Neuroscience Vol. 34, No. 3, pp. 771-775, 1990 Printed in Great Britain
0306-4522/90 $3.00 + 0.00 Pergamon Press plc 0 1990 IBRO
EFFECT OF CORTICOTROPIN RELEASING HORMONE AND NEUROPEPTIDE Y ON ELECTROPHYSIOLOGICAL ACTIVITY OF SYMPATHETIC NERVES TO INTERSCAPULAR BROWN ADIPOSE TISSUE M. EGAWA, H. YOSHIMATSU and Department
of Medicine, of Southern
G. A. BRAY*
Section of Diabetes and Clinical Nutrition, USC School of Medicine, California, 2025 Zonal Avenue, Los Angeles, CA 90033, U.S.A.
University
Abstract-It has been proposed that there is a reciprocal relationship between food intake and sympathetic activity. To test this hypothesis corticotropin releasing hormone, which suppresses feeding behavior, and neuropeptide Y, which stimulates it, were injected intracerebroventricularly and sympathetic nerve activity to interscapular brown adipose tissue measured in anesthetized rats. Multi-unit discharges of sympathetic nerves to interscapular brown adipose tissue were recorded electrophysiologically. The intracerebroventricular injection of corticotropin releasing hormone (250 and 500pmol) increased sympathetic nerve activity and the intracerebroventricular injection of neuropeptide Y (25(rSOO pmol) suppressed sympathetic nerve activity in a dose-dependent manner. The intracerebroventricular injection of vehicle did not affect sympathetic nerve activity. The result is consistent with the hypothesis that these brain peptides are neuromodulators of the sympathetic nervous system which may control energy expenditure in interscapular brown adipose tissue. The effects of these two brain peptides on sympathetic nerve activity are opposite to their effects on feeding behavior suggesting that sympathetic activity and food intake may be reciprocally related.
brown adipose tissue (IBAT) plays a significant role in diet-induced thermogenesis in small mammals.” The activation of heat production by IBAT is believed to be controlled mainly by sympathetic nerves from the central nervous system.14 The focus of this paper is the function of two brain peptides, corticotropin releasing hormone (CRH) and neuropeptide Y (NPY), in controlling the activity of sympathetic nerves to IBAT. CRH and NPY show opposite effects on food intake. CRH is a peptide hormone located in high concentrations in the paraventricular nucleus which stimulates the secretion of adrenocorticotrophic hormone (ACTH) from the pituitary.26 In addition, injection of CRH intracerebroventricularly suppresses feeding behavior. I6 It also affects the autonomic nervous system including the cardiovascular system8 and adrenal medulla.’ This is shown by the fact that icv injection of CRH increases guanosine S-diphosphate-binding to mitochondria in IBAT’ as well as increasing norepinephrine concentrations in the plasma.’ This suggests that icv injection of CRH should stimulate sympathetic nerve activity to IBAT. Conversely, icv injection of NPY markedly enhances food intake; indeed it is one of the most potent neurochemical stimulators of food intake known.”
Chronic repeated injections of NPY into the hypothalamus have been reported to increase body weight.22 In contrast to CRH, there are no reports on the effects of NPY on IBAT. If our hypothesis that food intake and sympathetic activity are reciprocally related is correct, then NPY would be expected to suppress sympathetic activity to IBAT. To investigate the effect of NPY and CRH on sympathetic nerve activity to IBAT, we have administered these peptides icv and measured electrical firing of sympathetic nerves to IBAT.
