P sycho pharmacology
Psychopharmacology 54, 6t - 6 5 (t 977)
9 by Springer-Verlag1977
Single-Dose Tolerance to the Behavioral Effects of Dibutyryl Cyclic AMP in Mice MYRON WEINER and JACK W. OLSON PharmacodynamicsSection,Universityof SouthernCalifornia, Schoolof Pharmacy,Los Angeles,California90033, U.S.A.
Abstract. The behavioral effects of varying doses of intraperitoneally administered dibutyryl cyclic AMP, cyclic AMP, adenosine, 5'-AMP, and butyric acid were studied in male ICR mice. Behavioral parameters 25 min following treatment included measurement of spontaneous locomotor activity (SLMA) and rotarod performance, the latter providing an indication of neuromuscular coordination. Dibutyryl cyclic AMP produced a dose-related inhibition of SLMA with the largest dose, 75 mg/kg, decreasing activity by 89%. Adenosine and 5'-AMP produced maximal inhibition of approximately 5 0 - 80 % of SLMA at doses ranging from 75-250 mg/kg, while cyclic AMP decreased SLMA by 58 % at only the highest dose, 250 mg/kg. Butyric acid failed to produce alterations in SLMA at doses ranging from 25-250 mg/kg. No compound altered neuromuscular coordination. Single-dose tolerance to the inhibitory effect of dibutyryl cyclic AMP on SLMA developed within 3 h and lasted at least 7 days. Adenosine failed to produce tolerance while cyclic AMP and 5'-AMP exhibited only a slightly reduced effect following a second injection at intervals of 4 and 24 h. These results suggest that exogenous administration of dibutyryl cyclic AMP and its metabolites exert centrally mediated behavioral effects with selective development of single-dose tolerance to the dibutyryl derivative. Key words: Spontaneous locomotor activity - Singledose tolerance -- Dibutyryl cyclic AMP - Cyclic AMP - 5'-AMP - Adenosine - Mice
Numerous reports and extensive reviews (Daly, 1975; Rall, 1975) have recently appeared on the relationship of cyclic adenosine 3 ',5'-monophosphate (cyclic AMP) to many aspects of brain function. Different types of
brain preparations (e.g., cell-fl'ee systems, cultured cells, brain slices, and intact brain) have provided evidence for the involvement of cyclic AMP in the pharmacological effects of psychoactive drugs (Uzunov and Weiss, 1971) or in the physiological control of postsynaptic events (Weiss and Costa, 1967). Although approaches to the complex problems of CNS regulation have generally included in vitro analyses, some in vivo studies have attempted to correlate cyclic AMP to spontaneous and drugelicited behavioral parameters. While various aspects of behavior have been investigated, spontaneous locomotor activity (SLMA) has been shown either to increase or to decrease following administration of the dibutyryl derivative of cyclic AMP. Drowsiness in mice was induced by intraperitoneal injection of dibutyryl cyclic AMP, 500 mg/kg (Henion et al., 1967). Intravenous administration of the dibutyryl derivative resulted in a dose-dependent (10-100 mg/kg) hypoactivity in dogs while similar doses of cyclic AMP, sodium butyrate, and butyric acid lacked effectiveness (Ono et al., 1976). Sleep and catatonia were produced in cats by injection of dibutyryl cyclic AMP into the cerebellum and mesencephalic reticular formation, respectively (Gessa et al., 1971). In addition, Beer et al. (1972) have observed that dibutyryl cyclic AMP (100-400 mg/kg, i.p.) had anxiety-reducing properties when a behavioral conflict test was utilized in rats 30 min following injection. In contrast, intracerebrat administration of dibutyryl cyclic AMP increased SLMA in immature rats (Breckenridge and Lisk, 1959), cats (Gessa et al., 1971), and chickens (Asakawa and Yoshida, 1971). Herman (1973) noted behavioral excitation in rats following intraventricular administration of dibutyryl cyclic AMP; however, a stuporous state and period of immobility was noticed between periods of excitation. Thus, these qualitatively different responses apparently depend on the species and the route of administration.
