Clonidine suppression and its adrenoreceptor mediation in schedule-induced polydipsia

Physiology&Behavior, Vol. 40, pp. 317-322. Copyright©Pergamon Journals Ltd., 1987. Printed in the U.S.A.

0031-9384/87 $3.00 + .00

Clonidine Suppression and Its Adrenoreceptor Mediation in Schedule-Induced Polydipsia' CHE-SE TUNG z AND TSAI-HSIN YIN

Department of Pharmacology, National Defense Medical Center and Neuroscience Laboratory, Clinical Research Center Tri-service General Hospital Taipei, Taiwan, Republic o f China R e c e i v e d 20 J u n e 1986 TUNG, C.-S. AND T.-H. YIN. Clonidine suppression and its adrenoreceptor mediation in schedule-inducedpolydipsia. PHYSIOL BEHAV 40(3)317-322, 1987.--Rats were trained in a fixed-interval, one-minute (FI 1 rain) food reinforcement schedule for 1 hour daily at reduced body weight until their lever presses, licks and water intake all became stabilized for 6 days. Two experiments were performed to examine the function of sympathetic activity in schedule-induced polydipsia. In experiment 1, intracerebroventricular injection of clonidine (0.75-37.5 nmol) produced a dose-related suppression of schedule-induced drinking and licking and schedule-dependent lever pressing; these effects were later attenuated by yohimbine (5 nmol) pretreatment. Prazosin (10 nmol) also decreased clonidine-induced suppression of lever pressing, whereas neither prazosin (10 nmol) nor naloxone (10 nmol) caused any alteration in the suppression effects of clonidine on drinking and licking. None of these antagonists alone changed an individual rat's preestablished behavioral baselines. In experiment 2, the endogenous catacholamine levels were determined in frontal cortex, hypothalamus, brainstem, dorsal obex area and adrenal glands. During the SIP situation, both the epinephrine level in adrenal glands and the norepinephrine level in hypothalamus were elevated. Schedule-induced polydipsia Prazosin Naloxone

Sympathetic activity

Catecholamines

Clonidine

Yohimbine

tonomic effects both in man and animals [10]. The alpha2 adrenoreceptors located postsynaptically in central medullary sites impede sympathetic outflow during activation [21]. In homeostatic drinking such as extracellular dehydrationinduced drinking, the antidipsogenic effect of the alpha2 adrenergic agonist, clonidine, has been attributed to its action on central sites [8]. Non-homeostatic drinking, such as SIP behavior, has been shown to be greatly facilitated in its establishment when it was preceded by an increase in central catecholamine neuronal activity [15]. Both chronic amphetamine administration [28] and lesion or antagonists on central dopamine system [18,19] can selectively by disrupt the acquisition of SIP. Furthermore, different degrees of adrenal hypertrophy have been found to be associated with high SIP, and adrenal demedullation, but not adrenalectomy, depresses SIP [4,27]. All these lines of evidence indicate that both central and peripheral catecholamine neurons are important in the control of drinking behavior and in particular SIP. In the present studies, clonidine was used as a probe to

T H E phenomenon of schedule-induced polydipsia (SIP) was introduced by Falk in 1961 [6]. It occurs when food-deprived animals are subjected to an intermittent food reinforcement schedule and is categorized as a type of animal adjunctive behavior. Although many theories have been postulated, the basic mechanisms still remain to be determined. Evidence indicates that SIP elicitation is under non-homeostatic control rather than by osmotic or hypovolemic thirst stimulation [7,16]. In addition to central and peripheral mechanisms, sensory input from visceral to central nervous structures also plays a critical role in SIP establishment [16,22]. It is important that the nature of central-peripheral signal coupling be better understood. Central catecholamines are generally recognized to be involved in behavioral activation [2,9]. The very interesting reports on the interactions between the function of catecholamine neurons and drinking behaviors [8, 11, 18] intrigued us to study the relation between adrenergic activity and SIP establishment. Administration of a selective alpha2 adrenergic agonist and an antagonist induces psychomotor and au-

1This research was supported by grants from the National Science Council, R.O.C., (NSC-74-0412-B016-01) and in part by the Tri-service General Hospital Grant-in-Aid program (1-7501) for the faculty of National Defense Medical Center. ~Requests for reprints should be addressed to Dr. C. S. Tung, National Defense Medical Center, P.O. Box 8244, Taipei, Taiwan, Republic of China.

