- Email: [email protected]
Rapid changes in cerebellar adenosine receptors following experimental seizures
Europem
Jounwl
01 Phumwcolog~~. 75 ( 198 I ) 79-80
Elsevier/North-Holland
Biomedical
19
Press
Rapid communication RAPID CHANGES SEIZURES MILTON
P. WYBENGA.
Deportmetlt
Received
IN CEREBELLAR
MARY
G. MURPHY
of Phormucologv, Faculty of Medicine,
8 September
1981. accepted
9 September
ADENOSINE
and HAROLD
0014-2999/g
I /OOOO-0000/$02.75
A. ROBERTSON
Dulhousie University.
Hulifux,
Now
FOLLOWING
EXPERIMENTAL
* Scotiu,
Cunudu
B3H 4H7
1981
Adenosine is a putative inhibitory neurotransmitter or neuromodulator in the mammalian central nervous system (Bruns et al., 1980). It has been shown to have sedative and anticonvulsant activities when injected directly into the cerebral ventricles (Maitre et al., 1974), actions not unlike those of the benzodiazepines. Both electrically and chemically induced seizures produce a rapid increase in the number of cortical benzodiazepine binding sites, with no alteration in the apparent affinity of the ligand for these sites (Paul and Skolnick, 1978). We now report that chemically induced seizures result in a significant decrease in the number of cerebellar adenosine binding sites (B,,) without altering the apparent affinity of the receptor. Adult male Sprague-Dawley rats were convulsed by a single injection of pentylenetetrazol (Sigma Chemical Co., St. Louis, MO.) (50 mg/kg in 0.9% saline, i.p.). Control animals received saline only. Only animals showing toxic-clonic seizures within 4min of injection were included in the study. Rats were killed by decapitation 1 h after injection, and cerebral cortex (not including hippocampus or basal ganglia) and cerebellum dissected on ice. Tissue from 3 rats was pooled, homogenized in 10 vol of ice-cold 0.32 M sucrose, and centrifuged 1000 X g (10 min). A crude Pz fraction (including synaptosomes) was isolated from the supernatant by centrifugation (38000 X g, 20 min) and resuspended in 10 vol of buffer containing: 10 mM Tris-HCl, pH 7.4 (37’C), 11 mM NaCl, 4.7 mM KCl, 1.2 mM NaH,PO,, 26.2
* To whom all correspondence
RECEPTORS
should be addressed ,b 19X I Elsevier/North-Holland
mM NaHCO,, 1.8 mM CaCl,, 1.2 mM MgCl, and glucose (0.2%). Prior to binding assays preparations were incubated (30 min 37’C) with adenosine deaminase (2 U/ml) to remove endogenous adenosine. N6 -cyclohexyl-[ 3Hladenosine ([ 3H]CHA) (New England Nuclear, spec. act. 11.5 Ci/mmol) binding was carried out on fresh preparations as described by Bruns et al. (1980) but at 37°C. Specific (displaceable) binding was defined using 10 PM 2-chloroadenosine (Sigma Chemical Co. St. Louis, MO.). Scatchard analysis was carried out on data obtained from determinations (performed in duplicate) at final concentrations of 1.25, 2.5, 5, 10 and 20 nM [ 3H]CHA. There was a significant (20.7%) decrease in the number of [‘H]CHA binding sites (B,,) in cerebellar cortex of rats 1 h after a pentylenetetrazolinduced convulsion (table 1). There was no change in the apparent affinity of the binding site. In the cerebral cortex, by contrast, there was no change in either the number of binding sites or in the affinity of the receptor. The rapid change observed in [3H]CHA binding in cerebellum after pentylenetetrazol-induced seizures is temporally similar (but opposite in direction) to the changes seen in cerebral cortical benzodiazepine receptors after such seizure activity (Paul and Skolnick, 1978). Adenosine levels in brain increase rapidly after seizures or anoxia (Winn et al., 1980), and down-regulation of adenosine receptors might be expected under these conditions. However, the differential effect of the seizures on cerebral and cerebellum cortical adenosine receptor suggests that the decrease in receptor density observed in the cerebellum is Biomedical
Press
80 TABLE 1 Effect of pentylenetetrazol-induced convulsions on N 6-cyclohexyl-[3H]adenosine binding. Brain region
Control
Pentylenetetrazol
Cerebellum
Bma~ a Kr-) ~'
690 ± 30 4.26 -+ 1.0
547 : 31 c 3 . 1 8+- 0.4NS
Cortex
Bmax ~ KD ~'
462 ± 1.59 5.5 -+ 0.3
482 -+ 12 NS 6.5 ± 0.2NS
a Expressed as f m o l / m g p r o t e i n+- S.E.M. of three Scatchard determinations, each done with tissue pooled from 3 animals. h nM-+ S.E.M., 3 determinations as above. c P<0.05, NS not significant when compared with control (Student's t-test).