Interscapular
EXPERIMENTAL
Thirty-six adult female Sprague-Dawley rats were purchased from Harlan-Sprague-Dawley Co. (Indianapolis, IN) weighing about 250g. Prior to surgery rats received pelleted laboratory chow (Wayne Lablox, Allied Industries, Chicago, IL) and tap water ad libifum. Surgical procedures One week before nerve recording, a 15-mm-long 23 gauge stainless steel guide cannula was implanted stereotaxically into the third cerebral ventricle, (icv) under pentobarbital sodium anesthesia (35 mg/kg body weight, ip) with supplemental inhalation anesthesia of methoxyfluorane. Cannulae were fixed to the skull by dental cement anchored with three small stainless steel screws. Rats were anesthetized with alpha-chloralose (80 mg/kg) and urethane (800 mg/kg) solution by giving 75% ip and 25% SC injection. Additional sc injections assured the stability of anesthesia for at least 120 min. After thoracotomy, a transverse incision was made on the back. IBAT is supplied with fine nerves which branch from the five posterior intercostal nerves on each side.” One of the fine nerves was cut out from the connective tissue on
*Present address: 6400 Perkins Road, Baton Rouge, LA 70808, U.S.A. Abbreviations:
ACTH,
adrenocoreticotrophic
CRH, corticotropin
releasing hormone;
scapular
brown
adipose
tissue; NPY,
PROCEDURES
Animals
hormone; IBAT, interneuropeptide Y. 771
112
M. ECAWA
et al. la
CRHicv
250pmol
lb
NPY icv
300-
SALINE
250pmol
icv 10min
Fig. 1. Examples of the effects of icv injection of CRH, NPY or saline on sympathetic nerve activity to IBAT in urethane-chlorarose anesthetized rats. Rats were injected in the third cerebroventricle. (a) CRH (250 pmol dissolved into 5 pl distilled water). (b) NPY (250 pmol dissolved into 5 ~1 distilled water). (c) Vehicle (5 pl of 0.15 M NaCI). the capsule of IBAT and the distal side of the nerve was dissected from IBAT with the aid of microscopic visualization. Multi-unit discharges, which showed a burst-like activity (I-2 bursts/s), were recorded through a pair of tungsten wire electrodes immersed in heavy white mineral oil. They were then amplified and converted to stand square pulse by an event detector. The standard pulses were counted by a
microcomputer (Apple IIe, Cupertino, CA) which integrated pulses for 5 s. The time course of response was calculated as the percentage of the 20 min pre-injected basal level. Icv injection was carried out through a 15.5mm-long 30 gauge stainless steel injector, which was connected to a 25-~1 microsyringe by PE50 polyethylene tubing. Six vehicle-treated rats were injected with 5 PI of 0.15 M NaCl. Four groups of six rats each were injected over 10 min with 250 pmol or 500 pmol of CRH and 250 pmol or 500 pmol NPY dissolved in 5 11 of distilled water. Following completion of the experiments, 0.5 ~1 of Cresyl Violet solution was injected icv through the same injector. All rats were killed with overdoses of anesthesia and were perfused transcardially with 200 ml of a 10% formalin solution. The brains were frozen and lOO+m-sections were obtained. All icv injections were verified under light microscopy. Statistical differences were computed by Scheffe’s post-hoc test after one-way ANOVA. RESULTS E&t
qf corticotropin
releasing
hormone
An example of the effect of icv injection of CRH on the sympathetic nerve activity to IBAT is shown
in Fig. la. Electrical activity was increased after i.c.v. injection of CRH with the increase of nerve activity beginning during injection. The peak appeared 2040 min after the end of the CRH injection. At 30 min after the injection of 250 or 500 pmol CRH, the firing rate increased by 56.4 k 15.1% and by 195.3 + 44.3% from pre-injection basal level (Fig. 2). The responses were significantly larger than that of the saline injected rats (CRF 250 pmol; P < 0.05 compared with saline, CRF 500 pmol; P < 0.01 compared with saline). After injection of the lower dose of CRH, a gradual recovery followed. The stimulatory effects of CRH were dose-dependent. Effkt
of‘ neuropeptide
Y
An example of the effect of an icv injection of NPY on the sympathetic nerve activity to IBAT is shown in Fig. lb. The decrease of activity started during the IO-min injection (Fig. 3). At 30 min after 250 or 500 pmol injection of NPY, the activities were suppressed by 59.0 + 15.9% and by 90.5 + 6.0% (Fig. 3). The suppressions were significantly larger than that of the saline injected rats (NPY 250 pmol; P < 0.01 compared with saline, NPY 500 pmol; P < 0.001 compared with saline). Then a recovery was observed about 4060min after the end of injection. The icv injection of saline did not affect the
