- Email: [email protected]
dy2J dystrophic mouse
Brain Research 788 Ž1998. 262–268
Research report
Electrophysiology of the neuromuscular junction of the laminin-2 Ž merosin. deficient C57 BLr6J dy 2 J r dy 2 J dystrophic mouse Jonathan P. Edwards, Paul A. Hatton, Anthony C. Wareham
)
DiÕision of Neuroscience, School of Biological Sciences, 1.124 Stopford Building, UniÕersity of Manchester, Oxford Road, Manchester M13 9PT, UK Accepted 23 December 1997
Abstract The C57 BLr6J dy 2 J r dy 2 J dystrophic mouse expresses an abnormal truncated form of the a 2 subunit of the protein laminin-2 Žor merosin., which is unable to form a stable link between the extracellular matrix and the dystrophin-associated proteins, resulting in muscular dystrophy. Morphological abnormalities of the peripheral nervous system and neuromuscular junction have also been reported. The electrophysiological properties of the neuromuscular junctions of diaphragm, extensor digitorum longus ŽEDL., and soleus from C57 BLr6J dy 2 J r dy 2 J mice and controls are described. No evidence for the presence of denervated fibres were found. Mean MEPP amplitudes were significantly increased in EDL and soleus but reduced in the diaphragm from affected mice. Mean MEPP frequencies were raised in all the dy 2 J r dy 2 J muscles studied. dy 2 J r dy 2 J muscles were paralysed by low concentrations of m-conotoxin suggesting that embryonic Žtetrodotoxin and m-conotoxin resistant. sodium channels are not widespread on dy 2 J r dy 2 J muscle as has previously been reported. EPP latencies were significantly prolonged in the diaphragm and EDL but not soleus from dy 2 J r dy 2 J mice. Quantal contents were higher in all dy 2 J r dy 2 J muscles. In the dy 2 J r dy 2 J diaphragm failures in neurotransmission occurred and a faster rate of rundown of EPPs was apparent. Some changes appear from a direct effect of dystrophy, whilst increased MEPP frequency and quantal content, and failures in neurotransmission indicate neuronal abnormalities. q 1998 Elsevier Science B.V. Keywords: dy 2 J r dy 2 J ; Laminin-2; Muscular dystrophy; Neuromuscular transmission; Neuromuscular junction
1. Introduction Laminin is an important structural component of the basal lamina. It is made up of three subunits a , b and g w31x, differing forms of these subunits being expressed in different cell types which may also vary within a cell type during development. In muscle the a subunit of laminin binds to a-dystroglycan of the dystroglycan complex thereby forming a link between the basal lamina and the actin filament of the sarcolemma Žfor reviews see w6,31x.. Laminin-2 Žmade up of a 2 b 1g 1 subunits. is the predominant form of laminin in adult striated muscle. It has recently been demonstrated that deficiencies in laminin-2 can cause muscular dystrophy in humans and Abbreviations: miniature endplate digitorum longus; Tetrodotoxin ) Corresponding
RMP s resting membrane potential; MEPP s potential; EPPsendplate potential; EDL sextensor CMD s congenital muscular dystrophy; TTX s author. Fax: q44-161-275-5363.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 1 0 - 9
mice. Patients with Fukyama congenital muscular dystrophy, the most common form of congenital muscular dystrophy ŽCMD. in Japan, have been found to have muscles and peripheral nerves deficient in laminin-2 w14x. There have recently been reports that some patients with other forms of CMD exhibit deficiencies in laminin-2 w32,36x. Previously described strains of dystrophic mice are now considered to be animal models of laminin-2 deficiency. The ReJ dy r dy dystrophic mouse, originally described by Michelson et al. in 1955 w27x has been found to lack laminin-2 by a number of labs w35,37x. The C57 BLr6J dy 2 J r dy 2 J dystrophic mouse, originally described by Meir and Southard in 1970 w26x, has recently been shown to express an abnormal truncated form of the a 2 subunit of laminin-2 w38x. This truncated form of a 2 subunit is believed to lack many of the qualities of the wild-type protein, most importantly the ability to form a stable link between the extracellular matrix and the dystrophin associated proteins required to maintain the structural integrity of the muscle.
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
The dy 2 J r dy 2 J mutant mouse has a normal life span and displays a less severe dystrophy than the REJ 129 dy r dy mouse, which is more severely affected and dies at around 3–6 months of unknown causes w7,38x. dy 2 J r dy 2 J mice develop a progressive weakness of the hindlimbs and a histological picture of muscle degeneration and regeneration observable from about 3 weeks of age w5x. Agbenyega et al. w1x using the Manchester colony of mice have shown that the soleus and EDL of affected dy 2 J r dy 2 J mice contain an increased percentage of small and large fibres with a high proportion of spherical fibres and a lot of connective tissue infiltration between the fibres. The distribution of the dystrophic changes was very different in soleus and EDL; in soleus dystrophic fibres were found throughout the muscle whereas in EDL only certain discreet areas were affected. Morphological abnormalities of peripheral nerve w3,34x and neuromuscular junctions w9x have also been described in dy 2 J r dy 2 J mice. Reports concerning neuromuscular structure and transmission in the ReJ dy r dy mice have frequently provided conflicting information Žfor review see Ref. w13x.. A preliminary report of the mechanical and electrical properties of dystrophic muscle in the C57 BLr6J dy 2 J r dy 2 J mouse was provided by Harris and Montgomery w12x followed by an account of spontaneous transmitter release in mice from the Manchester colony w33x. No detailed analysis of neurally evoked neuromuscular transmission in these mice has been done, however, despite the description of ultrastructural abnormalities of the neuromuscular junction. Noakes et al. w30x have recently shown that b 2-containing laminins Žor s-laminins. play an important role in the regulation of synaptic differentiation in the mouse. We, therefore, have investigated the properties of the neuromuscular junction in skeletal muscle of C57 BLr6J dy 2 J r dy 2 J mice to determine the effect of laminin-2 deficiency on neuromuscular function and differentiation.
