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Blockade of the picrotoxin-induced in vivo release of dopamine in the cat caudate nucleus by diazepam
Life Sciences Vol, 20, pp . 811-816, 1977 . Printed in the U.S .A .
Pergasron Prees
BLOCKADE OF THE PICROTOXIN-INDUCED IN VIVO RELEASE OF DOPAMINE IN THE CAT CAUDATE NUCLEUS BY DIAZEPAM A . Chéramy, A. Nieoullon and J . Glowinski Groupe NB, INSERM U .114, Collège de France 75231 - Paris cedex 05 (Received in final form January 17, 1977) Summary
S
The effects of picrotoxin and diazepam on the in vivo release of DA in the caudate nucleus were examlEeT"encéphale isolé" cats . A push-pull cannula was introduced into the left caudate nucleus and the structure was continuously superfused with L-3,5- 3H-tyrosine . The 3H-DA endogenously synthesized and released in the superfusates was estimated in successive serial fractions . Picrotoxin (2 .5 mg/kg) markedly enhanced the release of 3H-DA, as previously shown. Diazepam (10 mg/kg) had no effect on the spontaneous release of the labelled transmitter, but it did prevent the stimulating effect of picrotoxin . Picrotoxin stimulates the activity o£ nigrostriatal dopaminergic neurones when injected in the periphery . This was first suggested by experiments using rats, in which striatal homovanillic acid levels were slightly enhanced after the intraperitoneal injection of the drug(1,2) . Furthermore, picrotoxin inhibits the activity of striatal cholinergic neurones through its stimulating effect on the nigrostriatal dopaminergic pathway. In fact, the picrotoxininduced rise in striatal acetylcholine(ACh) levels was no longer detected after the destruction of the nigrostriatal dopaminergic pathway (3,4) or after the inhibition of dopamine (DA) synthesis . byo,.-methylparatyrosine(2) . Since dopaminergic neurones exert a tonic inhibitory control upon striatal cholinergic neurones(5,9), their activation by picrotoxin is responsible for the increase in striatal ACh levels (2-4) . More directly, it has been recently demonstrated that picrotoxin, injected intraperitoneally, markedly enhanced the release of DA in the caudate nucleus of the cat(10) . This was shown by using a push-pull cannula and by measuring the release of H-DA during the continuous superfusion of the structure with L-3,5- 3 H-tyrosine . Similar effects were observed when picrotoxin was introduced for a short time into the substantia nigra or the caudate nucleus (10) . It is generally assumed that the picrotoxin effect upon dopaminergic neurones is related to the blockade of gabaergic receptors located on dopaminergic neurones, since electrophysiological studies have suggested that nigral neurones were under the inhibitory control of a descending striato-nigral gabaergic pathway (11-13) . In various reports,it has been suggested that diazepam and other benzodiazepines were
812
Diazepam and Striatal DA Release
Vol 20, No . 5, 1977
exerting a GABA-like action (14,15) . The present study was undertaken to examine the effect of diazepam on the spontaneous and picrotoxin-evoked release of DA in the cat caudate nucleus . Methods Twenty-one adult cats of either sexes weighing from 2 to.3 .5kg were used . In all cases experiments were carried out on "encéphals isold"- animals prepared by transecting the spinal cord at the cervical level Cl-C2 under short halothane anaesthesia as described previously . Alveolar C0 2 (maintained at 3 .98 by the artificial ventilation), mean arterial blood pressure, electrocardiogram, body temperature and, in some cases,EEG were monitored and recorded (16) . A push-pull cannula, specially designed with an open-pulling system(16) was introduced stereotaxically into the left caudate nucleus, using a lateral approach to avoid the lateral ventricle, at the following coordinates A = 16 ; L - 5 ; H - +5 according to Snider and Niemer (1961)(17) .