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Depressant effect of forskolin on spontaneous locomotor activity in mice
Gen. Pharmac. Vol. 16, No. 5, pp. 521-524, 1985 Printed in Great Britain. All rights reserved
0306-3623/85 $3.00+ 0.00 Copyright © 1985 Pergamon Press Ltd
D E P R E S S A N T E F F E C T OF F O R S K O L I N ON S P O N T A N E O U S L O C O M O T O R ACTIVITY IN MICE ROBIN A. BARRACO1, JOHN W. PHILLIS1 and HARVEY J. ALTMAN2 IDepartment of Physiology, and 2Department of Psychiatry and the Lafayette Clinic, Wayne State University, School of Medicine 540 E. Canfield, Detroit, MI 48201, U.S.A. (Received 22 January 1985)
Abstract--1. Forskolin, an activator of adenylate cyclase, depressed spontaneous locomotor activity when injected intracerebroventricularly into mice in doses of 10 and 100pg. 2. The findings suggest that increases in brain cAMP levels may be associated with a reduction in excitability.
INTRODUCTION The concept of extracellular adenosine receptors has been generally accepted for CNS tissues where adenosine has been shown to exert potent inhibitory actions on neuronal firing rates (Kostopoulos and Phillis, 1977), on synaptic transmission (Okada and Kuroda, 1980) and on release of neurotransmitters (Fredholm and Hedqvist, 1980; Hollins and Stone, 1980). A well established effect of adenosine at the biochemical level is its capacity to modulate adenylate cyclase activity via more than one membraneassociated receptor. A high affinity receptor (A~) was inhibitory to adenylate cyclase while a lower affinity receptor (A0 was stimulatory (for review, see Daly, 1982). At the behavioral level, adenosine and its analogs exert marked depressant effects on behavior, including pronounced sedative (Barraco et al., 1983; Katims et al., 1983), hypnogenic (Radulovacki et al., 1982) and anticonvulsant actions (Dunwiddie and Worth, 1982). These behavioral effects were blocked by methylxanthines such as caffeine and theophylline. The methylxanthines have also been shown to block the modulation of adenylate cyclase (Daly, 1979), the depressant effects on neuronal firing rates (Phillis and Wu, 1981) and the inhibitory effects of adenosine on the release of neurotransmitters (Stone et al., 1981). The competitive blockade of methylxanthines in these studies, as well as their displacement of [3H]adenosine analogs in binding studies (Snyder et al., 1981), suggest that these effects are mediated by the PI (R or A) adenosine receptor (Burnstock, 1978; Daly, 1982). Nevertheless, the question remains whether or not the receptor mediated influences of adenosine and its analogs on adenylate cyclase has direct behavioral consequences. For example, it has been suggested that the inhibitory effect of adenosine on transmitter release in the peripheral nervous system and hippocampal slices is unlikely to be mediated via cyclic A M P since this would presume that transmitter release is critically dependent upon cyclic A M P (Fredholm, 1982). Send correspondence and reprint requests to John W. Phillis. 521
Even less is known about the coupling of the adenosine receptor and adenylate cyclase to other types of effectors (e.g. calcium channels). Furthermore, efforts to establish specific physiological roles for cyclic nucleotides in the regulation of spontaneous behaviors following direct injections into the whole animal have yielded conflicting findings, possibly since exogenous cyclic nucleotides penetrate cell membranes with great difficulty. Direct intracerebral or intraventricular injections of dibutyryl cyclic A M P (dBcAMP) and, less effectively, of cyclic A M P (cAMP) have elicited an increase in spontaneous motor activity in rats (Herman, 1973), sleep and catatonia in cats (Gessa et al., 1971), or a decrease in activity and hypothermia in rats (Wachtel, 1982), whereas intraperitoneal injections produced locomotor depression in mice (Weiner and Olson, 1973) and sedation in rats (Beer et al., 1972); intravenous administration of dBcAMP resulted in dosedependent hypoactivity in dogs (Ono et al., 1976). Apparently these qualitatively different responses vary depending on the species and the route of administration; it has also been pointed out that cAMP, dBcAMP and other analogues may exert actions not related to the cyclic phosphate moiety (Breckenridge, 1970). On the other hand, manipulations of in vivo cAMP levels in the brain have produced more characteristic behavioral symptomatology. For example, parenteral administration of phosphodiesterase (PDE) inhibitors such as RO 20-1724 and rolipram, which have been shown to possess a high selectivity towards cAMP rather than cGMP PDE, produce hypoactivity and hypothermia in rats and mice and these behavioral effects are mimicked by systemically or intracerebrally administered dBcAMP, but not dBcGMP (Wachtel, 1982, 1983; Dascombe et al., 1980; Dascombe and Parkes, 1981). In fact, the order of potency and effective dose range of a series of PDE inhibitors to induce behavioral alterations corresponded with the efficacy of the drugs to elevate brain cAMP in vivo (Wachtel, 1983). Similar behavioral effects were seen with other PDE inhibitors, suggesting that depression of spontaneous locomotor activity is a potential in vivo correlate of enhanced availability of brain cAMP (Wachtel, 1982, 1983).
