Brain Research, 530 (1990) 353-357 Elsevier
353
BRES 24344
Chronic benzodiazepine treatment and cortical responses to adenosine and GABA Judit Mally*, J.H. Connick and T.W. Stone Department of Pharmacology, University of Glasgow, Glasgow (U.K.) (Accepted 10 July 1990)
Key words: Hippocampus; Adenosine; 7-Aminobutyricacid; Purine; Benzodiazepine; Clonazepam; Diazepam
The effects of chronic treatment of mice with clonazepam have been examined on the responses of neocortical slices to adenosine, 5-hydroxytryptamine (5-HT) and ~,-aminobutyric acid (GABA). Responses to these agonists were measured as changes in the depolarisation induced by N-methyl-D-aspartate (NMDA). Added to the superfusion medium diazepam blocked responses to adenosine but not 5-HT; this effect was not observed with 2-chioroadenosineor in the presence of 2-hydroxynitrobenzylthioguanosine.GABA was inactive in control slices but chronic treatment with clonazepam induced responses to GABA and enhanced responses to adenosine but not 5-HT. It is suggested that the induction of GABA responses may reflect the up-regulation of GABA receptors, but the increase of adenosine responses by clonazepam implies that there is no simple relationship between adenosine receptor binding and functional responses. In previous work we have used the superfused mouse neocortical slice preparation to determine the effects of theophylline, both acutely and after chronic administration, on responses to 5-hydroxytryptamine (5-HT) and adenosine 6. That study revealed that the neocortical slice could be used to detect consistent and reproducible changes of transmitter responses after chronic treatments and the present project was therefore designed to examine possible changes of sensitivity to ~-aminobutyric acid (GABA) and adenosine following the administration of benzodiazepines. It was hypothesised that changes of G A B A and adenosine responses might occur if these mediators were involved in the actions of benzodiazepines on central neurons. Male mice were killed by cervical dislocation and the brain removed into cold bicarbonate buffered medium of the following composition (mM): NaCI 115, KCI 3, KH2PO 4 1.5, CaC12 2.5, MgSO 4 1.2, NaHCO 3 25, glucose 10. Sections of the brain were cut at 500/~m using a vibratome, in a coronal plane to include regions of frontoparietal and cingulate cortex as described in detail previously 1. These slices were then trimmed and transferred to two-compartment chambers where they were positioned across a greased slot in the dividing wall so that most of the grey matter lay on one side while the white matter extended into the second chamber. Silver/silver chloride electrodes in contact with the solutions on the two sides of the dividing wall were used
to record DC potentials which were displayed on digital oscilloscopes and chart recorders. The amplitude of induced DC potentials was used to quantify the drug effects. Both compartments were superfused with the bicarbonate buffer. N-Methyl-D-aspartate (NMDA) was added into the fluid superfusing the pial side of the slice for one minute periods. Other agonists were applied for two minute periods beginning one minute before NMDA. After obtaining control responses to agonists, antagonists were superfused for at least 20 rain before further applications of agonists. Statistical comparisons were made using Student's t-test. For chronic administration, theophylline was dissolved fresh in dilute sodium hydroxide (pH 5.0) and injected immediately. Propranolol was dissolved in saline and clonazepam was administered from ampoules for injection (Rivotril, Roche). All injections were given i.p. Slices were prepared from mice 24 h after the final injection of drug. When applied alone to cortical slices neither G A B A nor adenosine up to 1 mM concentrations had any effect on the recorded DC potential of the slices. The same protocol was therefore adopted as in the previous paper 6, the amino acid N M D A being used to induce control depolarising DC shifts of slice potential and the changes of NMDA responses produced by other agents being used to quantify their effects. Even using this model, however, G A B A had no effect on N M D A responses in
* Present address: County Hospital Fejer, Szekesfehervar, Hungary. Correspondence: T.W. Stone, Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
354 200-
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tJ 11}0-
Iii
51}-
121
GABA
-
Oiazepam lquM
Adenosine
÷
-
+
-
5HI 4
Fig. 1. Histogram showing the size of responses obtained using NMDA in the presence of GABA (100 pM), adenosine (20 ~M) or 5-HT (10 pM) and the presence or absence of diazepam 10 #M. The columns indicate the mean _+ 1 S.E.M. and the enclosed figures indicate the number of experiments. **P < 0.01 compared with NMDA controls.
