This work was supported by the Federal Ministry of Education and Research (BMBF DysTract to C.W.I.) and by the Interdisciplinary Center for Clinical Research (IZKF) at the University of W&#252;rzburg (Z2-CSP3 to L.R.). The authors thank Louisa Frie&#223;, Keali R&#246;hm, Veronika Senger and Heike Menzel and for their technical assistance as well as Helga Br&#252;nner for the animal care.

All procedures were performed in accordance with applicable international, national, and/or institutional guidelines for care and use of animals. The local authorities at the Regierung von Unterfranken, W&#252;rzburg, Germany, approved all animal experiments.
1. Priming of osmotic pumps
NOTE: This step has to be performed at least 48 h prior to surgery. ALZET osmotic pumps need to be prefilled in order to ensure that the pumping rate reaches a steady state before implantation.
<ol>
	<li>Prepare the desired solution for chronic perfusion beforehand. Ensure that the solvent and agent are compatible with the osmotic pumps and catheters. For the project herein, thaw a 10x stock solution of ouabain (stored at -20 &#176;C) at room temperature and vortex for 10 s. 
	CAUTION: Ouabain is toxic: it should be handled only with gloves and opened only under a sterile hood to avoid inhalation.</li>
	<li>Turn the sterile hood on; disinfect the hood as well as all the instruments and material needed before putting them under the hood.</li>
	<li>Put on surgical gloves before handling the osmotic pumps. Separately prepare the ouabain-solution designated for the pumps of the striatum and the cerebellum. 
	NOTE: Consider that the concentration of the solution in the pump for the striatum has to be doubled compared to the concentration of the solution in the pump designated for the cerebellum. This is due to the fact that the pump for the striatum is connected to a double cannula, the flow rate, however, is the same as for the pump of the cerebellum, which is connected to a single cannula.</li>
	<li>Calculate the amount of solution needed using the following formula: 
	mass delivery rate (ko) = volume delivery rate (Q) x concentration of the agent in the vehicle (Cd) 
	For the present project, the osmotic pumps were primed and filled with either ouabain solution at a concentration of 11.2&#8239;ng/h or 0.9% saline for the control group.</li>
	<li>Vortex both ouabain solutions for 10 s and sterile filter (i.e., 0.22 &#181;m syringe-end filter) them into separate, new microcentrifuge tubes, using a different filter for each solution.</li>
	<li>Weigh the empty osmotic pumps together with the flow moderator (about 0.4 g).</li>
	<li>Fill a 1 mL syringe with a 27 G filling cannula (preferably with a blunt tip) with sterile filtered ouabain-solution or vehicle solution.</li>
	<li>Get all the air bubbles out of the syringe, hold the osmotic pump in an upright position, insert the cannula all the way into the pump and slowly fill the reservoir until excess solution appears on top (avoid rapid filling as this can lead to air bubbles in the pump). Next, insert the flow moderator into the pump (excess solution should appear on top).</li>
	<li>Pull the flow moderator out (about 5 mm), carefully take off the white flange with scissors and connect a piece of vinyl tubing to the flow moderator. Ensure that the catheter has a length of approximately 2 cm to allow proper mobility of the animal.</li>
	<li>Weigh the filled osmotic pump once more to ensure that the difference in weight between filled and empty pump is concordant with the expected weight of the solution loaded. 
	NOTE: For most aqueous solutions this weight is equal to the volume in microliters. If the weight does not correspond to the expected volume, air might be trapped inside the pump, which needs to be emptied and refilled.</li>
	<li>For the priming of the pumps, prepare two 2 mL microcentrifuge tubes, one tube for the striatum and one for the cerebellum.</li>
