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Neuromuscular pathology in mice lacking alpha-synuclein
Neuroscience Letters 487 (2011) 350–353
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuromuscular pathology in mice lacking alpha-synuclein Anssi Pelkonen ∗ , Leonid Yavich School of Pharmacy, University of Eastern Finland, Kuopio, Finland
a r t i c l e
i n f o
Article history: Received 17 August 2010 Received in revised form 19 October 2010 Accepted 19 October 2010 Keywords: Alpha-synuclein Skeletal muscles Neuromuscular junction Electromyography Knockout mice
a b s t r a c t This work was undertaken in order to study the possible role of alpha-synuclein in the function of the neuro-muscular junction in skeletal muscles. Repeated stimulation of skeletal muscle motor neurons revealed signs of neuromuscular pathology in alpha-synuclein null mutated (C57Bl/6JOlaHsd) and knockout (B6;129X1-Sncatm1Rosl /J) mice. This stimulation produced repetitive compound muscle action potentials in both lines of alpha-synuclein deficient mice. Muscle strength and muscle coordination during ambulation were unaffected, though motor learning was slower in alpha-synuclein deficient mice in the Rotarod test. We conclude that alpha-synuclein may play a role in acetylcholine compartmentalization at the neuromuscular junction, and in the fine control of activity of skeletal muscles. © 2010 Elsevier Ireland Ltd. All rights reserved.
Alpha-synuclein (␣-syn) is a 140 amino acid and a 14 kDa protein that is best known for its involvement in serious neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, amyotrophic lateral sclerosis and some other pathological states [5]. Recent experiments have examined the involvement of ␣-syn in the control of compartmentalization and pre-synaptic release of neurotransmitters such as dopamine [2,26], glutamate [14] and norepinephrine [27]. ␣-Syn is expressed not only in the central nervous system but also in muscles [4] and peripheral neurons [17,19], though it is not essential for their survival. Recent results also indicate that ␣-syn may be involved in the fine tuning of acetylcholine (ACh) dependent muscle contractility of smooth muscle cells in mice [17]. So far no clear behavioral pathology has been observed in ␣-syn deficient mice [2,10]. We speculated that if ␣-syn possesses a universal role in the storage of neurotransmitters in the pre-synaptic terminals, then repeated stimulation at a level sufficient to exhaust the pre-synaptic storage pool should reveal its role in the compartmentalization of ACh in the neuromuscular synaptic terminals. Repetitive nerve stimulation is widely used to diagnose neuromuscular disorders [1]. Thus, we tested the functionality of skeletal muscles in ␣-syn deficient mice with repeated stimulation of the motor nerves. In some pathological conditions with impaired sig-
Abbreviations: ␣-syn, alpha-synuclein; ACh, acetylcholine; CMAP, compound muscle action potential; R-CMAP, repetitive compound muscle action potential. ∗ Corresponding author at: School of Pharmacy, University of Eastern Finland, Yliopistonranta 1 C, P.O. Box 1627, FIN-70211 Kuopio, Finland. Tel.: +358 403 553 714. E-mail addresses: anssi.pelkonen@uef.fi (A. Pelkonen), leonid.yavich@uef.fi (L. Yavich). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.10.054
nalling in the neuromuscular junction electrical stimulation may yield compound muscle action potentials (CMAPs) accompanied by late components of relatively high amplitude [13,20,25]. These CMAPs are known as repetitive CMAPs (R-CMAPs). The effect of ␣syn deficiency on muscle strength and coordination was also briefly assessed in this work. Three mouse lines were used in this study. The C57BL/6J subpopulation (Charles River Wiga, Sulzfeld, Germany) expresses ␣-syn (referred to as b6+). The C57BL/6J subpopulation of mice from Harlan Olac (Bicester, UK, referred to as b6−) have a spontaneous chromosomal deletion of the ␣-syn and multimerin-1 loci [23,24]. The B6;129X1-Sncatm1Rosl /J is a gene-targeted ␣-syn knockout line [2]. A breeding pair of these mice (referred to as b6-Ros), were obtained from the Jackson Laboratory, cleaned by embryo transfer and bred together with the other two lines in the National Laboratory Animal Center (Kuopio, Finland). The animals used for electromyography (5 per line) were 10–23 weeks of age and animals used for behavioral experiments (6 per line) were 8–15 weeks of age during the experiments. Male b6+, b6− and b6-Ros mice were group housed at an ambient temperature of 22–23 ◦ C and on a 12/12 h dark/light cycle (lights on 7 a.m.). Animals had food and water available ad libitum. The experiments were conducted with a minimized number of animals according to the Council of Europe (Directive 86/609) and Finnish guidelines. The animals were anesthetized with chloral hydrate (450 mg/kg i.p., 10 ml/kg) and the anesthesia was maintained by injecting one quarter of the initial dose after every 45-60 min. The animals were fixed to a heating pad by their limbs and tail with a tape. The electrodes were 26-gauge stainless steel needles. The registration of the CMAPs in the caudal muscles was undertaken as described previously [6] with modifications. The stimulating cathode was placed against the caudal motor axons by puncturing the skin at the base
A. Pelkonen, L. Yavich / Neuroscience Letters 487 (2011) 350–353
