified from the atlas of Paxinos and Watson (17). The location is in agreement with Skinner and Garcia-Rill (21). The typical sequence of events during MLR stimulation at lowlevel currents consisted of flexion of the hindlimbs until the feet were solidly placed on the wheel surface. All limbs then extended, and the locomotor gait appropriate to the level of stimulation was initiated. Gait patterns ranged in sequence as the stimulus level increased from fast walking, to running, to galloping (Fig. 1). Unlike the dog (unpublished observations) and cat (2, 4), the rat easily supports its body weight during stimulation of the MLR locomotor region. Locomotion was stimulus bound
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in the majority of cases, whereas at some currents, locomotion spontaneously ended even though stimulation continued. Animals sometimes showed poststimulus locomotion for a few cycles. This phenomenon has also been noted in the cat (2, 4) and the dog (unpublished observations). EMG activity increased with increased levels of stimulation as shown in Figs. 1 and 2. The threshold for locomotion was 20 t 2.2 (SE) PA (n = 10). Stepping frequency ranged from 2.9 to 5.0 Hz over the range of stimulation levels as shown in Fig. 3. If stepping frequencies were grouped according to the gait, then the frequency for fast walking was 3.1 t 0.25 Hz, 4.6 t 0.21 Hz for running, and 5.3 t 0.30 Hz for galloping. It is likely that the criterion of self-supported hindlimb locomotion to indicate the threshold skewed the stepping frequency toward faster gait patterns in which the animal was able to self-support. Stepping movements without self-support could be elicited by turning the wheel during subthreshold stimulation. Resting blood pressure was 124 t 6 mmHg, which is similar to values in intact conscious rats of 107-119 mmHg (3, 16). Resting heart rate was 450 t 18 beats/ min, which was elevated compared with 389 t 15 beats/ min in conscious resting rats (16). Blood pressure increased with increased levels of stimulation and concomitantly with locomotion as shown in Figs. 1, 2, and 4. Blood pressure increased 47 mmHg above the control pressure at the highest level of MLR stimulation. The blood pressure rise from stimulation level 1 to 6 (from ~20 to 45 PA stimulation current) was only 25 mmHg. This latter pressure increase is similar to that observed in conscious rats running on a treadmill in which the increase in blood pressure is ~27 mmHg over the lowest work load (11). The electrical threshold for blood pressure increases of 210 mmHg was 18.5 t 2.5 PA. In Fig. 2, blood pressure and EMG values are normalized to the percentage of the maximal response for each measurement. This provides a comparison between blood pressure and EMG changes at all levels of stimulation. Blood pressure increased relatively more than EMG at the two lowest stimulation levels. Heart rate increased with stimulation and running on the wheel to 503 t 29 beats/min (Fig. 4). Resting control blood pressure after paralysis was 136 t 13 mmHg and heart rate was 5Olk 19 beats/min. Blood pressure increased during stimulation of the physiologically identified MLR after muscular paralysis as shown in Fig. 5, At lower levels of stimulation, blood pressure changes were nearly the same as before paralysis. At stimulation EeueZs5 and 6, blood pressure increased significantly more after paralysis. At the highest level of stimulation, blood pressure increased 120 t 16 mmHg. Heart rate did not change during stimulation, but considering the high resting heart rate this is not surprising. Microinjection of 0.5 M GABA (0.5 ~1) into the MLR (as verified by electrically induced locomotion) was followed by elevated electrical thresholds for both locomotion and pressor responses. The peak change in thresholds occurred during the first time period of 5-14 min postinjection. Thereafter the thresholds gradually re-
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1. Digitized (1,000 Hz) record of decerebrate rat stimulated (60 Hz, 1 ms) in mesencephalic locomotor region to evoke locomotion on a freely turning wheel. Time lines at bottom represent 1 s. Animal rested between each stimulation. EMG is from each hindlimb. SAP, systemic arterial pressure. Right: brain section from the same animal; dark region represents electrolytic lesion. FIG.