ROBIN A. BARRACO et al.
522
The purpose of the following study was to examine the behavioral consequences of elevating brain c A M P levels via stimulation of adenylate cyclase. Forskolin, a diterpene which reversibly activates adenylate cyclase in intact tissues, including the brain (for review, see Seamon and Daly, 1981) was administered into the lateral cerebral ventricle of mice and the effects of spontaneous locomotor activity were assessed. Forskolin has recently been demonstrated to prevent the seizures induced in mice by pentylenetetrazol, whilst simultaneously elevating brain c A M P levels (Sano et al., 1984). MATERIALS AND METHODS
Adult male Swiss Webster mice (ICR strain, Harlan Industries) approximately l0 weeks old (30-40g) were implanted with permanent indwelling stainless-steel guide cannulas for injection into the lateral ventricle of the brain as previously described (Barraco et al., 1983). They were housed 6 per cage in 18 × 24 x 15 cm polypropylene cages, on a 12 hr/12 hr light cycle (lights on 07:00). Animals were maintained on ad libitum food and water throughout the experiment. Mice were permitted to recover from surgery and adapt to their housing for a minimum of l0 days. Drugs or control solutions administered intracerebroventricularly (i.c.v.t.) were injected slowly in a 5/~1 volume over a 30 sec period during which time the animals were able to freely move around the bench top. Six mice were placed in each of six testing chambers 08 × 24 × 15 cm) and were monitored simultaneously. Locomotor activity was measured with a Stoelting 31410 Modular Electronic Activity Monitor with 6 sensors. The sensors were placed in a dark compartment (2 x 3 × 2.4 m high) equipped with a fan for ventilation and noise attenuation. All sensors were adjusted to equal sensitivity with a pendulum. Each sensor was activated by a uniform, low power, radio-frequency field extending several inches above the surface. Sensitivity of the sensors was adjusted to accumulate counts for gross movement. Immediately following injections, there was a l0 min pretest period, prior to the 30 min test period for locomotor activity. Locomotor activity scores were analyzed by the Kruskal-Wallis ranked one-way analysis of variance and Mann-Whitney U post hoc one-tailed paired comparisons. Forskolin (Tfl-acetoxy-8, 13-epoxy-lct,6fl,9~t-trihydroxylabd-14-en-ll-one) (Calbiochem) was dissolved in dimethylsulfoxide (DMSO) (Sigma) and injected i.c.v.t. DMSO was used for the control injections.
