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Tolerance to hexobarbital and supersensitivity to pilocarpine after chronic barbital treatments in the rat
European Journal of Pharmacology, 38 (1976) 123--129
123
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
T O L E R A N C E TO H E X O B A R B I T A L AND SUPERSENSITIVITY TO PILOCARPINE A F T E R CHRONIC BARBITAL TREATMENTS IN THE RAT GORAN WAHLSTROM and TORSTEN EKWALL
Department of Pharmacology, University o f Umet}, S-901 8 7 Umeti, Sweden Received 25 September 1975, revised MS received 24 February 1976, accepted 18 March 1976
G. WAHLSTROM and T. EKWALL, Tolerance to hexobarbital and supersensitivity to pilocarpine after chronic babital treatments in the rat, European J. Pharmacol. 38 (1976) 123--129. Tolerance and physical dependence induced by chronic treatments with depressant drugs has been proposed as being due to denervation or disuse supersensitivity in the central nervous system. To illustrate this hypothesis, tolerance to barbiturates and the sensitivity to central cholinergic stimulation were recorded in the same male rats. Tolerance to barbiturates was recorded with a hexobarbital threshold. The dose needed to obtain an EEG criterion was measured. Sensitivity in central cholinergic structures was recorded by a temperature threshold method. A subthreshold dose of pilocarpine (3.5 mg/kg) was given s.c. together with 1.0 mg/kg methylscopolamine i.p. An increase in sensitivity to pilocarpine was recorded as a decrease in body temperature. In the abstinence period after 42 weeks of oral treatment with barbital, tolerance was recorded with the hexobarbital threshold method. The tolerance had two peaks, one after the end of the treatment and one approximately 3 weeks later. An increased sensitivity to pilocarpine was also recorded in the abstinence period. It had the same biphasic time pattern as the tolerance. Cholinergic neurones are t h u s probably involved in the changes induced by chronic barbital treatment. The denervation supersensitivity hypothesis of tolerance was n o t refuted by the present experiments.
Supersensitivity
Pilocarpine
Tolerance
1. Introduction
The law of denervation formulated by Cannon (1939) states that an increased irritability to chemical agents develops in structures where the efferent neurones are destroyed. This denervation supersensitivity is present in the neuromuscular junction (Thesleff, 1960) and in various peripheral neuroeffector junctions (Sharpless, 1964; Emmelin, 1965; Trendelenburg, 1966). A similar phenomenon can also be induced in the periphery by a pharmacological depression of activity i.n the efferent nerves (Sharpless, 1964; Emmelin, 1965). It is thus not surprising that denervation supersensitivity has been discussed several times as a theoretical model to explain tol-
Hexobarbital
Abstinence
Barbiturates
Rat
erance and physical dependence to drugs acting on the central nervous system (Sharpless, 1964; Jaffe and Sharpless, 1968; Collier, 1968). Denervation supersensitivity in the central nervous system has been directly implicated as a possible explanation for the changes recorded after chronic barbiturate treatment in the cat (Jaffe and Sharpless, 1965) and also more specifically as a factor in the changes in central cholinergic neurones after chronic scopolamine treatment (Friedman et al., 1969). The possibility that changes in central cholinergic neurones could be involved in barbiturate tolerance has been studied by McBride and Turnbull (1970). They found a change in temperature sensitivity to choliner-
124 gic stimulation after the barbiturate treatment, but no data were given. The basal barbiturate tolerance was n o t measured. Since the tolerance to a hexobarbital threshold in the abstinence after chronic barbital treatments is well d o c u m e n t e d (Wahlstr~m, 1974), the question whether this tolerance is accompanied by a supersensitivity to cholinergic stimulation could easily be studied. As a measure of sensitivity to cholinergic stimulation in the central nervous system the t e m p e r a t u r e response to pilocarpine after peripheral blockade (Friedman and Jaffe, 1969) was chosen. The technique had to be modified to fit the present experiments. The results have been briefly discussed earlier (Ekwall and WahlstrOm 1972).
2. Materials and m e t h o d s
2.1. Materials Male Sprague--Dawley rats (Nih/Han/Mol) were obtained from M~llegaard, Li. Skensved, Denmark. The initial b o d y weight was around 300 g. The animals were kept in a r oom at a t e m p e r a t u r e of 25°C. External light was excluded. Artificial light was t ur ne d on at 01.00 and o f f at 13.00. The reason for this shift from normal light conditions was to make the activity of the animals occur at a m o r e suitable time for the experimental work. There were 2--3 rats in each cage. F o o d was available ad libitum. The drinking fluid was used as the vehicle for barbiturate administration (see 2.2.).
2. 2. Barbital treatment Sodium barbital was administered in the drinking fluid. The c o n c e n t r a t i o n was 3.33 mg/ml, e x c e p t during the first week of the t r e a t m e n t when it was 1.67 mg/ml. Controls were given tap water. If n o t otherwise stated there was free access to the drinking fluid. At the end of the t r e a t m e n t the barbital solution was replaced by tap water. The daily dose in
G. WAHLSTROM, T. EKWALL mg/kg/day was based on weekly c o n s u m p t i o n data from all cages. For details regarding the t r e a t m e n t see Wahlstr6m (1974).
2.3. Hexobarbital threshold determinations Sodium hexobarbital was infused continuously in a tail vein with an infusion rate of 0.25 mg/kg/sec (volume rate 0.1 ml/min). During the infusion the animals were restrained and the electroencephalogram was recorded. Stainless steel sutures were used as electrodes. The first burst suppression of one second or more (the 'silent second') was taken as the threshold criterion and the dose of hexobarbital needed was determined. All threshold determinations were p e r f o r m e d during the first half of the dark period. Prior to the barbital t r e a t m e n t two threshold determinations were performed. These were used to calculate a pre-experimental values for each individual. The average of these pre-experimental values was 57.1 + 1.1 mg/kg (-+ S.E., n = 12) in the experimental animals and 62.5 + 2.0 mg/kg (n = 13) in the control animals. Since 'the individual rats tend to keep to a certain threshold the experimental data are given as percent of the pre-experimental value. Only the animals which survived the barbital t r e a t m e n t are included. For furt her details of the m e t h o d see Wahlstr~m (1966).