	<li>Submerge the pumps into the microcentrifuge tubes filled halfway with 0.9% saline solution. Do not submerge the open end of the catheter. Put the microcentrifuge tubes into a thermocycler for 48 h at 37 &#176;C in order to ensure that the pump has reached a steady pumping rate before implantation and the catheters are prefilled.</li>
</ol>
2. Cannula and osmotic pump implantation
<ol>
	<li>Place the mouse (male, C57Bl/6N, 11-12 weeks of age) into a chamber designed for inhalation anesthesia; set the flow rate of isoflurane to 2-3% and the flow rate of oxygen to 2 L/min.</li>
	<li>After the mouse is deeply anaesthetized according to approved protocols, shave the top of the animal&#8217;s head, dorsal neck and proximal third of the back.</li>
	<li>Place the animal into a stereotactic frame and continue the anesthesia with isoflurane via a mouse anesthesia mask designed for the stereotactic instrument (isoflurane flow rate 1.5-2%, 2 L/min oxygen).</li>
	<li>Using rubber tips or non-rupture ear bars, fix the head of the animal in the stereotactic frame, taking care that the head is leveled.</li>
	<li>In order to prevent hypothermia, place a heating pad under the animal, insert a rectal temperature probe and set the temperature to 37 &#176;C. Protect the eyes of the animal from drying out by using a drop of ophthalmic ointment on each eye.</li>
	<li>Apply an analgesic, such as Carprofen (5 mg/kg bodyweight), subcutaneously before beginning surgery.</li>
	<li>With a syringe subcutaneously inject up to 0.2&#160;mL of bupivacaine 0.25% and wait 30 s for the local anesthesia to take effect.</li>
	<li>Disinfect the shaved areas thoroughly with an antiseptic, such as octenidine dihydrochloride.</li>
	<li>After thorough disinfection of the surgical area, use a scalpel to place an incision at the top of the head and use scissors to continue the incision down to the fore limbs. Expose the skull with the help of bulldog clamps and wipe the periosteum with a sterile cotton-tipped applicator.</li>
	<li>Align either a pen or the tip of a cannula stained in black ink with bregma and use the appropriate coordinates to mark the three entry points for the brain cannulas on the skull (coordinates for the bilateral holes needed for perfusion of the basal ganglia: anterior/posterior: + 0.74&#8239;mm, medial/lateral: +/- 1.50&#8239;mm (+ indicating the right side, - the left side relative to bregma); coordinates for the hole at the midline of the cerebellum: anterior/posterior: - 6.90 mm). Next, carefully drill the holes for the double cannula designated for the basal ganglia and the single cannula designated for the cerebellum (Figure 1A).</li>
	<li>Drill a fourth hole for a small screw in-between the striatum and the cerebellum. This screw will eventually be embedded in dental cement and provide additional hold for the cannulas. Using fine forceps and a screw driver, carefully introduce the screw into the small hole until it is firmly fixed in the skull. Do not implant the screw too deeply as this damages the brain tissue. 
	NOTE: It is recommended to continue with the osmotic pump implantation before inserting the osmotic cannulas. This avoids causing any accidental damage to the cannulas when implanting the pumps.</li>
	<li>In order to create a small pocket for the osmotic pump on each side of the animals back, use tissue forceps to separate the subcutaneous tissue layers. Advance the forceps towards one hind leg and open the forceps slightly in order to widen the subcutaneous pocket. Repeat the same procedure for the other side, first removing the forceps from the incision and then pushing them gently towards the second hind leg. 
	NOTE: The pocket should permit the pump to easily slide in, however, it should not have too much room to move under the skin.</li>
	<li>With the help of tissue forceps take the first pump together with the connected piece of tubing and slide it into one subcutaneous pocket. Repeat the same procedure with the second pump.</li>
