Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

Summary

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

Abstract

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

Introduction

Epilepsy is a major neurological disorder with significant economic and human burdens. NINDS estimates there are 3 million Americans with epilepsy. Approximately 0.6 million of these patients have temporal lobe epilepsy (TLE)¹. Unfortunately, medical treatment of TLE fails in one-third of the patients because of ineffectiveness, development of drug resistance, or intolerance to side effects². Clearly, there is a significant need to develop novel therapies for TLE, a conclusion shared by the American Epilepsy Society Basic Science Committee, the International League Against Epilepsy Working Group for Preclinical Epilepsy ....

Protocol

Animal use followed ARRIVE¹² guidelines and was approved by the Animal Care and Use Committee of the University of Virginia.

1. Making headsets with two bipolar electrodes (Figure 1)

Figure 1: Key steps in EEG headset fabrication. (A) Appearance of the electrodes at the various steps in the protocol (numbers in the left match step). (

Results

Animals
The model was originally developed using VGAT-Cre mice (Slc32a1^tm2(cre)Lowl/J)¹³ on a mixed background. However, it has also been applied to the VGAT-Cre strain that is congenic with C57BL/6J. No difference has been observed in epilepsy that develops between the strains. Both strains express Cre recombinase under the control of the vesicular GABA transporter promoter. These mice were generated by knocking in an IRES-Cre cassette after the stop codon in the.......

Discussion

This report describes a protocol where electrical kindling of mice leads to epilepsy. Since the stimulating electrode is placed in the hippocampus, this is a focal limbic epilepsy that models temporal lobe epilepsy (TLE) in patients. A critical step in this protocol is to use VGAT-Cre mice, which due to insertion of an IRES-Cre recombinase cassette in the Vgat gene, shows impaired inhibitory GABA currents¹¹. C57BL/6 do not develop epilepsy after kindling with this protocol, although it is.......

Materials

Name	Company	Catalog Number	Comments
16 Channel Extracellular Differential AC Amplifier (115V/60Hz)	AD Instruments	AM3500-115-60	Alternate EEG amplifier
363/CP PLUG COLLAR, PINS SLEEVE	P1 Technologies	363SLEEVPIN0NL	For electrode holder
Cable, 363-363 5CM - 100CM W/MESH 6TCM	P1 Technologies	363363XXXXCM004	mouse-to-commutator cable
CCTV cameras Qcwox HD Sony IR LED	Sony	QC-SP316
Commutator SL6C/SB (single brush)	P1 Technologies	8BSL6CSBC0MT	formerly Plastics One, Inc.
Current amplifier	A-M Systems	Model 2100
Dental cement	Stoelting	51459
Drill bits, #75, OD  0.310" LOC 130 PT	Kyocera	105-0210.310
E363/0 SOCKET CONTACT SKEWED	P1 Technologies	8IE3630XXXXE	pins for connector
iBond Self Etch glue	Kulzer	CE0197
MS363 PEDESTAL 2298 6 PIN WHITE	P1 Technologies	8K000229801F	EEG headset connector
Ohmeter	Simpson	260	High sensitivity
PowerLab 16/35 and LabChart Pro	AD Instruments	PL3516/P	Alternate EEG software
SomnoSuite	Kent Scientific Corp.	SS-01	anesthesia unit & RightTemp monitoring
Stereotactic drill and micromotor kit	Foredom Electric Co.	K.1070
Stereotactic frame	David Kopf Instruments	Model 940
Teflon-coated wire for depth electrode, OD 0.008'	A-M Systems	791400
VGAT-Cre mice on congenic C57BL/6J background	The Jackson Laboratory	000664