316
Brain Research, 524 (1990) 316-318 Elsevier
BRES 24208
Chronic caffeine exposure reduces the excitant action of acetylcholine on cerebral cortical neurons Y. Lin and J.W. Phillis Department of Physiology, WayneState University School of Medicine, Detroit, MI 48201 (U.S.A.) (Accepted 24 April 1990) Key words: Caffeine; Acetylcholine; Receptor; Cerebral cortex
Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10-70 mg/kg) decreased the sensitivity of rat cerebral cortical neurons to the excitant action of microiontophoretically applied acetylcholine. The sensitivity of spontaneously firing rat cerebral cortical neurons in caffeine-treated animals was compared with that of saline-treated controls using the same multiple barrel micropipettes tested on the same day. Acetylcholine sensitivity was determined by the E.Tso method. The E.T50 for 71 neurons in the caffeine-treated rats of 224.0 + 11.3 (S.E.M.) was significantly (P < 0.001) greater than that of 65 neurons in the saline-treated control rats (153.8 + 6.9), indicating a reduction in the excitant action of acetylcholine on neurons which had been chronically exposed to caffeine. The level of spontaneous activity was also reduced in the caffeine-treated animals. A down-regulation of acetylcholine receptors is a possible cause for these effects. Chronic administration of the methylxanthines, caffeine and theophylline, leads to the development of tolerance to some of their effects, both in humans and experimental animals 6'9. This is likely to be the result of an increase in adenosine receptors, which has been shown to occur after long-term treatment with caffeine or theophylline 1'3'4"8"12"19. Regional differences in xanthineinduced up-regulation of adenosine have been demonstrated in rodents 11'18. Increases in binding were observed in the cerebral cortex and cerebellum, but not in the hippocampus. The effects of methylxanthine-induced up-regulation of adenosine receptors have only recently been studied experimentally. Chronic exposure to caffeine led to the development of tolerance to the ability of caffeine to increase the rate of firing of mesencephalic reticular neurons, whilst causing an increase in the density of [3H]cyclohexyladenosine binding sites in the same area of the brain 2. Adenosine-evoked inhibition of the spontaneous firing of rat cerebral cortical neurons was significantly enhanced in animals treated chronically with caffeine 2°. Other animal studies also suggest that central adenosine receptors may be involved in the development of the tolerance and dependence phenomena that have been associated with the central effects of chronic caffeine administration in animals and humans 6'9. There are reports that other putative neurotransmitter receptors can also be affected by chronic treatment with adenosine antagonists, fl-Adrenoceptors in the rat hippocampus
were down-regulated as a consequence of chronic theophylline treatment 5,7. The possibility exists that cholinergic receptors could also be affected by the chronic administration of adenosine antagonists. As the spontaneous firing of rat corticospinal neurons is driven by acetylcholine (ACh) 17, these neurons offered a convenient model on which to evaluate the effects of chronic caffeine administration on the sensitivity of muscarinic cholinergic receptors to iontophoretically applied ACh. Rats, injected daily with caffeine for a period of 14 days, were used to ascertain whether there was an alteration in the responsiveness of cerebral cortical neurons to ACh. Eight male Sprague-Dawley rats (300-350 g) were housed in individual cages and kept on a 12 h light-dark cycle. Four of the rats were injected daily with caffeine subcutaneously and the remaining animals were injected with an equal volume (2-2.5 ml) of 0.9% sterile sodium chloride (U.S.P.). The initial dose of caffeine was 10 mg/kg, which was increased by 10 mg/kg daily, until the animals were receiving 70 mg/kg on the 7th day. This dosage rate was continued during the second week. No injections were given on the 15th and 16th days and the animals were tested for A C h sensitivity on the 17th day. The rats were anesthetized with halothane, and after insertion of a tracheal cannula, anesthesia was maintained with a mixture of methoxyflurane in nitrous oxide (60%) and oxygen. Body temperature was maintained at 37 °C with an abdominal heating pad controlled by a
Correspondence: J.W. Phillis, Department of Physiology, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
317 TABLE I Effect of chronic caffeine administration on the spontaneous activity of, and sensitivity of rat cerebral cortical neurons to the excitant effects of acetylcholine
Values are mean + S.E.M. Control
Number of neurons Spontaneous firing rate (spikes/min) E-Tso
65 35.8 + 2.2 153.8 + 6.9
Caffeine-treated
71 28.4 + 2.3* 224.0 + 11.3"**
Significantly different from corresponding value for saline-treated animals: *P < 0.05; ***P < 0.01 by Student's t-test.