control slices at concentrations up to 1 mM. Further experiments were conducted with G A B A at a single concentration of 100 HM. Adenosine, 20/~M increased responses to N M D A to 148.8% + 10.9 (n = 21; P < 0.01) of control size. This concentration of adenosine was selected on the basis of the dose response relationships presented previously. 5 HT, 10 HM, was included for comparative purposes in some of these experiments and
*
t
yielded a potentiation of N M D A responses of 148.4% _+ 8.8 (n = 7; P < 0.01) of control. Perfusion with diazepam. The inclusion of diazepam, 10 HM, in the superfusion medium had no effect alone upon responses to N M D A and a combination of 100 pM G A B A and 10 HM diazepam, did not cause any significant change in the response size (Fig. 1). Similarly diazepam did not modify the potentiation of N M D A responses produced by 5-HT. However diazepam did appear to prevent the potentiation of N M D A responses produced by adenosine, 20 aM, reducing the response size to control levels (Fig. 1). Chronic treatment with clonazeparn. Groups of mice were treated chronically with clonazepam 2 mg/kg/day for 14 days. Clonazepam was selected for use in this section of the study partly because of its more selective action on the CNS and partly because of its longer duration of action following single daily injections. Clonazepam treatment did not itself modify responses to N M D A but, as illustrated in Fig. 2, G A B A now induced a significant enhancement of N M D A responses following the treatment. Similarly the increase of N M D A responses produced by adenosine was further significantly increased after chronic clonazepam to 193.7% + 23.2 (n = 7) of control. In contrast no change was detectable in the ability of 5-HT to change N M D A depolarisation (Fig. 2). Interaction of GABA and adenosine. Because of the unexpected observation that diazepam superfused in vitro could reduce responses to adenosine additional experiments were performed in an attempt to clarify the mechanism. Firstly it was confirmed that the observed effects of adenosine were being mediated at least partly through conventional A1 receptors by the fact that they
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511-
+
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treatment
Fig. 2. Histogram showing the size of responses obtained using NMDA in the presence of GABA (100 ~M), adenosine (20 aM) or 5-HT (10 aM) with or without chronic treatment with clonazepam 2 mg/kg/day for 14 days. The columns indicate the mean + 1 S.E.M. and the enclosed figures indicate the number of experiments. *P < 0.05 compared with NMDA controls. **P < 0.01 compared with NMDA controls. +P < 0.05 compared with preparations from drug naive animals.
10 Adenosine
I
t-lhe0ph,-I I--SP T----I 10 100 0.1 1 ~M Fig. 3. Histogram showing the size of responses obtained using NMDA in the presence of adenosine (20 HM) and either theophylline 10 or 100#M or 8-phenyltheophylline 0.1 or 1/~M. The columns indicate the mean 4- 1 S.E.M. and the enclosed figures indicate the number of experiments. *P < 0.05 compared with NMDA controls. **P < 0.01 compared with NMDA controls.
355 could be partially blocked either by theophylline (10-100 /~M) or 8-phenyltheophyUine (0.1 - 1/~M) (Fig. 3). Secondly, in the presence of G A B A , adenosine was no longer able to modify the size of responses to N M D A , implying that G A B A might be acting as a functional antagonist of adenosine in this system (Fig. 4). A similar reduction of responses to 2-chloroadenosine was also observed. Since one explanation of the effects of both G A B A and diazepam in reducing adenosine responses might be that they were increasing the uptake or metabolism of adenosine, the effect of diazepam was also examined on responses to 2-chloroadenosine (1 /~M) which is not removed readily by either of these processes. This combination potentiated N M D A depolarisation by 127.1% + 6.1 (n = 6) compared with the potentiation seen by 2-chloroadenosine alone of 140.3% + 8.3 (n = 5). This difference was not significant (Fig. 4). This result also indicates that diazepam was not able to antagonise responses via adenosine receptors. Thirdly the effect of diazepam, 10/~M, on adenosine responses was tested in the presence of a selective inhibitor of the nucleoside transporter, 2-hydroxynitrobenzylthioguanosine (HNBTG) (5/~M). In the presence of this combination the response size reached 141.0% + 10.9 (n = 8). This was not significantly different from the effect of adenosine itself (Fig. 4). Application of H N B T G alone potentiated the effect of adenosine from 136% + 11.2 (n = 4) to 159.4% + 7.6 (n = 4) but this was not statistically significant. For comparison with the clonazepam treatment several animals were pretreated with theophylline, 10 or 100
200-
v
NS
r
NS'--I
mg/kg/day or propranolol, 5 or 25 mg/kg/day as in the previous study. After treatment with the higher dose of theophylline and both doses of propranolol G A B A was able to induce significant elevations in the size of responses to N M D A (Fig. 5). In the absence of G A B A , N M D A responses were no different from controls. Some of the observed interactions are difficult to explain. It might be expected that G A B A and adenosine would reduce responses to N M D A , yet the former was inactive in acute experiments and the latter potentiated adenosine, as reported previously 6. The most likely explanation for this may lie in the complexity of the neocortical slices, in which the net effect on N M D A sensitivity may depend on the balance of excitatory and inhibitory influences. The potentiation of N M D A by adenosine for example may result from a net disinhibition of pyramidal cells. Many of the central effects of benzodiazepines are believed to involve a potentiation of G A B A at the receptor-ionophore complex, leading to an increase in the frequency of chloride channel openings 11. An alternative possibility, that potentiation of endogenous adenosine might be involved, was proposed on the basis that some correlation could be demonstrated between the clinical potency of a series of benzodiazepines and their ability to inhibit the uptake, and thereby potentiate the effects of, adenosine 14'21. Strong arguments have been made against this view, for example from the realisation that antagonists of the behavioural effects of benzodiazepines did not prevent the inhibition of nucleoside uptake 9. Indeed, some of those antagonists actually shared the inhibitory effects of the 'agonist' benzodiazepines 1°. Nevertheless, there remain valid reasons for believing in some close relationship between benzodiazepines and adenosine, including demonstrations that dipyridamole can inhibit benzodi-
150/-/ //
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8 GABA
Aden.
