- Email: [email protected]
56. Neuroprotection from traumatic injury in the hippocampal slice with inhibitors of nitric oxide and ADP-ribosylation
A24
Abstracts/Journal of Neuroscience Methods (1994) A 1-1428
hydraulic manipulators improves their applicability to brain slice studies by easily converting them into stepping manipulators.
54. FURTHER IMPROVEMENT OF THE METHOD OF INTERNAL PERFUSION OF N E U R O N E S IN BRAIN SLICES
A.A. Velumian, L. Zhang, P.L. Carlen, P. Pennefather
Playfair Neuroscience Unit, Toronto Hospital Research Institute, MRC Group "Nerve Cells and Synapses", Departments of Physiology, Medicine (Neurology) and Pharmacology, Bloorview Epilepsy Research Program, Faculty of Pharmacy, University of Toronto, Toronto M5T 2S8, Canada The replacement of solutions in the patch pipette and thereby in the recorded cell is important for studies of various aspects of internal regulation of ion channels and other neuronal functions. Recently we have developed a simple method allowing multiple and fast solution exchanges in the patch pipette specifically designed for studies in brain slices. We are presenting an improved version of our internal perfusion method which has the following advantages: (i) loading and perfusion of various solutions immediately during recording without affecting the recording stability; (ii) an easy and fast procedure of replacement of patch pipettes with simultaneous positioning of the perfusing tube tip at a preset distance from the patch pipette tip (in our experiments, 250-300 IxM); (iii) a complete plastic perfusing tube with a tip pulled to 25-30 t.~M o.d. which can be used in many experiments without damage; (iv) minimized effects on the input capacitance and prevention of essential mixing of the perfused solution with the previously perfused solution. We will present experimental data demonstrating the effectiveness of internal perfusion in rat hippocampal CA1 neurones: up to 3 reversible complete replacements of K + by Cs + in a single neuron, perfusion of TEA and calcium ion chelators BAPTA and EGTA.
55. TYROSINE KINASES PLAY A ROLE IN LONGTERM POTENTIATION AND NOREPINEPHRINEI N D U C E D LONG-LASTING POTENTIATION IN RAT HIPPOCAMPAL DENTATE GYRUS
P.J. Voulalas, J.M. Sarvey
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA Tyrosine kinase inhibitors have been demonstrated previously to block long-term potentiation (LTP) in
CA1 and dentate gyrus. Both/3-adrenergic and NMDA receptors are required for induction of norepinephrine-induced long-lasting potentiation (NELLP) and LTP in the dentate gyrus. We examined (1) the effects of tyrosine kinase inhibitors on NELLP and (2) the effect of either high frequency stimulation (HFS) or bath-application of isoproterenol (IP) on tyrosine phosphorylation. Rat hippocampal slices were exposed in an interface chamber to either of the tyrosine kinase inhibitors genistein (110/xM) or lavendustin (5 izM) 30 min prior to, during, and after 20-min bath-application of l ~tM IP. Potentiation of the medial perforant path-granule cell population spike (PS) in the presence of IP was not significantly affected by either genistein or lavendustin. However, potentiation of the PS after 30 min wash-out of IP was completely blocked in genistein-treated slices; in lavendustin-treated slices, potentiation was reduced to 34% of potentiation after IP alone. IP (10 /xM) and HFS each transiently increased tyrosine phosphorylation of at least two proteins, detected by Western blot with an anti-phosphotyrosine antibody. The profile of proteins phosphorylated after IP differed from that after HFS. Furthermore, the onset of increased phosphorylation directly paralleled the increase in the amplitude of the PS; peak phosphorylation occurred 5-10 min after exposure to IP, but 30-60 s after HFS. Phosphorylation decreased by 20 min, when the PS was still maximally potentiated. These results imply involvement of tyrosine kinases in induction of both LTP and NELLP. Future studies will determine whether any of the phosphorylated substrates are necessary for both NELLP and LTP, or whether each form of potentiation utilizes a unique set of substrates. 56. NEUROPROTECTION FROM TRAUMATIC INJURY IN THE HIPPOCAMPAL SLICE WITH INHIBITORS OF NITRIC OXIDE AND ADP-RIBOSYLATION