rectal probe. A small opening was drilled through the parietal bone to expose the sensorimotor cortex in the hindlimb area and a narrow slit was made in the dura mater to allow access to the cerebral cortical neurons. Recording of the spontaneous activity of deep (800-1400 ~m) cortical neurons was achieved with a central 2 M NaCl-filled barrel of a multiple-barrel microelectrode (fabricated with fiber-fill capillary tubing). Another barrel of the microelectrode was filled with 2 M NaCI for automatic current neutralization and two barrels were filled by acetylcholine chloride (0.1 M, pH 4.8). To eliminate any possibility of variability in the apparent excitant potency of ACh due to electrode barrel characteristics, pairs of rats were tested with same electrode on the same day. In two instances, the caffeine-injected rat was anesthetized first, with the saline-injected animal following several hours later. This order was reversed for the remaining rats. Groups of neurons were tested with ACh applied alternately from each barrel in both the caffeine-treated and control animals. The use of two ACh containing barrels minimized the difficulties associated with continuous barrel usage and blocking of the passage of current. The potency of ACh as an excitant of cerebral cortical neurons was estimated by the E.Tso (current in nA x time in seconds for 50% increase of firing) method. A constant application current of 40 or 50 nA was selected for each ACh barrel and the time for which this current had to be applied to increase neuronal firing rate by 50% measured for each neuron. Multiple E.T5o estimates were made on every cell, from which an average E.Tso value for the cell could be calculated. The same ACh barrels were used to test all neurons in the pairs of caffeine- and saline-treated animals. The results of this survey are presented in Table I. The mean spontaneous firing rate for neurons in the caffeine treated rats was 28.4 + 2.3 (S.E.M.) spikes/s, and that for neurons in the saline-treated controls was 35.8 + 2.2 spikes/s (P < 0.05). The mean E.Ts0 value for the 71
spontaneously active cortical neurons tested in the caffeine treated animals was 224.0 + 11.3 (S.E.M.), which was significantly greater (P < 0.001) than the E.Ts0 of 153.8 + 6.9 recorded for 65 neurons in the saline-treated control animals. The present results demonstrate that the excitant effects of ACh on cerebral cortical neurons in chronic caffeine-treated rats are less pronounced than those in normal rats. The cholinergically mediated spontaneous firing of neurons in the caffeine-treated animals was also significantly lower than that of the saline-treated controls. The explanation for these differences may lie in a down-regulation of the ACh receptors on cerebral cortical neurons. The depressant action of adenosine at cholinergic synapses appears to be primarily mediated by receptors on the presynaptic nerve terminals. Adenosine decreases ACh release from electrically stimulated cortical slices 13 and from cortical and hippocampal K ÷depolarized synaptosomes 14. As adenosine inhibits the cholinergic synaptic excitation of spinal cord Renshaw cells by antidromic volleys in the ventral roots, without affecting the excitant actions of iontophoretically applied ACh, it presumably acts by inhibiting the presynaptic release of ACh 1°. Similar studies on cerebral cortical neurons have confirmed the susceptibility of cholinergically evoked spontaneous activity 17 to inhibition by adenosine, even though iontophoretically applied ACh continued to elicit excitation 15'16. Blockade of adenosine receptors on cholinergic nerve terminals in the cerebral cortex by caffeine would release these terminals from the inhibitory action of adenosine, with a resultant elevation of ACh levels in the extracellular space 14. This could, in turn, cause a down-regulation of receptors on cholinoceptive cortical neurons, with decreased sensitivity to iontophoretically applied ACh and a reduced level of spontaneous firing. A down-regulation of fl-adrenoceptors in the rat hippocampus has previously been demonstrated following chronic theophylline administration 5"7. Alternatively, chronic caffeine administration may cause a general reduction in brain excitability, as a result of the supersensitivity of inhibitory adenosine receptors 2°. Such an explanation would be consistent with the observation that the increase in Bmax for adenosine ligand binding following chronic theophylline administration in rats is associated with a reduced sensitivity to convulsant agents 18. In conclusion, the development of tolerance to chronic methylxanthine administration and the resultant physical dependence may involve receptors for other transmitters, in addition to those for adenosine.
Supported by NIH Grant RR 08167-10, D.R.R.