8; GABA +
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Aden.
Aden. ÷
2CA
2CA +
DlazeDam
Dlazeoam
Fig. 4. Histogram showing the size of responses obtained using NMDA in the presence of GABA (100pM), adenosine (20pM), or a combination of adenosine with GABA, and adenosine with NBTG (5 pM) and diazepam (10 pM). Also shown is the effect of 2-chloroadenosine (1 pM) and its combination with diazepam (10 pM). Details as for Fig. 2.
'GABA
I-------TheoPhyll|ne----.I 10 100
I /----- Pro0ran010 I 5
25
mg/kglday
Fig. 5. Histogram showing the size of responses obtained using NMDA in the presence of GABA 100/~M in slices from drug naive animals and preparations from animals treated with theophylline 10 or 100 mg/kg/day or propranolol 5 or 25 mg/kg/day for 14 days. Details as for Fig. 2.
356 azepine binding 2 and antagonise some effects of these drugs 3. In addition, xanthine antagonists of adenosine are able to antagonise some effects of benzodiazepines s" 11.13,15.18. These various interactions may be related more to the sedative than the anxiolytic activity of benzodiazepines ~s. Some of these interactions have been covered in recent reviews 17'18. A particularly striking argument for a purine/benzodiazepine relationship is that the chronic administration of benzodiazepines can modify adenosine receptors. Chronic treatment with diazepam for example induces a down-regulation of adenosine receptor number in the CNS 4. Conversely, if animals are treated chronically with an adenosine antagonist such as caffeine, several studies have revealed an up-regulation of binding sites for benzodiazepine ligands 19'2°. The present study represents an attempt to examine adenosine sensitivity in a functional electropharmacological system. The results indicate that chronic treatment with the CNS specific benzodiazepine clonazepam results in an increased sensitivity of cortical slices to both adenosine and GABA. The absence of any change of 5 HT responses would seem to exclude the possibility that the increases of sensitivity observed are the result of some non-specific effect of clonazepam on cortical function. The induction of responses to G A B A would be entirely consistent with evidence that chronic clonazepam increases the number of muscimol binding sites in the CNS 7. The enhancement of adenosine responses is contrary to that expected from a simple interpretation of the binding data, but in a previous study we have reported that chronic treatment with theophylline resulted in a decrease, not the predicted increase of adenosine responses 6. In that respect the present results are not inconsistent with the view that chronic benzodiazepines down-regulate adenosine receptors: there may not be a simple relationship between adenosine receptor density and physiological activity. In addition, the initial superfusion experiments provide a further explanation for the chronic results, since superfusion with diazepam was able to block the effect of adenosine. This unexpected result may indicate that benzodiazepines are able to increase adenosine uptake or 1 Burton, N.R., Smith, D.A.S. and Stone, T.W., The mouse neocortical slice: preparation and responses to excitatory amino acids, Comp. Biochern. Physiol., 88C (1987) 47-55. 2 Davies, L.P., Cook, A.F., Poonian, A.M. and Taylor, K.M., Displacement of [3H]-diazepam binding in rat brain by dipyridamole and by 1-methyl-isoguanosine.A natural marine product with muscle relaxant activity, Life Sci., 26 (1980) 1089-1097. 3 Davies, L.P., Chow, S.C. and Johnston, G.A.R., Interactions of purines and related compounds with photoaffinity labeled benzodiazepine receptors in rat brain membranes, Eur. J.
