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Increased number of muscarinic binding sites in brain following chronic barbiturate treatment to rat
Pergamon Press
Life Sciences, Vol . 26, pp . 231-237 Printed in the U .S .A .
INCREASED NUMBER OF MUSCARINIC BINDING SITES IN BRAIN FOLLOWING CHRONIC BARBITURATE TREATI~NT TO RAT Agneta Nordberg, Góran Wahlström and Christer Larsson Department of Pharmacology, University of Uppsala, 5-751 23 Uppsala and Department of Pharmacology, University of Ume~, 5-901 87 Ume~, Sweden (Received in final form December 4, 1979)
Summary Rats received as their only drinking fluid a solution of sodium barbital (3 .33 mg/ml) for more than 40 weeks . In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice . Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C) . Synaptosomes from different parts of the brain were incubated with radioactive quinuclidinyl benzilate ( 3 H-QNB)(0 .2 nM) for 60 min . A significantly increased number of 3 H-QNB binding sites was found in the striatum and midbrain + medulla oblongata + cerebellum of rats abstinent for 3 days (A3) in comparison with controls (C) . Saturation studies indicated that group A3 had significantly more receptors in the midbrain + medulla oblongata + cerebellum than group C, while there was no differences in receptor affinity . There are studies indicating that cholinergic mechanisms might be involved in the tolerance and physical dependence that develop after forced oral chronic barbiturate treatment (for reviews see 1, 2) . If rats were treated with barbital for 25-30 weeks no effect was found on the content of endogenous acetylcholine (ACh) in brain . However, 3 days after the barbital solution had been replaced by water (on the day when the number of convulsions were at a maximum) the endogenous ACh content was markedly reduced in the striatum and the decrease was still significant after 30 days of abstinence (3,4) . The biosynthesis of radioactive ACh from radioactive tracer dose of choline (Ch) given intravenously was increased in the midbrain + medulla oblongata + cerebellum of rats receiving barbital until sacrifice and rats abstinent for 3 days . In the hippocampus + cortex the biosynthesis of radioactive ACh was also increased on the 3rd day of abstinence in comparison with untreated animals . No significant effect on the ACh formation was found in the striatum (4) . The specific radio-
0024-3205/80/030231-07$02 .00/0 Copyright (c) 1980 Pergamon Prese Ltd
232
Barbiturate Treatment and Binding Sites
Vol . 26, No . 3, 1980
activity of ACh was increased in all brain regions indicating an increased turnover of ACh in brain in the abstinence (4) . These changes are the opposite to those found in acute experiments (5) . In the present study the effect of chronic barbiturate treatment on the number of muscarinic binding sites in different parts of the rat brain was investigated . It has been shown in several studies that the reversible muscarinic cholinergic antagonist quinuclidinyl benzilate (QNB) can be used as radioactive ligand in assay for the muscarinic cholinergic receptor in brain (6) . Recently Möhler et al . (7) have shown that 24-28 hours after a rather short pentobarbital treatment of 4 weeks there was a decrease in QNB binding in the cerebellum . In the present study QNB was incubated with synaptosomas and an increased number of muscarinic binding sites was found in the striatum . This change was recorded when the rats had been abstinent for 3 days after a considerably longer barbital treatment . Material and methods Male Sprague-Dawley rats (Nih/Han/Mol, Möllegaard, Li Skensved, Denmark) were used (initial weight around 300 g) . The artificial light in the animal room was on between 8 p.m . and 8 a . m . and the animals were supplied with food and drinking fluid ad libitum. During the barbital treatment the drinking fluid consisted of a solution of sodium barbital (3 .33 mg/ml) and the treatment lasted approximately 42 weeks with a daily consumption of about 200mg/kg . In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice respectively . Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C) . During the abstinence period convulsions were recorded by jiggle cages (8) . The recording was started on day 0 when the drinking fluid was changed from barbital solution to water . For further details regarding the chronic treatment see Wahlström (9) . The animals were killed by decapitation . The brains were rapidly taken out and dissected on an ice-cold glass plate into three parts : striatum, hippocampus + cortex, midbrain + medulla oblongata + cerebellum . The tissues were homogenized in 20 vol of icecold sucrose 0 .32 M, and centrifuged at 1000 g for 10 min . The pellet (P1) was discarded and the supernatant centrifuged at 17000 g for 15 min . The supernatant was discarded and the crude synaptosomal fraction (P2) was resuspended and homogenized in the original volume of ice-cold sucrose 0 .32 M . All operation were performed at +5°C . The P2 fractions were kept frozen until receptor binding studies were performed . A 100 ul aliquot of the crude synaptosomal fraction was incubated for 60 min at 25oC or 35oC in 900 ul of Na KPOy buffer, pH 7 .4, containing tritium labelled QNB ( 3H-QNB, SA 16 Ci/mmol)(0 .1-1 nM of (-)-3H-QNB)_ . Previous studies have shown that the 3 H-QNB binding is saturated after 60 min of incubation (10) . The incubation was terminated by cooling the samples to OoC and was followed by centrifugation in a Microfuge®, Beckman, for 5 min . The tip o= the tubes were cut and the pellet dissolved in soluen/toluen (1 :3) over night (11) . 14 ml of scintillation mixture containing
Barbiturate Treatment and Binding Sitea
Vol . 26, No . 3, 1980
233
toluen methanol and Permablend® were then added and the radioactivity meásured by liquid scintillation . The protein content of the P2 fraction was measured (12) using bovine albumin as the standard . Specific binding was determined by subtracting nonspecific binding in presence of 100 uA: oxotremorine from the total binding in absence of oxotremorine (6) . The specific binding amounted to about 90 $ of the total binding . Every determination of binding was performed in duplicate or triplicate . Results Figure 1 shows the number of spontaneous convulsions after the end of the barbital treatment . A maximal number of convulsions was seen on the 3rd day of abstinence and by the 12th day of abstinence no convulsions were seen . This is in accordance with previous findings after similar barbital treatments (3) . The number of 3H-QNB binding sites in different brain regions following chronic barbital treatment for a little more than 40 weeks is shown in figure 2 . In this first part of the study the crude synaptosomal fractions were incubated with 0 .2 nM 9 H-QPIB as described in methods . Compared with controls (group C) no significant change in number of 9 H-QNB binding sites was found in animals treated with barbital until sacrifice (group B) . 4
Q0
~3 Z O ~2 U u_ 01
1
2
3
4
5
6
7
8
9
10
DAYS AFTER END OF BARBITAL TREATMENT FIG . 1
11
12
Spontaneous convulsions in the abstinence period after the end of the barbital treatment . Open symbols indicate group A12, filled symbols indicate group A3 .
234
Barbiturate Treatment and Binding Sitea
Vol. 26, No . 3, 1980
3
H-QNB speclflcaly bound (pmolig protein)
700E
Striatum
Hippocartipus Cortex
500
Midbrein Medina oblongata Cerebellum
100 C B A3Á12
C B Á.3A12
C B Á3A12
FIG . 2
Number of 3 H-QNB binding sites in different rat brain regions following chronic barbital treatment . Crude synaptosomal fractions (P2 fraction) were incubated with 3 H-QNB 0 .2 nM at 25oC for 60 min . C = control ; B = barbital until sacrifice ; A3 = abstinent for 3 days ; A12 = abstinent for 12 days . **p<0 .01 *p<0 .05 ; 6 - 9 animals in each group .
MIDBRAW MEDULLA OBLONGATA CEREBELLUM ~ CONTROL ~~pmovg s~ aM Ko
~o
FIG . 3 Scatchard plot of 3 H-QNB binding to synaptosomas from the midbrein + medul1a oblongata + cerebellum of gróup C and A3 . Crude synaptosomal fractions were incubated with 9 H-QNB 0 .1-1 nM at 35oC (condition also used in the dissociation study) for 60 min . Receptor-ligand concentration (R L) is expressed in pmol/g protein . Ligand concentration (L) is expressed in pM and represents the free ligand concentration .