62 In a previous study (Weiner and Olson, 1973), we showed that i.p. administration of dibutyryl cyclic A M P (25 mg/kg) to male Institute of Cancer Research (ICR) mice decreased SLMA and exploratory behavior approximately 40 ~ without affecting motor coordination. The threshold dose for decreased SLMA was 10 mg/kg with near maximal response occurring with a dose of 75 mg/kg. This effect was almost immediate in onset and persisted for approximately 90 min. The failure of a second injection, 24 h later, of dibutyryl cyclic A M P to produce behavioral changes prompted us to characterize further the apparent tachyphylactic response to the cyclic nucleotide in the present investigation. In addition, we decided to study the effects of single and multiple injections of cyclic AMP, Y-AMP, adenosine, and butyric acid on SLMA and motor coordination.
MATERIALS AND METHODS Male ICR albino mice (Simonsen Labs, Gilman, California) weighing 20-25 g were used in all experiments. They were maintained in a constant temperature room (22 + 1~ with Purina Laboratory chow and water continuously available for at least 3 days beforeuse. All compounds, purchased from SigmaChemical Company, were dissolved in distilled water to prepare solutions so that 0.01 ml/g body weight was injected. Control mice received the same volume of saline. Mice were tested 25 rain after a single injection of the test compound. The behavioral activity of mice receivingmultiple injectionswas observed25 min after the last dose was administered. This time period between treatment and testing was chosen on the basis of maximum behavioral effectsobservedin a previous study (Weiner and Olson, 1973). SLMA, used to provide a measurement of nondirected movement, was measured for 15 min in groups of 3 mice in an Electronic Motility Meter (Fc 40, Motron Products, Stockholm, Sweden), a circular photocell activity cage previously described (Weiner and Olson, 1973). Coordination of movement was determined as performance on a rotating rod (rotarod). The number of falls in 5 min for mice placed on the rotarod was recorded. Mice were considered uncoordinated if they fell more than twice in the experiment. All determinations were made in the afternoon hours. The statisticalsignificanceof SLMAwas determinedby the Student's t-test and that of rotarod performanceby the Chi Square test.
RESULTS SLMAs of mice receiving varied doses of dibutyryl cyclic A M P or its metabolites are shown in Figure 1. The dibutyryl derivative produced a dose-related decrease in SLMA with 25 mg/kg reducing activity 55 ~ , while a 75 mg/kg dose almost totally eliminated activity (i.e., 89 ~ inhibition). Cyclic A M P failed to alter activity at doses similar to its dibutyryl derivative. However, a similar degree of inhibition was induced by cyclic A M P at 250 mg/kg, a dose tenfold greater than that of dibutyryl cyclic AMP. Butyric acid was
Psychopharmacology54 (1977) without significant effect at doses up to 250 mg/kg. Although neither adenosine nor 5'-AMP produced statistically significant changes in SLMA at 25 mg/kg, a pronounced inhibition but no dose-response effect was observed in the dosage range 7 5 - 250 mg/kg. No statistically significant differences were observed between treatment groups and control mice on rotarod performance at the same dosage levels used above, although several mice receiving 5'-AMP at higher doses were deemed uncoordinated. The effect of a second injection of dibutyryl cyclic A M P on SLMA was determined using the same dose, 25 mg/kg, which initially produced a significant but submaximal decrease in activity. In contrast to the effects of a single injection of dibutyryl cyclic AMP, the same dose of a second injection failed to produce a change in activity (Fig. 2). This tachyphylactic response was first observed when 3 h separated injections and persisted for at least 7 days. Other compounds that inhibited SLMA (Fig. 1) were tested for the phenomenon of tolerance to a second injection at intervals of 4 and 24 h. No tolerance was observed following submaximal doses of adenosine (75 mg/kg). Although not statistically significant, the effect of a second injection of cyclic A M P (150 mg/kg) and 5'-AMP (75 mg/kg) was less than that of the initial administration by 25 ~ and 20 ~ respectively.
