GABA(C) rho(1) subunits form functional receptors but not functional synapses in hippocampal neurons
| Autor: | Jay Yang, Paul M. Burkat, John C. Kulli, Qing Cheng |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Physiology
Drug Resistance Neurotransmission Inhibitory postsynaptic potential Bicuculline Hippocampus Synaptic Transmission gamma-Aminobutyric acid Rats Sprague-Dawley Receptors GABA Postsynaptic potential medicine Animals Humans Picrotoxin Protein Isoforms Receptor Cells Cultured gamma-Aminobutyric Acid Neurons Chemistry General Neuroscience Neural Inhibition Receptors GABA-A Ruthenium Red Cell biology Rats medicine.anatomical_structure nervous system Synapses GABAergic Indicators and Reagents Neuron Neuroscience medicine.drug Forecasting |
| Zdroj: | Journal of neurophysiology. 86(5) |
| ISSN: | 0022-3077 |
| Popis: | The ability to control the physiological and pharmacological properties of synaptic receptors is a powerful tool for studying neuronal function and may be of therapeutic utility. We designed a recombinant adenovirus to deliver either a GABACreceptor ρ1subunit or a mutant GABAAreceptor β2subunit lacking picrotoxin sensitivity [β2(mut)] to hippocampal neurons. A green fluorescent protein (GFP) reporter molecule was simultaneously expressed. Whole cell patch-clamp recordings demonstrated somatic expression of both bicuculline-resistant GABACreceptor-mediated and picrotoxin-resistant GABAAreceptor-mediated GABA-evoked currents in ρ1- and β2(mut)-transduced hippocampal neurons, respectively. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of 6-cyano-7-nitroquinoxalene-2,3-dione, Mg2+, and TTX revealed synaptic events with monoexponential activation and biexponential decay phases. Despite the robust expression of somatic GABACreceptors in ρ1-neurons, no bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch between the relatively brief presynaptic GABA release and slow-activating ρ1receptors or failure of the ρ1subunit to target properly to the subsynaptic membrane. Addition of ruthenium red, a presynaptic release enhancer, failed to unmask GABACreceptor-mediated mIPSCs. Short pulse (2 ms) application of 1 mM GABA to excised outside-out patches from ρ1neurons proved that a brief GABA transient is sufficient to activate ρ1receptors. The simulated-IPSC experiment strongly suggests that if postsynaptic GABACreceptors were present, bicuculline-resistant mIPSCs would have been observed. In contrast, in β2(mut)-transduced neurons, picrotoxin-resistant mIPSCs were observed; they exhibited a smaller peak amplitude and faster decay compared with control. Confocal imaging of transduced neurons revealed ρ1immunofluorescence restricted to the soma, whereas punctate β2(mut) immunofluorescence was seen throughout the neuron, including the dendrites. Together, the electrophysiological and imaging data show that despite robust somatic expression of the ρ1subunit, the GABACreceptor fails to be delivered to the subsynaptic target. On the other hand, the successful incorporation of β2(mut) subunits into subsynaptic GABAAreceptors demonstrates that viral transduction is a powerful method for altering the physiological properties of synapses. |
| Databáze: | OpenAIRE |
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