Modification of the actions of ethanol by centrally active peptides

Peptides, Vol. 2, Suppl. 1, pp. 99-106, 1981. Printed in the U.S.A.

Modification of the Actions of Ethanol by Centrally Active Peptides G E R A L D D. F R Y E , D A N I E L L U T T I N G E R , C H A R L E S B. N E M E R O F F , R I C H A R D A. V O G E L , A R T H U R J. P R A N G E , JR. A N D G E O R G E R. B R E E S E

Biological Sciences Research Center, Center for Alcohol Studies Departments o f Psychiatry, and Pharmacology, and the Neurobiology Program University of North Carolina, School of Medicine, Chapel Hill, NC 27514

FRYE, G. D., D. LUTTINGER, C. B. NEMEROFF, R. A. VOGEL, A. J. PRANGE, JR. AND G. R. BREESE.

Modification of the actions of ethanol by centrally active peptides. PEPTIDES 2: Suppl. 1,99-106, 1981.--Ethanol (2.0-5.0 g/kg, IP) caused a dose-related impairment of the aerial righting reflex of mice 60 min after injection. Ethanol (3.5 g/kg, IP) given simultaneously with neurotensin (30/zg, IC), bombesin (30/zg, IC) or fl-endorphin (20/zg, IC) caused a greater impairment of the reflex than ethanol alone. Simultaneous treatment with ethanol (4.0 g/kg, IP) and thyrotropin-releasing hormone (TRH, 3.0-30 p.g, IC) caused less impairment of this measure than ethanol alone. None of the peptides altered the height of aerial righting when administered alone, or when administered with ethanol no peptide altered blood or brain ethanol content. Unexpectedly, TRH (20 and 40 mg/kg, IP) potentiated the action of ethanol by increasing punished licking in water-deprived rats, rather than antagonizing this acute action of ethanol. Like ethanol (1.0 and 2.0 g/kg, IP), /3-endorphin (100 p.g, IC) suppressed ethanol-withdrawal tremor and audiogenic-seizure susceptibility in ethanol-dependent rats./3-Endorphin (1/zg) and bombesin (10 and 30/zg, IC) reduced only audiogenic-seizure susceptibility. TRH (10-100/xg, IC, or 1-40 mg/kg, IV) and neurotensin (10-100/xg, IC) had no effect on these ethanol-withdrawal signs. These findings suggest that centrally active peptides may play a role in certain acute and chronic actions of ethanol. Because TRH, neurotensin, bombesin and/3-endorphin do not alter all actions of ethanol in the same way, an interaction of ethanol with many functionally independent neuronal circuits is suggested. Ethanol Neuropeptides Thyrotropin-releasing hormone (TRH) Neurotensin /3-endorphin Aerial righting reflex Punished responding Ethanol withdrawal Audiogenic-seizure susceptibility

E T H Y L alcohol is now widely recognized as a potent psychotropic drug. Distinct changes in many central nervous system (CNS) functions have been identified which are characteristic of acute and chronic ethanol treatment in both man and animals [14, 32, 36]. The diverse nature of the many different behavioral and neurochemical actions of ethanol [14] as well as electrophysiological evidence demonstrating ethanol-induced changes in the electrical activity of most brain regions [36] lend support to the view that ethanol produces widespread effects involving many neural circuits throughout the CNS. In order to investigate the complex actions of ethanol, a variety of simple animal models have been developed so that specific behavioral actions of ethanol can be correlated with ethanol-related perturbations in neurochemical mechanisms [10]. This approach has been particularly useful in helping to define a specific interaction between ethanol and neurochemicals like the monoamines for which a neurotransmitter role is most clearly established. Thus far the catecholamines, serotonin, acetylcholine and gamma-aminobutyric acid have all been implicated as playing an important role in one or more of the behavioral actions of ethanol [36]. Another class of neurochemicals which could play an important role in the CNS actions of ethanol are the endoge-

Bombesin Tremor

nous neuropeptides. For example, neuropeptide-induced changes in behavior, neurochemistry and neurophysiology have contributed to the view that these endogenous peptides probably constitute a new class of neurotransmitter or neuromodulator substances [3, 23, 38, 40]. In addition, recent observations of interactions between CNS actions of opiate drugs and endogenous opiate-like peptides [1, 2, 19, 44] have strengthened considerably the hypothesis that endogenous neuropeptides could be involved in the central actions of ethanol. Whether ethanol exerts some of its acute behavioral actions through changes in brain neuropeptide systems is not known, but this is clearly plausible as suggested by recent reports. For example, intracisternal or systemic administration of thyrotropin releasing hormone (TRH), a tripeptide found heterogeneously distributed throughout the brain [38,50], was found to shorten sleeping time and to reverse hypothermia induced by ethanol in mice and rats [5, 9, 37, 39]. Antagonism of ethanol narcosis was also observed after intraventricular administration of histidyl-proline diketopiperazine, a cyclic dipeptide metabolite of TRH [39] and after treatment with MK-771, a synthetic tripeptide with some similarities to TRH [37]. In addition, systemic administration of TRH has been found to reverse decreases in loco-

C o p y r i g h t © 1981 A N K H O I n t e r n a t i o n a l Inc.--0196-9781/81/050099-08501.30/0

100

F R Y E ET AL.

