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Influence or metabotropic glutamate receptor agonists on the currents induced of AβP in rat cortical neurons
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IMMUNOCYTOCHEMICAL LOCALIZATION OF THE mGlnRlb METABOTROPIC GLUTAMATE RECEPTOR IN SYNAPTIC TERMINALS OF RAT HIPPOCAMPUS
Influence of metabotropic glutamate receptor agonists on the currents induced of Af3P in rat cortical neurons.
P. Grandes(l), J.M. Mat.eos(l), J. A&42), R Sarria(l), R. Benitez(lX D. Ruegg(3), B. Malitschek(3), R. Kuhn(3) and T. Knoepfel(1). Dept. Neuroaciencea, Faculty of Medicine and Dentistry, Basque Country University, 699-48080 Bilbao, Spain (1); Ii. Fhyaiology Institute, University of Heidelberg, D-69120, Heidelberg, Germany (2); Dept. of Cellular and Molecular Neurobiology, CNS Rosean%, Ciba K-125.6.12, CH-UX!, Bask, Switredarxi(3 j We used affinity purified antibodies specifically reacting with mGluRlb to investigate the expression of thia receptor in synaptic terminala of rat hippocampus. Immunocytochemical analysis at the light microscopical level revealed mGluRlb immunoreactivity within a distinct lamlnar staining pattern in the hippocampus. In the hilus, numemua strongly immunoreactive dota were placed in a band just underneath the granule cdl layer and between and dmely to CA4 pyramidal neurons. In the CA3, densely accumulating puncta heavily immunoreactive for mGluRlb were found mtricted to the stratum IucimUn To determine the identity of the mGluRlb itnmunoreactivity dots observed in the light microscope, a pmmbedding ABC and a p&embedding immunogold method for electron microscopy wets used. In the CA3 stratum lucidurn, strung mGluRlb immunoreactlvity was restricted to large synaptic boutons containing abundant synaptic vaicles and forming asymmetrical synaptic contacts onto dendritic thorns of CA3 pyramidal cells. These element could be clearly identified as masay fiber terminals. Supported by Eusko Jaurlaritza (J.M.M. BPI95.154, JA BF19J.017, and PI9407 grant).
V.V. Griporicv, I.G. Andronova. V.A. Ncmanova. V.V. Raguhn. S.O. Bachurin. Inslitutc of Physiologically Aclive Substances, Rus. Acad. Scl., Cliemogolovka,Moscow Region, Russia.
GROUP I mGLUR AGONISTS ALTER NEURONAL EXCITABILITY AND INTRACELLULAR CA*+ WA PHOSPHOLIPASE C (PLC) IN CULTURED PURKINJE NEURONS.
POTBNTIAYION OF mGloR RESPONSES BY BASIC PIBROBLAST GROWTE FACTOR IN HIPFOCAMPAL CULYURES J. Guiramand, E Blanc and M. Rccasena. CNRS ERS-5644 & Univeni!C Montpellier II - Place Eug&ne Bataillon - 34095
D.L. Gruel, J.G. Netzeband, K.L. Parsons and D.D. Sweeney Department of Neuropharmacology. The Scripps Research Inslitute. La Jolla, CA 92037 U.S.A. The purpose of the present studies was lo begin 10 define rhe transduction mechanisms underlying the changes in neuronal excitability produced in a defined neuronal type by metabotropic glutamate receptor (mGluR) agonists. To this end. Fura- microscopic Ca2+ imaging or current clamp recordings were used 10 record the Ca2+ signal or voltage response of cultured rat cerebellar Purkinje neurons (2 21 days in culture) to mGluR agonists applied by rapid microperfusion (I .5 s). The Group I agonists (IS,3R)- I aminocyclopentane-I,3-dicarboxylic acid (ACPD; 300 l.tM), 3.5-dihydroxyphenylglycine (DHPG; 200 FM). and quisqualate (5 l.tM) all increased intracellular Ca2+ and altered the membrane potential of Purkinje neurons, whereas Group II (L-CCG-I; IO PM) and Group 111(L-AP4 or L-SOP; 200-1000 pM) agonists were without effect. The Group I agonists also induced small, but consistent changes in the active membrane properties of Purkinje neurons as assessed by standard hyperpolarizing and depolarizing current test pulses. The actions of the Group I agonists were attenuated by bath application of the PLC inhibitor U-73122 (2 PM). but were relatively unaffected by the inactive analog U-73343 (2 FM). Preliminary evidence also suggests that the Group I mGluR-PLC pathway involves the Gq-protein. These results obtained from a defined neuronal type, the Purkinje neuron, confirm and extend biochemical and molecular studies of Group I mGluR transduction pathways studied in non-neuronal cells and whole-brain regions. Supported
by AA
06665
and
AA 05421.
