Depolarizing GABA-activated Cl- channels in embryonic rat spinal and olfactory bulb cells.
1. We have compared the electrical properties of the Cl- channels activated by GABA in cells acutely dissociated from embryonic (E) spinal cord (SC) and olfactory bulb (OB) regions at E15 using different configurations of the patch-recording technique. By in situ analysis these cells express GABAA receptor mRNAs encoding a common set of subunits (alpha 2, beta 2, and beta 3). SC cells also express alpha 3, alpha 5 and gamma 2s transcripts. 2. Whole-cell recordings revealed current responses to GABA (0.5 microM to 1 mM) in 242 out of 294 cells. In both SC and OB cells, currents evoked by 2 microM GABA could be potentiated by diazepam (DZP) in a dose-dependent manner with an EC50 of approximately 50 nM in both SC and OB. The maximal effect was approximately 300%. Both SC and OB cells exhibited GABA-activated currents that were only partially sensitive to zinc even at high micromolar concentrations. The effect of DZP and the relatively modest sensitivity to zinc suggest the presence of gamma subunits in both preparations. 3. Spectral analysis of current responses in twenty-six cells showed that power spectra could be fitted by three exponential components (tau 1-3) in the cells of both areas. The tau of the longest-lasting component (tau 3) was significantly different in the cells of the two areas: approximately 50 ms in OB and 80-100 ms in SC. No statistically significant differences in the average inferred unitary conductance between the two cell types could be resolved. 4. Single-channel properties were examined directly using the cell-attached configuration. GABA-activated channels could be recorded in only 89 out of well-sealed 984 patches and most of them exhibited multiple channel activity. The mean open time in the response to 10 microM GABA was significantly shorter in OB cells (12 ms) compared to SC cells (25 ms) while the average conductance values were not significantly different between the two cell types. 5. On average, Cl- channels reversed polarity when the on-cell patch pipette potential was approximately -30 mV. Thus, in these embryonic neurons, micromolar GABA activates Cl- channels, which, when open, effectively depolarize cells by approximately 30 mV. 6. Cl- channels activated by GABA are open longer in embryonic SC cells than in OB cells. This statistically significant difference in native GABAA receptor Cl- channel properties correlates with, and may be related to differences in subunit mRNA expression.
Serafini
R
R
Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Valeyev
A Y
AY
Barker
J L
JL
Poulter
M O
MO
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009830:Olfactory Bulb / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000096:biosynthesis; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D018118:Chloride Channels; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D015032:Zinc