298
Brain Research, 597 (1992) 298-303 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18309
Effects of cholecystokinin on sympathetic activity to interscapular brown adipose tissue H. Yoshimatsu, M. Egawa and G.A. Bray Department of Medicine, University of Southern California, Los Angeles) CA (USA) (Accepted 30 June 1992)
Key words: Ventromedial hypothalamic nucleus: Lateral hypothalamic area; Dorsomedial hypothalamic nucleus; Paraventricular nucleus; Third ventricle
The effects of injecting cholecystokinin (CCK) into the third ventricle or into selected hypothalamic sites on electrical firing rate of sympathetic nerves to interscapular brown fat (IBAT) has been investigated in anesthetized rats. The hypothesis for these experiments was that there was a reciprocal relationship between sympathetic activity and fond intake. Since CCK reduces food intake we predicted that CCK would stimulate sympathetic activity to IBAT. Following the injection of CCK into the third ventricle there was an increase in firing rate of sympathetic nerves to IBAT. When the peptide was injected into either the ventromedial hypothalamie nucleus (VMH) or lateral hypothalamic area (LHA), there was hkewise an increase in sympathetic firing rate. The injection of CCK into the paraventricular nucleus produced a small decrease in sympathetic firing rate. In contrast, no effect was seen following injection of CCK into the preoptic area or do~omedial hypothalamic nucleus. Thus, the VMH or LHA appear to be the principal hypothalamic areas mediating the stimulation of sympathetic activity to IBAT which is observed following the third ventrieular injection of CCK. These studies support the hypothesis of a reciprocal relationship between the effects of CCK on the thermogenic component of the sympathetic nervous system and food intake and identify the VMH and LHA as the primary sites for this effect.
INTRODUCTION The segmentation of the sympathetic activity which innervates interscapular brown adipose tissue (IBAT) and food intake are reciprocally related in a number of experimental settings~, including hypothalamic lesions, injection of peptides or treatment with several drugs. For example, lesions in the ventromedial hypothalamic nucleus (VMH) increase food intake and decrease sympathetic activity to IBAT ~,~,2~. In contrast, lesions of the lateral hypothalamic area (LHA) decrease food intake and increase sympathetic activity to IBAT whether measured by norepinephrine turnover of presynaptic vesicles in brown adipose tissue36, the electrical discharge rate of sympathetic nerves to IBAT 2, or GDP binding to mitochondria from brown adipose tissue 2~. Peptides also reciprocally affect food intake and this sympathetic activity. Neuropeptide Y (NPY) increases
food intake when injected into the third ventricle I°, into the paraventricular nucleus (PVN)"~4or into other hypothalamic nuclei. It has recently been reported to decrease sympathetic activity to IBAT when injected into the third ventricle or the PVN is,20. The effects of corticotropin-releasing hormone (CRH) are opposite to those of NPY. Injection of this peptide into the third ventricle decreases food intake4 and increases sympathetic activity to IBAT ~. This effect is localized to the preoptic area (PEA) m~. Two drugs also show a reciprocal relationship between food intake and sympathetic activity to IBAT. The anorexiant, fenfluramine, increases sympathetic activity 3 and decreases food intake '7. Conversely, 2-deOXy-D-glucose (2DG) is a stimulus to food intake whether injected peripherally or into the third ventricle'3. When injected into the third ventricle, this analog of glucose decreases sympathetic activity to IBATt6A7.
Correspondence: G.A. Bray. Present address: Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA. Fax (1) (504) 765-2825.
299 The present study was designed to examine the hypothesis of a reciprocal relationship between food intake and sympathetic activity to IBAT further by studying cholecystokinin. Choleeystokinin (CCK) occurs in the gastrointestinal tract as a 33 or 38 amino acid peptide and in the brain and gut as an octapeptide 30. Smith and his colleagues demonstrated that parenteral administration of cholecystokinin would decrease food intake 22. It was subsequently shown that cholecystokinin injected into the third ventricular system could decrease food intake ~, whereas injection of antibodies to this peptide increases food intake t2. Balhan has recently shown that the VMH is one locus for the suppressive effect of CCK on food intake 5. The peripheral effects of CCK are mediated by CCK-A receptors which may be located in the antral and pyloric region of the stomach 33. There are also CCK-B receptors in the brain TM. There is evidence to suggest that both CCK-A and CCK-B receptors may be involved in modulating the effects of CCK on food intake under various circumstances ~a'32. For the present studies, cholecystokinin was injected into the third ventricle and several specific hypothalamic nuclei and the sympathetic activity to brown adipose tissue examined. MATERIALS AND METHODS Female Sprague-Dawley rats were purchased from Haflan Sprague Dawley (Indianapolis, IN) weighing approximately 250 g. Under pentobarbital anesthesia, a 23-gauge stainless steel guide cannula was implanted into the third ventricle or unilaterally into desired hypothalamic regions. The tip of the cannula was 1 ram above the hypothalamic structure being studied except for 2 rats which were simultaneously implanted with 2 cannulas, one each in the PVN and the LHA. Stereotaxic coordinates of injection sites were as follows: the LHA: Ant = 2.8-3.0 mm posterior to the bregma; Lat = 1.6-1.9 mm lateral to the center medial sinus; Height ffi 8.0-8.5 mm below the surface of the skull at the bregma; the VMN: Ant = 3.0; Lat ffi 0.7; Height = 9.5; the PVN: Ant = 2.0; Lat = 0.4; Height = 7.8; the DMN: Ant = 3.0; Lat = 0.7; Height = 8.3; and the POA: Ant = 0,5; Lat = 0.4; Height ffi 8.02~. All measurements were in mm from the bregma mid-sagittal sinus or skull surface. After 7-10 days for recovery from surgery, experiments were carried out using a mixture of urethane (0.8 g/kg) and a-chloralose (80 mg/kg) as anesthesia. After dissection of the fine branches of the intercostal nerves supplying interscapular brown adipose tissue (IBAT), the nerves were cut in the region where they entered the IBAT. Electrical discharges were recorded from fine filaments proxinoi to the site of transection using bipolar tungsten wire electrodes immersed in heavy white mineral oil to prevent dehydration of the nerves. The discharges were amplified through a condenser-coupled differential input preamplifier and fed into a window discriminator to differentiate from background noise. The number of pulses per 5 s were integrated and printed out on a printer. During stable recording of neuronal activity, CCK was injected into the third ventricle (500 pmol/5 ~i saline, over 5 rain) through a 30-gauge cannula or was injected into each site (50 pmol/0.5/.tl saline, over 5 rain). The hypothalamic cannula was lowered 1 mm below the lower tip of the guide cannula. In controls, the same amount of saline was injected. Following completion of the recordings, 10% formalin solution was perfused into the left cardiac ventricle. The brains were removed and frozen sections were made of the injection site to verify histologically its location by light microscopy.