metabolism in the mouse neocortex; no comparable interaction was noted with 2-chloroadenosine, and diazepam no longer prevented adenosine's effect when tested in the presence of the uptake inhibitor NBTG. The mechanism of this acute effect of diazepam is unclear, but may involve a potentiation of endogenous G A B A since this amino acid was also found to reduce adenosine and 2-chloroadenosine responses. This effect may be quite independent of the direct inhibitory effect of diazepam on the nucleoside transporter 2~ but may be related to the finding that chronic xanthine treatment can reduce the GABA/benzodiazepine receptor interaction in neuronal cultures 16 such that benzodiazepines no longer modified G A B A binding. The receptor interaction could then be restored by performing the experiments in the presence of 2-chloroadenosine, emphasising further the existence of a complex interdependence between the adenosine, G A B A and benzodiazepine receptors. One of the incidental observations in this study was that chronic treatment with propranolol induced responses to G A B A whereas none were seen in drug naive mice. At present we have no explanation of this effect, but it is interesting to speculate on the possibility that it may contribute to the depressant effects of propranolol on CNS function. Chronic treatment of patients with either propranolol or theophylline has been found effective in the treatment of essential tremor 4, for example, and since both treatments here induced responses to G A B A it is possible that heightened sensitivity to this amino acid could be a common functional factor in the amelioration of tremor. Clearly the present data are difficult to reconcile with much of the binding work performed previously. However the results are internally consistent between this study and our previous report and may indicate major species differences which could be important for work on the mechanisms of action of benzodiazepines and the role of adenosine in the CNS. Alternatively the results may be highlighting the need for caution in extrapolating from binding data to functional responses.
We are grateful to the WellcomeTrust for grant support and the British Council for a travel grant to J.M. Pharmacol., 97 (1984) 325-329. 4 Hawkins, M., Pravica, M. and Radulovacki, M., Chronic administration of diazepam down-regulates adenosine receptors in the rat brain, Pharmacol. Biochem. Behav., 30 (1988) 303-308. 5 Maily, J., Aminophylline and essential tremor, Lancet, 2 (1989) 278-279. 6 Mally, J., Connick, J.H. and Stone, T.W., Theophylline downregulates adenosine receptor function, Brain Research, 509 (1990) 141-144.
357 7 Marangos, P.J. and Crawley, J.N., Chronic benzodiazepine treatment increases [3H]muscimol binding in mouse brain, Neuropharmacology, 21 (1982) 81-84. 8 Mattila, M.J. and Nuotto, E., Caffeine and theophylline counteract diazepam effects in man, Med. Biol., 61 (1983) 337-343. 9 Morgan, P.E, Lloyd, H.G.E. and Stone, T.W., Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists, Eur. J. Pharmacol., 87 (1983) 121-126. 10 Morgan, P.E, Lloyd, H.G.E. and Stone, T.W., Inhibition of adenosine accumulation by a CNS benzodiazepine antagonist (Ro15-1788) and a peripheral benzodiazepine receptor ligand (Ro05-4864), Neurosci. Lett., 41, 183-188. 11 Niemand, D., MartineU, S., Arvidsson, S., Svedmyr, N. and Ekstrom-Jodal, B., Aminophylline inhibition of diazepam sedation: is adenosine blockade of GABA receptors the mechanism? Lancet, 1 (1984) 463-464. 12 Olsen, R.W., Yang, J., King, R.G., Dilber, A., Stauber, G.B. and Ransom, R.W., Barbiturate and benzodiazepine moduation of GABA receptor binding and function, Life Sci., 39 (1986) 1969-1976. 13 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239.
14 Phillis, J.W. and Wu, P.H., Role of adenosine and adenine nucleotides in the CNS, In Adenosine Derivatives, J.W. Daly, K. Kuroda, J.W. Phillis, H. Shimizu, and M. Ui (Eds.), Raven, New York, 1983, pp. 219-236. 15 Polc, P., Bonetti, E.P., Pieri, L., Cumin, R., Angioi, R.M., Mohler, H. and Haefely, W.E., Caffeine antagonises several central effects of diazepam, Life Sci., 28 (1981) 2265-2275. 16 Roca, D.J., Schiller, G.D. and Farb, D.H., Chronic caffeine or theophyUine exposure reduces GABA/benzodiazepine site interactions, Mol. Pharmacol., 33 (1988) 481-485. 17 Stone, T.W., Purine receptors and their pharmacological roles, Adv. Drug Res., 18 (1989) 292-429. 18 Williams, M. and Yokoyama, N., Anxiolytics, anticonvulsants and sedative-hypnotics, Ann. Rep. Med. Chem., 21 (1986) 11-20.
19 Wu, P. and Coffin, V.L., Up-regulation of brain [3H]-diazepam binding sites in chronic caffeine treated rats, Brain Research, 294 (1984) 186-189. 20 Wu, P.H. and Phillis, J.W., Up-regulation of [3H]-diazepam binding sites in chronic caffeine treated rats, Gen. Pharmacol., 17 (1986) 501-504. 21 Wu, P.H., Phillis, J.W. and Bender, A.S., Do benzodiazepines bind at adenosine uptake sites in the CNS? Life Sci., 28 (1981) 1023-1031.