of the tail. The anode was placed 0.5 cm anterior to the cathode. The recording electrode was inserted into the caudal muscles on the lateral side of the tail 1.5 cm posterior to the cathode and a reference electrode was placed similarly 1 cm posterior to the active electrode. A needle implanted under the skin was used as a ground electrode. Sub-maximal electrical stimuli, 2–3 mA in amplitudes (these types of high currents were required to obtain effective stimulation) were produced using an A320 current isolation unit (World Precision Instruments, WPI, Sarasota, FL, USA). Pulse length (0.1 ms) and interval (10–300 ms) were set with Master-8 pulse stimulator (A.M.P.I., Jerusalem, Israel). The signals were amplified and filtered (300 Hz to 3 kHz) with DAM 70 Differential Amplifier (WPI). Signals were recorded to a personal computer using Digidata 1321A digitizer and Clampex 8.2 software (Axon Instruments, Sunnyvale, CA, USA). The sampling rate was set to 10 kHz. Five trains of six pulses each at different between-pulses intervals were applied to the caudal motor axons. The intervals between pulses were 300, 100, 50, 20 and 10 ms. Data was collected over a period of 2 s. Each train of stimulation at specific intervals was repeated three times per animal. There was a 1 min pause after each train. The animals were sacrificed after the measurements and their brains removed for assays in related projects. The motor coordination and balance were tested using an accelerating Rotarod (Ugo Basile, Comerio, Italy). The animals were allowed to become accustomed with the apparatus for 1 min before testing, 30 s on a motionless bar and 30 s on a bar that was rotating at a minimal speed (4 rpm). After this period of adaptation, the apparatus was set to accelerate (from 4 to 40 rpm) and the timing began. The time that the animals were able to run on the rotating bar without falling down from the rod was recorded. The mouse could grab hold on the bar for one or two consecutive turns, but after the third consecutive turn the animal was removed from the rod. The maximum time the animals were allowed to remain on the rod was 480 s (8 min). The test was repeated three times with two days between the experiments. We also assessed the gait of the mice with a standard footprint pattern test [8,11]. Muscle strength was evaluated with an adapted front paw grip strength test in which the animals grabbed a normal home cage roof (parallel round metal bars with 2 mm in diameter and 7 mm from each other) with their forepaws when they were lifted upwards at a steady speed while attached to a calibrated spring. The measured parameter was the maximal force against which the animals were able to hold onto the bars. The result of each animal was the average of three attempts repeated after every 30 s. The electromyography results were analyzed off-line with Clampfit, version 8.2 (Axon Instruments). CMAP amplitudes were calculated from the peak of the positive phase to the peak of the negative phase. When R-CMAPs were evaluated, waveforms with amplitude less than 5% of the primary CMAP amplitude were not analyzed to exclude possible A- and F-waves [15,18]. One-way ANOVA and ANOVA for repeated measures were used for statistical analysis of the results (SPSS 14.0 for Windows, SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard error of mean (S.E.M.). There were no differences between the lines in the absolute values of amplitudes of CMAPs calculated from the first pulse in the trains with 300 ms pulse intervals (3.0 ± 1.0 mV in b6+, 3.2 ± 1.7 mV in b6− and 2.6 ± 0.8 mV in b6-Ros mice). No ␣-syn dependent statistically significant differences were seen in the amplitudes of CMAPs in response to repetitive stimulation (data not shown). We detected repetitive CMAPs (R-CMAPs) in the electromyographic recordings in 4 out of 5 b6− mice and 3 out of 5 b6-Ros mice (Fig. 1), while only 1 out of 5 b6+ mice displayed R-CMAPs. Typically the R-CMAPs were detected in ␣-syn deficient mice after the first pulse in trains of 300 ms intervals (39.5% of all R-CMAPs).
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Fig. 1. Occurrence of R-CMAPs. (A) Example of a train with 300 ms intervals between pulses from a b6-Ros mouse showing R-CMAP after the first pulse. The insert shows the artifact of stimulation (the first small peak), the primary CMAP and three late components. (B) Number of detected R-CMAPs with different amounts of late components.
According to ANOVA for repeated measures, the lines demonstrated different behavior on the Rotarod (the effect of genotype: F (2, 15) = 10.5, p = 0.020). According to Bonferroni’s post hoc test, the time on the rod of b6-Ros mice differed significantly from that of b6− and b6+ mice (p = 0.004 and 0.003, respectively) but there was no significant difference between b6− and b6+ mice (p = 1.000). Both ␣-syn deficient lines showed deteriorated performance in the Rotarod test on the first day of testing (Fig. 2A). Training improved these results so that ␣-syn deficient mice were indistinguishable from the b6+ mice by the third day of testing. ANOVA for repeated measures revealed significant within-subject effects in training × genotype interactions (F (4, 30) = 3.2, p = 0.026). b6+ and b6− mice were able to stay on the non-slippery rod of the Rotarod apparatus during a full turn but in contrast to this behavior, b6-Ros mice could not cling on to the rod and dropped off. Clinging on to the bar was seen in 83% of all trials with b6+ mice, 89% with b6− mice but only in 6% with b6-Ros mice. We initially attributed the inability of b6-Ros mice to cling on the bar to a muscular deficiency. However, according to one way ANOVA, the differences between lines in the front paw grip strength test were not significant (Fig. 2B, F (2, 15) = 1.0, p = 0.396). The results of the footprint pattern test were similar to those reported in normal healthy mice [8], but no differences were seen between the lines (Table 1). Two lines of evidence point to neuromuscular pathology in ␣syn deficient mice. The first is the appearance of R-CMAPs following repetitive stimulation, and the second is the result of the Rotarod test. R-CMAPs are considered as a reliable indicator of neuromuscular pathology in clinical neurophysiology [25]. The deteriorated performance found during the first test on the Rotarod is a less conclusive indicator of muscular insufficiency but at present it is the only verified behavioral manifestation of the deletion of ␣-syn,
Fig. 2. Assessment of motor functions. (A) Performance of the alpha-synuclein deficient (b6− and b6-Ros) and wild-type mice (b6+) in the Rotarod test. The test was repeated three times every third day. (B) Front paw grip strength (N) relative to body weight (kg) in b6+, b6− and b6-Ros mice. n = 6.