	<li>Using a minipump holder, introduce a single osmotic cannula with a custom length of 3.0 mm into the hole drilled in the midline of the cerebellum. Detach the cannula head carefully and fix the cannula as well as the small screw with dental cement, taking care as not to cover the connecting piece for the tubing of the osmotic pump. Ensure that the dental cement surrounding the cannula has fully dried before continuing with surgery.</li>
	<li>Before inserting a double cannula with a center-to-center distance of 3.0&#8239;mm and custom-made length of 4.0&#8239;mm into the bilaterally drilled holes above the basal ganglia, attach two short pieces of vinyl tubing (0.5 cm) to the two connecting pieces of the double cannula. Connect the vinyl tubings with a bifurcation adaptor and carefully prefill the entire tubing system including the adaptor and cannula with ouabain solution or vehicle (room temperature, sterile filtered beforehand). This can be done best with a 1 mL syringe and a 27 G filling cannula introduced into the single rear end of the bifurcation adaptor (Figure 1B).</li>
	<li>Using a minipump holder, carefully insert the double cannula into the bilateral holes. Use a clamp to detach the cannula head and fix the double cannula with dental cement.</li>
	<li>Connect the catheters of the osmotic pumps to the bifurcation adaptor as well as the single cannula, respectively. Ensure that the catheters have a strong hold on the connecting pieces of the cannulas. 
	NOTE: Both pumps have an equal flow rate, so be careful to connect the osmotic pump with the double concentrated solution to the bifurcation adaptor to ensure that the same concentration reaches both the basal ganglia and the cerebellum.</li>
	<li>Close the incision on the back of the animals with stitches as far as possible in direction of the skull, being careful not to overstretch the skin (Figure 1C).</li>
	<li>Subcutaneously inject 0.5 mL of 0.9% saline, which should have body temperature, into a skin fold on each side of the back of the animals in order to avoid dehydration of the mice, carefully avoiding the pumps.</li>
</ol>
3. Motor challenge
<ol>
	<li>Subject ouabain- or vehicle-perfused mice to challenging motor tasks as a form of mild stress exposure 4 h post-surgery and repetitively every 24 h afterwards in order to induce dystonia-like movements. This does not include a behavioral characterization; its purpose is to induce a higher stress level compared to normal cage keeping.</li>
	<li>Place the mouse onto a 50 cm rough-surfaced, wooden pole with a diameter of 1 cm, nose facing downwards. Ensure that the pole is placed into a large cage with enough bedding in case of falls. It is not necessary to measure the time of descent, but look out for involuntary hyperextensions of front limbs and hindlimbs presented by ouabain-perfused animals while descending the pole. Let mice descend the pole three times and allow 2 min of recovery between descends. 
	NOTE: Ouabain-perfused mice should present first symptoms like bradykinesia 4 h post surgery. However, 24 h after surgery mice should start to present involuntary hyperextensions of front limbs and hindlimbs as a sign of dystonia-like movements during descend.</li>
	<li>For the second motor task, place mice on the rotating rod as done for the Rotarod performance test. The Rotarod apparatus is not used as a measure of the latency to fall, the aim is to subject mice to forced activity. Place the mice on the rotating rod three times and allow 2 min of recovery between tests. 
	NOTE: To increase stress exposure, use an accelerating Rotarod apparatus. For the project described herein, the rod accelerates from 5 to 50 rpm over a set time period of 300 sec.</li>
	<li>Let animals recover for 30 min in between the pole test and the Rotarod performance test and again another 30 min before scoring for dystonia-like movements as described under protocol step 4. In between the repetitive stress exposures, allow mice to recover for 24 h-intervals.</li>
