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An experimental model for Huntington's chorea?
Behavioural Brain Research 262 (2014) 31–34
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
An experimental model for Huntington’s chorea? Dagmar H. Zeef a,b,d , Ali Jahanshahi a,b,d , Rinske Vlamings a,d , João Casaca-Carreira a,d , Remco G. Santegoeds a,d , Marcus L.F. Janssen a,c,d , Mayke Oosterloo c , Yasin Temel a,b,d,∗ a
Department of Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands Department of Neurosurgery, Maastricht University Medical Center, Maastricht, The Netherlands Department of Neurology, Maastricht University Medical Center, Maastricht, The Netherlands d European Graduate School of Neuroscience (EURON) , The Netherlands b c
h i g h l i g h t s • Hyperkinetic movements in tgHD rats fulfilled the clinical criteria of chorea. • Administration of tetrabenazine reduced the number of hyperkinetic movements. • tgHD rat can be considered as an animal model for choreiform movement disorder.
a r t i c l e
i n f o
Article history: Received 8 September 2013 Received in revised form 23 December 2013 Accepted 27 December 2013 Available online 8 January 2014 Keywords: Transgenic rat model Huntington’s disease Chorea Dopamine Tetrabenazine
a b s t r a c t Clinically, Huntington’s disease (HD) is well known for the predominant motor symptom chorea, which is a hyperkinetic motor disorder. The only experimental model currently described in the literature, as far as we are aware of, exhibiting hyperkinetic movements is the transgenic rat model of HD. We assessed and characterized these hyperkinetic movements in detail and investigated the effect of tetrabenazine (TBZ) treatment. TBZ is an effective drug in the treatment of chorea in HD patients. Our results showed that the hyperkinetic movements fulfilled the clinical-behavioral criteria of a choreiform movement. Administration of TBZ reduced the number of these hyperkinetic movements substantially. These findings suggest that the hyperkinetic movements observed in this animal model can be considered as a choreiform movement disorder. This makes these animals unique and provides opportunities for chorea-research. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Huntington’s disease (HD) is an inherited fatal neurodegenerative disorder. The disease is caused by a pathologically increased number of CAG trinucleotides within the IT15 gene, located on the short arm of the fourth chromosome. The result is the formation of the mutant form of the huntingtin (htt) protein. Clinically, HD is well known for the predominant motor symptom chorea. A chorea, which is the Greek word for dance, is defined as a rapid, involuntary, non-repetitive movement involving the face, trunk, and/or limbs [1]. Many patients are unaware of these movements or may incorporate the chorea into purposeful movements. A characteristic feature of chorea is that the movements are unpredictable in timing,
∗ Corresponding author at: Department of Neurosurgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Tel.: +31 43 3882048; fax: +31 43 3876038. E-mail address: [email protected] (Y. Temel). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.12.036
direction and the body parts affected. These unpredictable movements may contribute to a poor balance and gait abnormalities in patients. Like most forms of involuntary movement disorders, the severity of the chorea can be worsened by stress and mood disorders, and is typically absent during sleep. Additionally, the severity of the chorea increases with disease progression, but eventually decreases and a hypokinetic rigid syndrome appears [2]. To investigate mechanisms of disease and to develop new therapeutic strategies a number of animal models of HD have been developed in the last years. Most models accurately mimic the neuropathological features of human HD [3–6] the cognitive and emotional symptoms [7–9], and the late-stage hypokinetic motor feature [10,11]. The question arises whether an experimental model exists, which shows hyperkinetic movements resembling the chorea. The only experimental model currently described in the literature, as far as we are aware of, exhibiting hyperkinetic movements is the transgenic rat model, originally described by Von Hörsten and co-workers [6]. This transgenic HD rat (tgHD) carries a truncated huntingtin cDNA fragment with 51 CAG repeats
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D.H. Zeef et al. / Behavioural Brain Research 262 (2014) 31–34
under control of the native rat huntingtin promoter and shows typical neuropathological, neurophysiological, and behavioral changes comparable with those seen in humans with HD [12,13]. Whether the hyperkinetic movement disorder of the tgHD rats fulfills the clinical criteria of a choreiform movement is not well documented. We addressed this question and systematically analyzed the hyperkinetic movement disorder of the tgHD rats. In addition, we performed a pharmacological evaluation by administering tetrabenazine (TBZ). TBZ, a reversible inhibitor of the vesicular monoamine transporter type 2, is effective in reducing the chorea in HD patients [14]. We hypothesized that if the observed hyperkinetic movements of the tgHD rats respond to TBZ treatment; this would provide, besides the clinical-behavioral evaluations, additional evidence that these movements are choreiform.
