Name: preparation and implantation of electrodes for electrically kindling vgat-cre mice to generate a model for temporal lobe epilepsy
Uploaded: 2021-08-17T14:00:00
Description: Watch this Scientific Journal Video about Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy at JoVE.com

The authors thank John Williamson for helpful discussions on this protocol. This work was supported by NIH/NINDS grant NS112549.

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)
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62929/62929fig01.jpg" /> 
Figure 1: Key steps in EEG headset fabrication. (A</stron...

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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16 Channel Extracellular Differential AC Amplifier (115V/60Hz)</td>
			<td>AD Instruments</td>
			<td>AM3500-115-60</td>
			<td>Alternate EEG amplifier</td>
		</tr>
		<tr>
			<td>363/CP PLUG COLLAR, PINS SLEEVE</td>
			<td>P1 Technologies</td>
			<td>363SLEEVPIN0NL</td>
			<td>For electrode holder</td>
		</tr>
		<tr>
			<td>Cable, 363-363 5CM - 100CM W/MESH 6TCM</td>
			<td>P1 Technologies</td>
			<td>363363XXXXCM004</td>
			<td>mouse-to-commutator cable</td>
		</tr>
		<tr>
			<td>CCTV cameras Qcwox HD Sony IR LED</td>
			<td>Sony</td>
			<td>QC-SP316</td>
			<td></td>
		</tr>
		<tr>
			<td>Commutator SL6C/SB (single brush)</td>
			<td>P1 Technologies</td>
			<td>8BSL6CSBC0MT</td>
			<td>formerly Plastics One, Inc.</td>
		</tr>
		<tr>
			<td>Current amplifier</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Stoelting</td>
			<td>51459</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill bits, #75, OD&#160; 0.310&#34; LOC 130 PT</td>
			<td>Kyocera</td>
			<td>105-0210.310</td>
			<td></td>
		</tr>
		<tr>
			<td>E363/0 SOCKET CONTACT SKEWED</td>
			<td>P1 Technologies</td>
			<td>8IE3630XXXXE</td>
			<td>pins for connector</td>
		</tr>
		<tr>
			<td>iBond Self Etch glue</td>
			<td>Kulzer</td>
			<td>CE0197</td>
			<td></td>
		</tr>
		<tr>
			<td>MS363 PEDESTAL 2298 6 PIN WHITE</td>
			<td>P1 Technologies</td>
			<td>8K000229801F</td>
			<td>EEG headset connector</td>
		</tr>
		<tr>
			<td>Ohmeter</td>
			<td>Simpson</td>
			<td>260</td>
			<td>High sensitivity</td>
		</tr>
		<tr>
			<td>PowerLab 16/35 and LabChart Pro</td>
			<td>AD Instruments</td>
			<td>PL3516/P</td>
			<td>Alternate EEG software</td>
		</tr>
		<tr>
			<td>SomnoSuite</td>
			<td>Kent Scientific Corp.</td>
			<td>SS-01</td>
			<td>anesthesia unit &#38; RightTemp monitoring</td>
		</tr>
		<tr>
			<td>Stereotactic drill and micromotor kit</td>
			<td>Foredom Electric Co.</td>
			<td>K.1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>David Kopf Instruments</td>
			<td>Model 940</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon-coated wire for depth electrode, OD 0.008&#39;</td>
			<td>A-M Systems</td>
			<td>791400</td>
			<td></td>
		</tr>
		<tr>
			<td>VGAT-Cre mice on congenic C57BL/6J background</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and implantation of electrodes for electrically kindling vgat-cre mice to generate a model for temporal lobe epilepsy

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.

Animals 
The model was originally developed using VGAT-Cre mice (Slc32a1tm2(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...

Watch this Scientific Journal Video about Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy at JoVE.com

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

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.

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 ...

