Antisense oligonucleotide-induced block of individual GABAA receptor alpha subunits in cultured visual cortex slices reduces amplitude of evoked inhibitory postsynaptic currents.
Whole cell patch clamp recordings were made in layer II-IV from organotypic slices of rat primary visual cortex, explanted at postnatal day 6 and maintained in a serum-free medium. Neurons evinced current clamp characteristics typical for stellate cells. Between 7 and 21 days in culture, both glutamate- and GABA-mediated postsynaptic currents were observed. Long-term culturing in the presence of a degenerate 15-mer antisense oligonucleotide directed against the transcripts of all alpha subunits genes of the GABAA receptor resulted in a dose dependent reduction of evoked GABA synaptic currents. This reduction was maximal (80%) at 20 microM. A randomized control oligo had no effect. Evoked glutamatergic excitatory postsynaptic currents were unaffected following oligo treatment. A 15-mer antisense oligo directed against the alpha 1 subunit gave variable effects: in some cells the amplitude of evoked GABAergic inhibitory postsynaptic currents (IPSCs) was reduced by 50-75%, while in other cells recorded from the same slices, there was little or no effect. An antisense oligo, directed against the alpha 2 subunit, however, gave a consistent and robust 80% reduction of the amplitude of evoked IPSCs. A 15-mer 3-base mismatch oligo against alpha 2 had no effect. We conclude that the alpha 2 subunit functions in postsynaptic GABAA receptors located on or close to the cell bodies of stellate cells. The role of the alpha 1 subunit is less clear, but this subunit seems spatially differentiated. The in situ antisense oligo technique should provide further insight into the biophysical and pharmacological consequences of the subunit composition of ligand gated channels at functional synapses.
Brussaard
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Graduate School Neurosciences Amsterdam, Research Institute Neurosciences, Vrije Universiteit, Faculty of Biology, The Netherlands.
Baker
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D000818:Animals; D001483:Base Sequence; D004594:Electrophysiology; D005074:Evoked Potentials, Visual / Q000502:physiology; D058787:GABA-A Receptor Antagonists; D007150:Immunohistochemistry; D015640:Ion Channel Gating / Q000187:drug effects; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000187:drug effects / Q000378:metabolism*; D014793:Visual Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D058787:GABA-A Receptor Antagonists; D016376:Oligonucleotides, Antisense