RESULTS CCK injection into the third cerebral ventricle Fig. 1 shows a typical response to the injection of CCK into the third cerebral ventricle (ICV). There was an initial rise in electrical firing rate of sympathetic nerves as the CCK was being infused. This reached a peak approximately 20 rain later and then began a gradual decline. However, the firing rate remained higher than the baseline values at the end of the 1-h recording period. Injection of saline into the ICV had no effect. Of 10 rats tested with CCK, 7 showed an increase, 2 showed a decrease and 1 had no response (Table I). Five control rats showed no remarkable changes of nerve activity in response to injection of 0.15 M NaCl solution. The stimulatory effects of CCK versus control infusion were statistically significant (P < 0.01, Fisher's exact probability test). CCK injection into the VMH The injection of CCK into the VMH produced a gradual increase in electrical firing rate of sympathetic nerves to brown adipose tissue (Fig. 2). The rise began at or just following the completion of the CCK injection and continued for the entire period of observation which lasted up to 60 min. Injections of saline into this area were without effect. Of 13 rats tested, CCK injection produced an increase in 11 and no effect in 2 (Table I). Control injection in 5 rats showed no effect on nerve activity (Table I). The increase produced by CCK versus control injection was statistically significant (P < 0.01, Fisher's exact probability test).
,r. rrv
300
Imp. 0 /5sec lOmln
200
imp. 0 /Ssec m
lOmin Fig. 1. Rate meter plots of BAT sympathetic nerve activity following CCK injection into the third cerebral ventricle. Upper panel: CCK injection increased nerve activity. Lower panel: injection of saline (SA) induced no remarkable change. ICV, into the third cerebral ventricle, imp., number of pulses.
3OO ~
TABLE !
z
A
"
"~
300
Effects of cholecystokinin injection into the third ventricle and the hypothalamus on BAT sympathetic nerve activi~.
ICV VMH LHA DMH PVN PeA
CCK control CCK control CCK control CCK control CCK control CCK control
"[
,[
~
total
7* 0 11 * 0 9* 0 1 0 0 0 1 0
2 0 0 0 0 0 1 0 4 0 2 0
1 5 2 5 3 5 8 5 7 5 8 5
10 5 13 5 12 5 10 5 1i 5 11 5
imp. 0 /Ssec lOrnin
200
imp. o /Ssec lOmln
* P < 0.01, Fisher's exact probability test. ICV, into third ventricle.
CCK injection into the LHA Fig. 3 shows the effects of injecting CCK into the LHA. Following injection into the LHA, there was a gradual increase in firing rate which was similar to that observed following the injection of CCK into the VMH. The firing rate increased linearly for as long as observations were continued. When saline was injected into the same area, there was no effect. In 12 rats which did not include the 2 rats used for repeated injection of CCK into the PVN and the LHA, 9 showed an increase and 3 were without effect (Table I). Control injection in 5 rats showed no effect on nerve activity (Table I). The increase in responses to CCK was statistically significantly greater than in control (P < 0.01, Fisher's exact probability test), CCK injection into the DMH Fig. 4 shows the effect of injecting CCK or saline into the DMH. In contrast with the VMH or LHA,
Fig. 3. Rate meter plots of BAT sympathetic nerve activity following CCK in.iection into the LHA. Upper panel: CCK injection increased nerve activity. Lower panel: injection of saline induced no remarkable change.
CCK had no effect following its injection into the DMH. Eight out of the 10 rats tested showed ao remarkable change of nerve activity whereas one each showed an increase or decrease (Table I).
CCK injection into the PPN Fig. 5 shows the effect of injecting CCK into the paraventricular nucleus and LHA. Two patterns are observed. In the upper panel, the injection of CCK into the PVN produced a decrease in firing rate which reached a nadir at approximately the end of the injection. The rate of firing had decreased by more than 50% following the injection of CCK. In the middle panel, the first injection of CCK into the PVN had no effect on nerve activity, but when a second injection was given into the LHA of the same animal it produced an increase in firing rate. In the bottom, saline injection showed no effect. Out of 11 rats tested includ-
300
~DMH CCK
imp. O /5see 1"0rain
VMH SA
mp. '5sec 10rain
200
200
imp. 0 /Sse¢
imp. 0 /5see
lOmtn
Fig. 2, Rate meter plots of BAT sympathetic nerve activity following CCK injection into the VMH. Upper panel: CCK injection increased nerve activity. Lower panel: injection of saline induced no remarkable change.