353
BRES 24344
Chronic benzodiazepine treatment and cortical responses to adenosine and GABA Judit Mally*, J.H. Connick and T.W. Stone Department of Pharmacology, University of Glasgow, Glasgow (U.K.) (Accepted 10 July 1990)
Key words: Hippocampus; Adenosine; 7-Aminobutyricacid; Purine; Benzodiazepine; Clonazepam; Diazepam
The effects of chronic treatment of mice with clonazepam have been examined on the responses of neocortical slices to adenosine, 5-hydroxytryptamine (5-HT) and ~,-aminobutyric acid (GABA). Responses to these agonists were measured as changes in the depolarisation induced by N-methyl-D-aspartate (NMDA). Added to the superfusion medium diazepam blocked responses to adenosine but not 5-HT; this effect was not observed with 2-chioroadenosineor in the presence of 2-hydroxynitrobenzylthioguanosine.GABA was inactive in control slices but chronic treatment with clonazepam induced responses to GABA and enhanced responses to adenosine but not 5-HT. It is suggested that the induction of GABA responses may reflect the up-regulation of GABA receptors, but the increase of adenosine responses by clonazepam implies that there is no simple relationship between adenosine receptor binding and functional responses. In previous work we have used the superfused mouse neocortical slice preparation to determine the effects of theophylline, both acutely and after chronic administration, on responses to 5-hydroxytryptamine (5-HT) and adenosine 6. That study revealed that the neocortical slice could be used to detect consistent and reproducible changes of transmitter responses after chronic treatments and the present project was therefore designed to examine possible changes of sensitivity to ~-aminobutyric acid (GABA) and adenosine following the administration of benzodiazepines. It was hypothesised that changes of G A B A and adenosine responses might occur if these mediators were involved in the actions of benzodiazepines on central neurons. Male mice were killed by cervical dislocation and the brain removed into cold bicarbonate buffered medium of the following composition (mM): NaCI 115, KCI 3, KH2PO 4 1.5, CaC12 2.5, MgSO 4 1.2, NaHCO 3 25, glucose 10. Sections of the brain were cut at 500/~m using a vibratome, in a coronal plane to include regions of frontoparietal and cingulate cortex as described in detail previously 1. These slices were then trimmed and transferred to two-compartment chambers where they were positioned across a greased slot in the dividing wall so that most of the grey matter lay on one side while the white matter extended into the second chamber. Silver/silver chloride electrodes in contact with the solutions on the two sides of the dividing wall were used
to record DC potentials which were displayed on digital oscilloscopes and chart recorders. The amplitude of induced DC potentials was used to quantify the drug effects. Both compartments were superfused with the bicarbonate buffer. N-Methyl-D-aspartate (NMDA) was added into the fluid superfusing the pial side of the slice for one minute periods. Other agonists were applied for two minute periods beginning one minute before NMDA. After obtaining control responses to agonists, antagonists were superfused for at least 20 rain before further applications of agonists. Statistical comparisons were made using Student's t-test. For chronic administration, theophylline was dissolved fresh in dilute sodium hydroxide (pH 5.0) and injected immediately. Propranolol was dissolved in saline and clonazepam was administered from ampoules for injection (Rivotril, Roche). All injections were given i.p. Slices were prepared from mice 24 h after the final injection of drug. When applied alone to cortical slices neither G A B A nor adenosine up to 1 mM concentrations had any effect on the recorded DC potential of the slices. The same protocol was therefore adopted as in the previous paper 6, the amino acid N M D A being used to induce control depolarising DC shifts of slice potential and the changes of NMDA responses produced by other agents being used to quantify their effects. Even using this model, however, G A B A had no effect on N M D A responses in
* Present address: County Hospital Fejer, Szekesfehervar, Hungary. Correspondence: T.W. Stone, Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
354 200-
150o
tJ 11}0-
Iii
51}-
121
GABA
-
Oiazepam lquM
Adenosine
÷
-
+
-
5HI 4
Fig. 1. Histogram showing the size of responses obtained using NMDA in the presence of GABA (100 pM), adenosine (20 ~M) or 5-HT (10 pM) and the presence or absence of diazepam 10 #M. The columns indicate the mean _+ 1 S.E.M. and the enclosed figures indicate the number of experiments. **P < 0.01 compared with NMDA controls.