2. Materials and methods Eighteen 6- to 8-week-old affected dy 2 J r dy 2 J mice and 19 age-matched unaffected Ž DY r dy or DY r DY . littermate controls were obtained from the Biological Service Unit of Manchester University. The colony was originally obtained from the MRC Laboratory Animal Centre in Carshalton, Surrey. Mice were killed by cervical dislocation. Recordings from muscles were carried out in vitro at room temperature using standard intracellular techniques. Resting membrane potentials ŽRMP., miniature endplate potentials ŽMEPPs., and endplate potentials ŽEPPs. were recorded from the diaphragm, soleus, and extensor digitorum longus ŽEDL. muscles. Intracellular micro electrodes Ž10–15 M V resistance. were connected to a Neurolog NL102 preamplifier ŽDigitimer Research Instruments.. The resultant signals were digitised at 20 kHz using a CED 1401 ŽCambridge Electronic Design. and captured in event
263
detector mode Žwith the trigger set at 0.1 mV. using Whole Cell Electrophysiology Program software supplied by Dr. J. Dempster of Strathclyde University. This software was also used for the subsequent analysis of MEPPs and EPPs. The bathing media contained ŽmM. 135 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 15 NaHCO 3 , 1 Na 2 HPO4 , 11 glucose, pH 7.2 and was bubbled with 95% O 2r5% CO 2 . To facilitate the recording of EPPs 2–4 m grml of m-conotoxin w15x ŽSigma Chemical, Poole, Dorset, UK. was added to the bathing media and indirect stimulation at 2 Hz was applied. After fibre impalement with a microelectrode the RMP was allowed to stabilise for 5 min before recording was started. During recording most fibres depolarised by 0–2 mV. Any fibre whose RMP depolarized by more than 10 mV after impalement was not recorded from. The analysis of MEPP amplitudes was further restricted to fibres where the mean MEPP rise times were less than 1 ms to ensure the recordings were focal to the endplate region. Similarly, the analysis of EPPs was restricted to fibres where the mean EPP rise time Žmeasured using Whole Cell Electrophysiological program. was less than 1.5 ms. Quantal contents of EPPs were calculated using the direct method and corrected for nonlinear summation w22,23x using the empirical constant of 0.8 suggested by McLachlan and Martin w24x for mouse muscle. The mean amplitude of approximately 40 MEPPs and EPPs was used for each calculation of quantal content in each fibre. In a further set of experiments a range of stimulation frequencies were applied to the diaphragm to monitor the effects of ‘rundown’ at control and dystrophic neuromuscular junctions. Indirect stimulation at 2, 10, 20, 40 and 60 Hz was applied for 30 s to the same muscle fibre, with a 2-min rest between each stimulation frequency and 10 min between each fibre. The resultant EPPs were recorded on a Gould 2200 pen recorder ŽGould Electronics.. At 0 s, 10 s, 20 s, and 30 s after the start of the stimulation, five EPPs were measured and expressed as a % of the amplitude of the EPPs at time 0 Žstandardised to 100%.. Muscles were also stained for endplate cholinesterase activity using the method of McIsaac and Kiernan w25x. 2.1. Statistical analysis The results are expressed as means " S.E.M. All data sets were initially tested for normality using the Kolmogorov–Smirnov goodness-of-fit test. Data for the EPP latency in soleus and EDL and the quantal content data from all muscles were found to be normally distributed. All remaining data were found to be non-normally distributed. Student’s t-test was used to compare the means of normally distributed data sets, whereas non-normally distributed data were compared using the Mann–Whitney U-test. Repeated-measure multiple-factorial ANOVA was employed to analyse the data produced in the ‘rundown’ experiments.
264
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
3. Results 3.1. Clinical and morphological obserÕations It was possible to differentiate between affected dy 2 J r dy mice and unaffected Ž DY r dy or DY r DY . littermate 2J
controls at approximately three weeks of age. Affected mice walked with a wobbling gait and were unable to grasp with the toes of their hind paws when placed on the wire lid of their cages. The hind limbs were more severely affected than the fore limbs. The distribution of endplates
Fig. 1. Amplitude distributions of spontaneous MEPPS recorded in Ža. control diaphragm, Žb. dystrophic diaphragm, Žc. control EDL, Žd. dystrophic EDL, Že. control soleus, and Žf. dystrophic soleus. Each histogram represents the results pooled from five muscles, from more than 170 fibres, where N f 2000.
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
265
Table 1 The passive properties and spontaneous transmitter release recorded in control and dy 2 J r dy 2 J muscle MMuscle
RMP ŽmV. Ž n s 200, 5. MEPP rise time Žms. Ž n ) 2000, 5. MEPP amplitude ŽmV. Ž n ) 2000, 5. MEPP frequency ŽHz. Ž n ) 150, 5.
Diaphragm Control 69.5 " 0.68 dy 2 J r dy 2 J 64.5 " 0.58 p - 0.0001
0.9 " 0.006 0.81 " 0.01 p - 0.0001
1.32 " 0.01 1.27 " 0.07 p - 0.0001
0.58 " 0.03 0.76 " 0.03 p - 0.0001
Soleus Control 67.2 " 0.58 dy 2 J r dy 2 J 69.8 " 0.63 p - 0.01
0.85 " 0.005 0.87 " 0.02 p - 0.0001
0.92 " 0.006 1.31 " 0.01 p - 0.0001
0.95 " 0.04 1.35 " 0.05 p - 0.0001
EDL Control 76.1 " 0.73 dy 2 J r dy 2 J 71.1 " 0.66 p - 0.01
0.78 " 0.006 0.89 " 0.007 p - 0.0001
0.97 " 0.008 1.10 " 0.01 p - 0.0001
1.16 " 0.06 1.17 " 0.07 NS
The values are means" S.E.M. The figures in parentheses are the number of observations on the left Ž n for the statistical analysis. and number of animals on the right. The results obtained from the dy 2 J r dy 2 J muscles were compared with their respective controls using the Mann–Whitney U-test, the significance of any differences between the means are shown, NS s not significant.