</ol>
4. Scoring systems for the assessment of dystonia-like movements
NOTE: The experimenter should be blinded to the group assignment analyzed to prevent bias. The behavioral tests used to characterize the phenotype of the mice are two scoring systems: a dystonia rating scale scoring abnormal, dystonia-like movements and a behavioral score using the tail suspension test. Assess the dystonia-like movements after a recovery time of 30 min following the exposure to mild stress.
<ol>
	<li>Dystonia rating scale 
	NOTE: Due to the lack of predefined behavioral tasks, the dystonia rating scale was established as an observer-based scoring system similar to the clinical rating scales of human dystonia. It is a modified version of the dystonia rating scale used by Calderon et al.13.
	<ol>
		<li>Record posture and gait of animals over a period of 4 min after animals have been placed in a plastic or wooden box.</li>
		<li>Score for frequency and distribution of dystonia-like movements from 0 to 4 points: (0) normal motor behavior; (1) abnormal motor behavior, no dystonia-like movements; (2) mild motor impairment with mild focal dystonia-like movements; (3) moderate motor impairment with severe focal dystonia-like movements; (4) severe impairment, with sustained, generalized dystonia-like movements (Figure 2). Consider the following movements or postures as dystonia-like: hyperextension of front limbs, wide stance or hyperextension of hindlimbs as well as kyphosis. Consider dystonia as focal in case a single body part is affected and as generalized in case the trunk and at least two other body parts are affected.</li>
	</ol>
	</li>
	<li>Tail suspension test 
	NOTE: The tail suspension test is often used to observe and score for hindlimb clasping. This is however a highly unspecific phenotype indicating a motor impairment. The following protocol proposes a scoring system specific for dystonia-like movements. Due to the generalization of dystonia in DYT/PARK-ATP1A3 patients, dystonia-like movements should be assessed in front limbs, trunk and hindlimbs. A newly-developed scoring system from 0-8 points was developed, a total score &#60; 2 indicated that no dystonia-like movements were present (Figure 3).
	<ol>
		<li>Pick the mouse up by the tail near its base and lift the animal up. Record a 2 min video of the tail suspension test and assign a score in a subsequent, thorough analysis of the recording.</li>
		<li>Score the front limbs from 0 to 4 points, where repeated or sustained tonic retractions of one or both front limbs, as well as a hyperextension combined with crossing of the front limbs, were classified as dystonia-like: (0) no abnormal movements; (1) reduced movement of front limbs with hyperextension of paws seen &#8805; 50% of the recorded time; (2) mild dystonia-like movements of front limb(s) &#60; 50% of the recorded time; (3) mild dystonia-like movements of front limb(s) &#8805; 50% of the recorded time or severe &#60; 50% of the recorded time; (4) severe dystonia-like movements of front limb(s) &#8805; 50% of the recorded time. 
		NOTE: Hindlimb clasping is an abnormal movement that should not be scored as dystonia-like.</li>
		<li>Score the hindlimbs from 0 to 3 points, where retraction and clenching of rear limbs as well as sustained hyperextension were assessed as dystonia-like: (0) no abnormal movements; (1) reduced movement of hindlimbs with hyperextension of paws seen &#8805; 50% of the recorded time; (2) dystonia-like movements of one hindlimb; (3) dystonia-like movements of both hindlimbs.</li>
		<li>In case of truncal distortion &#62; 80% of the recorded time, an additional point is added to the score.</li>