318 1 Boulenger, J.-P., Patel, J., Post, R.M., Parma, A.M. and Marangos, P.J., Chronic caffeine consumption increases the number of brain adenosine receptors, Life Sci., 32 (1983) 1135-1142. 2 Chou, D.T., Khan, S., Forde, J. and Hirsch, K.R., Caffeine tolerance: behavioral, electrophysiological and neurochemical evidence, Life Sci., 36 (1985) 2347-2358. 3 Daval, J.-L., Deckert, J., Weiss, S.R.B., Post, R.M. and Marangos, P.J., Upregulation of adenosine A 1 receptors and forskolin binding sites following chronic treatment with caffeine or carbamazepine: a quantitative autoradiographic study, Epilepsia, 30 (1989) 26-33. 4 Fredholm, B.B., Adenosine actions and adenosine receptors after 1 week treatment with caffeine, Acta Physiol. Scand., 115 (1982) 283-286. 5 Fredholm, B.B., Jonzon, B. and Lindgrcn, E., Changes in noradrenalinc release and in beta receptor number in rat hippocampus following long term treatment with theophylline or L-phenylisopropyladenosine, Acta Physiol. Scand., 122 (1984) 55-60. 6 Gilbert, R.M., Caffeine as a drug of abuse. In R.J. Gibbins, Y. Israel, H. Kalant, R.E. Popham, W. Schmidt and R.G. Smart (Eds.), Research Advances in Alcohol and Drug Problems, Vol. 3, Wiley, New York, 1976, pp. 49-176. 7 Goldberg, M.R., Curatolo, P.W., Tung, C.-S. and Robertson, D., Caffeine down-regulates fl-adrenoceptors in rat forebrain, Neurosci. Lett., 31 (1982) 47-52. 8 Hawkins, N., Dugich, M.M., Porter, N.M., Urbancic, M. and Radulovacki, M., Effects of chronic administration of caffeine on adenosine A~ and A 2 receptors in rat brain, Brain Res. Bull., 21 (1988) 479-482. 9 Hirsh, K., Central nervous system pharmacology of the dietary methylxanthines. In G.A. Spiller (Ed.), The Methylxanthine Beverages and Foods: Chemistry, Consumption, and Health Effects, Liss, New York, 1984, pp. 235-301. 10 Lekic, D., Presynaptic depression of synaptic responses of Renshaw cells by adenosine 5"-monophosphate, Can. J. Physiol. Pharmacol., 55 (1977) 1391-1393.
11 Marangos, EJ., Boulenger, J.P. and Patel, J., Effects of chronic caffeine on brain adenosine receptors: regional and ontogenetic studies, Life Sci., 34 (1984) 899-907. 12 Murray, T.E, Up-regulation of rat cortical adenosine receptors following chronic administration of theophylline, Eur. J. Pharmacol., 82 (1982) 113-114. 13 Pcdata, E, Antonelli, T., Lambertini, L., Beani, L. and Pepeu, G., Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electrically-stimulated brain slices, Neuropharmacology, 22 (1983) 609-614. 14 Pcdata, E, Giovannelli, L., De Samo, E and Pepeu, G., Effect of adenosine, adenosine derivatives and caffeine on acetylcholine release from brain synaptosomes: interaction with muscarinic autoregulatory mechanisms, J. Neurochem., 46 (1986) 1593-1598. 15 Phillis, J.W., Edstrom, J.P., Kostopoulos, G.K. and Kirkpatrick, J.R., Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex, Can. J. Physiol. Pharmacol., 57 (1979) 1289-1312. 16 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Progr. Neurobiol., 16 (1981) 187-239. 17 Swanson, T.H. and Phillis, J.W., Effects of muscarinic antagonists pirenzepine and gallamine on spontaneous and evoked responses of rat cerebral cortical neurons, Br. J. PharmacoL, 94 (1988) 192-198. 18 Szot, P., Sanders, R.C. and Murray, T.E, Theophyiline-induced upregulation of Al-adenosine receptors associated with reduced sensitivity to convulsants, Neuropharmacology, 26 (1987) 11731180. 19 Wu, P.H. and Coffin, V.L., Up-regulation of brain [3H]diazepam binding sites in chronic caffeine-treated rats, Brain Research, 294 (1984) 186-189. 20 Lin, Y. and Phillis, J.W., Chronic caffeine exposure enhances adenosinergic inhibition of cerebral cortical neurons, Brain Research, in press.
