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Concomitant regulation of running activity and metabolic change by the ventromedial nucleus of the hypothalamus
BRAIN RESEARCH ELSEVIER
Brain Research 642 (1994) 290-296
Research Report
Concomitant regulation of running activity and metabolic change by the ventromedial nucleus of the hypothalamus Kazumi Narita, Masugi Nishihara, Michio Takahashi * Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113, Japan (Accepted 21 December 1993)
Abstract
The aim of this study was to elucidate the involvement of kainate (KA) type glutaminergic, GABAergic and adrenergic receptors in the ventromedial nucleus of the hypothalamus (VMH) in inducing running activity and metabolic adaptations. Injection of either KA or bicuculline methiodide (BM), a GABA A receptor antagonist, into the VMH of conscious rats resulted in an increase in plasma glucose, norepinephrine, epinephrine and corticosterone, as well as running activity. KA or BM increased plasma glucose and catecholamines even under urethane anesthesia. Co-injection of either a- or/3-adrenergic receptor antagonist, i.e. phentolamine or timolol, respectively, with KA into the VMH of conscious rats elicited only a slight increase in plasma glucose and catecholamines, though it successfully induced hyper-running. However, plasma corticosterone was higher in the animals injected with adrenergic blockers, suggesting that an insufficient supply of energy substrates would enhance the activity of the hypothalamo-pituitary-adrenal system. We conclude that: (1) KA type glutaminergic and GABAergic receptors in the VMH are involved in regulating running activity and the sympathetic nervous system; (2) the brain noradrenergic system may mediate the KA action on the sympathetic nervous system. Key words: Ventromedial nucleus of the hypothalamus; Running activity; Blood glucose; Kainate; GABA; Norepinephrine
1. Introduction
During exercise such as running, the sympathetic nervous and the hypothalamo-pituitary-adrenal (HPA) systems are activated to increase the energy substrates in the blood [3]. The activation of these systems includes an increase in the concentrations of norepinephrine (NE), epinephrine (E), and glucocorticoid in plasma. These changes in peripheral hormonal levels are known to be under the control of the hypothalamus including the ventromedial nucleus of the hypothalamus (VMH) [4,16]. Further, the central noradrenergic system is also suggested to be involved in regulating energy metabolism [8,18]. The blockade of both a- and fl-adrenergic transmission in the V M H during exercise has been shown to decrease the plasma levels of N E and E [15], with a resultant decrease in the plasma
* Corresponding author. Fax: (81) (3) 3815-4266. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) 0 0 0 2 1 - 4
concentrations of glucose and free fatty acid (FFA) [14]. It is generally accepted that changes in energy metabolism during exercise are caused by a feedback mechanism, depending on the metabolic demands of the exercising muscles and the ambient glucose and FFA concentrations [1,2,5,6]. However, there is increasing evidence that the central nervous system commands the initiation and regulation of the mobilization of energy substrates independently of the feedback mechanisms [13]. For example, it was reported that, at the onset of exercise, an excess energy substrate mobilization occurred irrespective of the peripheral glucose concentration [17]. Although this 'central command' mechanism is suggested to reside in the limbic system [13], knowledge of the central control of the metabolism during exercise is still limited. Recently, we have revealed that the VMH is involved in inducing running activity [10,21,22]. Injection of bicuculline methiodide (BM), G A B A A receptor antagonist [22], or kainate (KA), a KA type glutaminergic
K Narita et al./Brain Research 642 (1994) 290-296
receptor agonist [10], into the VMH of the rat exclusively elicited running activity. Furthermore, the presynaptic GABAergic system inhibiting glutamate release in the VMH seems to be involved in the inhibition of the spontaneous occurrence of running activity [10]. Since the VMH is involved in metabolic changes during exercise as mentioned above, we hypothesize that the neuronal system in the VMH which controls the running activity concomitantly commands the mobilization of energy substrates to support the intended running activity. To examine this hypothesis, the present study was performed to elucidate the involvement of KA type glutaminergic, GABAergic and adrenergic receptors in controlling energy metabolism during hyper-running originating in the VMH.
2. Materials and methods 2.1. Animals Male Wistar strain rats weighing 280-320 g were maintained under controlled conditions of room temperature (23+1°C) and lighting (lights on 05.00-19.00 h). Laboratory chow diet and water were available ad libitum. The animals were subjected to hypothalamic implantation of cannulae as described below, and kept in individual cages until the day of the experiment. After hypothalamic implantation, they were handled and habituated to the experimental room every day until they were subjected to the following experiments. At the day of experiments, food was removed two hours before the experiments.
2.Z Vehicle and drugs Water-absorbent polymer (WAP) (Sanwet IM-1000, Sanyo Kasei Co., Kyoto), which consisted of bridged amylogen, was allowed to form a fully swollen hydrogel in physiological saline in this study [10]. When used as a drug injection vehicle, WAP was swollen in physiological saline containing KA (20, 100 and 500/zM), or BM (500/zM). Except for the experiment of dose-dependency for KA, the dose of KA used in this study was 100/zM. KA (100 ~M) was also absorbed in WAP in combination with a KA antagonist 6,7-dinitroquioxalline2,3-dione (DNQX; 1 mM), an a-adrenergic receptor antagonist phentolamine (Phent; 4.5 raM), a /3-adrenergic receptor antagonist timolol (Timol; 4.5 mM) or with GABA (25 mM). All drugs except WAP were purchased from Sigma Chemical (St. Louis, MO).
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microsyringe with an aid of polyethylene tubing. Drug injection procedures were performed without disturbing the animal movement. Following the injection, the cannula was kept in place for an additional 1 min and then replaced by the stylet. In all the experiments, the single injection of each drug in WAP of 1.5/zl was done. Injection of this volume of WAP containing KA in the VMH has been shown to induce hyper-running in a region-specific manner [10].