Al4
Patch-clamp method was used for recording of amyioid-()protein-induced currtmts in acutely isolated rat cortical neumns. lnput current induced by APP:,.,I (IO-100 fl) rcachcd fast (during 0.5-5.0 s) a certain lcvcl and remained constant during IS-20 min of registration. Under influence of agonist melatiopic glutamate rcccplors L-AP-l(jx I O”M - 4x I OAM) in doscdcpendtnr mode this current was decreased to 80% from its maximum value. Increase of L-APJ dose t?om 2~10~ to 4~10~ M did not lead to growth of blocking of current. Thus it is possible that dose of 2xlc’ M was the maximal enc. In system witbout L-APJ the inilial lcvcl of tbe current was recovered. (DL)-para-(phosphonometbyl)phenyl-alanine (DL)-p-PMPA) and (L)-ortho(phosphonomcthyI)phcnyl-alaninc (L)-o-PMPA) which may considcrcd as cyclic analogs of L-API decreased APP-induced CIUT~II as well. (DL)p-PMPA was 6.S-fold and (L)+PMPA was 3-fold etTcctivc then L-AP-I. Both compounds blocked the current up to IUO%. It was shown in onr preliminary experiments that L2-amino-2-methyl+hosphonobutanoic acid (L-2-Me-AP4) IS also able to decrease APP:c.,s induced current. Its activily was about 5-7 times more than L-API one. Thus (DL)-gPMPA. (L)-oPMPA, and L-2-Me-AP4 are supposed to bc now eITective ago&s of metabotropic glutamate receptors of CNS.
MontpelIier C&x 5 - Prurc. The role of phospholipwe C (PIX)-linked metabotropic glutamate receptora (mOIuRS) In &veIopmentaI plasticity, was investigated in bippocampaI ll~uro~ in p&nay culhue. We have shown that mOluR agonists stimuI& in&to1 phosphate formation via hvo distinct rexeptom, OI)Cpr&rentIally rtinted by quiequalate (QA) the other me by IS.3R.unitto-cyclopropaopan dIc&oxyI~te (IS.3R.ACPD). These receptors present different PIE activation pattern during the in virro development of thee IIC(slur et& 1995, lnt. J. Dov. Neurcsi-, 13, 723-737). lltia differewe could be relata5 to apeciftc mgulationr of these receptora by protein kinuc C (!dlrmc et 11.1995, Neurccbem. Int.. 26. 623.633). To further demon&ate the putative Iink lxhvnn developmental plasticity and mCIuR activities. the in vitro development of neuronal cults was modulated by buic fibroblast gnwth factor @POP) which is known to promote both mu-vival and neurlte outgrowth of hippo+ IIOIMM. bPOP added 2 hours after plating &mngIy potentiata QA-indwsd incait phosphate fomution at and 2 DIV without affecting the ISfR-ACPD mwotme at these days. Convently. this treatment potcntiatcs the 1&3R-ACPD stimulation of PLC meuured at IO DIV. without affecting the QA rcnpoNe. These reaulta hwther nlppwt the hypothesis of the existence of two PLC-lb&cd mOLm in our aperbnenul model. Puthermo~=. the early QA nsponscpotmti~tion in concomitant to bPGP effect on mtuonll survival. l’hio suggata a prot4tva mls of bPGP and a function of mOluR in nammaI death, u recatIy propcwd @icolctti et al.. 1995. J. NeurochmL. 64. IOI-lo(1). The Iaterswcitic wtentition of-& li,fR-ACPD r&& might & linked to ;he &ndotion by bPGF of utmcyte proliterarion &or up.regulation of m&R in these cells. In fact. It bu been mcently demormtnted that bPt3P up-regulates mGluR5 in cortical ~trocyta (Miller et al.. 1995. J. NeumacI., IS. 6103-6109). Moreover. we have &avn by Northem blot uvlysir that mGhlR5 expprasion incrcllsss dting the in vitro dsvsl~ment. in a way ~bnilar to the IS,fR-ACPD-lndund bwsitolo phaphte raponu. The QA response wan not inhibited by &a&al mGIuR antagonists, Buggeating the poarible praem of an u yet unidentified m0IuR in hippocal@ cultlue.