K.L. Panizzon, J.M. Girard, R.A. Wallis
UCLA School of Medicine, Sepulveda VAMC, 16111 Plummer, Sepulveda, CA 91343, USA Traumatic neuronal injury appears to be mediated in large part through activation of NMDA receptors. Because neuronal injury from NMDA receptor activation has been shown to occur through nitric oxide production, and since ADP-ribosylation inhibitors have been shown to protect against direct nitric oxide application, we postulated that inhibitors of ADP-ribosylation might also be protective against traumatic neuronal injury. To test this hypothesis, we examined the neuroprotective potential of nitric oxide and ADP-ribosylation inhibitors against traumatic neuronal injury, using a
Abstracts/Journal of Neuroscience Methods (1994) AI-A28
fluid percussion model of traumatic injury with rat hippocampal slices. Slice trauma produced evidence of severe neuronal injury, with CA1 orthodromic and antidromic population spike recovering after one hour to only 20 + 7% and 15 ___8% of original amplitude. Treatment with the ADP-ribosylation inhibitor, nicotinamide (10 mM) for 35 min. after trauma, increased these recoveries to 96 _+4% and 98 _+2%. Similarly, another ADP-ribosylation inhibitor, 3'-aminobenzamide (1 mM), improved recoveries after trauma to 89 +_3% and 90 + 3%. In
A25
addition, protection was seen with post-traumatic application of 170 ~M methyl arginine which produced CA1 orthodromic and antidromic PS recoveries of 100 + 0 % and 91 _+2%. Likewise, 50 /zM hemoglobin which binds nitric oxide radicals, provided good protection against traumatic neuronal injury with recoveries of 87 _+ 1% and 92 +_ 1%. These results suggest that nitric oxide and ADPribosylation play a role in traumatic neuronal injury, and that ADP-ribosylation inhibition may represent a therapeutic strategy for head trauma.
Abstracts/Journal of Neuroscience Methods (1994) A 1-1428
hydraulic manipulators improves their applicability to brain slice studies by easily converting them into stepping manipulators.
54. FURTHER IMPROVEMENT OF THE METHOD OF INTERNAL PERFUSION OF N E U R O N E S IN BRAIN SLICES
A.A. Velumian, L. Zhang, P.L. Carlen, P. Pennefather
Playfair Neuroscience Unit, Toronto Hospital Research Institute, MRC Group "Nerve Cells and Synapses", Departments of Physiology, Medicine (Neurology) and Pharmacology, Bloorview Epilepsy Research Program, Faculty of Pharmacy, University of Toronto, Toronto M5T 2S8, Canada The replacement of solutions in the patch pipette and thereby in the recorded cell is important for studies of various aspects of internal regulation of ion channels and other neuronal functions. Recently we have developed a simple method allowing multiple and fast solution exchanges in the patch pipette specifically designed for studies in brain slices. We are presenting an improved version of our internal perfusion method which has the following advantages: (i) loading and perfusion of various solutions immediately during recording without affecting the recording stability; (ii) an easy and fast procedure of replacement of patch pipettes with simultaneous positioning of the perfusing tube tip at a preset distance from the patch pipette tip (in our experiments, 250-300 IxM); (iii) a complete plastic perfusing tube with a tip pulled to 25-30 t.~M o.d. which can be used in many experiments without damage; (iv) minimized effects on the input capacitance and prevention of essential mixing of the perfused solution with the previously perfused solution. We will present experimental data demonstrating the effectiveness of internal perfusion in rat hippocampal CA1 neurones: up to 3 reversible complete replacements of K + by Cs + in a single neuron, perfusion of TEA and calcium ion chelators BAPTA and EGTA.