adenylate cyclase in intact tissues including the brain, it is possible that the depression of locomotor activity observed in this study might reflect the enhanced availability of c A M P in the brain. In this context, it is of interest that forskolin can also prevent pentylenetetrazol-induced seizures in mice (Sano et aL, 1984). Our findings therefore are consistent with the behavioral effects of c A M P elevations induced by P D E inhibitors referred to in the Introduction. Furthermore, the effect of forskolin on adenylate cyclase appears to be specific since it has not been reported to affect the activity of a variety of enzymes examined including P D E , N a + / K + ATPase, cAMP-dependent protein kinase and guanylate cyclase (for review, see Seamon and Daly, 1981). Moreover, although forskolin activates adenylate cyclases normally stimulated by different agonists, its effect is not dependent on receptor stimulation, but apparently involves a direct activation of the enzyme. In fact, it has been suggested that one potential criterion for assessing the possible role of c A M P in a physiological response is whether forskolin is able to elicit the same response and, collaterally, potentiate the response of a given agonist on a specific adenylate cyclase-linked receptor. However, the question of whether or not the depressant effects of adenosine and its analogs on behavior are mediated by in vivo alterations in c A M P levels in the nervous system is still not resolved. It is conceivable both mechanisms could operate in parallel with adenosine depressing neuronal activity via presynaptic inhibition of neurotransmitter release (for references, see Fredholm and Hedqvist, 1980) whilst c A M P mediates inhibitory neurotransmission by hyperpolarizing neurons through phosphorylation of the postsynaptic membrane (Greengard, 1976). Nevertheless, a number of studies have shown a remarkable correlation between depression of excitatory transmission in the hippocampus and olfactory cortex and the accumulation of c A M P by adenosine and its derivatives (Kuroda et al., 1976; Okada and Saito, 1979; Fredholm et al., 1982; Motley and I10 100
RESULTS The effect of i.c.v.t injections of forskolin on spontaneous locomotor activity (expressed as percent of D M S O controls) is shown in Fig. 1. An overall analysis of variance indicated that forskolin produced a dose-related decrease in locomotor activity (H = 17.28, d.f. = 3; P <0.001). Significance levels determined by post hoc comparison for individual doses are indicated by asterisks in Fig. 1. Forskolin produced significant reductions in locomotor activity at the 10/~g (P < 0.05) and 100/lg (P < 0.01) doses when compared with D M S O controls. DISCUSSION These results show that i.c.v.t, injections of forskolin produced a dose-related depression of spontaneous locomotor activity. Since it is known that forskolin causes a rapid, reversible, and dosedependent increase in c A M P levels via stimulation of
,,¢
"6
90 80 7'0
.3 60 "6 50 (_)
40 "6 46 30 E #_ 20 10
].0
10 100 F0rskolin(pg/mouse) Fig. 1. Effect ofi.c.v.t injections of forskolin on spontaneous locomotor activity expressed as percent of controls. Values represent the mean + SEM. Overall analysis of variance showed dose-related decreases in locomotor activity (P <0.001). Significance levels determined by post hoc comparisons with controls are indicated (*P < 0.05; **P < 0.01).