in dystrophic muscles appeared to be more diffuse than those from the controls. This was observed with both the electrophysiological recordings and the cholinesterase staining although no attempt was made to quantify this observation. 3.2. PassiÕe electrophysiological properties It can been seen from Table 1 that the RMP in dy 2 J r dy diaphragm and EDL was significantly depolarised compared to the controls whereas in the soleus the RMP was significantly hyperpolarised in the dy 2 J r dy 2 J muscle compared to the controls. The value of the RMP in the control soleus was significantly smaller than that of the control EDL Ž p - 0.0001. a finding previously noted to occur by Harris w11x. The mean RMP of the control diaphragm was slightly smaller than that reported by Hong and Chang w15x, but higher than that reported by Nagel et al. w29x for the murine diaphragm. 2J
3.3. Spontaneous transmitter release MEPPs were recorded from over 90% of fibres impaled in both the dy 2 J r dy 2 J and control muscles. Fibrillation potentials were not seen in any muscles, indicating that fibres were not denervated. Mean MEPP amplitudes were significantly greater in the dy 2 J r dy 2 J EDL and soleus. In the soleus the mean MEPP amplitude in the dy 2 J r dy 2 J muscle was over 40% greater than the controls. The MEPP amplitude distribution showed a greater amount of skewing to the right in the dy 2 J r dy 2 J soleus than in the control soleus Žthe value of skewness for the control soleus data being 1.27 " 0.05 compared to 1.33 " 0.04 in the dy 2 J r dy 2 J muscles.. Similarly, the EDL data were also more skewed to the right in the dy 2 J r dy 2 J muscles Ž2.81 " 0.05 vs. 2.11 " 0.04. than in the controls. In the dy 2 J r dy 2 J
diaphragm, however, the mean MEPP amplitudes was significantly lower than in the controls. The MEPP amplitude histograms were also more skewed to the right in the control diaphragms Ž1.38 " 0.03. than the dy 2 J r dy 2 J diaphragms Ž0.64 " 0.04. in contrast to the soleus and EDL. The MEPP amplitude distributions for all muscle types are shown in Fig. 1. The MEPP amplitudes recorded in the control diaphragm are in agreement to those reported previously w15,29x. Likewise the MEPP amplitudes recorded in control soleus and EDL are similar to those recorded by Edwards et al. w8x. The mean MEPP rise times were also increased in the dy 2 J r dy 2 J soleus and EDL but not in the diaphragm when compared to the controls. Table 2 Transmitter release in dy 2 J r dy 2 J muscle Muscle
EPP latency Žms. Ž n)90, 5. Quantal content Ž n) 75, 5.
Diaphragm Control 1.75"0.03 dy 2 J r dy 2 J 2.27"0.06 p- 0.0001
15.83"0.71 17.31"0.74 NS
Soleus Control 2.01"0.04 dy 2 J r dy 2 J 1.93"0.05 NS
19.71"0.89 30.48"1.21 p- 0.0001
EDL Control 1.54"0.03 dy 2 J r dy 2 J 1.63"0.03 p- 0.05
19.74"1.00 20.21"1.02 NS
The values are means"S.E.M. The figures in parentheses are the number of observations on the left Ž n for the statistical analysis. and number of animals on the right. The results obtained from the dy 2 J r dy 2 J muscles were compared with their respective controls using Student’s t-test for normally distributed data and the Mann–Whitney U-test for non-normally distributed data, the significance of any differences between the means are shown, NSs not significant.
266
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
Higher MEPP frequencies were recorded from the diaphragm, soleus and EDL of dy 2 J r dy 2 J mice, although only in the soleus did this increase reach statistical significance. In the diaphragm and soleus the mean MEPP frequencies in the dy 2 J r dy 2 J were over 30% higher than their respective controls. 3.4. EÕoked transmitter release EPPs were recorded from over 90% of both control and dy 2 J r dy 2 J muscles fibres impaled, confirming that few, if any, fibres were denervated ŽTable 2.. EPP latencies were found to be significantly prolonged in the dy 2 J r dy 2 J diaphragm and EDL. In soleus, however, there was no significant difference in EPP latency between dy 2 J r dy 2 J and controls. The mean EPP latency in the control soleus and EDL were very similar to those previously published w8x. The length of nerve aspirated into the suction electrode was of approximately the same in both dystrophic and control preparations to prevent any apparent increases in latency due to using a longer length of nerve. In soleus preparations the length of nerve used was always far shorter than that of the EDL and diaphragm. Quantal contents of EPPs were very significantly increased Žby 55%. in the dy 2 J r dy 2 J soleus. In the diaphragm and EDL the quantal contents appeared to be slightly higher in the dy 2 J r dy 2 J muscles, but these differences were not statistically significant. The quantal contents recorded in the control diaphragm are in close agreement with those calculated by the direct method in the mouse diaphragm paralysed using 2–4 m grml m-conotoxin w15x.
Fig. 2. Rundown in diaphragm EPPs evoked by indirect stimulation at 2 Žsquare., 10 Žtriangle., 20 Žhexagon., 40 Ždiamond., and 60 Žstar. Hz. The results for the control fibres are shown by a complete line and closed symbols whereas the dystrophics have a dashed line and open symbols. Muscle contractions were blocked using m-conotoxin 2–4 m grml. The results are the means"S.E.M. of 18 fibres from four control preparations and 14 fibres from three dystrophic muscles. Each point represents the EPP amplitude expressed as a percentage of the EPP amplitude at time 0 Žstandardised to 100%..