Vol . 26, No . 3, 1980
Barbiturate Treatment and Binding Sítes
235
However, an increased number of muscarinic binding sites was found in the striatum and midtrain preparations of rats abstinent for 3 days (group A3) . In the hippocampus + cortex there was a. tendency towards a similar change but the difference was not significant (p>0 .05) . A clear return to the control value was seen in the ~H-QNB binding on day 12 (group A12) for all brain regions . Since too little material was available from the striatum preparation the effect of increasing concentrations of 9 H-QNB (0 .1-1nM) was studied on crude synaptosomal fractions from the midtrain + medulla oblongata + cerebellum of group A3 and C . A saturation of the binding sites was obtained . A Scatchard plot of the data is shown in figure 3 and the maximal binding (Borax) was calculated to be 89 pmol/g protein for controls and 139 pool/g protein for rats abstinent for 3 days (A3) . The confidence limits for p less than 0 .05 calculated according to Diem and Lentner (13) were in these two cases 78-151 pmol/g (group C) and 122-249 pmol/g (group A3) respectively, which indicate that the two values were well separated . This is further indicated if the two regression 11nes are tested on identity (13) . This hypothesis can be rejected on a p value less than 0 .001 with 5 degrees of freedom. The dissociation constant, RD was 57 pM for controls and 51 pM for group A3 . Extrapolation of KD to infinitely low receptor concentration gave the same values . The rate of dissociation of the 9 H-QNB receptor complex was also measured . Oxotremorine (100 uM) was added to the incubation media when synaptosomes from the midtrain + medulla oblongata + cerebellum had been incubated with 9 H-QNB (0 .2 nM) for 60 min . Since the dissociation process is slow at 25oC the temperature was increased to 35oC during the 9H-QNB incubation . After different time intervals this incubation with oxotremorine was then terminated by cooling . T~ for the dissociation of the receptor complex was 65 min for the control and 66 min for rats abstinent for 3 days (A3) . Discussion In the present study it was found that the number of muscarinic binding sites were markedly increased on the 3rd day of abstinence in the striatum and midtrain + medulla oblongata + cerebellum after long-term barbital administration . The Borax for the binding in the midtrain + medulla oblongata + cerebellum is 56 8 higher in abstinent rats in comparison with control rats . However, the KD has almost the same value in the control (57 pM) and the abstinent (51 pM) rats . Thus on the third day of abstinence the number of muscarinic binding sites seem to be increased without any certain change in receptor affinity in the part of the brain, which was studied . In the present experiments the 3rd day of abstinence was the time when we found the maximal number of convulsions (FIG . 1) . At the same time a maximal tolerance to hexobarbital has also beén recorded earlier (9) . Such a tolerance is probably another measure of the increased excitation found in the abstinence . A supersensitivity towards the temperature reducing effect of pilocarpine had been found during abstinence after a comparable barbitale treatment (14) . This supersensitivity had a maximum
236
Barbiturate Treatment and Binding Sites
Vol. 26, No . 3, 1980
on day 3 of the abstinence and no supersensitivity was found on day 12 . This time pattern corresponds to the change found in the i H-QNB binding in the present experiments and a possible explanation to the supersensitivity to pilocarpine is thus an increased number of muscarinic receptors . The results of recent experiments, including the present one, indicate that there might at least be two different changes in cholinergic neurotransmission during the abstinence from chronic barbital treatments . The number of muscarinic binding sites which reached a maximum on day 3 had returned to control level after 12 days of abstinence . A corresponding time relation was also found by Nordberg and Wahlsträm with regard to some changes in regional biosynthesis of acetylcholine (4) . However, the content of endogenous ACh which was markedly reduced in the striatum on the 3rd abstinence day was still decreased on day 12 and day 30 of the abstinence (3, 4) . In contrast to the ~H-QNB binding the high affinity Ch uptake into synaptosomes in vitro was not changed on the 3rd day of abstinence . Later in the abstinence period (day 12) there was an increase in both Vmax and Km in synaptosomes from the striatum (15) . The sensitivity to the convulsive effects of a Ch infusion did correspond to these changes in the in vitro Ch uptake in that no change was found on day 3 while an increased sensitivity was found on day 12 of the abstinence (16) . In the present study the number of muscarinic binding sites was the same in the striatum (369 ± 23 pmol/g protein) and hippocampus + cortex (343 ± 21 pmol/g protein) of untreated rats . Such a rather even distribution of receptor binding sites has also been found in the striatum and cortex of mouse (17) . However, in the mouse there is a distinct difference in ACh turnover and high affinity Ch uptake between the striatum and cortex . Thus in the mouse there seems to be no obvious relationship between ACh turnover or Ch uptake and number of muscarinic binding sites (5, 17) . This conclusion is also sup~orted in the rat by the time difference in the change of the H-QNB binding which occurred on day 3 and the change in high affinity Ch uptake into isolated synaptosomes which was changed on day 12 of the abstinence (15) . Acknowledgement This study was supported by the Swedish Tobacco Company, the Swedish Medical Research Council (project 2879, 3771) and the "IF :s Stiftelse för farmaceutisk forskning" . The skilful assistance of Miss Maria Astin, Mr Roland Larsson, Mrs Elisabeth Ljungblad and Mrs Kerstin Wahlström is gratefully acknowledged . References 1 . 2.