2.4. Implantation of jugular catheter and blood sampling One day before the experiments, the animals were provided with a silastic catheter inserted through the jugular vein to reach the right atrium under ether anesthesia. The distal end of the catheter was tunneled subcutaneously to the back of the neck. All experiments were performed between 10.00 and 16.00 h. A 350 ~.1 blood sample was taken through the jugular catheter for the determination of plasma glucose, NE, E and corticosterone. An equal volume of heparinized saline (10 IU/ml) was replaced after each sampling. The time of the start of drug injection into the VMH was designated 0 min, and the injection was completed within 1 min. In the experiment with conscious animals, blood samples were taken at - 2 , 10, 20, 30, 60, 120 and 180 min. In the experiment with anesthetized rats, animals were anesthetized with urethane (1.0 g/kg i.p.) at - 3 0 rain. Blood samples were taken at - 3 2 , - 2 , 10, 20, 30, 60, 120 and 180 min. In the experiment examining dose-dependency and regional-specificity for KA in inducing activation of the sympa= thetic nervous system, blood samples were taken at - 2 and 30 min.
2. 5. Chemical determination The blood samples were immediately transferred to a chilled (0°C) centrifuge tube and centrifuged at 4°C. 20 ~1 of the plasma was used for the glucose assay, 20/~1 for corticosterone assay and 100 p.1 for the determination of NE and E. Plasma glucose was measured with a commercial kit (Wako, Tokyo). Plasma corticosterone was determined by radioimmunoassay with a specific antibody generated in this laboratory by corticosterone conjugated to bovine serum albumin as an antigen. The relative cross-reactivities of the antibody to testosterone, progesterone and deoxycorticosterone were less than 0.1%, 4% and 10%, respectively. For the corticosterone assay, intraand interassay coefficients of variance (CVs) were 5.3% and 7.2%, respectively, and sensitivity was 1 /~g/dl in plasma. Plasma NE and E were determined by high-pressure liquid chromatography in combination with electrochemical detection (HPLC-ECD, Eicom, Kyoto). As the internal standard, 3,4-dihydroxybenzylamine was used in each sample. The plasma was deproteinized, and the catecholamines were purified with activated alumina and then injected into the HPLCECD system. Detection limits for NE and E were 25 pg/ml of plasma and the interassay CV was 3.5%.
2.6. Analysis of locomotor activity 2.3. Implantation of Brain Cannula and Drug Injection One week before the experiments, a chronic stainless steel cannula (0.6 mm o.d., 0.4 mm i.d., 15 mm length) for drug injection was stereotaxically implanted unilaterally into the VMH according to the coordinates given in the atlas of Pellegrino and Cushman [11] during pentobarbital anesthesia (45 mg/kg i.p.). The cannula was fixed to the skull with anchor screws and dental cement. A sterile stainless steel stylet, 0.3 mm in diameter, was inserted into the cannula to prevent the lumen from clotting. Each drug trapped in WAP was injected into the VMH by means of a sterile injection cannula (0.3 mm o.d., 0.15 mm i.d., 25 mm length) which extended 0.2 mm beyond the tip of the guide cannula. The distal end of the injection cannula was connected to a 10-p.1
The locomotor activity of the animals was analyzed by means of a tremor detecting system made in this laboratory. The tremor detector was composed of two parallel electrodes and mercury. When the mercury contacted the two electrodes simultaneously due to the tremor induced by the movement of the rat, a flow of electricity occurred and the output was analyzed and stored in a microcomputer. In the experiments with conscious animals, the rats in individual cages where they had been kept were put on the system at - 3 0 min and analysis of locomotor activity was continued until the end of the experiment. As for the evaluation of the running activity of the rats, this system gave virtually the same results as those obtained with the running wheel system that we used in previous experiments [10,221.
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2. Z Histology At the end of the experiment, each animal was anesthetized with ether and sacrificed by decapitation, and the brain was excised and frozen at -20°C. Coronal sections (25 /xm thick) were cut on a freezing microtome and stained with thionin, and the injection site was identified by microscopic histological examination. Animals with the implantation sites outside of the VMH were excluded from the results. 2.8. Statistics The data were first statistically analyzed by analysis of variance. In experiments with conscious animals, differences between the total counts of running activity were further analyzed by Duncan's multiple range test. In all experiments, differences between the level of plasma substrate measured at a certain time during the experiments and the basal value for the - 2 min samples in the vehicle injected group were further analyzed by Duncan's multiple range test. Unpaired t-test was applied at each sample point to determine the difference between the level of plasma components in animals injected with drugs and that in animals injected with vehicle. Unpaired t-test was also applied at each sample point to analyze the difference between the level of plasma components in the animals in which DNQX, GABA, Phent or Timol was co-injected with KA and those .in the animals in which KA alone was injected. Differences were considered to be significant at P < 0.05. In the experiment to determine the regional specificity of KA in activating the sympathetic nervous system, the effects of KA were considered to be effective if both plasma NE and E levels at 30 min exceeded mean + 2S.D. of the values of vehicle injected animals.