Effect of CRH and NPY on electrophysiological m
CRHicv
250pmol
M
CRHicv
500pmol
M
SALlNEicv
l*
activity
113
sympathetic nerve activity compared with pre-injection level (Fig. lc). Histology showed that all icv cannulations were into the third ventricle. DISCUSSION
CRH is a neuropeptide containing 41 amino acids which was initially isolated from ovine hypothalamus.26 CRH has been suggested to be clinically important, because the concentration of CRH is increased in the cerebral spinal fluid in anorectic patients with depression” and in those with anorexia nervosa.i5 Although CRH is now accepted as being a physiological stimulator of pituitary ACTH secretion,26 icv injection of CRH into the rat or dog elicits a dose-related elevation of plasma epinephrine and norepinephrine concentration.’ Furthermore, CRH is CRH Icv suspected to act within the CNS to influence the sympathetic nervous system since it affects blood pressure, heart rate* and gastric acid secretion.24 I In the present study, icv injection of CRH showed I 0 -10 0 10 30 50 a potent stimulatory effect on the sympathetic nerve T I ME lminl activity to IBAT. This increased sympathetic nerve activity may account for the thermogenesis in IBAT Fig. 2. Effects of the intracerebroventricular (icv) injection produced by this neuropeptide since icv injection of of CRH on sympathetic nerve activity to IBAT. CRH CRH increases the thermogenesis in IBAT as as(250 pmol/rat or 500 pmol/rat) was injected over 10 min. The injection of vehicle did not affect the activity. Each sessed by GDP-binding’ and increases the systemic point represents the mean + S.E.M. for five rats. *P < 0.05, oxygen consumption.* CRH has also been shown to **P < 0.01, ***P < 0.001compared with the saline injected reduce food intake’,6 when injected into the cerebral group. ventricular system (icv). Since chronic icv injection of CRH reduced body weight,’ both the increased sympathetic nerve activity and the reduction of food intake could contribute to the reduction in body 300 weight. These findings suggest that CRH may play a role in inducing negative energy balance. CRH may act directly or indirectly on neurons which modulate ti NPYicv 250pmol sympathetic nervous activity. Shimizu and Bray” A--4 NPYicv SOOpmol have microinjected CRH into the lateral hypothalao---O SALlNEicv mus of fasted rats and found that the norepinephrine turnover and DOPAC concentrations fall to levels similar to those found in fed animals. This suggests that CRH may have its effect on sympathetic activity by modulating monoamine metabolism. NPY is a 36 amino acid peptide which is similar in NPYicv structure to pancreatic polypeptide and peptide YY.25 Injection of NPY into the hypothalamus of rats markedly enhances food intake and appears to be one of the most potent neurochemical stimulators of feeding behavior. ‘“.‘6,22.23However, we are unaware of any report on the thermogenic effects of this neuropeptide on IBAT. In the present study injections of NPY consistently reduced the activity of sympathetic nerves to IBAT. This suggested that NPY might induce positive energy balance in part by T I ME lminl increasing energy intake and in part by reducing Fig. 3. Effects of the icv injection of NPY on sympathetic energy expenditure in IBAT. Furthermore, repeated nerve activity to IBAT. Effect of the icv injection of NPY injections of NPY into the hypothalamus increase (250 pmol/rat or 500 pmol/rat) on sympathetic nerve activbody weight.22 ity. Each point represents the mean + S.E.M. for five rats. Intraventricular injection of CRH and NPY pro*P -c0.05, **P < 0.01,***P < 0.001 compared with the saline injected group. duce a variety of metabolic and hormonal changes.
1
tlzszzl
774
M. EGAWA et ul.
Injection of either CRH or NPY into the paraventricular nucleus stimulates the secretion of ACTH and corticosterone.‘3,27 CRH given icv acts within the CNS to increase plasma concentration of catecholamines.’ Icv injection of NPY induces a biphasic change in catecholamine utilization in hypothalamus.” These changes might suppress sympathetic nerve activity. However, the effect of these two peptides on nerve activity appeared rapidly during their icv injection. Since NPY administration centrally increases CRH content in the median eminence13 and since NPY neurons have been shown to terminate in the paraventricular nucleus which is the major site of CRH synthesis4 NPY and CRH may have an intimate relationship in this system. The reciprocal relationship between the effects on food intake and sympathetic activity deserves additional
comment.’