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A. Pelkonen, L. Yavich / Neuroscience Letters 487 (2011) 350–353
Table 1 Results of the footprint pattern test. b6+ Hindpaw stride length Forepaw stride length Hindpaw base Forepaw base Forepaw/hindpaw overlap
5.9 6.2 2.5 1.6 0.7
b6− ± ± ± ± ±
0.3 0.4 0.1 0.1 0.1
6.1 6.4 2.6 1.3 0.8
± ± ± ± ±
b6-Ros 0.4 0.4 0.1 0.1 0.1
5.5 5.8 2.3 1.6 0.7
± ± ± ± ±
0.3 0.3 0.1 0.0 0.1
F (2, 15)
p
1.3 3.1 2.3 0.6 0.5
0.304 0.076 0.132 0.542 0.618
The results are presented in centimetres. The two columns on the right are the results of one-way ANOVA.
because neither muscle strength nor gait were significantly affected in these lines. R-CMAPs occurred almost exclusively in the ␣-syn deficient mice but not in the b6+ animals. The test on the Rotarod also detected differences between the ␣-syn deficient mouse lines, with the worst behavioral outcome in b6-Ros mice and better performance in b6− animals. It is interesting that another line of ␣-syn knockout mice described in the literature [9] demonstrated much milder signs of synaptic pathology in an indirect comparison with b6-Ros line, which may indicate that some other downstream alteration in protein expression had occurred in b6-Ros mice. This line of knockout mice differs from the line with spontaneous deletion of ␣-syn in their sensitivity to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, indicative that the genetic background is a critical factor in expression of the effects of the deletion [22]. A similar conclusion can be drawn from the present comparison between b6− and b6-Ros mice. ␣-syn belongs to a three-member protein family of synucleins (alpha, beta and gamma), which was first found from the cholinergic nerve terminals of the Torpedo californica electric organ [16], and it appears to exist only in vertebrates [12]. There are fundamental differences between vertebrates and invertebrates in the innervations of their muscles [21]. The evolutionary co-occurrence of the appearance of synucleins in vertebrates and the changes occurring in the innervations of their skeletal muscles makes it tempting to speculate that this family of proteins may play a role in fine neuromuscular control. If one of the postulated functions of ␣-syn in the pre-synaptic terminal is correct compartmentalization of neurotransmitter [2,14,26,27], and this function is universal for any type of neuron, then a lack of the protein may disrupt the formation of the storage and readily releasable pools of ACh. R-CMAPs were detected in both lines of ␣-syn deficient animals. It has been proposed that R-CMAPs are caused by a prolongation of the duration of the endplate potential [25]. The most common reason for this prolongation is excessive ACh release which is in line with data on increased neurotransmitter release in response to burst stimulation obtained in the synapses of central nervous system in ␣-syn deficient mice [2,26,27]. However, other factors cannot be excluded, e.g. acetylcholinesterase inhibition or repeated excitation of the axon in response to a single shock which is known in several neuromuscular disorders with excess motor unit activity [25]. It is unclear whether the suggested role of ␣-syn in muscle development could in some way give rise to the R-CMAPs [4]. The role of ␣-syn in Ca2+ channel function is an unlikely source for the R-CMAPs since ␣-syn activates Ca2+ influx and thus the lack of the protein should diminish the probability of R-CMAPs [3]. The initial performance of ␣-syn deficient mice in the Rotarod test was worse than the performance of the wild-type animals. The significant improvement in performance in consecutive tests and the lack of significant differences between the lines in the footprint pattern and grip strength suggests that the initial differences did not result from weaker muscle strength. It is possible that the ␣-syn deficiency which causes abnormalities in the signaling of the neuromuscular junction makes motor control more difficult for these mice. This could be reflected as slower motor learning. It is also possible that the problems in motor learning arise from the cen-