vehicle injected rats showed no significant increase in running activity from the pretreatment level. The effect of drugs injection on metabolic changes were shown in Fig. 2. Vehicle injected animals did not show significant change in any of the parameters in plasma in this experiment. Injection of KA into the VMH elicited significant increases in plasma glucose, NE and E concentrations during almost the entire 3-h period, with peak values between 30 and 60 min. The plasma corticosterone level also increased between 60 and 180 min in KA injected animals. In the rats injected with DNQX in combination with KA, similar plasma parameters to those in the control group were observed. Injection of BM into the VMH elicited significant increases in plasma glucose and E, with peak values between 10 and 20 min. As for the corticosterone level, however, BM injection produced similar change to vehicle injection. Though the plasma
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3. Results
3.1. Experiment 1 First, to evaluate the involvement of KA type glutaminergic and GABAergic receptors in the VMH in regulating the mobilization of the energy substrate during hyper-running, BM and KA alone or in combination with DNQX were injected into the VMH of conscious animals. Further, to examine the involvement of the central adrenergic system in the metabolic changes and running activity induced by KA injection, Phent or Timol was injected simultaneously with KA into the VMH of conscious rats. The effects of drug injection on running activity are summarized in Fig. 1A in terms of the total counts for a 3-h period. Typical patterns of the time course of locomotor activity following drug injection, as depicted by the microcomputer, are shown in Fig. lB. The injection of KA into the VMH induced the hyper-running, and this effect of KA on running activity was completely blocked by DNQX. BM injected into the VMH also significantly increased running activity. These results are consistent with our previous ones. Simultaneous injection of either Phent or Timol with KA did not significantly affect the level of running activity induced by the injection of KA alone. The
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I£ Narita et al. ~Brain Research 642 (1994) 290-296
N E level rose from the basal value in rats injected with BM, the difference did not reach statistical significance. Co-injection of either Phent or Timol with K A into the V M H elicited a slight, but not significant increase in plasma glucose. The co-injection increased plasma E, but not NE, significantly, but the increases were much smaller than those in animals injected with KA. Conversely, the plasma corticosterone level was significantly higher in animals injected with Phent or Timol concomitantly with KA than in those injected with either K A or vehicle alone. 3.2. Experiment 2
To evaluate further the involvement of K A type glutaminergic receptors in the V M H in regulating energy metabolism, various doses of K A were injected into the V M H under urethane anesthesia. U n d e r this condition, the effect of exercise on metabolic change
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should be eliminated. In the - 2 min values of plasma N E and E, there were no significant difference among each experimental group. The differences of N E and E concentrations between - 2 min and 30 min were shown in Fig. 3. While the injection of the vehicle induced no change in both N E and E, that of K A increased the plasma concentrations of both N E and E in a dose-dependent manner up to the concentration of 100/xM. Fig. 4 shows the site where the injection of 100 ~ M K A successfully induced an increase in t h e plasma N E and E level. The rats with K A injected outside the V M H showed no significant changes in the plasma N E and E levels. BM and K A alone or in combination with G A B A were injected into the V M H under urethane anesthesia (Fig. 5). The urethane anesthesia did not cause any significant differences between the parameters for plasma obtained before and after treatment in the vehicle injected group. Injection of K A into the V M H
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Fig. 2. Effects of the injections of vehicle, BM (500/xM), KA (100 p,M) alone or in combination with DNQX (1 mM) (A), or KA (100/zM) in combination with either Phent (4.5 raM) or Timol (4.5 mM) (B) into the VMH of conscious rats on plasma concentrations of glucose (upper), corticosterone (middle upper), NE (middle lower) and E (lower). The qhanges of plasma components of vehicle or KA injected rat are shown in both A and B. Each symbol indicates the mean + S.E.M., and the number of animals in each group is given in parentheses. Solid symbols indicate the values that are significantlydifferent from the control value (P < 0.05). * P < 0.05 vs. injection of KA alone.
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elicited significant increases in plasma glucose, NE and E concentrations during almost the entire 3-h period, with peak values between 20 and 60 min. Plasma corticosterone also tended to increase, but differences
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Fig. 5. Effects of the injections of vehicle, BM (500 /zM), KA (100 tzM) alone or in combination with GABA (25 mM) into the VMH under urethane anesthesia on plasma concentrations of glucose (upper), corticosterone (middle upper), NE (middle lower) and E (lower). Each symbol indicates the mean_+ S.E.M., and the number of animals in each group is given in parentheses. Solid symbols indicate the values that are significantly different from the control value ( P < 0.05). * P < 0.05 vs. injection of KA alone.
reaching statistical significance were observed only at 120 and 180 min. Following a simultaneous injection of GABA with KA into the VMH, significant increases in plasma glucose, NE and E, but not in corticosterone, levels were observed. However, the increases in plasma glucose, NE and E in animals injected with both GABA and KA were significantly smaller than those in animals injected with KA only. In animals injected with BM, plasma glucose, NE and E had significantly increased by 60 min after injection. The plasma corticosterone level was not affected by BM injection into the VMH.
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Fig. 4. Schematic coronal section of the rat brain adapted from the atlas by Pellegrino and Cushman showing the injection sites of KA (100 ~M). Filled circle, effective site for the activation of the sympathetic nervous system; open circle, ineffective site.
4. D i s c u s s i o n
We have previously shown that the effective site in inducing running activity by KA was limited only in the
I~ Narita et al. / Brain Research 642 (1994) 290-296