2.4. Temperature recording The rectal t em perat ure was recorded with a thermistor probe as one part of a Wheatstone bridge. The probe was inserted 5 cm in the rectum. Prior to any injection the basal temperature level was always recorded. The animals were then given the drugs. In m ost cases both methylscopolamine and pilocarpine were administered. Two different injection schedules were used (see 3.1.). All injections were p e r f o r m e d under sterile conditions during the first part of the dark period.
2. 5. Experimental procedure After the pre-experimental values on threshold dose had been determined the tempera-
BARBITURATES AND PILOCARPINE
ture experiments seen in fig. 1 were performed. When the liminal dose of pilocarpine had been determined and the final injection schedule tested in all participating animals, the cages were randomly divided into the two treatment groups. After 4 weeks of barbital treatment a single temperature test was performed. After 42 weeks of treatment the barbital solution was replaced with water. The same animals participated in the temperature and threshold determinations. The schedule during the abstinence period is best seen in figs. 1 and 4. There was never less than a 2 day interval between two different experimental sessions.
2.6. Drugs Sodium hexobarbital (Evipan ®) was kindly supplied by Bayer AG through Mr. R. Arensberg. Methylscopolamine nitrate was kindly supplied by Pharmacia AB, Uppsala, Sweden. Pilocarpine chloride and sodium barbital were obtained from a local pharmacy. All doses are calculated as the salt.
2. 7. Statistical treatment Differences were tested with Student's ttest if not otherwise stated.
3. Results
3.1. The effects o f pilocarpine on body temperature in normal rats The body temperature of the rat kept under the experimental conditions used is given in fig. 1A. All rats which participated in the experiments were used. As expected there was an increase in body temperature at the start of the dark period. The gradual changes during the dark period were of such a magnitude that controls run in parallel were necessary in the critical experiments. Fig. 1B shows the effects of methylscopolamine alone and combined with different
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Fig. 1. Recordings of b o d y temperature prior to the barbital treatment. A. Normal b o d y t e m p e r a t u r e of the rat kept under the experimental conditions described. All rats started in the e x p e r i m e n t (n = 32) were tested at every point. B. B o d y temperature after m e t h y l s c o p o l a m i n e alone (filled circles) and in combination with different doses o f pilocarpine. The doses of pilocarpine and the n u m b e r of animals tested were: 3.1 m g / k g (squares) n = 9, 6.25 mg/kg (triangles down) n = 11, 12.5 mg/kg (triangles up) n = 12, and 25 mg/kg (circles) n = 9. The arrows indicate the times of administration of 1.0 m g / k g m e t h y l s c o p o l amine (MS) i.p. and pilocarpine (P) s.c.C. The effects on the b o d y t e m p e r a t u r e of 3.5 m g / k g pilocarpine given s.c. i m m e d i a t e l y prior to 1.0 m g / k g methylscopolamine i.p.; 32 rats participated. The times of administration of the drugs are indicated by the arrows.
doses of pilocarpine on the body temperature of the rat. After 1 mg/kg of methylscopolamine i.p. there was an increase in body temperature compared with the pre-experimental basal level. The relative magnitude is uncertain since no untreated controls were included. However, the temperature was above the basal level recorded in fig. 1A. If 3.1 mg/kg of pilocarpine was given s.c. 0.5 h after the methylscopolamine no definite effect on body temperature was seen. Compared with the effect of methylscopolamine alone the level was slightly depressed. If the dose of pilocarpine was increased a dose-dependent decrease in body temperature was obtained. Based upon these data 3.5 mg/kg s.c. was
126 c h o s e n as a s u b t h r e s h o l d dose o f pilocarpine. A slight e f f e c t o f this dose on the b o d y temp e r a t u r e is seen in fig. 1C with pilocarpine given s.c. i m m e d i a t e l y prior to m e t h y l s c o p o l amine (i.p.) t o all participating animals. This new schedule was i n t r o d u c e d to r e d u c e the time n e e d e d to r e c o r d the t e m p e r a t u r e minim u m in the critical e x p e r i m e n t s (see 3.4.).
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3.2. Barbital treatment The t r e a t m e n t with barbital in the drinking w a t e r lasted 42 weeks. During the s e c o n d half o f the t r e a t m e n t , 4 barbital-treated and one c o n t r o l animal died. T h e average b o d y weight at the end o f the t r e a t m e n t was 518 g in the barbital-treated animals and 558 g in the controls. The average c o n s u m p t i o n o f barbital was 222 m g / k g / d a y during the last 5 weeks o f the barbital t r e a t m e n t . These values are close t o the values r e p o r t e d earlier in a m o r e syst e m a t i c s t u d y on the e f f e c t o f similar barbital t r e a t m e n t s (WahlstrSm, 1 9 7 4 ) .
3.3. Hexobarbital threshold determinations in the abstinence period after the end o f the barbital treatment The results o f the h e x o b a r b i t a l t h r e s h o l d d e t e r m i n a t i o n s after the barbital t r e a t m e n t are given in fig. 2. T h e figure clearly indicates t h a t t h e r e were at least t w o peaks o f decreased sensitivity t o h e x o b a r b i t a l during the a b s t i n e n c e p e r i o d a f t e r the barbital t r e a t m e n t . With the p r e s e n t testing schedule the t w o peaks app e a r e d a r o u n d day 5 and s o m e t i m e s after day 21. All values e x c e p t the one o f day 17 had a p value less than 0.001 if c o m p a r e d with the c o r r e s p o n d i n g c o n t r o l value. T h e p value was less than 0.02 on day 17.