Neuromuscular rundown is shown in Fig. 2. Statistical analysis accounted for three independent variables: group Žnormal vs. dystrophic., stimulation frequency and time. Overall the effect of increasing stimulation frequency, the effect of time and the interaction between the two Ži.e., increasing frequency over time. were highly significant Ž p - 0.001.. The overall effect of dystrophy was not significant, highlighting the fact that there is very little separation between the datasets as a whole ŽFig. 2.. However, the three-way interaction between group, stimulation frequency and time was significant Ž p s 0.01., indicating differences between controls and dystrophics in the time course of the response to increasing frequency Ži.e., the effect of increasing frequency over time was different between controls and dystrophics.. For example, it can be seen that at the lower stimulation frequencies Ž2, 10 and 20 Hz. the dy 2 J r dy 2 J neuromuscular junctions showed a slightly increased rate of ‘rundown’ to the controls. Also, at the higher stimulation frequencies Ž40 and 60 Hz., the dy 2 J r dy 2 J neuromuscular junctions showed a much faster initial rate of ‘rundown’ than was seen in the controls, although by 30-s rundown was similar. Failures in neurotransmission were also seen, prior to complete ‘rundown’ in 4 out of 14 dy 2 J r dy 2 J muscle fibres at stimulation frequencies above 20 Hz a feature never seen in any of the 18 control muscle fibres sampled.
4. Discussion The clinical observations of the affected dy 2 J r dy 2 J mice concur with previous descriptions of this form of murine dystrophy w12,26,33x. The smaller values for RMP found in the dy 2 J r dy 2 J diaphragm and EDL are in agreement with previous work on the mdx mouse w20,29x, the REJ 129 dy r dy mouse w13x, and the C57 BLr6J dy 2 J r dy 2 J mouse w12,33x. The reasons for the increased RMP in dy 2 J r dy 2 J soleus compared to the control are unclear, particularly as the soleus is histologically more severely affected w5x. It is possible that small regenerating fibres were more frequently impaled in soleus, which shows a more generalised distribution of affected fibres than the EDL w1x. The smaller RMP in dystrophic muscle is thought to be caused by changes in the sarcolemmal permeability to sodium or potassium ions brought about by membrane defects w29x. The larger MEPP amplitudes in dy 2 J r dy 2 J soleus and EDL probably reflect a higher fibre input resistance of affected muscle fibres. Dystrophic murine muscle fibres have previously been shown to have a higher mean input resistance w13,21x. The MEPP amplitude distribution for the dy 2 J r dy 2 J diaphragm shows a very different pattern from the control diaphragm indicating MEPP amplitudes are more variable in the dy 2 J r dy 2 J diaphragm. It should be noted, however, that pooling of data from five muscles could have contributed to an apparent
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
skew distribution. Smaller MEPP amplitudes have also been recorded the diaphragm of the mdx mouse w29x which might suggest that this is a characteristic of murine dystrophy. Developing muscle fibres and reinnervated fibres have a large proportion of small-skewed MEPPs w28x and it would have been interesting to determine if these were present in the dy 2 J r dy 2 J diaphragm. However, since an anticholinesterase was not used in the present experiments and our sampling method excluded events - 0.1 mV, the small-skewed MEPPs described in normal mouse muscle by Kriebel et al. w17x would not have been detected. The MEPP amplitudes quoted in this paper have not been corrected to a predetermined RMP. If the values for the dy 2 J r dy 2 J fibres are corrected w16x to the RMP of their respective controls, the value for the dy 2 J r dy 2 J diaphragm increases to 1.37 mV Žnow significantly larger than the control., whereas for the EDL the corrected value is 1.17 mV, and the soleus value is 1.25 mV Žboth still significantly larger than their controls.. It would appear from the results that dystrophic muscle fibres are found in all three of the muscles examined Žthe slow twitch, oxidative soleus, fast twitch, glycolytic, EDL and fast, oxidative, diaphragm. in 6- to 8-week-old mice and not just in the slow twitch, oxidative muscles, such as the soleus, as reported to occur early in the condition w5x. Agbenyega et al. w1x have recently reported that in this particular colony of mice dystrophic changes are seen in both the EDL and SOL of 12-week-old mice. Increased MEPP frequencies, compared with the controls, have previously been recorded from dy 2 J r dy 2 J soleus and EDL w33x. High MEPP frequencies, and increased quantal content are indicative of presynaptic abnormalities which could be the functional correlates of morphological changes. Reduced numbers of synaptic vesicles and retraction of the axolemma away from the postsynaptic specialisations of the sarcolemma have been observed w9x. It seems unlikely that a reduction in synaptic vesicles and retraction of the axolemma would lead to an increase in MEPP frequency and quantal content, however. The fact that the largest increases in MEPP frequency and quantal content are in the soleus which is histologically the most severely affected muscle suggests these changes are directly related to the dystrophy in the muscle. Increases in mean MEPP frequency and quantal content could also be caused by the sampling of the large neuromuscular junctions of hypertrophied fibres, which is more likely to occur in SOL which has a more generalised distribution of spherical hypertrophied fibres than the EDL w1x. It has previously been demonstrated that MEPP frequency and quantal content increase with endplate size w17,18x and endplates increase in size as the muscle fibre grows w2x. Sampling of the large endplates of hypertrophied fibres would increase the mean MEPP frequency and quantal content. We were surprised that the dy 2 J r dy 2 J muscle were as susceptible to m-conotoxin as the controls and could be
267
completely paralysed by a concentrations as low as 2 m grml Žthe same concentrations as controls.. Previous work had shown that distal hind limb muscles of C57 BLr6J dy 2 J r dy 2 J mice show tetrodotoxin ŽTTX. resistant action potentials w12x indicative of the widespread distribution of the embryonic form of sodium channel along the sarcolemma. It has been demonstrated that mconotoxin ŽGeographutoxin II. distinguishes more clearly between TTX-sensitive Žadult. and TTX-insensitive Žembryonic. sodium channels than TTX itself and may have no action at all at TTX-insensitive sodium channels w10x. We therefore expected that the dystrophic muscle would be difficult to paralyse with m-conotoxin, as we find in developing rat muscle Žunpublished observations., where embryonic sodium channels predominate w20x. This finding suggests that the TTX-insensitive sodium channels in dystrophic muscle differ from those found in the developing rat muscle. The increased latency of the EPP seen in the dy 2 J r dy 2 J EDL and diaphragm and the increased rate of rundown and incidence of failures in evoked neurotransmission at the dystrophic diaphragm are indicative of neuronal abnormalities. Extensive widening of the nodes of Ranvier, retraction of Schwann cell cytoplasm, and paranodal thinning of the myelin sheaths of peripheral nerves have been reported w4x. It is unclear why the latency of EPP in the dy 2 J r dy 2 J soleus did not increase. These results demonstrate that neuromuscular transmission is largely intact in the C57 BLr6J dy 2 J r dy 2 J mouse since no evidence of denervation Žmuscles failing to show MEPPs or EPPs and fibrillation potentials. were seen. Some of the abnormalities in neurotransmission observed, notably, the changes in mean MEPP amplitude and MEPP amplitude distribution, reflect muscle fibre changes. Increases in MEPP frequency, EPP quantal content, and the increased latency of the EPP are indicative of neuronal abnormalities. It appears that laminin-2 Žmerosin. is not of major importance for neuromuscular differentiation, unlike b 2-containing laminins Žs-laminin. w30x. This is perhaps not surprising as laminin-2 Žmerosin. is expressed late in muscle development w19x making it an extremely unlikely candidate for a primary role in regulating the development of the neuromuscular junction. However, the inability of the truncated form of the a 2 subunit of the dy 2 J r dy 2 J mice to form a stable link with membrane associated glycoproteins such as a-dystroglycan does causes a breakdown in the structural integrity of the muscle and affects the properties of the neuromuscular junction. Acknowledgements We wish to thank Mr. Alan Nevitt of the Department of Medical Computation for his help with the statistical analysis. J.P. Edwards is supported by the Medical Research Council. P.A. Hatton holds a Wellcome Trust Prize Studentship.