2.7 mm posteriorly, 2.5 mm laterally, and 7.7 mm ventrally (Paxinos, 1998) and stainless steel screw was implanted in the skull on the vertex. Connectors for the electrodes were fixed on the skull using a dental acrylic. Bipolar recordings were performed with the PowerLab 8/35 data acquisition system connected to a Dual Bio Amp amplifier (ADInstruments, Castle Hill, Australia). Recordings were made with a sample frequency of 100 Hz, frequency band 0,3 Hz – 100 Hz. EEG was recorded before, during and after the movements [16]. Data are presented as means and standard errors of means (S.E.M.). The quantitative data of the total number of choreiform movements at different assessment time points were analyzed using the Independent Samples T test. All statistical analyses were performed with SPSS 20.0 version for Mac. P-values smaller than 0.05 were considered significant.
2. Methods A total of 21 male animals (11 transgenic subjects and 10 controls) of 17 months of age were included in this study. The onset of the hyperkinetic movements is typically at an age of 6–8 months and their severity increases with disease progression. At the age of 17 months the animals reveal clear-cut hyperkinetic movements of the neck with a relatively high frequency, allowing convenient analysis of the movements in a systematic fashion. The control subjects were wildtype (WT) littermates. All animals were bred at the Central Animal facility of Maastricht University (Maastricht, The Netherlands). Dr. S. Von Hörsten provided the breeding pair from the Friedrich-Alexander University ErlangenNürnberg, Erlangen, Germany [6]. All litters were chipped and tail tipped at weaning, and genotypes determined by qPCR [8]. Animals were housed individually in standard MakrolonTM cages on sawdust bedding in an air-conditioned room (±20 ◦ C; ±60% humidity) under a 12 h reversed light/dark cycle (lights on from 19:00–07:00 h) with standard laboratory rat chow (Hopefarms, Woerden, The Netherlands) and acidified water ad libitum. The Animal Experiments and Ethics Committee of Maastricht University approved all experimental procedures. All animals where videotaped in their home cages. All video recordings where performed in the first hours of the dark phase (08.00–12.00 h). The recording started 15 min before and continued until 2 h after receiving a subcutaneous injection with TBZ 5 mg/kg (Sigma–Aldrich, Zwijndrecht, The Netherlands) or the vehicle solution. TBZ was first dissolved in 1.5 ml 100% ethanol with 2% Tween 80. Following the extraction of ethanol by means of vaporization with N2 gas, the remaining compounds were dissolved in 0.5% methylcellulose. The vehicle solution was prepared with the same protocol, but excluding TBZ. The TBZ dosage of 5 mg/kg was based on clinical data and a previous study in the 128YAC transgenic mouse model for HD [15]. Each animal served as its own control by receiving both conditions; twice TBZ and once the vehicle solution. The order in which the animals received the conditions was randomized. Between the different experimental conditions the animals were given a washout period of one week. Controls received no injections but underwent the same videotaping protocol. The videotapes were systematically analyzed by a, for the conditions, blinded investigator. The assessment of the movements was done every 15 min, starting 15 min pre-injection until 2 h postinjection. Only, the first 5 min of each time point was assessed resulting in a total of 10 assessment periods. To verify the non epileptic nature of the neck movements in tgHD rats EEG recording was performed during these movements using multichannel scalp. Six tgHD rats were stereotactically (Dual Manipulator Lab Standard Stereotact, Stoelting Inc., Wood Dale, USA) implanted by A bipolar recording needle electrode (core − and shell: +) in the Globus pallidus (GP) (coordinates relative to bregma:
3. Results We found that the tgHD rats show typical hyperkinetic movements at the level of the neck/head. As previously described, these movements were not observed in the WT animals [10,12,17]. The hyperkinetic movements exhibited by the tgHD rats were extension-rotation movements of the neck/head, purposeless and rapid. These purposeless actions were not observed in other parts of the body. The movements occurred in a repetitive way, but were not predictable. Importantly these movements were not linked to grooming, gnawing, rearing, and/or sniffing. The activity often had a short-duration (1–3 s) and sometimes the animals showed a prolonged hyperkinesia lasting for several seconds. The animals seemed unable to interrupt the movement (video 