The
present
experiments
were
food intake and activity of the thermogenic component of the sympathetic nervous system, using activation of brown stimulated
by
the
hypothesis
that
adipose tissue as an example, are reciprocally related. This implies that hormones which stimulate food intake would reduce sympathetic activity and vice versa. Lesions in the ventromedial hypothalamus produce hyperphagia and reduce electrical firing of sympathetic nerves to brown adipose tissue.*’ Conversely, lesions in the lateral hypothalamus reduce food intake and increase sympathetic activity.*,** Chemical agents may also fit this model. 2-Deoxy-Dglucose increases food intake and reduces sympathetic activity.’ Fenfluramine, an appetite suppressant, has the opposite effect stimulating sympathetic activity and reducing food intake.” The two neuropeptides used in the present experiments extend this hypothesis to physiological compounds which may be important in regulating food intake. In addition to providing the first direct measures of electrophysiological activity following icv injection, the present studies are consistent with the hypothesis that there is a reciprocal relationship between food intake and sympathetic activity under a variety of circumstances.
REFERENCES I. Arase
2. 3. 4.
5. 6. 7. 8. 9. IO. I I. 12.
13. 14. 15.
16.
K.. York D. A., Shimizu H., Shargill N. and Bray Cl. A. (1988) Effects of corticotropin-releasing factor on food intake and brown adiuose tissue thermoeenesis in rats. Am. J. Phvsiol. 255. E255-E259. Arase K., Sakaguchi T. and Bray G. A. yl987) Lateral hypothalamic lesions and activity of the sympathetic nervous system. Life Sri. 41, 657662. Arase K., York D. A. and Bray G. A. (1987) Corticosterone inhibition of the intracerebroventricular effects of 2-deoxy-u-glucose on brown adipose tissue thermogenesis. Phy.siol. Behac. 40, 489495. Bai F. L., Yamano M., Shiotani Y., Emson P. C., Smith A. D., Powell J. F. and Tohyama M. (1985) An arcuato-paraventricular and dorsomedial hypothalamic neuropeptide Y-containing system which lacks noradrenaline in the rat. Brain Res. 331, 1722175. Bray G. A. (1990) Obesity-a state of reduced sympathetic activity and normal or high adrenal activity (the autonomic and adrenal hypothesis revisted). Inr. J. Obesity (in press). Britton D.. Koob G.. Rivier J. and Vale W. (1982) Intraventricular corticotropin-releasing factor enhances behavioral effects of novelty. Life Sci. 31, 3633367. Brown M. R. and Fisher L. A. (1982) Corticotropin-releasing factor: effects on the sympathetic nervous system and oxygen consumption. L$ Sci. 30, 2077210. Brown M. R. and Fisher L. A. (1984) Brain peptide regulation of adrenal epinephrine secretion. Am. J. PhrJiol. 247, E4 I-E46. Brown M. R., Fisher L. A., Spiess J., Rivier J., Rivier C. and Vale W. (1982) Corticotropin releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology 111, 9288931. Clark J. T., Kalra P. S., Crowly W. R. and Kalra S. P. (1984) Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115, 427~429. Foster D. 0.. Depocas F. and Zuker M. (1982) Heterogeneity of the sympathetic innervation of rat interscapular brown adipose tissue via intercostal nerves. Can. J. Physiol.-Pharkuc. 