DISCUSSION These results confirm previous observations that i.p. administration ofdibutyryl cyclic A M P to mice evoked behavioral changes characterized by drowsiness (Henion et al., 1967) and a decreased SLMA (Weiner and Olson, 1973), and extend these results to cyclic AMP, adenosine, and 5'-AMP. The parent cyclic nucleotide was the least potent compound tested, while the dibutyryl derivative was the most potent. In addition to the greater activity of the dibutyryl or monobutyryl moiety, the difference in potency may be explained by dibutyryl cyclic AMP's increased ability to penetrate cellular membranes or its resistance to and inhibition of phosphodiesterase, the enzyme responsible for the catalysis of cyclic A M P to 5'-AMP. Only butyric acid was completely without effect even at a dose as high as 250 mg/kg, an order of magnitude greater on a molar basis than the dibutyryl cyclic nucleotide. Thus, the possibility that butyrate deacylated from dibutyryl cyclic A M P was responsible for the latter compound's behavioral effects was essentially ruled out, a conclusion supported by Ono et al. (1976). Although no definitive explanation can be provided for the decrease in SLMA while coordination remained
M. Weiner and J. W. Olson: Effects of Cyclic Nucleotides
63
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1500
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1000
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25
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butyric
125250 acid
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adenosine
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Fig. 1. SLMA of mice treated with varying doses of dibutyryl cyclic AMP, cyclic AMP, butyric acid, adenosine, and Y-AMP, administered i.p. 25 min before testing. Columns represent mean + SE of 3 - 7 groups of 3 mice. Percent change from saline control (0 dose) shown within columns. Significance expressed as *, P < 0.05
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2 d~
i6 4 do~.
9
7
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Fig.2. Time-response relationship for single-dose tolerance to dibutyryl cyclic AMP. SLMA was tested 25 rain following the second i.p. injection of saline or nucleotide. Each result is the mean • SE of groups of 3 mice, the total numbers of groups being shown in the column, and is expressed as a percentage of SLMA in in the corresponding saline controls. Significance expressed as *, P < 0.05
the same in these experiments, it seems reasonable to speculate, as have others (Beer et al., 1972; Ho et al., 1973; Ono et al., 1976), that a central effect may be involved in behavioral alterations produced by exogenous cyclic adenine nucleotides. In this regard, mice treated with antipsychotic agents (e.g., chlorpromazine) are also inactive and tranquil in an unstimulated environment but are capable of responding to stimulated activity as demonstrated by the ability to maintain balance on the rotarod (Carr, 1963). Since the i.p. route was utilized, the compounds tested in these experiments most likely passed the blood-brain barrier to produce changes in SLMA. However, it has generally been considered that exogenous cyclic nucleotides penetrate cell membranes with great difficulty. Recent evidence suggests that the dibutyryl cyclic nucleotide has access to the brain, although the fraction that permeates is very small (Tachizawa et al., 1974). Indeed, in the pharmacological doses (25 to 75 mg/kg) that were administered in our experiments, only a fraction of the dose would be necessary to enter the brain to approach physiological brain concentrations of approximately 10 .7 M. The hypoactivity produced by adenosine and 5'-AMP may result from increases in intracellular cyclic AMP concentration. Regardless of the species
64 or brain region tested, small amounts of adenosine have been shown consistently to cause accumulation of cyclic A M P in brain slices (Sattin and Rall, 1970; M a h and Daly, 1976), presumably as a result of activation of external receptors linked to adenylate cyclase (Daly, 1976). Although usually considered an inactive metabolite of cyclic A M P , 5 ' - A M P has been reported to increase cyclic A M P levels in brain slices following prior extracellular hydrolyses to adenosine (Sattin and Rall, 1970; M a h and Daly, 1976). Moreover, following central injection, both adenosine and 5'-AMP have potent depressant actions on neurons in the cerebral cortex, cerebellar Purkinje cells, and