Fig. 4. Rate meter plots of BAT sympathetic nerve activity following CCK injection into the DMH. Upper panel: CCK injection induced no remarkable change. Lower panel: injection of saline induced no remarkable change.
301 m,M . ~
200
~A I.:oOo,
Imp. 0 /Ssec
0.3
1Omin 200
~,HA,
'
,
/
o,
.""
i
,
-1
3
O.~.t~, -
'::::,I~::." .~
imp. 0 /5sec
~AHA~II!.[ i ',,._.,,; .' .i ",,,,..,,'
lOmin r---i PVN SA
POA
'
200
--1.B
-2.B
imp, Jo/ssec
-3.3
m
lOmin
Fig. 5. Rate meter plots of BAT sympathetic nerve activity following
CCK injectioninto the PVN. Upper panel: CCK injection decreased nerve activity.Middle panel: CCK injectioninto the PVN inducedno remarkable change but followinginjection into the LHA increased nerve activity.Lower panel injection of saline induced no remarkable change. ing 2 rats which received repeated injection into the PVN and the LHA, 4 showed a decrease and 7 showed no effect (Table I). Two rats for repeated injections showed no respdnse to the first injection into the PVN and an increase in firing rate in the second injection into the LHA as shown i~ Fig. 5 (middle).
CCK injection into the P04 The data in Fig. 6 show that the injection of CCK into the preoptie area produced no difference from an injection of saline into the same area. ,.---1Dn&~K
200
imp, 0 /5see lOmln
np.
5sec
lOmin
Fig. 6. Rate meter plots of BAT sympathetic nerve activity following CCK injection into the POA. Upper panel: CCK injection induced no remarkable change, Lower panel: injection of saline induced no remarkable change.
Fig. 7. Diagrammatic representation of CCK injection sites. (*), increase; (v), decrease; (o), no change. AC, anterior commissura; POA, preoptic area; ALIA, anterior hypothalamicarea; PVN, paraventrieular nucleus; DMH, dorsomedial hypothalamic nucleus; LHA, lateral hypothalamicarea; VMH, ventromediai hypothalamic nucleus. Injection sites were distributed from -0.3 to -1,3 in the POA, from -2.8 to 3.3 in the DMH, LHA and VMH, and localized to the plane at -1.8 in the PVN according to the atlas of Paxinos and Watson2a.
Histological examination Fig. 7 shows diagrammatically the injection sites. Injection sites for the VMH, the LHA and the DMH distributed in the range from - 2 . 8 to - 3 . 3 according to the atlas of Paxinos and Watson 29. There were no specific responses and localization of injection site within each region. Injection sites for the PVN and the POA were localized to the restricted region in - 0 . 3 for POA and - 1.8 for PVN. No specific localization of effective and non-effective sites by CCK injections could be observed within the PVN. DISCUSSION The present experiments have explored the role of CCK on the activity of the sympathetic nervous system, innervating interscapular brown adipose tissue (IBAT). The data clearly show that CCK, whether injected into the third ventricle, into the VMH or into the LHA was associated with an increase in sympathetic firing rate. In contrast, no effects were observed when CCK was injected into the POA, into the PVN or into the DMH. Data from several laboratories suggests that the sympathetic nervous system has several functionally
302 separate parts s'2s. Fasting decreases sympathetic activity to IBAT, heart, liver and pancreas 36'as but not to the limbs ls'~s. 2-Deoxy-D-glucose (2DG) is an analogue of glucose which impairs intracellular glucose metabolism and this produces glucopenia and reduces sympathetic activity to IBAT 16a7 but stimulates the adrenal nerve to release norepinephrine and epinephrine a6. Interleukin-1/3 stimulates sympathetic activity to lung and spleen but not elsewhere 3t. The sympathetic nerves innervating IBAT are thus but one part of the sympathetic nervous system. These sympathetic nerves to IBAT subserve the activation of this tissue which increases heat production. This part of the sympathetic nervous system might best be referred to as the 'thermogenic' component of the sympathetic nervous system. CCK fits a consistent pattern which has been observed for several other peptides as well as monoamines, drugs and hypothalamic lesions 9. Both NPY and /]-endorphin, which increase food intake have been ~hown to decrease sympathetic activity to IBAT whereas peptides such as CRH and CCK which decrease food intake s'22, increase thermogenic sympathetic activity. The anatomic basis for this robust physiologic relationship is unclear. Anatomic mapping studies for the effects of peptides on sympathetic activity have only been reported ih a few cases. Neuropeptide Y decreases sympathetic activity when injected into the PVN 2° and increases sympathetic activity when injected into the preoptic area, but not into other hypothalamic regions 2°. CRH increases sympathetic activity when injected into the POA but not elsewhere tg. CCK, the subject of this study, increased sympathetic activity when injected into either the VMN or LHA, but was without effect in the PVN, POA or DMH. Thus peptide receptors which can modulate sympathetic efferent activity appear to exist in several regions of the hypothalamus. Injection of peptides into several different regions of the hypothalamus can modulate sympathetic activity in a reciprocal fashion to food intake. This would suggest that when the sympathetic activity is changed, a component of the common final response which arises after these various sympathetic sites have been activated must then serve as a feedback element modulating food intake. This pattern of multiple sites for activating sympathetic activity being funnelled into a common pathway which generates a signal for modulating food intake, would be consistent with known ~ and fl receptor modulation of feeding which occurs in two different hypothalamic areas, a2-Adrenergic receptors appear to be activated in the paraventricular nucleus to increase food intake 24. Conversely,/3-adrenergic recep-