VMH when KA was administered in WAP [10]. The solution trapped in the gel is considered to be stoichiometrically exchanged with the fluid outside and, therefore, the drug delivery rate from WAP must be much slower than that from a simple water solution. Furthermore, WAP is deposited at the injection site, while a simple water solution easily spreads out. Thus, an effective distribution area of the drug from WAP is much restricted. The results of experiment 1 showed that the injection of either KA or BM into the VMH elicited increases in the concentrations of plasma catecholamines and glucose, as well as running activity, though the former was more potent than the latter. As shown in experiment 2, the effect of KA was dose-dependent and regional-specific. Further, the effect of KA was significantly attenuated by concomitant injection with DNQX, suggesting that the effect of KA on both metabolic changes and running activity was mediated by specific KA type glutaminergic receptors in the VMH. Noradrenergic system in the VMH was shown to be involved in the metabolic changes during exercise [15]. In experiment 1, although the KA-induced running was not affected by simultaneous injection of either Phent or Timol, the increases in plasma catecholamines were profoundly attenuated. These observation suggest that the noradrenergic transmission in the VMH mediates the stimulatory effect of KA on the activity of the sympathetic nervous system, but not on the running activity. The significant increase in plasma corticosterone following KA injection into the VMH in conscious and anesthetized animals was observed 60-180 min and 120-180 min, respectively. This is compatible with a report that plasma corticosterone increases during exercise, and this increase is attributed to the feedback information resulting from the running activity [3]. It has been reported that the injection of neither a- nor fl-adrenergic blockers into the VMH changed the level of plasma corticosterone [15]. In the present study, however, the plasma concentration of corticosterone of the rats injected KA with either Phent or Timol was significantly higher than that of the rats injected KA alone. To provide working muscles and the central nervous system with energy substrates, the HPA system might be activated further to compensate for the decreased activity of the sympathetic nervous system. In experiment 2, to prevent the occurrence of exercise, the animals were anesthetized with urethane. Although urethane itself did not influence the level of plasma glucose, NE and E, plasma levels of those substances were markedly increased by the injection of KA or BM into the VMH. These results suggest that either stimulation of the KA type glutaminergic receptors or blockade of GABAergic receptors in the VMH
295
can facilitate the activity of the sympathetic nervous system without the occurrence of the running activity. Although different response in plasma E level between conscious and anesthetized animals was observed after KA injection, the reason of this difference is currently unknown. In previous reports, it was suggested that GABA presynaptically inhibited the glutaminergic system which could induce hyper-running in the VMH [10]. In experiment 2, GABA was simultaneously injected with KA to examine the effect of GABA on metabolic changes. The results indicated that GABA significantly, but not completely, attenuated the effect of KA on the levels of plasma catecholamines and glucose. Although the dose of GABA used in the present study was relatively high, it was shown that GABA at this dose had no inhibitory effect on KA-induced running activity [10], suggesting that GABA does not have non-specific inhibitory effect. It is therefore possible that G A B A has, at least in part, direct inhibitory effect on the neurons involved in stimulating the sympathetic nervous system ih the VMH. The VMH plays an important role in regulating energy metabolism [16,19] as well as instinctive behavior [9,12]. A series of experiments in this laboratory have provided evidence that the VMH also participates in running activity [10,21,22]. The present study suggests that the VMH also issues commands to supply energy substrates during the hyper-running and thus can integrate both exercise and energy metabolism at the same time. It is possible that the VMH is involved in the 'central command' and control both running activity and metabolic change. In summary we propose that: (1) KA type glutaminergic receptors in the VMH play a role in inducing running activity and, simultaneously, in activating the sympathetic nervous system with a resultant increase in blood glucose; (2) the GABAergic system in the VMH suppresses the activity of the sympathetic nervous system and running activity; (3) the noradrenergic system in the VMH participates in the KA action on the sympathetic nervous system.
5. Acknowledgements This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
6. References [1] Bagby,G.J., Green, H.J., Katsuta,S. and Gollnick,P.D., Glycogen depletion in exercisingrats infusedwith glucose,lactate, or pyruvate, J. Appl. Physiol., 45 (1978) 425-429.
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[2] Felig, P. and Wahren, J., Role of insulin and glucagon in the regulation of hepatic glucose production during exercise, Diabetes, 28, Suppl. 1 (1979) 71-75. [3] Galbo, H., Hormonal and Metabolic Adaptation to Exercise, Georg Thieme, New York, 1983. [4] Gillies, G.E., and Lowry, P.J., Adrenal function. In S.L. Lightman and B.J. Everitt (Eds.), Neuroendocrinology, Blackwell, Oxford, 1986, pp. 360-388. [5] Issekutz, B.J., Effects of glucose infusion on hepatic and muscle glycogenolysis in exercising dogs, Am. J. Physiol., 240 (1981) E451-E457. [6] Jenkins, A.B., Chisholm, D.J., James, D.E., Ho, K.Y. and Kraegen, E.W., Exercise-induced hepatic glucose output is precisely sensitive to the rate of systemic glucose supply, Metabolism, 34 (1985) 431-436. [7] Luiten, P.G.M., Ter Horst, G.J. and Steffens, A.B., The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism, Prog. Neurobiol., 28 (1987) 1-54. [8] Matsushita, H. and Shimazu, T., Chemical coding of the hypothalamic neurones in metabolic control. 2. Norepinephrinesensitive neurones and glycogen breakdown in liver, Brain Res., 183 (1980) 79-87. [9] Nakao, H., Emotional behavior produced by hypothalamic stimulation, Am. J. Physiol., 194 (1958) 411-418. [10] Narita, K., Yokawa, T., Nishihara, M. and Takahashi, M., Interaction between excitatory and inhibitory amino acids in the ventromedial nucleus of the hypothalamus in inducing hyperrunning, Brain Res., 603 (1993) 243-247. [11] PeUegrino, L.J. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Meredith, New York, 1967. [12] Pfaff, D.W. and Sakuma, Y., Facilitation of the lordosis reflex of female rats from the ventromedial nucleus of the hypothalamus, J. Physiol., 288 (1979) 189-202. [13] Scheurink, A.J.W. and Steffens, A.B., Central and peripheral
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
control of sympathoadrenal activity and energy metabolism in rats, Physiol. Behav., 48 (1990) 909-920. Scheurink, A.J.W., Steffens, A.B. and Benthem, L., Central and peripheral adrenoceptors affect glucose, free fatty acids, and insulin in exercising rats, Am. J. Physiol., 255 (1998) R547-R556. Scheurink, A.J.W., Steffens, A.B. and Gaykema, R.P.A., Hypothalamic adrenoceptors mediate sympathoadrenal activity in exercising rats, Am. J. Physiol., 259 (1990) R470-R477. Shimazu, T., Intermediate metabolism. In S.L. Lightman and B.J. Everitt (Eds.), Neuroendocrinology, Blackwell, Oxford, 1986, pp. 304-330. Sonne, B. and Galbo, H., Carbohydrate metabolism during and after exercise in rats: studies with radioglucose, J. AppL Physiol., 59 (1985) 1627-1639. Steffens, A.B., Damsma, G., Gugten, J.V.D. and Luiten, P.G.M., Circulating free fatty acids, insulin, and glucose during chemical stimulation of hypothalamus in rats, Am. J. Physiol., 247 (1984) E765-E771. Steffens, A.B. and Strubbe, J.H., CNS regulation of glucagon secretion. In A.J. Szabo (Eds.), Advances in Metabolic Disorders. CNS Regulation of Carbohydrate Metabolism, Vol. 10, Academic, New York, 1983, pp. 221-258. Swanson, L.W. and Hartman, B.K., The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-/3-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506. Yokawa, T., Mitsushima, D., Itoh, C., Konishi, H., Shiota, K. and Takahashi, M., The ventromedial nucleus of the hypothalamus outputs long-lasting running in rats, Physiol. Behav., 46 (1989) 713-717. Yokawa, T., Shiota, K. and Takahashi, M., Hyper-running activity originating from the hypothalamus is blocked by GABA, Physiol. Behav., 47 (1990) 1261-1264.