corticospinal cells (Phillis and Kostopoulos, 1975). While it is attractive to speculate that a central effect is involved in the differential effects on S L M A and rotarod performance, the possibility that the reductions in S L M A are the consequence of peripherally mediated, nonspecific effects exists and needs to be explored further. In addition, evidence demonstrating that these compounds enter the brain after i.p. administration needs to be provided. A puzzling aspect of this study concerns the refractoriness to the inhibition of the S L M A of mice following readministration of dibutyryl cyclic AMP. This effect was observed within 3 h of the first injection and lasted at least 7 days, the longest time utilized. The single-dose tolerance m a y represent an adaptive event within the CNS rather than a disposition tolerance since the effect lasts longer than the nucleotide would be expected to be present in the brain. Single-dose tolerance has also been observed with other compounds, e.g., ethanol (LeBlanc et al., 1975) and morphine (Kornetsky and Bain, 1968). Moreover, intravenous cyclic A M P (10 mg/kg) enhanced tolerance development in I C R mice after morphine pellet implantation (Ho et al., 1973) and after a single dose of morphine (Huidobro et al., 1976), although the latter effect was not statistically significant at the only morphine dose used. Refractoriness of adenylate cyclase systems has also been observed following initial stimulation by histamine, norepinephrine, or a histamine-norepinephrine combination, washing, and attempted restimulation in rabbit cerebellar slices (Kakiuchi and Rall, 1968) and guinea pig cortical slices (Schultz and Daly, 1973). Refractoriness to these amines was not observed in the presence of adenosine. In our studies adenosine failed to produce single-dose tolerance while second doses of cyclic A M P and 5'-AMP produced nonsignificant decreases in activity from the first dose. Much more sophisticated approaches to the correlation of behavioral aspects and neurochemical parameters are necessary to establish specific physiological roles for adenosine, 5'-AMP, cyclic A M P , and its
Psychopharmacology54 (1977) dibutyryl derivatives in the regulation of behavior as suggested by our observations and those of other investigators. In one such approach, a positive correlation of S L M A in rats with norepinephrine-induced accumulation of cyclic A M P in midbrain striatum was observed (Skolnick and Daly, 1974). However, a negative correlation was obtained between spontaneous behavioral activity and the responses of the norepinephrine-sensitive cyclic AMP-generating system in the cerebral cortex, a brain region associated with inhibitory pathways (Siggins et al., 1971). Moreover, it would be of interest to extend these studies to comparisons of behavioral and neurochemical alterations induced by single doses of both dibutyryl cyclic A M P and morphine.
REFERENCES Asakawa, T., Yoshida, H.: Studies on the functional role of adenosine Y,5'-monophosphate, histamine, and prostaglandin E1 in the central nervous system. Jp. J. Pharmacol. 21, 569-583 (1971) Beer, B., Chasin, M., Clody, D. E., Vogel, J. R., Horovitz, Z. P.: Cyclic adenosine monophosphate phosphodiesterase in brain: effect on anxiety. Science 176, 428-430 (1972) Breckenridge, B. McL., Lisk, R. D. : Cyclic adenylate and hypothalamic regulatory functions. Proc. Soc. Exp. Biol. Med. 131, 934-935 (1969) Carr, C. J. : An appraisal of animal drug screening techniques used in psychopharmacology.J. New Drugs 3, J 35-146 (1963) Daly, J.W.: Cyclic adenosine 3',5'-monophosphate role in the physiology and pharmacology of the central nervous system. Biochem. Pharmacol. 24, 159-164 (1975) Daly, J. W. : The nature of receptors regulating the formation of cyclicAMP in brain tissue. Life Sci. 18, 1349-1358 (1976) Gessa, G. L., Krishna, G., Forn, J., Tagliamonte, A., Brodie, B. B. : Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain. In: Role of cyclic AMP in cell function, P. Greengard, E. Costa, eds., pp. 371 -381. New York: Raven Press 1971 Henion, W. F., Sutherland, E. W., Posternak, T.: Effects of derivatives of adenosine 3',5'-phosphate on liver slices and intact animals. Biochem. Biophys. Acta 148, 106-- 119 (1967) Herman, Z. S. : Behavioral effects of dibutyryl cyclic 3',5'-AMP, noradrenaline and cyclic3',5'-AMP in rats. Neuropharmacology 12, 705--709 (1973) Ho, I. K., Loh, H. H., Way, E. L. : Effects of cyclic 3',5'-adenosine monophosphate on morphine tolerance and physical dependence. J. Pharmacol. Exp. Ther. 185, 347-357 (1973) Huidobro, F., Huidobro-Toro, J. P., Way, E. L. : Studies on tolerance development to single doses of morphine in mice. J. Pharmacol. Exp. Ther. 198, 318-329 (1976) Kakiuchi, S., Rall, T. W. : The influence of chemical agents on the accumulation of adenosine 3',5'-phosphate in slices of rabbit cerebellum. Mol. Pharmacol. 