tral effects of ␣-syn depletion. Earlier experiments with knockout mice detected no changes in motor performance in the Rotarod test [7]. However, in the present study we used a different protocol. Typically normal healthy mice should improve their results in the Rotarod as the test is repeated, but with the current protocol, the wild type mice were able to reach their maximal level of performance already on the first trial. We conclude that ␣-syn may play a role in ACh compartmentalization in the neuromuscular junction, and in the fine control of activity of skeletal muscles. Acknowledgments Authors thank Dr. Ewen MacDonald for revising the language of the manuscript and Saara Stavén for advice concerning the Rotarod test. This work was supported by the Academy of Finland grants 122397 and 125653 to L.Y. References [1] AAEM Quality Assurance Committee, American Association of Electrodiagnostic Medicine, Literature review of the usefulness of repetitive nerve stimulation and single fiber EMG in the electrodiagnostic evaluation of patients with suspected myasthenia gravis or Lambert–Eaton myasthenic syndrome, Muscle Nerve 24 (2001) 1239–1247. [2] A. Abeliovich, Y. Schmitz, I. Farinas, D. Choi-Lundberg, W.H. Ho, P.E. Castillo, N. Shinsky, J.M. Verdugo, M. Armanini, A. Ryan, M. Hynes, H. Phillips, D. Sulzer, A. Rosenthal, Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system, Neuron 25 (2000) 239–252. [3] A. Adamczyk, J.B. Strosznajder, Alpha-synuclein potentiates Ca2+ influx through voltage-dependent Ca2+ channels, Neuroreport 17 (2006) 1883–1886. [4] V. Askanas, W.K. Engel, R.B. Alvarez, J. McFerrin, A. Broccolini, Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions, J. Neuropathol. Exp. Neurol. 59 (2000) 592–598. [5] M.C. Bennett, The role of alpha-synuclein in neurodegenerative diseases, Pharmacol. Ther. 105 (2005) 311–331. [6] D. Boërio, L. Greensmith, H. Bostock, Excitability properties of motor axons in the maturing mouse, J. Peripher. Nerv. Syst. 14 (2009) 45–53. [7] D.E. Cabin, K. Shimazu, D. Murphy, N.B. Cole, W. Gottschalk, K.L. McIlwain, B. Orrison, A. Chen, C.E. Ellis, R. Paylor, B. Lu, R.L. Nussbaum, Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein, J. Neurosci. 22 (2002) 8797–8807. [8] R.J. Carter, L.A. Lione, T. Humby, L. Mangiarini, A. Mahal, G.P. Bates, S.B. Dunnett, A.J. Morton, Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation, J. Neurosci. 19 (1999) 3248–3257. [9] S. Chandra, F. Fornai, H.B. Kwon, U. Yazdani, D. Atasoy, X. Liu, R.E. Hammer, G. Battaglia, D.C. German, P.E. Castillo, T.C. Sudhof, Double-knockout mice for alpha- and beta-synucleins: effect on synaptic functions, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 14966–14971. [10] P.E. Chen, C.G. Specht, R.G. Morris, R. Schoepfer, Spatial learning is unimpaired in mice containing a deletion of the alpha-synuclein locus, Eur. J. Neurosci. 16 (2002) 154–158. [11] J.N. Crawley, What’s Wrong With My Mouse?, 2nd ed., John Wiley & Sons, Inc., New York, 2007. [12] J.M. George, The synucleins, Genome. Biol. 3 (2002), REVIEWS3002. [13] C.M. Gomez, R. Maselli, J.E. Gundeck, M. Chao, J.W. Day, S. Tamamizu, J.A. Lasalde, M. McNamee, R.L. Wollmann, Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction, J. Neurosci. 17 (1997) 4170–4179. [14] I. Gureviciene, K. Gurevicius, H. Tanila, Role of alpha-synuclein in synaptic glutamate release, Neurobiol. Dis. 28 (2007) 83–89. [15] M.E. Kornhuber, C. Bischoff, H. Mentrup, B. Conrad, Multiple A waves in Guillain–Barre syndrome, Muscle Nerve 22 (1999) 394–399.
A. Pelkonen, L. Yavich / Neuroscience Letters 487 (2011) 350–353 [16] L. Maroteaux, J.T. Campanelli, R.H. Scheller, Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal, J. Neurosci. 8 (1988) 2804–2815. [17] V.G. Marrachelli, F.J. Miranda, J.A. Alabadi, M. Milan, M. Cano-Jaimez, M. Kirstein, E. Alborch, I. Farinas, F. Perez-Sanchez, Perivascular nerve fiber alpha-synuclein regulates contractility of mouse aorta: a link to autonomic dysfunction in Parkinson’s disease, Neurochem. Int. 56 (2010) 991–998. [18] F. Mesrati, M.F. Vecchierini, F-waves: neurophysiology and clinical value, Neurophysiol. Clin. 34 (2004) 217–243. [19] K. Papachroni, N. Ninkina, J. Wanless, A.T. Kalofoutis, N.V. Gnuchev, V.L. Buchman, Peripheral sensory neurons survive in the absence of alpha- and gamma-synucleins, J. Mol. Neurosci. 25 (2005) 157–164. [20] A.R. Punga, R. Flink, H. Askmark, E.V. Stalberg, Cholinergic neuromuscular hyperactivity in patients with myasthenia gravis seropositive for MuSK antibody, Muscle Nerve 34 (2006) 111–115. [21] P.J. Russell, S.L. Wolfe, P.E. Hertz, C. Starr, Biology: The Dynamic Science, Thompson Brooks/Cole, Canada, 2008.