Brain Research 642 (1994) 290-296
Research Report
Concomitant regulation of running activity and metabolic change by the ventromedial nucleus of the hypothalamus Kazumi Narita, Masugi Nishihara, Michio Takahashi * Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113, Japan (Accepted 21 December 1993)
Abstract
The aim of this study was to elucidate the involvement of kainate (KA) type glutaminergic, GABAergic and adrenergic receptors in the ventromedial nucleus of the hypothalamus (VMH) in inducing running activity and metabolic adaptations. Injection of either KA or bicuculline methiodide (BM), a GABA A receptor antagonist, into the VMH of conscious rats resulted in an increase in plasma glucose, norepinephrine, epinephrine and corticosterone, as well as running activity. KA or BM increased plasma glucose and catecholamines even under urethane anesthesia. Co-injection of either a- or/3-adrenergic receptor antagonist, i.e. phentolamine or timolol, respectively, with KA into the VMH of conscious rats elicited only a slight increase in plasma glucose and catecholamines, though it successfully induced hyper-running. However, plasma corticosterone was higher in the animals injected with adrenergic blockers, suggesting that an insufficient supply of energy substrates would enhance the activity of the hypothalamo-pituitary-adrenal system. We conclude that: (1) KA type glutaminergic and GABAergic receptors in the VMH are involved in regulating running activity and the sympathetic nervous system; (2) the brain noradrenergic system may mediate the KA action on the sympathetic nervous system. Key words: Ventromedial nucleus of the hypothalamus; Running activity; Blood glucose; Kainate; GABA; Norepinephrine
1. Introduction
During exercise such as running, the sympathetic nervous and the hypothalamo-pituitary-adrenal (HPA) systems are activated to increase the energy substrates in the blood [3]. The activation of these systems includes an increase in the concentrations of norepinephrine (NE), epinephrine (E), and glucocorticoid in plasma. These changes in peripheral hormonal levels are known to be under the control of the hypothalamus including the ventromedial nucleus of the hypothalamus (VMH) [4,16]. Further, the central noradrenergic system is also suggested to be involved in regulating energy metabolism [8,18]. The blockade of both a- and fl-adrenergic transmission in the V M H during exercise has been shown to decrease the plasma levels of N E and E [15], with a resultant decrease in the plasma
* Corresponding author. Fax: (81) (3) 3815-4266. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) 0 0 0 2 1 - 4
concentrations of glucose and free fatty acid (FFA) [14]. It is generally accepted that changes in energy metabolism during exercise are caused by a feedback mechanism, depending on the metabolic demands of the exercising muscles and the ambient glucose and FFA concentrations [1,2,5,6]. However, there is increasing evidence that the central nervous system commands the initiation and regulation of the mobilization of energy substrates independently of the feedback mechanisms [13]. For example, it was reported that, at the onset of exercise, an excess energy substrate mobilization occurred irrespective of the peripheral glucose concentration [17]. Although this 'central command' mechanism is suggested to reside in the limbic system [13], knowledge of the central control of the metabolism during exercise is still limited. Recently, we have revealed that the VMH is involved in inducing running activity [10,21,22]. Injection of bicuculline methiodide (BM), G A B A A receptor antagonist [22], or kainate (KA), a KA type glutaminergic
K Narita et al./Brain Research 642 (1994) 290-296
receptor agonist [10], into the VMH of the rat exclusively elicited running activity. Furthermore, the presynaptic GABAergic system inhibiting glutamate release in the VMH seems to be involved in the inhibition of the spontaneous occurrence of running activity [10]. Since the VMH is involved in metabolic changes during exercise as mentioned above, we hypothesize that the neuronal system in the VMH which controls the running activity concomitantly commands the mobilization of energy substrates to support the intended running activity. To examine this hypothesis, the present study was performed to elucidate the involvement of KA type glutaminergic, GABAergic and adrenergic receptors in controlling energy metabolism during hyper-running originating in the VMH.
2. Materials and methods 2.1. Animals Male Wistar strain rats weighing 280-320 g were maintained under controlled conditions of room temperature (23+1°C) and lighting (lights on 05.00-19.00 h). Laboratory chow diet and water were available ad libitum. The animals were subjected to hypothalamic implantation of cannulae as described below, and kept in individual cages until the day of the experiment. After hypothalamic implantation, they were handled and habituated to the experimental room every day until they were subjected to the following experiments. At the day of experiments, food was removed two hours before the experiments.
2.Z Vehicle and drugs Water-absorbent polymer (WAP) (Sanwet IM-1000, Sanyo Kasei Co., Kyoto), which consisted of bridged amylogen, was allowed to form a fully swollen hydrogel in physiological saline in this study [10]. When used as a drug injection vehicle, WAP was swollen in physiological saline containing KA (20, 100 and 500/zM), or BM (500/zM). Except for the experiment of dose-dependency for KA, the dose of KA used in this study was 100/zM. KA (100 ~M) was also absorbed in WAP in combination with a KA antagonist 6,7-dinitroquioxalline2,3-dione (DNQX; 1 mM), an a-adrenergic receptor antagonist phentolamine (Phent; 4.5 raM), a /3-adrenergic receptor antagonist timolol (Timol; 4.5 mM) or with GABA (25 mM). All drugs except WAP were purchased from Sigma Chemical (St. Louis, MO).
291
microsyringe with an aid of polyethylene tubing. Drug injection procedures were performed without disturbing the animal movement. Following the injection, the cannula was kept in place for an additional 1 min and then replaced by the stylet. In all the experiments, the single injection of each drug in WAP of 1.5/zl was done. Injection of this volume of WAP containing KA in the VMH has been shown to induce hyper-running in a region-specific manner [10].