268
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
References w1x E.T. Agbenyega, R.H. Morton, P.A. Hatton, A.C. Wareham, Effect of the b 2-adrenergic agonist clenbuterol on the growth of fast- and slow-twitch skeletal muscle of the dystrophic ŽC57Bl6J dy 2 J r dy 2 J . mouse, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 112 Ž1995. 397–403. w2x R.J. Balice-Gordon, S.M. Breedlove, S. Bernstein, J.W. Lichtman, Neuromuscular junctions shrink and expand as muscle fiber size is manipulated: in vivo observations in the androgen-sensitive bulbocavernosus muscle of mice, J. Neurosci. 10 Ž1990. 2660–2671. w3x W.G. Bradley, M. Jenkinson, Neural abnormalities in the dystrophic mouse, J. Neurol. Sci. 25 Ž2. Ž1975. 249–255. w4x W.G. Bradley, E. Jaros, M. Jenkinson, The nodes of Ranvier in the nerves of mice with muscular dystrophy, J. Neuropathol. Exp. Neurol. 36 Ž5. Ž1977. 797–806. w5x J. Butler, E. Cosmos, Histochemical and structural analyses of the phenotypic expression of the dystrophic gene in the 129rReJ dyr dy and the C57BLr6J dy 2 J r dy 2 J mice, Exp. Neurol. 57 Ž1977. 661– 681. w6x S. Carbonetto, M. Lindenbaum, The basement membrane at the neuromuscular junction: a synaptic mediatrix, Curr. Opin. Neurobiol. 5 Ž1995. 596–605. w7x L.R. Dribin, S.B. Simpson, Histochemical and morphological study of dystrophic ŽC57BLr6J dy 2 J r dy 2 J . and normal muscle, Exp. Neurol. 56 Ž1977. 480–497. w8x J.P. Edwards, C.-C. Lee, L.W. Duchen, The evolution of an experimental distal motor axonopathy, physiological studies of changes in neuromuscular transmission caused by cycloleucine, an inhibitor of methionine adenosyltransferase, Brain 117 Ž1994. 959–974. w9x J.J. Gilbert, M.C. Steinberg, B.Q. Banker, Ultrastructural alterations of the motor endplate in myotonic dystrophy of the mouse Ž dy 2 J r dy 2 J ., J. Neuropathol. Exp. Neurol. 32 Ž3. Ž1973. 345–362. w10x T. Gonoi, Y. Ohizumi, H. Nakamura, J. Kobayashi, W.A. Catterall, The Conus toxin geographutoxin II distinguishes two functional sodium channel subtypes in rat muscle cells developing in vitro, J. Neurosci. 7 Ž6. Ž1987. 1728–1731. w11x J.B. Harris, The resting membrane potential of fibres of fast and slow twitch muscles in normal and dystrophic mice, J. Neurol. Sci. 12 Ž1971. 45–52. w12x J.B. Harris, A. Montgomery, Some mechanical and electrical properties of distal hind limb muscles of genetically dystrophic mice ŽC57BLr6J dy 2 J r dy 2 J ., Exp. Neurol. 48 Ž1975. 569–585. w13x J.B. Harris, R.R. Ribchester, Muscular dystrophy in the mouse: neuromuscular transmission and the concept of functional denervation, Ann. New York Acad. Sci. 317 Ž1979. 152–170. w14x Y.K. Hayashi, E. Engvall, E. Arikawa-Hiraswa, K. Goto, R. Koga, I. Nonaka, H. Sugita, K. Arahata, Abnormal localization of laminin subunits in muscular dystrophies, J. Neurol. Sci. 119 Ž1993. 53–64. w15x S.J. Hong, C.C. Chang, Use of geographutoxin II Ž m-conotoxin. for the study of neuromuscular transmission in mouse, Br. J. Pharmacol. 97 Ž1989. 934–940. w16x B. Katz, S. Thesleff, On factors which determine the amplitude of the ‘miniature endplate potentials’, J. Physiol. ŽLondon. 137 Ž1957. 267–278. w17x M.E. Kriebel, R. Hanna, C. Muniak, Synaptic vesicle diameters and synaptic cleft widths at the mouse diaphragm in neonates and adults, Dev. Brain Res. 27 Ž1986. 19–29. w18x M. Kuno, S.A. Turkanis, J.N. Weakley, Correlation between nerve
