title: Upregulation of high-affinity GABA(A) receptors in cultured rat dorsal root ganglion neurons.
abstract: Despite evidence that high-affinity GABA(A) receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low-affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high-affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABA(A) currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 h (acute) and 24 h (cultured) after plating with whole-cell patch-clamp techniques, Western blot, and semiquantitative reverse transcriptase polymerase chain reaction (sqRT-PCR) analysis. GABA(A) current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50's of 0.25±0.01 μM and 3.2±0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16±10.1 μM to 9.56±1.3 μM with time in culture. A fraction of the high-affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute, but the membrane fraction in cultured, DRG neurons, with no detectable change in δ-subunit mRNA. However, the emergence of a high-affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2- and ρ3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit-containing GABA(A) receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution.
authors.affiliation: Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: K Y
authors.initials: KY
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Charbonnet
authors.forename: M S
authors.initials: MS
authors.lastname: Gold
mesh_terms: D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003437:Crotonates / Q000494:pharmacology; D055724:Electrophysiological Phenomena; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007555:Isoxazoles / Q000494:pharmacology; D008297:Male; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D011963:Receptors, GABA-A / Q000096:biosynthesis*; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D003437:Crotonates; D018755:GABA Agonists; D018756:GABA Antagonists; D007555:Isoxazoles; D011963:Receptors, GABA-A; C055331:4-aminocrotonic acid; C015542:gaboxadol; D001640:Bicuculline