Án artificial CSF (18) containing L3,5- 3H-tyrosine(20 to 25pCi/500 pl ; specific activity 40 to 50C1/ mmole, from the Radiochemical Centre,Amersham, England) and previously purified by alumina adsorption and ion-exchange chromatography (18),was continuously introduced into the push-pull cannula at a rate of 500 pl/15 min. Superfusates were collected in successive 15 min fractions in tubes containing a frozen s_tgbilizing solutXon (final concentration in superfusates : D3: 10 M ; tyrosine : 10 -3M ; etthyleiediamine-tetra-acetic acid : 10-3M ; thioglycolic acid : 10-2M) . H-DA in superfusates was first isolated by ionexchange chromatography on amberlite CG 50 and adsorption on alumina as described by Giorguieff et al .(1976X18) and then estimated by liquid scintillation counting . Results were correcte for recovery (758) . At the steady state level, the quantity of H-DA released in one fraction (15 min) was about 100 times the blank value which corresponded to the counter background . Saline (10ml, isotonic NaCl), diazepam (Roche, 10 mg/kg) or picrotoxin(Sigma, 2 .5mg/kg) were injected intraperitoneally . Drugs were dissolved in saline, however the dissolution of diazepam required acidification with hydrochloric acid . The solvent without the drug did not affect by itsblf the spontaneous release of 3H-DA when compared with saline treated cats . The mean of the 3H-DA quantities recovered in five fractions preceding the drug administration, corresponding to the mean spontane~us release, was used as an individual control value (1008) . The H-DA in each fraction was then expressed as a percentage of this basal control value . This procedure allowed the determination of the mean + SEM of data obtained In .groups of 4 to 8 cats . Differences between the quantities of H-DA released in .corresponding fractions of control and treated cats were analyzed: using Student's t test . A value of p < 0 .05 was considered statistically significant .
Vol. 20, No . 5, 1977
Diazepam and Striatal DA Release
813
Results As shown previously (J0) picrotoxin (2 .5 mg/kg,ip) markedly enhanced the release of H-DA endogenously synthesized from L-3, 5- '3H-tyrosine when compared to control (fig .lA,C) . Diazepam (10 mg/kg,ip) alone had no effect on the spontaneous release of FIG 1
Effect of intraperitoneal injection of diazepam and picrotoxin on the release of newly synthesized 3H-DA in the caudate nucleus
B00 7001
N .5
SALINE
15min.
9W z
4M
a 300
0 0 N-8 .
~~I~oïïïïiiïi
DIAZEP14F1 ~Lp. .'Smin .
200
0
[lV
A discrete area of the caudate nucleus of "encéphale tdolé" cats was superfused continuously with L-3,5- 3H-tyrosine (20-25 pCi/500 p1/ 15 min) using a push-pull cannula . Tritiated DA was estimated in serial 15 min fractions . A)Saline or B)Diazepam (10 mg/kg,ip) or C) Picrotoxin (2 .5 mg/kg, ip) was injected 3 hours after the onset of the superfusion (black arrows) . The data are the mean value + SEN of results obtained in N experiments . They are expressed as a percentage of the mean spontaneous release qs described in Methods . Differences between the quantities o£ H-nA released in corresponding fractions of control and treated cats were analysed using Student's t test .lpG0 .05 was considered statistically significant .
Diazepam and Striatal DA Release
814
Vol . 20, No . 5, 1977
the labelled transmitter (fig .1B) . When diazepam (10 mg/kg,ip)was injected one hour before the administration of picrotoxin (2 .5 mg/ kg, ip) it completely prevented the stimulating effect of the GAGA antagonist on 3H-DA release (fig .2) . In one experiment, diazepam (10 mg/kg,ip) was injected two hours after picrotoxin (2 .5mg/kg,ip) . The benzodiazepine progressivgly reversed the pronounced stimulatory effect of picrotoxin on H-DA release in the caudate nucleus . In this experiment, the release of 3 H-DA which was of 4 .3 + O .inCi (mean + SEM of 4 successive 15min fractions) from 60 to 120 min after tKe picrotoxin injection was decreased to 2 .3 + 0 .2 nCi from 60 to 120 min after diazepam injection. The spontaneous release before picrotoxin injection(mean + SEM of 4 successive fractions)was 2 .5 + 0 .2nCi . The EEG seizurés induced by picrotoxin were prevented or blocked by diazepam under our experimental conditions . FIG 2 Prevention of the effect of picrotoxin on the release of newly synthesized 3H-DA by diazepam
3 H-DA
2
01 W Z Q IZ O O
000
0 N=5
YIIiI~IÏÎiÎÎÎÎÎÎÎ DIAZEPAM
10mg/kg i.p.