This protocol generates a new model for temporal lobe epilepsy in which mice developed spontaneous seizures, and it's been very useful for probing the mechanisms of epileptogenesis and also for screening novel therapies. This procedure in VGAT-Cre mice reliably leads to spontaneous recurring electrographic and motor and motor composite seizures without causing extensive hippocampal cell death that is often seen after status epilepticus triggered with chemoconvulsants or continuous electrical stimulation. To start making headsets, cut 3.5-centimeter-long polytetrafluoroethylene-coated stainless steel wire and strip about one millimeter of the insulation coat off both ends of the wire.Put two pins on a vice holder, with the bottom part of the pin having a longer slit facing down. Then, apply flux onto the stripped ends of the wire and the tops of the pins. Tin the stripped part of the wire and the top of the pins with just enough solder to coat it without overflowing onto the sides.Place one of the stripped ends of the wire into the pin as deep as possible while the solder is melting. Repeat the process for the second pin with the other stripped end of the wire. After cooling the pins and solder for 10 seconds, remove the pins from the vice holder and then pull onto the pins to ensure that the connection between the wire and the pins is strong and holds.Rinse the pins in cold water followed by drying. Later, use an ohmmeter to verify conductance between pin one and pin two. Bring the pins at the end of the wire together to hold them parallel and close.Clamp a hemostat to the center of the wire and rotate the hemostat to twist the wire around itself tightly. After removing the hemostat, clamp the forceps onto the twisted wire at two millimeters below the pins to bend the wire at 90 degrees. Push the wire 90 degrees back over the forceps, creating one more bend at one millimeter from the first.Use small, sharp scissors to cut the twisted wire at 45 degrees and 3.5 millimeters below the bend. Prepare two bipolar or double-pin twisted electrodes for each headset. To prepare one reference electrode, cut a wire with pin soldered on both ends into two.Then, cut the wire at seven millimeters and bend the end of the wire at one millimeter below the tip. Cover the bent one-millimeter tip of the wire with forceps and rotate the wire tight around the forceps to create a small loop with one millimeter in diameter. Then, bend the loop perpendicular to the straight part of the wire to make the wire tip point outward again.Assemble all the electrodes into the six-pin pedestal side by side with a six-millimeter distance between the two bipolar electrodes. Place the reference electrode in the middle outer hole. Before proceeding with the surgery, record the weight of the eight-week-old VGAT-Cre mice.Then, place the animal in an induction chamber to induce anesthesia. Place the anesthetized mouse on a heating pad at 37 degrees Celsius. If using a feedback-controlled temperature system, insert the temperature probe into the rectum of the animal for temperature monitoring during the surgery.To start the surgery, redirect the inhalent anesthesia flow to the nose cone and mount the animal in the stereotaxic frame by gently placing the front upper teeth into the incisor bar and move the cone onto its nose. Then, place the ear bars into the ears and affix to the stereotaxic frame. Ensure that the animal's head is leveled and centered and cannot be moved when slightly probed.Once the animal is mounted, inject 0.5 milliliters of Normosol onto the mice for hydration and apply ocular lubricant to prevent corneal drying. Monitor the depth of anesthesia by pinching a hind limb paw. If the withdrawal reflex is absent in the animal, proceed to remove the scalp hair of the mouse around the surgical area.When done, disinfect the clean skin area thrice with an alternating application of ethanol and iodine, finishing with iodine, and inject 0.05 milliliters of 0.25%bupivacaine subcutaneously. Make an incision on the skull using a scalpel and then cut out a part of the skin with surgical scissors, exposing the skull. Push the skin aside before cleaning the skull from all underlying tissues with a cotton swab.Clean the skull with hydrogen peroxide using sterile cotton swabs and make the skull sutures, bregma, and lambda visible. With the stereotaxically-mounted drill, move the drill tip onto bregma in zero X, Y, and Z coordinates. Then, raise and move the drill to lambda to determine its coordinates to determine if the head is level.After drying the skull thoroughly, apply one drop of self-etching dental adhesive with the applicator. Wait 60 seconds before curing the dental adhesive with a dental UV light for 40 seconds. A glossy surface indicates that the adhesive has been effectively cross-linked with the skull.With the help of a 0.031-inch drill bit, carefully drill two burr holes bilaterally into the skull at 5, 000 rotations per minute, or RPM, avoiding drilling into the brain. Apply drop of saline to enhance the drilling of the burr holes. For implantation of hippocampal-depth electrodes, drill at three millimeters posterior and three millimeters lateral from bregma.Drill one burr hole for the reference electrode above the cerebellum behind the lambda at six millimeters posterior and zero millimeters lateral from bregma. Next, mount the six-pin pedestal with assembled electrodes into the electrode holder on the stereotaxic frame. By aligning the electrodes above the corresponding burr holes, slowly lower and navigate the headset to locate the two bipolar electrode tips right on top of the left and the right hippocampal burr holes, and if needed, adjust the reference electrode to hover above hole over the cerebellum.When the hippocampal twist electrodes are right above the holes, zero the Z axis and slowly lower the electrode to minus three millimeters into the right and left hippocampus and guide the reference electrode into the hole above the cerebellum. Prepared the dental cement by mixing the powder and liquid in a mixing bowl. Then, cover the skull surface, the electrodes, and the space between the skull surface and the bottom of the pedestal with dental cement.Once the cement dries and hardens, detach the electrode holder from the stereotaxic arm, raise arm, and remove the holder from the pedestal. After administering the mouse subcutaneously with ketoprofen and Normosol, remove the animal from the stereotaxic frame to move the animal onto the heated pad in the vivarium cage. Once fully awake, return the single animal to the vivarium cage, feed the animal with soft food, and monitor the weight loss daily up to 72 hours post-surgery.In the representative violin plot, the number of stimulations required by mice to the kindled state criterion is shown. The mice typically reached the kindled state or experiences five bilateral tonic-clonic motor seizures after 15 stimulations. The latency to spontaneous recurring seizures, or SRS, was found to be 10.7, and the average frequency of the seizures was 1.3 seizures per day.The reliable SRS distribution showed that the mice were epileptic for approximately 23 days. In the representative images, the number of anti-NeuN positive neurons or neuronal deaths in various subfields of the hippocampus and entorhinal cortex can be seen. It is important to have the two bipolar electrodes smoothly enter the two burr holes, so they need to be right above the burr holes.And also, it's important to carefully apply the dental cement and let it harden completely before removing the electrode holder. This technique allows us to generate epileptic mice and continuously monitor them with EEG recording and also video recording, simultaneously. And with the experiments we do, we can then test novel treatments strategies such as gene therapy to reduce the frequency, the duration, and the severity of the seizures.

preparation-implantation-electrodes-for-electrically-kindling-vgat

Research

JoVE Journal

Neuroscience

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the &#945;3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the &#945;3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...