3. 4.
G . WAHLSTRÖM, Drug and Alcohol Dependence _4 221 (1979) . A. NORDBERG and G . WAHLSTRÛM, in : Proceedin s of the Inter national S osium on Biplo ical Researc in Alco olism . Eds : H. Beg Biter and B . Kissen, Plenum Press, New Yor (in press) . A. NORDBERG and G . WAHLSTR~M, European J . Pharmacol . _43 237 (1977) . A. NORDBERG and G . WAHLSTR~M, J . Neurochem. 32 371 (1979) .
Vol . 26, No . 3, 1980
5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
Barbiturate Treatment and Binding Sitea
A . NORDBERG, Acta Physiol . Scand . Suppl . 445 1 (1977) . H .I . YAMAMURA and S .H . SNYDER, Proc . Nat . P .cad . Sci . (Wash) 71 1725 (1974) . A . MZSHLER, T . ORADA and S .J . ENNA, Brain Research 156 391 (1978) . G . WAHLSTRÛM, Life Sci . 19 1817 (1976) . G . WAHLSTRtíM, Acta Pharmacol . Toxicol . 35 131 (1974) . A . NORDBERG and C . LARSSON, Acta Physiol . Scand . (1979) (in press) . L . TERENIUS and A. WAHLSTRÖM, Acta Physiol . Scand . 94 74 (1975) . O .H . LOWRY, N .J . ROSENBROUGH, A.L . FARR and R .J . RANDALL, Biol . Chem . 193 265 (1951) . K . DIEM and . C LENTNER, Documenta Gei scientific tables 7 ed, Geigy SA, Basel, 177-179 1970 . G . WAHLSTRÛM and T . EKWALL, European J . Pharmacol . 38 123 (1976) . G . WAHLSTRSM and A. NORDBERG, Acta Physiol . Scand . Suppl . 473 65 (1979) . .WAHLSTRSM, European J . Pharmacol . 51 219 (1978) . G A. NORDBERG and C . LARSSON, Acta Physiól . Scand . Suppl . 473 65 (1979) . .NORDBERG, Life Sci . 23 937 (1978) . A
237
Life Sciences, Vol . 26, pp . 231-237 Printed in the U .S .A .