PItROTOXIN
2.5 mg/kg i.p.
15min.
Animals were treated as described in fig .l excepted that picrotoxin (2 .5 mg/kg,ip) was administered one hour after the injection of diazepam (10 mg/kg,ip) . Data are expressed as described in fig .l . There is no statistical difference when compared to fig .1A and 1B . * p-4- 0 .05 when compared to corresponding fraction of picrotoxin-treated cats (fig .1C) .
Vol. 20, No . 5, 1977
Diazepam and Striatal DA Release
.815
Discussion The peripheral injection of picrotoxin stimulated the release of H-DA in the caudate nucleus of the cat . A similar effect has been shown. after the local introduction of the drug into the substantia nigra(10) . Therefore, the stimulation of 3H-DA release appears to be related to an activation of the dopaminergic neurones . Such an activation could increase the release as well as the synthesis ~f the H-transmitter from its precursor . The marked activation of 3H-DA release observed in the caudate nucleus after picrotoxin peripheral injection can be blocked(or reversed)by diazepam . This suggests that the benzodiazepine is able to prevent or suppress the activation of the dopaminergic neurones induced by the gabaergic antagonist . This interaction observed in vivo by measuring directly the changes in the transmitter release-7lo -In agreement with that detected in previous studies made with the rat. It has already been shown that the picrotoxin induced rise in striatal ACh levels in the rat was related to the activation of nigrostriatal dopaminergic neurones (2-4) .This change in ACh levels, corresponding to a decreased activity of the striatal cholinergic neurones, was no longer seen when diazepam was injected shortly before picrotoxin(3,4) . Diazepam was also able to reduce the activation of nigrostriatal dopaminergic neurones induced by neuröleptics as revealed by the changes in striatal levels of homovanillic acid (1) . From our present knowledge, it can be proposed that diazepam acts mainly by a GARA-like effect . The drug could reinforce the tonic inhibitory influence of the striato-nigral gabaergic neuro nes on the activity of nigrostriatal dopaminergic neurones by facilitating gabaergic transmission . Other authors have suggested that diazepam could indirectly compete with picrotoxin at gabaergic receptor sites by increasing the synaptic GARA concentration in the substantia nigra (15,19) . This view is supported by electrophysiological studies .Indeed, it was initially shown that diazepam prevented the suppressing effect of bicuculline on the 'inhibitory potential evoked in the substantia nigra by the stimulation of the caudate nucleus in the cat (20) . Such an interaction of diazepam with gabaergic antagonists has also been observed in gabaergic synapses located in other areas of the CNS(21,22) . When injected alone, diazepam failed to reduce the release of endogenously synthesized 3 H-DA in the caudate nucleus . In the rat, diazepam only slightly reduced the levels of homovanillic acid(1) and had no significant effect on ACh in the striatum(3,4) . This may suggest that the activity of nigrostriatal dopaminergic neurones is already tonically inhibited in normal states and that diazepam mainly prevents their activation . The substantia nigra appeared to be the principal site of action of diazepam since as already mentioned we have observed that 3H-DA release in the caudate nucleus is not only stimulated by the peri pheral injection of picrotoxin but also by the introduction of the gabaergic antagonist into t e substantia nigra using a second pushpull cannula(10) . However, H-DA release from dopaminergic terminals was also enhanced, when picrotoxin was locally applied in the caudate nucleus(10,23) .Therefore, under our . experimental condi-