1; supplementary materials). The level of extension/rotation varied between brief movements to a more than 90◦ extension/rotation of the neck (Fig. 1). Since there was no significant difference between the effect of two TBZ treatment sessions (both reduced the number of hyperkinetic movements substantially, P < 0.01) the data was pooled. Our results revealed that at the age of 17 months the tgHD animals have, on average, 11 choreiform movements of the neck in a 5-min assessment period, comparable to data reported previously [17]. Following the administration of TBZ the number of hyperkinetic movements was significantly reduced (Fig. 1). On average a 55% reduction of the number of hyperkinetic movements in a 5 min of assessment period was found comparing the two conditions at 45, 60 and 90 min post administration of TBZ or the vehicle (P < 0.01). Besides tgHD animals revealed a decreased amount of grooming and overall activity 30 min post-injection with TBZ. There were no signs of immobility. EEG recordings did not show any epileptic activity before, during or after these movements (Fig. 2). 4. Discussion Our behavioral observations revealed that the hyperkinetic movements exhibited by the tgHD rats fulfill the criteria of a choreiform movement. The effects of TBZ treatment, which substantially reduced the number of hyperkinetic movements, support this statement. Potential side effects of TBZ include immobility and somnolence. For instance, complete immobility was found when high a concentration (2.5 mg/kg) of TBZ was administered to BACHD mice [18]. Here, we observed reduced grooming behavior, but no immobility. TBZ treatment resulted in approximately 50% reduction in the number of the choreiform movements, which is comparable to the effect seen in patients. Some investigators (personal communications) suggested that these movements could be epileptic. With multichannel scalp EEG
D.H. Zeef et al. / Behavioural Brain Research 262 (2014) 31–34
33
Fig. 1. A–F: Serial photographs of a typical choreiform movement by a tgHD rat during 3 s. G: Graph represents the mean ± SEM of the number of choreiform movements per 5 min assessed before and 45, 60 and 90 min after administration of TBZ, *P < 0.05.
recordings in these animals during these movements, we found no signs of epileptic activity (Fig. 2). The question arises why specifically in this animal model choreiform movements are observed. In other models of HD a hypermobile phenotype has been observed. In our opinion, a major difference is that the hyperkinetic features observed in this model are periodic/intermittent involuntary movements, and not a generalized hypermobility. The latter has also been observed in this model [13]. What is different in this animal model, when compared to other models of HD? The answer may lay in the fact that specifically this animal model shows a hyperdopaminergic status. In our previous studies, we found an increased number of dopamine containing cells in the midbrain of tgHD rats, which was accompanied by an enhanced dopamine expression in the striatum [19,20]. These results are in line with the early findings by Bird and Spokes in the 1970s and 1980s. They found high increases in dopamine concentrations in the corpus striatum in post-mortem
brain tissue from choreic patients [21]. In a recent study using fast-scan cycling voltammetry in brain slices of 20–23 months old tgHD rats, decreased DA release was found in the striatum [22]. These findings are not in line with our neuropathological findings in the same animal model. The main difference between the studies is the end-stage versus intermediate-disease stage. Furthermore, it is possible that DA release only changes during a choreiform movement, which will not be detectable using ex vivo neurochemistry. Interestingly, the commonly used transgenic mouse models of HD (R6/2 and R6/1 models), which do not display choreiform hyperkinetic movements, show decreased level of dopamine in the striatum [23,24]. Altogether, tgHD rats show choreiform movements and histopathological investigations propose a link with the dopaminergic system. This makes these animals unique and provides opportunities for future chorea-research.
Fig. 2. Electrographic example of the EEG recorded from a transgenic HD rat. First lead shows the EEG registered from the electrode core versus the vertex, the second the electrode shell versus the vertex, and the third lead represents the EEG from the core versus the shell of the electrode. Note that, the general pattern of EEG does not change before and after the choreic movement. Scale: 0.1 mV × 40 ms for A and B; 0.0002 mV × 40 ms for C.
