Mouse spinal cord and cortical neurons in primary dissociated cell culture will be used to investigate the actions of benzodiazepine (BDZ) receptor ligands and barbiturates on GABA-activated whole cell currents and single channel currents. A major goal of these studies is to understand the organization of the GABA receptor and its associated ion channel and the mechanisms whereby clinically used anticonvulsant drugs modify GABAergic inhibition. To perform these studies, intracellular, voltage clamp and patch clamp techniques will be applied to the mouse neurons in cell culture. Pharmacological studies of BDZ receptor ligands and barbiturates will be performed using intracellular recording and voltage clamp techniques. Spinal cord and cortical neurons will be impaled and the voltage or current responses to the application of GABA will be recorded. BDZ receptor ligands and barbiturates will be applied to neurons using local superfusion techniques. Control GABA responses will be obtained prior to drug application. BDZ receptor ligands or barbiturates will be applied using independently controlled micropipettes. A major goal will be to characterize the actions of barbiturates and BDZ receptor ligands on GABA-activated chloride currents and voltages to the full range of agonist, antagonist and inverse agonist BDZ receptor ligands and the full range of anticonvulsant and anesthetic barbiturates. A second phase of these investigations will involve application of the patch clamp technique to spinal cord and cortical neurons. Excised outside out patches will be obtained using symmetrical chloride medium and nonpermeant cations in the patch clamp pipette. Under these circumstances, GABA activates single channel chloride currents of 2-4 picoamps in the + 100 mV range. These channels have conductances of 10-50 picosiemens. Barbiturates and BDZ receptor ligands characterized in the first part of the studies will be applied to the excised outside out patches and their effect on the GABA-activated single chloride currents will be determined. Major emphasis will be placed upon obtaining a kinetic scheme which will describe the mechanism whereby barbiturates and BDZ receptor ligands modify the gating characteristics of the GABA-activated single channel chloride currents. For example, it has been proposed that BDZs enhance the probability for activation of single channel chloride currents but do not modify the mean duration of channel opening. Barbiturates, however, have been proposed to prolong channel duration. We will test these hypotheses directly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08NS001019-01
Application #
3083670
Study Section
Neurological Disorders Program Project Review B Committee (NSPB)
Project Start
1985-07-01
Project End
1990-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Wiley, J W; Gross, R A; MacDonald, R L (1993) Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite actions on rat nodose neuron calcium currents. J Neurophysiol 70:324-30
Wiley, J W; Gross, R A; MacDonald, R L (1992) The peptide CGRP increases a high-threshold Ca2+ current in rat nodose neurones via a pertussis toxin-sensitive pathway. J Physiol 455:367-81
Kelly, K M; Gross, R A; Macdonald, R L (1991) Tetrahydroaminoacridine (THA) reduces voltage-dependent calcium currents in rat sensory neurons. Neurosci Lett 132:247-50
Gross, R A; Uhler, M D; Macdonald, R L (1990) The reduction of neuronal calcium currents by ATP-gamma-S is mediated by a G protein and occurs independently of cyclic AMP-dependent protein kinase. Brain Res 535:214-20
Gross, R A; Moises, H C; Uhler, M D et al. (1990) Dynorphin A and cAMP-dependent protein kinase independently regulate neuronal calcium currents. Proc Natl Acad Sci U S A 87:7025-9
Kelly, K M; Gross, R A; Macdonald, R L (1990) Valproic acid selectively reduces the low-threshold (T) calcium current in rat nodose neurons. Neurosci Lett 116:233-8
Gross, R A; Uhler, M D; Macdonald, R L (1990) The cyclic AMP-dependent protein kinase catalytic subunit selectively enhances calcium currents in rat nodose neurones. J Physiol 429:483-96
Gross, R A; Wiley, J W; Ryan-Jastrow, T et al. (1990) Regulation by GTP and its stable thiol derivatives of calcium current components in rat nodose ganglion neurons. Mol Pharmacol 37:546-53
Gross, R A; Macdonald, R L; Ryan-Jastrow, T (1989) 2-Chloroadenosine reduces the N calcium current of cultured mouse sensory neurones in a pertussis toxin-sensitive manner. J Physiol 411:585-95
Gross, R A; Macdonald, R L (1988) Reduction of the same calcium current component by A and C kinases: differential pertussis toxin sensitivity. Neurosci Lett 88:50-6

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