816
Diazepam and Striatal DA Release
Vol . 20, No . S, 1977
tions (injection of the drug in the periphery), it cannot be excluded that part cqf the effect of diazepam on the picrotoxin induced release of H-DA might also be mediated by an interaction occurring in the caudate nucleus . References 1 . H.H . KELLER, R . SCHAFFNER and W. HAEFELY, Naunyn Schmiedeberg ' s Arch .Pharmacol . 294 1-8 (1976) 2 . H. LADINSKY, S . CONSOLO, S.BIANCHI and A. JORI, Brain Research 108 351-361 (1976) 3. F .JAVOY, C . EUVRARD, A. HERSET and J. GLOWINSKI ,Brain Research (in press) 4 . F . JAVOY, C . EUVRARD, J . BOCKAERT and J . GLOWINSKI, Collegium Intern . Neuro-Psychopharmacologicum (p .92)Quebec (1976) 5 . H. STADLER, K .G . LLOYD, M . GADEA-CIRIA and G . BARTHOLINI, Brain Research 55 476-480 (1973) 6 . V.H . SETHY an M.H . VON WOERT, Nature 251 528-530 (1974) 7 . P .G . GUYENET, Y . AGID, F . JAVOY, J .C . BÉAUJOUAN, J . ROSSIER and J . GLOWINSKI, Brain Research 84 227-244 (1975) 8 . S . CONSOLO, H . LADINSKY an S . BIAHCHI, European J . Pharmacol . 33 345-351 (1975) 9. M . TRABUCCHI, D.L . CHENEY, G. RACAGNI and E . COSTA, Brain Research 85 131-134 (1975) 10 . A. CHERAMY, A. NIEOULLON and J . GLOWINSKI, Naunyn Schmiedeber 's Arch .Pharmacol . (in press) 11 . P. FELTZ, Can .J .P siol .Pharmacol . 49 1113-1115 (1971) 12 . W. PRECHT an M. YOSHIDA, Bra Research 32 229-233 (1971) 13 . A.R . CROSSMAN, R.J . WALKER an G.N . WOODRUFF, Br .J .Pharmacol . 49 696-698 (1973) . COSTA, A . GUIDOTTI, C .C . MAO and A . SURIA, Life Sci 17 14 . L 167-186 (1973) 15 . W. HAEFELY, A . KULCZAR, H . MOHLER, L . PIERI and P . POLO, Mechanism of action of benzodiaze inas (p .131)Raven Press, New-York (1975) 16 . A. NIEOULLON, A. CHERAMY and J . GLOWINSKI, J . Neurochem an press) 17 . R.S . SNIDER and W .T . NIEMER, The University of Chicago Press , Chicago (1961) 18 . M.F . GIORGUIEFF, M.L . LE FLOC'H, T.C . WESTFALL, J . GLOWINSKI and M.J . BESSON, Brain Research .106 117-132 (1976) 19 . W. HAEFELY, P . POLO, H .H . KELLER, R. SCHAFFNER, H . MOHLER, A. KULCZAR and L . PIERI, Collegium intern . Neuro- s ho harmacologicum (p ;47) Quebec (1976) 20 . R. SCHAFFNER and W. HAEFELY, Experientia (Basel) 31 732 (1975) 21 . P . POLO, H . MOHLER and W. HAEFELY, Naunyn Schmiedëberg's Arch . Pharmacol . 284 319-337 (1974) 22 . P . POLC andW . HAEFELY, Naunyn Schmiedeberg's Arch .Pharmacol . 294, 121-131 (1976) 23 . G .BARTHOLINI and H . STADLER, Chemical Tools in Catecholamtne Research , vol .i l (p .235) North Ho an .Co . 19
Pergasron Prees
BLOCKADE OF THE PICROTOXIN-INDUCED IN VIVO RELEASE OF DOPAMINE IN THE CAT CAUDATE NUCLEUS BY DIAZEPAM A . Chéramy, A. Nieoullon and J . Glowinski Groupe NB, INSERM U .114, Collège de France 75231 - Paris cedex 05 (Received in final form January 17, 1977) Summary
S
The effects of picrotoxin and diazepam on the in vivo release of DA in the caudate nucleus were examlEeT"encéphale isolé" cats . A push-pull cannula was introduced into the left caudate nucleus and the structure was continuously superfused with L-3,5- 3H-tyrosine . The 3H-DA endogenously synthesized and released in the superfusates was estimated in successive serial fractions . Picrotoxin (2 .5 mg/kg) markedly enhanced the release of 3H-DA, as previously shown. Diazepam (10 mg/kg) had no effect on the spontaneous release of the labelled transmitter, but it did prevent the stimulating effect of picrotoxin . Picrotoxin stimulates the activity o£ nigrostriatal dopaminergic neurones when injected in the periphery . This was first suggested by experiments using rats, in which striatal homovanillic acid levels were slightly enhanced after the intraperitoneal injection of the drug(1,2) . Furthermore, picrotoxin inhibits the activity of striatal cholinergic neurones through its stimulating effect on the nigrostriatal dopaminergic pathway. In fact, the picrotoxininduced rise in striatal acetylcholine(ACh) levels was no longer detected after the destruction of the nigrostriatal dopaminergic pathway (3,4) or after the inhibition of dopamine (DA) synthesis . byo,.-methylparatyrosine(2) . Since dopaminergic neurones exert a tonic inhibitory control upon striatal cholinergic neurones(5,9), their activation by picrotoxin is responsible for the increase in striatal ACh levels (2-4) . More directly, it has been recently demonstrated that picrotoxin, injected intraperitoneally, markedly enhanced the release of DA in the caudate nucleus of the cat(10) . This was shown by using a push-pull cannula and by measuring the release of H-DA during the continuous superfusion of the structure with L-3,5- 3 H-tyrosine . Similar effects were observed when picrotoxin was introduced for a short time into the substantia nigra or the caudate nucleus (10) . It is generally assumed that the picrotoxin effect upon dopaminergic neurones is related to the blockade of gabaergic receptors located on dopaminergic neurones, since electrophysiological studies have suggested that nigral neurones were under the inhibitory control of a descending striato-nigral gabaergic pathway (11-13) . In various reports,it has been suggested that diazepam and other benzodiazepines were
812
Diazepam and Striatal DA Release
Vol 20, No . 5, 1977
exerting a GABA-like action (14,15) . The present study was undertaken to examine the effect of diazepam on the spontaneous and picrotoxin-evoked release of DA in the cat caudate nucleus . Methods Twenty-one adult cats of either sexes weighing from 2 to.3 .5kg were used . In all cases experiments were carried out on "encéphals isold"- animals prepared by transecting the spinal cord at the cervical level Cl-C2 under short halothane anaesthesia as described previously . Alveolar C0 2 (maintained at 3 .98 by the artificial ventilation), mean arterial blood pressure, electrocardiogram, body temperature and, in some cases,EEG were monitored and recorded (16) . A push-pull cannula, specially designed with an open-pulling system(16) was introduced stereotaxically into the left caudate nucleus, using a lateral approach to avoid the lateral ventricle, at the following coordinates A = 16 ; L - 5 ; H - +5 according to Snider and Niemer (1961)(17) .Án artificial CSF (18) containing L3,5- 3H-tyrosine(20 to 25pCi/500 pl ; specific activity 40 to 50C1/ mmole, from the Radiochemical Centre,Amersham, England) and previously purified by alumina adsorption and ion-exchange chromatography (18),was continuously introduced into the push-pull cannula at a rate of 500 pl/15 min. Superfusates were collected in successive 15 min fractions in tubes containing a frozen s_tgbilizing solutXon (final concentration in superfusates : D3: 10 M ; tyrosine : 10 -3M ; etthyleiediamine-tetra-acetic acid : 10-3M ; thioglycolic acid : 10-2M) . H-DA in superfusates was first isolated by ionexchange chromatography on amberlite CG 50 and adsorption on alumina as described by Giorguieff et al .