Brain Research, 524 (1990) 316-318 Elsevier
BRES 24208
Chronic caffeine exposure reduces the excitant action of acetylcholine on cerebral cortical neurons Y. Lin and J.W. Phillis Department of Physiology, WayneState University School of Medicine, Detroit, MI 48201 (U.S.A.) (Accepted 24 April 1990) Key words: Caffeine; Acetylcholine; Receptor; Cerebral cortex
Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10-70 mg/kg) decreased the sensitivity of rat cerebral cortical neurons to the excitant action of microiontophoretically applied acetylcholine. The sensitivity of spontaneously firing rat cerebral cortical neurons in caffeine-treated animals was compared with that of saline-treated controls using the same multiple barrel micropipettes tested on the same day. Acetylcholine sensitivity was determined by the E.Tso method. The E.T50 for 71 neurons in the caffeine-treated rats of 224.0 + 11.3 (S.E.M.) was significantly (P < 0.001) greater than that of 65 neurons in the saline-treated control rats (153.8 + 6.9), indicating a reduction in the excitant action of acetylcholine on neurons which had been chronically exposed to caffeine. The level of spontaneous activity was also reduced in the caffeine-treated animals. A down-regulation of acetylcholine receptors is a possible cause for these effects. Chronic administration of the methylxanthines, caffeine and theophylline, leads to the development of tolerance to some of their effects, both in humans and experimental animals 6'9. This is likely to be the result of an increase in adenosine receptors, which has been shown to occur after long-term treatment with caffeine or theophylline 1'3'4"8"12"19. Regional differences in xanthineinduced up-regulation of adenosine have been demonstrated in rodents 11'18. Increases in binding were observed in the cerebral cortex and cerebellum, but not in the hippocampus. The effects of methylxanthine-induced up-regulation of adenosine receptors have only recently been studied experimentally. Chronic exposure to caffeine led to the development of tolerance to the ability of caffeine to increase the rate of firing of mesencephalic reticular neurons, whilst causing an increase in the density of [3H]cyclohexyladenosine binding sites in the same area of the brain 2. Adenosine-evoked inhibition of the spontaneous firing of rat cerebral cortical neurons was significantly enhanced in animals treated chronically with caffeine 2°. Other animal studies also suggest that central adenosine receptors may be involved in the development of the tolerance and dependence phenomena that have been associated with the central effects of chronic caffeine administration in animals and humans 6'9. There are reports that other putative neurotransmitter receptors can also be affected by chronic treatment with adenosine antagonists, fl-Adrenoceptors in the rat hippocampus
were down-regulated as a consequence of chronic theophylline treatment 5,7. The possibility exists that cholinergic receptors could also be affected by the chronic administration of adenosine antagonists. As the spontaneous firing of rat corticospinal neurons is driven by acetylcholine (ACh) 17, these neurons offered a convenient model on which to evaluate the effects of chronic caffeine administration on the sensitivity of muscarinic cholinergic receptors to iontophoretically applied ACh. Rats, injected daily with caffeine for a period of 14 days, were used to ascertain whether there was an alteration in the responsiveness of cerebral cortical neurons to ACh. Eight male Sprague-Dawley rats (300-350 g) were housed in individual cages and kept on a 12 h light-dark cycle. Four of the rats were injected daily with caffeine subcutaneously and the remaining animals were injected with an equal volume (2-2.5 ml) of 0.9% sterile sodium chloride (U.S.P.). The initial dose of caffeine was 10 mg/kg, which was increased by 10 mg/kg daily, until the animals were receiving 70 mg/kg on the 7th day. This dosage rate was continued during the second week. No injections were given on the 15th and 16th days and the animals were tested for A C h sensitivity on the 17th day. The rats were anesthetized with halothane, and after insertion of a tracheal cannula, anesthesia was maintained with a mixture of methoxyflurane in nitrous oxide (60%) and oxygen. Body temperature was maintained at 37 °C with an abdominal heating pad controlled by a
Correspondence: J.W. Phillis, Department of Physiology, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
317 TABLE I Effect of chronic caffeine administration on the spontaneous activity of, and sensitivity of rat cerebral cortical neurons to the excitant effects of acetylcholine
Values are mean + S.E.M. Control
Number of neurons Spontaneous firing rate (spikes/min) E-Tso
65 35.8 + 2.2 153.8 + 6.9
Caffeine-treated
71 28.4 + 2.3* 224.0 + 11.3"**
Significantly different from corresponding value for saline-treated animals: *P < 0.05; ***P < 0.01 by Student's t-test.
