Brain Research, 520 (1990) 322-323 Elsevier
322 BRES 24123
Chronic caffeine exposure enhances adenosinergic inhibition of cerebral cortical neurons Yu Lin and J.W. Phillis Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.) (Accepted 27 February 1990)
Key words: Chronic caffeine; Adenosine; Inhibition; Up-regulation; Cerebral cortex
Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10-70 mg/kg) increased the sensitivity of rat cerebral cortical neurons to the inhibitory action of microiontophoretically applied adenosine. 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. Adenosine sensitivity was determined by the 1.1"5omethod. The I.Tso value for 134 neurons in the caffeine-treated rats of 130.77 + 4.33 (S.E.M.) was significantly (P < 0.001) different to that of 136 neurons in the saline-treated control rats (222.16 + 6.68), indicating a supersensitivity to adenosine in neurons which had been chronically exposed to caffeine. Caffeine is widely used as a psychoactive agent and it is now generally accepted that its central stimulant actions are mediated through a competitive inhibition of adenosine receptors in the brain s-l°. Chronic exposure to caffeine produces an up-regulation of adenosine A 1 and A 2 receptors in the rat forebrain 1'3'5'6'12. In these reports, however, no effort was made to evaluate the effects of this up-regulation of adenosine receptors on the depressant action of the purine on neuronal activity. The present study represents an attempt to ascertain whether the increase in adenosine receptor numbers resulting from chronic caffeine administration is paralleled by increases in neuronal sensitivity to adenosine-elicited inhibition. Rats injected daily with caffeine for a period of 14 days were used to ascertain whether there was an altered sensitivity of cerebral cortical neurons to purinergic inhibition. Ten male Sprague-Dawley rats (300-350 g) were housed in individual cages and kept on a 12-h light-dark cycle. Five 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 adenosine 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 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 the central 2 M NaC1 filled barrel of a multiple-barrel microelectrode (fabricated with fiber-fill capillary tubing). A n o t h e r barrel of the microelectrode was filled with 2 M NaCI for automatic current neutralization and two barrels were filled with adenosine hemisulphate (0.1 M, p H 3.67). To eliminate any possibility of variability in the apparent potency of adenosine due to electrode barrel characteristics, pairs of rats were tested with the same electrode on the same day. On 3 days 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 adenosine applied alternately from each barrel in both the caffeine-treated and control animals. The use of two adenosine-containing barrels minimized the difficulties associated with continuous barrel usage and blocking of the passage of current. The potency of adenosine as a depressant of the
Correspondence: J.W. Phillis, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A.
323 TABLE I Effect of chronic caffeine administration on the sensitivity of rat cerebral cortical neurons to the depressant effects of adenosine
Number of neurons I.'1"5o(mean + S.E.M.)
Control
Caffeine-treated
136 222.16 + 6.68
134 130.77+ 4.33***
*** Significantly different from corresponding value for salinetreated animals, P < 0.001 by the Student's t-test.
spontaneous activity of cerebral cortical neurons was estimated by the I'Ts0 (current in n A x time in seconds for 50% inhibition of firing) method 2. A n application current in the range of 20-50 n A was selected for each adenosine barrel and the time for which this current had to be applied to reduce neuronal firing rate by 50% measured for each neuron. Multiple I.Ts0 estimates were made on every cell, from which an average 1.'I"50value for the cell could be calculated. The same adenosine 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 I.Tso value for the 134 spontaneously active cortical neurons tested in the caffeine-treated animals was 130.7 + 4.33 (S.E.M.), which was significantly different (P < 0.001) to the I.T5o of 222.16 + 6.68 recorded for 136 neurons in the saline-treated control animals. The mean spontaneous firing rate for neurons in the caffeinetreated rats was 21.8 + 10.45 (S.E.M.) spikes/s, and that for neurons in the saline-treated controls was 19.4 + 9.4 spikes/s. Earlier studies involving the chronic administration of caffeine have revealed an up-regulation of the number of
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 Brunel, S. and DeMontigny, C., Validation of the I-Tso method for assessing neuronal responsiveness to microiontophoretic applications: a single cell recording study, J. Pharm. Methods, 19 (1988) 23-30. 3 Daval, J.-L,, Deckert, J., Weiss, S.R.B., Post, R.M. and Marangos, P.J., Up-regulation of adenosine A~ receptors and forskolin binding sites following chronic treatment with caffeine or carbamazepine: a quantitative autoradiographic study, Epilepsia, 30 (1989) 26-33. 4 Green, M.R. and Stiles, G.L., Chronic caffeine ingestion sensitizes the A 1 adenosine receptor-adenylate cyclase system in rat cerebral cortex, J. Clin. Invest., 77 (1986) 222-227. 5 Fredholm, B.B., Adenosine actions and adenosine receptors after 1 week with caffeine, Acta Physiol. Scand., 115 (1982) 283-286. 6 Hawkins, M., Dugich, M.M., Porter, N.M., Urbancic, M. and