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D.H. Zeef et al. / Behavioural Brain Research 262 (2014) 31–34
Acknowledgement This study was supported by a research grant from the Cure Huntington’s Disease Initiative (CHDI). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2013.12.036. References [1] Walker FO. Huntington’s disease. Lancet 2007;369:218–28. [2] Roos RA. Huntington’s disease: a clinical review. Orphanet J Rare Dis 2010;5:40. [3] Gray M, Shirasaki DI, Cepeda C, Andre VM, Wilburn B, Lu XH, et al. Fulllength human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. J Neurosci 2008;28:6182–95. [4] Kantor O, Temel Y, Holzmann C, Raber K, Nguyen HP, Cao C, et al. Selective striatal neuron loss and alterations in behavior correlate with impaired striatal function in Huntington’s disease transgenic rats. Neurobiol Dis 2006;22:538–47. [5] Reddy PH, Williams M, Charles V, Garrett L, Pike-Buchanan L, Whetsell Jr WO, et al. Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA. Nat Genet 1998;20:198–202. [6] von Horsten S, Schmitt I, Nguyen HP, Holzmann C, Schmidt T, Walther T, et al. Transgenic rat model of Huntington’s disease. Hum Mol Genet 2003;12:617–24. [7] Van Raamsdonk JM, Pearson J, Slow EJ, Hossain SM, Leavitt BR, Hayden MR. Cognitive dysfunction precedes neuropathology and motor abnormalities in the YAC128 mouse model of Huntington’s disease. J Neurosci 2005;25:4169–80. [8] Nguyen HP, Kobbe P, Rahne H, Worpel T, Jager B, Stephan M, et al. Behavioral abnormalities precede neuropathological markers in rats transgenic for Huntington’s disease. Hum Mol Genet 2006;15:3177–94. [9] Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 1996;87:493–506.
[10] Carreira JC, Jahanshahi A, Zeef D, Kocabicak E, Vlamings R, von Horsten S, et al. Transgenic rat models of Huntington’s disease. Curr Top Behav Neurosci 2013. Epub ahead of print. [11] Kim J, Bordiuk OL, Ferrante RJ. Experimental models of HD and reflection on therapeutic strategies. Int Rev Neurobiol 2011;98:419–81. [12] Vlamings R, Zeef DH, Janssen ML, Oosterloo M, Schaper F, Jahanshahi A, et al. Lessons learned from the transgenic Huntington’s disease rats. Neural Plast 2012;2012:682712. [13] Zeef DH, Vlamings R, Lim LW, Tan S, Janssen ML, Jahanshahi A, et al. Motor and non-motor behaviour in experimental Huntington’s disease. Behav Brain Res 2012;226:435–9. [14] Huntington Study Group. Tetrabenazine as antichorea therapy in Huntington disease: a randomized controlled trial. Neurology 2006;66:366–72. [15] Wang H, Chen X, Li Y, Tang TS, Bezprozvanny I. Tetrabenazine is neuroprotective in Huntington’s disease mice. Mol Neurodegener 2010;5:18. [16] Rijkers K, Aalbers M, Hoogland G, van Winden L, Vles J, Steinbusch H, et al. Acute seizure-suppressing effect of vagus nerve stimulation in the amygdala kindled rat. Brain Res 2010;1319:155–63. [17] Temel Y, Cao C, Vlamings R, Blokland A, Ozen H, Steinbusch HW, et al. Motor and cognitive improvement by deep brain stimulation in a transgenic rat model of Huntington’s disease. Neurosci Lett 2006;406:138–41. [18] Andre VM, Cepeda C, Fisher YE, Huynh M, Bardakjian N, Singh S, et al. Differential electrophysiological changes in striatal output neurons in Huntington’s disease. J Neurosci 2011;31:1170–82. [19] Jahanshahi A, Vlamings R, Kaya AH, Lim LW, Janssen ML, Tan S, et al. Hyperdopaminergic status in experimental Huntington disease. J Neuropathol Exp Neurol 2010;69:910–7. [20] Jahanshahi A, Vlamings R, van Roon-Mom WM, Faull RL, Waldvogel HJ, Janssen ML, et al. Changes in brainstem serotonergic and dopaminergic cell populations in experimental and clinical Huntington’s disease. Neuroscience 2013;238:71–81. [21] Spokes EG. Neurochemical alterations in Huntington’s chorea: a study of postmortem brain tissue. Brain 1980;103:179–210. [22] Ortiz AN, Osterhaus GL, Lauderdale K, Mahoney L, Fowler SC, von Horsten S, et al. Motor function and dopamine release measurements in transgenic Huntington’s disease model rats. Brain Res 2012;1450:148–56. [23] Ortiz AN, Kurth BJ, Osterhaus GL, Johnson MA. Dysregulation of intracellular dopamine stores revealed in the R6/2 mouse striatum. J Neurochem 2010;112:755–61. [24] Ortiz AN, Kurth BJ, Osterhaus GL, Johnson MA. Impaired dopamine release and uptake in R6/1 Huntington’s disease model mice. Neurosci Lett 2011;492:11–4.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
An experimental model for Huntington’s chorea? Dagmar H. Zeef a,b,d , Ali Jahanshahi a,b,d , Rinske Vlamings a,d , João Casaca-Carreira a,d , Remco G. Santegoeds a,d , Marcus L.F. Janssen a,c,d , Mayke Oosterloo c , Yasin Temel a,b,d,∗ a
Department of Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands Department of Neurosurgery, Maastricht University Medical Center, Maastricht, The Netherlands Department of Neurology, Maastricht University Medical Center, Maastricht, The Netherlands d European Graduate School of Neuroscience (EURON) , The Netherlands b c
h i g h l i g h t s • Hyperkinetic movements in tgHD rats fulfilled the clinical criteria of chorea. • Administration of tetrabenazine reduced the number of hyperkinetic movements. • tgHD rat can be considered as an animal model for choreiform movement disorder.
