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In vivo extracellular recordings from rat posterior cingulate and retrosplenial cortex after MK-801 injections
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87 EFFECT OF EXCITATIORY AMINO ACIDS ON THE LEVEL O~ FREE [Ca++]i IN CORTICAL ASTROCYTES L.A. McNaughton*, M. Socolovsky, K. Martin, S.P. Hunt* & P.A. McNaughton. Physiological Laboratory, Downing St, and *MRC Molecular Neurobiology Unit, Hills Road, Cambridge CB2 2QH. Cortical astrocytes were cultured for 8-12 days before loading with the fluorescent Ca indicator Pluo-B (Molecular Probes). Free [Ca++]i was imaged using a confocal microscope. Glutamate and quisqualate (all concs 100~M unless noted) elicited a series of rapid spike-like elevations in [Ca++]i , which were unaffected in the short term by removal of external Ca ++ , and persisted in the presence of the non-NMDA blocker CNQX. Kainate caused a slow elevation in [Ca++]i, often followed by slow oscillations. The slow rise, but not spike generation, was abolished by removal of [Ca++lo. We find that Ins(l,4,5)P 3 is increased ~everal fold by a 15 sec exposure to both quis and kain. There seems to be no simple relationship between spike frequency and IP 3 levels, though, because quis and kain were almost equally potent in elevating IP3, while quis is clearly more potent in eliciting spike activity. Factors other than IP 3 level may be involved in the generation of calcium spikes in astrocytes. Application of the phorbol ester PMA (100nM) completely abolishes spike production by glutamate and quisqualate, but the response to kainate is not affected by PMA. These data show that glutamate and analogues elevate clacium in astrocytes by two distinct mechanisms: (i) by repetitive spike-like elevations in [Ca++]i , caused by release from an internal store, and (2) by an influx o:~ calcium from the external medium which causes a more maintained rise in [Ca++]i. Glutamate and quis are potent agonists for (i), and kain for (2).
88 COMPARISON OF COMPETITIVE VS NON-COMPETITIVE N-METHYL-D ASPARTATE (NMDA) ANTAGONISTS IN THE TREATMENT OF EXPERIMENTAL BRAIN INJURY IN THE RAT Tracy K. Mclntosh, Douglas Smith, Mark Thomas and Roger P. Simon. University of Connecticut Health Center, Farmington CT 06032 and University of California, San Francisco, CA 94121 U.S.A. Treatment with NMDA receptor antagonists protects against cell death in experimental cerebral ischemia and spinal cord injury. We examined the effects of dextromethorphan (DM, a non-competitive NMDA receptor antagonist) and Cis-4-(phosphomethyl)2-piperidine-carboxylic acid (CGS 19755, a competitive NMDA receptor antagonist) on neurologic function, cation homeostasis and cerebral edema following lateral fluid-percussion (FP) brain injury in rats. Anesthetized male Sprague-Dawley rats (350-450g) were subjected to FP brain injury (2.4atm) centered over the left parietal cortex. Fifteen minutes after injury the animals received either DM (lOmg/kg IV n=24), CGS 19755 (10mg/kg IV n=24) or saline (IV n=24). At 48 hours postinjury, a subgroup of animals (n=12/treatment) was sacrificed, brains removed, dissected and assayed for water content using specific gravimetric techniques. Tissue samples were then analyzed for regional cation concentrations using atomic absorption spectrophotometry. Neurologic motor deficits were measured in a second group of animals (n=12/treatment) at 24 h, 1 wk and 2 wks post-injury. Animals treated with DM showed significant improvement in neurologic scores compared to saline treated animals. A worsening of neurologic function was observed after CGS 19755 administration. There were no significant differences in regional cerebral edema between drug treated and control animals. Surprisingly, DM caused a significant increase in total tissue calcium and sodium (p < 0.05) and a decrease in potassium concentrations (p < 0.05) at 48 h postinjury when compared to saline-treated controls. These results suggest that treatment with non-competitive antagonists may be beneficial in traumatic brain injury. (Supported in part by NIH ROI NS26818, the Sunny von Bulow Coma and Head Injury Foundation, and a HCRAC Faculty Grant).
