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Upregulation of high-affinity GABA(A) receptors in cultured rat dorsal root ganglion neurons



Upregulation of high-affinity GABA(A) receptors in cultured rat dorsal root ganglion neurons



Neuroscience 208: 133-142



Despite evidence that high-affinity GABAAreceptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low affinity currents dominate those detected in acutely dissociated DRG neuronsin 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 GABAAcurrents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 hrs (acute) and 24 hrs (cultured) after plating with whole cell patch clamp techniques, western blot and qRT-PCR analysis. GABAAcurrent density increases dramatically with time in culture in association with the emergence of two persistent currents with EC5's of .25 .1 ?M and 3.2 .2 ?M 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 EC5of 78.16 1.1 ?M to 9.56 1.3 ?M with time in culture. A fraction of the high affinity current was potentiated by ?-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). ?-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute but the membrane fraction in cultured DRG neurons with no detectable change in ?-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 ?2and3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity ?-subunit containing GABAAreceptors 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.?GABAAcurrents in DRG neurons change with time in culture. High affinity extrasynaptic GABAAcurrents are increased. ?-subunit containing receptors already present are trafficked to the membrane. An increase in ?-subunit expression and functional receptors is also detected.

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Accession: 056780934

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PMID: 22366297

DOI: 10.1016/j.neuroscience.2012.01.050


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