(MLR)
turned to control values by 35-50 min. These changes are shown in Fig. 6. The increase in thresholds for the first time period was 15 t 3 PA for blood pressure and 22 t 5 PA for locomotion. These values were not significantly different from each other. There were no significant changes from control in threshold for locomotion or pressor responses when saline (0.5 ~1) was injected (locomotion 20.5 t 3.8 vs. 22.0 t 3.3 PA, pressor response 17.3 t 2.2 vs. 18.5 t 1.8 PA). When the blood pressure response to 30% suprathreshold stimulation was compared with control values, there was no change in resting blood pressure after injection of GABA [87.4 t 9.2 mmHg
(control) vs. 83.4 t 11.3 mmHg (GABA)], but the pressor response to stimulation was significantly reduced by 76% [39.4 k 11.9 mmHg (control) vs. 9.6 t 6.9 mmHg (GABA)]. The brain sites of GABA injection are illustrated in Fig. 7. The lesion areas correspond to the pedunculopontine tegmentum nucleus, which is a portion of the MLR (21). Estimation of spread of injectate (0.5 ~1) was made by successively sectioning those areas of brain tissue that demonstrated stain (Fig. 8). The size and location of the stained areas agree with Garcia-Rill et al. (6) for MLR
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5. Comparison of blood pressure changes to stimulation before and after muscular paralysis. *Significant difference between pre- and postparalysis blood pressure response at a given level of stimulation, n = 10 for levels 1-3, n = 9 for ZeveZ4, n = 8 for level 5, and n = 6 for level 6. FIG.
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localization. There was no stain in the inferior colliculus, an area that also elicits blood pressure-locomotion responses (5, 13). Furthermore there was no stain in the parabrachial nucleus, which has been shown to elevate blood pressure when stimulated (24). DISCUSSION
The rat has served as a useful model for investigation of the cardiovascular response to dynamic exercise and the effects of training (10,19). The decerebrate rat locomotor preparation offers another investigative approach to study physiological responses to exercise and exercise training. The preparation exhibited several characteristics of the dynamic exercise response. Muscle recruitment is very similar to that in normal rats running at
15-24 25-34 35-50 Minutes post-injection FIG. 6. Change in threshold current after injection of GABA into M&R, which was confirmed physiologically by locomotion and anatomically by location of electrolytic lesions (n = 5). Increases in threshold current for blood pressure and locomotion during first time period were not significantly different from each other.
different speeds (15). Because the animals were able to support themselves over most levels of stimulation and achieve high stepping frequencies, muscle metabolism probably increased. Arterial blood pressure and heart rate increased and respiration was also noted to increase during stimulation. These observations and fklings suggest that the decerebrate rat locomotor preparation offers another method to study many integrative aspects of dynamic exercise. Two maneuvers were performed with the preparation to illustrate its potential use for cardiovascular studies, which is our focus. Paralysis was used to separate the contribution of muscle chemo- and mechanoreceptors and muscle vasodilation from CNS mechanisms to the pressor response during locomotion. A pressor response
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was obtained after paralysis during MLR stimulation and suggests a CNS contribution. This is a new finding concerning the MLR. Blood pressure increased significantly more at the highest levels of stimulation compared with that obtained during locomotion. The exaggerated pressor response during paralysis may reflect the absence of massive muscle vasodilation, which would oppose vasoconstriction. The second maneuver was performed to determine whether the pressor response observed during paralysis was an artifact of electrical stimulation. If the model is to be realistic, blood pressure responses should not be an artifact resulting from stimulation of unrelated neuronal elements in the CNS. Electrical stimulation activates both axons and cells (18). Thus it is impossible to state whether only cell bodies and/or axons of passage were activated, which resulted in pressor responses. There is strong evidence that the locomoti .on component of MLR electrical/chemical stimulation is due to activation of cell bodies and not axonal fibers of passage (47). The blood pressure component of the response to electrical stimulation was tested by microinjection of GABA into the MLR. After injection, electrical stimulation thresholds for blood pressure and locomotion increased in parallel. In addition, there was a 76% reduction of the pressor response to a standard suprathreshold stimulation. These new findings strongly suggest that the blood pressure response to electrical stimulation of the MLR during paralysis was not an artifact. Microinjection of dye enabled an estimate of the spread of GABA to various surrounding neuronal groups (Fig. 8). Spread in the caudal direction did not reach the
ANT, 0.5 FIG. 7, Brain section taken through MLR with extent of electrolytic lesions outlined on right. Each lesion represents a GABA injection site. Lesions are in pedunculopontine tegmental nucleus, which is part of MLR. Brain section is 0.5 anterior, and lesions are ~1.9 mm lateral. Abbreviations from Paxinos and Watson (17); Aq, aqueduct; CG, central gray; CNF, cuneiform nucleus; IC, inferior colliculus; PPTg, pedunculopontine tegmental nucleus.