a r t i c l e
i n f o
Article history: Received 8 September 2013 Received in revised form 23 December 2013 Accepted 27 December 2013 Available online 8 January 2014 Keywords: Transgenic rat model Huntington’s disease Chorea Dopamine Tetrabenazine
a b s t r a c t Clinically, Huntington’s disease (HD) is well known for the predominant motor symptom chorea, which is a hyperkinetic motor disorder. The only experimental model currently described in the literature, as far as we are aware of, exhibiting hyperkinetic movements is the transgenic rat model of HD. We assessed and characterized these hyperkinetic movements in detail and investigated the effect of tetrabenazine (TBZ) treatment. TBZ is an effective drug in the treatment of chorea in HD patients. Our results showed that the hyperkinetic movements fulfilled the clinical-behavioral criteria of a choreiform movement. Administration of TBZ reduced the number of these hyperkinetic movements substantially. These findings suggest that the hyperkinetic movements observed in this animal model can be considered as a choreiform movement disorder. This makes these animals unique and provides opportunities for chorea-research. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Huntington’s disease (HD) is an inherited fatal neurodegenerative disorder. The disease is caused by a pathologically increased number of CAG trinucleotides within the IT15 gene, located on the short arm of the fourth chromosome. The result is the formation of the mutant form of the huntingtin (htt) protein. Clinically, HD is well known for the predominant motor symptom chorea. A chorea, which is the Greek word for dance, is defined as a rapid, involuntary, non-repetitive movement involving the face, trunk, and/or limbs [1]. Many patients are unaware of these movements or may incorporate the chorea into purposeful movements. A characteristic feature of chorea is that the movements are unpredictable in timing,
∗ Corresponding author at: Department of Neurosurgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Tel.: +31 43 3882048; fax: +31 43 3876038. E-mail address: [email protected] (Y. Temel). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.12.036
direction and the body parts affected. These unpredictable movements may contribute to a poor balance and gait abnormalities in patients. Like most forms of involuntary movement disorders, the severity of the chorea can be worsened by stress and mood disorders, and is typically absent during sleep. Additionally, the severity of the chorea increases with disease progression, but eventually decreases and a hypokinetic rigid syndrome appears [2]. To investigate mechanisms of disease and to develop new therapeutic strategies a number of animal models of HD have been developed in the last years. Most models accurately mimic the neuropathological features of human HD [3–6] the cognitive and emotional symptoms [7–9], and the late-stage hypokinetic motor feature [10,11]. The question arises whether an experimental model exists, which shows hyperkinetic movements resembling the chorea. The only experimental model currently described in the literature, as far as we are aware of, exhibiting hyperkinetic movements is the transgenic rat model, originally described by Von Hörsten and co-workers [6]. This transgenic HD rat (tgHD) carries a truncated huntingtin cDNA fragment with 51 CAG repeats
32
D.H. Zeef et al. / Behavioural Brain Research 262 (2014) 31–34
under control of the native rat huntingtin promoter and shows typical neuropathological, neurophysiological, and behavioral changes comparable with those seen in humans with HD [12,13]. Whether the hyperkinetic movement disorder of the tgHD rats fulfills the clinical criteria of a choreiform movement is not well documented. We addressed this question and systematically analyzed the hyperkinetic movement disorder of the tgHD rats. In addition, we performed a pharmacological evaluation by administering tetrabenazine (TBZ). TBZ, a reversible inhibitor of the vesicular monoamine transporter type 2, is effective in reducing the chorea in HD patients [14]. We hypothesized that if the observed hyperkinetic movements of the tgHD rats respond to TBZ treatment; this would provide, besides the clinical-behavioral evaluations, additional evidence that these movements are choreiform.