317

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TUN() AND YIN

test the hypothesis that central sympathetic outflow increases during SIP. Catecholamine levels in different brain areas and adrenal medulla in rats were also assessed with or without SIP training. The results revealed that clonidine suppressed all parameters of SIP including drinking, licking, lever pressing and pellet intake, and these effects were partially or entirely reversed by pretreatment with yohimbine, an alphae antagonist.

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Animals. Male Sprague-Dawley rats (300-350 g) were housed individually in standard stainless-steel cages with a wire mesh floor on a 12-12 light/dark cycle at room temperature for 10 days to adapt to the environment. Rats had coninuous access to Charles River pellets and tap water ad lib except during the periods of controlled deprivation and of experimentation, Apparatus. Two rodent operant chambers (LVE 1469, medium size) with matching sound attenuation cubicles and 45 mg food pellet dispensers were used. A 100-ml glass graduated cyclinder was fixed on the back wall outside the chamber with its stainless steel spout protruding 2.0 cm into the chamber and 4.0 cm above the grid floor. The lever for pellet activation was positioned on the wall 3.0 cm above the floor adjacent to a food chute and to the right of the water spout. Procedure. Body weight recorded on the tenth day was considered as the 100% ad lib feeding weight for each animal. Rats were gradually reduced to 80%, of these weights by restricting daily food rations over one week. For the next 4 days, animals were trained in the operant chamber to press a lever for food pellets on a continuous reinforcement schedule. On the fifth day, the schedule was changed to a fixed interval 1-min food reinforcement for 1 hr per day until SIP fully developed. SIP developed usually after six sessions of fixed interval food reinforcement schedule training in 6 days (one session per day for six days). The number of licks and presses were recorded by individual counters, and water consumption was determined by weighing the water bottles before and after the session. At the end of each session, the animal was returned to its home cage and was given about 5-10 g of food pellets for the rest of the day. Sessions were conducted between 0830-1630, 6 days a week. Rats with established SIP after training were selected for intracerebroventricular cannulation. Under anesthesia with pentobarbital, 40 mg/kg, a stainless steel cannula (C313 G, Roanoke, VA) was chronically implanted into the right lateral cerebroventricle (ICV) according to the coordinates: posterior 1.0 ram, lateral 1.25 ram, depth 4-5 mm in reference to the bregma as zero-point. The proper positioning of the cannula in the ventricle was verified by the injection of methylene blue into the ventricle at the sacrifice of each animal. When the rat had recovered from the operation and its presses, licks and water consumption became stabilized and had sustained over a period of 6 days, these measures were taken as baseline data. Then, eight daily sessions of experiment with either saline or one type of drug followed. Drugs used were clonidine (0.75, 7.5 and 37.5 nmol), yohimbine (1 and 5 nmol), prazosin (10 nmol) and naloxone (10 nmol). Twenty minutes before the session, one of the above agents in 2 txl of saline or, in controls, only the vehicle was introduced through the cannula. Because of the lengthy time span of the experiment, the

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Experiment I

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FIG, 1. Effect of drugs administered intracerebroventricularly on water consumption during 1 hr daily session in the test chamber. Data were net differences from the baseline value as a function of 4 daily sessions in 4-7 animals for each trial. Values are mean_+SEM. C (Saline, 0.9~), Y, (Yohimbine, l nmol), Y~ (Yohimbine, 5 nmol), P (Prazosin, 10 nmol), N (Naloxone, 10 nmol), CII (Clonidine, 0.75 nmol), CI., (Clonidine, 7.5 nmol), CI:~(Clonidine 37.5 nmol).