Forskolin depresses motor activity Collins, 1983). The fact that these effects are inhibited by methylxanthines, potentiated by adenosine uptake inhibitors, and the relative activity of adenosine analogs in these studies correlates well with their potency in binding studies, suggests that the extracellular adenosine receptors mediating depression of neuronal activity and accumulation of c A M P have similar characteristics. It is plausible these receptorlinked phenomena correspond to the A2 receptor subtype since (1) certain findings in hippocampal slices and striatal membrane preparations raise the possibility that the biological effects of A1 receptor agonists in the CNS may be related to inhibition of c A M P accumulation (Fredholm et al., 1983; Wojcik and Neff, 1983), (2) N E C A , a selective A2 agonist (Daly, 1982), was by far the most potent of a series of analogs in depressing spontaneous locomotor activity in mice (Barraco et al., 1983) and (3) N E C A was also the most potent in a series of analogs for depressing the activity of cerebral cortical neurons (Phillis, 1982). Finally, although it is possible that many of the effects of adenosine are not mediated via adenylate cyclase, the results of the present study do not contradict the hypothesis that the adenosine receptor mediating behavioral depression and accumulation of c A M P may be the A 2 receptor. Acknowledgements--The authors gratefully acknowledge
the technical skills of Mr George Smoot. This research was supported in part by Grant RR-08167-OX from the Division of Research Resources, NIH. REFERENCES Barraco R. A., Coffin V. L., Altman H. J. and Phillis J. W. (1983) Central effects of adenosine analogs on locomotor activity in mice and antagonism of caffeine. Brain Res. 272, 392-395. Beer B., Chasin M., Clody D. E., Vogel J. R. and Horovitz Z. P. (1972) Cyclic adenosine monophosphate phosphodiesterase in brain: effect on anxiety. Science 176, 428-430. Breckenridge B. McL. (1970) Cyclic AMP and drug action. A. Rev. Pharmac. 10, 19-34. Burnstock G. (1978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Edited by Bolis L. and Straub R. W.) pp. 107-118. Raven Press, New York. Daly J. W. (1979) Adenosine and cyclic adenosine monophosphate generating systems in brain tissue. In Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides (Edited by Baer H. P. and Drummond
G. I.) pp. 229-241. Raven Press, New York. Daly J. W. (1982) Adenosine receptors: Targets for future drugs. J. reed. Chem. 25, 197-207. Dascombe M. J. and Parkes J. (1981) Effects of N6-2'-O-dibutyryl 3',5'-monophosphate on body temperature in the restrained rat. Br. J. Pharmac. 72, 565-566. Dascombe M. J., Milton A. S., Nyemitei-Addo I. and Pertwee R. G. (1980) Thermoregulatory effects of N6-2'-O-dibutyryl adenosine 3',5'-monophosphate in the restrained mouse. Br. J. Pharmac. 70, 453-459. Dunwiddie T. W. and Worth T. (1982) Sedative and anticonvulsive effects of adenosine analogs in mouse and rat. J. Pharmac. exp. Ther. 220, 70-76. Fredholm B. B. (1982) Adenosine receptors. Med. Biol. 60, 289-293. Fredholm B. B. and Hedqvist P. (1980) Modulation of
523
neurotransmission by purine nucleotides and nucleosides. Biochem. Pharmac. 29, 1635-1643.
Fredholm B. B., Jonzon B. and Lindstrom K. (1983) Adenosine receptor mediated increases and decreases in cyclic AMP in hippocampal slices treated with forskolin. Acta physiol, scand. 117, 461-463. Fredholm B. B., Jonzon B., Lindgren E. and Lindstrom K. (1982) Adenosine receptors mediating cyclic AMP production in the rat hippocampus. J. Neurochem. 39, 165-175. Gessa G. L., Krishna G., Forn J., Tagliamonte A. and Brodie B. B. (1971) Behavioral and vegetative effects produced by dibutyryl cyclic AMP injected into different areas of the brain. In Role of Cyclic A M P in Cell Function (Edited by Greengard P. and Costa E.) pp. 371-381. Raven Press, New York. Greengard P. (1976) Possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters. Nature (Lond.) 260, 101-108. Herman Z. S. (1973) Behavioral effects of dibutyryl cyclic 3',5'-AMP, noradrenaline and cyclic Y,5'-AMP in rats. Neuropharmacology 12, 705-709. Hollins C. and Stone T. W. (1980) Adenosine inhibition of 7-aminobutyric acid release from slices of rat cerebral cortex. Br. J. Pharmac. 