motor activity and in the content of cerebellar guanosine 3',5'-monophosphate (cGMP) caused by treating rats with a depressant dose of ethanol [26]. These actions of TRH are apparently independent of the neuroendocrine action of this peptide [9,27]. In contrast to these actions of TRH to reduce the CNS depression caused by ethanol, recent investigations indicate that certain other peptides can increase the depressant effects of ethanol. Bombesin,/3-endorphin and neurotensin are three neuropeptides which, like TRH, have been identified in mammalian CNS, and which produce a variety of physiological and behavioral alterations, suggesting an important regulatory role in CNS function [6, 31, 34]. Recent studies have shown that intracisternal administration of neurotensin, bombesin and/3-endorphin lengthened the anesthetic effect of ethanol and potentiated the hypothermia caused by ethanol in mice [25,35]. In addition to peptide-induced modification of acute ethanol actions, it also seems likely that neuropeptides can influence the chronic actions of ethanol. F o r example, the rate of development of tolerance to ethanol [20, 21, 41], the severity of ethanol withdrawal syndrome [41] and alcohol consumption [11,33] were all increased by administration of vasopressin-like peptides. Measurement of arginine vasopressin in human plasma also clearly indicated that intravenous ethanol infusion inhibited the secretion of this peptide from the hypothalamic-neurohypophyseal axis [51]. Furthermore, acute and chronic ethanol treatment has been reported to increase and decrease respectively the concentration of methionine-enkephalin and/3-endorphin in rat brain [43]. These findings and those with TRH, neurotensin, bombesin and arginine vasopressin, suggest that neuropeptides, like the classical monoamine neurotransmitters, may participate in some of the central neurochemical mechanisms underlying both the acute and chronic actions of ethanol treatment. In the present studies, the interaction of ethanol with TRH, neurotensin, bombesin and/3-endorphin, is examined further using three behavioral models of specific ethanol actions not previously studied. These included a test of ethanol-induced motor impairment, a measure of punishment attenuation caused by ethanol and evaluation of two distinct signs of ethanol withdrawal. The results of these studies may be useful in developing a conceptual framework of potential neuropeptide-ethanol interactions in the brain. METHOD Male Sprague-Dawley rats (140-160 g) or male and female CD-1 and Swiss Webster mice (15-30 g) were purchased from Charles River Laboratories (Somerville, MA), housed for several days in groups of 4-6 animals under environmentally controlled conditions (lights 700-1900 hr; 22-25°C) and fed Wayne Blox Rodent Chow and water ad lib. Ethanol was administered acutely by intraperitoneal (IP) injection as a 10% w/v solution in sterile saline. Physical dependence was induced by feeding rats a nutritionally complete liquid diet containing ethanol [45] for 12 days as previously described [14,15]. In most studies neuropeptides were administered intracisternally (IC) dissolved in 10 tzl of sterile saline as previously reported for mice [9] and rats [8]. However, TRH was also administered by IP or intravenous (IV) injection to rats when its actions were studied on ethanol-induced impairment of the aerial righting reflex, attenuation of punished responding or precipitation of ethanol-withdrawal signs.

Changes in motor coordination induced by ethanol or TRH in rats were monitored by determining an "aerial righting reflex score" as previously described [15,24,]. This test was also modified for use with mice. The test apparatus consisted of a meter stick mounted vertically above a foam rubber pad. A removable stainless steel bar inserted into the meter stick at 3 cm intervals for mice and 5 cm intervals for rats served as an indicator of distance to the mat below. Mice or rats were held in an inverted position by grasping the back of the neck and the base of the tail, at a specified height above the mat. A "successful" landing required that all four feet of the animal were in fiat contact with the foam rubber pad at the time of first contact on the pad, on two consecutive releases. The minimum height required for a successful landing was used as an index of motor impairment. Mice given saline or no treatment consistently required only 3 cm for this maneuver while rats required only 5 cm. Mice were not dropped from heights greater than 42 cm, or rats from heights greater than 50 cm. In order to evaluate the effects of ethanol and TRH on punished responding, a conflict test [46,47] was utilized. During a 3 min habituation period, 24 hr water-deprived rats had access to water via a drinking spout. After an additional 24 hr of water-deprivation, a 3 rain conflict test was carried out. Every 20th lick on the spout resulted in the application of a one mA electric current to the water spout for two sec. The number of shocks elicited by the rat was used as an index of punished responding. Drug-induced changes in jump-flinch pain thresholds were evaluated in rats as previously reported [4,47] using scrambled shock which increased in intensity over a range of 0.6 to 4.0 mA. The effect of ethanol and neuropeptides on ethanol withdrawal tremors and audiogenically-induced seizures was measured at the time of withdrawal of the ethanol-containing liquid diet or 7.0-7.5 hr later. Tremors were evaluated in rats by a modification [16] of the "convulsions on handling" method for mice [18]. Audiogenic seizure susceptibility was evaluated by exposing the rats to sound generated by a 98 db electric bell for up to one min; the occurrence of wild running, clonic and tonic seizures was recorded by a trained observer [14]. Blood and brain ethanol concentrations were determined as previously described [30]. Twenty microliter samples of blood or brain supernatant were diluted 10-fold in distilled water and analyzed by direct injection of 1-5 ~1 into a Varian 2440 gas chromatograph, using tert-butanol as an internal standard [16]. Data are expressed as the mean_+the standard error of the mean (S.E.M.) except for data on the frequency of audiogenically-induced seizures. The data obtained from the aerial righting reflex test and scoring of ethanol-withdrawal tremor were evaluated using the Mann-Whitney U test. Data for audiogenic seizure frequency was evaluated with the Chi square test, while blood alcohol concentrations and punished responding data were analyzed with Dunnett's test for multiple comparisons. RESULTS Ethanol and Peptide Effects on Aerial Righting in Mice

