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>

<ol>
	<li>DeAndrade, M. P., Yokoi, F., van Groen, T., Lingrel, J. B., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Atp1a3+mutant+mice+as+a+model+of+rapid-onset+dystonia+with+parkinsonism.">Characterization of Atp1a3 mutant mice as a model of rapid-onset dystonia with parkinsonism.</a> Behavioural Brain Research. 216 (2), 659-665 (2011).</li><li>Dang, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+Dyt1+DeltaGAG+knock-in+mouse+as+a+model+for+early-onset+dystonia.">Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.</a> Experimental Neurology. 196 (2), 452-463 (2005).</li><li>Kreiner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+mechanisms+in+genetic+models+of+neurodegeneration:+are+the+mice+better+than+humans.">Compensatory mechanisms in genetic models of neurodegeneration: are the mice better than humans.</a> Frontiers in Cellular Neuroscience. 9, 56 (2015).</li><li>Albanese, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenomenology+and+classification+of+dystonia:+a+consensus+update.">Phenomenology and classification of dystonia: a consensus update.</a> Movement Disorders. 28 (7), 863-873 (2013).</li><li>Marsden, C. D., Obeso, J. A., Zarranz, J. J., Lang, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomical+basis+of+symptomatic+hemidystonia.">The anatomical basis of symptomatic hemidystonia.</a> Brain. 108, 463-483 (1985).</li><li>Carbon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+sensorimotor+network+activity+in+DYT1+dystonia:+a+functional+imaging+study.">Increased sensorimotor network activity in DYT1 dystonia: a functional imaging study.</a> Brain. 133, 690-700 (2010).</li><li>Carbon, M., Su, S., Dhawan, V., Raymond, D., Bressman, S., Eidelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+metabolism+in+primary+torsion+dystonia:+effects+of+penetrance+and+genotype.">Regional metabolism in primary torsion dystonia: effects of penetrance and genotype.</a> Neurology. 62 (8), 1384-1390 (2004).</li><li>de Carvalho Aguiar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+Na+/K++-ATPase+alpha3+gene+ATP1A3+are+associated+with+rapid-onset+dystonia+parkinsonism.">Mutations in the Na+/K+ -ATPase alpha3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism.</a> Neuron. 43 (2), 169-175 (2004).</li><li>Brashear, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phenotypic+spectrum+of+rapid-onset+dystonia-parkinsonism+(RDP)+and+mutations+in+the+ATP1A3+gene.">The phenotypic spectrum of rapid-onset dystonia-parkinsonism (RDP) and mutations in the ATP1A3 gene.</a> Brain. 130 (3), 828-835 (2007).</li><li>Barbano, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+triggers+and+non-motor+findings+in+a+family+with+rapid-onset+dystonia-parkinsonism.">New triggers and non-motor findings in a family with rapid-onset dystonia-parkinsonism.</a> Parkinsonism &amp; Related Disorders. 18 (6), 737-741 (2012).</li><li>Isaksen, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia-induced+dystonia+and+abnormal+cerebellar+activity+in+a+mouse+model+with+a+single+disease-mutation+in+the+sodium-potassium+pump.">Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump.</a> PLOS Genetics. 13 (5), 1006763 (2017).</li><li>Sugimoto, H., Ikeda, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygous+mice+deficient+in+Atp1a3+exhibit+motor+deficits+by+chronic+restraint+stress.">Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress.</a> Behavioural Brain Research. 272, 100-110 (2014).</li><li>Calderon, D. P., Fremont, R., Kraenzlin, F., Khodakhah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+substrates+of+rapid-onset+Dystonia-Parkinsonism.">The neural substrates of rapid-onset Dystonia-Parkinsonism.</a> Nature Neuroscience. 14 (3), 357-365 (2011).</li><li>Cutsforth-Gregory, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+exercise+dystonia:+A+difficult+to+treat+hazard+of+runner+and+non-runner+athletes.">Repetitive exercise dystonia: A difficult to treat hazard of runner and non-runner athletes.</a> Parkinsonism &amp; Related Disorders. 27, 74-80 (2016).</li><li>Pizoli, C. E., Jinnah, H. A., Billingsley, M. L., Hess, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+Cerebellar+Signaling+Induces+Dystonia+in+Mice.">Abnormal Cerebellar Signaling Induces Dystonia in Mice.</a> The Journal of Neuroscience. 22 (17), 7825-7833 (2002).</li><li>Jinnah, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+agonists+and+dystonia+in+the+mouse.">Calcium channel agonists and dystonia in the mouse.</a> Movement Disorders. 15 (3), 542-551 (2000).</li><li>Rauschenberger, L., Knorr, S., Al-Zuraiqi, Y., Tovote, P., Volkmann, J., Ip, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+dopaminergic+dysregulation+and+dystonia-like+movements+induced+by+sensorimotor+stress+in+a+pharmacological+mouse+model+of+rapid-onset+dystonia-parkinsonism.">Striatal dopaminergic dysregulation and dystonia-like movements induced by sensorimotor stress in a pharmacological mouse model of rapid-onset dystonia-parkinsonism.</a> Experimental Neurology. 323, 113109 (2020).</li><li>Fernagut, P. O., Diguet, E., Bioulac, B., Tison, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MPTP+potentiates+3-nitropropionic+acid-induced+striatal+damage+in+mice:+reference+to+striatonigral+degeneration.">MPTP potentiates 3-nitropropionic acid-induced striatal damage in mice: reference to striatonigral degeneration.</a> Experimental Neurology. 185 (1), 47-62 (2004).</li><li>Guyenet, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+composite+phenotype+scoring+system+for+evaluating+mouse+models+of+cerebellar+ataxia.">A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia.</a> Journal of Visualized Experiments. 39, 1787 (2010).</li><li>Ip, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tor1a+/-+mice+develop+dystonia-like+movements+via+a+striatal+dopaminergic+dysregulation+triggered+by+peripheral+nerve+injury.">Tor1a+/- mice develop dystonia-like movements via a striatal dopaminergic dysregulation triggered by peripheral nerve injury.</a> Acta Neuropathologica Communications. 4, 108 (2016).</li></ol>