adenosine receptor binding sites in the brain. In another binding study 4 the A t receptors in control rat cortical membranes were shown to exist in both high and low affinity states. After treatment with caffeine, all A 1 receptors were shifted to the high affinity state. In addition, membranes from caffeine-treated rats displayed enhanced inhibition of adenylate cyclase as mediated by the A 1 receptor 4. Thus the altered radioligand binding following caffeine administration is associated with functional changes in the receptor coupling system. A similar increase in the Bma x for adenosine ligand binding has also been demonstrated following chronic theophylline administration, which was associated with a reduced sensitivity to convulsants H. These results were interpreted as suggesting that the reduction in seizures in animals chronically treated with theophylline represented a functional change resulting from supersensitivity to adenosine. The results of the present experiments support this interpretation of the functional consequences of an up-regulation of adenosine receptors, in that the I.T50 for adenosine inhibition of cortical neuronal firing was reduced by 41% following chronic caffeine administration. The data reported here can be compared with that of Wu and Coffin 12 who administered a comparable dose of caffeine (75 mg/kg) for 12 days and observed a 123% increase in the Brnax for R-[3H]PIA binding to rat forebrain membranes. Considered together these results suggest that chronic exposure to methylxanthines results both in an increase in the number of adenosine receptors and an enhancement of the effects of activation of these receptors on membrane excitability. Supported by NIH Grant RR 08167-09, D.R.R.
7 8 9 10 11
12
Radulovacki, M., Effects of chronic administration of caffeine on adenosine A 1 and A 2 receptors in rat brain, Brain Res. Bull., 21 (1988) 479-482. Murray, T.F., Up-regulation of rat cortical adenosine receptors following chronic administration of theophylline, Eur. J. Pharmacol., 82 (1982) 113-114. Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239. Snyder, S.H., Adenosine as a neuromodulator, Ann. Rev. Neurosci., 8 (1985) 103-124. Stone, T.W,, Purine receptors and their pharmacological roles, Adv. Drug Res., 18 (1989) 292-429. Szot, P., Sanders, R.C. and Murray, T.E, Theophylline-induced up-regulation of Aradenosine receptors associated with redued sensitivity to convulsants, Neuropharmacology, 26 (1987) 11731180. 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.
322 BRES 24123
Chronic caffeine exposure enhances adenosinergic inhibition of cerebral cortical neurons Yu Lin and J.W. Phillis Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.) (Accepted 27 February 1990)
Key words: Chronic caffeine; Adenosine; Inhibition; Up-regulation; Cerebral cortex
Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10-70 mg/kg) increased the sensitivity of rat cerebral cortical neurons to the inhibitory action of microiontophoretically applied adenosine. 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. Adenosine sensitivity was determined by the 1.1"5omethod. The I.Tso value for 134 neurons in the caffeine-treated rats of 130.77 + 4.33 (S.E.M.) was significantly (P < 0.001) different to that of 136 neurons in the saline-treated control rats (222.16 + 6.68), indicating a supersensitivity to adenosine in neurons which had been chronically exposed to caffeine. Caffeine is widely used as a psychoactive agent and it is now generally accepted that its central stimulant actions are mediated through a competitive inhibition of adenosine receptors in the brain s-l°. Chronic exposure to caffeine produces an up-regulation of adenosine A 1 and A 2 receptors in the rat forebrain 1'3'5'6'12. In these reports, however, no effort was made to evaluate the effects of this up-regulation of adenosine receptors on the depressant action of the purine on neuronal activity. The present study represents an attempt to ascertain whether the increase in adenosine receptor numbers resulting from chronic caffeine administration is paralleled by increases in neuronal sensitivity to adenosine-elicited inhibition. Rats injected daily with caffeine for a period of 14 days were used to ascertain whether there was an altered sensitivity of cerebral cortical neurons to purinergic inhibition. Ten male Sprague-Dawley rats (300-350 g) were housed in individual cages and kept on a 12-h light-dark cycle. Five 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 adenosine 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 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 the central 2 M NaC1 filled barrel of a multiple-barrel microelectrode (fabricated with fiber-fill capillary tubing). A n o t h e r barrel of the microelectrode was filled with 2 M NaCI for automatic current neutralization and two barrels were filled with adenosine hemisulphate (0.1 M, p H 3.67). To eliminate any possibility of variability in the apparent potency of adenosine due to electrode barrel characteristics, pairs of rats were tested with the same electrode on the same day. On 3 days 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 adenosine applied alternately from each barrel in both the caffeine-treated and control animals. The use of two adenosine-containing barrels minimized the difficulties associated with continuous barrel usage and blocking of the passage of current. The potency of adenosine as a depressant of the
Correspondence: J.W. Phillis, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A.
