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Different types of glutamate receptors contribute to the presynaptic regulation of dopamine release from synaptosomes of the rat striatum
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3O DIFFERE/~f TYPES OF GI/IEAMATE RECEFIORS CONTRIBUTE 2~9 2~E PREo~£NAIrflC REGUIA'flON OF DOPAMINE RELEASE FRC~I SYI~M~IX3SC~2S OF %]~E RAT b~£R/.A'IUM Desce, J.bi., Godeheu, G., Ch~ramy, A. & GlcwiJ',~ki, J. Neurophanr~acologie, INSETS4 U.II4, Coll~ge de France, 75231 Paris cedex 05, France The effects of several agonists of glutanwaterc3{c receptors on the release of 3H-dopamine (3H-DA) continuously synthesized frcra ~H-tyrosine w ~ e examined using purified synaptoscrnes isolated from the rat striattma and prepared with percoll'" gradients. This study was undertaken since we have previously shown t/~t cortico-striatal glut~,~tergic neurons are involved in the presynaptic control of DA release in halothane-anaesthetizcd cats ir~planted witl* push-pull cannulae. Indeed, a quisqualate/kainate (QUI/KAI) receptor s~>£y[~ was shown to mediate this direct in viva presynap_tic regulation of DA release. KAI (10 6 to i0 2/~) was sho~a% to increase t~,e release of 3H-DA /]i a concentration-dependent manner. This latter effect could not be antagonize~ by glutamate-dietJayl ester but was abolisht4 in the presence of riluzole (i0 5M), a new anti-glutanatergic, comlxguaad. QUI alone_ (10 5M and 5x10 5M) was devoid of activity but it prevented the stimulatory effect of KAI (5x10 5M) on 3II-DA release. This is in agre~nent wit]1 our in viva data indicating that QUI induced a rapid desensitization of the receptors and that receptors of a QUI/KAI subtype are present on DA ne.rve terlnlnals. S~DA receptors are also involved in t/~e pres~aak~tic regulation of DA r e l ~ s e since h~0A (5x10 5M) had a slight sta/nulatory effect on 3 H ~ A release in t/~e presence of M g S . T ~ s effect was n~ch more pronounced in the absence of Mg and [~)tentlated by glycine (10 6M, In the presence of st~chnine I0 6M). The latter response could b~. ozltagonized by MK 801 (i0 6M) or the addition of b ~ g (0.83 ~I) into the superfusion mu~itzn. 31 C H R O N I C N M D A E X P O S U R E O F D E V E L O P I N G C E R E B E I , L A R G R A N U L E CELLS DECREASES T H E I R SENSITIVITY T O NMDA. M.Didier 1, F.A.Rassendren2,J.M.Mienville 1, P.Soubri61 , J.Bockaert .~ and J.P.Pin 3. 1SANOFI R E C H E R C H E ligne neuropsychiatrie, 2CNRS CRBM [IPR41, INSERM U249, 3CCIPE Montpellier France. The high K ÷ concentration (30 mM) necessary for the in vitro survival and development of cerebellar granule cells can be obviated by addition of N M D A to the culture medium (i.e., 100 uM N M D A * 12.5 mM K÷). On the other hand, N M D A neurotoxicity has been demonstrated in mature cells grown in 30 mM K ÷. In the present work, we compared the N M D A receptor sensitivity of such cells with those grown in 100 uM N M D A ÷ 12.5 mM K ÷. 45Ca2* uptake experiments and maximum current amplitude measured with the wholecell patch-clamp technique indicated that the sensitivity of control cells (K ÷ 30 mM)was low before 6 days in culture (DIC), was maximal at 10-14 DIC and decreased thereafter. This was correlated with a strong neurotoxicity at 10 DIC (45.4" 6.3% of dead cells, as revealed by the double labelling technique with fluorescein diacetate (FDA) and propidium iodide (PI)) measured 24 h after a 10 rain. exposure of neurons to 100 uM N M D A in the absence of Mg 2÷. In neurons growing in the presence of NMDA, N M D A responses remained low until at least 18 DIC (30% and 15% of control ,-esponses at 14 DIC determined in Ca 2÷ influx and patch-clamp experiments, respectively). Moreover, these cells were less sensitive to N M D A neurotoxicity than neurons cultured in normal conditions (25.6 ÷ 4.1% of neuronal death in FDA-PI double labelling). These results clearly demonstrate a decrease in NMDA response of cerebellar granule cells grown in the presence of NMDA wich could be related to several types of modifications involving
receptor expression, desensitization or "down-regulation". These hypotheses are currently under investigation.
