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Development of glutamate receptors involved in visual cortical synaptic transmission studied by organotypic slice culture preparations
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POTENTIATING EFFECTS OF A NOOTROPIC DRUG ANIRACETAM ON THE GLUTAMATE RECEPTOR SUBUNITS, GLURl AND GLURZ, EXPRESSED IN XENOPUS OOCYTES. KEISUKE TSUZUKI, ’ TOSHIYUKI TAKEUCHI,2AND SEIJI OZAWA, 1 'Department of Physiology, School of Medicine, and Division of Molecular Endocrinology, Institute for Endocrinology, Gunma University, 3-39-22 Showa-machi, Maebashi-shi, Gunma 371, Japan. GluRl and GluR2 cDNAs encoding non-NMDA subtypes of glutamate receptor were isolated from a rat brain cDNA library by Boulter et al. (Science -249:1033-1037, 1990). Functional receptors activated by kainate, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and glutamate were expressed in Xenopus oocytes injected with GluRl, GluR2 or mixture of GluRl and GluR2 RNAs. In oocytes injected with GluRl alone, 1 mM aniracetam potentiated AMPA-activated currents by 99 ? 18 % (mean + S.D., n=5) and glutamate-activated currents by 140 ?r11 % (n=4), but little affected kainate-activated currents. The effect of aniracetam was detected at 0.1 mM, and it increased in a concentration-dependent manner in the range of 0.1 - 5 mM. GluRZ-injected oocytes showed no response to these agonists. In both GluRl and GluRZ-injected oocytes, aniracetam more markedly potentiated current responses to AMPA and glutamate than in GluRl-injected oocytes. For example, 1 mM aniracetam potentiated AMPA-activated currents by 396 t 107 % (n=4) and glutamateactivated currents by 970 ? 130 % (n=5) in oocytes injected with 10 % GluRl RNA and 90 % GluR2 RNA. In these oocytes, the potentiation of kainate-activated currents in 1 mM aniracetam was only 8 * 7 % (n=4). It is therefore concluded that the potentiation of kainate/AMPA receptors by aniracetam depends on both species of agonists and subunit composition of receptors.
SPANTIDE-SENSITIVE FACILITATORY EFFECT OF THIN FIBER STIMULATIONS ON NMDA RECEPTOR-MEDIATED MONOSYNAPTIC REFLEX IN THE NEWBORN RAT SPINAL CORD. YOSHIO HARADA. Deuartment of Phvsiolopv, Ninnon Medical School. l-l-5 Sendaei. Bunkvo-ku. Tokvo 113, Jm In the spinal cord, it has been thought that Ia primary afferents release L-glutamate and Ia EPSP is mediated by non-NMDA receptors on motoneurones. In an isolated spinal cord preparation from newborn rats ( 2-5 days old ), monosynaptic reflexes ( MSRs ) evoked by dorsal root stimulations at l/15 set were eliminated completely by DNQX ( 2.5 PM ) and depressed partially by APV ( 20 PM ). If voltage dependent blockade of NMDA receptors by Mg+ + is taken into consideration, these results suggest both NMDA and non-NMDA receptors mediate MSRs. When the stimulation rate was increased to l/set, the amplitudes of MSRs were greatly reduced initially and, then, increased gradually. In solutions containing APV, MSRs ( mediated by non-NMDA receptors ) were suppressed significantly at l/set. In solutions containing DNQX and no Mg++, MSRs ( mediated by NMDA receptors ) decreased initially and increased gradually at l/set, to the same magnitude as those seen in the normal solution. This facilitation of the NMDA component at l/set required stronger stimulations known to activate c-fibers in this preparation, and was eliminated by spantide ( 8 PM ), a tachykinin antagonist. It is concluded that thin sensory fibers can enhance NMDA receptor-mediated Ia monosynaptic transmissions through tachykinin receptors activation, in order to improve synaptic efficacy during repetitive activations in newborn rats.