I
54
IMMUNOCYTOCHEMICAL LOCALIZATION OF THE mGlnRlb METABOTROPIC GLUTAMATE RECEPTOR IN SYNAPTIC TERMINALS OF RAT HIPPOCAMPUS
Influence of metabotropic glutamate receptor agonists on the currents induced of Af3P in rat cortical neurons.
P. Grandes(l), J.M. Mat.eos(l), J. A&42), R Sarria(l), R. Benitez(lX D. Ruegg(3), B. Malitschek(3), R. Kuhn(3) and T. Knoepfel(1). Dept. Neuroaciencea, Faculty of Medicine and Dentistry, Basque Country University, 699-48080 Bilbao, Spain (1); Ii. Fhyaiology Institute, University of Heidelberg, D-69120, Heidelberg, Germany (2); Dept. of Cellular and Molecular Neurobiology, CNS Rosean%, Ciba K-125.6.12, CH-UX!, Bask, Switredarxi(3 j We used affinity purified antibodies specifically reacting with mGluRlb to investigate the expression of thia receptor in synaptic terminala of rat hippocampus. Immunocytochemical analysis at the light microscopical level revealed mGluRlb immunoreactivity within a distinct lamlnar staining pattern in the hippocampus. In the hilus, numemua strongly immunoreactive dota were placed in a band just underneath the granule cdl layer and between and dmely to CA4 pyramidal neurons. In the CA3, densely accumulating puncta heavily immunoreactive for mGluRlb were found mtricted to the stratum IucimUn To determine the identity of the mGluRlb itnmunoreactivity dots observed in the light microscope, a pmmbedding ABC and a p&embedding immunogold method for electron microscopy wets used. In the CA3 stratum lucidurn, strung mGluRlb immunoreactlvity was restricted to large synaptic boutons containing abundant synaptic vaicles and forming asymmetrical synaptic contacts onto dendritic thorns of CA3 pyramidal cells. These element could be clearly identified as masay fiber terminals. Supported by Eusko Jaurlaritza (J.M.M. BPI95.154, JA BF19J.017, and PI9407 grant).
V.V. Griporicv, I.G. Andronova. V.A. Ncmanova. V.V. Raguhn. S.O. Bachurin. Inslitutc of Physiologically Aclive Substances, Rus. Acad. Scl., Cliemogolovka,Moscow Region, Russia.
GROUP I mGLUR AGONISTS ALTER NEURONAL EXCITABILITY AND INTRACELLULAR CA*+ WA PHOSPHOLIPASE C (PLC) IN CULTURED PURKINJE NEURONS.
POTBNTIAYION OF mGloR RESPONSES BY BASIC PIBROBLAST GROWTE FACTOR IN HIPFOCAMPAL CULYURES J. Guiramand, E Blanc and M. Rccasena. CNRS ERS-5644 & Univeni!C Montpellier II - Place Eug&ne Bataillon - 34095
D.L. Gruel, J.G. Netzeband, K.L. Parsons and D.D. Sweeney Department of Neuropharmacology. The Scripps Research Inslitute. La Jolla, CA 92037 U.S.A. The purpose of the present studies was lo begin 10 define rhe transduction mechanisms underlying the changes in neuronal excitability produced in a defined neuronal type by metabotropic glutamate receptor (mGluR) agonists. To this end. Fura- microscopic Ca2+ imaging or current clamp recordings were used 10 record the Ca2+ signal or voltage response of cultured rat cerebellar Purkinje neurons (2 21 days in culture) to mGluR agonists applied by rapid microperfusion (I .5 s). The Group I agonists (IS,3R)- I aminocyclopentane-I,3-dicarboxylic acid (ACPD; 300 l.tM), 3.5-dihydroxyphenylglycine (DHPG; 200 FM). and quisqualate (5 l.tM) all increased intracellular Ca2+ and altered the membrane potential of Purkinje neurons, whereas Group II (L-CCG-I; IO PM) and Group 111(L-AP4 or L-SOP; 200-1000 pM) agonists were without effect. The Group I agonists also induced small, but consistent changes in the active membrane properties of Purkinje neurons as assessed by standard hyperpolarizing and depolarizing current test pulses. The actions of the Group I agonists were attenuated by bath application of the PLC inhibitor U-73122 (2 PM). but were relatively unaffected by the inactive analog U-73343 (2 FM). Preliminary evidence also suggests that the Group I mGluR-PLC pathway involves the Gq-protein. These results obtained from a defined neuronal type, the Purkinje neuron, confirm and extend biochemical and molecular studies of Group I mGluR transduction pathways studied in non-neuronal cells and whole-brain regions. Supported
by AA
06665
and
AA 05421.
