Gabac ?1 subunits form functional receptors but not functional synapses in
hippocampal neurons.
Cheng, Qing, Paul M Burkat, John C Kulli, Jay Yang.
Departments of Pharmacology / Physiology1 and Anesthesiology2, University of
Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14641
APStracts 8:0235J, 2001.
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 GABAC receptor ?1
subunit or a mutant GABAA receptor ?? subunit lacking picrotoxin sensitivity (???mut???
to hippocampal neurons. A GFP reporter molecule was simultaneously expressed.
Whole-cell patch clamp recordings demonstrated somatic expression of both bicuculline-
resistant GABAC receptor-mediated and picrotoxin-resistant GABAA receptor-mediated
GABA-evoked currents in ?1- and ?2(mut)-transduced hippocampal neurons,
respectively. GABAergic mIPSCs recorded in the presence of CNQX, Mg2+, and TTX
revealed synaptic events with monoexponential activation and biexponential decay
phases. Despite the robust expression of somatic GABAC receptors in ?1-neurons, no
bicuculline-resistant mIPSCs were observed. This suggested either a kinetic mismatch
between the relatively brief presynaptic GABA release and slow-activating ?1 receptors,
or failure of the ?1 subunit to target properly to the subsynaptic membrane. Addition of
ruthenium red, a presynaptic release enhancer, failed to unmask GABAC receptor-
mediated mIPSCs. Short pulse (2 ms) application of 1mM GABA to excised outside-out
patches from ?1-neurons proved that a brief GABA transient is sufficient to activate ?1
receptors. The simulated-IPSC experiment strongly suggests that if post-synaptic
GABAC receptors were present, bicuculline-resistant mIPSCs would have been
observed. In contrast, in ?2(mut)-transduced neurons, picrotoxin-resistant mIPSCs were
observed; they exhibited a smaller peak amplitude and faster decay compared to control.
Confocal imaging of transduced neurons revealed ?1-immunofluorescence restricted to
the soma while punctate ?2(mut) immunofluorescence was seen throughout the neuron,
including the dendrites. Together, the electrophysiological and imaging data show that,
despite robust somatic expression of the ?1 subunit, the GABAC receptor fails to be
delivered to the subsynaptic target. On the other hand, the successful incorporation of
?2(mut) subunits into subsynaptic GABAA receptors demonstrates that viral transduction
is a powerful method for altering the physiological properties of synapses.
Received 7 March 2001; accepted in final form 30 May 2001
APS Manuscript Number J188-1.
Article publication pending Am J Physiol
ISSN 1080-4757 Copyright 2001 The American Physiological Society.
Published in APStracts on 31 July 2001