32 EXCITATORY SULPHUR AMINO ACIDS EVOKE A RELEASE OF [3H]GABA FROM CORTICAL NEURONS BY DISTINCT MECHANISMS COMPRISING RECEPTOR ACTIVATION AND REVERSAL OF THE PLASMA MEMBRANE TRANSPORTER John Dunlop *, Ame Schousboe and Roger Griffiths *,Department of Biochemistry *, University of St. Andrews, KY16 9AL, Scotland and PharmaBiotec Research Center, Panum Institute, Copenhagen, Denmark. The endogenous molecules which activate all excitatory amino acid (EAA) receptors in the CNS under both normal and pathologic conditions have yet to be definitively identified. Although the majority of evidence points toward glutamate (and aspartate), additional evidence supports a role for the acidic sulphur amino acids (SAAs) as transmitter candidates and/or neuroregulators. In order to probe further the actions of the SAAs, a continuous superfusion system has been used to characterise the SAA-evoked release of [3H]GABA from primary cultures of mouse cerebral cortex neurons. All SAAs were shown to ; (i) evoke a dose-dependent, saturable release, (ii) depolarize the neuronal plasma membrane (measured by intracellular accumulation of the lipophilic cation [3H]tetraphenylpbosphonium, (iii) evoke both a calcium-dependent and -independent component of release. In the presence of calcium ions, SAA-evoked release was attenuated by a variety of EAA receptor antagonists, viz. CPP, APV, DNQX, and kynurenate. All SAA responses were sensitive to the presence of magnesium ions. Under calcium-free conditions, SAA-evoked release was unaffected by EAA antagonists, ttowever, this calcium-independent component of release was abolished in the presence of SKF 89976-A, a potent inhibitor of the GABA transporter, and, when sodium ions in the superfusion medium were replaced by choline. These observations suggest that: (1) the calcium-dependent SAA-evoked release of [3H]GABA is a wholly receptor-mediated event, thereby enhancing the transmitter candidacy oftbe SAAs. The evoked release appears to be mediated by activation of both NMDA and non-NMDA receptors, although on the basis of antagonist potency, a predominant non-NMDA receptor involvement is inferred, and (2) the calcium independent component of release arises from reversal of the GABA transporter due to reuptake-indueed depolarization following electrogenic cotransport of SAA with sodium ions. This observation is of significance especially with regard to neuropathologic conditions in which high extracellular levels of EAAs may be encountered.
3O DIFFERE/~f TYPES OF GI/IEAMATE RECEFIORS CONTRIBUTE 2~9 2~E PREo~£NAIrflC REGUIA'flON OF DOPAMINE RELEASE FRC~I SYI~M~IX3SC~2S OF %]~E RAT b~£R/.A'IUM Desce, J.bi., Godeheu, G., Ch~ramy, A. & GlcwiJ',~ki, J. Neurophanr~acologie, INSETS4 U.II4, Coll~ge de France, 75231 Paris cedex 05, France The effects of several agonists of glutanwaterc3{c receptors on the release of 3H-dopamine (3H-DA) continuously synthesized frcra ~H-tyrosine w ~ e examined using purified synaptoscrnes isolated from the rat striattma and prepared with percoll'" gradients. This study was undertaken since we have previously shown t/~t cortico-striatal glut~,~tergic neurons are involved in the presynaptic control of DA release in halothane-anaesthetizcd cats ir~planted witl* push-pull cannulae. Indeed, a quisqualate/kainate (QUI/KAI) receptor s~>£y[~ was shown to mediate this direct in viva presynap_tic regulation of DA release. KAI (10 6 to i0 2/~) was sho~a% to increase t~,e release of 3H-DA /]i a concentration-dependent manner. This latter effect could not be antagonize~ by glutamate-dietJayl ester but was abolisht4 in the presence of riluzole (i0 5M), a new anti-glutanatergic, comlxguaad. QUI alone_ (10 5M and 5x10 5M) was devoid of activity but it prevented the stimulatory effect of KAI (5x10 5M) on 3II-DA release. This is in agre~nent wit]1 our in viva data indicating that QUI induced a rapid desensitization of the receptors and that receptors of a QUI/KAI subtype are present on DA ne.rve terlnlnals. S~DA receptors are also involved in t/~e pres~aak~tic regulation of DA r e l ~ s e since h~0A (5x10 5M) had a slight sta/nulatory effect on 3 H ~ A release in t/~e presence of M g S . T ~ s effect was n~ch more pronounced in the absence of Mg and [~)tentlated by glycine (10 6M, In the presence of st~chnine I0 6M). The latter response could b~. ozltagonized by MK 801 (i0 6M) or the addition of b ~ g (0.83 ~I) into the superfusion mu~itzn. 31 C H R O N I C N M D A E X P O S U R E O F D E V E L O P I N G C E R E B E I , L A R G R A N U L E CELLS DECREASES T H E I R SENSITIVITY T O NMDA. M.Didier 1, F.A.Rassendren2,J.M.Mienville 1, P.Soubri61 , J.Bockaert .