Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Our protocol takes advantage of the local administration of the naturally occurring inhibitory neurotransmitter GABA using a very novel device, which could propose a new solution in the treatment of epileptic seizures. The microaerophilic ion pump allows us to have very precise control over exactly when and where we're delivering drugs. Precisely threaded electrophoretic delivery could be modified to target other neurological diseases.

Another benefit of this drug delivery technique is that there's a very negligible pressure increase at the drug delivery outlet because there's no fluid that's actually being delivered. Rather, it's just the drug molecules themselves. Since close localization of the probes will pose the greatest issue, it's very advisible to test their alignment angles and positioning with dummy probes in the stereotaxic frame.

Begin by fixing the head of an anesthetized mouse in a stereotaxic frame and using a 30 gauge needle to inject a local analgesic subcutaneously at the planned incision site. After five minutes, use a scalpel to make a midline incision in the skull. And use fine forceps to gently retract the skin.

Clamp the skin with bulldog Serofine clamps. And clean the exposed skull surface of fascia. Using a dental drill with a fine, round 0.4 millimeter drill bit, drill a hole in the skull above the cerebellum until the dura is visible.

Place the ground screw into the hole and use a precision screwdriver to turn the screw until it reaches the top of the cerebellum. Using the stereotaxic frame, measure the stereotaxic coordinates for the desired brain region and use the drill set at a fast speed to thin an approximately one to two millimeter diameter area of the skull, about the target region, until a thin, well-polished transparent bone membrane remains. Then, use a custom-made hooked-tip needle to remove the dura and add a drop of saline into the resulting hole.

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 dorsoventral 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 micromanipulator arm before connecting electrical leads to the source measurement unit. With the stereotaxic arm at a 20-degree medio-lateral 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 dorsoventral coordinate negative 1200 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 lateral-medial angle. Use an automated microinjection pump to load 500 to 1000 nanoliters of 50 millimolar full aminopyridine in artificial cerebrospinal fluid into the syringe and lower the glass micropipette to the dorsoventral 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 one volt between the source and the target for 100 seconds on and one second off for 30 cycles to deliver the GABA solution. The precise localization of the implants helps in recording the hippocampal local field potentials, inducing the small hippocampal seizures, and delivering GABA at the seizure onset.

Further, the localization of the implants can be verified after each experiment by post hoc histology. Here, an example of when seizure-like events could be stopped with the delivery of GABA by the novel neuronal probe, incorporating the microfluidic probe is shown. When four aminopyridine was injected into a larger area or onto the top of the cortex, the epileptic seizures became generalized, so the delivered GABA was not able to modify the extent of the epileptic seizures.

Notably, the delivery of sodium ions did not have a significant effect on the four aminopyridine-induced activity. So this protocol can be useful for any stereotaxic surgery that involves implanting multiple fragile probes. With the help of the microfluidic ion pump, other species in charged molecules could be introduced to treat not only epilepsy, but other neurological diseases.

Electrophoretic delivery in tissue interfacing presents a new approach in general of delivering species into any soft tissue. Take special care when applying the 4AP, since it's a toxin and induces epilepsy.