Assessment of the height necessary for aerial righting has been used successfully to monitor ethanol-induced (1.5-3.5 g/kg) impairment of motor coordination in the rat [15,24]. The present studies revealed that this measure is also useful in monitoring motor impairment induced by ethanol adminis-

ETHANOL-PEPTIDE INTERACTIONS

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FIG. 1. Effect of ethanol dose on blood ethanol content and impairment of the aerial righting reflex of mice. Mice were administered ethanol by IP injection 60 min before testing the aerial righting reflex and sampling of venous trunk blood. Values for the aerial righting reflex and for blood ethanol content represent the mean_+SEM for 6 or more animals. All doses of ethanol studied except 1.0 g/kg produced detectable levels of ethanol in the blood. *p<0.01 when compared with saline (3.0_+0.0 cm).

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Neur0pepfide tration to mice. A dose-related impairment of the aerial righting reflex was observed one hr after treatment with doses equal to or larger than 3.0 g/kg of ethanol (Fig. 1), while complete inhibition of the response occurred when the dose exceeded 5.0 g/kg (data not shown). As expected, the doserelated impairment of the aerial righting reflex by ethanol was paralleled by an increase in blood ethanol concentrations. The aerial righting reflex of mice used in the present study was much less sensitive to impairment by ethanol than that of Sprague-Dawley rats ([16] EDs0 ethanol dose and estimated blood ethanol content, respectively, in mice=3.75 g/kg, 2.48 mg/ml and in rats=2.40 g/kg, 1.80 mg/ml). To evaluate the effects of neurotensin, bombesin, /3-endorphin and TRH on motor incoordination due to ethanol, doses of these peptides were selected which previously have been shown to significantly alter ethanol-induced sleep in mice [9, 25, 35]. As shown in Fig. 2 (top), none of the peptides increased the height necessary for successful aerial righting when administered alone by IC injection to mice. Intraperitoneal treatment with ethanol (3.5 g/kg) increased this measure to 18.8_+1.8 cm. However, when neurotensin (30/~g, IC),/3-endorphin (20 ~g, IC) or bombesin (30/zg, IC) was administered immediately before ethanol (3.5 g/kg), each peptide significantly increased the height necessary for successful aerial righting 60 min post-injection. In contrast to the increase in ethanol impairment caused by these three peptides, TRH reduced motor incoordination due to ethanol in a dose-related manner. The largest dose of TRH (30/zg, IC) decreased the height necessary for successful righting after

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FIG. 2. Effect of intracisternal administration of neuropeptides on ethanol-induced impairment of the aerial righting reflex of mice. Swiss Webster mice were administered neurotensin (NT), /3-endorphin (/3-E) or bombesin (Bom) by IC injection followed immediately by IP injection of saline or ethanol (3.5 g/kg). CD-1 mice were administered thyrotropin-releasing hormone (TRH) by IC injection followed immediately by IP injection of saline or ethanol (4.0 g/kg). The aerial righting reflex was measured 60 min later. Bars represent the mean_+SEM for 6 or more animals. *p <0.05 compared with saline plus ethanol. +p<0.05 compared with saline plus saline.

ethanol (4.0 g/kg) by approximately 40 % (Fig. 2, bottom). Table 1 indicates that none of the peptides altered either brain or blood ethanol concentrations in mice one hour after treatment. Effect o f T R H on the Punishment Attenuating Action o f Ethanol

Although data from the aerial righting test (Fig. 2) and other reports [9,26] suggest that TRH reduces several actions of ethanol, antagonism of ethanol by TRH is not always observed. For example, our laboratory has been interested in the attenuating action of ethanol on acute punishment. In these studies, an acute conflict paradigm, previously shown to be sensitive to the punishment-attenuating action of

FRYE E T AL.

102 u~ 2o

TABLE 1 E F F E C T OF I N T R A C I S T E R N A L L Y A D M I N I S T E R E D PEPTIDES ON B L O O D A N D BRAIN E T H A N O L C O N C E N T R A T I O N IN MICE

Treatment

,~u 50

Ethanol Concentrations Blood (~ Control)

~_~ 6o

100.0 ± 3.5

100.0 ± 6.8 97.1 99.7 93.5 97.7

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20

12 6 6 6 6

Mice were treated with ethanol (5.2 g/kg, IP) and 10 min later were administered intracisternally 10/~1 of saline or 5.98 nmoles of one of the peptides. Sixty min after ethanol treatment trunk blood and brain samples were collected. No significant differences in blood or brain ethanol content were observed following peptide treatments. For Swiss Webster mice used in the neurotensin, bombesin and /~-endorphin studies, saline blood and brain ethanol concentrations were 5.6 _+ 0.2 mg/ml and 3.1 _+ 0.1 mg/g, respectively; for CD-1 mice used in the studies with thyrotropin-releasing hormone, saline blood and brain ethanol concentrations were 4.2 + 0.1 mg/ml and 2.3 ± 0.2 mg/g, respectively.