control slices at concentrations up to 1 mM. Further experiments were conducted with G A B A at a single concentration of 100 HM. Adenosine, 20/~M increased responses to N M D A to 148.8% + 10.9 (n = 21; P < 0.01) of control size. This concentration of adenosine was selected on the basis of the dose response relationships presented previously. 5 HT, 10 HM, was included for comparative purposes in some of these experiments and
*
t
yielded a potentiation of N M D A responses of 148.4% _+ 8.8 (n = 7; P < 0.01) of control. Perfusion with diazepam. The inclusion of diazepam, 10 HM, in the superfusion medium had no effect alone upon responses to N M D A and a combination of 100 pM G A B A and 10 HM diazepam, did not cause any significant change in the response size (Fig. 1). Similarly diazepam did not modify the potentiation of N M D A responses produced by 5-HT. However diazepam did appear to prevent the potentiation of N M D A responses produced by adenosine, 20 aM, reducing the response size to control levels (Fig. 1). Chronic treatment with clonazeparn. Groups of mice were treated chronically with clonazepam 2 mg/kg/day for 14 days. Clonazepam was selected for use in this section of the study partly because of its more selective action on the CNS and partly because of its longer duration of action following single daily injections. Clonazepam treatment did not itself modify responses to N M D A but, as illustrated in Fig. 2, G A B A now induced a significant enhancement of N M D A responses following the treatment. Similarly the increase of N M D A responses produced by adenosine was further significantly increased after chronic clonazepam to 193.7% + 23.2 (n = 7) of control. In contrast no change was detectable in the ability of 5-HT to change N M D A depolarisation (Fig. 2). Interaction of GABA and adenosine. Because of the unexpected observation that diazepam superfused in vitro could reduce responses to adenosine additional experiments were performed in an attempt to clarify the mechanism. Firstly it was confirmed that the observed effects of adenosine were being mediated at least partly through conventional A1 receptors by the fact that they
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50-
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+
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treatment
Fig. 2. Histogram showing the size of responses obtained using NMDA in the presence of GABA (100 ~M), adenosine (20 aM) or 5-HT (10 aM) with or without chronic treatment with clonazepam 2 mg/kg/day for 14 days. The columns indicate the mean + 1 S.E.M. and the enclosed figures indicate the number of experiments. *P < 0.05 compared with NMDA controls. **P < 0.01 compared with NMDA controls. +P < 0.05 compared with preparations from drug naive animals.
10 Adenosine
I
t-lhe0ph,-I I--SP T----I 10 100 0.1 1 ~M Fig. 3. Histogram showing the size of responses obtained using NMDA in the presence of adenosine (20 HM) and either theophylline 10 or 100#M or 8-phenyltheophylline 0.1 or 1/~M. The columns indicate the mean 4- 1 S.E.M. and the enclosed figures indicate the number of experiments. *P < 0.05 compared with NMDA controls. **P < 0.01 compared with NMDA controls.
355 could be partially blocked either by theophylline (10-100 /~M) or 8-phenyltheophyUine (0.1 - 1/~M) (Fig. 3). Secondly, in the presence of G A B A , adenosine was no longer able to modify the size of responses to N M D A , implying that G A B A might be acting as a functional antagonist of adenosine in this system (Fig. 4). A similar reduction of responses to 2-chloroadenosine was also observed. Since one explanation of the effects of both G A B A and diazepam in reducing adenosine responses might be that they were increasing the uptake or metabolism of adenosine, the effect of diazepam was also examined on responses to 2-chloroadenosine (1 /~M) which is not removed readily by either of these processes. This combination potentiated N M D A depolarisation by 127.1% + 6.1 (n = 6) compared with the potentiation seen by 2-chloroadenosine alone of 140.3% + 8.3 (n = 5). This difference was not significant (Fig. 4). This result also indicates that diazepam was not able to antagonise responses via adenosine receptors. Thirdly the effect of diazepam, 10/~M, on adenosine responses was tested in the presence of a selective inhibitor of the nucleoside transporter, 2-hydroxynitrobenzylthioguanosine (HNBTG) (5/~M). In the presence of this combination the response size reached 141.0% + 10.9 (n = 8). This was not significantly different from the effect of adenosine itself (Fig. 4). Application of H N B T G alone potentiated the effect of adenosine from 136% + 11.2 (n = 4) to 159.4% + 7.6 (n = 4) but this was not statistically significant. For comparison with the clonazepam treatment several animals were pretreated with theophylline, 10 or 100
200-
v
NS
r
NS'--I
mg/kg/day or propranolol, 5 or 25 mg/kg/day as in the previous study. After treatment with the higher dose of theophylline and both doses of propranolol G A B A was able to induce significant elevations in the size of responses to N M D A (Fig. 5). In the absence of G A B A , N M D A responses were no different from controls. Some of the observed interactions are difficult to explain. It might be expected that G A B A and adenosine would reduce responses to N M D A , yet the former was inactive in acute experiments and the latter potentiated adenosine, as reported previously 6. The most likely explanation for this may lie in the complexity of the neocortical slices, in which the net effect on N M D A sensitivity may depend on the balance of excitatory and inhibitory influences. The potentiation of N M D A by adenosine for example may result from a net disinhibition of pyramidal cells. Many of the central effects of benzodiazepines are believed to involve a potentiation of G A B A at the receptor-ionophore complex, leading to an increase in the frequency of chloride channel openings 11. An alternative possibility, that potentiation of endogenous adenosine might be involved, was proposed on the basis that some correlation could be demonstrated between the clinical potency of a series of benzodiazepines and their ability to inhibit the uptake, and thereby potentiate the effects of, adenosine 14'21. Strong arguments have been made against this view, for example from the realisation that antagonists of the behavioural effects of benzodiazepines did not prevent the inhibition of nucleoside uptake 9. Indeed, some of those antagonists actually shared the inhibitory effects of the 'agonist' benzodiazepines 1°. Nevertheless, there remain valid reasons for believing in some close relationship between benzodiazepines and adenosine, including demonstrations that dipyridamole can inhibit benzodi-
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DlazeDam
Dlazeoam
Fig. 4. Histogram showing the size of responses obtained using NMDA in the presence of GABA (100pM), adenosine (20pM), or a combination of adenosine with GABA, and adenosine with NBTG (5 pM) and diazepam (10 pM). Also shown is the effect of 2-chloroadenosine (1 pM) and its combination with diazepam (10 pM). Details as for Fig. 2.