4, 367-378 (1968) Kornetsky, C., Bain, G. : Morphine: Single-dose tolerance. Science 162, 1011- 1012 (1968) LeBlanc, A. E., Kalant, H., Gibbins, R. J. : Acute tolerance to ethanol in the rat. Psychopharmacologia (Berl.) 41, 43-46 (1975) Mah, H. D., Daly, J. W. : Adenosine-dependent formation of cyclic AMP in brain slices. Pharmacol. Res. Commun. 8, 65 -- 79 (1976)
M. Weiner and J. W. Olson : Effects of Cyclic Nucleotides Ono, H., Taira, N., Hashimoto, K.: Behavioral and vegetative effects of dibutyryl cyclic AMP on conscious dogs. Neuropharmacology 15, 571-575 (1976) Phillis, J. W., Kostopoulos, G. K. : Adenosine as a putative transmitter in the cerebral cortex. Studies with potentiators and antagonists. Life Sci. 17, 1085 1094 (1975) Rall, T. W. : On the importance of cyclic AMP in neurobiology: an essay. Metabolism 24, 245 - 2 4 7 (1975) Sattin, A., Rail, T. W. : The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',5'-monophosphate content of guinea pig cortical slices. Mol. Pharmacol. 6, 13--23 (t970) Schultz, J., Daly, J. W.: Cyclic adenosine 3',5'-monophosphate in guinea pig cerebral cortical slices. J. Biol. Chem. 248, 860-866 (1973) Siggins, G. R., Oliver, A. P., Hoffer, B. J., Bloom, F. E.: Cyclic adenosine monophosphate and norepinephrine: effects on transmembrane properties of cerebellar purkinje cells. Science 171, 192-194 (1971)
NOTE ADDED IN PROOF Dr. Myron Weiner's present address: Department of Pharmaco-
logy and Toxicology, School of Pharmacy, University of Maryland, 636 W. Lombard St., Baltimore, MD 21201, U.S.A.
65 Skolnick, P., Daly, J. W.: Norepinephrine-sensitive adenylate cyclase in rat brain: relation to behavior and tyrosine hydroxylase. Science 184, 175-177 (I974) Tachizawa, H., Saito, T., Akimoto, T.: Metabolism of N6-O2'dibutyryl-3',5'-cyclic-AMP (DBcAMP). Jp. J. Pharmacol. 24, Suppl. 51 (1974) Uzunov, P., Weiss, B. : Effects of phenothiazine tranquilizers on the cyclic 3',5'-adenosine monophosphate system of rat brain. Neuropharmacology 10, 697-708 (1971) Weiner, M., Olson, J. W. : The behavioral effects of dibutyryl cyclic AMP in mice. Life Sci. 12, 345- 356 (1973) Weiss, B., Costa, E. : Adenyl cyclase activity in rat pineal gland: effects of chronic denervation and norepinephrine. Science 156, 1750-1752 (1967)
Received November 16, 1976
Psychopharmacology 54, 6t - 6 5 (t 977)
9 by Springer-Verlag1977
Single-Dose Tolerance to the Behavioral Effects of Dibutyryl Cyclic AMP in Mice MYRON WEINER and JACK W. OLSON PharmacodynamicsSection,Universityof SouthernCalifornia, Schoolof Pharmacy,Los Angeles,California90033, U.S.A.
Abstract. The behavioral effects of varying doses of intraperitoneally administered dibutyryl cyclic AMP, cyclic AMP, adenosine, 5'-AMP, and butyric acid were studied in male ICR mice. Behavioral parameters 25 min following treatment included measurement of spontaneous locomotor activity (SLMA) and rotarod performance, the latter providing an indication of neuromuscular coordination. Dibutyryl cyclic AMP produced a dose-related inhibition of SLMA with the largest dose, 75 mg/kg, decreasing activity by 89%. Adenosine and 5'-AMP produced maximal inhibition of approximately 5 0 - 80 % of SLMA at doses ranging from 75-250 mg/kg, while cyclic AMP decreased SLMA by 58 % at only the highest dose, 250 mg/kg. Butyric acid failed to produce alterations in SLMA at doses ranging from 25-250 mg/kg. No compound altered neuromuscular coordination. Single-dose tolerance to the inhibitory effect of dibutyryl cyclic AMP on SLMA developed within 3 h and lasted at least 7 days. Adenosine failed to produce tolerance while cyclic AMP and 5'-AMP exhibited only a slightly reduced effect following a second injection at intervals of 4 and 24 h. These results suggest that exogenous administration of dibutyryl cyclic AMP and its metabolites exert centrally mediated behavioral effects with selective development of single-dose tolerance to the dibutyryl derivative. Key words: Spontaneous locomotor activity - Singledose tolerance -- Dibutyryl cyclic AMP - Cyclic AMP - 5'-AMP - Adenosine - Mice