69, 107-112. Katims J. J., Annau Z. and Snyder S. H. (1983) Interactions in the behavioral effects of methylxanthines and adenosine derivatives. J. Pharmac. exp. Ther. 227, 167-173. Kostopoulos G. K. and Phillis J. W. (1977) Purinergic depression of neurons in different areas of the rat brain. Exp. Neurol. 55, 719-724. Kuroda Y., Saito M. and Kobayashi K. (1976) Concomitant changes in cyclic AMP level and postsynaptic potentials by olfactory cortex slices induced by adenosine derivatives. Brain Res. 109, 196-201. Motley S. J. and Collins G. G. S. (1983) Endogenous adenosine inhibits excitatory transmission in the rat olfactory cortex slice. Neuropharmacology 22, 1081-1086. Okada Y. and Kuroda Y. (1980) Inhibitory action of adenosine and adenosine analogs on neurotransmission in the olfactory cortex slice of guinea pig--structure activity relationships. Eur. J. Pharmac. 61, 137-146. Okada Y. and Saito M. (1979) Inhibitory action of adenosine, 5-HT (serotonin) and GABA (7-aminobutyric acid) on the postsynaptic potential (PSP) of slices from olfactory and superior colliculus in correlation to the level of cyclic AMP. Brain Res. 160, 368-371. Ono H., Taira N. and Hashimoto K. (1976) Behavioral and vegetative effects of dibutyryl cyclic AMP on conscious dogs. Neuropharmacology 15, 571-575. Phillis J. W. (1982) Evidence for an A2-1ike adenosine receptor on cerebral cortical neurons. J. Pharm. Pharmac. 34, 453-454. Phillis J. W. and Wu P. H. (1981) The role of adenosine and its nucleotides in central synaptic transmission. Prog. Neurobiol. 16, 187-239. Radulovacki M., Miletich R. S. and Green R. D. (1982) N6(L-phenylisopropyl) adenosine (L-PIA) increases slowwave sleep ($2) and decreases wakefulness in rats. Brain Res. 246, 178-180. Sano M., Seto-Ohshima A. and Mizutani A. (1984) Forskolin suppresses seizures induced by pentylenetetrazol in mice. Experientia 40, 1270-1271. Seamon K. B. and Daly J. W. (1981) Forskolin: a unique diterpine activator of cyclic AMP-generating systems. J. cyclic Nucleotide Res. 7, 201-224. Snyder S. H., Katims J. J., Annau Z., Bruns R. F. and Daly J. W. (1981) Adenosine receptors and behavioral actions of methylxanthines. Proc. natn. Acad. Sci. U.S.A. 78, 3260-6264. Stone T. W., Hollins C. and Lloyd H. (1981) Methylxanthines modulate adenosine release from slices of cerebral cortex. Brain Res. 207, 421-431.
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Wachtel H. (1982) Characteristic behavioral alterations in rats induced by rolipram and other selective adenosine cyclic 3',5' monophosphate phosphodiesterase inhibitors. Psychopharmacology 77, 309-316. Wachtel H. (1983) Species differences in behavioral effects of rolipram and other adenosine cyclic 3',5'-monophosphate phosphodiesterase inhibitors. J. Neural Transm. 56, 139-152. Wachtel H. (1983) Potential antidepressant activity of ro-
lipram and other selective cyclic 3',5'-monophosphate phosphodiesterase inhibitors. Neuropharmacology 22, 267-272. Weiner N. and Olson J. W. (1973) Single dose tolerance to the behavioral effects of dibutyryl cyclic AMP in mice. Life Sci. 12, 345-356. Wojcik W. J. and Neff N. H. (1983) Differential location of adenosine A~ and A 2 receptors in striatum. Neurosci. Lett. 41, 55-60.
0306-3623/85 $3.00+ 0.00 Copyright © 1985 Pergamon Press Ltd
D E P R E S S A N T E F F E C T OF F O R S K O L I N ON S P O N T A N E O U S L O C O M O T O R ACTIVITY IN MICE ROBIN A. BARRACO1, JOHN W. PHILLIS1 and HARVEY J. ALTMAN2 IDepartment of Physiology, and 2Department of Psychiatry and the Lafayette Clinic, Wayne State University, School of Medicine 540 E. Canfield, Detroit, MI 48201, U.S.A. (Received 22 January 1985)
Abstract--1. Forskolin, an activator of adenylate cyclase, depressed spontaneous locomotor activity when injected intracerebroventricularly into mice in doses of 10 and 100pg. 2. The findings suggest that increases in brain cAMP levels may be associated with a reduction in excitability.