large number of animals involved and the varying conditions of the individual rats, no strict protocol could be followed. Therefore, the following guidelines and procedures were adopted: (a) Besides control treatment with saline, each animal was limited to one type of drug treatment as much as possible. If the animal's condition would follow for further testing with another agent, the baseline was first reestablished for reference. (b) In determining dose and response relationship for clonidine and yohimbine, rats were tested in either ascending or descending order. (c) The dosage of 7.5 nmol for clonidine and that of 5 nmol for yohimbine were selected for testing the interaction of these two drugs. Two other antagonists tested were the alpha1 antagonist, prazosin (10 nmol), and opiate antagonist, naioxone (10 nmol). One of the antagonists was given 10 minutes before clonidine administration. It should be noted that t h e " n " of each treatment in Figs, 1-4 represents the number of animals so treated, and the data of 4 sessions were averaged. Data analysis. In order to facilitate comparisons among the experimental groups, the net differences between the measured values and the baseline values were used as data, The transformed data were analyzed, where appropriate, by one-way analysis of variance with Duncan's t-test. Data were reported as mean+--standard error of the mean (SEM). A p value of 0.05 was selected as the upper limit for statistical significance.

Experiment 2 Animals and apparatus. Twenty eight male SpragueDawley rats were housed individually under the same conditions as described in experiment 1 and were maintained at

C L O N I D I N E S U P P R E S S I O N IN SIP

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FIG. 2. Effect of drugs administered intracerebroventricularly on licks during 1 hr daily test in the test chamber. Data were net differences from the baseline value as a function of 4 daily sessions in 4--7 animals for each trial. Values are mean±SEM. Abbreviations and symbols as in Fig. 1.

FIG. 3. Effect of drugs administered intracerebroventricularly on lever presses during 1 hr daily test in the test chamber. Data were net differences from the baseline value as a function of 4 daily sessions in 4--7 animals for each trial. Values are mean_SEM. Abbreviations and symbols as in Fig. 1.

80% body weight. The apparatus for SIP testing was the same. Procedure. Fourteen rats randomly selected from 28 were trained for SIP (G2) as described in experiment 1. When presses, licks, and water consumption became stabilized over a 6-day period, 6 consecutive daily sessions were followed. At the end of the last session, animals were sacrificed by decapitation. The 14 remaining rats served as the control group (G1) with body weight also reduced to 80% without any SIP training. The brain and the adrenal glands were immediately removed and the frontal cortex, hypothalamus and dorsal obex area were dissected out from a series of cross sections of the fresh brain. The samples were frozen in dry ice for preservation until analysis. Protein concentrations of tissue homogenate were determined by the method of Lowry et al. [14]. Catecholamine quantification was performed by the following procedure. Tissue extracts. Tissue dissections were homogenized in 1.5 ml of 0.4 M HC104 which contained sodium bisulfite (0.1%) and disodium EDTA (0.005-0.05%). After the tissue had been homogenized at 0°C, the sample was centrifuged at 15,000 × g for 10 minutes. Then to the supernatant, 50 mg of A1203 was added and the volume brought up to 2.5 ml with 0.5 M Tris buffer, p H 8.6. Catecholamines were absorbed to the A1203 by shaking the tubes thoroughly for about 15 min. The A1203 was allowed to fall to the bottom of the tube and the supernatant then aspirated off. Triple washes of 2 ml of deionized water were then applied to the A1203 and later aspirated off completely after shaking. Catecholamines were then eluded from the A1203 into 100/xl of HCIO4, 0.4 M.

Liquid chromatography with electrochemical detection (LCEC) was then applied to the supernatant. Chromatographic conditions. The LCEC apparatus consisted of a BAS Model LC-304D with a temperature control solvent delivery system and a Biophase ODS 5/xm (250×4.6 mm) column. The flow rate was 1.0 ml/min. This equipment was connected to a dual parallel electrochemical detector, which was set at +850 mV and +600 mV versus the Ag/AgCI/3 M NaCI reference electrode in this study. Sensitivity of the detector was set at the 5 nA testing range. The mobile phase was 8% acetonitrile/92% 0.15 M monochloroacetate, p H 3.0, containing 0,7 mM Na2EDTA and 0.14 mM sodium octyl sulfate. The LCEC method permits the resolution of norepinephrine (NE), epinephrine (E) and dopamine (DA) with capacity factors (K') of 0.75, 1.98, and 3.50 respectively and recovery rates of 60%-70% in this study. External standards were used to calibrate the instrument. The range of linearity of the integrator was from 200 pg to 20 ng with NE r=0.99, E r=0.99 and DA r=0.97. RESULTS Experiment 1 Figures 1, 2, 3, 4 represent control (0.9% NaC1) and drug effects on water consumption, licks, lever presses and pellet intake which are illustrated as the 4 day means of net differences to the baseline value. The means of total water intake (ml), licks and lever presses for each antagonism trial are also presented in Table 1. No differences for all parameters