Development of glutamate receptors involved in visual cortical synaptic transmission studied by organotypic slice culture preparations. K. F. Sladeczek . of . . . . . K. Tova~~a,mment Phvsiolggv. Kvoto Pmfc&@ral Umvm. 465 mo-ku. Ju Recent rat neocortical slice studies have suggested that the ability of exhibiting LTP in the presence of intact inhibitory synaptic mechanisms is limited to a critical period in supragranular layers while infragranular layers retain the ability in ad&hood. Also the relevance of developmental time course of NMDA receptor activation with these plastic changes have been suggested. The use of organotypic cocultures allows the study of specific elements of cortical circuitry in isolation from others. In the present study, development of ionotropic and metabotropic glutamate receptors were investigated using different types (lateral geniculate nucleus (LGN)-visual cortex (VC), VC-VC) of these coculturcs. The relative contributions of NMDA and non-NMDA receptors in generating synaptic potentials were assessed by measuring the initial EPSP slopes at the same membrane potential in the absence and presence of DNQX (20 uM). In layer III-IV cells of LGN-VC cocultures which received monosynaptic excitation from LGN, the NMDA component remaining in the presence of DNQX was relatively small (5.5k2.896) in young (8-11 days in vitro (DIV)), peaked (11.7&5.2%) in juvenile (12-15DIV), and was smallest (2.2*1.5%) in old (18-33DIV) cultures. In contrast, layer V cells of VC-VC cocultures which received monosynaptic excitanon from another VC explant showed no significant change of the NMDA component, (14.5+7.7%), (16.5+5.4%), and (12.1&3.1%), for young, juvenile, and old cultures respectively. These results might have implications for the remaining plasticity of intiagranular layers in adulthood. Activation of metabotropic receptors (mGluR) by the selective agonist t-ACPD (3OOuM) was also studied. t-ACPD induced a strong depmssion of BPSPs in layer III-IV cells of LGN-VC cocultums. The depression was stronger in young (80.3k8.396) than old (61.2k5.496) cultures suggesting a more prominent role of mGluR during synaptogenesis.
POTENTIATING EFFECTS OF A NOOTROPIC DRUG ANIRACETAM ON THE GLUTAMATE RECEPTOR SUBUNITS, GLURl AND GLURZ, EXPRESSED IN XENOPUS OOCYTES. KEISUKE TSUZUKI, ’ TOSHIYUKI TAKEUCHI,2AND SEIJI OZAWA, 1 'Department of Physiology, School of Medicine, and Division of Molecular Endocrinology, Institute for Endocrinology, Gunma University, 3-39-22 Showa-machi, Maebashi-shi, Gunma 371, Japan. GluRl and GluR2 cDNAs encoding non-NMDA subtypes of glutamate receptor were isolated from a rat brain cDNA library by Boulter et al. (Science -249:1033-1037, 1990). Functional receptors activated by kainate, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and glutamate were expressed in Xenopus oocytes injected with GluRl, GluR2 or mixture of GluRl and GluR2 RNAs. In oocytes injected with GluRl alone, 1 mM aniracetam potentiated AMPA-activated currents by 99 ? 18 % (mean + S.D., n=5) and glutamate-activated currents by 140 ?r11 % (n=4), but little affected kainate-activated currents. The effect of aniracetam was detected at 0.1 mM, and it increased in a concentration-dependent manner in the range of 0.1 - 5 mM. GluRZ-injected oocytes showed no response to these agonists. In both GluRl and GluRZ-injected oocytes, aniracetam more markedly potentiated current responses to AMPA and glutamate than in GluRl-injected oocytes. For example, 1 mM aniracetam potentiated AMPA-activated currents by 396 t 107 % (n=4) and glutamateactivated currents by 970 ? 130 % (n=5) in oocytes injected with 10 % GluRl RNA and 90 % GluR2 RNA. In these oocytes, the potentiation of kainate-activated currents in 1 mM aniracetam was only 8 * 7 % (n=4). It is therefore concluded that the potentiation of kainate/AMPA receptors by aniracetam depends on both species of agonists and subunit composition of receptors.