353
[22] O.M. Schlüter, F. Fornai, M.G. Alessandri, S. Takamori, M. Geppert, R. Jahn, T.C. Sudhof, Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced parkinsonism in mice, Neuroscience 118 (2003) 985–1002. [23] C.G. Specht, R. Schoepfer, Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice, BMC Neurosci. 2 (2001) 11. [24] C.G. Specht, R. Schoepfer, Deletion of multimerin-1 in alpha-synucleindeficient mice, Genomics 83 (2004) 1176–1178. [25] J.G. van Dijk, G.J. Lammers, A.R. Wintzen, P.C. Molenaar, Repetitive CMAPs: mechanisms of neural and synaptic genesis, Muscle Nerve 19 (1996) 1127–1133. [26] L. Yavich, H. Tanila, S. Vepsäläinen, P. Jäkälä, Role of alpha-synuclein in presynaptic dopamine recruitment, J. Neurosci. 24 (2004) 11165– 11170. [27] L. Yavich, P. Jäkälä, H. Tanila, Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in alpha-synuclein knockout and A30P transgenic mice, J. Neurochem. 99 (2006) 724–732.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuromuscular pathology in mice lacking alpha-synuclein Anssi Pelkonen ∗ , Leonid Yavich School of Pharmacy, University of Eastern Finland, Kuopio, Finland
a r t i c l e
i n f o
Article history: Received 17 August 2010 Received in revised form 19 October 2010 Accepted 19 October 2010 Keywords: Alpha-synuclein Skeletal muscles Neuromuscular junction Electromyography Knockout mice
a b s t r a c t This work was undertaken in order to study the possible role of alpha-synuclein in the function of the neuro-muscular junction in skeletal muscles. Repeated stimulation of skeletal muscle motor neurons revealed signs of neuromuscular pathology in alpha-synuclein null mutated (C57Bl/6JOlaHsd) and knockout (B6;129X1-Sncatm1Rosl /J) mice. This stimulation produced repetitive compound muscle action potentials in both lines of alpha-synuclein deficient mice. Muscle strength and muscle coordination during ambulation were unaffected, though motor learning was slower in alpha-synuclein deficient mice in the Rotarod test. We conclude that alpha-synuclein may play a role in acetylcholine compartmentalization at the neuromuscular junction, and in the fine control of activity of skeletal muscles. © 2010 Elsevier Ireland Ltd. All rights reserved.
Alpha-synuclein (␣-syn) is a 140 amino acid and a 14 kDa protein that is best known for its involvement in serious neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, multiple system atrophy, amyotrophic lateral sclerosis and some other pathological states [5]. Recent experiments have examined the involvement of ␣-syn in the control of compartmentalization and pre-synaptic release of neurotransmitters such as dopamine [2,26], glutamate [14] and norepinephrine [27]. ␣-Syn is expressed not only in the central nervous system but also in muscles [4] and peripheral neurons [17,19], though it is not essential for their survival. Recent results also indicate that ␣-syn may be involved in the fine tuning of acetylcholine (ACh) dependent muscle contractility of smooth muscle cells in mice [17]. So far no clear behavioral pathology has been observed in ␣-syn deficient mice [2,10]. We speculated that if ␣-syn possesses a universal role in the storage of neurotransmitters in the pre-synaptic terminals, then repeated stimulation at a level sufficient to exhaust the pre-synaptic storage pool should reveal its role in the compartmentalization of ACh in the neuromuscular synaptic terminals. Repetitive nerve stimulation is widely used to diagnose neuromuscular disorders [1]. Thus, we tested the functionality of skeletal muscles in ␣-syn deficient mice with repeated stimulation of the motor nerves. In some pathological conditions with impaired sig-
Abbreviations: ␣-syn, alpha-synuclein; ACh, acetylcholine; CMAP, compound muscle action potential; R-CMAP, repetitive compound muscle action potential. ∗ Corresponding author at: School of Pharmacy, University of Eastern Finland, Yliopistonranta 1 C, P.O. Box 1627, FIN-70211 Kuopio, Finland. Tel.: +358 403 553 714. E-mail addresses: anssi.pelkonen@uef.fi (A. Pelkonen), leonid.yavich@uef.fi (L. Yavich). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.10.054
nalling in the neuromuscular junction electrical stimulation may yield compound muscle action potentials (CMAPs) accompanied by late components of relatively high amplitude [13,20,25]. These CMAPs are known as repetitive CMAPs (R-CMAPs). The effect of ␣syn deficiency on muscle strength and coordination was also briefly assessed in this work. Three mouse lines were used in this study. The C57BL/6J subpopulation (Charles River Wiga, Sulzfeld, Germany) expresses ␣-syn (referred to as b6+). The C57BL/6J subpopulation of mice from Harlan Olac (Bicester, UK, referred to as b6−) have a spontaneous chromosomal deletion of the ␣-syn and multimerin-1 loci [23,24]. The B6;129X1-Sncatm1Rosl /J is a gene-targeted ␣-syn knockout line [2]. A breeding pair of these mice (referred to as b6-Ros), were obtained from the Jackson Laboratory, cleaned by embryo transfer and bred together with the other two lines in the National Laboratory Animal Center (Kuopio, Finland). The animals used for electromyography (5 per line) were 10–23 weeks of age and animals used for behavioral experiments (6 per line) were 8–15 weeks of age during the experiments. Male b6+, b6− and b6-Ros mice were group housed at an ambient temperature of 22–23 ◦ C and on a 12/12 h dark/light cycle (lights on 7 a.m.). Animals had food and water available ad libitum. The experiments were conducted with a minimized number of animals according to the Council of Europe (Directive 86/609) and Finnish guidelines. The animals were anesthetized with chloral hydrate (450 mg/kg i.p., 10 ml/kg) and the anesthesia was maintained by injecting one quarter of the initial dose after every 45-60 min. The animals were fixed to a heating pad by their limbs and tail with a tape. The electrodes were 26-gauge stainless steel needles. The registration of the CMAPs in the caudal muscles was undertaken as described previously [6] with modifications. The stimulating cathode was placed against the caudal motor axons by puncturing the skin at the base
A. Pelkonen, L. Yavich / Neuroscience Letters 487 (2011) 350–353