Numerous reports and extensive reviews (Daly, 1975; Rall, 1975) have recently appeared on the relationship of cyclic adenosine 3 ',5'-monophosphate (cyclic AMP) to many aspects of brain function. Different types of
brain preparations (e.g., cell-fl'ee systems, cultured cells, brain slices, and intact brain) have provided evidence for the involvement of cyclic AMP in the pharmacological effects of psychoactive drugs (Uzunov and Weiss, 1971) or in the physiological control of postsynaptic events (Weiss and Costa, 1967). Although approaches to the complex problems of CNS regulation have generally included in vitro analyses, some in vivo studies have attempted to correlate cyclic AMP to spontaneous and drugelicited behavioral parameters. While various aspects of behavior have been investigated, spontaneous locomotor activity (SLMA) has been shown either to increase or to decrease following administration of the dibutyryl derivative of cyclic AMP. Drowsiness in mice was induced by intraperitoneal injection of dibutyryl cyclic AMP, 500 mg/kg (Henion et al., 1967). Intravenous administration of the dibutyryl derivative resulted in a dose-dependent (10-100 mg/kg) hypoactivity in dogs while similar doses of cyclic AMP, sodium butyrate, and butyric acid lacked effectiveness (Ono et al., 1976). Sleep and catatonia were produced in cats by injection of dibutyryl cyclic AMP into the cerebellum and mesencephalic reticular formation, respectively (Gessa et al., 1971). In addition, Beer et al. (1972) have observed that dibutyryl cyclic AMP (100-400 mg/kg, i.p.) had anxiety-reducing properties when a behavioral conflict test was utilized in rats 30 min following injection. In contrast, intracerebrat administration of dibutyryl cyclic AMP increased SLMA in immature rats (Breckenridge and Lisk, 1959), cats (Gessa et al., 1971), and chickens (Asakawa and Yoshida, 1971). Herman (1973) noted behavioral excitation in rats following intraventricular administration of dibutyryl cyclic AMP; however, a stuporous state and period of immobility was noticed between periods of excitation. Thus, these qualitatively different responses apparently depend on the species and the route of administration.
62 In a previous study (Weiner and Olson, 1973), we showed that i.p. administration of dibutyryl cyclic A M P (25 mg/kg) to male Institute of Cancer Research (ICR) mice decreased SLMA and exploratory behavior approximately 40 ~ without affecting motor coordination. The threshold dose for decreased SLMA was 10 mg/kg with near maximal response occurring with a dose of 75 mg/kg. This effect was almost immediate in onset and persisted for approximately 90 min. The failure of a second injection, 24 h later, of dibutyryl cyclic A M P to produce behavioral changes prompted us to characterize further the apparent tachyphylactic response to the cyclic nucleotide in the present investigation. In addition, we decided to study the effects of single and multiple injections of cyclic AMP, Y-AMP, adenosine, and butyric acid on SLMA and motor coordination.
MATERIALS AND METHODS Male ICR albino mice (Simonsen Labs, Gilman, California) weighing 20-25 g were used in all experiments. They were maintained in a constant temperature room (22 + 1~ with Purina Laboratory chow and water continuously available for at least 3 days beforeuse. All compounds, purchased from SigmaChemical Company, were dissolved in distilled water to prepare solutions so that 0.01 ml/g body weight was injected. Control mice received the same volume of saline. Mice were tested 25 rain after a single injection of the test compound. The behavioral activity of mice receivingmultiple injectionswas observed25 min after the last dose was administered. This time period between treatment and testing was chosen on the basis of maximum behavioral effectsobservedin a previous study (Weiner and Olson, 1973). SLMA, used to provide a measurement of nondirected movement, was measured for 15 min in groups of 3 mice in an Electronic Motility Meter (Fc 40, Motron Products, Stockholm, Sweden), a circular photocell activity cage previously described (Weiner and Olson, 1973). Coordination of movement was determined as performance on a rotating rod (rotarod). The number of falls in 5 min for mice placed on the rotarod was recorded. Mice were considered uncoordinated if they fell more than twice in the experiment. All determinations were made in the afternoon hours. The statisticalsignificanceof SLMAwas determinedby the Student's t-test and that of rotarod performanceby the Chi Square test.