60, 747-754. Harfstrand A.. Eneroth P.. Aanati L. and Fuxe K. (1987) Further studies on the effects of central administration of neuropeptide Y on neuroendoirine function in the male rat: relationship to hypothalamic catecholamines. Regul. Pep. 17, 167.--179. Haas D. A. and George S. R. (1987) Neuropeptide Y administration acutely increases hypothalamic corticotropinreleasing factor immunoreactivity: lack of effect in other rat brain regions. Life Sci. 41, 2725-2731. HimmsrHagen J. (1985) Brown adipose tissue metabolism and thermogenesis. A. Rev. Nurr. 5, 69994. Hotta M.. Shibasaki T.. Masuda A.. Imaki T.. Demura H.. Lina N. and Shizume M. (1986) The response of plasma adrenocorticotropin and cortisol to corticotropin releasing hormone (CRH) and cerebrospinal fluid ‘immunoreactive CRH in anorexia nervosa patients. J. C/in. Endocrinol. Merab. 62, 319-324. Levine A. S. and Morley J. E. (1984) Neuropeptide Y: a potent inducer of consummatory behavior in rats. Peprides
5, 1025-1030. 17. Lupien J. R. and Bray G. A. (1988) Nicotine
increases thermogenesis in brown adipose tissue in rats. Phurmuc. Biochem. Behao. 29, 33337. 18. Nemeroff C. B.. Widerlov E., Bissette G., Walleus H., Karlsson I., Eklund K.. Kilts C. D., Loosen P. T. and Vale W. (1984) Elevated concentration of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science 226, l342- 1343. 19. Rothwell N. J. and Stock M. J. (1979) A role for brown adipose tissue in diet-induced thermogenesis. Nature 281,3lI35. 20. Sakaguchi T.. Arase K. and Bray G. A. (1988) Sympathetic activity and food intake of rats with ventromedial hypothalamic lesions. Int. J. Obesi/,r 12, 43349.
Effect of CRH and NPY on electrophysiological
activity
775
21. Shimizu H. and Bray G. A. (1990) Modulation by CRH of monoamine metabolism in the lateral hypothalamus. Neurosci. Left (in press). 22. Stanley B. G., Kyrkouli S. E., Lampert S. and Leibowitz S. F. (1987) Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Pepfides 7, 1189-I 192. 23. Stanley B. G. and Leibowitz S. F. (1984) Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci. 35, 2635-2642. 24. Tache Y., Goto Y. and Gunion M. W. (1983) Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor. Science 222, 935-937. 25. Tatemoto K., Carloquist M. and Mutt V. (1982) Neuropeptide Y-a novel brain peptide with structural similarities to peptide YV and pancreatic polypeptide. Nature 2%, 6599660. 26. Vale W., Spiess J. and Rivier J. (1981) Characterization of a 41-residue ovine hypothalamic peptides that stimulates secretion of corticosterone and beta-endorphin. Science 213, 13941397. 27. Wahlestedt C., Skagerberg G., Ekman R., Heilig M., Sundler F. and Hakanson R. (1987) Neuropeptide Y (NPY) in the area of hypothalamic paraventricular nucleus activates the pituitary-adrenocortical axis in the rat. Brain Res. 417, 33338. 28. Yoshida T., Kemnitz J. W. and Bray G. A. (1983) Lateral hypothalamic lesions and norepinephrine turnover in rats. J. clin. Invesf. 72, 919-921. (Accepted 18 July 1989)
0306-4522/90 $3.00 + 0.00 Pergamon Press plc 0 1990 IBRO
EFFECT OF CORTICOTROPIN RELEASING HORMONE AND NEUROPEPTIDE Y ON ELECTROPHYSIOLOGICAL ACTIVITY OF SYMPATHETIC NERVES TO INTERSCAPULAR BROWN ADIPOSE TISSUE M. EGAWA, H. YOSHIMATSU and Department
of Medicine, of Southern
G. A. BRAY*
Section of Diabetes and Clinical Nutrition, USC School of Medicine, California, 2025 Zonal Avenue, Los Angeles, CA 90033, U.S.A.