55. TYROSINE KINASES PLAY A ROLE IN LONGTERM POTENTIATION AND NOREPINEPHRINEI N D U C E D LONG-LASTING POTENTIATION IN RAT HIPPOCAMPAL DENTATE GYRUS
P.J. Voulalas, J.M. Sarvey
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA Tyrosine kinase inhibitors have been demonstrated previously to block long-term potentiation (LTP) in
CA1 and dentate gyrus. Both/3-adrenergic and NMDA receptors are required for induction of norepinephrine-induced long-lasting potentiation (NELLP) and LTP in the dentate gyrus. We examined (1) the effects of tyrosine kinase inhibitors on NELLP and (2) the effect of either high frequency stimulation (HFS) or bath-application of isoproterenol (IP) on tyrosine phosphorylation. Rat hippocampal slices were exposed in an interface chamber to either of the tyrosine kinase inhibitors genistein (110/xM) or lavendustin (5 izM) 30 min prior to, during, and after 20-min bath-application of l ~tM IP. Potentiation of the medial perforant path-granule cell population spike (PS) in the presence of IP was not significantly affected by either genistein or lavendustin. However, potentiation of the PS after 30 min wash-out of IP was completely blocked in genistein-treated slices; in lavendustin-treated slices, potentiation was reduced to 34% of potentiation after IP alone. IP (10 /xM) and HFS each transiently increased tyrosine phosphorylation of at least two proteins, detected by Western blot with an anti-phosphotyrosine antibody. The profile of proteins phosphorylated after IP differed from that after HFS. Furthermore, the onset of increased phosphorylation directly paralleled the increase in the amplitude of the PS; peak phosphorylation occurred 5-10 min after exposure to IP, but 30-60 s after HFS. Phosphorylation decreased by 20 min, when the PS was still maximally potentiated. These results imply involvement of tyrosine kinases in induction of both LTP and NELLP. Future studies will determine whether any of the phosphorylated substrates are necessary for both NELLP and LTP, or whether each form of potentiation utilizes a unique set of substrates. 56. NEUROPROTECTION FROM TRAUMATIC INJURY IN THE HIPPOCAMPAL SLICE WITH INHIBITORS OF NITRIC OXIDE AND ADP-RIBOSYLATION
K.L. Panizzon, J.M. Girard, R.A. Wallis
UCLA School of Medicine, Sepulveda VAMC, 16111 Plummer, Sepulveda, CA 91343, USA Traumatic neuronal injury appears to be mediated in large part through activation of NMDA receptors. Because neuronal injury from NMDA receptor activation has been shown to occur through nitric oxide production, and since ADP-ribosylation inhibitors have been shown to protect against direct nitric oxide application, we postulated that inhibitors of ADP-ribosylation might also be protective against traumatic neuronal injury. To test this hypothesis, we examined the neuroprotective potential of nitric oxide and ADP-ribosylation inhibitors against traumatic neuronal injury, using a
Abstracts/Journal of Neuroscience Methods (1994) AI-A28
fluid percussion model of traumatic injury with rat hippocampal slices. Slice trauma produced evidence of severe neuronal injury, with CA1 orthodromic and antidromic population spike recovering after one hour to only 20 + 7% and 15 ___8% of original amplitude. Treatment with the ADP-ribosylation inhibitor, nicotinamide (10 mM) for 35 min. after trauma, increased these recoveries to 96 _+4% and 98 _+2%. Similarly, another ADP-ribosylation inhibitor, 3'-aminobenzamide (1 mM), improved recoveries after trauma to 89 +_3% and 90 + 3%. In
A25
addition, protection was seen with post-traumatic application of 170 ~M methyl arginine which produced CA1 orthodromic and antidromic PS recoveries of 100 + 0 % and 91 _+2%. Likewise, 50 /zM hemoglobin which binds nitric oxide radicals, provided good protection against traumatic neuronal injury with recoveries of 87 _+ 1% and 92 +_ 1%. These results suggest that nitric oxide and ADPribosylation play a role in traumatic neuronal injury, and that ADP-ribosylation inhibition may represent a therapeutic strategy for head trauma.