2.7 mm posteriorly, 2.5 mm laterally, and 7.7 mm ventrally (Paxinos, 1998) and stainless steel screw was implanted in the skull on the vertex. Connectors for the electrodes were fixed on the skull using a dental acrylic. Bipolar recordings were performed with the PowerLab 8/35 data acquisition system connected to a Dual Bio Amp amplifier (ADInstruments, Castle Hill, Australia). Recordings were made with a sample frequency of 100 Hz, frequency band 0,3 Hz – 100 Hz. EEG was recorded before, during and after the movements [16]. Data are presented as means and standard errors of means (S.E.M.). The quantitative data of the total number of choreiform movements at different assessment time points were analyzed using the Independent Samples T test. All statistical analyses were performed with SPSS 20.0 version for Mac. P-values smaller than 0.05 were considered significant.
2. Methods A total of 21 male animals (11 transgenic subjects and 10 controls) of 17 months of age were included in this study. The onset of the hyperkinetic movements is typically at an age of 6–8 months and their severity increases with disease progression. At the age of 17 months the animals reveal clear-cut hyperkinetic movements of the neck with a relatively high frequency, allowing convenient analysis of the movements in a systematic fashion. The control subjects were wildtype (WT) littermates. All animals were bred at the Central Animal facility of Maastricht University (Maastricht, The Netherlands). Dr. S. Von Hörsten provided the breeding pair from the Friedrich-Alexander University ErlangenNürnberg, Erlangen, Germany [6]. All litters were chipped and tail tipped at weaning, and genotypes determined by qPCR [8]. Animals were housed individually in standard MakrolonTM cages on sawdust bedding in an air-conditioned room (±20 ◦ C; ±60% humidity) under a 12 h reversed light/dark cycle (lights on from 19:00–07:00 h) with standard laboratory rat chow (Hopefarms, Woerden, The Netherlands) and acidified water ad libitum. The Animal Experiments and Ethics Committee of Maastricht University approved all experimental procedures. All animals where videotaped in their home cages. All video recordings where performed in the first hours of the dark phase (08.00–12.00 h). The recording started 15 min before and continued until 2 h after receiving a subcutaneous injection with TBZ 5 mg/kg (Sigma–Aldrich, Zwijndrecht, The Netherlands) or the vehicle solution. TBZ was first dissolved in 1.5 ml 100% ethanol with 2% Tween 80. Following the extraction of ethanol by means of vaporization with N2 gas, the remaining compounds were dissolved in 0.5% methylcellulose. The vehicle solution was prepared with the same protocol, but excluding TBZ. The TBZ dosage of 5 mg/kg was based on clinical data and a previous study in the 128YAC transgenic mouse model for HD [15]. Each animal served as its own control by receiving both conditions; twice TBZ and once the vehicle solution. The order in which the animals received the conditions was randomized. Between the different experimental conditions the animals were given a washout period of one week. Controls received no injections but underwent the same videotaping protocol. The videotapes were systematically analyzed by a, for the conditions, blinded investigator. The assessment of the movements was done every 15 min, starting 15 min pre-injection until 2 h postinjection. Only, the first 5 min of each time point was assessed resulting in a total of 10 assessment periods. To verify the non epileptic nature of the neck movements in tgHD rats EEG recording was performed during these movements using multichannel scalp. Six tgHD rats were stereotactically (Dual Manipulator Lab Standard Stereotact, Stoelting Inc., Wood Dale, USA) implanted by A bipolar recording needle electrode (core − and shell: +) in the Globus pallidus (GP) (coordinates relative to bregma:
3. Results We found that the tgHD rats show typical hyperkinetic movements at the level of the neck/head. As previously described, these movements were not observed in the WT animals [10,12,17]. The hyperkinetic movements exhibited by the tgHD rats were extension-rotation movements of the neck/head, purposeless and rapid. These purposeless actions were not observed in other parts of the body. The movements occurred in a repetitive way, but were not predictable. Importantly these movements were not linked to grooming, gnawing, rearing, and/or sniffing. The activity often had a short-duration (1–3 s) and sometimes the animals showed a prolonged hyperkinesia lasting for several seconds. The animals seemed unable to interrupt the movement (video 1; supplementary materials). The