University
Abstract-It has been proposed that there is a reciprocal relationship between food intake and sympathetic activity. To test this hypothesis corticotropin releasing hormone, which suppresses feeding behavior, and neuropeptide Y, which stimulates it, were injected intracerebroventricularly and sympathetic nerve activity to interscapular brown adipose tissue measured in anesthetized rats. Multi-unit discharges of sympathetic nerves to interscapular brown adipose tissue were recorded electrophysiologically. The intracerebroventricular injection of corticotropin releasing hormone (250 and 500pmol) increased sympathetic nerve activity and the intracerebroventricular injection of neuropeptide Y (25(rSOO pmol) suppressed sympathetic nerve activity in a dose-dependent manner. The intracerebroventricular injection of vehicle did not affect sympathetic nerve activity. The result is consistent with the hypothesis that these brain peptides are neuromodulators of the sympathetic nervous system which may control energy expenditure in interscapular brown adipose tissue. The effects of these two brain peptides on sympathetic nerve activity are opposite to their effects on feeding behavior suggesting that sympathetic activity and food intake may be reciprocally related.
brown adipose tissue (IBAT) plays a significant role in diet-induced thermogenesis in small mammals.” The activation of heat production by IBAT is believed to be controlled mainly by sympathetic nerves from the central nervous system.14 The focus of this paper is the function of two brain peptides, corticotropin releasing hormone (CRH) and neuropeptide Y (NPY), in controlling the activity of sympathetic nerves to IBAT. CRH and NPY show opposite effects on food intake. CRH is a peptide hormone located in high concentrations in the paraventricular nucleus which stimulates the secretion of adrenocorticotrophic hormone (ACTH) from the pituitary.26 In addition, injection of CRH intracerebroventricularly suppresses feeding behavior. I6 It also affects the autonomic nervous system including the cardiovascular system8 and adrenal medulla.’ This is shown by the fact that icv injection of CRH increases guanosine S-diphosphate-binding to mitochondria in IBAT’ as well as increasing norepinephrine concentrations in the plasma.’ This suggests that icv injection of CRH should stimulate sympathetic nerve activity to IBAT. Conversely, icv injection of NPY markedly enhances food intake; indeed it is one of the most potent neurochemical stimulators of food intake known.”
Chronic repeated injections of NPY into the hypothalamus have been reported to increase body weight.22 In contrast to CRH, there are no reports on the effects of NPY on IBAT. If our hypothesis that food intake and sympathetic activity are reciprocally related is correct, then NPY would be expected to suppress sympathetic activity to IBAT. To investigate the effect of NPY and CRH on sympathetic nerve activity to IBAT, we have administered these peptides icv and measured electrical firing of sympathetic nerves to IBAT.
Interscapular
EXPERIMENTAL
Thirty-six adult female Sprague-Dawley rats were purchased from Harlan-Sprague-Dawley Co. (Indianapolis, IN) weighing about 250g. Prior to surgery rats received pelleted laboratory chow (Wayne Lablox, Allied Industries, Chicago, IL) and tap water ad libifum. Surgical procedures One week before nerve recording, a 15-mm-long 23 gauge stainless steel guide cannula was implanted stereotaxically into the third cerebral ventricle, (icv) under pentobarbital sodium anesthesia (35 mg/kg body weight, ip) with supplemental inhalation anesthesia of methoxyfluorane. Cannulae were fixed to the skull by dental cement anchored with three small stainless steel screws. Rats were anesthetized with alpha-chloralose (80 mg/kg) and urethane (800 mg/kg) solution by giving 75% ip and 25% SC injection. Additional sc injections assured the stability of anesthesia for at least 120 min. After thoracotomy, a transverse incision was made on the back. IBAT is supplied with fine nerves which branch from the five posterior intercostal nerves on each side.” One of the fine nerves was cut out from the connective tissue on
*Present address: 6400 Perkins Road, Baton Rouge, LA 70808, U.S.A. Abbreviations:
ACTH,
adrenocoreticotrophic
CRH, corticotropin
releasing hormone;
scapular
brown
adipose
tissue; NPY,
PROCEDURES
Animals
hormone; IBAT, interneuropeptide Y. 771
112
M. ECAWA
et al. la
CRHicv
250pmol
lb
NPY icv
300-
SALINE
250pmol
icv 10min