		<li>Place the animal back into its cage.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This DYT/PARK-ATP1A3 pharmacological mouse model allows for the detailed analysis of intracerebral structural and neurochemical changes induced solely by inhibition of the sodium-potassium ion pump in the basal ganglia and cerebellum as well as alterations related to stress exposure. In case of mice, a maximum of two osmotic pumps can be subcutaneously implanted. We herein present a method detailing chronic drug delivery to multiple brain structures by implementing a double cannula connected to a bifurcation adaptor in addition to a single cannula. This methodology can be used for any application requiring multiple brain structures to be perfused simultaneously and chronically.
We present a mouse model of a rare movement disorder, where patients develop permanent symptoms after exposure to stress. This presumed gene-environmental interaction is still not well understood, but might represent one of the key pathomechanisms in DYT/PARK-ATP1A3 development. Different methods of exposing mice to stress have been published in the past and include electric foot shocks, restraining, cold or warm environment and exposure to various odors11,12,13. In an effort to expose mice to a mild stress factor with translational value, we herein describe the repetitive subjection of mice to challenging motor tasks. For the pole test, ouabain-perfused animals revealed involuntary hyperextension of front limbs and hindlimbs. These movements were very similar to the dystonia-like movements observed during the 4-min video recording of the animals as well as the tail suspension test. The application of mild stress in form of challenging motor tasks might prove useful in other mouse models showing motor symptoms or neurodegeneration, where gene-environmental interactions massively influence the degree of disease progression.
There is a general lack of predefined behavioural tasks as well as rating scales to classify abnormal movements and postures in mice. Most of the available motor tasks reveal unspecific abnormalities, such as hindlimb clasping, which is a well-known phenomenon in many mouse models of movement disorders with neurodegeneration18,19. For the proper characterization of a phenotype, it is however necessary to analyze whether the mouse model recapitulates the salient features of the disease. Herein, we present the modified version of a dystonia rating scale used previously for the assessment of motor disability in dystonia mouse models15,16. We additionally developed an observer-based scoring system for the tail suspension test, which was established similarly to the clinical rating scales of human dystonia. Both rating scales show a significantly higher score in ouabain-perfused, stressed mice compared to ouabain-perfused, non-stressed animals as well as vehicle-perfused animals. The drawbacks of any observer-based scoring system are the necessary training of raters to ensure consistent scoring and to reduce observer variability as well as the danger of a possible bias of the rater if not fully blinded to the group analysed. However, observer-based scoring systems still present an easily accessible method to characterize a phenotype and can be adapted to the mouse model analyzed, as done in the present project for the assessment of dystonia-like movements. To ensure consistent scoring among different raters, training videos should be made available. To reduce any potential bias, it is recommended that different raters score the same video clips and that the individual scores are averaged. Both scoring systems mentioned within this work record the presence of dystonia-like movements in animals. The rating scales can be adapted according to the specific requirements within a project, as done previously by Ip et al., where solely the hindlimbs were scored for dystonia-like movements in a mouse model for dystonia 1 (DYT-TOR1A)20. The rating scales can be complemented by other previously published scoring systems, assessing for example the degree of bradykinesia in rodents as done with the locomotion disability score by Calderon et al.13.