SPANTIDE-SENSITIVE FACILITATORY EFFECT OF THIN FIBER STIMULATIONS ON NMDA RECEPTOR-MEDIATED MONOSYNAPTIC REFLEX IN THE NEWBORN RAT SPINAL CORD. YOSHIO HARADA. Deuartment of Phvsiolopv, Ninnon Medical School. l-l-5 Sendaei. Bunkvo-ku. Tokvo 113, Jm In the spinal cord, it has been thought that Ia primary afferents release L-glutamate and Ia EPSP is mediated by non-NMDA receptors on motoneurones. In an isolated spinal cord preparation from newborn rats ( 2-5 days old ), monosynaptic reflexes ( MSRs ) evoked by dorsal root stimulations at l/15 set were eliminated completely by DNQX ( 2.5 PM ) and depressed partially by APV ( 20 PM ). If voltage dependent blockade of NMDA receptors by Mg+ + is taken into consideration, these results suggest both NMDA and non-NMDA receptors mediate MSRs. When the stimulation rate was increased to l/set, the amplitudes of MSRs were greatly reduced initially and, then, increased gradually. In solutions containing APV, MSRs ( mediated by non-NMDA receptors ) were suppressed significantly at l/set. In solutions containing DNQX and no Mg++, MSRs ( mediated by NMDA receptors ) decreased initially and increased gradually at l/set, to the same magnitude as those seen in the normal solution. This facilitation of the NMDA component at l/set required stronger stimulations known to activate c-fibers in this preparation, and was eliminated by spantide ( 8 PM ), a tachykinin antagonist. It is concluded that thin sensory fibers can enhance NMDA receptor-mediated Ia monosynaptic transmissions through tachykinin receptors activation, in order to improve synaptic efficacy during repetitive activations in newborn rats.
Development of glutamate receptors involved in visual cortical synaptic transmission studied by organotypic slice culture preparations. K. F. Sladeczek . of . . . . . K. Tova~~a,mment Phvsiolggv. Kvoto Pmfc&@ral Umvm. 465 mo-ku. Ju Recent rat neocortical slice studies have suggested that the ability of exhibiting LTP in the presence of intact inhibitory synaptic mechanisms is limited to a critical period in supragranular layers while infragranular layers retain the ability in ad&hood. Also the relevance of developmental time course of NMDA receptor activation with these plastic changes have been suggested. The use of organotypic cocultures allows the study of specific elements of cortical circuitry in isolation from others. In the present study, development of ionotropic and metabotropic glutamate receptors were investigated using different types (lateral geniculate nucleus (LGN)-visual cortex (VC), VC-VC) of these coculturcs. The relative contributions of NMDA and non-NMDA receptors in generating synaptic potentials were assessed by measuring the initial EPSP slopes at the same membrane potential in the absence and presence of DNQX (20 uM). In layer III-IV cells of LGN-VC cocultures which received monosynaptic excitation from LGN, the NMDA component remaining in the presence of DNQX was relatively small (5.5k2.896) in young (8-11 days in vitro (DIV)), peaked (11.7&5.2%) in juvenile (12-15DIV), and was smallest (2.2*1.5%) in old (18-33DIV) cultures. In contrast, layer V cells of VC-VC cocultures which received monosynaptic excitanon from another VC explant showed no significant change of the NMDA component, (14.5+7.7%), (16.5+5.4%), and (12.1&3.1%), for young, juvenile, and old cultures respectively. These results might have implications for the remaining plasticity of intiagranular layers in adulthood. Activation of metabotropic receptors (mGluR) by the selective agonist t-ACPD (3OOuM) was also studied. t-ACPD induced a strong depmssion of BPSPs in layer III-IV cells of LGN-VC cocultums. The depression was stronger in young (80.3k8.396) than old (61.2k5.496) cultures suggesting a more prominent role of mGluR during synaptogenesis.