Fig. 1. Examples of the effects of icv injection of CRH, NPY or saline on sympathetic nerve activity to IBAT in urethane-chlorarose anesthetized rats. Rats were injected in the third cerebroventricle. (a) CRH (250 pmol dissolved into 5 pl distilled water). (b) NPY (250 pmol dissolved into 5 ~1 distilled water). (c) Vehicle (5 pl of 0.15 M NaCI). the capsule of IBAT and the distal side of the nerve was dissected from IBAT with the aid of microscopic visualization. Multi-unit discharges, which showed a burst-like activity (I-2 bursts/s), were recorded through a pair of tungsten wire electrodes immersed in heavy white mineral oil. They were then amplified and converted to stand square pulse by an event detector. The standard pulses were counted by a
microcomputer (Apple IIe, Cupertino, CA) which integrated pulses for 5 s. The time course of response was calculated as the percentage of the 20 min pre-injected basal level. Icv injection was carried out through a 15.5mm-long 30 gauge stainless steel injector, which was connected to a 25-~1 microsyringe by PE50 polyethylene tubing. Six vehicle-treated rats were injected with 5 PI of 0.15 M NaCl. Four groups of six rats each were injected over 10 min with 250 pmol or 500 pmol of CRH and 250 pmol or 500 pmol NPY dissolved in 5 11 of distilled water. Following completion of the experiments, 0.5 ~1 of Cresyl Violet solution was injected icv through the same injector. All rats were killed with overdoses of anesthesia and were perfused transcardially with 200 ml of a 10% formalin solution. The brains were frozen and lOO+m-sections were obtained. All icv injections were verified under light microscopy. Statistical differences were computed by Scheffe’s post-hoc test after one-way ANOVA. RESULTS E&t
qf corticotropin
releasing
hormone
An example of the effect of icv injection of CRH on the sympathetic nerve activity to IBAT is shown
in Fig. la. Electrical activity was increased after i.c.v. injection of CRH with the increase of nerve activity beginning during injection. The peak appeared 2040 min after the end of the CRH injection. At 30 min after the injection of 250 or 500 pmol CRH, the firing rate increased by 56.4 k 15.1% and by 195.3 + 44.3% from pre-injection basal level (Fig. 2). The responses were significantly larger than that of the saline injected rats (CRF 250 pmol; P < 0.05 compared with saline, CRF 500 pmol; P < 0.01 compared with saline). After injection of the lower dose of CRH, a gradual recovery followed. The stimulatory effects of CRH were dose-dependent. Effkt
of‘ neuropeptide
Y
An example of the effect of an icv injection of NPY on the sympathetic nerve activity to IBAT is shown in Fig. lb. The decrease of activity started during the IO-min injection (Fig. 3). At 30 min after 250 or 500 pmol injection of NPY, the activities were suppressed by 59.0 + 15.9% and by 90.5 + 6.0% (Fig. 3). The suppressions were significantly larger than that of the saline injected rats (NPY 250 pmol; P < 0.01 compared with saline, NPY 500 pmol; P < 0.001 compared with saline). Then a recovery was observed about 4060min after the end of injection. The icv injection of saline did not affect the
Effect of CRH and NPY on electrophysiological m
CRHicv
250pmol
M
CRHicv
500pmol
M
SALlNEicv
l*
activity
113
sympathetic nerve activity compared with pre-injection level (Fig. lc). Histology showed that all icv cannulations were into the third ventricle. DISCUSSION
CRH is a neuropeptide containing 41 amino acids which was initially isolated from ovine hypothalamus.26 CRH has been suggested to be clinically important, because the concentration of CRH is increased in the cerebral spinal fluid in anorectic patients with depression” and in those with anorexia nervosa.i5 Although CRH is now accepted as being a physiological stimulator of pituitary ACTH secretion,26 icv injection of CRH into the rat or dog elicits a dose-related elevation of plasma epinephrine and norepinephrine concentration.’ Furthermore, CRH is CRH Icv suspected to act within the CNS to influence the sympathetic nervous system since it affects blood pressure, heart rate* and gastric acid secretion.24 I In the present study, icv injection of CRH showed I 0 -10 0 10 30 50 a potent stimulatory effect on the sympathetic nerve T I ME lminl activity to IBAT. This increased sympathetic nerve activity may account for the thermogenesis in IBAT Fig. 2. Effects of the intracerebroventricular (icv) injection produced by this neuropeptide since icv injection of of CRH on sympathetic nerve activity to IBAT. CRH CRH increases the thermogenesis in IBAT as as(250 pmol/rat or 500 pmol/rat) was injected over 10 min. The injection of vehicle did not affect the activity. Each sessed by GDP-binding’ and increases the systemic point represents the mean + S.E.M. for five rats. *P < 0.05, oxygen consumption.