2.4. Implantation of jugular catheter and blood sampling One day before the experiments, the animals were provided with a silastic catheter inserted through the jugular vein to reach the right atrium under ether anesthesia. The distal end of the catheter was tunneled subcutaneously to the back of the neck. All experiments were performed between 10.00 and 16.00 h. A 350 ~.1 blood sample was taken through the jugular catheter for the determination of plasma glucose, NE, E and corticosterone. An equal volume of heparinized saline (10 IU/ml) was replaced after each sampling. The time of the start of drug injection into the VMH was designated 0 min, and the injection was completed within 1 min. In the experiment with conscious animals, blood samples were taken at - 2 , 10, 20, 30, 60, 120 and 180 min. In the experiment with anesthetized rats, animals were anesthetized with urethane (1.0 g/kg i.p.) at - 3 0 rain. Blood samples were taken at - 3 2 , - 2 , 10, 20, 30, 60, 120 and 180 min. In the experiment examining dose-dependency and regional-specificity for KA in inducing activation of the sympa= thetic nervous system, blood samples were taken at - 2 and 30 min.
2. 5. Chemical determination The blood samples were immediately transferred to a chilled (0°C) centrifuge tube and centrifuged at 4°C. 20 ~1 of the plasma was used for the glucose assay, 20/~1 for corticosterone assay and 100 p.1 for the determination of NE and E. Plasma glucose was measured with a commercial kit (Wako, Tokyo). Plasma corticosterone was determined by radioimmunoassay with a specific antibody generated in this laboratory by corticosterone conjugated to bovine serum albumin as an antigen. The relative cross-reactivities of the antibody to testosterone, progesterone and deoxycorticosterone were less than 0.1%, 4% and 10%, respectively. For the corticosterone assay, intraand interassay coefficients of variance (CVs) were 5.3% and 7.2%, respectively, and sensitivity was 1 /~g/dl in plasma. Plasma NE and E were determined by high-pressure liquid chromatography in combination with electrochemical detection (HPLC-ECD, Eicom, Kyoto). As the internal standard, 3,4-dihydroxybenzylamine was used in each sample. The plasma was deproteinized, and the catecholamines were purified with activated alumina and then injected into the HPLCECD system. Detection limits for NE and E were 25 pg/ml of plasma and the interassay CV was 3.5%.
2.6. Analysis of locomotor activity 2.3. Implantation of Brain Cannula and Drug Injection One week before the experiments, a chronic stainless steel cannula (0.6 mm o.d., 0.4 mm i.d., 15 mm length) for drug injection was stereotaxically implanted unilaterally into the VMH according to the coordinates given in the atlas of Pellegrino and Cushman [11] during pentobarbital anesthesia (45 mg/kg i.p.). The cannula was fixed to the skull with anchor screws and dental cement. A sterile stainless steel stylet, 0.3 mm in diameter, was inserted into the cannula to prevent the lumen from clotting. Each drug trapped in WAP was injected into the VMH by means of a sterile injection cannula (0.3 mm o.d., 0.15 mm i.d., 25 mm length) which extended 0.2 mm beyond the tip of the guide cannula. The distal end of the injection cannula was connected to a 10-p.1
The locomotor activity of the animals was analyzed by means of a tremor detecting system made in this laboratory. The tremor detector was composed of two parallel electrodes and mercury. When the mercury contacted the two electrodes simultaneously due to the tremor induced by the movement of the rat, a flow of electricity occurred and the output was analyzed and stored in a microcomputer. In the experiments with conscious animals, the rats in individual cages where they had been kept were put on the system at - 3 0 min and analysis of locomotor activity was continued until the end of the experiment. As for the evaluation of the running activity of the rats, this system gave virtually the same results as those obtained with the running wheel system that we used in previous experiments [10,221.
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2. Z Histology At the end of the experiment, each animal was anesthetized with ether and sacrificed by decapitation, and the brain was excised and frozen at -20°C. Coronal sections (25 /xm thick) were cut on a freezing microtome and stained with thionin, and the injection site was identified by microscopic histological examination. Animals with the implantation sites outside of the VMH were excluded from the results. 2.8. Statistics The data were first statistically analyzed by analysis of variance. In experiments with conscious animals, differences between the total counts of running activity were further analyzed by Duncan's multiple range test. In all experiments, differences between the level of plasma substrate measured at a certain time during the experiments and the basal value for the - 2 min samples in the vehicle injected group were further analyzed by Duncan's multiple range test. Unpaired t-test was applied at each sample point to determine the difference between the level of plasma components in animals injected with drugs and that in animals injected with vehicle. Unpaired t-test was also applied at each sample point to analyze the difference between the level of plasma components in the animals in which DNQX, GABA, Phent or Timol was co-injected with KA and those .in the animals in which KA alone was injected. Differences were considered to be significant at P < 0.05. In the experiment to determine the regional specificity of KA in activating the sympathetic nervous system, the effects of KA were considered to be effective if both plasma NE and E levels at 30 min exceeded mean + 2S.D. of the values of vehicle injected animals.