INCREASED NUMBER OF MUSCARINIC BINDING SITES IN BRAIN FOLLOWING CHRONIC BARBITURATE TREATI~NT TO RAT Agneta Nordberg, Góran Wahlström and Christer Larsson Department of Pharmacology, University of Uppsala, 5-751 23 Uppsala and Department of Pharmacology, University of Ume~, 5-901 87 Ume~, Sweden (Received in final form December 4, 1979)
Summary Rats received as their only drinking fluid a solution of sodium barbital (3 .33 mg/ml) for more than 40 weeks . In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice . Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C) . Synaptosomes from different parts of the brain were incubated with radioactive quinuclidinyl benzilate ( 3 H-QNB)(0 .2 nM) for 60 min . A significantly increased number of 3 H-QNB binding sites was found in the striatum and midbrain + medulla oblongata + cerebellum of rats abstinent for 3 days (A3) in comparison with controls (C) . Saturation studies indicated that group A3 had significantly more receptors in the midbrain + medulla oblongata + cerebellum than group C, while there was no differences in receptor affinity . There are studies indicating that cholinergic mechanisms might be involved in the tolerance and physical dependence that develop after forced oral chronic barbiturate treatment (for reviews see 1, 2) . If rats were treated with barbital for 25-30 weeks no effect was found on the content of endogenous acetylcholine (ACh) in brain . However, 3 days after the barbital solution had been replaced by water (on the day when the number of convulsions were at a maximum) the endogenous ACh content was markedly reduced in the striatum and the decrease was still significant after 30 days of abstinence (3,4) . The biosynthesis of radioactive ACh from radioactive tracer dose of choline (Ch) given intravenously was increased in the midbrain + medulla oblongata + cerebellum of rats receiving barbital until sacrifice and rats abstinent for 3 days . In the hippocampus + cortex the biosynthesis of radioactive ACh was also increased on the 3rd day of abstinence in comparison with untreated animals . No significant effect on the ACh formation was found in the striatum (4) . The specific radio-
0024-3205/80/030231-07$02 .00/0 Copyright (c) 1980 Pergamon Prese Ltd
232
Barbiturate Treatment and Binding Sites
Vol . 26, No . 3, 1980
activity of ACh was increased in all brain regions indicating an increased turnover of ACh in brain in the abstinence (4) . These changes are the opposite to those found in acute experiments (5) . In the present study the effect of chronic barbiturate treatment on the number of muscarinic binding sites in different parts of the rat brain was investigated . It has been shown in several studies that the reversible muscarinic cholinergic antagonist quinuclidinyl benzilate (QNB) can be used as radioactive ligand in assay for the muscarinic cholinergic receptor in brain (6) . Recently Möhler et al . (7) have shown that 24-28 hours after a rather short pentobarbital treatment of 4 weeks there was a decrease in QNB binding in the cerebellum . In the present study QNB was incubated with synaptosomas and an increased number of muscarinic binding sites was found in the striatum . This change was recorded when the rats had been abstinent for 3 days after a considerably longer barbital treatment . Material and methods Male Sprague-Dawley rats (Nih/Han/Mol, Möllegaard, Li Skensved, Denmark) were used (initial weight around 300 g) . The artificial light in the animal room was on between 8 p.m . and 8 a . m . and the animals were supplied with food and drinking fluid ad libitum. During the barbital treatment the drinking fluid consisted of a solution of sodium barbital (3 .33 mg/ml) and the treatment lasted approximately 42 weeks with a daily consumption of about 200mg/kg . In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice respectively . Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C) . During the abstinence period convulsions were recorded by jiggle cages (8) . The recording was started on day 0 when the drinking fluid was changed from barbital solution to water . For further details regarding the chronic treatment see Wahlström (9) . The animals were killed by decapitation . The brains were rapidly taken out and dissected on an ice-cold glass plate into three parts : striatum, hippocampus + cortex, midbrain + medulla oblongata + cerebellum . The tissues were homogenized in 20 vol of icecold sucrose 0 .32 M, and centrifuged at 1000 g for 10 min . The pellet (P1) was discarded and the supernatant centrifuged at 17000 g for 15 min . The supernatant was discarded and the crude synaptosomal fraction (P2) was resuspended and homogenized in the original volume of ice-cold sucrose 0 .32 M . All operation were performed at +5°C . The P2 fractions were kept frozen until receptor binding studies were performed . A 100 ul aliquot of the crude synaptosomal fraction was incubated for 60 min at 25oC or 35oC in 900 ul of Na KPOy buffer, pH 7 .4, containing tritium labelled QNB ( 3H-QNB, SA 16 Ci/mmol)(0 .1-1 nM of (-)-3H-QNB)_ . Previous studies have shown that the 3 H-QNB binding is saturated after 60 min of incubation (10) . The incubation was terminated by cooling the samples to OoC and was followed by centrifugation in a Microfuge®, Beckman, for 5 min . The tip o= the tubes were cut and the pellet dissolved in soluen/toluen (1 :3) over night (11) . 14 ml of scintillation mixture containing
Barbiturate Treatment and Binding Sitea
Vol . 26, No . 3, 1980
233
toluen methanol and Permablend® were then added and the radioactivity meásured by liquid scintillation . The protein content of the P2 fraction was measured (12) using bovine albumin as the standard . Specific binding was determined by subtracting nonspecific binding in presence of 100 uA: oxotremorine from the total binding in absence of oxotremorine (6) . The specific binding amounted to about 90 $ of the total binding . Every determination of binding was performed in duplicate or triplicate . Results Figure 1 shows the number of spontaneous convulsions after the end of the barbital treatment . A maximal number of convulsions was seen on the 3rd day of abstinence and by the 12th day of abstinence no convulsions were seen . This is in accordance with previous findings after similar barbital treatments (3) . The number of 3H-QNB binding sites in different brain regions following chronic barbital treatment for a little more than 40 weeks is shown in figure 2 . In this first part of the study the crude synaptosomal fractions were incubated with 0 .2 nM 9 H-QPIB as described in methods . Compared with controls (group C) no significant change in number of 9 H-QNB binding sites was found in animals treated with barbital until sacrifice (group B) . 4
Q0
~3 Z O ~2 U u_ 01
1
2
3
4
5
6
7
8
9
10
DAYS AFTER END OF BARBITAL TREATMENT FIG . 1
11
12
Spontaneous convulsions in the abstinence period after the end of the barbital treatment . Open symbols indicate group A12, filled symbols indicate group A3 .
