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 Drug Discovery, and the National Advisory Neurological Disorders and Stroke Council3,4.
Current animal models of temporal lobe epilepsy use either chemoconvulsants (e.g., kainate, pilocarpine) or prolonged electrical stimulation to induce a long-lasting status epilepticus5,6,7. Many animals die during the procedure (10%-30% in rats, up to 90% in mice8). Animals that survive and develop epilepsy show extensive neuronal death throughout the brain9,10. This death triggers a cascade of responses, beginning with the activation of microglia, astrocytes, and infiltrating monocytes. Neuronal responses include circuit reorganization (e.g., mossy fiber sprouting), birth of new neurons that fail to integrate properly into circuits (e.g., ectopic granule cells), and intrinsic changes that lead to hyperexcitability (e.g., upregulation of Na+ channels). An epilepsy model without significant neuronal death will facilitate the search for new antiepileptic drugs.
While testing the GABA hypothesis of epilepsy, it was discovered that treating VGAT-Cre mice with a mild electrical kindling protocol led to the spontaneous motor and electrographic seizures11. In general, the electrical kindling of rodents does not lead to spontaneous seizures that define epilepsy, although it can, in cases of over-kindling11. VGAT-Cre mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT) gene, which is specifically expressed in GABAergic inhibitory neurons. It was found that insertion of Cre disrupted the expression of VGAT at the mRNA and protein levels, thus impairing GABAergic synaptic transmission in the hippocampus. It was concluded that kindled VGAT-Cre mice could be useful to study the mechanisms involved in epileptogenesis and for screening novel therapeutics11. The present report provides the methods used in generating the model in detail.
Protocol
Representative Results
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank John Williamson for helpful discussions on this protocol. This work was supported by NIH/NINDS grant NS112549.
Materials
| 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 |
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