~ and J.P.Pin 3. 1SANOFI R E C H E R C H E ligne neuropsychiatrie, 2CNRS CRBM [IPR41, INSERM U249, 3CCIPE Montpellier France. The high K ÷ concentration (30 mM) necessary for the in vitro survival and development of cerebellar granule cells can be obviated by addition of N M D A to the culture medium (i.e., 100 uM N M D A * 12.5 mM K÷). On the other hand, N M D A neurotoxicity has been demonstrated in mature cells grown in 30 mM K ÷. In the present work, we compared the N M D A receptor sensitivity of such cells with those grown in 100 uM N M D A ÷ 12.5 mM K ÷. 45Ca2* uptake experiments and maximum current amplitude measured with the wholecell patch-clamp technique indicated that the sensitivity of control cells (K ÷ 30 mM)was low before 6 days in culture (DIC), was maximal at 10-14 DIC and decreased thereafter. This was correlated with a strong neurotoxicity at 10 DIC (45.4" 6.3% of dead cells, as revealed by the double labelling technique with fluorescein diacetate (FDA) and propidium iodide (PI)) measured 24 h after a 10 rain. exposure of neurons to 100 uM N M D A in the absence of Mg 2÷. In neurons growing in the presence of NMDA, N M D A responses remained low until at least 18 DIC (30% and 15% of control ,-esponses at 14 DIC determined in Ca 2÷ influx and patch-clamp experiments, respectively). Moreover, these cells were less sensitive to N M D A neurotoxicity than neurons cultured in normal conditions (25.6 ÷ 4.1% of neuronal death in FDA-PI double labelling). These results clearly demonstrate a decrease in NMDA response of cerebellar granule cells grown in the presence of NMDA wich could be related to several types of modifications involving
receptor expression, desensitization or "down-regulation". These hypotheses are currently under investigation.
32 EXCITATORY SULPHUR AMINO ACIDS EVOKE A RELEASE OF [3H]GABA FROM CORTICAL NEURONS BY DISTINCT MECHANISMS COMPRISING RECEPTOR ACTIVATION AND REVERSAL OF THE PLASMA MEMBRANE TRANSPORTER John Dunlop *, Ame Schousboe and Roger Griffiths *,Department of Biochemistry *, University of St. Andrews, KY16 9AL, Scotland and PharmaBiotec Research Center, Panum Institute, Copenhagen, Denmark. The endogenous molecules which activate all excitatory amino acid (EAA) receptors in the CNS under both normal and pathologic conditions have yet to be definitively identified. Although the majority of evidence points toward glutamate (and aspartate), additional evidence supports a role for the acidic sulphur amino acids (SAAs) as transmitter candidates and/or neuroregulators. In order to probe further the actions of the SAAs, a continuous superfusion system has been used to characterise the SAA-evoked release of [3H]GABA from primary cultures of mouse cerebral cortex neurons. All SAAs were shown to ; (i) evoke a dose-dependent, saturable release, (ii) depolarize the neuronal plasma membrane (measured by intracellular accumulation of the lipophilic cation [3H]tetraphenylpbosphonium, (iii) evoke both a calcium-dependent and -independent component of release. In the presence of calcium ions, SAA-evoked release was attenuated by a variety of EAA receptor antagonists, viz. CPP, APV, DNQX, and kynurenate. All SAA responses were sensitive to the presence of magnesium ions. Under calcium-free conditions, SAA-evoked release was unaffected by EAA antagonists, ttowever, this calcium-independent component of release was abolished in the presence of SKF 89976-A, a potent inhibitor of the GABA transporter, and, when sodium ions in the superfusion medium were replaced by choline. These observations suggest that: (1) the calcium-dependent SAA-evoked release of [3H]GABA is a wholly receptor-mediated event, thereby enhancing the transmitter candidacy oftbe SAAs. The evoked release appears to be mediated by activation of both NMDA and non-NMDA receptors, although on the basis of antagonist potency, a predominant non-NMDA receptor involvement is inferred, and (2) the calcium independent component of release arises from reversal of the GABA transporter due to reuptake-indueed depolarization following electrogenic cotransport of SAA with sodium ions. This observation is of significance especially with regard to neuropathologic conditions in which high extracellular levels of EAAs may be encountered.