INTRODUCTION The concept of extracellular adenosine receptors has been generally accepted for CNS tissues where adenosine has been shown to exert potent inhibitory actions on neuronal firing rates (Kostopoulos and Phillis, 1977), on synaptic transmission (Okada and Kuroda, 1980) and on release of neurotransmitters (Fredholm and Hedqvist, 1980; Hollins and Stone, 1980). A well established effect of adenosine at the biochemical level is its capacity to modulate adenylate cyclase activity via more than one membraneassociated receptor. A high affinity receptor (A~) was inhibitory to adenylate cyclase while a lower affinity receptor (A0 was stimulatory (for review, see Daly, 1982). At the behavioral level, adenosine and its analogs exert marked depressant effects on behavior, including pronounced sedative (Barraco et al., 1983; Katims et al., 1983), hypnogenic (Radulovacki et al., 1982) and anticonvulsant actions (Dunwiddie and Worth, 1982). These behavioral effects were blocked by methylxanthines such as caffeine and theophylline. The methylxanthines have also been shown to block the modulation of adenylate cyclase (Daly, 1979), the depressant effects on neuronal firing rates (Phillis and Wu, 1981) and the inhibitory effects of adenosine on the release of neurotransmitters (Stone et al., 1981). The competitive blockade of methylxanthines in these studies, as well as their displacement of [3H]adenosine analogs in binding studies (Snyder et al., 1981), suggest that these effects are mediated by the PI (R or A) adenosine receptor (Burnstock, 1978; Daly, 1982). Nevertheless, the question remains whether or not the receptor mediated influences of adenosine and its analogs on adenylate cyclase has direct behavioral consequences. For example, it has been suggested that the inhibitory effect of adenosine on transmitter release in the peripheral nervous system and hippocampal slices is unlikely to be mediated via cyclic A M P since this would presume that transmitter release is critically dependent upon cyclic A M P (Fredholm, 1982). Send correspondence and reprint requests to John W. Phillis. 521
Even less is known about the coupling of the adenosine receptor and adenylate cyclase to other types of effectors (e.g. calcium channels). Furthermore, efforts to establish specific physiological roles for cyclic nucleotides in the regulation of spontaneous behaviors following direct injections into the whole animal have yielded conflicting findings, possibly since exogenous cyclic nucleotides penetrate cell membranes with great difficulty. Direct intracerebral or intraventricular injections of dibutyryl cyclic A M P (dBcAMP) and, less effectively, of cyclic A M P (cAMP) have elicited an increase in spontaneous motor activity in rats (Herman, 1973), sleep and catatonia in cats (Gessa et al., 1971), or a decrease in activity and hypothermia in rats (Wachtel, 1982), whereas intraperitoneal injections produced locomotor depression in mice (Weiner and Olson, 1973) and sedation in rats (Beer et al., 1972); intravenous administration of dBcAMP resulted in dosedependent hypoactivity in dogs (Ono et al., 1976). Apparently these qualitatively different responses vary depending on the species and the route of administration; it has also been pointed out that cAMP, dBcAMP and other analogues may exert actions not related to the cyclic phosphate moiety (Breckenridge, 1970). On the other hand, manipulations of in vivo cAMP levels in the brain have produced more characteristic behavioral symptomatology. For example, parenteral administration of phosphodiesterase (PDE) inhibitors such as RO 20-1724 and rolipram, which have been shown to possess a high selectivity towards cAMP rather than cGMP PDE, produce hypoactivity and hypothermia in rats and mice and these behavioral effects are mimicked by systemically or intracerebrally administered dBcAMP, but not dBcGMP (Wachtel, 1982, 1983; Dascombe et al., 1980; Dascombe and Parkes, 1981). In fact, the order of potency and effective dose range of a series of PDE inhibitors to induce behavioral alterations corresponded with the efficacy of the drugs to elevate brain cAMP in vivo (Wachtel, 1983). Similar behavioral effects were seen with other PDE inhibitors, suggesting that depression of spontaneous locomotor activity is a potential in vivo correlate of enhanced availability of brain cAMP (Wachtel, 1982, 1983).
ROBIN A. BARRACO et al.