w19x
w20x
w21x w22x w23x w24x
w25x
w26x w27x
w28x
w29x w30x
w31x
w32x
w33x
w34x w35x
w36x
w37x
w38x
terminal size and transmitter release at the neuromuscular junction of the frog, J. Physiol. ŽLondon. 213 Ž1971. 545–556. I. Leiro, E. Engvall, Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblasts, is expressed late in nerve and muscle development, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 1544–1548. M.T. Lupa, D.M. Krzemien, K.L. Schaller, J.H. Caldwell, Aggregation of sodium channels during development of the neuromuscular junction, J. Neurosci. 13 Ž3. Ž1993. 1326–1336. P.R. Lyons, C.R. Slater, Structure and function of the neuromuscular junction in young adult mdx mice, J. Neurocytol. 20 Ž1991. 969–981. A.R. Martin, A further study of the statistical composition of the end-plate potential, J. Physiol. ŽLondon. 130 Ž1955. 114–122. A.R. Martin, Quantal nature of synaptic transmission, Physiol. Rev. 46 Ž1966. 51–66. E.M. McLachlan, A.R. Martin, Non-linear summation of end-plate potentials in the frog and mouse, J. Physiol. ŽLondon. 311 Ž1981. 307–324. G. McIsaac, J.A. Kiernan, Complete staining of neuromuscular innervation with bromoindigo and silver, Stain. Technol. 49 Ž4. Ž1974. 211–214. H. Meier, J.L. Southard, Muscular dystrophy in the mouse caused by an allele at the dy-locus, Life Sci. 9 Ž1970. 137–144. A.M. Michelson, E.S. Russell, P.J. Harman, Dystrophia muscularis: a hereditary primary myopathy in the house mouse, Proc. Natl. Acad. Sci. U.S.A. 41 Ž1955. 1079–1084. C.G. Muniak, M.E. Kriebel, C.G. Carlsom, Changes in MEPP and EPP amplitude distributions in the mouse diaphragm during synapse formation and degeneration, Brain Res. Ž1982. 123–138. A. Nagel, F. Lehmann-Horn, A.G. Engel, Neuromuscular transmission in the mdx mouse, Muscle Nerve 13 Ž1990. 742–749. P.G. Noakes, M. Gautam, J. Mudd, J.R. Sanes, J.P. Merlie, Aberrant differentiation of neuromuscular junctions in mice lacking slamininrlaminin Beta2, Nature ŽLondon. 374 Ž1995. 258–262. E. Ozawa, M. Yoshida, A. Suzuki, Y. Mizuno, Y. Hagiwara, S. Noguchi, Dystrophin-associated proteins in muscular dystrophy, Hum. Mol. Genet. 4 Ž1995. 1711–1716. C.A. Sewry, J. Philpot, D. Mahony, L.A. Wilson, F. Muntoni, V. Dubowitz, Expression of laminin subunits in congenital muscular dystrophy, Neuromuscular Disord. 5 Ž4. Ž1995. 307–316. P.M. Shalton, A.C. Wareham, Some factors affecting spontaneous transmitter release in dystrophic mice, Muscle Nerve 3 Ž1980. 120–127. C.A. Stirling, Abnormalities in Schwann cell sheaths in spinal nerve roots of dystrophic mice, J. Anat. 119 Ž1975. 169–180. Y. Sunuda, S.M. Bernier, C.A. Kozak, Y. Yamada, K.P. Cambell, Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin m chain gene to dy locus, J. Biol. Chem. 269 Ž19. Ž1994. 13729–13732. F.M.S. Tome, T. Evangelista, A. Leclerc, Y. Sunada, E. Manole, B. Estournet, A. Bardis, K.P. Campbell, M. Fardeau, Congenital muscular dystrophy with merosin deficiency, C. R. Acad. Sci. Ser. III 317 Ž1994. 351–357. H. Xu, P. Christmas, X.R. Wu, U.M. Wewer, E. Engvall, Defective muscle basement and lack of M-laminin in the dystrophic dyr dy mouse, Proc. Natl. Acad. Sci. U.S.A. 91 Ž12. Ž1994. 5572–5576. H. Xu, X.R. Wu, U.M. Wewer, E. Engvall, Murine muscular dystrophy caused by a mutation in the laminin a 2 Ž Lama2 . gene, Nat. Genet. 8 Ž1994. 297–302.