320

T U N G A N D YIN

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TABLE 1 MEANS ± SEM OF WATER INTAKE (ml), LICKS, AND LEVER PRESSES FOR ANTAGONISM TEST SESSIONS FOLLOWING INTRACEREBROVENTRICULAR ADMINISTRATION OF CLONIDINE 7.5 nmol r--1

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Licks

Lever Presses

23.0 _+ 2.6 13.8 _+ 2.2 23.8 -+ 2.9

5591 +_ 545 4430 + 812 5118 _+ 880

540 _+ 73 378 -+ 60 724 _+ 234

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FIG. 4. Effect of drugs administered intracerebroventricularly on pellet intake during 1 hr daily test in the test chamber. Data were net differences from the baseline value as a function of 4 daily sessions in 4-7 animals for each trial. Values are mean-+SEM. Abbreviations and symbols as in Fig. 1.

TABLE 2 MEANS +- SEM OF FRONTAL CORTEX, HYPOTHALAMUS. BRAINSTEM DORSAL OBEX AREA AND ADRENAL GLAND CATECHOLAMINE CONCENTRATIONS AFTER 6 CONSECUTIVE DAYS OF TRAINING SESSIONS G1 =CONTROL GROUP, G2=SIP GROUP Frontal Cortex

Hypothalamus

G1

G2

G1

G2

G1

G2

G1

G2

Norepinephrine

2.09 _+0.42

2.01 _+0.40

7.75a _+2.24

12.69" _+2.24

14.42 _+2.12

17.99 _+4.91

1.49 _+0.21

2.01 +_0.48

Epinephrine

0.24 -+0.06

0.38 -+0.46

0.91 -+0.22

2.35 _+0.98

5.03 _+1.67

10.96 _+4.89

2.09 _+0.48

3.71" _+0.59

Dopamine

0.37 _+0.16

0.49 _+0.16

1.76 _+0.65

0.99 _+0.42

0.91 _+0.42

0.21" _+0.09

0.12 _+0.05

0.03* _+0.01

Catecholamines (ng/mg protein)

Brainstem

Adrenal Gland

*p <0.05.

w e r e o b s e r v e d a m o n g trials d u r i n g t h e initial 6 d a y s o f the b a s e l i n e period. C e n t r a l a d m i n i s t r a t i o n o f c l o n i d i n e at d o s e s o f 0.75 to 37.5 n m o l s u p p r e s s e d w a t e r c o n s u m p t i o n , F ( 3 , 3 4 ) = 3 2 . 1 6 , p < 0 . 0 1 , licks, F ( 3 , 3 4 ) = 6 . 4 5 , p < 0 . 0 5 , a n d p r e s s e s , F ( 3 , 3 4 ) = 8 . 2 3 , p < 0 . 0 1 , in a d o s e - r e l a t e d f a s h i o n . A larger d o s e o f c l o n i d i n e , 37.5 n m o l , also significantly supp r e s s e d pellet i n t a k e at p < 0 . 0 1 level. F u r t h e r m o r e , treatment with doses of antagonists with yohimbine 1 nmol and 5 n m o l , p r a z o s i n 10 n m o l , a n d n a l o x o n e 10 n m o l , r e v e a l e d n o d i f f e r e n c e in a n y p a r a m e t e r m e a s u r e d . T h e a n a l y s i s o f w a t e r c o n s u m p t i o n w h e n the a n t a g o n i s t was g i v e n 10 m i n u t e s b e f o r e t h e a g o n i s t a d m i n i s t r a t i o n rev e a l e d t h a t y o h i m b i n e 5 n m o l , c l o n i d i n e 7.5 n m o l , y o h i m b i n e