3.4. The effects o f pilocarpine on body temperature in the abstinence period after the barbital treatment T w o examples o f the results o b t a i n e d with the pilocarpine t e m p e r a t u r e test during the abstinence p e r i o d a f t e r the barbital t r e a t m e n t
5
DAYS AFTER
10
~5
20
END OF BARBITAL TREATMENT
Fig. 2. The results of the hexobarbita] threshold de-
terminations during the abstinence after the barbital treatment. The thresholds are given in percent of a pre-experimental value obtained prior to any of the experiments. The number of experimental animals was 6--9 (filled circles) and the number of controls was 10--13 (unfilled circles). 2 times the standard error is indicated by the bars.
are given in fig. 3. On the third d a y o f abstin e n c e the e f f e c t o f pilocarpine (3.5 m g / k g s.c.) was increased in t.he barbital-treated animals as c o m p a r e d t o the c o n t r o l s . On the 1 5 t h d a y t h e r e was n o difference. O n l y the b o d y t e m p e r a t u r e s at the time o f the minim u m in the barbital-treated animals were used. These values are indicated by the filled circles in the e x a m p l e s s h o w n in fig. 3. The c o r r e s p o n d i n g t e m p e r a t u r e values in the controls were used for c o m p a r i s o n instead o f the minimal values. In the c o n t r o l s the d i f f e r e n c e b e t w e e n the value used and this minimal value varied b e t w e e n 0 and 0.19°C. One separate t e m p e r a t u r e e x p e r i m e n t was p e r f o r m e d a f t e r 4 weeks o f barbital treatm e n t . At t h a t time t h e r e was n o d i f f e r e n c e in the e f f e c t o f pilocarpine b e t w e e n the barbitalt r e a t e d animals and the controls. All animals had been w i t h o u t drinking fluid for 28 h p r i o r to the test. The minimal values o f b o d y t e m p e r a t u r e a f t e r pilocarpine in the a b s t i n e n c e p e r i o d after the barbital t r e a t m e n t are given in fig. 4. On no occasion was there any significant difference b e t w e e n the basal values r e c o r d e d in
BARBITURATES AND PILOCARPINE AFTER BARBITAL 3 DAYS
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Fig. 4. Minimal b o d y t e m p e r a t u r e s r e c o r d e d a f t e r p i l o c a r p i n e 3.5 m g / k g s.c. ! m m e d i a t e l y f o l l o w e d b y m e t h y l s c o p o l a m i n e 1.0 m g / k g i.p. d u r i n g t h e abstin e n c e a f t e r b a r b i t a l t r e a t m e n t . M i n i m a l values as s h o w n in fig. 3 were used. T h e n u m b e r o f barbitalt r e a t e d a n i m a l s was b e t w e e n 7 - - 1 1 o n each o c c a s i o n a n d is i n d i c a t e d b y filled circles a n d t h e n u m b e r o f c o n t r o l s was b e t w e e n 1 1 - - 1 3 a n d is i n d i c a t e d b y unfilled circles. 2 t i m e s t h e s t a n d a r d e r r o r is i n d i c a t e d b y t h e bars.
TIME AFTER LAST INJECTION Fig. 3. 2 e x a m p l e s o f the t e m p e r a t u r e recordings obt a i n e d a f t e r p i l o c a r p i n e 3.5 m g / k g s.c. f o l l o w e d b y 1.0 m g / k g o f m e t h y l s e o p o l a m i n e i.p. in the abstinence a f t e r t h e b a r b i t a l t r e a t m e n t . T h e c o n t r o l s are i n d i c a t e d b y a C a n d a solid line. T h e b a r b i t a l - t r e a t e d a n i m a l s are i n d i c a t e d b y a n E a n d a b r o k e n line. The h o r i z o n t a l lines i n d i c a t e t h e p r e - e x p e r i m e n t a l levels. The filled s y m b o l s i n d i c a t e t h e t i m e o f m i n i m a l b o d y t e m p e r a t u r e in t h e e x p e r i m e n t a l animals. T h e n u m bers i n d i c a t e t h e n u m b e r s of animals.
the controls and those recorded in the barbital-treated animals prior to injection of methylscopolamine. The range of these average basal temperature recordings was 38.0-39.3°C. Fig. 4 shows that there were two time periods in the abstinence period after barbital when the sensitivity to the temperature reducing effect of pilocarpine was increased in animals previously treated with barbital. The p values were less than 0.001 on days 3 and 7 and less than 0.01 on days 20 and 24. There was thus a clear increase in sensitivity to the temperature-reducing effect of pilocarpine in the abstinence after long barbital treatment.
4. Discussion The hexobarbital threshold provides a measure for tolerance during the abstinence after barbital treatments (Wahlstr6m, 1968). This tolerance can be biphasic after repeated or long term oral treatments (Wahlstrbm, 1971a; WahlstrSm, 1974). If the tolerance in the present study (fig. 2) is compared with the earlier report of tolerance recorded during abstinence after oral barbital treatment of similar duration (WahlstrSm, 1974}, the changes in the dose of hexobarbital were similar. Inherent in the threshold technique is a reduction of the amount of the tested substance to a dose level just necessary to obtain the change studied. The risk of influencing the tolerance by the test is reduced as much as possible. A threshold method must be regarded as essential in the present situation when the same animals were used in two kinds of tests. The hexobarbital threshold test can be performed at least every second day without influencing subsequent determinations (WahlstrSm, 1966). The test required an easily recorded function
128 which was influenced by cholinergic mechanisms in the central nervous system. Temperature changes induced by cholinergic stimulation after peripheral blockade with quaternary substances were deemed suitable (Everett, 1964; Spencer, 1965; Lomax and Jenden, 1966; Friedman and Jaffe, 1969). Methylscopolamine is a potent quaternary substance with little effect on the central nervous system which could be used in the present experiments (Visscher et al., 1954). Pilocarpine was chosen as the cholinomimetic drug (Friedman and Jaffe, 1969). The test pattern was developed so as to keep the dose of pilocarpine as low as possible. In this way the influence which the test could have on the abstinence was reduced as much as possible. As already stated the pattern of the tolerance to hexobarbital in the present experiments was not essentially different from that recorded in earlier experiments (WahlstrSm, 1974). A more dramatic effect of repeated testing with pilocarpine on the barbiturate tolerance as such can thus be excluded. Recording of body temperature after pilocarpine has been used by Friedman et al. (1969) to study changes in the central nervous system induced by chronic scopolamine treatments in mice. After dose--response studies they could state that the sensitivity to pilocarpine was increased after the treatment. Due to differences in species and technique, no direct comparison can be made but the similarity between the results of Friedman et al. (1969) and the present results (section 3.4. ) is nevertheless evident. The hexobarbital threshold (fig. 2 ) a n d the minimal body temperature after pilocarpine (fig. 4) both showed a biphasic pattern of changes during the abstinence period. The two different determinations were not performed on the same day. Correlations can thus not be calculated. However, the covariation with time between changes in the threshold determinations and the changes in the temperature sensitivity are clearly detectable if fig. 2 and fig. 4 are compared. The tolerance to hexobarbital is thus followed by a concom-
G. WAHLSTR(]M, T. EKWALL itant increase in sensitivity to a central effect of pilocarpine. The depressant effects of the barbiturates on the central nervous system probably are unspecific. Barbiturates have been shown, in acute experiments, to influence several neurotransmittors, e.g. acetylcholine (Crossland, 1953; Nordberg and Sundwall, 1975) and catecholamines (Corrodi et al., 1966; Persson and Waldeck, 1971) in the manner expected to result from decreased central nervous system activity. The selection of cholinergic drugs for the present comparison was based on results obtained in acute experiments. Stimulation of monoaminergic mechanisms decreased the hexobarbital threshold and depression by means of reserpine increased the threshold (WahlstrSm, 1971b). Increased activity in monoaminergic neurones during abstinence was thus an unlikely explanation for the tolerance to hexobarbital. Pilocarpine in doses above 25 mg/kg increased the hexobarbital threshold (WahlstrSm, 1975) and atropine in doses above 4 mg/kg deerased it (WahlstrSm, 1976). It. was thus more likely that increased activity in cholinergic neurones was involved. The conclusion to be drawn from t h i s study is that cholinergic neurones probably are involved in the tolerance seen after chronic barbital treatment. The present data are not extensive enough to state whether the change in the sensitivity of the central nervous system is identical with the changes in sensitivity seen after denervation in the peripheral nervous system. The hypothesis as such however, has survived the present assault. Acknowledgement
This study was supported by a grant from the Swedish Medical Research Council (No. 3771). References
Cannon, W.B., 1939, A law of denervation, Amer. J. Med. Sci. 198,737. Collier, H.O.J., 1968, Supersensitivity and dependence, Nature 220, 228.