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><tbody><tr><td>0.9% saline</td><td>Fresenius Kabi</td><td></td><td>PZN06178437</td></tr><tr><td>Alzet osmotic pumps</td><td>Durect</td><td>4317</td><td>model 1002, flowrate 0.25 &amp;mu;L/h</td></tr><tr><td>Anchor Screws</td><td>AgnTho&#039;s</td><td>MCS1x2</td><td>2 mm long with a thread of 1mm O.D.</td></tr><tr><td>Bulldog Clamps</td><td>AgnTho&#039;s</td><td>13-320-035</td><td>straight, 3.5 cm</td></tr><tr><td>Bupivacain 0.25% Jenapharm</td><td>mibe GmbH Arzneimittel</td><td></td><td></td></tr><tr><td>Cannula and Minipump Holder</td><td>Stoelting Co.</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></tr><tr><td>Cannula tubing</td><td>Plastics One</td><td>C312VT/PKG</td><td>vinyl, 0.69 mm x 1.14 mm</td></tr><tr><td>Dumont #5SF forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>fine forceps</td></tr><tr><td>eye cream Bepanthen</td><td>Bayer Vital GmbH</td><td></td><td></td></tr><tr><td>Gas Anesthesia Mask for Stereotaxic, Mouse</td><td>Stoelting Co.</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&amp;auml;ger Vapor 19.3</td><td>Dr. Wilfried M&amp;uuml;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><tr><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&amp;uuml;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 &amp;amp; Sohn</td><td>PFB 6000-1</td><td></td></tr><tr><td>Rectal Thermal Probe</td><td>Stoelting Co.</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 Co.</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&#039;s</td><td>30090-6</td><td></td></tr><tr><td>Stainless Steel Burrs</td><td>AgnTho&#039;s</td><td>HM71009</td><td>0.9 mm &amp;Oslash; burr</td></tr><tr><td>Stainless Steel Burrs</td><td>AgnTho&#039;s</td><td>HM71014</td><td>1.4 mm &amp;Oslash; burr</td></tr><tr><td>StereoDrive</td><td>Neurostar</td><td></td><td>software</td></tr><tr><td>Stereotaxic instrument</td><td>Stoelting Co.</td><td></td><td>custom made by Neurostar</td></tr><tr><td>Stereotaxic robot</td><td>Neurostar</td><td></td><td></td></tr><tr><td>suture: coated vicryl, polyglatin 910</td><td>Ethicon</td><td>V797D</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf AG</td><td>5382000015</td><td></td></tr></tbody></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

Research

JoVE Journal

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

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Behavior

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Developmental Biology

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

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 ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Medicine

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics stress-induced cardiomyopathy, the chronic model, administered through an osmotic pump, mimics advanced heart failure in humans. The purpose of the described protocol is to create the chronic ISO-induced heart failure model in mice using an implanted mini-pump. This protocol has been used to induce heart failure in 100+ strains of inbred mice. Techniques on surgical pump implantation are described in detail and may be relevant to anyone interested in creating a heart failure model in mice. In addition, the weekly cardiac remodeling changes based on echocardiographic parameters for each strain and expected time to model development are presented. In summary, the method is simple and reproducible. Continuous ISO administered via the implanted mini-pump over 3 to 4 weeks is sufficient to induce cardiac remodeling. Finally, the success for ISO model creation may be assessed in vivo by serial echocardiography demonstrating hypertrophy, ventricular dilation, and dysfunction.

The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics ...

Polycystic Ovary Syndrome Induction in Mice Through Controlled Letrozole Release 

A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of PCOS and for related drug development. Letrozole-induced mouse models replicate the metabolic and reproductive phenotypes of patients with PCOS. The traditional method of letrozole treatment in PCOS mice requires daily dosing over a certain period, which can be labor-intensive and cause significant stress to the mice. This study describes a simple and effective method for inducing PCOS in mice by implanting a controlled letrozole-releasing mini-pump. A mini-pump capable of stable, continuous release of a quantitative amount of letrozole was fabricated and implanted subcutaneously in mice under anesthesia. This study demonstrated that the mouse model successfully mimicked PCOS features after letrozole mini-pump implantation. The materials and equipment used in this study are readily available to most laboratories, requiring no special customization. Collectively, this article provides a unique, easy-to-perform method for inducing PCOS in mice.

Author Spotlight: Investigating the Mechanisms and Inducing Models of Polycystic Ovary Syndrome

This protocol introduces a technique for inducing polycystic ovary syndrome (PCOS) models in mice through controlled letrozole release using mini-pumps. Under adequate anesthesia, the mini-pump was implanted subcutaneously, and PCOS was successfully induced in the mice after a certain period of the mini-pump release.

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

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

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice