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 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 α3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the α3-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.
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 ATP1α3 gene, which encodes for the α3-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 α3-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 °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 (“pole test”) and three runs on a Rotarod apparatus (“Rotarod performance test”). 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.
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, Würzburg, 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.
2. Cannula and osmotic pump implantation
3. Motor challenge
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.
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.
Figure 1: The main surgical steps for cannula and osmotic pump implantation. (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. Please click here to view a larger version of this figure.
Figure 2: Assessment of dystonia-like movements with a dystonia rating scale. 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. Please click here to view a larger version of this figure.
Figure 3: Assessment of dystonia-like movements during a tail suspension test. 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. Please click here to view a larger version of this figure.
Figure 4: Representative graphs of the dystonia-rating scale and the tail suspension test. (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 ± 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 (§) 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. Please click here to view a larger version of this figure.
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 authors have nothing to disclose.
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 Würzburg (Z2-CSP3 to L.R.). The authors thank Louisa Frieß, Keali Röhm, Veronika Senger and Heike Menzel and for their technical assistance as well as Helga Brünner for the animal care.
0.9% saline | Fresenius Kabi | PZN06178437 | |
Alzet osmotic pumps | Durect | 4317 | model 1002, flowrate 0.25 μL/h |
Anchor Screws | AgnTho's | MCS1x2 | 2 mm long with a thread of 1mm O.D. |
Bulldog Clamps | Agntho's | 13-320-035 | straight, 3.5 cm |
Bupivacain 0.25% Jenapharm | mibe GmbH Arzneimittel | ||
Cannula and Minipump Holder | Stoelting | 51636 | designed to hold 3.4 mm cannula heads |
Cannula Bifurcation | Plastics One | 21Y | custom made |
Cannula tubing | Plastics One | C312VT/PKG | vinyl, 0.69 mm x 1.14 mm |
Dumont #5SF forceps | Fine Science Tools | 11252-00 | fine forceps |
eye cream Bepanthen | Bayer Vital GmbH | ||
Gas Anesthesia Mask for Stereotaxic, Mouse | Stoelting | 56109M | |
Hardened fine scissors | Fine Science Tools | 14090-09 | |
High Speed Rotary Micromotor Kit | Foredom | K.1070-2 | |
Isoflurane CP 1 mL/mL, 250 mL | cp-pharma | 1214 | prescription needed |
Isoflurane System Dräger Vapor 19.3 | Dr. Wilfried Müller GmbH | ||
Kallocryl A/C | Speiko | 1615 | dental cement, liquid |
Kallocryl CPGM rot | Speiko | 1692 | dental cement, red powder |
Mouse and neonates adaptor | Stoelting Co. | 51625 | adaptor for mice for a traditional U-frame |
needle holder | KLS Martin Group | 20-526-14 | |
Non-Rupture Ear Bars and Rubber Tips f/ Mouse Stereotaxic | Stoelting Co. | 51649 | |
Octenisept | Schülke | 118211 | |
Osmotic Pump Connector Cannula for Mice, double | Plastics One | 3280PD-3.0/SPC | 28 Gauge, length 4.0 mm, c/c distance 3.0 mm |
Osmotic Pump Connector Cannula for Mice, single | Plastics One | 3280PM/SPC | 28, Gauge, custom length 3.0 mm |
Ouabain octahydrate 250 mg | Sigma-Aldrich | 03125-250MG | CAUTION: toxic |
Precision balance | Kern & Sohn | PFB 6000-1 | |
Rectal Thermal Probe | Stoelting | 50304 | |
Rimadyl 50 mg/mL, injectable | Zoetis | Carprofen, prescription needed | |
Rodent Warmer X1 with Mouse Heating Pad | Stoelting | 53800M | |
RotaRod Advanced | TSE Systems | ||
screw driver set | Agntho's | 30090-6 | |
Stainless Steel Burrs | Agntho's | HM71009 | 0.9 mm Ø burr |
Stainless Steel Burrs | Agntho's | HM71014 | 1.4 mm Ø burr |
StereoDrive | Neurostar | software | |
Stereotaxic instrument | Stoelting Co. | custom made by Neurostar | |
Stereotaxic robot | Neurostar | ||
suture: coated vicryl, polyglatin 910 | Ethicon | V797D | |
ThermoMixer C | Eppendorf AG | 5382000015 |