rectal probe. A small opening was drilled through the parietal bone to expose the sensorimotor cortex in the hindlimb area and a narrow slit was made in the dura mater to allow access to the cerebral cortical neurons. Recording of the spontaneous activity of deep (800-1400 ~m) cortical neurons was achieved with a central 2 M NaCl-filled barrel of a multiple-barrel microelectrode (fabricated with fiber-fill capillary tubing). Another barrel of the microelectrode was filled with 2 M NaCI for automatic current neutralization and two barrels were filled by acetylcholine chloride (0.1 M, pH 4.8). To eliminate any possibility of variability in the apparent excitant potency of ACh due to electrode barrel characteristics, pairs of rats were tested with same electrode on the same day. In two instances, the caffeine-injected rat was anesthetized first, with the saline-injected animal following several hours later. This order was reversed for the remaining rats. Groups of neurons were tested with ACh applied alternately from each barrel in both the caffeine-treated and control animals. The use of two ACh containing barrels minimized the difficulties associated with continuous barrel usage and blocking of the passage of current. The potency of ACh as an excitant of cerebral cortical neurons was estimated by the E.Tso (current in nA x time in seconds for 50% increase of firing) method. A constant application current of 40 or 50 nA was selected for each ACh barrel and the time for which this current had to be applied to increase neuronal firing rate by 50% measured for each neuron. Multiple E.T5o estimates were made on every cell, from which an average E.Tso value for the cell could be calculated. The same ACh barrels were used to test all neurons in the pairs of caffeine- and saline-treated animals. The results of this survey are presented in Table I. The mean spontaneous firing rate for neurons in the caffeine treated rats was 28.4 + 2.3 (S.E.M.) spikes/s, and that for neurons in the saline-treated controls was 35.8 + 2.2 spikes/s (P < 0.05). The mean E.Ts0 value for the 71
spontaneously active cortical neurons tested in the caffeine treated animals was 224.0 + 11.3 (S.E.M.), which was significantly greater (P < 0.001) than the E.Ts0 of 153.8 + 6.9 recorded for 65 neurons in the saline-treated control animals. The present results demonstrate that the excitant effects of ACh on cerebral cortical neurons in chronic caffeine-treated rats are less pronounced than those in normal rats. The cholinergically mediated spontaneous firing of neurons in the caffeine-treated animals was also significantly lower than that of the saline-treated controls. The explanation for these differences may lie in a down-regulation of the ACh receptors on cerebral cortical neurons. The depressant action of adenosine at cholinergic synapses appears to be primarily mediated by receptors on the presynaptic nerve terminals. Adenosine decreases ACh release from electrically stimulated cortical slices 13 and from cortical and hippocampal K ÷depolarized synaptosomes 14. As adenosine inhibits the cholinergic synaptic excitation of spinal cord Renshaw cells by antidromic volleys in the ventral roots, without affecting the excitant actions of iontophoretically applied ACh, it presumably acts by inhibiting the presynaptic release of ACh 1°. Similar studies on cerebral cortical neurons have confirmed the susceptibility of cholinergically evoked spontaneous activity 17 to inhibition by adenosine, even though iontophoretically applied ACh continued to elicit excitation 15'16. Blockade of adenosine receptors on cholinergic nerve terminals in the cerebral cortex by caffeine would release these terminals from the inhibitory action of adenosine, with a resultant elevation of ACh levels in the extracellular space 14. This could, in turn, cause a down-regulation of receptors on cholinoceptive cortical neurons, with decreased sensitivity to iontophoretically applied ACh and a reduced level of spontaneous firing. A down-regulation of fl-adrenoceptors in the rat hippocampus has previously been demonstrated following chronic theophylline administration 5"7. Alternatively, chronic caffeine administration may cause a general reduction in brain excitability, as a result of the supersensitivity of inhibitory adenosine receptors 2°. Such an explanation would be consistent with the observation that the increase in Bmax for adenosine ligand binding following chronic theophylline administration in rats is associated with a reduced sensitivity to convulsant agents 18. In conclusion, the development of tolerance to chronic methylxanthine administration and the resultant physical dependence may involve receptors for other transmitters, in addition to those for adenosine.
Supported by NIH Grant RR 08167-10, D.R.R.