The advantages of a continuous drug delivery directly into the brain are numerous. Repetitive and frequent injections, which represent an unnecessary stress factor for animals, can be avoided and a more constant intracerebral concentration of the drug can be achieved. This is especially valid when systemically administered drugs fail to easily penetrate the blood brain barrier. Moreover, chronic drug delivery via osmotic pumps allows for the localized delivery of substrates that would otherwise have system-wide side effects. The drugs can be delivered in a targeted manner to the desired brain structures, the resulting effect can thus be directly traced. This can be utilized for an array of applications, such as the study of therapeutic effects as well as the study of pathomechanisms. This last application was used in the project herein in order to create a pharmacological mouse model for dystonia.
The analysis and understanding of dystonic syndromes, which represent the third most common movement disorder, have been heavily limited by the fact that genetic animal models largely fail to reproduce the disease phenotype found in diseased human as well as the pathophysiology. This issue is not limited to dystonic syndromes, but in fact concerns many transgenic rodent models in the field of movement disorders1,2. The reason for the lack of a phenotype in transgenic rodent models might be based on highly effective compensatory mechanisms3. In the case of dystonia, the disease is characterized by involuntary muscle contractions causing twisting movements and abnormal postures4. The study of secondary causes (i.e., brain injury) of dystonic symptoms, has helped to identify the structures involved in the manifestation of these motor abnormalities, such as the basal ganglia5. Brain imaging studies of hereditary forms of dystonia have shown functional abnormalities in almost all brain regions responsible for motor control and sensorimotor integration6,7. However, rodent models are still needed to deepen the understanding of the neural dysfunctions on a molecular and large scale network level as well as for the development of therapeutic options. This is where pharmacological mouse models offer the possibility to replicate the clinical and pathological features of a disease in a more precise manner.
Rapid-onset dystonia-parkinsonism (DYT/PARK-ATP1A3; RDP; DYT12) is one of the hereditary forms of dystonia. It is caused by loss-of-function mutations in the ATP1&#945;3 gene, which encodes for the &#945;3-subunit of the Na+/K+-ATPase8. Furthermore, it is recognized that gene mutation carriers can be free of symptoms for years before acutely developing persistent generalized dystonia and Parkinsonism after exposure to a stressful event. Indeed, the penetrance of DYT/PARK-ATP1A3 is incomplete and stressful events acting as a trigger range from physical overexertion and extreme temperatures to overconsumption of alcohol and infections9,10. In order to study DYT/PARK-ATP1A3 and to find potential therapeutic interventions, it has been tried numerous times to imitate the stress-dependent disease development in rodent models. However, aside from the one existing genetic DYT/PARK-ATP1A3 mouse model, where transient abnormal and convulsion-like movements were induced by hypothermia, all published genetic mouse models for DYT/PARK-ATP1A3 have failed to produce dystonic symptoms1,11,12. Calderon et al. previously demonstrated that blocking the &#945;3-subunit bilaterally in the basal ganglia and the cerebellum via the cardiac glycoside ouabain in wild type mice results in mild gait disturbance13. The additional exposure to electrical foot shocks in a warm environment led to a dystonic and bradykinetic phenotype, thus demonstrating that chronic and targeted perfusion of ouabain followed by stress successfully imitates the DYT/PARK-ATP1A3 phenotype.
However, exposing animals to electrical foot shocks in a warm environment of 38-40 &#176;C over a two hour-period induces pain and anxiety in animals, which represent confounding factors, especially for the assessment of changes in the catecholamine system related to the development of dystonia. Thus, we herein describe a different kind of stress paradigm with high translational value, which relates back to the fact that mild to moderate exercise have been described as triggers in DYT/PARK-ATP1A3 patients9. Moreover, repetitive exercise is a well-known trigger for focal dystonia14. Mice were repeatedly subjected to challenging motor tasks comprised of three descends of a wooden pole (&#8220;pole test&#8221;) and three runs on a Rotarod apparatus (&#8220;Rotarod performance test&#8221;). The placement of animals on the top of a 50 cm wooden pole was used to coerce the animals to descend, the Rotarod apparatus was employed to subject mice to forced activity by placing them on a rotating rod.
The characterization of the motor phenotype of a mouse model for dystonia is particular challenging due to the lack of predefined tests and scores. However, one variation of a motor disability assessment has been repeatedly used over the last years in order to evaluate the severity and the distribution of dystonia-like movements in rodents13,15,16. We herein present a modified version of the dystonia rating scale, which proved to be effective in evaluating the dystonia-like phenotype of animals when observed over a time period of four minutes. As a second method of assessing dystonia-like movements, we present a newly-developed scoring system for the assessment of abnormal movements during a tail suspension test. It allows for the assessment of the frequency and duration of dystonia-like movements and postures of the front limbs, hindlimbs as well as trunk.