related to the seizure process rather than the accompanying anoxia. There has been relatively little investigation of the role of the cerebellum in seizures, largely because it is the one region of the brain where stimulation, no matter how strong of prolonged, will not elicit a seizure (Laxer et al., 1980). However, degenerative neuropathological changes occur in the cerebellum of epileptic patients and this region may be involved in the modulation of seizure activity (Laxer et al., 1980). Our finding of a large selective decrease in cerebellum [3 H]CHA binding following convulsion may cast light on the role of adenosine in seizure suppression by the cerebellum.
Acknowledgements Supported by grants from the Medical Research Council of Canada and the Savoy Foundation (to H.A.R.). M.G.M. is a Medical Research Council fellow. M. Wybenga acknowledges a summer research studentship.
References Bruns, R.F., J.W. Daly and S.H. Snyder, 1980, Adenosine receptors in brain membranes: binding of N6-cyclohexyl [3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine, Proc. Nat. Acad. Sci. U.S.A. 77, 5547. Laxer, K.D., L.T. Robertson, R.M. Julien and R.S. Down, 1980, Phenytoin: relationship between cerebellar function and epileptic discharges, in: Antiepileptic Drugs: Mechanisms of Action, eds. G.H. Glaser, J.K. Penry and D.M. Woodbury (Raven Press, New York) p. 415. Maitre, M., L. Ciesielski, A. Lehmann, E. Kempf and P. Mandel, 1974, Protective effect of adenosine and nicotinamide against audiogenic seizure, Biochem. Pharmacol. 23, 2807. Paul, S.M. and P. Skolnick, 1978, Rapid changes in brain benzodiazepine receptors after experimental seizures, Science 202, 892. Winn, H.R., J.E. Welsh, R. Rubio and R.M. Berne, 1980, Changes in brain adenosine during bicuculline-induced seizures in rats, Circ. Res. 47, 568.
Jounwl
01 Phumwcolog~~. 75 ( 198 I ) 79-80
Elsevier/North-Holland
Biomedical
19
Press
Rapid communication RAPID CHANGES SEIZURES MILTON
P. WYBENGA.
Deportmetlt
Received
IN CEREBELLAR
MARY
G. MURPHY
of Phormucologv, Faculty of Medicine,
8 September
1981. accepted
9 September
ADENOSINE
and HAROLD
0014-2999/g
I /OOOO-0000/$02.75
A. ROBERTSON
Dulhousie University.
Hulifux,
Now
FOLLOWING
EXPERIMENTAL
* Scotiu,
Cunudu
B3H 4H7
1981
Adenosine is a putative inhibitory neurotransmitter or neuromodulator in the mammalian central nervous system (Bruns et al., 1980). It has been shown to have sedative and anticonvulsant activities when injected directly into the cerebral ventricles (Maitre et al., 1974), actions not unlike those of the benzodiazepines. Both electrically and chemically induced seizures produce a rapid increase in the number of cortical benzodiazepine binding sites, with no alteration in the apparent affinity of the ligand for these sites (Paul and Skolnick, 1978). We now report that chemically induced seizures result in a significant decrease in the number of cerebellar adenosine binding sites (B,,) without altering the apparent affinity of the receptor. Adult male Sprague-Dawley rats were convulsed by a single injection of pentylenetetrazol (Sigma Chemical Co., St. Louis, MO.) (50 mg/kg in 0.9% saline, i.p.). Control animals received saline only. Only animals showing toxic-clonic seizures within 4min of injection were included in the study. Rats were killed by decapitation 1 h after injection, and cerebral cortex (not including hippocampus or basal ganglia) and cerebellum dissected on ice. Tissue from 3 rats was pooled, homogenized in 10 vol of ice-cold 0.32 M sucrose, and centrifuged 1000 X g (10 min). A crude Pz fraction (including synaptosomes) was isolated from the supernatant by centrifugation (38000 X g, 20 min) and resuspended in 10 vol of buffer containing: 10 mM Tris-HCl, pH 7.4 (37’C), 11 mM NaCl, 4.7 mM KCl, 1.2 mM NaH,PO,, 26.2
* To whom all correspondence
RECEPTORS
should be addressed ,b 19X I Elsevier/North-Holland