BARBITURATES AND PILOCARPINE Corrodi, H., K. Fuxe and T. HSkfelt, 1966, The effects of barbiturates on the activity of the catecholamine neurones in the rat brain, J. Pharm. Pharmacol. 18, 556. Crossland, J., 1953, The significance of brain acetylcholine, J. Mental Sci. 99, 247. Ekwall, T. and G. WahlstrSm, 1972, Tolerance to hexobarbital and supersensitivity to pilocarpine induced by chronic barbital treatment in the rat, Acta Pharmacol. Toxicol. 31, Suppl. I, 2. Emmelin, N., 1965, Action of transmitters on the responsiveness of effector cells, Experientia 21, 57. Everett~ G.M., 1964, Animal and clinical techniques for evaluating anti-parkinson agents, in: Animal and Clinical Pharmacologic Techniques in Drug Evaluation, eds. J.H. Nodine and P.E. Siegler (Year Book Medical Publ. Inc., Chicago) p. 359. Friedman, M.J. and J.H. Jaffe, 1969, A central hypothermic response to pilocarpine in the mouse, J. Pharmacol. Exptl. Therap. 167, 34. Friedman, M,J., J.H. Jaffe and S.K. Sharpless, 1969, Central nervous system supersensitivity to pilocarpine after withdrawal of chronically administered scopolamine, J. Pharmacol. Exptl. Therap. 167, 45. Jaffe, J.H. and S.K. Sharpless, 1965, The rapid development of physical dependence on barbiturates, J. Pharmacol. Exptl. Therap. 150, 140. Jaffe, J.H. and S.K. Sharpless, 1968, Pharmacological denervation supersensitivity in the central nervous system: a theory of physical dependence, in: The Addictive States, ed. A. Wikler, Res. Publ. Ass. Nerv. Ment. Dis. Vol. 46 (Williams & Wilkins Comp., Baltimore) p. 226. Lomax, P. and D.J. Jenden, 1966, Hypothermia following systematic and intracerebral injection of oxotremorine in the rat, Intern. J. Neuropharmacol. 5,353. McBride, A. and M.J. Turnbull, 1970, The brain acetylcholine system in barbitone-dependent and withdrawn rats, Brit. J. Pharmacol. 39, 210P. Nordberg, A. and A. Sundwall, 1975, Effect of pentobarbital on endogenous acetylcholine and biotransformation of radioactive choline in different brain regions, in: Cholinergic Mechanisms, ed. P.G. Waser (Raven Press, New York) p. 229.
129 Persson, T. and B. Waldeck, 1971, A reduced rate of turnover of brain noradrenaline during pentobarbitone anaesthesia, J. Pharm. Pharmacol. 23,377. Sharpless, S.K., 1964, Reorganization of function in the nervous system -- use and disuse, Ann. Rev. Physiol. 26, 357. Spencer, P.S.J., 1965, Activity of centrally acting and other drugs against tremor and hypothermia induced in mice by tremorine, Brit. J. Pharmacol. 25, 442. Thesleff, S., 1960, Effects of m o t o r innervation on the chemical sensitivity of skeletal muscle, Physiol. Rev. 40, 734. Trendelenburg, U., 1966, I. Mechanisms of supersensitivity and subsensitivity to sympathomimetic amines, Pharmacol. Rev. 18,629. Visscher, F.E., P.H. Seay, A.P. Tazelaar Jr., W. Veldkamp and M.J. Vander Brook, 1954, Pharmacology of pamine bromide, J. Pharmacol. Exptl. Therap. 110, 188. Wahlstr6m, G., 1966, Estimation of brain sensitivity to hexobarbitone (Enhexymal NFN) in rats by an EEG threshold, Acta Pharmacol. Toxicol. 24,404. Wahlstr6m, G., 1968, Hexobarbital (Enhexymalum NFN) sleeping times and EEG threshold doses as measurements of tolerance to barbiturates in the rat, Acta Pharmacol. Toxicol. 26, 64. Wahlstr6m, G., 1971a, Changes in a hexobarbital anaesthesia threshold in rats induced by repeated long-term treatment with barbital or ethanol, Psychopharmacologia 19, 366. Wahlstr~m, G., 1971b, Changes in the effects of hexobarbital in male rats induced by manipulations of monoamines through reserpine or pargyline combined with DOPA or 5-hydroxytryptophane, Acta Pharmacol. Toxicol. 30, 353. Wahlstr6m, G., 1974, Withdrawal in the rat after long-term forced oral barbital administration, Aeta Pharmacol. Toxicol. 35,131. Wahlstr6m, G., 1975, The acute interaction between cholinergic drugs and a barbiturate, Exptl. Brain Res., Suppl. to Vol. 23, 213. Wahlstr6m, G., 1976, The interaction between hexobarbital and atropine or methylatropine in male rats, Acta Pharmacol. Toxicol. 38, 72.