vehicle injected rats showed no significant increase in running activity from the pretreatment level. The effect of drugs injection on metabolic changes were shown in Fig. 2. Vehicle injected animals did not show significant change in any of the parameters in plasma in this experiment. Injection of KA into the VMH elicited significant increases in plasma glucose, NE and E concentrations during almost the entire 3-h period, with peak values between 30 and 60 min. The plasma corticosterone level also increased between 60 and 180 min in KA injected animals. In the rats injected with DNQX in combination with KA, similar plasma parameters to those in the control group were observed. Injection of BM into the VMH elicited significant increases in plasma glucose and E, with peak values between 10 and 20 min. As for the corticosterone level, however, BM injection produced similar change to vehicle injection. Though the plasma
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3. Results
3.1. Experiment 1 First, to evaluate the involvement of KA type glutaminergic and GABAergic receptors in the VMH in regulating the mobilization of the energy substrate during hyper-running, BM and KA alone or in combination with DNQX were injected into the VMH of conscious animals. Further, to examine the involvement of the central adrenergic system in the metabolic changes and running activity induced by KA injection, Phent or Timol was injected simultaneously with KA into the VMH of conscious rats. The effects of drug injection on running activity are summarized in Fig. 1A in terms of the total counts for a 3-h period. Typical patterns of the time course of locomotor activity following drug injection, as depicted by the microcomputer, are shown in Fig. lB. The injection of KA into the VMH induced the hyper-running, and this effect of KA on running activity was completely blocked by DNQX. BM injected into the VMH also significantly increased running activity. These results are consistent with our previous ones. Simultaneous injection of either Phent or Timol with KA did not significantly affect the level of running activity induced by the injection of KA alone. The
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I£ Narita et al. ~Brain Research 642 (1994) 290-296
N E level rose from the basal value in rats injected with BM, the difference did not reach statistical significance. Co-injection of either Phent or Timol with K A into the V M H elicited a slight, but not significant increase in plasma glucose. The co-injection increased plasma E, but not NE, significantly, but the increases were much smaller than those in animals injected with KA. Conversely, the plasma corticosterone level was significantly higher in animals injected with Phent or Timol concomitantly with KA than in those injected with either K A or vehicle alone. 3.2. Experiment 2
To evaluate further the involvement of K A type glutaminergic receptors in the V M H in regulating energy metabolism, various doses of K A were injected into the V M H under urethane anesthesia. U n d e r this condition, the effect of exercise on metabolic change
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should be eliminated. In the - 2 min values of plasma N E and E, there were no significant difference among each experimental group. The differences of N E and E concentrations between - 2 min and 30 min were shown in Fig. 3. While the injection of the vehicle induced no change in both N E and E, that of K A increased the plasma concentrations of both N E and E in a dose-dependent manner up to the concentration of 100/xM. Fig. 4 shows the site where the injection of 100 ~ M K A successfully induced an increase in t h e plasma N E and E level. The rats with K A injected outside the V M H showed no significant changes in the plasma N E and E levels. BM and K A alone or in combination with G A B A were injected into the V M H under urethane anesthesia (Fig. 5). The urethane anesthesia did not cause any significant differences between the parameters for plasma obtained before and after treatment in the vehicle injected group. Injection of K A into the V M H
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Fig. 2. Effects of the injections of vehicle, BM (500/xM), KA (100 p,M) alone or in combination with DNQX (1 mM) (A), or KA (100/zM) in combination with either Phent (4.5 raM) or Timol (4.5 mM) (B) into the VMH of conscious rats on plasma concentrations of glucose (upper), corticosterone (middle upper), NE (middle lower) and E (lower). The qhanges of plasma components of vehicle or KA injected rat are shown in both A and B. Each symbol indicates the mean + S.E.M., and the number of animals in each group is given in parentheses. Solid symbols indicate the values that are significantlydifferent from the control value (P < 0.05). * P < 0.05 vs. injection of KA alone.
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elicited significant increases in plasma glucose, NE and E concentrations during almost the entire 3-h period, with peak values between 20 and 60 min. Plasma corticosterone also tended to increase, but differences
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reaching statistical significance were observed only at 120 and 180 min. Following a simultaneous injection of GABA with KA into the VMH, significant increases in plasma glucose, NE and E, but not in corticosterone, levels were observed. However, the increases in plasma glucose, NE and E in animals injected with both GABA and KA were significantly smaller than those in animals injected with KA only. In animals injected with BM, plasma glucose, NE and E had significantly increased by 60 min after injection. The plasma corticosterone level was not affected by BM injection into the VMH.
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Fig. 4. Schematic coronal section of the rat brain adapted from the atlas by Pellegrino and Cushman showing the injection sites of KA (100 ~M). Filled circle, effective site for the activation of the sympathetic nervous system; open circle, ineffective site.
4. D i s c u s s i o n
We have previously shown that the effective site in inducing running activity by KA was limited only in the
I~ Narita et al. / Brain Research 642 (1994) 290-296
VMH when KA was administered in WAP [10]. The solution trapped in the gel is considered to be stoichiometrically exchanged with the fluid outside and, therefore, the drug delivery rate from WAP must be much slower than that from a simple water solution. Furthermore, WAP is deposited at the injection site, while a simple water solution easily spreads out. Thus, an effective distribution area of the drug from WAP is much restricted. The results of experiment 1 showed that the injection of either KA or BM into the VMH elicited increases in the concentrations of plasma catecholamines and glucose, as well as running activity, though the former was more potent than the latter. As shown in experiment 2, the effect of KA was dose-dependent and regional-specific. Further, the effect of KA was significantly attenuated by concomitant injection with DNQX, suggesting that the effect of KA on both metabolic changes and running activity was mediated by specific KA type glutaminergic receptors in the VMH. Noradrenergic system in the VMH was shown to be involved in the metabolic changes during exercise [15]. In experiment 1, although the KA-induced running was not affected by simultaneous injection of either Phent or Timol, the increases in plasma catecholamines were profoundly attenuated. These observation suggest that the noradrenergic transmission in the VMH mediates the stimulatory effect of KA on the activity of the sympathetic nervous system, but not on the running activity. The significant increase in plasma corticosterone following KA injection into the VMH in conscious and anesthetized animals was observed 60-180 min and 120-180 min, respectively. This is compatible with a report that plasma corticosterone increases during exercise, and this increase is attributed to the feedback information resulting from the running activity [3]. It has been reported that the injection of neither a- nor fl-adrenergic blockers into the VMH changed the level of plasma corticosterone [15]. In the present study, however, the plasma concentration of corticosterone of the rats injected KA with either Phent or Timol was significantly higher than that of the rats injected KA alone. To provide working muscles and the central nervous system with energy substrates, the HPA system might be activated further to compensate for the decreased activity of the sympathetic nervous system. In experiment 2, to prevent the occurrence of exercise, the animals were anesthetized with urethane. Although urethane itself did not influence the level of plasma glucose, NE and E, plasma levels of those substances were markedly increased by the injection of KA or BM into the VMH. These results suggest that either stimulation of the KA type glutaminergic receptors or blockade of GABAergic receptors in the VMH
295
can facilitate the activity of the sympathetic nervous system without the occurrence of the running activity. Although different response in plasma E level between conscious and anesthetized animals was observed after KA injection, the reason of this difference is currently unknown. In previous reports, it was suggested that GABA presynaptically inhibited the glutaminergic system which could induce hyper-running in the VMH [10]. In experiment 2, GABA was simultaneously injected with KA to examine the effect of GABA on metabolic changes. The results indicated that GABA significantly, but not completely, attenuated the effect of KA on the levels of plasma catecholamines and glucose. Although the dose of GABA used in the present study was relatively high, it was shown that GABA at this dose had no inhibitory effect on KA-induced running activity [10], suggesting that GABA does not have non-specific inhibitory effect. It is therefore possible that G A B A has, at least in part, direct inhibitory effect on the neurons involved in stimulating the sympathetic nervous system ih the VMH. The VMH plays an important role in regulating energy metabolism [16,19] as well as instinctive behavior [9,12]. A series of experiments in this laboratory have provided evidence that the VMH also participates in running activity [10,21,22]. The present study suggests that the VMH also issues commands to supply energy substrates during the hyper-running and thus can integrate both exercise and energy metabolism at the same time. It is possible that the VMH is involved in the 'central command' and control both running activity and metabolic change. In summary we propose that: (1) KA type glutaminergic receptors in the VMH play a role in inducing running activity and, simultaneously, in activating the sympathetic nervous system with a resultant increase in blood glucose; (2) the GABAergic system in the VMH suppresses the activity of the sympathetic nervous system and running activity; (3) the noradrenergic system in the VMH participates in the KA action on the sympathetic nervous system.