89 I N VIVO EXTRACELLULAR RECORDINGS FROM RAT POSTERIOR CINGULATE AND RETROSPLENIAL CORTEX AFTER MK-801 INJECTIONS. Luiz E. Mello, Aiko M. Tan and David M. Finch, Brain Research Institute, UCLA, Los Angeles, CA 90024 U.S.A. A recent report by Olney et al. (Science, 1989,244:1360-1362) showed that systemic application of MK-801 produces morphologic changes that are restricted to layers III and IV of the posterior cingulate and retrosplenial cortices. Since there is no information regarding the physiology underlying thes.~ - morphologic changes, the present study was undertaken to examine the effects ofMK-801 injections on the firing of neurons in this region. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate (400 mg/kg), and a micropipet filled with 1 M NaCl saturated with Fast Green dye was lowered through the posterior cingulate and retrosplenial cortices in order to obtain extracellular recordings. Baseline firing rates were obtained for up to 30 minutes, after which MK-801 was injected (i.p., 1 - 5 mg/kg). Recording continued for as long as possible after the injection (up to 2 h). Other cells were recorded at various times prior to and after MK-801 injections. Recording sites were confirmed in Cresyl Violet-stained sections. Within 5-10 minutes after MK-801 injection, a marked decrease in the spontaneous fidng rate of cells located in layers IV and VI of both posterior cingulate and retrosplenial cortices could be seen. In some cells, firing decreased to I/4 of the baseline rate by 20 min after injection. Firing rates were suppressed for up to 2 h after injection. Firing rates of most of the cells in other layers (I, II, III, V) were not affected by MK-801 injection. We suggest that there may be a relation between our findings and the ones described by Olney (see above), despite some differences concerning the affected layers in these brain areas after MK-801 injection. Supported by NS 16721 (NIH) and FAPESP (Brazil).
87 EFFECT OF EXCITATIORY AMINO ACIDS ON THE LEVEL O~ FREE [Ca++]i IN CORTICAL ASTROCYTES L.A. McNaughton*, M. Socolovsky, K. Martin, S.P. Hunt* & P.A. McNaughton. Physiological Laboratory, Downing St, and *MRC Molecular Neurobiology Unit, Hills Road, Cambridge CB2 2QH. Cortical astrocytes were cultured for 8-12 days before loading with the fluorescent Ca indicator Pluo-B (Molecular Probes). Free [Ca++]i was imaged using a confocal microscope. Glutamate and quisqualate (all concs 100~M unless noted) elicited a series of rapid spike-like elevations in [Ca++]i , which were unaffected in the short term by removal of external Ca ++ , and persisted in the presence of the non-NMDA blocker CNQX. Kainate caused a slow elevation in [Ca++]i, often followed by slow oscillations. The slow rise, but not spike generation, was abolished by removal of [Ca++lo. We find that Ins(l,4,5)P 3 is increased ~everal fold by a 15 sec exposure to both quis and kain. There seems to be no simple relationship between spike frequency and IP 3 levels, though, because quis and kain were almost equally potent in elevating IP3, while quis is clearly more potent in eliciting spike activity. Factors other than IP 3 level may be involved in the generation of calcium spikes in astrocytes. Application of the phorbol ester PMA (100nM) completely abolishes spike production by glutamate and quisqualate, but the response to kainate is not affected by PMA. These data show that glutamate and analogues elevate clacium in astrocytes by two distinct mechanisms: (i) by repetitive spike-like elevations in [Ca++]i , caused by release from an internal store, and (2) by an influx o:~ calcium from the external medium which causes a more maintained rise in [Ca++]i. Glutamate and quis are potent agonists for (i), and kain for (2).