Research report
Electrophysiology of the neuromuscular junction of the laminin-2 Ž merosin. deficient C57 BLr6J dy 2 J r dy 2 J dystrophic mouse Jonathan P. Edwards, Paul A. Hatton, Anthony C. Wareham
)
DiÕision of Neuroscience, School of Biological Sciences, 1.124 Stopford Building, UniÕersity of Manchester, Oxford Road, Manchester M13 9PT, UK Accepted 23 December 1997
Abstract The C57 BLr6J dy 2 J r dy 2 J dystrophic mouse expresses an abnormal truncated form of the a 2 subunit of the protein laminin-2 Žor merosin., which is unable to form a stable link between the extracellular matrix and the dystrophin-associated proteins, resulting in muscular dystrophy. Morphological abnormalities of the peripheral nervous system and neuromuscular junction have also been reported. The electrophysiological properties of the neuromuscular junctions of diaphragm, extensor digitorum longus ŽEDL., and soleus from C57 BLr6J dy 2 J r dy 2 J mice and controls are described. No evidence for the presence of denervated fibres were found. Mean MEPP amplitudes were significantly increased in EDL and soleus but reduced in the diaphragm from affected mice. Mean MEPP frequencies were raised in all the dy 2 J r dy 2 J muscles studied. dy 2 J r dy 2 J muscles were paralysed by low concentrations of m-conotoxin suggesting that embryonic Žtetrodotoxin and m-conotoxin resistant. sodium channels are not widespread on dy 2 J r dy 2 J muscle as has previously been reported. EPP latencies were significantly prolonged in the diaphragm and EDL but not soleus from dy 2 J r dy 2 J mice. Quantal contents were higher in all dy 2 J r dy 2 J muscles. In the dy 2 J r dy 2 J diaphragm failures in neurotransmission occurred and a faster rate of rundown of EPPs was apparent. Some changes appear from a direct effect of dystrophy, whilst increased MEPP frequency and quantal content, and failures in neurotransmission indicate neuronal abnormalities. q 1998 Elsevier Science B.V. Keywords: dy 2 J r dy 2 J ; Laminin-2; Muscular dystrophy; Neuromuscular transmission; Neuromuscular junction
1. Introduction Laminin is an important structural component of the basal lamina. It is made up of three subunits a , b and g w31x, differing forms of these subunits being expressed in different cell types which may also vary within a cell type during development. In muscle the a subunit of laminin binds to a-dystroglycan of the dystroglycan complex thereby forming a link between the basal lamina and the actin filament of the sarcolemma Žfor reviews see w6,31x.. Laminin-2 Žmade up of a 2 b 1g 1 subunits. is the predominant form of laminin in adult striated muscle. It has recently been demonstrated that deficiencies in laminin-2 can cause muscular dystrophy in humans and Abbreviations: miniature endplate digitorum longus; Tetrodotoxin ) Corresponding
RMP s resting membrane potential; MEPP s potential; EPPsendplate potential; EDL sextensor CMD s congenital muscular dystrophy; TTX s author. Fax: q44-161-275-5363.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 0 1 0 - 9
mice. Patients with Fukyama congenital muscular dystrophy, the most common form of congenital muscular dystrophy ŽCMD. in Japan, have been found to have muscles and peripheral nerves deficient in laminin-2 w14x. There have recently been reports that some patients with other forms of CMD exhibit deficiencies in laminin-2 w32,36x. Previously described strains of dystrophic mice are now considered to be animal models of laminin-2 deficiency. The ReJ dy r dy dystrophic mouse, originally described by Michelson et al. in 1955 w27x has been found to lack laminin-2 by a number of labs w35,37x. The C57 BLr6J dy 2 J r dy 2 J dystrophic mouse, originally described by Meir and Southard in 1970 w26x, has recently been shown to express an abnormal truncated form of the a 2 subunit of laminin-2 w38x. This truncated form of a 2 subunit is believed to lack many of the qualities of the wild-type protein, most importantly the ability to form a stable link between the extracellular matrix and the dystrophin associated proteins required to maintain the structural integrity of the muscle.
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
The dy 2 J r dy 2 J mutant mouse has a normal life span and displays a less severe dystrophy than the REJ 129 dy r dy mouse, which is more severely affected and dies at around 3–6 months of unknown causes w7,38x. dy 2 J r dy 2 J mice develop a progressive weakness of the hindlimbs and a histological picture of muscle degeneration and regeneration observable from about 3 weeks of age w5x. Agbenyega et al. w1x using the Manchester colony of mice have shown that the soleus and EDL of affected dy 2 J r dy 2 J mice contain an increased percentage of small and large fibres with a high proportion of spherical fibres and a lot of connective tissue infiltration between the fibres. The distribution of the dystrophic changes was very different in soleus and EDL; in soleus dystrophic fibres were found throughout the muscle whereas in EDL only certain discreet areas were affected. Morphological abnormalities of peripheral nerve w3,34x and neuromuscular junctions w9x have also been described in dy 2 J r dy 2 J mice. Reports concerning neuromuscular structure and transmission in the ReJ dy r dy mice have frequently provided conflicting information Žfor review see Ref. w13x.. A preliminary report of the mechanical and electrical properties of dystrophic muscle in the C57 BLr6J dy 2 J r dy 2 J mouse was provided by Harris and Montgomery w12x followed by an account of spontaneous transmitter release in mice from the Manchester colony w33x. No detailed analysis of neurally evoked neuromuscular transmission in these mice has been done, however, despite the description of ultrastructural abnormalities of the neuromuscular junction. Noakes et al. w30x have recently shown that b 2-containing laminins Žor s-laminins. play an important role in the regulation of synaptic differentiation in the mouse. We, therefore, have investigated the properties of the neuromuscular junction in skeletal muscle of C57 BLr6J dy 2 J r dy 2 J mice to determine the effect of laminin-2 deficiency on neuromuscular function and differentiation.
2. Materials and methods Eighteen 6- to 8-week-old affected dy 2 J r dy 2 J mice and 19 age-matched unaffected Ž DY r dy or DY r DY . littermate controls were obtained from the Biological Service Unit of Manchester University. The colony was originally obtained from the MRC Laboratory Animal Centre in Carshalton, Surrey. Mice were killed by cervical dislocation. Recordings from muscles were carried out in vitro at room temperature using standard intracellular techniques. Resting membrane potentials ŽRMP., miniature endplate potentials ŽMEPPs., and endplate potentials ŽEPPs. were recorded from the diaphragm, soleus, and extensor digitorum longus ŽEDL. muscles. Intracellular micro electrodes Ž10–15 M V resistance. were connected to a Neurolog NL102 preamplifier ŽDigitimer Research Instruments.. The resultant signals were digitised at 20 kHz using a CED 1401 ŽCambridge Electronic Design. and captured in event
