Electrophoretic Delivery of GABA into the Epileptic Focus of an Anesthetized Mouse

Take an anesthetized mouse prepared for brain implant placement.

The mouse has a skull-fixed ground screw in contact with the cerebellum for reference signal recording.

Insert the probe connected to the headstage and ground screw into the hippocampus to record neuronal activity.

Identify synchronized bursts of neuronal activity, confirming probe placement in the hippocampal pyramidal layer.

Next, insert a GABA-filled microfluidic ion pump into the hippocampus, with a source electrode in its channel. The skull-fixed ground screw acts as the target electrode.

Finally, insert a micropipette into the hippocampus, and inject a potassium channel blocker to induce neuronal hyperexcitability, triggering seizures.

Immediately apply voltage to the pump to generate an electric field, driving GABA ions across the ion exchange membrane to the epileptic focus.

GABA induces chloride influx, reducing neuronal excitability and preventing seizures, confirming the efficacy of electrophoretic delivery.

For multi-channel silicon probe insertion, place the probe on the stereotaxic arm attached to a magnetic holder. And place the arm next to the stereotaxic frame. Set the stereotaxic arms in a slight 20-degree anterior posterior angle to leave ample space for the positioning of the other two implants. And connect the probe to the head stage and the ground screw.

Use the micron precise stereotaxic arm to slowly lower the silicon probe into the hippocampus. And initiate the recording software to record the electric neuronal signals, while moving the multi-channel silicon probe from the top of the cortex until the targeted dorso ventral position is reached.

Then simultaneously record the local field potential signal from the layers of the cortex and the hippocampus during the penetration on the computer screen, and use the ripple activity in the pyramidal layer of the hippocampal formation in the recorded local field potential as a marker of the target zone.

For filling of the microfluidic ion pump, connect the tubes to the inlet of the pump, and fill the probe with 0.05 molar GABA solution. Remove the tubes and close the inlet with paraffin film wrapping, and ensure that the microfluidic ion pump is attached securely to the holder. Take care when mounting the holder to the micro manipulator arm before connecting electrical leads to the source measurement unit.

With the stereotaxic arm at a 20 degree mediolateral angle, lower the pump slowly with axial movements as close to the probe as possible, without letting the pump bend until the pump reaches the dorsal ventral coordinate, negative 1,200 micrometers from the cortical surface. In preparation for the seizure induction, remove the metal needle hub of a 10-microliter syringe and fix a borosilicate micropipette onto the syringe tip. Place the hub over the micropipette tip and position the syringe at a 20-degree lateromedial angle.

Use an automated micro injection pump to load 500 to 1,000 nanoliters of 50 millimolar full aminopyridine in artificial cerebrospinal fluid into the syringe and lower the glass micropipette to the dorso ventral coordinate position. Inject 250 nanoliters of the solution into the brain and initiate the recording in the software. Then watch the screen and wait for the first interictal spike to appear, before applying 1 volt between the source and the target for 100 seconds on and 1 second off for 30 cycles to deliver the GABA solution.