clinically-active anxiolytic benzodiazepines, has been utilized [46]. Ethanol treatments (0.5-1.5 g/kg, IP) 30 min before testing significantly increased the number of shocks accepted by water-deprived rats while drinking during the c o n f i c t test [48]. Quite unexpectedly, administration of TRH simultaneously with ethanol did not result in the antagonism of this action of ethanol, but actually increased the effect (Fig. 3, right). When tested alone, TRH exhibited a punishment-attenuating action that was as large as that of ethanol. The unexpected potentiation of the anticonflict action of ethanol by TRH was not explained by species differences since TRH was effective in decreasing ethanolinduced impairment of the aerial righting reflex in rats (Fig. 3, left). Because drug-induced changes in the motivation to drink water or in the sensitivity to the aversive properties of shock could be responsible for the punishment-attenuating actions of TRH and ethanol, their effect on unpunished drinking and on jump-flinch pain thresholds were examined. Neither TRH nor ethanol significantly altered unpunished drinking at doses which attenuated the effects of punishment (Table 2). Evaluation of jump-flinch pain thresholds shows that TRH did not alter the response of rats to pain. However, ethanol did significantly increase the pain threshold of rats. Thus the punishment-attenuating actions of TRH and ethanol are probably independent of changes in the motivation to drink. In addition, a reduced responsiveness to shock probably is not responsible for the anticonflict action of TRH. The role of increased pain thresholds in the anticonflict action of ethanol is less clear, since morphine, which also consistently elevates jump-flinch thresholds (Table 2), did not alter punished responding in this conflict task.

£ffect o f TRH, Neurotensin, ~-Endorphin and Bombesin on Ethanol Withdrawal Signs Since TRH, neurotensin, bombesin and/3-endorphin were

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FIG. 3. Effect of thyrotropin-releasing hormone on ethanol-induced impairment of the aerial righting reflex and increase of punished licking by rats. For the aerial righting reflex test rats were administered ethanol (3.0 g/kg) by IP injection followed 20 min later by IP injection of saline or TRH (20 mg/kg). Testing of the aerial righting reflex occurred 60 min after ethanol treatment. For the punishment test, ethanol (1.5 g/kg) was administered by IP injection followed by similar treatment with TRH (20 mg/kg). Punished responding was evaluated 30 min later. Bars represent mean±SEM for 6 or more animals. *p<0.05 when compared with the appropriate ethanol plus saline treatment, tp<0.05 when compared with the appropriate saline plus saline treatment.

found to alter several responses of rodents to acute ethanol treatment, the effects of these peptides on rats made physically dependent on ethanol were studied. Tremor and susceptibility to audiogenically-induced seizures which are well recognized signs of ethanol withdrawal in the rat [12,29] were measured in rats made physically dependent on ethanol [14,16]. In rats withdrawn from ethanol 7.5 hr earlier, acute ethanol treatment effectively suppressed both withdrawalinduced tremor and audiogenic seizure susceptibility in a dose-related manner (Fig. 4). These and other ethanolwithdrawal signs are also rapidly suppressed by the administration of a variety of sedative-hypnotic type CNS depressants such as the benzodiazepines [15,17] and barbiturates [49]. Although TRH reduced sedative, hypothermic [9] and motor incoordinating actions (Figs. 2, bottom; 3, left) of acute ethanol treatment, IV, TRH (1-40 mg/kg) treatment did not cause an increase in audiogenically-induced seizures in ethanol-dependent rats which were withdrawn from the ethanol diet for only a 5 min period. As expected, blood ethanol concentrations were still elevated in the rats at this time. Later, during a 7.5 hr post-withdrawal test when ethanol had been largely metabolized [13], 70% or more of the rats exhibited audiogenically-induced seizures (Table 3). TRH-induced shaking was observed in most rats during the 30 min interval post-injection, but no attempt was made to quantify the response. It is probable that the tremors were due to a direct action of TRH [42] rather than the unmasking of ethanol withdrawal tremors. Despite the observed similarity between TRH and ethanol in the conflict test, rats treated with TRH (1 and 20 mg/kg, IV) at the time of ethanol withdrawal were significantly more susceptible to seizure induction 7.5 hr post-withdrawal than were their saline-treated counterparts. TRH (10-100/xg, IC) administered 10 min before seizure test unlike ethanol did

ETHANOL-PEPTIDE

INTERACTIONS

103

TABLE 2 EFFECT OF TRH, ETHANOL AND MORPHINE ON PUNISHED DRINKING, UNPUNISHED DRINKING AND PAIN THRESHOLD Treatment

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3 min

7.1 ± 2.5

438 ± 22

0.20 ± 0.01

0.45 ± 0.04

13.7 ± 1.3"

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18.6 ± 2.2*

416 _+ 32

0.20 - 0.02

0.61 ÷ 0.06

7.0 ± 3.8

--

0.35 _+ 0.05*

1.28 _+ 0.39*

1-2 paws

3-4 paws

Rats were administered IP injections of either saline, ethanol, TRH or morphine 30 min before testing punished or unpunished drinking, or pain thresholds. Different animals were utilized for each test. During a 3 rain interval 48 hr water-deprived rats were allowed free access to a water spout (unpunished licking) or they were shocked on every 20th lick (punished licking; see Method). Pain thresholds were expressed as the intensity of the electric current which caused rats to pick up one-two paws, or three-four paws from an electrified grid floor. Values represent the mean ± SEM for 8 or more observations. *p<0.05 when compared with saline.