tors loca'.ed in the perifornical area inhibit feeding and it is these receptors to which the sympathetic nervous system may feed back 2~. If the descending sympathetic fibers modulated an ascending pathway from the hindbrain to the perifornical area, this could explain the reciprocal relationship reported here and previously. A recent study using L-glutamate, a stimulator of neuronal activity, has provided a~litional understanding of the system l'a7. Eiectrophoretic application of CCK has been shown to produce an excitatory effect on neuronal activity of VMH neurons 32. The injection of glutamate into the VMH uniformly increases sympathetic activity and this may occur through the effects of L-glutamate on these neurons. Injection of CCK into the LHA also produced an increase in activity of the sympathetic nerves to IBAT. However, the pattern of response of peripheral sympathetic activity to IBAT following glutamate injection in the LHA was complex suggesting subregional organization within this area where both inhibitory, stimulatory and biphasic patterns of response exist a7. A recent electrophysiological study has demonstrated a suppressive effect of CCK on neuronal activity of LHA neurons a2. Taken together, it can be suggested that CCK increases sympathetic nerve activity through suppression of the inhibitory component of LHA, and that it thus works through a disinhibition mechanism. The present study demonstrated that the VMH and the LHA, but not other hypothalamic regions, were effective sites for activation of sympathetic nerves to IBAT in response to CCK administration. Recent neuroanatomieal studies have shown the presence of CCK-containing neurons in the brain 6,3s. The VMH has been shown to be one of the hypothalamic nuclei which receives a rich innervation of CCK neurons as, and the CCK receptor has been shown to he distributed in this nucleus and the LHA2k These studies and the present findings indicate that CCK neurons innervating both regions play a role in the regulation of sympathetic nerve activity to IBAT. Acknowledgements. Supported in part by Grant DK 31988 from the NIH.
REFERENCES 1 Amir, S., lntraventromedial hypothalamic injection of glutamate stimulates brown adipose tissue thermogenesis in the rat (Technical Note), Brain Res., 511 (2) (1990) 341-344. 2 Arase, K., Sakaguchi, T. and Bray, G.A., Lateral hypothalamic lesions and activity of the sympathetic nervous system, Life Sci., 41 (5) (1987) 657-662. 3 Arase, K., Sakaguchi, T. and Bray, G.A., Effect of fenfluramin¢ on sympathetic firing rate, Pharmacol. Biochem. Behav., 29 (3) (1988) 675-680.
303 4 Arase, K., York, D.A., Shargill, N.S. and Bray, G.A., Effects of corticotropin releasing factor on food intake and brown adipose tissue thermogenesis in rats, Am. Y. Physiol., 255 (1988) E255E259. 5 Balkan, B., Autonomic Influences on Metabolism in the Development and Maintenance of Obesity, Thesis, University of Groningen, 1991, Chapter 4. 6 Beinfeld, M., Cholecystokinin in the central nervous system: a mini review, Neuropeptides, 3 (1983) 411-427. 7 Bray, G.A., Obesity, a disorder of nutrient partitioning: the MONA LISA hypothesis, J. Nutr., 121 (1991) 1146-1162. 8 Bray, G.A., Fisler, J.S. and York, D.A., Neuroendocrine control of the development of obesity: understanding gained from studies of experimental animal models, Front. Neuroendocrinol., 11 (2) (1990) 128-181. 9 Bray, G.A., Peptides affect the intake of specific nutrients and the sympathetic nervous system, Am. J. Clin. Nutr., 55 (1992) 265S-271S. 10 Clarke, JJ., Kalra, P.S., Crawley, W.R. and Kalra, S.P., New ropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology, 115 (1984) 427-429. 11 Della-Fera, M.A. and Baile, C.A., Cholecystokinin octapeptide: continuous picomole injections into the cerebral ventricles suppress feeding, Science, 206 (1979) 471-473. 12 Della-Fera, M.A., Baile, C.A., Schneider, B.S. and Grinker, J., CholeoJstokinin antibody injected in cerebral ventricles stimulates feeding in sheep, Science, 212 (1981) 687-6-39. 13 Dourish, C.T., Coughlan, J., Hawley, D., Clark, M. and Iversen, S.D., Blockade of CCK-induced hyperphagia and prevention of morphine tolerance by CCK-antagonist L-374,718. In R.Y. Wang and R. Schocnfeld (Eds.), Cholecystokinin Antagonists, Alan Liss, New York, 1988, pp. 307-325. 14 Dourish, C,, Rycroft, W. and Iversen, S., Postponemen', of satiety by blockade of brain cholecystokinin (CCK-B) receptors, Science, 9 (1989) 1509-1511. 15 Dullo, A.G., Young J.B. and Landsberg, L., Sympathetic nervous system responses to cold exposure and diet in rat skeletal muscle, Am. J. Physiol., 255 (1988) E180-E188. 16 Egawa, M., Yoshimatsu, H. and Bray, G.A., Effects of 2-deoxyD-glucose on sympathetic nerve activity to interscapular brown adipose tissue, Am. J. Physiol., 257 (1989) R1377-RI385. 17 Egawa, M., Yoshimatsu, H. and Bray, G., Lateral hypothalamic injection of 2.deoxy-D-glucose suppresses sympathetic activity, Am. J. Physiol., 257 (1989) RI386-RI392. 18 Egawa, M., Yoshimatsu, H. and Bray, G,A., Effect of corticotropin releasing hormone and neuropeptide Y on electrophysiological activity of sympathetic nerves to interscapular brown adipose tissue, Neuroscience, 34 (3) (1990) 771-775. 19 Egawa, M., Yoshimatsu, I-I. and Bray, G.A., Preoptic area injection of corticotropin-releasing hormone stimulates sympathetic activity, Am. J. Physiol., 259 (28) (1990) R799-R806. 20 Egawa, M., Yoshimatsu, H. and Bray, G.A., Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats, Am. J. Physiol., 260 (2) (1991) R328-R334. 21 Gaudreau, P., Ouirion, R., St. Pierre, S. and Pert, C.B., Tritiumsensitive film autoradiography of [3H]cholecystokinin-5/pen-