of the tail. The anode was placed 0.5 cm anterior to the cathode. The recording electrode was inserted into the caudal muscles on the lateral side of the tail 1.5 cm posterior to the cathode and a reference electrode was placed similarly 1 cm posterior to the active electrode. A needle implanted under the skin was used as a ground electrode. Sub-maximal electrical stimuli, 2–3 mA in amplitudes (these types of high currents were required to obtain effective stimulation) were produced using an A320 current isolation unit (World Precision Instruments, WPI, Sarasota, FL, USA). Pulse length (0.1 ms) and interval (10–300 ms) were set with Master-8 pulse stimulator (A.M.P.I., Jerusalem, Israel). The signals were amplified and filtered (300 Hz to 3 kHz) with DAM 70 Differential Amplifier (WPI). Signals were recorded to a personal computer using Digidata 1321A digitizer and Clampex 8.2 software (Axon Instruments, Sunnyvale, CA, USA). The sampling rate was set to 10 kHz. Five trains of six pulses each at different between-pulses intervals were applied to the caudal motor axons. The intervals between pulses were 300, 100, 50, 20 and 10 ms. Data was collected over a period of 2 s. Each train of stimulation at specific intervals was repeated three times per animal. There was a 1 min pause after each train. The animals were sacrificed after the measurements and their brains removed for assays in related projects. The motor coordination and balance were tested using an accelerating Rotarod (Ugo Basile, Comerio, Italy). The animals were allowed to become accustomed with the apparatus for 1 min before testing, 30 s on a motionless bar and 30 s on a bar that was rotating at a minimal speed (4 rpm). After this period of adaptation, the apparatus was set to accelerate (from 4 to 40 rpm) and the timing began. The time that the animals were able to run on the rotating bar without falling down from the rod was recorded. The mouse could grab hold on the bar for one or two consecutive turns, but after the third consecutive turn the animal was removed from the rod. The maximum time the animals were allowed to remain on the rod was 480 s (8 min). The test was repeated three times with two days between the experiments. We also assessed the gait of the mice with a standard footprint pattern test [8,11]. Muscle strength was evaluated with an adapted front paw grip strength test in which the animals grabbed a normal home cage roof (parallel round metal bars with 2 mm in diameter and 7 mm from each other) with their forepaws when they were lifted upwards at a steady speed while attached to a calibrated spring. The measured parameter was the maximal force against which the animals were able to hold onto the bars. The result of each animal was the average of three attempts repeated after every 30 s. The electromyography results were analyzed off-line with Clampfit, version 8.2 (Axon Instruments). CMAP amplitudes were calculated from the peak of the positive phase to the peak of the negative phase. When R-CMAPs were evaluated, waveforms with amplitude less than 5% of the primary CMAP amplitude were not analyzed to exclude possible A- and F-waves [15,18]. One-way ANOVA and ANOVA for repeated measures were used for statistical analysis of the results (SPSS 14.0 for Windows, SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard error of mean (S.E.M.). There were no differences between the lines in the absolute values of amplitudes of CMAPs calculated from the first pulse in the trains with 300 ms pulse intervals (3.0 ± 1.0 mV in b6+, 3.2 ± 1.7 mV in b6− and 2.6 ± 0.8 mV in b6-Ros mice). No ␣-syn dependent statistically significant differences were seen in the amplitudes of CMAPs in response to repetitive stimulation (data not shown). We detected repetitive CMAPs (R-CMAPs) in the electromyographic recordings in 4 out of 5 b6− mice and 3 out of 5 b6-Ros mice (Fig. 1), while only 1 out of 5 b6+ mice displayed R-CMAPs. Typically the R-CMAPs were detected in ␣-syn deficient mice after the first pulse in trains of 300 ms intervals (39.5% of all R-CMAPs).
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Fig. 1. Occurrence of R-CMAPs. (A) Example of a train with 300 ms intervals between pulses from a b6-Ros mouse showing R-CMAP after the first pulse. The insert shows the artifact of stimulation (the first small peak), the primary CMAP and three late components. (B) Number of detected R-CMAPs with different amounts of late components.
According to ANOVA for repeated measures, the lines demonstrated different behavior on the Rotarod (the effect of genotype: F (2, 15) = 10.5, p = 0.020). According to Bonferroni’s post hoc test, the time on the rod of b6-Ros mice differed significantly from that of b6− and b6+ mice (p = 0.004 and 0.003, respectively) but there was no significant difference between b6− and b6+ mice (p = 1.000). Both ␣-syn deficient lines showed deteriorated performance in the Rotarod test on the first day of testing (Fig. 2A). Training improved these results so that ␣-syn deficient mice were indistinguishable from the b6+ mice by the third day of testing. ANOVA for repeated measures revealed significant within-subject effects in training × genotype interactions (F (4, 30) = 3.2, p = 0.026). b6+ and b6− mice were able to stay on the non-slippery rod of the Rotarod apparatus during a full turn but in contrast to this behavior, b6-Ros mice could not cling on to the rod and dropped off. Clinging on to the bar was seen in 83% of all trials with b6+ mice, 89% with b6− mice but only in 6% with b6-Ros mice. We initially attributed the inability of b6-Ros mice to cling on the bar to a muscular deficiency. However, according to one way ANOVA, the differences between lines in the front paw grip strength test were not significant (Fig. 2B, F (2, 15) = 1.0, p = 0.396). The results of the footprint pattern test were similar to those reported in normal healthy mice [8], but no differences were seen between the lines (Table 1). Two lines of evidence point to neuromuscular pathology in ␣syn deficient mice. The first is the appearance of R-CMAPs following repetitive stimulation, and the second is the result of the Rotarod test. R-CMAPs are considered as a reliable indicator of neuromuscular pathology in clinical neurophysiology [25]. The deteriorated performance found during the first test on the Rotarod is a less conclusive indicator of muscular insufficiency but at present it is the only verified behavioral manifestation of the deletion of ␣-syn,
Fig. 2. Assessment of motor functions. (A) Performance of the alpha-synuclein deficient (b6− and b6-Ros) and wild-type mice (b6+) in the Rotarod test. The test was repeated three times every third day. (B) Front paw grip strength (N) relative to body weight (kg) in b6+, b6− and b6-Ros mice. n = 6.