318 1 Boulenger, J.-P., Patel, J., Post, R.M., Parma, A.M. and Marangos, P.J., Chronic caffeine consumption increases the number of brain adenosine receptors, Life Sci., 32 (1983) 1135-1142. 2 Chou, D.T., Khan, S., Forde, J. and Hirsch, K.R., Caffeine tolerance: behavioral, electrophysiological and neurochemical evidence, Life Sci., 36 (1985) 2347-2358. 3 Daval, J.-L., Deckert, J., Weiss, S.R.B., Post, R.M. and Marangos, P.J., Upregulation of adenosine A 1 receptors and forskolin binding sites following chronic treatment with caffeine or carbamazepine: a quantitative autoradiographic study, Epilepsia, 30 (1989) 26-33. 4 Fredholm, B.B., Adenosine actions and adenosine receptors after 1 week treatment with caffeine, Acta Physiol. Scand., 115 (1982) 283-286. 5 Fredholm, B.B., Jonzon, B. and Lindgrcn, E., Changes in noradrenalinc release and in beta receptor number in rat hippocampus following long term treatment with theophylline or L-phenylisopropyladenosine, Acta Physiol. Scand., 122 (1984) 55-60. 6 Gilbert, R.M., Caffeine as a drug of abuse. In R.J. Gibbins, Y. Israel, H. Kalant, R.E. Popham, W. Schmidt and R.G. Smart (Eds.), Research Advances in Alcohol and Drug Problems, Vol. 3, Wiley, New York, 1976, pp. 49-176. 7 Goldberg, M.R., Curatolo, P.W., Tung, C.-S. and Robertson, D., Caffeine down-regulates fl-adrenoceptors in rat forebrain, Neurosci. Lett., 31 (1982) 47-52. 8 Hawkins, N., Dugich, M.M., Porter, N.M., Urbancic, M. and Radulovacki, M., Effects of chronic administration of caffeine on adenosine A~ and A 2 receptors in rat brain, Brain Res. Bull., 21 (1988) 479-482. 9 Hirsh, K., Central nervous system pharmacology of the dietary methylxanthines. In G.A. Spiller (Ed.), The Methylxanthine Beverages and Foods: Chemistry, Consumption, and Health Effects, Liss, New York, 1984, pp. 235-301. 10 Lekic, D., Presynaptic depression of synaptic responses of Renshaw cells by adenosine 5"-monophosphate, Can. J. Physiol. Pharmacol., 55 (1977) 1391-1393.
11 Marangos, EJ., Boulenger, J.P. and Patel, J., Effects of chronic caffeine on brain adenosine receptors: regional and ontogenetic studies, Life Sci., 34 (1984) 899-907. 12 Murray, T.E, Up-regulation of rat cortical adenosine receptors following chronic administration of theophylline, Eur. J. Pharmacol., 82 (1982) 113-114. 13 Pcdata, E, Antonelli, T., Lambertini, L., Beani, L. and Pepeu, G., Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electrically-stimulated brain slices, Neuropharmacology, 22 (1983) 609-614. 14 Pcdata, E, Giovannelli, L., De Samo, E and Pepeu, G., Effect of adenosine, adenosine derivatives and caffeine on acetylcholine release from brain synaptosomes: interaction with muscarinic autoregulatory mechanisms, J. Neurochem., 46 (1986) 1593-1598. 15 Phillis, J.W., Edstrom, J.P., Kostopoulos, G.K. and Kirkpatrick, J.R., Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex, Can. J. Physiol. Pharmacol., 57 (1979) 1289-1312. 16 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Progr. Neurobiol., 16 (1981) 187-239. 17 Swanson, T.H. and Phillis, J.W., Effects of muscarinic antagonists pirenzepine and gallamine on spontaneous and evoked responses of rat cerebral cortical neurons, Br. J. PharmacoL, 94 (1988) 192-198. 18 Szot, P., Sanders, R.C. and Murray, T.E, Theophyiline-induced upregulation of Al-adenosine receptors associated with reduced sensitivity to convulsants, Neuropharmacology, 26 (1987) 11731180. 19 Wu, P.H. and Coffin, V.L., Up-regulation of brain [3H]diazepam binding sites in chronic caffeine-treated rats, Brain Research, 294 (1984) 186-189. 20 Lin, Y. and Phillis, J.W., Chronic caffeine exposure enhances adenosinergic inhibition of cerebral cortical neurons, Brain Research, in press.