'GABA
I-------TheoPhyll|ne----.I 10 100
I /----- Pro0ran010 I 5
25
mg/kglday
Fig. 5. Histogram showing the size of responses obtained using NMDA in the presence of GABA 100/~M in slices from drug naive animals and preparations from animals treated with theophylline 10 or 100 mg/kg/day or propranolol 5 or 25 mg/kg/day for 14 days. Details as for Fig. 2.
356 azepine binding 2 and antagonise some effects of these drugs 3. In addition, xanthine antagonists of adenosine are able to antagonise some effects of benzodiazepines s" 11.13,15.18. These various interactions may be related more to the sedative than the anxiolytic activity of benzodiazepines ~s. Some of these interactions have been covered in recent reviews 17'18. A particularly striking argument for a purine/benzodiazepine relationship is that the chronic administration of benzodiazepines can modify adenosine receptors. Chronic treatment with diazepam for example induces a down-regulation of adenosine receptor number in the CNS 4. Conversely, if animals are treated chronically with an adenosine antagonist such as caffeine, several studies have revealed an up-regulation of binding sites for benzodiazepine ligands 19'2°. The present study represents an attempt to examine adenosine sensitivity in a functional electropharmacological system. The results indicate that chronic treatment with the CNS specific benzodiazepine clonazepam results in an increased sensitivity of cortical slices to both adenosine and GABA. The absence of any change of 5 HT responses would seem to exclude the possibility that the increases of sensitivity observed are the result of some non-specific effect of clonazepam on cortical function. The induction of responses to G A B A would be entirely consistent with evidence that chronic clonazepam increases the number of muscimol binding sites in the CNS 7. The enhancement of adenosine responses is contrary to that expected from a simple interpretation of the binding data, but in a previous study we have reported that chronic treatment with theophylline resulted in a decrease, not the predicted increase of adenosine responses 6. In that respect the present results are not inconsistent with the view that chronic benzodiazepines down-regulate adenosine receptors: there may not be a simple relationship between adenosine receptor density and physiological activity. In addition, the initial superfusion experiments provide a further explanation for the chronic results, since superfusion with diazepam was able to block the effect of adenosine. This unexpected result may indicate that benzodiazepines are able to increase adenosine uptake or 1 Burton, N.R., Smith, D.A.S. and Stone, T.W., The mouse neocortical slice: preparation and responses to excitatory amino acids, Comp. Biochern. Physiol., 88C (1987) 47-55. 2 Davies, L.P., Cook, A.F., Poonian, A.M. and Taylor, K.M., Displacement of [3H]-diazepam binding in rat brain by dipyridamole and by 1-methyl-isoguanosine.A natural marine product with muscle relaxant activity, Life Sci., 26 (1980) 1089-1097. 3 Davies, L.P., Chow, S.C. and Johnston, G.A.R., Interactions of purines and related compounds with photoaffinity labeled benzodiazepine receptors in rat brain membranes, Eur. J.