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T O L E R A N C E TO H E X O B A R B I T A L AND SUPERSENSITIVITY TO PILOCARPINE A F T E R CHRONIC BARBITAL TREATMENTS IN THE RAT GORAN WAHLSTROM and TORSTEN EKWALL
Department of Pharmacology, University o f Umet}, S-901 8 7 Umeti, Sweden Received 25 September 1975, revised MS received 24 February 1976, accepted 18 March 1976
G. WAHLSTROM and T. EKWALL, Tolerance to hexobarbital and supersensitivity to pilocarpine after chronic babital treatments in the rat, European J. Pharmacol. 38 (1976) 123--129. Tolerance and physical dependence induced by chronic treatments with depressant drugs has been proposed as being due to denervation or disuse supersensitivity in the central nervous system. To illustrate this hypothesis, tolerance to barbiturates and the sensitivity to central cholinergic stimulation were recorded in the same male rats. Tolerance to barbiturates was recorded with a hexobarbital threshold. The dose needed to obtain an EEG criterion was measured. Sensitivity in central cholinergic structures was recorded by a temperature threshold method. A subthreshold dose of pilocarpine (3.5 mg/kg) was given s.c. together with 1.0 mg/kg methylscopolamine i.p. An increase in sensitivity to pilocarpine was recorded as a decrease in body temperature. In the abstinence period after 42 weeks of oral treatment with barbital, tolerance was recorded with the hexobarbital threshold method. The tolerance had two peaks, one after the end of the treatment and one approximately 3 weeks later. An increased sensitivity to pilocarpine was also recorded in the abstinence period. It had the same biphasic time pattern as the tolerance. Cholinergic neurones are t h u s probably involved in the changes induced by chronic barbital treatment. The denervation supersensitivity hypothesis of tolerance was n o t refuted by the present experiments.
Supersensitivity
Pilocarpine
Tolerance
1. Introduction
The law of denervation formulated by Cannon (1939) states that an increased irritability to chemical agents develops in structures where the efferent neurones are destroyed. This denervation supersensitivity is present in the neuromuscular junction (Thesleff, 1960) and in various peripheral neuroeffector junctions (Sharpless, 1964; Emmelin, 1965; Trendelenburg, 1966). A similar phenomenon can also be induced in the periphery by a pharmacological depression of activity i.n the efferent nerves (Sharpless, 1964; Emmelin, 1965). It is thus not surprising that denervation supersensitivity has been discussed several times as a theoretical model to explain tol-
Hexobarbital
Abstinence
Barbiturates
Rat
erance and physical dependence to drugs acting on the central nervous system (Sharpless, 1964; Jaffe and Sharpless, 1968; Collier, 1968). Denervation supersensitivity in the central nervous system has been directly implicated as a possible explanation for the changes recorded after chronic barbiturate treatment in the cat (Jaffe and Sharpless, 1965) and also more specifically as a factor in the changes in central cholinergic neurones after chronic scopolamine treatment (Friedman et al., 1969). The possibility that changes in central cholinergic neurones could be involved in barbiturate tolerance has been studied by McBride and Turnbull (1970). They found a change in temperature sensitivity to choliner-
124 gic stimulation after the barbiturate treatment, but no data were given. The basal barbiturate tolerance was n o t measured. Since the tolerance to a hexobarbital threshold in the abstinence after chronic barbital treatments is well d o c u m e n t e d (Wahlstr~m, 1974), the question whether this tolerance is accompanied by a supersensitivity to cholinergic stimulation could easily be studied. As a measure of sensitivity to cholinergic stimulation in the central nervous system the t e m p e r a t u r e response to pilocarpine after peripheral blockade (Friedman and Jaffe, 1969) was chosen. The technique had to be modified to fit the present experiments. The results have been briefly discussed earlier (Ekwall and WahlstrOm 1972).
2. Materials and m e t h o d s
2.1. Materials Male Sprague--Dawley rats (Nih/Han/Mol) were obtained from M~llegaard, Li. Skensved, Denmark. The initial b o d y weight was around 300 g. The animals were kept in a r oom at a t e m p e r a t u r e of 25°C. External light was excluded. Artificial light was t ur ne d on at 01.00 and o f f at 13.00. The reason for this shift from normal light conditions was to make the activity of the animals occur at a m o r e suitable time for the experimental work. There were 2--3 rats in each cage. F o o d was available ad libitum. The drinking fluid was used as the vehicle for barbiturate administration (see 2.2.).
2. 2. Barbital treatment Sodium barbital was administered in the drinking fluid. The c o n c e n t r a t i o n was 3.33 mg/ml, e x c e p t during the first week of the t r e a t m e n t when it was 1.67 mg/ml. Controls were given tap water. If n o t otherwise stated there was free access to the drinking fluid. At the end of the t r e a t m e n t the barbital solution was replaced by tap water. The daily dose in
G. WAHLSTROM, T. EKWALL mg/kg/day was based on weekly c o n s u m p t i o n data from all cages. For details regarding the t r e a t m e n t see Wahlstr6m (1974).
2.3. Hexobarbital threshold determinations Sodium hexobarbital was infused continuously in a tail vein with an infusion rate of 0.25 mg/kg/sec (volume rate 0.1 ml/min). During the infusion the animals were restrained and the electroencephalogram was recorded. Stainless steel sutures were used as electrodes. The first burst suppression of one second or more (the 'silent second') was taken as the threshold criterion and the dose of hexobarbital needed was determined. All threshold determinations were p e r f o r m e d during the first half of the dark period. Prior to the barbital t r e a t m e n t two threshold determinations were performed. These were used to calculate a pre-experimental values for each individual. The average of these pre-experimental values was 57.1 + 1.1 mg/kg (-+ S.E., n = 12) in the experimental animals and 62.5 + 2.0 mg/kg (n = 13) in the control animals. Since 'the individual rats tend to keep to a certain threshold the experimental data are given as percent of the pre-experimental value. Only the animals which survived the barbital t r e a t m e n t are included. For furt her details of the m e t h o d see Wahlstr~m (1966).