<table>
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			<td>0.9% saline</td>
			<td>Fresenius Kabi</td>
			<td></td>
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			<td>Alzet osmotic pumps</td>
			<td>Durect</td>
			<td>4317</td>
			<td>model 1002, &#64258;owrate 0.25 &#956;L/h</td>
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			<td>Anchor Screws</td>
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			<td>Bulldog Clamps</td>
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			<td>13-320-035</td>
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			<td>Bupivacain 0.25% Jenapharm</td>
			<td>mibe GmbH Arzneimittel</td>
			<td></td>
			<td></td>
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			<td>Cannula and Minipump Holder</td>
			<td>Stoelting</td>
			<td>51636</td>
			<td>designed to hold 3.4 mm cannula heads</td>
		</tr>
		<tr>
			<td>Cannula Bifurcation</td>
			<td>Plastics One</td>
			<td>21Y</td>
			<td>custom made</td>
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		<tr>
			<td>Cannula tubing</td>
			<td>Plastics One</td>
			<td>C312VT/PKG</td>
			<td>vinyl, 0.69 mm x 1.14 mm</td>
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			<td>Dumont #5SF forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>fine forceps</td>
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			<td>eye cream Bepanthen</td>
			<td>Bayer Vital GmbH</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Gas Anesthesia Mask for Stereotaxic, Mouse</td>
			<td>Stoelting</td>
			<td>56109M</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Rotary Micromotor Kit</td>
			<td>Foredom</td>
			<td>K.1070-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane CP 1 mL/mL, 250 mL</td>
			<td>cp-pharma</td>
			<td>1214</td>
			<td>prescription needed</td>
		</tr>
		<tr>
			<td>Isoflurane System Dr&#228;ger Vapor 19.3</td>
			<td>Dr. Wilfried M&#252;ller GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kallocryl A/C</td>
			<td>Speiko</td>
			<td>1615</td>
			<td>dental cement, liquid</td>
		</tr>
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			<td>Kallocryl CPGM rot</td>
			<td>Speiko</td>
			<td>1692</td>
			<td>dental cement, red powder</td>
		</tr>
		<tr>
			<td>Mouse and neonates adaptor</td>
			<td>Stoelting Co.</td>
			<td>51625</td>
			<td>adaptor for mice for a traditional U-frame</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>KLS Martin Group</td>
			<td>20-526-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Rupture Ear Bars and Rubber Tips f/ Mouse Stereotaxic</td>
			<td>Stoelting Co.</td>
			<td>51649</td>
			<td></td>
		</tr>
		<tr>
			<td>Octenisept</td>
			<td>Sch&#252;lke</td>
			<td>118211</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic Pump Connector Cannula for Mice, double</td>
			<td>Plastics One</td>
			<td>3280PD-3.0/SPC</td>
			<td>28 Gauge, length 4.0 mm, c/c distance 3.0 mm</td>
		</tr>
		<tr>
			<td>Osmotic Pump Connector Cannula for Mice, single</td>
			<td>Plastics One</td>
			<td>3280PM/SPC</td>
			<td>28, Gauge, custom length 3.0 mm</td>
		</tr>
		<tr>
			<td>Ouabain octahydrate 250 mg</td>
			<td>Sigma-Aldrich</td>
			<td>03125-250MG</td>
			<td>CAUTION: toxic</td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td>Kern &#38; Sohn</td>
			<td>PFB 6000-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectal Thermal Probe</td>
			<td>Stoelting</td>
			<td>50304</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl 50 mg/mL, injectable</td>
			<td>Zoetis</td>
			<td></td>
			<td>Carprofen, prescription needed</td>
		</tr>
		<tr>
			<td>Rodent Warmer X1 with Mouse Heating Pad</td>
			<td>Stoelting</td>
			<td>53800M</td>
			<td></td>
		</tr>
		<tr>
			<td>RotaRod Advanced</td>
			<td>TSE Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>screw driver set</td>
			<td>Agntho&#39;s</td>
			<td>30090-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Burrs</td>
			<td>Agntho&#39;s</td>
			<td>HM71009</td>
			<td>0.9 mm &#216; burr</td>
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		<tr>
			<td>Stainless Steel Burrs</td>
			<td>Agntho&#39;s</td>
			<td>HM71014</td>
			<td>1.4 mm &#216; burr</td>
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		<tr>
			<td>StereoDrive</td>
			<td>Neurostar</td>
			<td></td>
			<td>software</td>
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		<tr>
			<td>Stereotaxic instrument</td>
			<td>Stoelting Co.</td>
			<td></td>
			<td>custom made by Neurostar</td>
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		<tr>
			<td>Stereotaxic robot</td>
			<td>Neurostar</td>
			<td></td>
			<td></td>
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			<td>suture: coated vicryl, polyglatin 910</td>
			<td>Ethicon</td>
			<td>V797D</td>
			<td></td>
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			<td>ThermoMixer C</td>
			<td>Eppendorf AG</td>
			<td>5382000015</td>
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</table>