tagastrin receptors in rat brain, Eur. J. Pharmacol., 87 (1983) 173-174. 22 Gibbs, J., Young, R. and Smith, G.P., Cholecystokinin decreases food intake in rats, J. Comp. Physiol., 84 (1973) 488-493. 23 Gonzales, M.F. and Novin, D., Feedir induced by intracranial and intravenously administration of 2-deoxy-D-glucose, Physiol. Psychol., 2 (1974) 326-330. 24 Goldman, C.K., Marino, L. and Leibowitz, S.F., Postsynaptic a2-noradrenergic receptors in the paraventricular nucleus mediate feeding induced by norepinephrine and clonidine, Fur. J. Pharmacol., 115 0985) 1102-1105. 25 Kaufman, L.N., Young, J.D. and Landsberg, L., Differential catecholamine responses to dietary intake: effects of macronutrients on dopamine and epinephrine excretion in the rat, Clin. Exp. Metab., 38 (1) (1989) 91-99. 26 Leibowitz, S.F., Reciprocal hunger-regulating circuits involving a- and ~-adrenergic receptors located, respectively, in the ventromedial and lateral hypothalamus, Proc. Natl. Acad. Sci. USA, 67 (1970) 1053-1070. 27 Lupien, J.R., Tokunga, K., Kemnitz, J.W., Groos, E. and Bray, G.A., Lateral hypothalamic lesions and fenfluramine increase thermogenesis in brown adipose tissue, Physiol. Behal,., 38 (1) (1986) 15-20. 28 Niijima, A., Rohner-Jeanrenaud, F. and Jeanrenaud, B., Role of ventromedial hypothalamus on sympathetic efferents of brown adipose tissue, Am. J. Physiol., 247 (1984) R650-654. 29 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York~ 1982. 30 Peikin, S.R., Role of cholecystokiuin in the control of food intake, Gastroenterol. Endocrinol., 18 (1989) 757-775. 31 Saito, M., Akiyoshi, M. and Shimizu, Y., Possible role of the sympathetic nervous system in responses to lnterleukin-l, Brain Res. Bull., 27 (1991) 305-308. 32 Shiraishi, T., CCK as a central satiety factor: behavioral and electrophysiological evidence, Physiol. Behar., 48 (1990) 879-885. 33 Smith, G.P., Tyrka, A. and Gibbs, J., Type-A CCK receptors mediate the inhibition of food-intake and activity by CCK-8 in 9-day-old to 12-day-old rat pups (technical note), Pharmacol. Biochem. Behat,., 38 (1) (1991) 207-210. 34 Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus, Life Sci., 35 (1984) 2635-2642. 35 Vanderhaeghen, JJ., Lotstra, F., DeMey, J. and Gilles, C., lmmunohistochemical localization of cholecystokinin and gastrin-like peptides in the brain and hypophysis of the rat, Proc. Nail. Acad. Sci. USA, 77 (1980) 1190-1194. 36 Yoshida, T., Kemnitz, J. and Bray, G.A., Lateral hypothalamic lesions and norepinephrine turnover in rats, J. Clin. lm,est., 72 (1983) 919-927. 37 Yoshimatsu, H., Egawa, M. and Bray, G.A,, The influence of discrete hypothalamic regions on sympathetic nerve activity to interscapnlar brown adipose tissue (IBAT), FASEB J., 5 (1991) A1078. 38 Young, J.B. and Landsberg, L., Suppression of sympathetic nervous system during fasting, Science, 196 (1977) 1473-1475.
Brain Research, 597 (1992) 298-303 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 18309
Effects of cholecystokinin on sympathetic activity to interscapular brown adipose tissue H. Yoshimatsu, M. Egawa and G.A. Bray Department of Medicine, University of Southern California, Los Angeles) CA (USA) (Accepted 30 June 1992)
Key words: Ventromedial hypothalamic nucleus: Lateral hypothalamic area; Dorsomedial hypothalamic nucleus; Paraventricular nucleus; Third ventricle
The effects of injecting cholecystokinin (CCK) into the third ventricle or into selected hypothalamic sites on electrical firing rate of sympathetic nerves to interscapular brown fat (IBAT) has been investigated in anesthetized rats. The hypothesis for these experiments was that there was a reciprocal relationship between sympathetic activity and fond intake. Since CCK reduces food intake we predicted that CCK would stimulate sympathetic activity to IBAT. Following the injection of CCK into the third ventricle there was an increase in firing rate of sympathetic nerves to IBAT. When the peptide was injected into either the ventromedial hypothalamie nucleus (VMH) or lateral hypothalamic area (LHA), there was hkewise an increase in sympathetic firing rate. The injection of CCK into the paraventricular nucleus produced a small decrease in sympathetic firing rate. In contrast, no effect was seen following injection of CCK into the preoptic area or do~omedial hypothalamic nucleus. Thus, the VMH or LHA appear to be the principal hypothalamic areas mediating the stimulation of sympathetic activity to IBAT which is observed following the third ventrieular injection of CCK. These studies support the hypothesis of a reciprocal relationship between the effects of CCK on the thermogenic component of the sympathetic nervous system and food intake and identify the VMH and LHA as the primary sites for this effect.