2.4. Temperature recording The rectal t em perat ure was recorded with a thermistor probe as one part of a Wheatstone bridge. The probe was inserted 5 cm in the rectum. Prior to any injection the basal temperature level was always recorded. The animals were then given the drugs. In m ost cases both methylscopolamine and pilocarpine were administered. Two different injection schedules were used (see 3.1.). All injections were p e r f o r m e d under sterile conditions during the first part of the dark period.
2. 5. Experimental procedure After the pre-experimental values on threshold dose had been determined the tempera-
BARBITURATES AND PILOCARPINE
ture experiments seen in fig. 1 were performed. When the liminal dose of pilocarpine had been determined and the final injection schedule tested in all participating animals, the cages were randomly divided into the two treatment groups. After 4 weeks of barbital treatment a single temperature test was performed. After 42 weeks of treatment the barbital solution was replaced with water. The same animals participated in the temperature and threshold determinations. The schedule during the abstinence period is best seen in figs. 1 and 4. There was never less than a 2 day interval between two different experimental sessions.
2.6. Drugs Sodium hexobarbital (Evipan ®) was kindly supplied by Bayer AG through Mr. R. Arensberg. Methylscopolamine nitrate was kindly supplied by Pharmacia AB, Uppsala, Sweden. Pilocarpine chloride and sodium barbital were obtained from a local pharmacy. All doses are calculated as the salt.
2. 7. Statistical treatment Differences were tested with Student's ttest if not otherwise stated.
3. Results
3.1. The effects o f pilocarpine on body temperature in normal rats The body temperature of the rat kept under the experimental conditions used is given in fig. 1A. All rats which participated in the experiments were used. As expected there was an increase in body temperature at the start of the dark period. The gradual changes during the dark period were of such a magnitude that controls run in parallel were necessary in the critical experiments. Fig. 1B shows the effects of methylscopolamine alone and combined with different
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Fig. 1. Recordings of b o d y temperature prior to the barbital treatment. A. Normal b o d y t e m p e r a t u r e of the rat kept under the experimental conditions described. All rats started in the e x p e r i m e n t (n = 32) were tested at every point. B. B o d y temperature after m e t h y l s c o p o l a m i n e alone (filled circles) and in combination with different doses o f pilocarpine. The doses of pilocarpine and the n u m b e r of animals tested were: 3.1 m g / k g (squares) n = 9, 6.25 mg/kg (triangles down) n = 11, 12.5 mg/kg (triangles up) n = 12, and 25 mg/kg (circles) n = 9. The arrows indicate the times of administration of 1.0 m g / k g m e t h y l s c o p o l amine (MS) i.p. and pilocarpine (P) s.c.C. The effects on the b o d y t e m p e r a t u r e of 3.5 m g / k g pilocarpine given s.c. i m m e d i a t e l y prior to 1.0 m g / k g methylscopolamine i.p.; 32 rats participated. The times of administration of the drugs are indicated by the arrows.
doses of pilocarpine on the body temperature of the rat. After 1 mg/kg of methylscopolamine i.p. there was an increase in body temperature compared with the pre-experimental basal level. The relative magnitude is uncertain since no untreated controls were included. However, the temperature was above the basal level recorded in fig. 1A. If 3.1 mg/kg of pilocarpine was given s.c. 0.5 h after the methylscopolamine no definite effect on body temperature was seen. Compared with the effect of methylscopolamine alone the level was slightly depressed. If the dose of pilocarpine was increased a dose-dependent decrease in body temperature was obtained. Based upon these data 3.5 mg/kg s.c. was
126 c h o s e n as a s u b t h r e s h o l d dose o f pilocarpine. A slight e f f e c t o f this dose on the b o d y temp e r a t u r e is seen in fig. 1C with pilocarpine given s.c. i m m e d i a t e l y prior to m e t h y l s c o p o l amine (i.p.) t o all participating animals. This new schedule was i n t r o d u c e d to r e d u c e the time n e e d e d to r e c o r d the t e m p e r a t u r e minim u m in the critical e x p e r i m e n t s (see 3.4.).
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3.2. Barbital treatment The t r e a t m e n t with barbital in the drinking w a t e r lasted 42 weeks. During the s e c o n d half o f the t r e a t m e n t , 4 barbital-treated and one c o n t r o l animal died. T h e average b o d y weight at the end o f the t r e a t m e n t was 518 g in the barbital-treated animals and 558 g in the controls. The average c o n s u m p t i o n o f barbital was 222 m g / k g / d a y during the last 5 weeks o f the barbital t r e a t m e n t . These values are close t o the values r e p o r t e d earlier in a m o r e syst e m a t i c s t u d y on the e f f e c t o f similar barbital t r e a t m e n t s (WahlstrSm, 1 9 7 4 ) .
3.3. Hexobarbital threshold determinations in the abstinence period after the end o f the barbital treatment The results o f the h e x o b a r b i t a l t h r e s h o l d d e t e r m i n a t i o n s after the barbital t r e a t m e n t are given in fig. 2. T h e figure clearly indicates t h a t t h e r e were at least t w o peaks o f decreased sensitivity t o h e x o b a r b i t a l during the a b s t i n e n c e p e r i o d a f t e r the barbital t r e a t m e n t . With the p r e s e n t testing schedule the t w o peaks app e a r e d a r o u n d day 5 and s o m e t i m e s after day 21. All values e x c e p t the one o f day 17 had a p value less than 0.001 if c o m p a r e d with the c o r r e s p o n d i n g c o n t r o l value. T h e p value was less than 0.02 on day 17.
3.4. The effects o f pilocarpine on body temperature in the abstinence period after the barbital treatment T w o examples o f the results o b t a i n e d with the pilocarpine t e m p e r a t u r e test during the abstinence p e r i o d a f t e r the barbital t r e a t m e n t
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DAYS AFTER
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END OF BARBITAL TREATMENT
Fig. 2. The results of the hexobarbita] threshold de-
terminations during the abstinence after the barbital treatment. The thresholds are given in percent of a pre-experimental value obtained prior to any of the experiments. The number of experimental animals was 6--9 (filled circles) and the number of controls was 10--13 (unfilled circles). 2 times the standard error is indicated by the bars.