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FIG. 4. Effect of acute ethanol treatment on ethanol-withdrawal tremor and audiogenic-seizure susceptibility in the rat. Physically dependent rats were withdrawn from ethanol for 7.0 hr before IP administration of saline or ethanol. Tremor and audiogenic seizure susceptibility were monitored 30 rain later. Bars represent the mean_+SEM of 7 or more measurements. *p<0.05 when compared with saline.

not suppress susceptibility to audiogenic seizures in rats undergoing ethanol withdrawal for 7.5 hr (Fig. 5). Also, T R H did not significantly alter t r e m o r measures. B e c a u s e the severity o f ethanol-withdrawal t r e m o r and the f r e q u e n c y of audiogenic seizures in saline-treated control rats w e r e near m a x i m u m values in these tests, it was not possible to determine if these withdrawal signs were intensified by t r e a t m e n t with T R H . Doses of neurotensin (10-100 /~g, IC), which w e r e c o m p a r a b l e to those that potentiated the acute m o t o r impairing and sedative actions o f ethanol [25,35], w e r e also without effect on ethanol-withdrawal t r e m o r or audiogenicseizure susceptibility 7.5 hr post-withdrawal w h e n given 10 min before testing (Fig. 5).

In sharp contrast to the results obtained with T R H and neurotensin, bombesin ( 1 - 3 0 / z g ) caused a dose-related reduction in audiogenic-seizure susceptibility (Fig. 5). Although b o m b e s i n caused a dramatic reduction in susceptibility to audiogenic seizures, t r e a t m e n t with this peptide caused no reduction in the severity of ethanol-withdrawal tremor. During a 5 to 10 rain period immediately after IC administration of b o m b e s i n (30/xg), both alcohol-naive rats and those undergoing ethanol withdrawal for 7.5 hr w e r e found to exhibit m a r k e d spasticity, broad-based gait and frequent hind paw scratching of the back o f the neck. B o m b e s i n - i n d u c e d spasticity and broad-based gait w e r e quite similar to spontaneous withdrawal signs s o m e t i m e s o b s e r v e d in ethanol dependent rats [14,29]. fl-endorphin (1 and 100 tzg, IC) also significantly reduced susceptibility to audiogenic seizures (Fig. 5). Surprisingly, a dose of 30 /xg of fl-endorphin was ineffective in this test suggesting that the anti-convulsant action of the higher and l o w e r dose may involve different mechanisms. Although all four doses tested s e e m e d to reduce the t r e m o r associated with alcohol withdrawal, only the largest dose of /3-endorphin (100 ~g) significantly reduced this measure. All doses of/3-endorphin caused an occasional wet-dog shake in ethanol-withdrawn rats. All withdrawn animals that r e c e i v e d either 30 or 100/xg o f 13-endorphin exhibited at least one wet dog shake within the ten minute o b s e r v a t i o n period; however, no shaking was o b s e r v e d among six alcohol-naive rats treated similarly with 30/xg of/3-endorphin. DISCUSSION The hypothesis that neuropeptides may play important neuroregulatory roles in the C N S has been based in part on e v i d e n c e that the central effects of psychotropic drugs can be altered by certain peptides. Consistent with this hypothesis are the findings r e p o r t e d here, which d e m o n s t r a t e an interaction of T R H , neurotensin, b o m b e s i n and/3-endorphin

104

FRYE E T AL. TABLE 3

E F F E C T O F I N T R A V E N O U S T R H ON S E I Z U R E S U S C E P T I B I L I T Y IN E T H A N O L D E P E N D E N T RATS

Treatment (mg/kg)

Blood Ethanol Concentration at Withdrawal (mg/ml)

Saline TRH 1.0 20.0 40.0

2.66 _+ 0.21 2.64 + 0.19 2.83 _+ 0.18 2.72 _+ 0.23

Percent Exhibiting Clonic Seizures after Injection 5 min 7.5 hr 0 0 0 0

69.2 100.0" 100.0" 70.0

Physically dependent rats were withdrawn from ethanol and TRH was immediately administered by intravenous injection. Five min later tests for audiogenic seizure susceptibility were conducted and venous tail blood samples were collected. A second test for susceptibility to audiogenic seizures was conducted 7.5 hr after ethanol withdrawal. *p<0.01 when compared with saline-treated controls.

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Bom

(jug)

with several distinct actions of ethanol. Perhaps not unexpectedly, the interaction of ethanol with neuropeptides appears to be at least as complex as the interactions of ethanol with more traditionally recognized neurotransmitters which are only partially understood [36,49]. The mechanisms by which TRH, neurotensin, fl-endorphin and bombesin alter ethanol-induced motor incoordination, sedation or hypothermia remain to be defined. Peptide-induced changes in the pharmacokinetics of ethanol, which might result in an increased or decreased penetration of alcohol into brain tissue, do not seem to be responsible for the observed peptide-ethanol interactions (Table 1). Since/~g amount of the peptides were active when administered intracisternally, the interaction of TRH, neurotensin, bombesin and fl-endorphin with ethanol probably occurs within the CNS. However, the sharp contrast in the effects of TRH as compared with neurotensin, bombesin and flendorphin on ethanol-induced sedation, hypothermia, and motor incoordination is difficult to explain based on a single consistent action of ethanol on the function of peptidecontaining neurons. It seems clear that these effects are not non-specific effects of centrally administered peptides, but reflect an interaction at particular brain loci. For example, recent studies have found that the ability of TRH to antagonize pentobarbital narcosis is localized in a few specific areas in the brain, but not in other areas [22]. Ethanol has many well recognized acute and chronic actions [49], each of which probably reflects changes in the activity of a specific group of functionally integrated neuronal circuits within the CNS. Because neurotransmission within individual functional units probably utilizes only a few of the many transmitter candidates now recognized, it is not surprising that a specific neuropeptide does not consistently alter all actions of ethanol in the same manner. For example, neurotensin, bombesin and fl-endorphin all potentiated ethanol-induced motor incoordination (Fig. 2); both fl-endorphin and bombesin also substituted for ethanol in suppressing audiogenic-seizure susceptibility (Fig. 5). However, only fl-endorphin also reduced withdrawal tremor (Fig. 5). Other examples of the differential interaction of ethanol and neuropeptides are the actions of TRH to potentiate the punishment-attenuating action of ethanol on the one hand, while antagonizing ethanol-induced motor incoordination on the other. Furthermore, TRH has no effect on certain other actions of ethanol. TRH treatment did not precipitate ethanol-withdrawal seizures in physically-dependent rats (Table 3) or alter discriminative cues which allowed rats to distinguish between ethanol or saline treatment [7]. In some cases the central actions of TRH, neurotensin, bombesin and/3-endorphin, when administered alone, were very different from those of ethanol. For example, neurotensin, bombesin and fl-endorphin potentiated, but did not mimic the sedative and motor incoordinating actions of ethanol. Such observations suggest that the potentiating action of these neuropeptides may result from modulation of ongoing CNS activity which has been altered already by ethanol. However, other actions of TRH, neurotensin, bombesin and fl-endorphin do resemble certain acute or chronic actions of ethanol. For example, TRH, like ethanol, attenuated the effects of punishment (Fig. 3), stimulated locomotor activity [26] and also induced a tremor similar to that observed in rats undergoing ethanol withdrawal. Neurotensin, fl-endorphin and bombesin injections all cause hypothermia, as does ethanol, while bombesin also induces spasticity and broad based gait, both of which resemble signs