5. Acknowledgements This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
6. References [1] Bagby,G.J., Green, H.J., Katsuta,S. and Gollnick,P.D., Glycogen depletion in exercisingrats infusedwith glucose,lactate, or pyruvate, J. Appl. Physiol., 45 (1978) 425-429.
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[2] Felig, P. and Wahren, J., Role of insulin and glucagon in the regulation of hepatic glucose production during exercise, Diabetes, 28, Suppl. 1 (1979) 71-75. [3] Galbo, H., Hormonal and Metabolic Adaptation to Exercise, Georg Thieme, New York, 1983. [4] Gillies, G.E., and Lowry, P.J., Adrenal function. In S.L. Lightman and B.J. Everitt (Eds.), Neuroendocrinology, Blackwell, Oxford, 1986, pp. 360-388. [5] Issekutz, B.J., Effects of glucose infusion on hepatic and muscle glycogenolysis in exercising dogs, Am. J. Physiol., 240 (1981) E451-E457. [6] Jenkins, A.B., Chisholm, D.J., James, D.E., Ho, K.Y. and Kraegen, E.W., Exercise-induced hepatic glucose output is precisely sensitive to the rate of systemic glucose supply, Metabolism, 34 (1985) 431-436. [7] Luiten, P.G.M., Ter Horst, G.J. and Steffens, A.B., The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism, Prog. Neurobiol., 28 (1987) 1-54. [8] Matsushita, H. and Shimazu, T., Chemical coding of the hypothalamic neurones in metabolic control. 2. Norepinephrinesensitive neurones and glycogen breakdown in liver, Brain Res., 183 (1980) 79-87. [9] Nakao, H., Emotional behavior produced by hypothalamic stimulation, Am. J. Physiol., 194 (1958) 411-418. [10] Narita, K., Yokawa, T., Nishihara, M. and Takahashi, M., Interaction between excitatory and inhibitory amino acids in the ventromedial nucleus of the hypothalamus in inducing hyperrunning, Brain Res., 603 (1993) 243-247. [11] PeUegrino, L.J. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Meredith, New York, 1967. [12] Pfaff, D.W. and Sakuma, Y., Facilitation of the lordosis reflex of female rats from the ventromedial nucleus of the hypothalamus, J. Physiol., 288 (1979) 189-202. [13] Scheurink, A.J.W. and Steffens, A.B., Central and peripheral
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
control of sympathoadrenal activity and energy metabolism in rats, Physiol. Behav., 48 (1990) 909-920. Scheurink, A.J.W., Steffens, A.B. and Benthem, L., Central and peripheral adrenoceptors affect glucose, free fatty acids, and insulin in exercising rats, Am. J. Physiol., 255 (1998) R547-R556. Scheurink, A.J.W., Steffens, A.B. and Gaykema, R.P.A., Hypothalamic adrenoceptors mediate sympathoadrenal activity in exercising rats, Am. J. Physiol., 259 (1990) R470-R477. Shimazu, T., Intermediate metabolism. In S.L. Lightman and B.J. Everitt (Eds.), Neuroendocrinology, Blackwell, Oxford, 1986, pp. 304-330. Sonne, B. and Galbo, H., Carbohydrate metabolism during and after exercise in rats: studies with radioglucose, J. AppL Physiol., 59 (1985) 1627-1639. Steffens, A.B., Damsma, G., Gugten, J.V.D. and Luiten, P.G.M., Circulating free fatty acids, insulin, and glucose during chemical stimulation of hypothalamus in rats, Am. J. Physiol., 247 (1984) E765-E771. Steffens, A.B. and Strubbe, J.H., CNS regulation of glucagon secretion. In A.J. Szabo (Eds.), Advances in Metabolic Disorders. CNS Regulation of Carbohydrate Metabolism, Vol. 10, Academic, New York, 1983, pp. 221-258. Swanson, L.W. and Hartman, B.K., The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-/3-hydroxylase as a marker, J. Comp. Neurol., 163 (1975) 467-506. Yokawa, T., Mitsushima, D., Itoh, C., Konishi, H., Shiota, K. and Takahashi, M., The ventromedial nucleus of the hypothalamus outputs long-lasting running in rats, Physiol. Behav., 46 (1989) 713-717. Yokawa, T., Shiota, K. and Takahashi, M., Hyper-running activity originating from the hypothalamus is blocked by GABA, Physiol. Behav., 47 (1990) 1261-1264.