are given in fig. 3. On the third d a y o f abstin e n c e the e f f e c t o f pilocarpine (3.5 m g / k g s.c.) was increased in t.he barbital-treated animals as c o m p a r e d t o the c o n t r o l s . On the 1 5 t h d a y t h e r e was n o difference. O n l y the b o d y t e m p e r a t u r e s at the time o f the minim u m in the barbital-treated animals were used. These values are indicated by the filled circles in the e x a m p l e s s h o w n in fig. 3. The c o r r e s p o n d i n g t e m p e r a t u r e values in the controls were used for c o m p a r i s o n instead o f the minimal values. In the c o n t r o l s the d i f f e r e n c e b e t w e e n the value used and this minimal value varied b e t w e e n 0 and 0.19°C. One separate t e m p e r a t u r e e x p e r i m e n t was p e r f o r m e d a f t e r 4 weeks o f barbital treatm e n t . At t h a t time t h e r e was n o d i f f e r e n c e in the e f f e c t o f pilocarpine b e t w e e n the barbitalt r e a t e d animals and the controls. All animals had been w i t h o u t drinking fluid for 28 h p r i o r to the test. The minimal values o f b o d y t e m p e r a t u r e a f t e r pilocarpine in the a b s t i n e n c e p e r i o d after the barbital t r e a t m e n t are given in fig. 4. On no occasion was there any significant difference b e t w e e n the basal values r e c o r d e d in
BARBITURATES AND PILOCARPINE AFTER BARBITAL 3 DAYS
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Fig. 4. Minimal b o d y t e m p e r a t u r e s r e c o r d e d a f t e r p i l o c a r p i n e 3.5 m g / k g s.c. ! m m e d i a t e l y f o l l o w e d b y m e t h y l s c o p o l a m i n e 1.0 m g / k g i.p. d u r i n g t h e abstin e n c e a f t e r b a r b i t a l t r e a t m e n t . M i n i m a l values as s h o w n in fig. 3 were used. T h e n u m b e r o f barbitalt r e a t e d a n i m a l s was b e t w e e n 7 - - 1 1 o n each o c c a s i o n a n d is i n d i c a t e d b y filled circles a n d t h e n u m b e r o f c o n t r o l s was b e t w e e n 1 1 - - 1 3 a n d is i n d i c a t e d b y unfilled circles. 2 t i m e s t h e s t a n d a r d e r r o r is i n d i c a t e d b y t h e bars.
TIME AFTER LAST INJECTION Fig. 3. 2 e x a m p l e s o f the t e m p e r a t u r e recordings obt a i n e d a f t e r p i l o c a r p i n e 3.5 m g / k g s.c. f o l l o w e d b y 1.0 m g / k g o f m e t h y l s e o p o l a m i n e i.p. in the abstinence a f t e r t h e b a r b i t a l t r e a t m e n t . T h e c o n t r o l s are i n d i c a t e d b y a C a n d a solid line. T h e b a r b i t a l - t r e a t e d a n i m a l s are i n d i c a t e d b y a n E a n d a b r o k e n line. The h o r i z o n t a l lines i n d i c a t e t h e p r e - e x p e r i m e n t a l levels. The filled s y m b o l s i n d i c a t e t h e t i m e o f m i n i m a l b o d y t e m p e r a t u r e in t h e e x p e r i m e n t a l animals. T h e n u m bers i n d i c a t e t h e n u m b e r s of animals.
the controls and those recorded in the barbital-treated animals prior to injection of methylscopolamine. The range of these average basal temperature recordings was 38.0-39.3°C. Fig. 4 shows that there were two time periods in the abstinence period after barbital when the sensitivity to the temperature reducing effect of pilocarpine was increased in animals previously treated with barbital. The p values were less than 0.001 on days 3 and 7 and less than 0.01 on days 20 and 24. There was thus a clear increase in sensitivity to the temperature-reducing effect of pilocarpine in the abstinence after long barbital treatment.
4. Discussion The hexobarbital threshold provides a measure for tolerance during the abstinence after barbital treatments (Wahlstr6m, 1968). This tolerance can be biphasic after repeated or long term oral treatments (Wahlstrbm, 1971a; WahlstrSm, 1974). If the tolerance in the present study (fig. 2) is compared with the earlier report of tolerance recorded during abstinence after oral barbital treatment of similar duration (WahlstrSm, 1974}, the changes in the dose of hexobarbital were similar. Inherent in the threshold technique is a reduction of the amount of the tested substance to a dose level just necessary to obtain the change studied. The risk of influencing the tolerance by the test is reduced as much as possible. A threshold method must be regarded as essential in the present situation when the same animals were used in two kinds of tests. The hexobarbital threshold test can be performed at least every second day without influencing subsequent determinations (WahlstrSm, 1966). The test required an easily recorded function
128 which was influenced by cholinergic mechanisms in the central nervous system. Temperature changes induced by cholinergic stimulation after peripheral blockade with quaternary substances were deemed suitable (Everett, 1964; Spencer, 1965; Lomax and Jenden, 1966; Friedman and Jaffe, 1969). Methylscopolamine is a potent quaternary substance with little effect on the central nervous system which could be used in the present experiments (Visscher et al., 1954). Pilocarpine was chosen as the cholinomimetic drug (Friedman and Jaffe, 1969). The test pattern was developed so as to keep the dose of pilocarpine as low as possible. In this way the influence which the test could have on the abstinence was reduced as much as possible. As already stated the pattern of the tolerance to hexobarbital in the present experiments was not essentially different from that recorded in earlier experiments (WahlstrSm, 1974). A more dramatic effect of repeated testing with pilocarpine on the barbiturate tolerance as such can thus be excluded. Recording of body temperature after pilocarpine has been used by Friedman et al. (1969) to study changes in the central nervous system induced by chronic scopolamine treatments in mice. After dose--response studies they could state that the sensitivity to pilocarpine was increased after the treatment. Due to differences in species and technique, no direct comparison can be made but the similarity between the results of Friedman et al. (1969) and the present results (section 3.4. ) is nevertheless evident. The hexobarbital threshold (fig. 2 ) a n d the minimal body temperature after pilocarpine (fig. 4) both showed a biphasic pattern of changes during the abstinence period. The two different determinations were not performed on the same day. Correlations can thus not be calculated. However, the covariation with time between changes in the threshold determinations and the changes in the temperature sensitivity are clearly detectable if fig. 2 and fig. 4 are compared. The tolerance to hexobarbital is thus followed by a concom-
G. WAHLSTR(]M, T. EKWALL itant increase in sensitivity to a central effect of pilocarpine. The depressant effects of the barbiturates on the central nervous system probably are unspecific. Barbiturates have been shown, in acute experiments, to influence several neurotransmittors, e.g. acetylcholine (Crossland, 1953; Nordberg and Sundwall, 1975) and catecholamines (Corrodi et al., 1966; Persson and Waldeck, 1971) in the manner expected to result from decreased central nervous system activity. The selection of cholinergic drugs for the present comparison was based on results obtained in acute experiments. Stimulation of monoaminergic mechanisms decreased the hexobarbital threshold and depression by means of reserpine increased the threshold (WahlstrSm, 1971b). Increased activity in monoaminergic neurones during abstinence was thus an unlikely explanation for the tolerance to hexobarbital. Pilocarpine in doses above 25 mg/kg increased the hexobarbital threshold (WahlstrSm, 1975) and atropine in doses above 4 mg/kg deerased it (WahlstrSm, 1976). It. was thus more likely that increased activity in cholinergic neurones was involved. The conclusion to be drawn from t h i s study is that cholinergic neurones probably are involved in the tolerance seen after chronic barbital treatment. The present data are not extensive enough to state whether the change in the sensitivity of the central nervous system is identical with the changes in sensitivity seen after denervation in the peripheral nervous system. The hypothesis as such however, has survived the present assault. Acknowledgement
This study was supported by a grant from the Swedish Medical Research Council (No. 3771). References
Cannon, W.B., 1939, A law of denervation, Amer. J. Med. Sci. 198,737. Collier, H.O.J., 1968, Supersensitivity and dependence, Nature 220, 228.
