Name: electrophoretic delivery of &#x3b3;-aminobutyric acid (gaba) into epileptic focus prevents seizures in mice
Uploaded: 2019-05-16T13:00:00
Description: Watch this Scientific Journal Video about Electrophoretic Delivery of Î³-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice at JoVE.com

C.M.P. acknowledges funding from a Whitaker International Scholar grant administered by the Institute for International Education. A.K. was sponsored by the Marie Curie IEF (No. 625372). A.W. acknowledges funding from the European Research Council (ERC) under the European Union&#8217;s Horizon 2020 research and innovation programme (grant agreement No. 716867). A.W. additionally acknowledges the Excellence Initiative of Aix-Marseille University - A*MIDEX, a French &#8220;Investissements d&#8217;Avenir&#8221; programme. The authors acknowledge Dr. Ilke Uguz, Dr. Sahika Inal, Dr. Vincenzo Curto, Dr. Mary Donahue, Dr. Marc Ferro, and Zs&#243;fia Magl&#243;czky for their participation in fruitful discussions.

All experimental procedures were performed according to the ethical guidelines of the Institut de Neurosciences des Syst&#232;mes and approved by the local Ethical Committees and Veterinary Offices.
NOTE: Seventeen adult male OF1 mice were used for the experiments. Mice were entrained to a 12 h light/dark cycle with food and water available ad libitum.
1. Anesthesia
<ol>
	<li>Inject intraperitoneally a mixture of ketamine and xylazine (10...

<ol>
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By developing a new experimental protocol in an acute mouse model of epilepsy, SLEs could be successfully controlled with the help of a &#181;FIP implanted in the epileptic focus. Thanks to its capability to deliver GABA with temporal and spatial precision, 4AP-induced SLEs were controlled at the onset of the seizures. Treatment of epilepsy is theoretically possible if the control of the neural network discharges is achieved at the place of the seizure start. The presented protocol proved this possible if the localizatio...