level of extension/rotation varied between brief movements to a more than 90◦ extension/rotation of the neck (Fig. 1). Since there was no significant difference between the effect of two TBZ treatment sessions (both reduced the number of hyperkinetic movements substantially, P < 0.01) the data was pooled. Our results revealed that at the age of 17 months the tgHD animals have, on average, 11 choreiform movements of the neck in a 5-min assessment period, comparable to data reported previously [17]. Following the administration of TBZ the number of hyperkinetic movements was significantly reduced (Fig. 1). On average a 55% reduction of the number of hyperkinetic movements in a 5 min of assessment period was found comparing the two conditions at 45, 60 and 90 min post administration of TBZ or the vehicle (P < 0.01). Besides tgHD animals revealed a decreased amount of grooming and overall activity 30 min post-injection with TBZ. There were no signs of immobility. EEG recordings did not show any epileptic activity before, during or after these movements (Fig. 2). 4. Discussion Our behavioral observations revealed that the hyperkinetic movements exhibited by the tgHD rats fulfill the criteria of a choreiform movement. The effects of TBZ treatment, which substantially reduced the number of hyperkinetic movements, support this statement. Potential side effects of TBZ include immobility and somnolence. For instance, complete immobility was found when high a concentration (2.5 mg/kg) of TBZ was administered to BACHD mice [18]. Here, we observed reduced grooming behavior, but no immobility. TBZ treatment resulted in approximately 50% reduction in the number of the choreiform movements, which is comparable to the effect seen in patients. Some investigators (personal communications) suggested that these movements could be epileptic. With multichannel scalp EEG
D.H. Zeef et al. / Behavioural Brain Research 262 (2014) 31–34
33
Fig. 1. A–F: Serial photographs of a typical choreiform movement by a tgHD rat during 3 s. G: Graph represents the mean ± SEM of the number of choreiform movements per 5 min assessed before and 45, 60 and 90 min after administration of TBZ, *P < 0.05.
recordings in these animals during these movements, we found no signs of epileptic activity (Fig. 2). The question arises why specifically in this animal model choreiform movements are observed. In other models of HD a hypermobile phenotype has been observed. In our opinion, a major difference is that the hyperkinetic features observed in this model are periodic/intermittent involuntary movements, and not a generalized hypermobility. The latter has also been observed in this model [13]. What is different in this animal model, when compared to other models of HD? The answer may lay in the fact that specifically this animal model shows a hyperdopaminergic status. In our previous studies, we found an increased number of dopamine containing cells in the midbrain of tgHD rats, which was accompanied by an enhanced dopamine expression in the striatum [19,20]. These results are in line with the early findings by Bird and Spokes in the 1970s and 1980s. They found high increases in dopamine concentrations in the corpus striatum in post-mortem
brain tissue from choreic patients [21]. In a recent study using fast-scan cycling voltammetry in brain slices of 20–23 months old tgHD rats, decreased DA release was found in the striatum [22]. These findings are not in line with our neuropathological findings in the same animal model. The main difference between the studies is the end-stage versus intermediate-disease stage. Furthermore, it is possible that DA release only changes during a choreiform movement, which will not be detectable using ex vivo neurochemistry. Interestingly, the commonly used transgenic mouse models of HD (R6/2 and R6/1 models), which do not display choreiform hyperkinetic movements, show decreased level of dopamine in the striatum [23,24]. Altogether, tgHD rats show choreiform movements and histopathological investigations propose a link with the dopaminergic system. This makes these animals unique and provides opportunities for future chorea-research.
Fig. 2. Electrographic example of the EEG recorded from a transgenic HD rat. First lead shows the EEG registered from the electrode core versus the vertex, the second the electrode shell versus the vertex, and the third lead represents the EEG from the core versus the shell of the electrode. Note that, the general pattern of EEG does not change before and after the choreic movement. Scale: 0.1 mV × 40 ms for A and B; 0.0002 mV × 40 ms for C.
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