9- E

(#g)

FIG. 5. Effect of intracisternally administered neuropeptides on ethanol withdrawal tremor and audiogenic-seizure susceptibility. Physically dependent rats were withdrawn from ethanol for 7.5 hrs before IC administration of saline or neuropeptides. Tremor and audiogenic seizure susceptibility were monitored 10 min later. Bars represent the mean_+SEMof not less than 5 measurements. *p <0.05 when compared with saline. **p<0.01 when compared with saline.

ETHANOL-PEPTIDE

INTERACTIONS

105

o b s e r v e d d u r i n g e t h a n o l w i t h d r a w a l . S u c h a c t i o n s m a y supp o r t a d i r e c t role for t h e p e p t i d e s as c e n t r a l m e d i a t o r s o f t h e s e a c u t e o r c h r o n i c a c t i o n s o f e t h a n o l . H o w e v e r , t h e r e is as yet little e x p e r i m e n t a l e v i d e n c e o f a d i r e c t effect of ethanol on peptide-containing neurons. Recently, the cont e n t o f m e t h i o n i n e - e n k e p h a l i n a n d /3-endorphin-like imm u n o r e a c t i v e m a t e r i a l in b r a i n t i s s u e s w e r e f o u n d to b e increased by acute, and decreased by chronic administration o f e t h a n o l [43]. T h i s finding is c o m p a t i b l e w i t h t h e ability o f /3-endorphin to p o t e n t i a t e a c u t e d e p r e s s a n t a c t i o n s o f e t h a n o l a n d to r e d u c e e t h a n o l w i t h d r a w a l h y p e r e x c i t a b i l i t y . P r e l i m i n a r y e v i d e n c e f r o m o u r l a b o r a t o r y suggests t h a t the c o n c e n t r a t i o n o f a T R H - l i k e i m m u n o r e a c t i v e m a t e r i a l in t h e h y p o t h a l a m u s a n d the r e s t o f b r a i n is n o t c h a n g e d b y a c u t e e t h a n o l t r e a t m e n t [28]. F u t u r e studies d e s i g n e d to e v a l u a t e

a n d c o r r e l a t e e t h a n o l - i n d u c e d b e h a v i o r a l a c t i o n s with regional c h a n g e s in C N S n e u r o p e p t i d e h o m e o s t a s i s a n d recept o r f u n c t i o n s h o u l d p r o v e v e r y useful in u n r a v e l i n g t h e complex central mechanism of ethanol action.

ACKNOWLEDGEMENTS This work was supported by grants from the North Carolina Alcoholism Research Authority 7908 and 7915, and U.S. Public Health Service awards MH-32316, MH-34121, MH-22536, MH33127, HD-03110 and AA-02334. The authors express their appreciation to Robert Considine, Barbara Gau, Bill Hudson and David Knight for their excellent technical assistance and to Pam McLaurin and Faygele ben Miriam for their skillful assistance in the preparation of this manuscript.