BARBITURATES AND PILOCARPINE Corrodi, H., K. Fuxe and T. HSkfelt, 1966, The effects of barbiturates on the activity of the catecholamine neurones in the rat brain, J. Pharm. Pharmacol. 18, 556. Crossland, J., 1953, The significance of brain acetylcholine, J. Mental Sci. 99, 247. Ekwall, T. and G. WahlstrSm, 1972, Tolerance to hexobarbital and supersensitivity to pilocarpine induced by chronic barbital treatment in the rat, Acta Pharmacol. Toxicol. 31, Suppl. I, 2. Emmelin, N., 1965, Action of transmitters on the responsiveness of effector cells, Experientia 21, 57. Everett~ G.M., 1964, Animal and clinical techniques for evaluating anti-parkinson agents, in: Animal and Clinical Pharmacologic Techniques in Drug Evaluation, eds. J.H. Nodine and P.E. Siegler (Year Book Medical Publ. Inc., Chicago) p. 359. Friedman, M.J. and J.H. Jaffe, 1969, A central hypothermic response to pilocarpine in the mouse, J. Pharmacol. Exptl. Therap. 167, 34. Friedman, M,J., J.H. Jaffe and S.K. Sharpless, 1969, Central nervous system supersensitivity to pilocarpine after withdrawal of chronically administered scopolamine, J. Pharmacol. Exptl. Therap. 167, 45. Jaffe, J.H. and S.K. Sharpless, 1965, The rapid development of physical dependence on barbiturates, J. Pharmacol. Exptl. Therap. 150, 140. Jaffe, J.H. and S.K. Sharpless, 1968, Pharmacological denervation supersensitivity in the central nervous system: a theory of physical dependence, in: The Addictive States, ed. A. Wikler, Res. Publ. Ass. Nerv. Ment. Dis. Vol. 46 (Williams & Wilkins Comp., Baltimore) p. 226. Lomax, P. and D.J. Jenden, 1966, Hypothermia following systematic and intracerebral injection of oxotremorine in the rat, Intern. J. Neuropharmacol. 5,353. McBride, A. and M.J. Turnbull, 1970, The brain acetylcholine system in barbitone-dependent and withdrawn rats, Brit. J. Pharmacol. 39, 210P. Nordberg, A. and A. Sundwall, 1975, Effect of pentobarbital on endogenous acetylcholine and biotransformation of radioactive choline in different brain regions, in: Cholinergic Mechanisms, ed. P.G. Waser (Raven Press, New York) p. 229.
129 Persson, T. and B. Waldeck, 1971, A reduced rate of turnover of brain noradrenaline during pentobarbitone anaesthesia, J. Pharm. Pharmacol. 23,377. Sharpless, S.K., 1964, Reorganization of function in the nervous system -- use and disuse, Ann. Rev. Physiol. 26, 357. Spencer, P.S.J., 1965, Activity of centrally acting and other drugs against tremor and hypothermia induced in mice by tremorine, Brit. J. Pharmacol. 25, 442. Thesleff, S., 1960, Effects of m o t o r innervation on the chemical sensitivity of skeletal muscle, Physiol. Rev. 40, 734. Trendelenburg, U., 1966, I. Mechanisms of supersensitivity and subsensitivity to sympathomimetic amines, Pharmacol. Rev. 18,629. Visscher, F.E., P.H. Seay, A.P. Tazelaar Jr., W. Veldkamp and M.J. Vander Brook, 1954, Pharmacology of pamine bromide, J. Pharmacol. Exptl. Therap. 110, 188. Wahlstr6m, G., 1966, Estimation of brain sensitivity to hexobarbitone (Enhexymal NFN) in rats by an EEG threshold, Acta Pharmacol. Toxicol. 24,404. Wahlstr6m, G., 1968, Hexobarbital (Enhexymalum NFN) sleeping times and EEG threshold doses as measurements of tolerance to barbiturates in the rat, Acta Pharmacol. Toxicol. 26, 64. Wahlstr6m, G., 1971a, Changes in a hexobarbital anaesthesia threshold in rats induced by repeated long-term treatment with barbital or ethanol, Psychopharmacologia 19, 366. Wahlstr~m, G., 1971b, Changes in the effects of hexobarbital in male rats induced by manipulations of monoamines through reserpine or pargyline combined with DOPA or 5-hydroxytryptophane, Acta Pharmacol. Toxicol. 30, 353. Wahlstr6m, G., 1974, Withdrawal in the rat after long-term forced oral barbital administration, Aeta Pharmacol. Toxicol. 35,131. Wahlstr6m, G., 1975, The acute interaction between cholinergic drugs and a barbiturate, Exptl. Brain Res., Suppl. to Vol. 23, 213. Wahlstr6m, G., 1976, The interaction between hexobarbital and atropine or methylatropine in male rats, Acta Pharmacol. Toxicol. 38, 72.