Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons

• Published in: Frontiers in cellular neuroscience 2013, Vol.7, p.83-83
• Main Authors: Minlebaev, Marat, Valeeva, Guzel, Tcheremiskine, Vadim, Coustillier, Gaëlle, Khazipov, Rustem
• Format: Article
• Language: English
• Subjects: Brain slice preparation; Bumetanide; cell-attach; Chloride; Chloride transport; Depolarization; Engineering Sciences; GABA; Hippocampus; Inhibitory Postsynaptic Potentials; Laboratory animals; Neurons; Neuroscience; Neurosciences; Optics; patch-clamp; Photonic; Polarity; RuBi-GABA; uncaged GABA; Voltage; γ-Aminobutyric acid
• ISSN: 1662-5102; 1662-5102
• DOI: 10.3389/fncel.2013.00083

We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A) channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by RuBi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A) receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A) channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A)-Rs mediated signals in small cell compartments.