MODEL
FIG. 8. Extent of India ink-Alcian blue stain after 0.5~~1 injection into MLR. MLR was confirmed by inducing locomotion. Sagittal sections (1.9 mm lateral) indicate that stain did not enter inferior colliculus or parabrachial nucleus. Stain overlaps pedunculopontine tegmental nucleus (hatched area), which is part of MLR. Identification of nuclei from Paxinos and Watson (17): BC, brachium conjunctivum; CB, cerebellum; IC, inferior colliculus; PB, parabrachial nucleus; SC, superior colliculus; SN, substantia nigra.
parabrachial nucleus. This is important inasmuch as electrical and chemical stimulation of the parabrachial nucleus with glutamate (24) causes pressor responses. In the dorsal direction, stain did not reach the inferior colliculus, another region that causes locomotion and pressor responses (13 l). The bulk of the stain overlapped the pedunculopontine tegmental nucleus, which has been shown to be part of the MLR (4,6). The dye experiments corroborate the suggestion that GABA was acting on cells within the MLR and that electrical stimulation does not create pressor artifacts. In summary, a decerebrate locomotor preparation has been described in the rat that enables an alternate aPpreach to the study of exercise physiology. Electrical stimulation of the MLR in the absence of peripheral feedback elevated blood pressure. Parallel blockade of locomotion and pressor responses by microinjection of GABA suggests that cells within MLR are mediating the combined responses and that blood pressure changes are not an artifact of electrical stimulation. The authors acknowledge Susan Grieve and A. M. Sidek who participated in the experiments and R. Bremiller for assistance and use of the histology laboratory. Support for this study was provided by National Heart, Lung, and Blood Institute Grant R23-HL-39664. Address reprint requests to T. G. Bedford. Received 15 March 1991; accepted in final form 3 September 1991. REFERENCES 1. ARMSTRONG, D. M. Supraspinal contributions to the initiation and control of locomotion in the cat. Prog. Neurobt’ol. 26: 273-361,1986. 2. ELDRIDGE, F. L., D. E. MILLHORN, J. P. KILEY, AND T. G. WAL-
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DROP. Stimulation by central command of locomotion, respiration and circulation during exercise. Respir. Physiol. 59: 313-337, 1985, 3. FINK, G. D., W. J. BRYAN, M. PV3[ANN, J. OSBORN, AND A. WERBER. Continuous blood pressure measurement in rats with aortic baroreceptor deafferentation. Am. J. Physiol. 241 (Heart Circ. Physiol. 10): H268-H272,1981. 4. GARCIA-RILL, E. The basal ganglia and the locomotor regions. Brain Res. Rev. 11: 47-63, 1986. 5. GARCIA-RILL, E., C. R. HOUSER, R. D. SKINNER, W. SMITH, AND D. J. WOODWARD. Locomotion-inducing sites in the vicinity of the pedunculopontine nucleus. Brain Res. BuZZ. 18: 731-738, 1987. 6. GARCIA-RILL, E., N. KINJQ Y. ATSUTA, Y. ISHIKAWA, M. WEBBER, AND R. D. SKINNER, Posterior midbrain-induced locomotion. Brain Res. Bull. 24: 499-508, 1990. 