5 n m o l + c l o n i d i n e 7.5 n m o l , a n d 0.9% NaC1 yielded v a l u e s t h a t w e r e significantly different, F ( 3 , 3 5 ) = 1 1 . 6 , p < 0 . 0 1 . T r e a t m e n t w i t h p r a z o s i n 10 n m o i , c l o n i d i n e 7.5 n m o l , p r a z o s i n 10 n m o l + c l o n i d i n e 7.5 n m o l , a n d 0.9% NaC1 yielded v a l u e s t h a t w e r e also significantly different, F ( 3 , 3 8 ) = 8 . 9 , p < 0 . 0 1 . T r e a t m e n t w i t h n a l o x o n e 10 n m o l , c l o n i d i n e 7.5 n m o l , n a l o x o n e 10 n m o l + c l o n i d i n e 7.5 n m o l , a n d 0.9% N a C I yielded v a l u e s t h a t w e r e significantly differe n t as well, F ( 3 , 3 9 ) = 1 7 . 8 , p < 0 . 0 1 . T o d e t e r m i n e if t h e c h a n g e s are r e l a t e d to t h e a n t a g o n i s t a n d a g o n i s t i n t e r a c t i o n , D u n c a n ' s t - t e s t s w e r e p e r f o r m e d . Y o h i m b i n e c a u s e d a signifi c a n t a t t e n u a t i o n o f w a t e r intake s u p p r e s s i o n b y clonidine, p < 0 . 0 1 , w h e r e a s n e i t h e r p r a z o s i n n o r n a l o x o n e h a d a signifi-

CLONIDINE SUPPRESSION IN SIP cant effect on the clonidine drinking response, p>0.05. The analysis of licks indicated that trials of yohimbine 5 nmol, clonidine 7.5 nmol, yohimbine 5 nmol + clonidine 7.5 nmol, and 0.9% NaC1 were significantly different, F(3,35)=2.69, p<0.05; trials of prazosin 10 nmol, clonidine 7.5 nmol, prazosin 10 nmol + clonidine 7.5 nmol, and 0.9% NaC1 were also significantly different, F(3,38)=4.75, p<0.01; and that trials of naloxone 10 nmol, clonidine 7.5 nmol, naloxone 10 nmol + clonidine 7.5 nmol, and 0.9% NaCI was significantly different as well, F(3,39)=7.33, p<0.01. Duncan's t-tests revealed that suppression of licks with clonidine was signficantly attenuated by pretreatment of yohimbine, p<0.01, but not by either prazosin or naloxone pretreatment, p >0.05. The analysis of lever presses indicated that trials of yohimbine 5 nmol, clonidine 7.5 nmol, yohimbine 5 nmol + clonidine 7.5 nmol, and 0.9% NaC1 were signficantly different, F(3,35)=5.5, p<0.01; trials of prazosin 10 nmol, clonidine 7.5 nmol, prazosin 10 nmol + clonidine 7.5 nmol, and 0.9% NaC1 were also significantly different, F(3,38)=5.71, p<0.01; and that trials of naloxone 10 nmol, clonidine 7.5 nmol, naloxone 10 nmol + clonidine 7.5 nmol, and 0.9% NaCI were significantly different as well, F(3,39)=5.63, p<0.01. Duncan's t-tests revealed that lever suppression with clonidine was attenuated by both yohimbine or prazosin pretreatment, p<0.01 and p<0.05 respectively, but not by naloxone pretreatment, p>0.05.