234
Barbiturate Treatment and Binding Sitea
Vol. 26, No . 3, 1980
3
H-QNB speclflcaly bound (pmolig protein)
700E
Striatum
Hippocartipus Cortex
500
Midbrein Medina oblongata Cerebellum
100 C B A3Á12
C B Á.3A12
C B Á3A12
FIG . 2
Number of 3 H-QNB binding sites in different rat brain regions following chronic barbital treatment . Crude synaptosomal fractions (P2 fraction) were incubated with 3 H-QNB 0 .2 nM at 25oC for 60 min . C = control ; B = barbital until sacrifice ; A3 = abstinent for 3 days ; A12 = abstinent for 12 days . **p<0 .01 *p<0 .05 ; 6 - 9 animals in each group .
MIDBRAW MEDULLA OBLONGATA CEREBELLUM ~ CONTROL ~~pmovg s~ aM Ko
~o
FIG . 3 Scatchard plot of 3 H-QNB binding to synaptosomas from the midbrein + medul1a oblongata + cerebellum of gróup C and A3 . Crude synaptosomal fractions were incubated with 9 H-QNB 0 .1-1 nM at 35oC (condition also used in the dissociation study) for 60 min . Receptor-ligand concentration (R L) is expressed in pmol/g protein . Ligand concentration (L) is expressed in pM and represents the free ligand concentration .
Vol . 26, No . 3, 1980
Barbiturate Treatment and Binding Sítes
235
However, an increased number of muscarinic binding sites was found in the striatum and midtrain preparations of rats abstinent for 3 days (group A3) . In the hippocampus + cortex there was a. tendency towards a similar change but the difference was not significant (p>0 .05) . A clear return to the control value was seen in the ~H-QNB binding on day 12 (group A12) for all brain regions . Since too little material was available from the striatum preparation the effect of increasing concentrations of 9 H-QNB (0 .1-1nM) was studied on crude synaptosomal fractions from the midtrain + medulla oblongata + cerebellum of group A3 and C . A saturation of the binding sites was obtained . A Scatchard plot of the data is shown in figure 3 and the maximal binding (Borax) was calculated to be 89 pmol/g protein for controls and 139 pool/g protein for rats abstinent for 3 days (A3) . The confidence limits for p less than 0 .05 calculated according to Diem and Lentner (13) were in these two cases 78-151 pmol/g (group C) and 122-249 pmol/g (group A3) respectively, which indicate that the two values were well separated . This is further indicated if the two regression 11nes are tested on identity (13) . This hypothesis can be rejected on a p value less than 0 .001 with 5 degrees of freedom. The dissociation constant, RD was 57 pM for controls and 51 pM for group A3 . Extrapolation of KD to infinitely low receptor concentration gave the same values . The rate of dissociation of the 9 H-QNB receptor complex was also measured . Oxotremorine (100 uM) was added to the incubation media when synaptosomes from the midtrain + medulla oblongata + cerebellum had been incubated with 9 H-QNB (0 .2 nM) for 60 min . Since the dissociation process is slow at 25oC the temperature was increased to 35oC during the 9H-QNB incubation . After different time intervals this incubation with oxotremorine was then terminated by cooling . T~ for the dissociation of the receptor complex was 65 min for the control and 66 min for rats abstinent for 3 days (A3) . Discussion In the present study it was found that the number of muscarinic binding sites were markedly increased on the 3rd day of abstinence in the striatum and midtrain + medulla oblongata + cerebellum after long-term barbital administration . The Borax for the binding in the midtrain + medulla oblongata + cerebellum is 56 8 higher in abstinent rats in comparison with control rats . However, the KD has almost the same value in the control (57 pM) and the abstinent (51 pM) rats . Thus on the third day of abstinence the number of muscarinic binding sites seem to be increased without any certain change in receptor affinity in the part of the brain, which was studied . In the present experiments the 3rd day of abstinence was the time when we found the maximal number of convulsions (FIG . 1) . At the same time a maximal tolerance to hexobarbital has also beén recorded earlier (9) . Such a tolerance is probably another measure of the increased excitation found in the abstinence . A supersensitivity towards the temperature reducing effect of pilocarpine had been found during abstinence after a comparable barbitale treatment (14) . This supersensitivity had a maximum