RESULTS SLMAs of mice receiving varied doses of dibutyryl cyclic A M P or its metabolites are shown in Figure 1. The dibutyryl derivative produced a dose-related decrease in SLMA with 25 mg/kg reducing activity 55 ~ , while a 75 mg/kg dose almost totally eliminated activity (i.e., 89 ~ inhibition). Cyclic A M P failed to alter activity at doses similar to its dibutyryl derivative. However, a similar degree of inhibition was induced by cyclic A M P at 250 mg/kg, a dose tenfold greater than that of dibutyryl cyclic AMP. Butyric acid was
Psychopharmacology54 (1977) without significant effect at doses up to 250 mg/kg. Although neither adenosine nor 5'-AMP produced statistically significant changes in SLMA at 25 mg/kg, a pronounced inhibition but no dose-response effect was observed in the dosage range 7 5 - 250 mg/kg. No statistically significant differences were observed between treatment groups and control mice on rotarod performance at the same dosage levels used above, although several mice receiving 5'-AMP at higher doses were deemed uncoordinated. The effect of a second injection of dibutyryl cyclic A M P on SLMA was determined using the same dose, 25 mg/kg, which initially produced a significant but submaximal decrease in activity. In contrast to the effects of a single injection of dibutyryl cyclic AMP, the same dose of a second injection failed to produce a change in activity (Fig. 2). This tachyphylactic response was first observed when 3 h separated injections and persisted for at least 7 days. Other compounds that inhibited SLMA (Fig. 1) were tested for the phenomenon of tolerance to a second injection at intervals of 4 and 24 h. No tolerance was observed following submaximal doses of adenosine (75 mg/kg). Although not statistically significant, the effect of a second injection of cyclic A M P (150 mg/kg) and 5'-AMP (75 mg/kg) was less than that of the initial administration by 25 ~ and 20 ~ respectively.
DISCUSSION These results confirm previous observations that i.p. administration ofdibutyryl cyclic A M P to mice evoked behavioral changes characterized by drowsiness (Henion et al., 1967) and a decreased SLMA (Weiner and Olson, 1973), and extend these results to cyclic AMP, adenosine, and 5'-AMP. The parent cyclic nucleotide was the least potent compound tested, while the dibutyryl derivative was the most potent. In addition to the greater activity of the dibutyryl or monobutyryl moiety, the difference in potency may be explained by dibutyryl cyclic AMP's increased ability to penetrate cellular membranes or its resistance to and inhibition of phosphodiesterase, the enzyme responsible for the catalysis of cyclic A M P to 5'-AMP. Only butyric acid was completely without effect even at a dose as high as 250 mg/kg, an order of magnitude greater on a molar basis than the dibutyryl cyclic nucleotide. Thus, the possibility that butyrate deacylated from dibutyryl cyclic A M P was responsible for the latter compound's behavioral effects was essentially ruled out, a conclusion supported by Ono et al. (1976). Although no definitive explanation can be provided for the decrease in SLMA while coordination remained
M. Weiner and J. W. Olson: Effects of Cyclic Nucleotides
63
2000
1750
1500
-~ c
u
1250
--
1000
-J
750
500 250
o
Dose(mg/k9)
0
25
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75
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25
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Treatment
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cyclic A M P
25
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125250 acid
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125250
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75
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Fig. 1. SLMA of mice treated with varying doses of dibutyryl cyclic AMP, cyclic AMP, butyric acid, adenosine, and Y-AMP, administered i.p. 25 min before testing. Columns represent mean + SE of 3 - 7 groups of 3 mice. Percent change from saline control (0 dose) shown within columns. Significance expressed as *, P < 0.05
I
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Fig.2. Time-response relationship for single-dose tolerance to dibutyryl cyclic AMP. SLMA was tested 25 rain following the second i.p. injection of saline or nucleotide. Each result is the mean • SE of groups of 3 mice, the total numbers of groups being shown in the column, and is expressed as a percentage of SLMA in in the corresponding saline controls. Significance expressed as *, P < 0.05