Experiment 2 The levels of NE, E, and DA were determined in the brain regions and adrenal medulla for both control (G1) and SIP (G2) rats and are presented in Table 2. The fullblown SIP behavior was evidenced by the high water consumption of 22.0 m l - 1.1 (SEM) per session for G2 rats. SIP rats showed a marked elevation of NE in the hypothalamus (p<0.05), but a reduction of DA in brainstem dorsal obex area (p<0.05). An elevation of E (p<0.05) and a reduction of DA (p<0.05) were also found in the adrenal medulla after SIP practice. No differences were observed with any catecholamine in the frontal cortex between G 1 and G2. DISCUSSION Several lines of research have suggested that the development and maintenance of SIP has a neurophysiological basis and, in fact, is controlled or influenced by both central and peripheral structures [13, 17, 25, 26]. Wayner (1970) introduced the idea that a contingent schedule will build up in the subject a general motor excitability which is then released as adjunctive behavior, the manifestation of which depends upon the environmental conditions at the time [24]. The food-contingent stimulation of the gastrointestinal system serves as the origin of SIP with our experimental conditions. The vagal afferents were found to be essential in maintaining the heigtened central activity, mostly in the posterior lateral hypothalamus, since subdiaphragmatic vagotomy or bilateral lesions of this hypothalamic area abolished SIP [22,25]. The efferent limb for this behavior has been hypothesized by Wright and Kelso (1981) to include the hypothalamus-pituitary-adrenal axis, with emphasis on the adrenal medullary catecholamines [27]. In the present study, by altering sympathetic outflow with an agonist and several antagonists and by directly determining both brain and adrenal catecholamines, fresh evidence for the involvement of central sympathetic outflow in SIP establishment has been provided.

321 Central noradrenergic activity has been implicated in many pathophysiological conditions such as stress [9]. Selective pharmacological probes have been used for the classification of adrenoreceptors [10]. In intact animals and man, alpha2 agonists reduce sympathetic outflow while alphas antagonists, notably yohimbine, increase outflow [10,23]. Thus, there may be a role for central alpha2 adrenoreceptor in the pathophysiology of hyper- and hypoadrenergic conditions. The result of our first experiment indicates that clonidine suppresses schedule-induced drinking and licking and schedule-dependent lever pressing in a dose-related manner. The parallel suppressant effects of clonidine on both schedule-induced and schedule-dependent behaviors were significant and were attenuated by yohimbine pretreatment. Although we did not find one dosage of clonidine that will affect only one particular facet of these coexistent behaviors, our data support the previous report that clonidine is psychoactive, and its effects are due at least in part to selective alpha2 receptor stimulation [5]. An alternative interpretation of our data would be that the clonidineinduced reduction in SIP is secondary to the disruption of bar pressing and the resultant decrease in pallet intake should provide less drinking occations. Further studies based on evaluation of drug kinetic profiles on individual parameters could be useful to further clarify the drug selectivity on each behavior. Since administration of a high dose of clonidine may also affect other receptors [1, 12, 20], two antagonists of other receptors (prazosin, alpha1, and naloxone, opiate receptors) were also administered. Prazosin pretreatment attenuated clonidine effect on lever pressing, indicating that stimulation of central alpha2 receptor can not be the sole component of the drug's effect in schedule-dependent lever pressing behavior. Naloxone did not suppress either drinking, licking or lever pressing. This observation lends additional support to the previous report that adjunctive drinking is mediated by neural substrates rather than endogenous opioids [3]. Furthermore, naloxone failed to prevent the suppression effects of clonidine on any other parameters, indicating that central modulation of nociceptive action through opioid receptors by clonidine might not play a part in these studies. Our second experiment indicated that the adrenal medulla epinephrine level was significantly elevated, but that the dopamine level was reduced during SIP. Circulating levels of norepinephrine and epinephrine were also found to be elevated (unpublished observations of the authors). There was a significant increase in norepinephrine in the hypothalamus, and a decrease of dopamine in brainstem dorsal obex area during SIP. Although these changes do not necessarily reflect alterations in neuronal activity, they nevertheless suggest a relation between catecholaminergic function and the establishment of a chronic behavior. A tentative correlation between central noradrenergic function in hypothalamus and adjunctive behavior is thus provided. Further experiments to determine the relationship between the level of circulating catecholamines activated by sensory input and the establishment of adjunctive behayior will be interesting.

ACKNOWLEDGEMENTS We thank Ms. Yi-Fang, Juang and Yu-Ming, Ho for their technical assistance.

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