Name: gaba-activated single-channel and tonic currents in rat brain slices
Uploaded: 2011-07-17T14:03:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about GABA-activated Single-channel and Tonic Currents in Rat Brain Slices at JoVE.com

This work was funded by the Swedish Research Council, Uppsala University and the Fredrik and Ingrid Thurings Foundation. ZJ held a postdoctoral fellowship from EXODIAB and Svenska S&auml;llskapet f&ouml;r Medicinsk Forskning. We thank Frank Lee for technique assistance.

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No conflicts of interest declared.

The whole-cell current represents the ensemble of currents generated by active channels in the cell. The specific pharmacology and chloride 
permeability of the GABAA channels makes it possible to identify currents associated with these channels. The transient nature of activation of synaptic 
channels and the tonic activation of the extrasynaptic channels allows easy differentiation between these two populations of GABAA channels expressed in a neuron. 
However, only with single-channel recordings can one study the kinetic properties of the channel molecules that generate the tonic currents. Unlike synaptic 
channels that activate within 100 &mu;s, the tonic GABAA channels are activated with a latency in the range of minutes from the time GABA is applied 4,7,15,16,17,18. This delayed activation of the channels results in that many channel properties cannot be studied using whole-cell recordings. At the single-channel level the different sub-populations of GABAA channels can be identified based on varying functional and pharmacological properties. Thus, by taking an advantage of what the different patch-clamp configurations have to offer, it is possible to study in detail the molecular function and pharmacology of the GABA-activated channels that generate the tonic and synaptic currents in neurons.

Several patch-clamp hardware and software systems are available and they all operate using the same basic principles. In our laboratory we use the Axoclamp amplifiers and the PClamp software (Molecular Devices, USA) to record and analyze our experiments. Another popular brand is HEKA amplifiers and software (HEKA Elektronik , Germany) 
Microforge: Narishige MF-830 (Japan); Vibrotome: e.g. Leica VT1200S (Germany) or Campden Instruments vibrotome (UK); Microscope: Nikon Eclipse E600FN; Water bath; Micromanipulators: custom made and Narishige micromanipulators (Japan); Puller: DMZ-universal (Germany) or Narishige PC-10 (Japan). Electrode glass: GC150F-15 (Harvard Apparatus, UK).

gaba-activated single-channel and tonic currents in rat brain slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

Watch this Scientific Journal Video about GABA-activated Single-channel and Tonic Currents in Rat Brain Slices at JoVE.com

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

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