Epilepsy is the fourth most common neurological disorder: about 1% of the population suffers from epilepsy, and about one-third of the affected have recurrent seizures. In most cases, seizures can be controlled with medication. However, drug treatment needs to be set for every patient individually, where proper dosing can take years to find1,2. Additionally, most of the medication has serious side effects that reduce the quality of life3,4,5,6,7. Finally, in 30% of the cases patients are resistant to medication, and in case of a constant single seizure generator locus, only resective neurosurgery can attenuate the occurrence of seizures8. Therefore, a major initiative in modern epilepsy research is to discover new strategies which can prevent recurrent seizures in patients at risk, while reducing the necessity of strong drug therapies and invasive resective surgeries.
Epileptic seizures occur when there is an imbalance within excitatory and inhibitory circuits either throughout the brain (generalized epilepsy) or in a localized part of the brain (focal epilepsy), such that neurons discharge in an abnormal fashion9,10,11. Antiepileptic drugs can act in two different ways in seizure prevention: either decreasing excitation or enhancing inhibition12. Specifically, they can either modify the electrical activity of neuronal cells by affecting ion channels in the cell membrane13 or act on chemical transmission between neurons by affecting the inhibitory neurotransmitter GABA or the excitatory glutamate in the synapses14,15. For some medications, the mode of action is unknown18. Also, drug treatments have a continuous effect on patients and cannot adapt to the prevalence dynamics of seizures. Ideally, drugs with specific mechanisms of action would act on the underlying epileptic processes. An optimal treatment would not touch the brain interictally but would act immediately when a seizure starts developing. In contrast to that, in all cases of epilepsy, medication now means a systematic treatment, affecting the whole brain and the whole body of the patient9.
Epileptic seizures can appear many years after the initial insult such as brain trauma. The period between the initial insult and the occurrence of the first spontaneous seizures is characterized by considerable molecular and cellular reorganizations, including neuronal death with the disappearance of neuronal network connections and axonal sprouting/neosynaptogenesis with the appearance of new connections19,20,21. Once seizures become recurrent, their frequency and severity tend to increase, involving more brain regions. It is important to distinguish the sites of seizure onset (epileptogenic regions) from propagation networks, as the rules of seizure genesis and propagation may differ. Research performed on human tissue and experimental models of epilepsy have provided important data regarding the reorganization of circuits and their ability to generate seizures20,21,22,23. However, it is difficult to determine if these reorganizations are adaptive responses or whether they are causally related to epileptogenesis or seizure genesis and propagation12.
Therefore, localizing the epileptic focus and applying antiepileptic drugs locally are one of the main challenges in contemporary epilepsy research. Several experiments using animal models of epilepsy and some clinical studies aimed to find the onset of the seizure events and define the underlying mechanisms in the brain24,25,26,27. To this end, we developed a new experimental protocol using the 4AP-induced epilepsy model28,29,30,31 in an acute mouse preparation, which allows the precise insertion of three devices into the given area of the hippocampus, where network activity in vivo is manipulated in a highly localized manner. Localized 4AP injection by a glass micropipette helps to induce epileptic SLEs in a localized spot in the hippocampus, while with the help of the novel polymer-based &#181;FIP probe the control of the seizure activity is achieved simultaneously by recording the neuronal electrical activity with the device&#8217;s recording sites. Hippocampal local field activity is also monitored with a multichannel silicon probe in a layer-specific manner in the cortex and in the hippocampus simultaneously.
The recently invented &#181;FIP probes work by using an applied electric field to push charged drugs stored in a microfluidic channel across an ion exchange membrane (IEM) and out to the surrounding tissue (Figure 1). The IEM selectively transports only one type of ion (cation or anion) and, thus, works to limit both passive diffusion in the &#8220;off&#8221; state and&#160; transport of oppositely charged species from the surrounding tissue into the device. The electric field is created on demand by applying a small voltage (&#60;1 V) between the source electrode which is internal to the microfluidic channel and a target electrode which is external to the device (in this case, the head screw on the animal model). The rate of drug delivery is proportional to the applied voltage and the measured current between the source and target electrodes. The precise tunability of drug delivery is one of the primary advantages of the &#181;FIP. Another critical advantage, compared to fluidic or pressure-based drug delivery systems, is that in the &#181;FIP there is only a negligible pressure increase at the drug delivery outlet as drugs are delivered across the IEM without their carrier solution.
There is a small amount of passive leaking of GABA when the &#181;FIP is &#8220;off&#8221;, but this was found not to effect SLEs. The &#181;FIP are custom-made following conventional microfabrication methods that we reported previously31.
Since one way of preventing recurrent seizures is the blockade of network discharges at the very beginning or even before the first seizure event, the presented method for delivering the inhibitory neurotransmitter GABA into the epileptic focus has great therapeutic potential for seizure control in patients with focal epilepsy. Since GABA is an endogenous substrate, it leaves intrinsic neuronal properties unchanged in physiological concentrations. The local application of low levels of GABA will only affect cells naturally responsive to inhibition, and will only cause similar effects to physiological inhibition, contrary to deep brain stimulation (DBS), which has unspecific actions by stimulating all cells of the neuronal network in its environment, causing a mixed response involving both excitation and inhibition. In conclusion, the proposed method provides a more specific approach to seizure control than DBS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >4AP</td>
