Analysis by polymerase chain reaction of alpha 1 and alpha 6 GABAA receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings.
With the use of the single-cell polymerase chain reaction (PCR), the GABAA receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to alpha 1 and alpha 6 GABAA receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, alpha 1 subunit mRNA but not alpha 6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the alpha 1 and alpha 6 GABAA receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of alpha 6 subunit mRNA.
Department of Biology, Georgetown University, Washington, D.C. 20057.
M R
MR
Santi
S
S
Vicini
B
B
Eldadah
J H
JH
Neale
D001483:Base Sequence; D002531:Cerebellum / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004594:Electrophysiology; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011689:Purkinje Cells / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D018079:Receptors, GABA / Q000235:genetics*
D012333:RNA, Messenger; D018079:Receptors, GABA; D015252:Deoxyribonucleases, Type II Site-Specific; C067488:GTMKAC-specific type II deoxyribonucleases