323 TABLE I Effect of chronic caffeine administration on the sensitivity of rat cerebral cortical neurons to the depressant effects of adenosine
Number of neurons I.'1"5o(mean + S.E.M.)
Control
Caffeine-treated
136 222.16 + 6.68
134 130.77+ 4.33***
*** Significantly different from corresponding value for salinetreated animals, P < 0.001 by the Student's t-test.
spontaneous activity of cerebral cortical neurons was estimated by the I'Ts0 (current in n A x time in seconds for 50% inhibition of firing) method 2. A n application current in the range of 20-50 n A was selected for each adenosine barrel and the time for which this current had to be applied to reduce neuronal firing rate by 50% measured for each neuron. Multiple I.Ts0 estimates were made on every cell, from which an average 1.'I"50value for the cell could be calculated. The same adenosine 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 I.Tso value for the 134 spontaneously active cortical neurons tested in the caffeine-treated animals was 130.7 + 4.33 (S.E.M.), which was significantly different (P < 0.001) to the I.T5o of 222.16 + 6.68 recorded for 136 neurons in the saline-treated control animals. The mean spontaneous firing rate for neurons in the caffeinetreated rats was 21.8 + 10.45 (S.E.M.) spikes/s, and that for neurons in the saline-treated controls was 19.4 + 9.4 spikes/s. Earlier studies involving the chronic administration of caffeine have revealed an up-regulation of the number of
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 Brunel, S. and DeMontigny, C., Validation of the I-Tso method for assessing neuronal responsiveness to microiontophoretic applications: a single cell recording study, J. Pharm. Methods, 19 (1988) 23-30. 3 Daval, J.-L,, Deckert, J., Weiss, S.R.B., Post, R.M. and Marangos, P.J., Up-regulation of adenosine A~ receptors and forskolin binding sites following chronic treatment with caffeine or carbamazepine: a quantitative autoradiographic study, Epilepsia, 30 (1989) 26-33. 4 Green, M.R. and Stiles, G.L., Chronic caffeine ingestion sensitizes the A 1 adenosine receptor-adenylate cyclase system in rat cerebral cortex, J. Clin. Invest., 77 (1986) 222-227. 5 Fredholm, B.B., Adenosine actions and adenosine receptors after 1 week with caffeine, Acta Physiol. Scand., 115 (1982) 283-286. 6 Hawkins, M., Dugich, M.M., Porter, N.M., Urbancic, M. and
adenosine receptor binding sites in the brain. In another binding study 4 the A t receptors in control rat cortical membranes were shown to exist in both high and low affinity states. After treatment with caffeine, all A 1 receptors were shifted to the high affinity state. In addition, membranes from caffeine-treated rats displayed enhanced inhibition of adenylate cyclase as mediated by the A 1 receptor 4. Thus the altered radioligand binding following caffeine administration is associated with functional changes in the receptor coupling system. A similar increase in the Bma x for adenosine ligand binding has also been demonstrated following chronic theophylline administration, which was associated with a reduced sensitivity to convulsants H. These results were interpreted as suggesting that the reduction in seizures in animals chronically treated with theophylline represented a functional change resulting from supersensitivity to adenosine. The results of the present experiments support this interpretation of the functional consequences of an up-regulation of adenosine receptors, in that the I.T50 for adenosine inhibition of cortical neuronal firing was reduced by 41% following chronic caffeine administration. The data reported here can be compared with that of Wu and Coffin 12 who administered a comparable dose of caffeine (75 mg/kg) for 12 days and observed a 123% increase in the Brnax for R-[3H]PIA binding to rat forebrain membranes. Considered together these results suggest that chronic exposure to methylxanthines results both in an increase in the number of adenosine receptors and an enhancement of the effects of activation of these receptors on membrane excitability. Supported by NIH Grant RR 08167-09, D.R.R.
7 8 9 10 11
12
Radulovacki, M., Effects of chronic administration of caffeine on adenosine A 1 and A 2 receptors in rat brain, Brain Res. Bull., 21 (1988) 479-482. Murray, T.F., Up-regulation of rat cortical adenosine receptors following chronic administration of theophylline, Eur. J. Pharmacol., 82 (1982) 113-114. Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239. Snyder, S.H., Adenosine as a neuromodulator, Ann. Rev. Neurosci., 8 (1985) 103-124. Stone, T.W,, Purine receptors and their pharmacological roles, Adv. Drug Res., 18 (1989) 292-429. Szot, P., Sanders, R.C. and Murray, T.E, Theophylline-induced up-regulation of Aradenosine receptors associated with redued sensitivity to convulsants, Neuropharmacology, 26 (1987) 11731180. 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.