metabolism in the mouse neocortex; no comparable interaction was noted with 2-chloroadenosine, and diazepam no longer prevented adenosine's effect when tested in the presence of the uptake inhibitor NBTG. The mechanism of this acute effect of diazepam is unclear, but may involve a potentiation of endogenous G A B A since this amino acid was also found to reduce adenosine and 2-chloroadenosine responses. This effect may be quite independent of the direct inhibitory effect of diazepam on the nucleoside transporter 2~ but may be related to the finding that chronic xanthine treatment can reduce the GABA/benzodiazepine receptor interaction in neuronal cultures 16 such that benzodiazepines no longer modified G A B A binding. The receptor interaction could then be restored by performing the experiments in the presence of 2-chloroadenosine, emphasising further the existence of a complex interdependence between the adenosine, G A B A and benzodiazepine receptors. One of the incidental observations in this study was that chronic treatment with propranolol induced responses to G A B A whereas none were seen in drug naive mice. At present we have no explanation of this effect, but it is interesting to speculate on the possibility that it may contribute to the depressant effects of propranolol on CNS function. Chronic treatment of patients with either propranolol or theophylline has been found effective in the treatment of essential tremor 4, for example, and since both treatments here induced responses to G A B A it is possible that heightened sensitivity to this amino acid could be a common functional factor in the amelioration of tremor. Clearly the present data are difficult to reconcile with much of the binding work performed previously. However the results are internally consistent between this study and our previous report and may indicate major species differences which could be important for work on the mechanisms of action of benzodiazepines and the role of adenosine in the CNS. Alternatively the results may be highlighting the need for caution in extrapolating from binding data to functional responses.
We are grateful to the WellcomeTrust for grant support and the British Council for a travel grant to J.M. Pharmacol., 97 (1984) 325-329. 4 Hawkins, M., Pravica, M. and Radulovacki, M., Chronic administration of diazepam down-regulates adenosine receptors in the rat brain, Pharmacol. Biochem. Behav., 30 (1988) 303-308. 5 Maily, J., Aminophylline and essential tremor, Lancet, 2 (1989) 278-279. 6 Mally, J., Connick, J.H. and Stone, T.W., Theophylline downregulates adenosine receptor function, Brain Research, 509 (1990) 141-144.
357 7 Marangos, P.J. and Crawley, J.N., Chronic benzodiazepine treatment increases [3H]muscimol binding in mouse brain, Neuropharmacology, 21 (1982) 81-84. 8 Mattila, M.J. and Nuotto, E., Caffeine and theophylline counteract diazepam effects in man, Med. Biol., 61 (1983) 337-343. 9 Morgan, P.E, Lloyd, H.G.E. and Stone, T.W., Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists, Eur. J. Pharmacol., 87 (1983) 121-126. 10 Morgan, P.E, Lloyd, H.G.E. and Stone, T.W., Inhibition of adenosine accumulation by a CNS benzodiazepine antagonist (Ro15-1788) and a peripheral benzodiazepine receptor ligand (Ro05-4864), Neurosci. Lett., 41, 183-188. 11 Niemand, D., MartineU, S., Arvidsson, S., Svedmyr, N. and Ekstrom-Jodal, B., Aminophylline inhibition of diazepam sedation: is adenosine blockade of GABA receptors the mechanism? Lancet, 1 (1984) 463-464. 12 Olsen, R.W., Yang, J., King, R.G., Dilber, A., Stauber, G.B. and Ransom, R.W., Barbiturate and benzodiazepine moduation of GABA receptor binding and function, Life Sci., 39 (1986) 1969-1976. 13 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Prog. Neurobiol., 16 (1981) 187-239.
14 Phillis, J.W. and Wu, P.H., Role of adenosine and adenine nucleotides in the CNS, In Adenosine Derivatives, J.W. Daly, K. Kuroda, J.W. Phillis, H. Shimizu, and M. Ui (Eds.), Raven, New York, 1983, pp. 219-236. 15 Polc, P., Bonetti, E.P., Pieri, L., Cumin, R., Angioi, R.M., Mohler, H. and Haefely, W.E., Caffeine antagonises several central effects of diazepam, Life Sci., 28 (1981) 2265-2275. 16 Roca, D.J., Schiller, G.D. and Farb, D.H., Chronic caffeine or theophyUine exposure reduces GABA/benzodiazepine site interactions, Mol. Pharmacol., 33 (1988) 481-485. 17 Stone, T.W., Purine receptors and their pharmacological roles, Adv. Drug Res., 18 (1989) 292-429. 18 Williams, M. and Yokoyama, N., Anxiolytics, anticonvulsants and sedative-hypnotics, Ann. Rep. Med. Chem., 21 (1986) 11-20.
19 Wu, P. and Coffin, V.L., Up-regulation of brain [3H]-diazepam binding sites in chronic caffeine treated rats, Brain Research, 294 (1984) 186-189. 20 Wu, P.H. and Phillis, J.W., Up-regulation of [3H]-diazepam binding sites in chronic caffeine treated rats, Gen. Pharmacol., 17 (1986) 501-504. 21 Wu, P.H., Phillis, J.W. and Bender, A.S., Do benzodiazepines bind at adenosine uptake sites in the CNS? Life Sci., 28 (1981) 1023-1031.