7. GARCIA-RILL, E., AND R. D. SKINNER. Modulation of rhythmic function in the posterior midbrain. Neuroscience 27: 639-654,1988. 8. GARCIA-RILL, E., R. D. SKINNER, AND J. A. FITZGERALD. Chemical activation of the mesencephalic locomotor region. Brain Res. 330: 43-54,1985. 9. GRILLNER, S. Control of locomotion in bipeds, tetrapods, and fish. In: Handbook of Physiology. The Nervous System. Motor Control. Bethesda, MD: Am. Physiol. Sot., 1981, sect. 1, vol. II, pt. 2, chapt. 26. 10. LAUGHLIN, M. H. Skeletal muscle blood flow capacity: role of muscle pump in exercise hyperemia. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H993-H1004, 1987. 11. LAUGHLIN, M. H., AND R. B. ARMSTRONG. Muscular blood flow distribution patterns as a function of running speed in rats. Am. J. Physiol. 243 (Heart Circ. PhysioZ. 12): H296-H306, 1982. 12. LOEB, G. E., AND C. GANS. Electromyography for Experimental&s. Chicago, IL: University of Chicago Press, 1986, p. 115-117. 13. LOI, P. K., AND T. BEDFORD. GABA injections into the mesence-
phalic locomotor region (MLR) raises blood pressure and locomotor threshold in rats (Abstract). FASEB J. 4: A822, 1990.
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J. H., AND R. F. SCHMIDT. Cardiovascular control by afferent fibers from skeletal muscle receptors. In: Handbook of
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Physiology. The Cardiovascular Sys tern. Peripheral CircuZa tion and Organ Blood FZow, Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. III, pt. 2, chapt. 17, p. 623-658. 15. NICOLOPOULOS-STOURNAFUS, S., AND J. F. ILES. Hindlimb muscle activity during locomotion in the rat (Rattus norvegicus) (Rodentia:Muridae). J. Physiol. Land. 203: 427-440, 1984. 16. NORMAN, R. A., T. G. COLEMAN, AND A. C. DENT. Continuous
monitoring of arterial pressure indicates sinoaortic denervated rats are not hypertensive. Hypertension 3: 119-125, 1981. 17. PAXINOS, G., AND C. WATSON. The Rat Brain in Stereotaxic Coordinates. Orlando, FL: Academic, 1986. 18. RANCK, J. B. Which elements are excited in electrical stimulation of mammalian central nervous system: a review. Brain Res. 98: 417-440,1975. 19. SCHEUER, 20. 21. 22.
23.
J., AND C. M. TIPTON. Cardiovascular adaptations to physical training. Annu. Rev. Physiol. 39: 221-251, 1977. SHIK, M. L., AND G, N. ORLOVSKY. Neurophysiology of locomotor automatism. Physiol. Rev. 56: 465-500, 1976. SKINNER, R. J., AND E. GARCIA-RILL. The mesencephalic locomotor region (MLR) in the rat. Bruin Res. 323: 385-389,1984, SUNDARAM, K., J. MURAGAIAN, A. KRIEGER, AND H. SAPRU. Microinjections of cholinergic agonists into the intermediolateral cell column of the spinal cord at T,-T, increase heart rate and contractility. Brain Res. 503: 22-31, 1989. TAYLOR, I? Neuromuscular blocking agents. In: The Pharmacological Basis of Therapeutics, edited by A. G. Gillman, L. S. Goodman, T. W. Rall, and F. Murad. New York: Macmillan, 1985, chapt. 11, p.
230. 24. WARD,
D. G. Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure. Brain Res. 462: 383390,1988,
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