(1976X18) and then estimated by liquid scintillation counting . Results were correcte for recovery (758) . At the steady state level, the quantity of H-DA released in one fraction (15 min) was about 100 times the blank value which corresponded to the counter background . Saline (10ml, isotonic NaCl), diazepam (Roche, 10 mg/kg) or picrotoxin(Sigma, 2 .5mg/kg) were injected intraperitoneally . Drugs were dissolved in saline, however the dissolution of diazepam required acidification with hydrochloric acid . The solvent without the drug did not affect by itsblf the spontaneous release of 3H-DA when compared with saline treated cats . The mean of the 3H-DA quantities recovered in five fractions preceding the drug administration, corresponding to the mean spontane~us release, was used as an individual control value (1008) . The H-DA in each fraction was then expressed as a percentage of this basal control value . This procedure allowed the determination of the mean + SEM of data obtained In .groups of 4 to 8 cats . Differences between the quantities of H-DA released in .corresponding fractions of control and treated cats were analyzed: using Student's t test . A value of p < 0 .05 was considered statistically significant .
Vol. 20, No . 5, 1977
Diazepam and Striatal DA Release
813
Results As shown previously (J0) picrotoxin (2 .5 mg/kg,ip) markedly enhanced the release of H-DA endogenously synthesized from L-3, 5- '3H-tyrosine when compared to control (fig .lA,C) . Diazepam (10 mg/kg,ip) alone had no effect on the spontaneous release of FIG 1
Effect of intraperitoneal injection of diazepam and picrotoxin on the release of newly synthesized 3H-DA in the caudate nucleus
B00 7001
N .5
SALINE
15min.
9W z
4M
a 300
0 0 N-8 .
~~I~oïïïïiiïi
DIAZEP14F1 ~Lp. .'Smin .
200
0
[lV
A discrete area of the caudate nucleus of "encéphale tdolé" cats was superfused continuously with L-3,5- 3H-tyrosine (20-25 pCi/500 p1/ 15 min) using a push-pull cannula . Tritiated DA was estimated in serial 15 min fractions . A)Saline or B)Diazepam (10 mg/kg,ip) or C) Picrotoxin (2 .5 mg/kg, ip) was injected 3 hours after the onset of the superfusion (black arrows) . The data are the mean value + SEN of results obtained in N experiments . They are expressed as a percentage of the mean spontaneous release qs described in Methods . Differences between the quantities o£ H-nA released in corresponding fractions of control and treated cats were analysed using Student's t test .lpG0 .05 was considered statistically significant .
Diazepam and Striatal DA Release
814
Vol . 20, No . 5, 1977
the labelled transmitter (fig .1B) . When diazepam (10 mg/kg,ip)was injected one hour before the administration of picrotoxin (2 .5 mg/ kg, ip) it completely prevented the stimulating effect of the GAGA antagonist on 3H-DA release (fig .2) . In one experiment, diazepam (10 mg/kg,ip) was injected two hours after picrotoxin (2 .5mg/kg,ip) . The benzodiazepine progressivgly reversed the pronounced stimulatory effect of picrotoxin on H-DA release in the caudate nucleus . In this experiment, the release of 3 H-DA which was of 4 .3 + O .inCi (mean + SEM of 4 successive 15min fractions) from 60 to 120 min after tKe picrotoxin injection was decreased to 2 .3 + 0 .2 nCi from 60 to 120 min after diazepam injection. The spontaneous release before picrotoxin injection(mean + SEM of 4 successive fractions)was 2 .5 + 0 .2nCi . The EEG seizurés induced by picrotoxin were prevented or blocked by diazepam under our experimental conditions . FIG 2 Prevention of the effect of picrotoxin on the release of newly synthesized 3H-DA by diazepam
3 H-DA
2
01 W Z Q IZ O O
000
0 N=5
YIIiI~IÏÎiÎÎÎÎÎÎÎ DIAZEPAM
10mg/kg i.p.