522
The purpose of the following study was to examine the behavioral consequences of elevating brain c A M P levels via stimulation of adenylate cyclase. Forskolin, a diterpene which reversibly activates adenylate cyclase in intact tissues, including the brain (for review, see Seamon and Daly, 1981) was administered into the lateral cerebral ventricle of mice and the effects of spontaneous locomotor activity were assessed. Forskolin has recently been demonstrated to prevent the seizures induced in mice by pentylenetetrazol, whilst simultaneously elevating brain c A M P levels (Sano et al., 1984). MATERIALS AND METHODS
Adult male Swiss Webster mice (ICR strain, Harlan Industries) approximately l0 weeks old (30-40g) were implanted with permanent indwelling stainless-steel guide cannulas for injection into the lateral ventricle of the brain as previously described (Barraco et al., 1983). They were housed 6 per cage in 18 × 24 x 15 cm polypropylene cages, on a 12 hr/12 hr light cycle (lights on 07:00). Animals were maintained on ad libitum food and water throughout the experiment. Mice were permitted to recover from surgery and adapt to their housing for a minimum of l0 days. Drugs or control solutions administered intracerebroventricularly (i.c.v.t.) were injected slowly in a 5/~1 volume over a 30 sec period during which time the animals were able to freely move around the bench top. Six mice were placed in each of six testing chambers 08 × 24 × 15 cm) and were monitored simultaneously. Locomotor activity was measured with a Stoelting 31410 Modular Electronic Activity Monitor with 6 sensors. The sensors were placed in a dark compartment (2 x 3 × 2.4 m high) equipped with a fan for ventilation and noise attenuation. All sensors were adjusted to equal sensitivity with a pendulum. Each sensor was activated by a uniform, low power, radio-frequency field extending several inches above the surface. Sensitivity of the sensors was adjusted to accumulate counts for gross movement. Immediately following injections, there was a l0 min pretest period, prior to the 30 min test period for locomotor activity. Locomotor activity scores were analyzed by the Kruskal-Wallis ranked one-way analysis of variance and Mann-Whitney U post hoc one-tailed paired comparisons. Forskolin (Tfl-acetoxy-8, 13-epoxy-lct,6fl,9~t-trihydroxylabd-14-en-ll-one) (Calbiochem) was dissolved in dimethylsulfoxide (DMSO) (Sigma) and injected i.c.v.t. DMSO was used for the control injections.
adenylate cyclase in intact tissues including the brain, it is possible that the depression of locomotor activity observed in this study might reflect the enhanced availability of c A M P in the brain. In this context, it is of interest that forskolin can also prevent pentylenetetrazol-induced seizures in mice (Sano et aL, 1984). Our findings therefore are consistent with the behavioral effects of c A M P elevations induced by P D E inhibitors referred to in the Introduction. Furthermore, the effect of forskolin on adenylate cyclase appears to be specific since it has not been reported to affect the activity of a variety of enzymes examined including P D E , N a + / K + ATPase, cAMP-dependent protein kinase and guanylate cyclase (for review, see Seamon and Daly, 1981). Moreover, although forskolin activates adenylate cyclases normally stimulated by different agonists, its effect is not dependent on receptor stimulation, but apparently involves a direct activation of the enzyme. In fact, it has been suggested that one potential criterion for assessing the possible role of c A M P in a physiological response is whether forskolin is able to elicit the same response and, collaterally, potentiate the response of a given agonist on a specific adenylate cyclase-linked receptor. However, the question of whether or not the depressant effects of adenosine and its analogs on behavior are mediated by in vivo alterations in c A M P levels in the nervous system is still not resolved. It is conceivable both mechanisms could operate in parallel with adenosine depressing neuronal activity via presynaptic inhibition of neurotransmitter release (for references, see Fredholm and Hedqvist, 1980) whilst c A M P mediates inhibitory neurotransmission by hyperpolarizing neurons through phosphorylation of the postsynaptic membrane (Greengard, 1976). Nevertheless, a number of studies have shown a remarkable correlation between depression of excitatory transmission in the hippocampus and olfactory cortex and the accumulation of c A M P by adenosine and its derivatives (Kuroda et al., 1976; Okada and Saito, 1979; Fredholm et al., 1982; Motley and I10 100
RESULTS The effect of i.c.v.t injections of forskolin on spontaneous locomotor activity (expressed as percent of D M S O controls) is shown in Fig. 1. An overall analysis of variance indicated that forskolin produced a dose-related decrease in locomotor activity (H = 17.28, d.f. = 3; P <0.001). Significance levels determined by post hoc comparison for individual doses are indicated by asterisks in Fig. 1. Forskolin produced significant reductions in locomotor activity at the 10/~g (P < 0.05) and 100/lg (P < 0.01) doses when compared with D M S O controls. DISCUSSION These results show that i.c.v.t, injections of forskolin produced a dose-related depression of spontaneous locomotor activity. Since it is known that forskolin causes a rapid, reversible, and dosedependent increase in c A M P levels via stimulation of
,,¢
"6
90 80 7'0
.3 60 "6 50 (_)
40 "6 46 30 E #_ 20 10
].0
10 100 F0rskolin(pg/mouse) Fig. 1. Effect ofi.c.v.t injections of forskolin on spontaneous locomotor activity expressed as percent of controls. Values represent the mean + SEM. Overall analysis of variance showed dose-related decreases in locomotor activity (P <0.001). Significance levels determined by post hoc comparisons with controls are indicated (*P < 0.05; **P < 0.01).