REFERENCES

1. Berney, S. and O. Hornykiewicz. The effects of/3-endorphin and metenkephalin on striatal dopamine metabolism and catalepsy: Comparison with morphine. Communs Psychopharmac. 1: 597-604, 1977. 2. B1/isig, J. and A. Herz. Tolerance and dependence induced by morphine-like pituitary peptides in rats. Naunyn-Schmiedebergs's Arch. Pharmac. 294: 297-300, 1976. 3. Bloom, F. E. Peptide transmitters: Clues to the chemical cryptogram of interneuronal communication. Biosystems 8: 179-183, 1977. 4. Bonnet, K. A. and K. E. Peterson. A modification of the jumpflinch technique for measuring pain sensitivity in rats. Pharmac. Biochem. Behav. 3: 47-55, 1975. 5. Breese, G. R., J. M. Cott, B. R. Cooper, A. J. Prange, Jr. and M. A. Lipton. Antagonism of ethanol narcosis by thyrotropin releasing hormone. Lift, Sci. 14: 1053-1063, 1974. 6. Brown, M., J. Rivier and W. Vale. Bombesin: Potent effects on thermoregulation in the rat. Science 196: 998-1000, 1977. 7. Chipkin, R. E., J. M. Stewart and K. Channabasavaiah. The effects of peptides on the stimulus properties of ethanol. Pharmac. Bioehem. Behav. 12: 93-98, 1980. 8. Cooper, B. R., K. Viik, R. M. Ferris and H. L. White. Antagonism of the enhanced susceptibility to audiogenic seizures during alcohol withdrawal in the rat by GABA and "GABAmimetic" agents. J. Pharmac. exp. Ther. 209: 396-403, 1979. 9. Cott, J. M., G. R. Breese, B. R. Cooper, T. S. Barlow and A. J. Prange, Jr. Investigations into the mechanism of reduction of ethanol sleep by thyrotropin releasing hormone (TRH). J. Pharmac. exp. Ther. 196: 594-604, 1976. 10. Ellingboe, J. Effects of alcohol on neurochemical processes. In: Psychopharmacology: A Generation o f Progress, edited by M. A. Lipton, A. DiMascio and K. F. Killam. New York: Raven Press, 1978, pp. 1653-1664. 11. Finkelberg, F., H. Kalant and A. E. LeBlanc. Effect of vasopressin-like peptides on consumption of ethanol by the rat. Pharmac. Biochem. Behav. 9: 453-458, 1978. 12. Freund, G. Induction of physical dependence on alcohol in rodents. In: Biochemical Pharmacology o f Ethanol, edited by E. Majchrowicz. New York: Plenum Press, 1975, pp. 311-325. 13. Frye, G. D. and F. W. Ellis. Audiogenic evaluation of withdrawal excitability in rats after chronic ethanol feeding. Pharmacologist 17: 197, 1975. 14. Frye, G. D. and F. W. Ellis. Effects of 6-hydroxydopamine or 5,7-dihydroxytryptamine on the development of physical dependence on ethanol. Drug Alcohol Depend. 2: 34%359, 1977. 15. Frye, G. D., G. R. Breese, R. B. Mailman, R. A. Vogel, M. G. Ondrusek and R. A. Mueller. An evaluation of the selectivity of fenmetozole (DH-524) reversal of ethanol-induced changes in central nervous system function. Psychopharmacology 69: 149-155, 1980.

16. Frye, G. D., R. E. Chapin, R. A. Vogel, R. B. Mailman, C. D. Kilts, R. A. Mueller and G. R. Breese. Effects of acute and chronic 1,3-butanediol treatment on central nervous system function: A comparison with ethanol. J. Pharmac. exp. Ther. 216: 306-314, 1981. 17. Gessner, P. K. Drug therapy of the alcohol withdrawal syndrome. In: The Biochemistry and Pharmacology o f Ethanol, edited by E. Majchrowicz and E. Noble. New York: Plenum Press, 1979, pp. 375-435. 18. Goldstein, D. B. An animal model for testing effects on alcohol withdrawal reactions. J. Pharmac. exp. Ther. 183: 14-23, 1972. 19. Goldstein, A. Opiate receptors and opioid peptides: A ten-year overview. In: Psychopharmacology: A Generation o f Progress, edited by M. A. Lipton, A. DiMascio and K. F. Killalr.. New York: Raven Press, 1978, pp. 1557-1563. 20. Hoffman, P. L., R. F. Ritzmann, R. Walter and B. Tabakoff. Arginine vasopressin maintains ethanol tolerance. Nature 276: 614, 1978. 21. Hoffman, P. L., R. F. Ritzmann and B. Tabaokoff. The influence of arginine vasopressin and oxytocin on ethanol dependence and tolerance. In: Cttrrents in Alcoholism, Vol. 5, edited by M. Galanter. New York: Grune and Stratton, 1979, p. 5. 22. Kalivas, P. W. and A. Horita. Thyrotropin-releasing hormone: Central site of action in antagonism of pentobarbital narcosis. Nature 278: 461-463, 1979. 23. Kastin, A. J., R. D. Olson, A. V. Schally and D. H. Coy. CNS effects of peripherally administered brain peptides. Lift, Sei. 25: 401-414, 1979. 24. Leitch, G. J., D. J. Backes, F. S. Siegman and G. D. Guthrie. Possible role of GABA in the development of tolerance to alcohol. Experientia 33: 496-497, 1977. 25. Luttinger, D., C. B. Nemeroff, G. A. Mason, G. D. Frye, G. R. Breese and A. J. Prange, Jr. Enhancement of ethanol-induced sedation and hypothermia by centrally administered neurotensin, fl-endorphin and bombesin. Neuropharmacology 20: 305309, 1981. 26. Mailman, R. B., G. D. Frye, R. A. Mueller and G. R. Breese. Thyrotropin-releasing hormone (TRH): Reversal of ethanolinduced decreases in cerebellar cGMP. Nature 272: 832-833, 1978. 27. Mailman, R. B., G. D. Frye, R. A. Mueller and G. R. Breese. Change in brain guanosine Y,5'-monophosphate (cGMP) content by thyrotropin releasing hormone. J. Pharmac. exp. Ther. 208: 16%175, 1979. 28. Mailman, R. B., G. D. Frye, R. A. Mueller and G. R. Breese. The effects of thyrotropin-releasing hormone (TRH) and other drugs on the actions of alcohol. In: Biological Effects o f Alcohol, edited by H. Begleiter. New York: Plenum Publ. Co., 1980, pp. 50%522. 29. Majchrowicz, E. Induction of physical dependence on alcohol and associated behavioral changes in rats. Psychopharmacologia 43: 245-254, 1975.