the same in these experiments, it seems reasonable to speculate, as have others (Beer et al., 1972; Ho et al., 1973; Ono et al., 1976), that a central effect may be involved in behavioral alterations produced by exogenous cyclic adenine nucleotides. In this regard, mice treated with antipsychotic agents (e.g., chlorpromazine) are also inactive and tranquil in an unstimulated environment but are capable of responding to stimulated activity as demonstrated by the ability to maintain balance on the rotarod (Carr, 1963). Since the i.p. route was utilized, the compounds tested in these experiments most likely passed the blood-brain barrier to produce changes in SLMA. However, it has generally been considered that exogenous cyclic nucleotides penetrate cell membranes with great difficulty. Recent evidence suggests that the dibutyryl cyclic nucleotide has access to the brain, although the fraction that permeates is very small (Tachizawa et al., 1974). Indeed, in the pharmacological doses (25 to 75 mg/kg) that were administered in our experiments, only a fraction of the dose would be necessary to enter the brain to approach physiological brain concentrations of approximately 10 .7 M. The hypoactivity produced by adenosine and 5'-AMP may result from increases in intracellular cyclic AMP concentration. Regardless of the species
64 or brain region tested, small amounts of adenosine have been shown consistently to cause accumulation of cyclic A M P in brain slices (Sattin and Rall, 1970; M a h and Daly, 1976), presumably as a result of activation of external receptors linked to adenylate cyclase (Daly, 1976). Although usually considered an inactive metabolite of cyclic A M P , 5 ' - A M P has been reported to increase cyclic A M P levels in brain slices following prior extracellular hydrolyses to adenosine (Sattin and Rall, 1970; M a h and Daly, 1976). Moreover, following central injection, both adenosine and 5'-AMP have potent depressant actions on neurons in the cerebral cortex, cerebellar Purkinje cells, and corticospinal cells (Phillis and Kostopoulos, 1975). While it is attractive to speculate that a central effect is involved in the differential effects on S L M A and rotarod performance, the possibility that the reductions in S L M A are the consequence of peripherally mediated, nonspecific effects exists and needs to be explored further. In addition, evidence demonstrating that these compounds enter the brain after i.p. administration needs to be provided. A puzzling aspect of this study concerns the refractoriness to the inhibition of the S L M A of mice following readministration of dibutyryl cyclic AMP. This effect was observed within 3 h of the first injection and lasted at least 7 days, the longest time utilized. The single-dose tolerance m a y represent an adaptive event within the CNS rather than a disposition tolerance since the effect lasts longer than the nucleotide would be expected to be present in the brain. Single-dose tolerance has also been observed with other compounds, e.g., ethanol (LeBlanc et al., 1975) and morphine (Kornetsky and Bain, 1968). Moreover, intravenous cyclic A M P (10 mg/kg) enhanced tolerance development in I C R mice after morphine pellet implantation (Ho et al., 1973) and after a single dose of morphine (Huidobro et al., 1976), although the latter effect was not statistically significant at the only morphine dose used. Refractoriness of adenylate cyclase systems has also been observed following initial stimulation by histamine, norepinephrine, or a histamine-norepinephrine combination, washing, and attempted restimulation in rabbit cerebellar slices (Kakiuchi and Rall, 1968) and guinea pig cortical slices (Schultz and Daly, 1973). Refractoriness to these amines was not observed in the presence of adenosine. In our studies adenosine failed to produce single-dose tolerance while second doses of cyclic A M P and 5'-AMP produced nonsignificant decreases in activity from the first dose. Much more sophisticated approaches to the correlation of behavioral aspects and neurochemical parameters are necessary to establish specific physiological roles for adenosine, 5'-AMP, cyclic A M P , and its
Psychopharmacology54 (1977) dibutyryl derivatives in the regulation of behavior as suggested by our observations and those of other investigators. In one such approach, a positive correlation of S L M A in rats with norepinephrine-induced accumulation of cyclic A M P in midbrain striatum was observed (Skolnick and Daly, 1974). However, a negative correlation was obtained between spontaneous behavioral activity and the responses of the norepinephrine-sensitive cyclic AMP-generating system in the cerebral cortex, a brain region associated with inhibitory pathways (Siggins et al., 1971). Moreover, it would be of interest to extend these studies to comparisons of behavioral and neurochemical alterations induced by single doses of both dibutyryl cyclic A M P and morphine.
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NOTE ADDED IN PROOF Dr. Myron Weiner's present address: Department of Pharmaco-
logy and Toxicology, School of Pharmacy, University of Maryland, 636 W. Lombard St., Baltimore, MD 21201, U.S.A.
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Received November 16, 1976