* CRH has also been shown to **P < 0.01, ***P < 0.001compared with the saline injected reduce food intake’,6 when injected into the cerebral group. ventricular system (icv). Since chronic icv injection of CRH reduced body weight,’ both the increased sympathetic nerve activity and the reduction of food intake could contribute to the reduction in body 300 weight. These findings suggest that CRH may play a role in inducing negative energy balance. CRH may act directly or indirectly on neurons which modulate ti NPYicv 250pmol sympathetic nervous activity. Shimizu and Bray” A--4 NPYicv SOOpmol have microinjected CRH into the lateral hypothalao---O SALlNEicv mus of fasted rats and found that the norepinephrine turnover and DOPAC concentrations fall to levels similar to those found in fed animals. This suggests that CRH may have its effect on sympathetic activity by modulating monoamine metabolism. NPY is a 36 amino acid peptide which is similar in NPYicv structure to pancreatic polypeptide and peptide YY.25 Injection of NPY into the hypothalamus of rats markedly enhances food intake and appears to be one of the most potent neurochemical stimulators of feeding behavior. ‘“.‘6,22.23However, we are unaware of any report on the thermogenic effects of this neuropeptide on IBAT. In the present study injections of NPY consistently reduced the activity of sympathetic nerves to IBAT. This suggested that NPY might induce positive energy balance in part by T I ME lminl increasing energy intake and in part by reducing Fig. 3. Effects of the icv injection of NPY on sympathetic energy expenditure in IBAT. Furthermore, repeated nerve activity to IBAT. Effect of the icv injection of NPY injections of NPY into the hypothalamus increase (250 pmol/rat or 500 pmol/rat) on sympathetic nerve activbody weight.22 ity. Each point represents the mean + S.E.M. for five rats. Intraventricular injection of CRH and NPY pro*P -c0.05, **P < 0.01,***P < 0.001 compared with the saline injected group. duce a variety of metabolic and hormonal changes.
1
tlzszzl
774
M. EGAWA et ul.
Injection of either CRH or NPY into the paraventricular nucleus stimulates the secretion of ACTH and corticosterone.‘3,27 CRH given icv acts within the CNS to increase plasma concentration of catecholamines.’ Icv injection of NPY induces a biphasic change in catecholamine utilization in hypothalamus.” These changes might suppress sympathetic nerve activity. However, the effect of these two peptides on nerve activity appeared rapidly during their icv injection. Since NPY administration centrally increases CRH content in the median eminence13 and since NPY neurons have been shown to terminate in the paraventricular nucleus which is the major site of CRH synthesis4 NPY and CRH may have an intimate relationship in this system. The reciprocal relationship between the effects on food intake and sympathetic activity deserves additional
comment.’
The
present
experiments
were
food intake and activity of the thermogenic component of the sympathetic nervous system, using activation of brown stimulated
by
the
hypothesis
that
adipose tissue as an example, are reciprocally related. This implies that hormones which stimulate food intake would reduce sympathetic activity and vice versa. Lesions in the ventromedial hypothalamus produce hyperphagia and reduce electrical firing of sympathetic nerves to brown adipose tissue.*’ Conversely, lesions in the lateral hypothalamus reduce food intake and increase sympathetic activity.*,** Chemical agents may also fit this model. 2-Deoxy-Dglucose increases food intake and reduces sympathetic activity.’ Fenfluramine, an appetite suppressant, has the opposite effect stimulating sympathetic activity and reducing food intake.” The two neuropeptides used in the present experiments extend this hypothesis to physiological compounds which may be important in regulating food intake. In addition to providing the first direct measures of electrophysiological activity following icv injection, the present studies are consistent with the hypothesis that there is a reciprocal relationship between food intake and sympathetic activity under a variety of circumstances.
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