mM NaHCO,, 1.8 mM CaCl,, 1.2 mM MgCl, and glucose (0.2%). Prior to binding assays preparations were incubated (30 min 37’C) with adenosine deaminase (2 U/ml) to remove endogenous adenosine. N6 -cyclohexyl-[ 3Hladenosine ([ 3H]CHA) (New England Nuclear, spec. act. 11.5 Ci/mmol) binding was carried out on fresh preparations as described by Bruns et al. (1980) but at 37°C. Specific (displaceable) binding was defined using 10 PM 2-chloroadenosine (Sigma Chemical Co. St. Louis, MO.). Scatchard analysis was carried out on data obtained from determinations (performed in duplicate) at final concentrations of 1.25, 2.5, 5, 10 and 20 nM [ 3H]CHA. There was a significant (20.7%) decrease in the number of [‘H]CHA binding sites (B,,) in cerebellar cortex of rats 1 h after a pentylenetetrazolinduced convulsion (table 1). There was no change in the apparent affinity of the binding site. In the cerebral cortex, by contrast, there was no change in either the number of binding sites or in the affinity of the receptor. The rapid change observed in [3H]CHA binding in cerebellum after pentylenetetrazol-induced seizures is temporally similar (but opposite in direction) to the changes seen in cerebral cortical benzodiazepine receptors after such seizure activity (Paul and Skolnick, 1978). Adenosine levels in brain increase rapidly after seizures or anoxia (Winn et al., 1980), and down-regulation of adenosine receptors might be expected under these conditions. However, the differential effect of the seizures on cerebral and cerebellum cortical adenosine receptor suggests that the decrease in receptor density observed in the cerebellum is Biomedical
Press
80 TABLE 1 Effect of pentylenetetrazol-induced convulsions on N 6-cyclohexyl-[3H]adenosine binding. Brain region
Control
Pentylenetetrazol
Cerebellum
Bma~ a Kr-) ~'
690 ± 30 4.26 -+ 1.0
547 : 31 c 3 . 1 8+- 0.4NS
Cortex
Bmax ~ KD ~'
462 ± 1.59 5.5 -+ 0.3
482 -+ 12 NS 6.5 ± 0.2NS
a Expressed as f m o l / m g p r o t e i n+- S.E.M. of three Scatchard determinations, each done with tissue pooled from 3 animals. h nM-+ S.E.M., 3 determinations as above. c P<0.05, NS not significant when compared with control (Student's t-test).
related to the seizure process rather than the accompanying anoxia. There has been relatively little investigation of the role of the cerebellum in seizures, largely because it is the one region of the brain where stimulation, no matter how strong of prolonged, will not elicit a seizure (Laxer et al., 1980). However, degenerative neuropathological changes occur in the cerebellum of epileptic patients and this region may be involved in the modulation of seizure activity (Laxer et al., 1980). Our finding of a large selective decrease in cerebellum [3 H]CHA binding following convulsion may cast light on the role of adenosine in seizure suppression by the cerebellum.
Acknowledgements Supported by grants from the Medical Research Council of Canada and the Savoy Foundation (to H.A.R.). M.G.M. is a Medical Research Council fellow. M. Wybenga acknowledges a summer research studentship.
References Bruns, R.F., J.W. Daly and S.H. Snyder, 1980, Adenosine receptors in brain membranes: binding of N6-cyclohexyl [3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine, Proc. Nat. Acad. Sci. U.S.A. 77, 5547. Laxer, K.D., L.T. Robertson, R.M. Julien and R.S. Down, 1980, Phenytoin: relationship between cerebellar function and epileptic discharges, in: Antiepileptic Drugs: Mechanisms of Action, eds. G.H. Glaser, J.K. Penry and D.M. Woodbury (Raven Press, New York) p. 415. Maitre, M., L. Ciesielski, A. Lehmann, E. Kempf and P. Mandel, 1974, Protective effect of adenosine and nicotinamide against audiogenic seizure, Biochem. Pharmacol. 23, 2807. Paul, S.M. and P. Skolnick, 1978, Rapid changes in brain benzodiazepine receptors after experimental seizures, Science 202, 892. Winn, H.R., J.E. Welsh, R. Rubio and R.M. Berne, 1980, Changes in brain adenosine during bicuculline-induced seizures in rats, Circ. Res. 47, 568.