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.

Figure 4 has been modified from Rauschenberger et al.17. For data analysis of both the dystonia rating scale (A) and the tail suspension test (B), calculate the total score for each time point for each animal. The mean of each time point and each group should be plotted on an appropriate graph. The distribution of the values should be investigated and the appropriate statistical test should be applied to determine significance. With a sufficient number of animals, a motor phenotype can be detected both with the dystonia rating scale as well as with the assessment of abnormal movements in the tail suspension test. The dystonia-like phenotype is demonstrated by the significantly higher motor score in both assessments in the ouabain-perfused, stressed group compared to ouabain-perfused, non-stressed mice as well as the control mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61635/61635fig1v2.jpg" /> 
Figure 1: The main surgical steps for cannula and osmotic pump implantation.&#160;(A) For the indicated coordinates, holes need to be drilled bilaterally for the double cannula designated for the basal ganglia and for the single cannula placed at the midline of the cerebellum. The two fully-constructed osmotic pumps are shown on each side of the animal. (B) The picture shows an implanted, single cannula into the cerebellum, fixed with dental cement. The double cannula for the basal ganglia should be connected to the bifurcation adaptor and prefilled with ouabain before implantation. (C) Image of the finished procedure. <a href="https://www.jove.com/files/ftp_upload/61635/61635fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61635/61635fig02.jpg" /> 
Figure 2: Assessment of dystonia-like movements with a dystonia rating scale.&#160;During a 4 min video, dystonia-like movements were scored based on body distribution and duration. Involuntary hyperextension of the front limbs, a wide stance or hyperextension of hindlimbs as well as kyphosis were rated as dystonia-like. <a href="https://www.jove.com/files/ftp_upload/61635/61635fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61635/61635fig03.jpg" /> 
Figure 3: Assessment of dystonia-like movements during a tail suspension test.&#160;The newly-developed scoring system for abnormal movements during a 2 min tail suspension test evaluates dystonia-like movements in front limbs, hindlimbs and trunk from 0-8 points in total. For the front limbs, a hyperextension and crossing of the front limbs as well as a tonic flexion towards the trunk qualified as dystonia-like. For the hindlimbs the involuntary hyperextension as well as retraction with extension over the midline was scored as dystonia-like. A truncal distortion over 80% of the recorded time was scored with one point. <a href="https://www.jove.com/files/ftp_upload/61635/61635fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61635/61635fig04.jpg" /> 
Figure 4: Representative graphs of the dystonia-rating scale and the tail suspension test.&#160;(A) The graph depicts the dystonia-rating scale for NaCl-perfused, stressed mice (dotted black line), ouabain-perfused, non-stressed mice (dotted orange line) and ouabain-perfused, stressed mice (dark blue line). For each time point, the mean values &#177; standard error of the mean (SEM) are shown. (B) The diagram shows the assessment of abnormal movements during a 2-min tail suspension test for NaCl-perfused, stressed mice (dotted black line), ouabain-perfused, non-stressed mice (dotted orange line) and ouabain-perfused, stressed mice (dark blue line). Statistical analysis was done for the dystonia rating scale and the tail suspension test scoring using the two-tailed Mann-Whitney test. Bonferroni-Holm correction (&#167;) of the p-values showed a significant difference for the observational period of 72 h. Dark blue * denote significant differences between ouabain-perfused, stressed mice and ouabain-perfused, non-stressed mice mice, black * denote significant differences between NaCl-perfused, stressed mice and ouabain-perfused, stressed mice as well as between NaCl-perfused, stressed mice and ouabain-perfused, non-stressed mice. <a href="https://www.jove.com/files/ftp_upload/61635/61635fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia at JoVE.com

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

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-osmotic-pumps-induction-stress-to-establish-symptomatic

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Neuroscience

7.2K Views.  University Hospital of Würzburg. 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.

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

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Measurement &amp; Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being asynchronous (typically 30-50 ms). It has been shown to be abnormal (prolonged) in neurological disorders, including cervical dystonia, a phenotype of adult onset idiopathic isolated focal dystonia. The TDT is a quantitative measure of the ability to perceive rapid changes in the environment and is considered indicative of the behavior of the visual neurons in the superior colliculus, a key node in covert attentional orienting. This article sets out methods for measuring the TDT (including two hardware options and two modes of stimuli presentation). We also explore two approaches of data analysis and TDT calculation. The application of the assessment of temporal discrimination to the understanding of the pathogenesis of cervical dystonia and adult onset idiopathic isolated focal dystonia is also discussed.

Methods for the measurement and analysis of the temporal discrimination threshold are presented, and its application to the study of the pathogenesis of cervical dystonia are discussed.

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being ...

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

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. ...

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.

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 ...