INTRODUCTION The segmentation of the sympathetic activity which innervates interscapular brown adipose tissue (IBAT) and food intake are reciprocally related in a number of experimental settings~, including hypothalamic lesions, injection of peptides or treatment with several drugs. For example, lesions in the ventromedial hypothalamic nucleus (VMH) increase food intake and decrease sympathetic activity to IBAT ~,~,2~. In contrast, lesions of the lateral hypothalamic area (LHA) decrease food intake and increase sympathetic activity to IBAT whether measured by norepinephrine turnover of presynaptic vesicles in brown adipose tissue36, the electrical discharge rate of sympathetic nerves to IBAT 2, or GDP binding to mitochondria from brown adipose tissue 2~. Peptides also reciprocally affect food intake and this sympathetic activity. Neuropeptide Y (NPY) increases
food intake when injected into the third ventricle I°, into the paraventricular nucleus (PVN)"~4or into other hypothalamic nuclei. It has recently been reported to decrease sympathetic activity to IBAT when injected into the third ventricle or the PVN is,20. The effects of corticotropin-releasing hormone (CRH) are opposite to those of NPY. Injection of this peptide into the third ventricle decreases food intake4 and increases sympathetic activity to IBAT ~. This effect is localized to the preoptic area (PEA) m~. Two drugs also show a reciprocal relationship between food intake and sympathetic activity to IBAT. The anorexiant, fenfluramine, increases sympathetic activity 3 and decreases food intake '7. Conversely, 2-deOXy-D-glucose (2DG) is a stimulus to food intake whether injected peripherally or into the third ventricle'3. When injected into the third ventricle, this analog of glucose decreases sympathetic activity to IBATt6A7.
Correspondence: G.A. Bray. Present address: Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA. Fax (1) (504) 765-2825.
299 The present study was designed to examine the hypothesis of a reciprocal relationship between food intake and sympathetic activity to IBAT further by studying cholecystokinin. Choleeystokinin (CCK) occurs in the gastrointestinal tract as a 33 or 38 amino acid peptide and in the brain and gut as an octapeptide 30. Smith and his colleagues demonstrated that parenteral administration of cholecystokinin would decrease food intake 22. It was subsequently shown that cholecystokinin injected into the third ventricular system could decrease food intake ~, whereas injection of antibodies to this peptide increases food intake t2. Balhan has recently shown that the VMH is one locus for the suppressive effect of CCK on food intake 5. The peripheral effects of CCK are mediated by CCK-A receptors which may be located in the antral and pyloric region of the stomach 33. There are also CCK-B receptors in the brain TM. There is evidence to suggest that both CCK-A and CCK-B receptors may be involved in modulating the effects of CCK on food intake under various circumstances ~a'32. For the present studies, cholecystokinin was injected into the third ventricle and several specific hypothalamic nuclei and the sympathetic activity to brown adipose tissue examined. MATERIALS AND METHODS Female Sprague-Dawley rats were purchased from Haflan Sprague Dawley (Indianapolis, IN) weighing approximately 250 g. Under pentobarbital anesthesia, a 23-gauge stainless steel guide cannula was implanted into the third ventricle or unilaterally into desired hypothalamic regions. The tip of the cannula was 1 ram above the hypothalamic structure being studied except for 2 rats which were simultaneously implanted with 2 cannulas, one each in the PVN and the LHA. Stereotaxic coordinates of injection sites were as follows: the LHA: Ant = 2.8-3.0 mm posterior to the bregma; Lat = 1.6-1.9 mm lateral to the center medial sinus; Height ffi 8.0-8.5 mm below the surface of the skull at the bregma; the VMN: Ant = 3.0; Lat ffi 0.7; Height = 9.5; the PVN: Ant = 2.0; Lat = 0.4; Height = 7.8; the DMN: Ant = 3.0; Lat = 0.7; Height = 8.3; and the POA: Ant = 0,5; Lat = 0.4; Height ffi 8.02~. All measurements were in mm from the bregma mid-sagittal sinus or skull surface. After 7-10 days for recovery from surgery, experiments were carried out using a mixture of urethane (0.8 g/kg) and a-chloralose (80 mg/kg) as anesthesia. After dissection of the fine branches of the intercostal nerves supplying interscapular brown adipose tissue (IBAT), the nerves were cut in the region where they entered the IBAT. Electrical discharges were recorded from fine filaments proxinoi to the site of transection using bipolar tungsten wire electrodes immersed in heavy white mineral oil to prevent dehydration of the nerves. The discharges were amplified through a condenser-coupled differential input preamplifier and fed into a window discriminator to differentiate from background noise. The number of pulses per 5 s were integrated and printed out on a printer. During stable recording of neuronal activity, CCK was injected into the third ventricle (500 pmol/5 ~i saline, over 5 rain) through a 30-gauge cannula or was injected into each site (50 pmol/0.5/.tl saline, over 5 rain). The hypothalamic cannula was lowered 1 mm below the lower tip of the guide cannula. In controls, the same amount of saline was injected. Following completion of the recordings, 10% formalin solution was perfused into the left cardiac ventricle. The brains were removed and frozen sections were made of the injection site to verify histologically its location by light microscopy.