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Table 1 Results of the footprint pattern test. b6+ Hindpaw stride length Forepaw stride length Hindpaw base Forepaw base Forepaw/hindpaw overlap
5.9 6.2 2.5 1.6 0.7
b6− ± ± ± ± ±
0.3 0.4 0.1 0.1 0.1
6.1 6.4 2.6 1.3 0.8
± ± ± ± ±
b6-Ros 0.4 0.4 0.1 0.1 0.1
5.5 5.8 2.3 1.6 0.7
± ± ± ± ±
0.3 0.3 0.1 0.0 0.1
F (2, 15)
p
1.3 3.1 2.3 0.6 0.5
0.304 0.076 0.132 0.542 0.618
The results are presented in centimetres. The two columns on the right are the results of one-way ANOVA.
because neither muscle strength nor gait were significantly affected in these lines. R-CMAPs occurred almost exclusively in the ␣-syn deficient mice but not in the b6+ animals. The test on the Rotarod also detected differences between the ␣-syn deficient mouse lines, with the worst behavioral outcome in b6-Ros mice and better performance in b6− animals. It is interesting that another line of ␣-syn knockout mice described in the literature [9] demonstrated much milder signs of synaptic pathology in an indirect comparison with b6-Ros line, which may indicate that some other downstream alteration in protein expression had occurred in b6-Ros mice. This line of knockout mice differs from the line with spontaneous deletion of ␣-syn in their sensitivity to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, indicative that the genetic background is a critical factor in expression of the effects of the deletion [22]. A similar conclusion can be drawn from the present comparison between b6− and b6-Ros mice. ␣-syn belongs to a three-member protein family of synucleins (alpha, beta and gamma), which was first found from the cholinergic nerve terminals of the Torpedo californica electric organ [16], and it appears to exist only in vertebrates [12]. There are fundamental differences between vertebrates and invertebrates in the innervations of their muscles [21]. The evolutionary co-occurrence of the appearance of synucleins in vertebrates and the changes occurring in the innervations of their skeletal muscles makes it tempting to speculate that this family of proteins may play a role in fine neuromuscular control. If one of the postulated functions of ␣-syn in the pre-synaptic terminal is correct compartmentalization of neurotransmitter [2,14,26,27], and this function is universal for any type of neuron, then a lack of the protein may disrupt the formation of the storage and readily releasable pools of ACh. R-CMAPs were detected in both lines of ␣-syn deficient animals. It has been proposed that R-CMAPs are caused by a prolongation of the duration of the endplate potential [25]. The most common reason for this prolongation is excessive ACh release which is in line with data on increased neurotransmitter release in response to burst stimulation obtained in the synapses of central nervous system in ␣-syn deficient mice [2,26,27]. However, other factors cannot be excluded, e.g. acetylcholinesterase inhibition or repeated excitation of the axon in response to a single shock which is known in several neuromuscular disorders with excess motor unit activity [25]. It is unclear whether the suggested role of ␣-syn in muscle development could in some way give rise to the R-CMAPs [4]. The role of ␣-syn in Ca2+ channel function is an unlikely source for the R-CMAPs since ␣-syn activates Ca2+ influx and thus the lack of the protein should diminish the probability of R-CMAPs [3]. The initial performance of ␣-syn deficient mice in the Rotarod test was worse than the performance of the wild-type animals. The significant improvement in performance in consecutive tests and the lack of significant differences between the lines in the footprint pattern and grip strength suggests that the initial differences did not result from weaker muscle strength. It is possible that the ␣-syn deficiency which causes abnormalities in the signaling of the neuromuscular junction makes motor control more difficult for these mice. This could be reflected as slower motor learning. It is also possible that the problems in motor learning arise from the cen-
tral effects of ␣-syn depletion. Earlier experiments with knockout mice detected no changes in motor performance in the Rotarod test [7]. However, in the present study we used a different protocol. Typically normal healthy mice should improve their results in the Rotarod as the test is repeated, but with the current protocol, the wild type mice were able to reach their maximal level of performance already on the first trial. We conclude that ␣-syn may play a role in ACh compartmentalization in the neuromuscular junction, and in the fine control of activity of skeletal muscles. Acknowledgments Authors thank Dr. Ewen MacDonald for revising the language of the manuscript and Saara Stavén for advice concerning the Rotarod test. This work was supported by the Academy