Al4
Patch-clamp method was used for recording of amyioid-()protein-induced currtmts in acutely isolated rat cortical neumns. lnput current induced by APP:,.,I (IO-100 fl) rcachcd fast (during 0.5-5.0 s) a certain lcvcl and remained constant during IS-20 min of registration. Under influence of agonist melatiopic glutamate rcccplors L-AP-l(jx I O”M - 4x I OAM) in doscdcpendtnr mode this current was decreased to 80% from its maximum value. Increase of L-APJ dose t?om 2~10~ to 4~10~ M did not lead to growth of blocking of current. Thus it is possible that dose of 2xlc’ M was the maximal enc. In system witbout L-APJ the inilial lcvcl of tbe current was recovered. (DL)-para-(phosphonometbyl)phenyl-alanine (DL)-p-PMPA) and (L)-ortho(phosphonomcthyI)phcnyl-alaninc (L)-o-PMPA) which may considcrcd as cyclic analogs of L-API decreased APP-induced CIUT~II as well. (DL)p-PMPA was 6.S-fold and (L)+PMPA was 3-fold etTcctivc then L-AP-I. Both compounds blocked the current up to IUO%. It was shown in onr preliminary experiments that L2-amino-2-methyl+hosphonobutanoic acid (L-2-Me-AP4) IS also able to decrease APP:c.,s induced current. Its activily was about 5-7 times more than L-API one. Thus (DL)-gPMPA. (L)-oPMPA, and L-2-Me-AP4 are supposed to bc now eITective ago&s of metabotropic glutamate receptors of CNS.
MontpelIier C&x 5 - Prurc. The role of phospholipwe C (PIX)-linked metabotropic glutamate receptora (mOIuRS) In &veIopmentaI plasticity, was investigated in bippocampaI ll~uro~ in p&nay culhue. We have shown that mOluR agonists stimuI& in&to1 phosphate formation via hvo distinct rexeptom, OI)Cpr&rentIally rtinted by quiequalate (QA) the other me by IS.3R.unitto-cyclopropaopan dIc&oxyI~te (IS.3R.ACPD). These receptors present different PIE activation pattern during the in virro development of thee IIC(slur et& 1995, lnt. J. Dov. Neurcsi-, 13, 723-737). lltia differewe could be relata5 to apeciftc mgulationr of these receptora by protein kinuc C (!dlrmc et 11.1995, Neurccbem. Int.. 26. 623.633). To further demon&ate the putative Iink lxhvnn developmental plasticity and mCIuR activities. the in vitro development of neuronal cults was modulated by buic fibroblast gnwth factor @POP) which is known to promote both mu-vival and neurlte outgrowth of hippo+ IIOIMM. bPOP added 2 hours after plating &mngIy potentiata QA-indwsd incait phosphate fomution at and 2 DIV without affecting the ISfR-ACPD mwotme at these days. Convently. this treatment potcntiatcs the 1&3R-ACPD stimulation of PLC meuured at IO DIV. without affecting the QA rcnpoNe. These reaulta hwther nlppwt the hypothesis of the existence of two PLC-lb&cd mOLm in our aperbnenul model. Puthermo~=. the early QA nsponscpotmti~tion in concomitant to bPGP effect on mtuonll survival. l’hio suggata a prot4tva mls of bPGP and a function of mOluR in nammaI death, u recatIy propcwd @icolctti et al.. 1995. J. NeurochmL. 64. IOI-lo(1). The Iaterswcitic wtentition of-& li,fR-ACPD r&& might & linked to ;he &ndotion by bPGF of utmcyte proliterarion &or up.regulation of m&R in these cells. In fact. It bu been mcently demormtnted that bPt3P up-regulates mGluR5 in cortical ~trocyta (Miller et al.. 1995. J. NeumacI., IS. 6103-6109). Moreover. we have &avn by Northem blot uvlysir that mGhlR5 expprasion incrcllsss dting the in vitro dsvsl~ment. in a way ~bnilar to the IS,fR-ACPD-lndund bwsitolo phaphte raponu. The QA response wan not inhibited by &a&al mGIuR antagonists, Buggeating the poarible praem of an u yet unidentified m0IuR in hippocal@ cultlue.
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