RESULTS CCK injection into the third cerebral ventricle Fig. 1 shows a typical response to the injection of CCK into the third cerebral ventricle (ICV). There was an initial rise in electrical firing rate of sympathetic nerves as the CCK was being infused. This reached a peak approximately 20 rain later and then began a gradual decline. However, the firing rate remained higher than the baseline values at the end of the 1-h recording period. Injection of saline into the ICV had no effect. Of 10 rats tested with CCK, 7 showed an increase, 2 showed a decrease and 1 had no response (Table I). Five control rats showed no remarkable changes of nerve activity in response to injection of 0.15 M NaCl solution. The stimulatory effects of CCK versus control infusion were statistically significant (P < 0.01, Fisher's exact probability test). CCK injection into the VMH The injection of CCK into the VMH produced a gradual increase in electrical firing rate of sympathetic nerves to brown adipose tissue (Fig. 2). The rise began at or just following the completion of the CCK injection and continued for the entire period of observation which lasted up to 60 min. Injections of saline into this area were without effect. Of 13 rats tested, CCK injection produced an increase in 11 and no effect in 2 (Table I). Control injection in 5 rats showed no effect on nerve activity (Table I). The increase produced by CCK versus control injection was statistically significant (P < 0.01, Fisher's exact probability test).
,r. rrv
300
Imp. 0 /5sec lOmln
200
imp. 0 /Ssec m
lOmin Fig. 1. Rate meter plots of BAT sympathetic nerve activity following CCK injection into the third cerebral ventricle. Upper panel: CCK injection increased nerve activity. Lower panel: injection of saline (SA) induced no remarkable change. ICV, into the third cerebral ventricle, imp., number of pulses.
3OO ~
TABLE !
z
A
"
"~
300
Effects of cholecystokinin injection into the third ventricle and the hypothalamus on BAT sympathetic nerve activi~.
ICV VMH LHA DMH PVN PeA
CCK control CCK control CCK control CCK control CCK control CCK control
"[
,[
~
total
7* 0 11 * 0 9* 0 1 0 0 0 1 0
2 0 0 0 0 0 1 0 4 0 2 0
1 5 2 5 3 5 8 5 7 5 8 5
10 5 13 5 12 5 10 5 1i 5 11 5
imp. 0 /Ssec lOrnin
200
imp. o /Ssec lOmln
* P < 0.01, Fisher's exact probability test. ICV, into third ventricle.
CCK injection into the LHA Fig. 3 shows the effects of injecting CCK into the LHA. Following injection into the LHA, there was a gradual increase in firing rate which was similar to that observed following the injection of CCK into the VMH. The firing rate increased linearly for as long as observations were continued. When saline was injected into the same area, there was no effect. In 12 rats which did not include the 2 rats used for repeated injection of CCK into the PVN and the LHA, 9 showed an increase and 3 were without effect (Table I). Control injection in 5 rats showed no effect on nerve activity (Table I). The increase in responses to CCK was statistically significantly greater than in control (P < 0.01, Fisher's exact probability test), CCK injection into the DMH Fig. 4 shows the effect of injecting CCK or saline into the DMH. In contrast with the VMH or LHA,
Fig. 3. Rate meter plots of BAT sympathetic nerve activity following CCK in.iection into the LHA. Upper panel: CCK injection increased nerve activity. Lower panel: injection of saline induced no remarkable change.
CCK had no effect following its injection into the DMH. Eight out of the 10 rats tested showed ao remarkable change of nerve activity whereas one each showed an increase or decrease (Table I).
CCK injection into the PPN Fig. 5 shows the effect of injecting CCK into the paraventricular nucleus and LHA. Two patterns are observed. In the upper panel, the injection of CCK into the PVN produced a decrease in firing rate which reached a nadir at approximately the end of the injection. The rate of firing had decreased by more than 50% following the injection of CCK. In the middle panel, the first injection of CCK into the PVN had no effect on nerve activity, but when a second injection was given into the LHA of the same animal it produced an increase in firing rate. In the bottom, saline injection showed no effect. Out of 11 rats tested includ-
300
~DMH CCK
imp. O /5see 1"0rain
VMH SA
mp. '5sec 10rain
200
200
imp. 0 /Sse¢
imp. 0 /5see
lOmtn
Fig. 2, Rate meter plots of BAT sympathetic nerve activity following CCK injection into the VMH. Upper panel: CCK injection increased nerve activity. Lower panel: injection of saline induced no remarkable change.
Fig. 4. Rate meter plots of BAT sympathetic nerve activity following CCK injection into the DMH. Upper panel: CCK injection induced no remarkable change. Lower panel: injection of saline induced no remarkable change.
301 m,M . ~
200
~A I.:oOo,
Imp. 0 /Ssec
0.3
1Omin 200
~,HA,
'
,
/
o,
.""
i
,
-1
3
O.~.t~, -
'::::,I~::." .~
imp. 0 /5sec
~AHA~II!.[ i ',,._.,,; .' .i ",,,,..,,'
lOmin r---i PVN SA
POA
'
200
--1.B
-2.B
imp, Jo/ssec
-3.3
m
lOmin
Fig. 5. Rate meter plots of BAT sympathetic nerve activity following
CCK injectioninto the PVN. Upper panel: CCK injection decreased nerve activity.Middle panel: CCK injectioninto the PVN inducedno remarkable change but followinginjection into the LHA increased nerve activity.Lower panel injection of saline induced no remarkable change. ing 2 rats which received repeated injection into the PVN and the LHA, 4 showed a decrease and 7 showed no effect (Table I). Two rats for repeated injections showed no respdnse to the first injection into the PVN and an increase in firing rate in the second injection into the LHA as shown i~ Fig. 5 (middle).
CCK injection into the P04 The data in Fig. 6 show that the injection of CCK into the preoptie area produced no difference from an injection of saline into the same area. ,.---1Dn&~K
200
imp, 0 /5see lOmln
np.
5sec
lOmin
Fig. 6. Rate meter plots of BAT sympathetic nerve activity following CCK injection into the POA. Upper panel: CCK injection induced no remarkable change, Lower panel: injection of saline induced no remarkable change.