of Finland grants 122397 and 125653 to L.Y. References [1] AAEM Quality Assurance Committee, American Association of Electrodiagnostic Medicine, Literature review of the usefulness of repetitive nerve stimulation and single fiber EMG in the electrodiagnostic evaluation of patients with suspected myasthenia gravis or Lambert–Eaton myasthenic syndrome, Muscle Nerve 24 (2001) 1239–1247. [2] A. Abeliovich, Y. Schmitz, I. Farinas, D. Choi-Lundberg, W.H. Ho, P.E. Castillo, N. Shinsky, J.M. Verdugo, M. Armanini, A. Ryan, M. Hynes, H. Phillips, D. Sulzer, A. Rosenthal, Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system, Neuron 25 (2000) 239–252. [3] A. Adamczyk, J.B. Strosznajder, Alpha-synuclein potentiates Ca2+ influx through voltage-dependent Ca2+ channels, Neuroreport 17 (2006) 1883–1886. [4] V. Askanas, W.K. Engel, R.B. Alvarez, J. McFerrin, A. Broccolini, Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions, J. Neuropathol. Exp. Neurol. 59 (2000) 592–598. [5] M.C. Bennett, The role of alpha-synuclein in neurodegenerative diseases, Pharmacol. Ther. 105 (2005) 311–331. [6] D. Boërio, L. Greensmith, H. Bostock, Excitability properties of motor axons in the maturing mouse, J. Peripher. Nerv. Syst. 14 (2009) 45–53. [7] D.E. Cabin, K. Shimazu, D. Murphy, N.B. Cole, W. Gottschalk, K.L. McIlwain, B. Orrison, A. Chen, C.E. Ellis, R. Paylor, B. Lu, R.L. Nussbaum, Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein, J. Neurosci. 22 (2002) 8797–8807. [8] R.J. Carter, L.A. Lione, T. Humby, L. Mangiarini, A. Mahal, G.P. Bates, S.B. Dunnett, A.J. Morton, Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation, J. Neurosci. 19 (1999) 3248–3257. [9] S. Chandra, F. Fornai, H.B. Kwon, U. Yazdani, D. Atasoy, X. Liu, R.E. Hammer, G. Battaglia, D.C. German, P.E. Castillo, T.C. Sudhof, Double-knockout mice for alpha- and beta-synucleins: effect on synaptic functions, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 14966–14971. [10] P.E. Chen, C.G. Specht, R.G. Morris, R. Schoepfer, Spatial learning is unimpaired in mice containing a deletion of the alpha-synuclein locus, Eur. J. Neurosci. 16 (2002) 154–158. [11] J.N. Crawley, What’s Wrong With My Mouse?, 2nd ed., John Wiley & Sons, Inc., New York, 2007. [12] J.M. George, The synucleins, Genome. Biol. 3 (2002), REVIEWS3002. [13] C.M. Gomez, R. Maselli, J.E. Gundeck, M. Chao, J.W. Day, S. Tamamizu, J.A. Lasalde, M. McNamee, R.L. Wollmann, Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction, J. Neurosci. 17 (1997) 4170–4179. [14] I. Gureviciene, K. Gurevicius, H. Tanila, Role of alpha-synuclein in synaptic glutamate release, Neurobiol. Dis. 28 (2007) 83–89. [15] M.E. Kornhuber, C. Bischoff, H. Mentrup, B. Conrad, Multiple A waves in Guillain–Barre syndrome, Muscle Nerve 22 (1999) 394–399.
A. Pelkonen, L. Yavich / Neuroscience Letters 487 (2011) 350–353 [16] L. Maroteaux, J.T. Campanelli, R.H. Scheller, Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal, J. Neurosci. 8 (1988) 2804–2815. [17] V.G. Marrachelli, F.J. Miranda, J.A. Alabadi, M. Milan, M. Cano-Jaimez, M. Kirstein, E. Alborch, I. Farinas, F. Perez-Sanchez, Perivascular nerve fiber alpha-synuclein regulates contractility of mouse aorta: a link to autonomic dysfunction in Parkinson’s disease, Neurochem. Int. 56 (2010) 991–998. [18] F. Mesrati, M.F. Vecchierini, F-waves: neurophysiology and clinical value, Neurophysiol. Clin. 34 (2004) 217–243. [19] K. Papachroni, N. Ninkina, J. Wanless, A.T. Kalofoutis, N.V. Gnuchev, V.L. Buchman, Peripheral sensory neurons survive in the absence of alpha- and gamma-synucleins, J. Mol. Neurosci. 25 (2005) 157–164. [20] A.R. Punga, R. Flink, H. Askmark, E.V. Stalberg, Cholinergic neuromuscular hyperactivity in patients with myasthenia gravis seropositive for MuSK antibody, Muscle Nerve 34 (2006) 111–115. [21] P.J. Russell, S.L. Wolfe, P.E. Hertz, C. Starr, Biology: The Dynamic Science, Thompson Brooks/Cole, Canada, 2008.
353
[22] O.M. Schlüter, F. Fornai, M.G. Alessandri, S. Takamori, M. Geppert, R. Jahn, T.C. Sudhof, Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced parkinsonism in mice, Neuroscience 118 (2003) 985–1002. [23] C.G. Specht, R. Schoepfer, Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice, BMC Neurosci. 2 (2001) 11. [24] C.G. Specht, R. Schoepfer, Deletion of multimerin-1 in alpha-synucleindeficient mice, Genomics 83 (2004) 1176–1178. [25] J.G. van Dijk, G.J. Lammers, A.R. Wintzen, P.C. Molenaar, Repetitive CMAPs: mechanisms of neural and synaptic genesis, Muscle Nerve 19 (1996) 1127–1133. [26] L. Yavich, H. Tanila, S. Vepsäläinen, P. Jäkälä, Role of alpha-synuclein in presynaptic dopamine recruitment, J. Neurosci. 24 (2004) 11165– 11170. [27] L. Yavich, P. Jäkälä, H. Tanila, Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in alpha-synuclein knockout and A30P transgenic mice, J. Neurochem. 99 (2006) 724–732.