106 30. Manno, B. R. and J. E. Manno. A simple approach to gas chromatographic microanalysis of alcohols in blood and urine by a direct-injection technique. J. A n a l Tox. 2: 257-261, 1978. 3 I. Meglio, M., Y. Hosobuchi, H. H. Loh, J. E. Adams and C. H. Li. /3-Endorphin: Behavioral and analgesic activity in cats. Proc. natn. Acad. Sci. U.S.A. 74: 774-776, 1977. 32. Mello, N. K. A review of methods to induce alcohol addiction in animals. Pharmac. Bioehem. Behav. 1: 8%101, 1973. 33. Mucha, R. F. and H. Kalant. Effects of desglycinamide(~) lysine(S)-vasopressin and prolyl-leucyl-glycinamide on oral ethanol intake in the rat. Pharmae. Biochem. Behav. 10: 22% 234, 1979. 34. Nemeroff, C. B., A. J. Osbahr, III, P. J. Manberg, G. N. Ervin and A. J. Prange, Jr. Alterations in nociception, and body temperature after intracisternally administered neurotensin, /3-endorphin, other endogenous peptides and morphine. Proc. natn. Acad. Sci. U.S.A. 76: 5368-5371, 1979. 35. Nemeroff, C. B., D. Luttinger, G. A. Mason, G. D. Frye, G. R. Breese and A. J. Prange, Jr. Effects of intracisternal neurotensin,/3-endorphin or bombesin on ethanol-induced sedation and hypothermia. Drug Alcohol Depend. 6:118, 1980. 36. Okamoto, M. Barbiturates and alcohol: Comparative overviews on neurophysiology and neurochemistry. In: Psychopharmacology: A Generation o f Progress. edited by M. A. Lipton, A. DiMascio and K. F. Killam. New York: Raven Press, 1978, pp. 1575-1590. 37. Porter, C. C., V. J. Lotti and M, J. DeFelice. The effect of TRH and related tripeptide, 1-N-(2-oxopiperidin-6-yl-carbonyl)-lHistidyl-l-thiazolidine-4-carboxamide (MK-771, OHT), on the depressant action of barbiturates and alcohol in mice and rats. Life Sci. 21: 811-820, 1977. 38. Prange, A. J., Jr., C. B. Nemeroff, M. A. Lipton, G. R. Breese and I. C. Wilson. Peptides and the central nervous system. In: Handbook o f Psychopharmacology, Vol. 13, edited by L. L. Iversen, S. D. Iversen and S. H. Snyder. New York: Plenum Publishing Co., 1978, pp. 1-107. 39. Prasad, C., T. Matsui and A. Peterkofsky. Antagonism of ethanol narcosis by histidyl-proline diketopiperazine. Nature 268: 142-144, 1977.

FRYE ET AL. 40. Renaud, L. P. Peptides as neurotransmitters or neuromodulators. In: Psychopharmacology: A Generation o f Progress, edited by M. A. Lipton, A. DiMascio and K. F. Killam. New York: Raven Press, 1978, pp. 423-430. 41. Rigter, H., H. Rijk and J. C. Crabbe. Tolerance to ethanol and severity of withdrawal in mice are enhanced by a vasopressin fragment. Eur. J. Pharmac. 64: 53-68, 1980. 42. Schenkel, L., W. P. Hulliger, A. Koella, A. Hartmann and L. Maitre. Tremorogenic effects of thyrotropin releasing hormone in rats. Experientia 30:1168-1170, 1974. 43. Schulz, R., M. Wuster, T. Duka and A. Herz. Acute and chronic ethanol treatment changes endorphin levels in brain and pituitary. Psychopharmacoh>gy 68: 221-227, 1980. 44. Szekely, J. I., A. Z. Ronai, Z. Dumai-Kovacs, E. Miglecz, S. Bajusz and L. Graf. Cross tolerance between morphine and /3-endorphin in vivo. LiJ~J Sci. 20: 125%1264, 1977. 45. Thompson, J. A. and R. C. Reitz. Effects of ethanol ingestion and dietary fat levels on mitochondrial lipids in male and female rats. Lipids 13: 540-550, 1978. 46. Vogel, J. R., B. Beer and D. E. Clody. A simple and reliable conflict procedure for testing anti-anxiety agents. Psychopharmacologia 21: 1-7, 1971. 47. Vogel, R. A., G. D. Frye, J. H. Wilson, C. M. Kuhn, R. B. Mailman, R. A. Mueller and G. R. Breese. Attenuation of the effect of punishment by thyrotropin-releasing hormone: Comparisons with chlordiazepoxide. J. Pharmac. exp. Ther. 212: 153-161, 1980. 48. Vogel, R. A., G. D. Frye, J. H. Wilson, C. M. Kuhn, K. M. Koepke, R. B. Mailman, R. A. Mueller and G. R. Breese. Attenuation of the effects of punishment by ethanol: Comparisons with chlordiazepoxide. Psy<'hopharmaeology 71: 123-129, 1980. 49. Wallgren, H. and H. Barry, III. Actions o f Ethanol, Vol. 1 and 2. New York: Elsevier, 1970. 50. Yarbrough, G. G. On the neuropharmacology of thyrotropinreleasing hormone (TRH). Prog. Neurobiol. 12: 291-312, 1979. 51. Helderman, J. H., R. E. Vestal, J. W. Rowe, J. D. Tobin, R. Andres and G. L. Robertson. The response of arginine vasopressin to intravenous ethanol and hypertonic saline in man: The impact of aging. J. Gerontol. 33" 3%47, 1978.