PItROTOXIN
2.5 mg/kg i.p.
15min.
Animals were treated as described in fig .l excepted that picrotoxin (2 .5 mg/kg,ip) was administered one hour after the injection of diazepam (10 mg/kg,ip) . Data are expressed as described in fig .l . There is no statistical difference when compared to fig .1A and 1B . * p-4- 0 .05 when compared to corresponding fraction of picrotoxin-treated cats (fig .1C) .
Vol. 20, No . 5, 1977
Diazepam and Striatal DA Release
.815
Discussion The peripheral injection of picrotoxin stimulated the release of H-DA in the caudate nucleus of the cat . A similar effect has been shown. after the local introduction of the drug into the substantia nigra(10) . Therefore, the stimulation of 3H-DA release appears to be related to an activation of the dopaminergic neurones . Such an activation could increase the release as well as the synthesis ~f the H-transmitter from its precursor . The marked activation of 3H-DA release observed in the caudate nucleus after picrotoxin peripheral injection can be blocked(or reversed)by diazepam . This suggests that the benzodiazepine is able to prevent or suppress the activation of the dopaminergic neurones induced by the gabaergic antagonist . This interaction observed in vivo by measuring directly the changes in the transmitter release-7lo -In agreement with that detected in previous studies made with the rat. It has already been shown that the picrotoxin induced rise in striatal ACh levels in the rat was related to the activation of nigrostriatal dopaminergic neurones (2-4) .This change in ACh levels, corresponding to a decreased activity of the striatal cholinergic neurones, was no longer seen when diazepam was injected shortly before picrotoxin(3,4) . Diazepam was also able to reduce the activation of nigrostriatal dopaminergic neurones induced by neuröleptics as revealed by the changes in striatal levels of homovanillic acid (1) . From our present knowledge, it can be proposed that diazepam acts mainly by a GARA-like effect . The drug could reinforce the tonic inhibitory influence of the striato-nigral gabaergic neuro nes on the activity of nigrostriatal dopaminergic neurones by facilitating gabaergic transmission . Other authors have suggested that diazepam could indirectly compete with picrotoxin at gabaergic receptor sites by increasing the synaptic GARA concentration in the substantia nigra (15,19) . This view is supported by electrophysiological studies .Indeed, it was initially shown that diazepam prevented the suppressing effect of bicuculline on the 'inhibitory potential evoked in the substantia nigra by the stimulation of the caudate nucleus in the cat (20) . Such an interaction of diazepam with gabaergic antagonists has also been observed in gabaergic synapses located in other areas of the CNS(21,22) . When injected alone, diazepam failed to reduce the release of endogenously synthesized 3 H-DA in the caudate nucleus . In the rat, diazepam only slightly reduced the levels of homovanillic acid(1) and had no significant effect on ACh in the striatum(3,4) . This may suggest that the activity of nigrostriatal dopaminergic neurones is already tonically inhibited in normal states and that diazepam mainly prevents their activation . The substantia nigra appeared to be the principal site of action of diazepam since as already mentioned we have observed that 3H-DA release in the caudate nucleus is not only stimulated by the peri pheral injection of picrotoxin but also by the introduction of the gabaergic antagonist into t e substantia nigra using a second pushpull cannula(10) . However, H-DA release from dopaminergic terminals was also enhanced, when picrotoxin was locally applied in the caudate nucleus(10,23) .Therefore, under our . experimental condi-
816
Diazepam and Striatal DA Release
Vol . 20, No . S, 1977
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