88 COMPARISON OF COMPETITIVE VS NON-COMPETITIVE N-METHYL-D ASPARTATE (NMDA) ANTAGONISTS IN THE TREATMENT OF EXPERIMENTAL BRAIN INJURY IN THE RAT Tracy K. Mclntosh, Douglas Smith, Mark Thomas and Roger P. Simon. University of Connecticut Health Center, Farmington CT 06032 and University of California, San Francisco, CA 94121 U.S.A. Treatment with NMDA receptor antagonists protects against cell death in experimental cerebral ischemia and spinal cord injury. We examined the effects of dextromethorphan (DM, a non-competitive NMDA receptor antagonist) and Cis-4-(phosphomethyl)2-piperidine-carboxylic acid (CGS 19755, a competitive NMDA receptor antagonist) on neurologic function, cation homeostasis and cerebral edema following lateral fluid-percussion (FP) brain injury in rats. Anesthetized male Sprague-Dawley rats (350-450g) were subjected to FP brain injury (2.4atm) centered over the left parietal cortex. Fifteen minutes after injury the animals received either DM (lOmg/kg IV n=24), CGS 19755 (10mg/kg IV n=24) or saline (IV n=24). At 48 hours postinjury, a subgroup of animals (n=12/treatment) was sacrificed, brains removed, dissected and assayed for water content using specific gravimetric techniques. Tissue samples were then analyzed for regional cation concentrations using atomic absorption spectrophotometry. Neurologic motor deficits were measured in a second group of animals (n=12/treatment) at 24 h, 1 wk and 2 wks post-injury. Animals treated with DM showed significant improvement in neurologic scores compared to saline treated animals. A worsening of neurologic function was observed after CGS 19755 administration. There were no significant differences in regional cerebral edema between drug treated and control animals. Surprisingly, DM caused a significant increase in total tissue calcium and sodium (p < 0.05) and a decrease in potassium concentrations (p < 0.05) at 48 h postinjury when compared to saline-treated controls. These results suggest that treatment with non-competitive antagonists may be beneficial in traumatic brain injury. (Supported in part by NIH ROI NS26818, the Sunny von Bulow Coma and Head Injury Foundation, and a HCRAC Faculty Grant).
89 I N VIVO EXTRACELLULAR RECORDINGS FROM RAT POSTERIOR CINGULATE AND RETROSPLENIAL CORTEX AFTER MK-801 INJECTIONS. Luiz E. Mello, Aiko M. Tan and David M. Finch, Brain Research Institute, UCLA, Los Angeles, CA 90024 U.S.A. A recent report by Olney et al. (Science, 1989,244:1360-1362) showed that systemic application of MK-801 produces morphologic changes that are restricted to layers III and IV of the posterior cingulate and retrosplenial cortices. Since there is no information regarding the physiology underlying thes.~ - morphologic changes, the present study was undertaken to examine the effects ofMK-801 injections on the firing of neurons in this region. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate (400 mg/kg), and a micropipet filled with 1 M NaCl saturated with Fast Green dye was lowered through the posterior cingulate and retrosplenial cortices in order to obtain extracellular recordings. Baseline firing rates were obtained for up to 30 minutes, after which MK-801 was injected (i.p., 1 - 5 mg/kg). Recording continued for as long as possible after the injection (up to 2 h). Other cells were recorded at various times prior to and after MK-801 injections. Recording sites were confirmed in Cresyl Violet-stained sections. Within 5-10 minutes after MK-801 injection, a marked decrease in the spontaneous fidng rate of cells located in layers IV and VI of both posterior cingulate and retrosplenial cortices could be seen. In some cells, firing decreased to I/4 of the baseline rate by 20 min after injection. Firing rates were suppressed for up to 2 h after injection. Firing rates of most of the cells in other layers (I, II, III, V) were not affected by MK-801 injection. We suggest that there may be a relation between our findings and the ones described by Olney (see above), despite some differences concerning the affected layers in these brain areas after MK-801 injection. Supported by NS 16721 (NIH) and FAPESP (Brazil).