Fig. 7. Diagrammatic representation of CCK injection sites. (*), increase; (v), decrease; (o), no change. AC, anterior commissura; POA, preoptic area; ALIA, anterior hypothalamicarea; PVN, paraventrieular nucleus; DMH, dorsomedial hypothalamic nucleus; LHA, lateral hypothalamicarea; VMH, ventromediai hypothalamic nucleus. Injection sites were distributed from -0.3 to -1,3 in the POA, from -2.8 to 3.3 in the DMH, LHA and VMH, and localized to the plane at -1.8 in the PVN according to the atlas of Paxinos and Watson2a.
Histological examination Fig. 7 shows diagrammatically the injection sites. Injection sites for the VMH, the LHA and the DMH distributed in the range from - 2 . 8 to - 3 . 3 according to the atlas of Paxinos and Watson 29. There were no specific responses and localization of injection site within each region. Injection sites for the PVN and the POA were localized to the restricted region in - 0 . 3 for POA and - 1.8 for PVN. No specific localization of effective and non-effective sites by CCK injections could be observed within the PVN. DISCUSSION The present experiments have explored the role of CCK on the activity of the sympathetic nervous system, innervating interscapular brown adipose tissue (IBAT). The data clearly show that CCK, whether injected into the third ventricle, into the VMH or into the LHA was associated with an increase in sympathetic firing rate. In contrast, no effects were observed when CCK was injected into the POA, into the PVN or into the DMH. Data from several laboratories suggests that the sympathetic nervous system has several functionally
302 separate parts s'2s. Fasting decreases sympathetic activity to IBAT, heart, liver and pancreas 36'as but not to the limbs ls'~s. 2-Deoxy-D-glucose (2DG) is an analogue of glucose which impairs intracellular glucose metabolism and this produces glucopenia and reduces sympathetic activity to IBAT 16a7 but stimulates the adrenal nerve to release norepinephrine and epinephrine a6. Interleukin-1/3 stimulates sympathetic activity to lung and spleen but not elsewhere 3t. The sympathetic nerves innervating IBAT are thus but one part of the sympathetic nervous system. These sympathetic nerves to IBAT subserve the activation of this tissue which increases heat production. This part of the sympathetic nervous system might best be referred to as the 'thermogenic' component of the sympathetic nervous system. CCK fits a consistent pattern which has been observed for several other peptides as well as monoamines, drugs and hypothalamic lesions 9. Both NPY and /]-endorphin, which increase food intake have been ~hown to decrease sympathetic activity to IBAT whereas peptides such as CRH and CCK which decrease food intake s'22, increase thermogenic sympathetic activity. The anatomic basis for this robust physiologic relationship is unclear. Anatomic mapping studies for the effects of peptides on sympathetic activity have only been reported ih a few cases. Neuropeptide Y decreases sympathetic activity when injected into the PVN 2° and increases sympathetic activity when injected into the preoptic area, but not into other hypothalamic regions 2°. CRH increases sympathetic activity when injected into the POA but not elsewhere tg. CCK, the subject of this study, increased sympathetic activity when injected into either the VMN or LHA, but was without effect in the PVN, POA or DMH. Thus peptide receptors which can modulate sympathetic efferent activity appear to exist in several regions of the hypothalamus. Injection of peptides into several different regions of the hypothalamus can modulate sympathetic activity in a reciprocal fashion to food intake. This would suggest that when the sympathetic activity is changed, a component of the common final response which arises after these various sympathetic sites have been activated must then serve as a feedback element modulating food intake. This pattern of multiple sites for activating sympathetic activity being funnelled into a common pathway which generates a signal for modulating food intake, would be consistent with known ~ and fl receptor modulation of feeding which occurs in two different hypothalamic areas, a2-Adrenergic receptors appear to be activated in the paraventricular nucleus to increase food intake 24. Conversely,/3-adrenergic recep-
tors loca'.ed in the perifornical area inhibit feeding and it is these receptors to which the sympathetic nervous system may feed back 2~. If the descending sympathetic fibers modulated an ascending pathway from the hindbrain to the perifornical area, this could explain the reciprocal relationship reported here and previously. A recent study using L-glutamate, a stimulator of neuronal activity, has provided a~litional understanding of the system l'a7. Eiectrophoretic application of CCK has been shown to produce an excitatory effect on neuronal activity of VMH neurons 32. The injection of glutamate into the VMH uniformly increases sympathetic activity and this may occur through the effects of L-glutamate on these neurons. Injection of CCK into the LHA also produced an increase in activity of the sympathetic nerves to IBAT. However, the pattern of response of peripheral sympathetic activity to IBAT following glutamate injection in the LHA was complex suggesting subregional organization within this area where both inhibitory, stimulatory and biphasic patterns of response exist a7. A recent electrophysiological study has demonstrated a suppressive effect of CCK on neuronal activity of LHA neurons a2. Taken together, it can be suggested that CCK increases sympathetic nerve activity through suppression of the inhibitory component of LHA, and that it thus works through a disinhibition mechanism. The present study demonstrated that the VMH and the LHA, but not other hypothalamic regions, were effective sites for activation of sympathetic nerves to IBAT in response to CCK administration. Recent neuroanatomieal studies have shown the presence of CCK-containing neurons in the brain 6,3s. The VMH has been shown to be one of the hypothalamic nuclei which receives a rich innervation of CCK neurons as, and the CCK receptor has been shown to he distributed in this nucleus and the LHA2k These studies and the present findings indicate that CCK neurons innervating both regions play a role in the regulation of sympathetic nerve activity to IBAT. Acknowledgements. Supported in part by Grant DK 31988 from the NIH.
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