			<td >Sigma</td>
			<td >275875</td>
			<td ></td>
		</tr>
		<tr >
			<td >Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab15007</td>
			<td></td>
		</tr>
		<tr >
			<td >Amplifier</td>
			<td>Neuralynx, Montana, USA</td>
			<td >Digital Lynx 4SX</td>
			<td ></td>
		</tr>
		<tr >
			<td >Amplifier</td>
			<td >Ampliplex</td>
			<td >KJE-1001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Atlas Stereotaxique&#160;</td>
			<td>Allen Atlas</td>
			<td>978-0470054086</td>
			<td></td>
		</tr>
		<tr >
			<td >Borosilica glass pipette</td>
			<td >Sutter</td>
			<td >BF120-69-15</td>
			<td ></td>
		</tr>
		<tr >
			<td >Brain Matrix</td>
			<td >WPI&#160;</td>
			<td >RBMA-200C</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bone trimmer</td>
			<td >FST</td>
			<td >16109-14</td>
			<td ></td>
		</tr>
		<tr >
			<td >Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td ></td>
		</tr>
		<tr >
			<td >Connector</td>
			<td>INSTECH</td>
			<td>SC20/15</td>
			<td ></td>
		</tr>
		<tr >
			<td >Coton tige</td>
			<td>Monoprix</td>
			<td>EMD 6107OD</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover slip</td>
			<td >Menzel-Glass</td>
			<td >15747592</td>
			<td ></td>
		</tr>
		<tr >
			<td >DiI Stain&#160;</td>
			<td >Thermo Fisher</td>
			<td >D282</td>
			<td ></td>
		</tr>
		<tr >
			<td >DMSO</td>
			<td >Sigma</td>
			<td >11412-11</td>
			<td ></td>
		</tr>
		<tr >
			<td >Drill</td>
			<td>FOREDOM</td>
			<td>K1070</td>
			<td></td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >F.S.T.</td>
			<td >11412-11</td>
			<td ></td>
		</tr>
		<tr >
			<td >GABA</td>
			<td>Sigma</td>
			<td>A2129</td>
			<td></td>
		</tr>
		<tr >
			<td >GFAP Monoclonal Antibody</td>
			<td>Thermofisher</td>
			<td>53-9892-80</td>
			<td></td>
		</tr>
		<tr >
			<td >GOPS</td>
			<td>Sigma</td>
			<td>440167-100M</td>
			<td></td>
		</tr>
		<tr >
			<td >Hamilton seringe&#160;</td>
			<td>Hamilton&#160;</td>
			<td>80330</td>
			<td></td>
		</tr>
		<tr >
			<td >Headscrew</td>
			<td>Component Supply</td>
			<td>TX00-2FH</td>
			<td></td>
		</tr>
		<tr >
			<td >Heating pad&#160;</td>
			<td>Harvard apparatus</td>
			<td>341446</td>
			<td></td>
		</tr>
		<tr >
			<td >Injection Pump</td>
			<td>WPI&#160;</td>
			<td>UMP3-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Keithley</td>
			<td >Tektoronix</td>
			<td >216A</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td>Renaudin</td>
			<td>5787419</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnetic holder</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Mice</td>
			<td>Charles River</td>
			<td>612</td>
			<td ></td>
		</tr>
		<tr >
			<td >Motoric manipulator</td>
			<td>Scientifica, UK</td>
			<td >IVM</td>
			<td ></td>
		</tr>
		<tr >
			<td >Na2HPO4</td>
			<td >Sigma</td>
			<td >255793</td>
			<td ></td>
		</tr>
		<tr >
			<td >NaH2PO4</td>
			<td >Sigma</td>
			<td >7558807</td>
			<td ></td>
		</tr>
		<tr >
			<td >NeuroTrace DiI&#160;</td>
			<td>Thermofisher</td>
			<td>N22880</td>
			<td></td>
		</tr>
		<tr >
			<td >Paper towel</td>
			<td>KIMBERLY CLARK</td>
			<td>7552000</td>
			<td></td>
		</tr>
		<tr >
			<td >PB</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr >
			<td >PEDOT:PSS</td>
			<td>CLEVIOS</td>
			<td>81076212</td>
			<td></td>
		</tr>
		<tr >
			<td >PFA</td>
			<td>Acros Organic</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Rectal temperature probe</td>
			<td>Harvard apparatus</td>
			<td>521591</td>
			<td></td>
		</tr>
		<tr >
			<td >Ropivacaine&#160;</td>
			<td>KABI</td>
			<td>1260216</td>
			<td></td>
		</tr>
		<tr >
			<td >Saline</td>
			<td>Sigma</td>
			<td>7982</td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel</td>
			<td>F.S.T</td>
			<td>AUST R195806</td>
			<td></td>
		</tr>
		<tr >
			<td >Seringue&#160;</td>
			<td>BD Medical</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr >
			<td >Serrefine clamp</td>
			<td>F.S.T</td>
			<td>18050-28</td>
			<td>4 is recommended</td>
		</tr>
		<tr >
			<td >Silicon probe</td>
			<td>NeuroNexus, Michigan, USA</td>
			<td>A2x16-10mm-50-500-177 or A1x16-5mm-150-703</td>
		</tr>
		<tr >
			<td >Stereotoxic frame</td>
			<td>Stoelting</td>
			<td>51733U</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Slide</td>
			<td >ThermoScientific</td>
			<td >J38000AMNZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tubing</td>
			<td >INSTECH</td>
			<td >LS20</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vaseline&#160;</td>
			<td>Laboratoire Gilbert</td>
			<td>3518646126611</td>
			<td></td>
		</tr>
		<tr >
			<td >Vectashield DAPI</td>
			<td>Vector Laboratories, California, USA</td>
			<td>H-1200-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Vibratome, Leica VT1200S</td>
			<td>Leica Microsystems</td>
			<td>1491200S001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Xylazine&#160;</td>
			<td>Bayer</td>
			<td>4007221032311</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophoretic delivery of &#x3b3;-aminobutyric acid (gaba) into epileptic focus prevents seizures in mice

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk.
As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (&#181;FIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The &#947;-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control.
As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Using the procedure presented here with a 4AP epilepsy model in anesthetized mice, control of epileptic seizures can be achieved in the epileptic focus. The precise localization of the implants (Figure 2) helped to record hippocampal local field potentials (LFPs, Figure 4), to induce small hippocampal seizures and to deliver GABA at the seizure onset. The localization of the implants was verified after each experiment by post hoc...

Watch this Scientific Journal Video about Electrophoretic Delivery of Î³-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice at JoVE.com

Electrophoretic Delivery of &#947;-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice

The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol&#8212;with the help of an implantable drug delivery system&#8212;we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus.

Electrophoretic Delivery of &#x3B3;-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the ...

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.

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