Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

The overall goal of this video is to illustrate a screening cascade that allows the discovery of novel GABA-A receptor ligands. The advantage of using radioligand binding, electrophysiological recording in xenopus oocytes and in rodent brain slices in a literative manner is that the profile of the compound can be improved. Ultimately, potent subtype, selective and efficacious compounds are identified.

This demonstration will be performed by Kumico Kambara and Jenna Tognaccini as well as Sonia and Daniel Bertrand from HiQScreen and Marie Claire Pflimlin from Roche. Inject 10 to 50 nanoliters of the solution containing plasmid using a glass microinjection needle with a tip diameter ranging up to 100 micrometers mounted on a micromanipulator equipped with a pressure ejection system or with an automated injection system. To begin this procedure, keep the oocytes at 17 degrees Celsius to prevent the expression of heat-shock proteins.

Store the microplate in a thermal-controlled storage area. Then, dissolve the test compounds, which were tested positive in the binding assay in OR2, at 0.1 and 1000 micromolar for electrophysiological recordings and dispose them in a 96-well flat-bottom polypropylene plate. To perform two electrode voltage clamp recording, place a plate containing the oocytes on the automated system.

Program the automated recording system using the icon-based interface with this scheme for the appropriate determination of the concentration activity relationship. For curve fitting, using the illustrated concentration activation curve, plot the current amplitude as a function of the logarithm of the agonist concentration. For electrophysiological recording, keep the brain in dACSF solution bubbled with carbogen at room temperature.

Then, dissect the left hippocampal formation with a fine spatula. Subsequently, section transverse slides of 400 micrometer thickness from the median part of the hippocampus with a tissue chopper. Using a painting brush, transfer the slices to the recording chamber and maintain them at room temperature for 45 minutes.

Afterward, perfuse the slices with rACSF bubbled with carbogen at 35 degrees Celsius and at a rate of 1.5 milliliters per minute. To record single population spike, place a brain slice in the microscope mounted chamber. Perfuse the slice with rACSF at a rate of three milliliters per minute.

Using the pipette puller, pull a borosilicate glass micropipette with a resistance of about two megaohms. Fill the micropipette with a solution containing two molar sodium chloride and place it into the pipette holder. Position the recording micropipette in the stratum pyramidale in the CA1 region of the hippocampal slice using the right micromanipulator.

Afterward, place an insulated bipolar platinum iridium electrode into the holder on the left micromanipulator. Position the stimulation electrode in the Schaffer collaterals in the CA1 region of the hippocampal slice using the left micromanipulator. Using the stimulus generator, deliver a current pulse to the stimulation electrode every 30 seconds and gradually increase the stimulation strength until a population spike appears.

Adjust the stimulus strength to evoke a population spike corresponding to 45%of the maximum amplitude that can be obtained. To perform paired pulse inhibition, deliver two current pulses to the stimulation electrode every 30 seconds using the stimulus generator. Set the stimulus strength to evoke a population spike corresponding to 45%of the maximum amplitude.

To test the compounds, make dilutions of the compounds to be tested in ACSF so that the final concentration of DMSO is not higher than 0.1%Add DMSO to the control solution at the same concentration as that in the compound solution. Record a single or a paired pulse population spike evoked by Schaffer collateral stimulations every 30 seconds for at least 30 minutes. The population spike shape should be stable during this baseline period.

Next, prepare a beaker with carbogenated rACSF containing a fixed concentration of the compound to be tested and perfuse the hippocampal slice with the solution during the recording of single or paired pulse population spikes. Also, evaluate the recovery from the compound effect by perfusing the slice with carbogenated rACSF without the compound. Shown here is a schematic representation of a rat hippocampal slice, the Schaffer collaterals originating from the CA3 pyramidal cell axons projecting onto the dendritic arborization of the CA1 pyramidal neurons.

Micropipettes were placed in the stratum pyramidale to record population spikes and in the stratum radiatum for dendritic recordings of field excitatory postsynaptic potentials. The stimulation electrode was placed within the Schaffer collaterals. This figure shows population spikes evoked by the paired stimuli applied through the same stimulating electrode at a 20 millisecond interval.

The population response to the second stimulus is of smaller amplitude than that of the response to the first stimulus. Population spikes were recorded in the absence and presence of beta CCM, a non-selective GABA A receptor NAM. Beta CCM enhanced the amplitude of the second population spike by partially blocking any feed-forward gabaergic inhibition.

Following this procedure, other methods as in pharmacokinetics, receptor occupancy and efficacy in vivo and safety pharmacology can be performed in order to answer additional questions like potential for clinical development of the compounds identified.