236
Barbiturate Treatment and Binding Sites
Vol. 26, No . 3, 1980
on day 3 of the abstinence and no supersensitivity was found on day 12 . This time pattern corresponds to the change found in the i H-QNB binding in the present experiments and a possible explanation to the supersensitivity to pilocarpine is thus an increased number of muscarinic receptors . The results of recent experiments, including the present one, indicate that there might at least be two different changes in cholinergic neurotransmission during the abstinence from chronic barbital treatments . The number of muscarinic binding sites which reached a maximum on day 3 had returned to control level after 12 days of abstinence . A corresponding time relation was also found by Nordberg and Wahlsträm with regard to some changes in regional biosynthesis of acetylcholine (4) . However, the content of endogenous ACh which was markedly reduced in the striatum on the 3rd abstinence day was still decreased on day 12 and day 30 of the abstinence (3, 4) . In contrast to the ~H-QNB binding the high affinity Ch uptake into synaptosomes in vitro was not changed on the 3rd day of abstinence . Later in the abstinence period (day 12) there was an increase in both Vmax and Km in synaptosomes from the striatum (15) . The sensitivity to the convulsive effects of a Ch infusion did correspond to these changes in the in vitro Ch uptake in that no change was found on day 3 while an increased sensitivity was found on day 12 of the abstinence (16) . In the present study the number of muscarinic binding sites was the same in the striatum (369 ± 23 pmol/g protein) and hippocampus + cortex (343 ± 21 pmol/g protein) of untreated rats . Such a rather even distribution of receptor binding sites has also been found in the striatum and cortex of mouse (17) . However, in the mouse there is a distinct difference in ACh turnover and high affinity Ch uptake between the striatum and cortex . Thus in the mouse there seems to be no obvious relationship between ACh turnover or Ch uptake and number of muscarinic binding sites (5, 17) . This conclusion is also sup~orted in the rat by the time difference in the change of the H-QNB binding which occurred on day 3 and the change in high affinity Ch uptake into isolated synaptosomes which was changed on day 12 of the abstinence (15) . Acknowledgement This study was supported by the Swedish Tobacco Company, the Swedish Medical Research Council (project 2879, 3771) and the "IF :s Stiftelse för farmaceutisk forskning" . The skilful assistance of Miss Maria Astin, Mr Roland Larsson, Mrs Elisabeth Ljungblad and Mrs Kerstin Wahlström is gratefully acknowledged . References 1 . 2.
3. 4.
G . WAHLSTRÖM, Drug and Alcohol Dependence _4 221 (1979) . A. NORDBERG and G . WAHLSTRÛM, in : Proceedin s of the Inter national S osium on Biplo ical Researc in Alco olism . Eds : H. Beg Biter and B . Kissen, Plenum Press, New Yor (in press) . A. NORDBERG and G . WAHLSTR~M, European J . Pharmacol . _43 237 (1977) . A. NORDBERG and G . WAHLSTR~M, J . Neurochem. 32 371 (1979) .
Vol . 26, No . 3, 1980
5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .
Barbiturate Treatment and Binding Sitea
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