Ethanol withdrawal hyper-responsiveness mediated by NMDA receptors in spinal cord motor neurons.
1. Following ethanol (EtOH) exposure, population excitatory postsynaptic potentials (pEPSPs) in isolated spinal cord increase to a level above control (withdrawal hyper-responsiveness). The present studies were designed to characterize this phenomenon and in particular to test the hypothesis that protein kinases mediate withdrawal. 2. Patch-clamp studies were carried out in motor neurons in rat spinal cord slices. Currents were evoked by brief pulses of glutamate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartic acid (NMDA). 3. Of 15 EtOH-sensitive neurons in which currents were evoked by glutamate, four (27%) displayed withdrawal hyper-responsiveness in the washout period. Mean current area after washout was 129.6+/-5% of control. 4. When currents were evoked by AMPA, two of 10 neurons (20%) displayed withdrawal hyper-responsiveness, with a mean current area 122+/-8% of control on washout. 5. Of a group of 11 neurons in which currents were evoked by NMDA, nine (82%) displayed withdrawal hyper-responsiveness. Mean increase in current area at the end of the washout period was to 133+/-6% of control (n=9, P<0.001). When NMDA applications were stopped during the period of EtOH exposure, mean area of NMDA-evoked responses on washout was only 98.0+/-5% of control (n=6, P>0.05). 6. The tyrosine kinase inhibitor genistein (10-20 microM) blocked withdrawal hyper-responsiveness. Of six EtOH-sensitive neurons, the mean NMDA-evoked current area after washout was 89+/-6% of control, P>0.05. 7 The protein kinase A (PKA) inhibitor Rp-cAMP (20-500 microM) did not block withdrawal hyper-responsiveness. On washout, the mean NMDA-evoked current area was 124+/-6% of control (n=5, P<0.05). 8 Two broad-spectrum specific protein kinase C (PKC) inhibitors, GF-109203X (0.3 microM) and chelerythrine chloride (0.5-2 nM), blocked withdrawal hyper-responsiveness. Responses on washout were 108+/-7%, n=5 and 88+/-4%, n=4 of control, respectively, P>0.05. 9 NMDA activation during EtOH exposure is necessary for withdrawal hyper-responsiveness. Both tyrosine kinase and PKC, but not PKA, appear to be essential for EtOH withdrawal hyper-responsiveness mediated by postsynaptic NMDA receptors in spinal cord motor neurons.
Department of Anesthesia, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, U.S.A. huifang@stanford.edu
Hui-Fang
HF
Li
Joan J
JJ
Kendig
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases; D000431:Ethanol / Q000009:adverse effects*; D019054:Evoked Potentials, Motor / Q000187:drug effects / Q000502:physiology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D009046:Motor Neurons / Q000187:drug effects / Q000502:physiology*; D016202:N-Methylaspartate / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000819:agonists / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000502:physiology*; D013116:Spinal Cord / Q000187:drug effects / Q000502:physiology*; D013375:Substance Withdrawal Syndrome / Q000503:physiopathology*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D018690:Excitatory Amino Acid Agonists; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D000431:Ethanol; D018698:Glutamic Acid; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C