Forskolin depresses motor activity Collins, 1983). The fact that these effects are inhibited by methylxanthines, potentiated by adenosine uptake inhibitors, and the relative activity of adenosine analogs in these studies correlates well with their potency in binding studies, suggests that the extracellular adenosine receptors mediating depression of neuronal activity and accumulation of c A M P have similar characteristics. It is plausible these receptorlinked phenomena correspond to the A2 receptor subtype since (1) certain findings in hippocampal slices and striatal membrane preparations raise the possibility that the biological effects of A1 receptor agonists in the CNS may be related to inhibition of c A M P accumulation (Fredholm et al., 1983; Wojcik and Neff, 1983), (2) N E C A , a selective A2 agonist (Daly, 1982), was by far the most potent of a series of analogs in depressing spontaneous locomotor activity in mice (Barraco et al., 1983) and (3) N E C A was also the most potent in a series of analogs for depressing the activity of cerebral cortical neurons (Phillis, 1982). Finally, although it is possible that many of the effects of adenosine are not mediated via adenylate cyclase, the results of the present study do not contradict the hypothesis that the adenosine receptor mediating behavioral depression and accumulation of c A M P may be the A 2 receptor. Acknowledgements--The authors gratefully acknowledge
the technical skills of Mr George Smoot. This research was supported in part by Grant RR-08167-OX from the Division of Research Resources, NIH. REFERENCES Barraco R. A., Coffin V. L., Altman H. J. and Phillis J. W. (1983) Central effects of adenosine analogs on locomotor activity in mice and antagonism of caffeine. Brain Res. 272, 392-395. Beer B., Chasin M., Clody D. E., Vogel J. R. and Horovitz Z. P. (1972) Cyclic adenosine monophosphate phosphodiesterase in brain: effect on anxiety. Science 176, 428-430. Breckenridge B. McL. (1970) Cyclic AMP and drug action. A. Rev. Pharmac. 10, 19-34. Burnstock G. (1978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Edited by Bolis L. and Straub R. W.) pp. 107-118. Raven Press, New York. Daly J. W. (1979) Adenosine and cyclic adenosine monophosphate generating systems in brain tissue. In Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides (Edited by Baer H. P. and Drummond
G. I.) pp. 229-241. Raven Press, New York. Daly J. W. (1982) Adenosine receptors: Targets for future drugs. J. reed. Chem. 25, 197-207. Dascombe M. J. and Parkes J. (1981) Effects of N6-2'-O-dibutyryl 3',5'-monophosphate on body temperature in the restrained rat. Br. J. Pharmac. 72, 565-566. Dascombe M. J., Milton A. S., Nyemitei-Addo I. and Pertwee R. G. (1980) Thermoregulatory effects of N6-2'-O-dibutyryl adenosine 3',5'-monophosphate in the restrained mouse. Br. J. Pharmac. 70, 453-459. Dunwiddie T. W. and Worth T. (1982) Sedative and anticonvulsive effects of adenosine analogs in mouse and rat. J. Pharmac. exp. Ther. 220, 70-76. Fredholm B. B. (1982) Adenosine receptors. Med. Biol. 60, 289-293. Fredholm B. B. and Hedqvist P. (1980) Modulation of
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neurotransmission by purine nucleotides and nucleosides. Biochem. Pharmac. 29, 1635-1643.
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