263
detector mode Žwith the trigger set at 0.1 mV. using Whole Cell Electrophysiology Program software supplied by Dr. J. Dempster of Strathclyde University. This software was also used for the subsequent analysis of MEPPs and EPPs. The bathing media contained ŽmM. 135 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 15 NaHCO 3 , 1 Na 2 HPO4 , 11 glucose, pH 7.2 and was bubbled with 95% O 2r5% CO 2 . To facilitate the recording of EPPs 2–4 m grml of m-conotoxin w15x ŽSigma Chemical, Poole, Dorset, UK. was added to the bathing media and indirect stimulation at 2 Hz was applied. After fibre impalement with a microelectrode the RMP was allowed to stabilise for 5 min before recording was started. During recording most fibres depolarised by 0–2 mV. Any fibre whose RMP depolarized by more than 10 mV after impalement was not recorded from. The analysis of MEPP amplitudes was further restricted to fibres where the mean MEPP rise times were less than 1 ms to ensure the recordings were focal to the endplate region. Similarly, the analysis of EPPs was restricted to fibres where the mean EPP rise time Žmeasured using Whole Cell Electrophysiological program. was less than 1.5 ms. Quantal contents of EPPs were calculated using the direct method and corrected for nonlinear summation w22,23x using the empirical constant of 0.8 suggested by McLachlan and Martin w24x for mouse muscle. The mean amplitude of approximately 40 MEPPs and EPPs was used for each calculation of quantal content in each fibre. In a further set of experiments a range of stimulation frequencies were applied to the diaphragm to monitor the effects of ‘rundown’ at control and dystrophic neuromuscular junctions. Indirect stimulation at 2, 10, 20, 40 and 60 Hz was applied for 30 s to the same muscle fibre, with a 2-min rest between each stimulation frequency and 10 min between each fibre. The resultant EPPs were recorded on a Gould 2200 pen recorder ŽGould Electronics.. At 0 s, 10 s, 20 s, and 30 s after the start of the stimulation, five EPPs were measured and expressed as a % of the amplitude of the EPPs at time 0 Žstandardised to 100%.. Muscles were also stained for endplate cholinesterase activity using the method of McIsaac and Kiernan w25x. 2.1. Statistical analysis The results are expressed as means " S.E.M. All data sets were initially tested for normality using the Kolmogorov–Smirnov goodness-of-fit test. Data for the EPP latency in soleus and EDL and the quantal content data from all muscles were found to be normally distributed. All remaining data were found to be non-normally distributed. Student’s t-test was used to compare the means of normally distributed data sets, whereas non-normally distributed data were compared using the Mann–Whitney U-test. Repeated-measure multiple-factorial ANOVA was employed to analyse the data produced in the ‘rundown’ experiments.
264
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
3. Results 3.1. Clinical and morphological obserÕations It was possible to differentiate between affected dy 2 J r dy mice and unaffected Ž DY r dy or DY r DY . littermate 2J
controls at approximately three weeks of age. Affected mice walked with a wobbling gait and were unable to grasp with the toes of their hind paws when placed on the wire lid of their cages. The hind limbs were more severely affected than the fore limbs. The distribution of endplates
Fig. 1. Amplitude distributions of spontaneous MEPPS recorded in Ža. control diaphragm, Žb. dystrophic diaphragm, Žc. control EDL, Žd. dystrophic EDL, Že. control soleus, and Žf. dystrophic soleus. Each histogram represents the results pooled from five muscles, from more than 170 fibres, where N f 2000.
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
265
Table 1 The passive properties and spontaneous transmitter release recorded in control and dy 2 J r dy 2 J muscle MMuscle
RMP ŽmV. Ž n s 200, 5. MEPP rise time Žms. Ž n ) 2000, 5. MEPP amplitude ŽmV. Ž n ) 2000, 5. MEPP frequency ŽHz. Ž n ) 150, 5.
Diaphragm Control 69.5 " 0.68 dy 2 J r dy 2 J 64.5 " 0.58 p - 0.0001
0.9 " 0.006 0.81 " 0.01 p - 0.0001
1.32 " 0.01 1.27 " 0.07 p - 0.0001
0.58 " 0.03 0.76 " 0.03 p - 0.0001
Soleus Control 67.2 " 0.58 dy 2 J r dy 2 J 69.8 " 0.63 p - 0.01
0.85 " 0.005 0.87 " 0.02 p - 0.0001
0.92 " 0.006 1.31 " 0.01 p - 0.0001
0.95 " 0.04 1.35 " 0.05 p - 0.0001
EDL Control 76.1 " 0.73 dy 2 J r dy 2 J 71.1 " 0.66 p - 0.01
0.78 " 0.006 0.89 " 0.007 p - 0.0001
0.97 " 0.008 1.10 " 0.01 p - 0.0001
1.16 " 0.06 1.17 " 0.07 NS
The values are means" S.E.M. The figures in parentheses are the number of observations on the left Ž n for the statistical analysis. and number of animals on the right. The results obtained from the dy 2 J r dy 2 J muscles were compared with their respective controls using the Mann–Whitney U-test, the significance of any differences between the means are shown, NS s not significant.
in dystrophic muscles appeared to be more diffuse than those from the controls. This was observed with both the electrophysiological recordings and the cholinesterase staining although no attempt was made to quantify this observation. 3.2. PassiÕe electrophysiological properties It can been seen from Table 1 that the RMP in dy 2 J r dy diaphragm and EDL was significantly depolarised compared to the controls whereas in the soleus the RMP was significantly hyperpolarised in the dy 2 J r dy 2 J muscle compared to the controls. The value of the RMP in the control soleus was significantly smaller than that of the control EDL Ž p - 0.0001. a finding previously noted to occur by Harris w11x. The mean RMP of the control diaphragm was slightly smaller than that reported by Hong and Chang w15x, but higher than that reported by Nagel et al. w29x for the murine diaphragm. 2J
3.3. Spontaneous transmitter release MEPPs were recorded from over 90% of fibres impaled in both the dy 2 J r dy 2 J and control muscles. Fibrillation potentials were not seen in any muscles, indicating that fibres were not denervated. Mean MEPP amplitudes were significantly greater in the dy 2 J r dy 2 J EDL and soleus. In the soleus the mean MEPP amplitude in the dy 2 J r dy 2 J muscle was over 40% greater than the controls. The MEPP amplitude distribution showed a greater amount of skewing to the right in the dy 2 J r dy 2 J soleus than in the control soleus Žthe value of skewness for the control soleus data being 1.27 " 0.05 compared to 1.33 " 0.04 in the dy 2 J r dy 2 J muscles.. Similarly, the EDL data were also more skewed to the right in the dy 2 J r dy 2 J muscles Ž2.81 " 0.05 vs. 2.11 " 0.04. than in the controls. In the dy 2 J r dy 2 J
diaphragm, however, the mean MEPP amplitudes was significantly lower than in the controls. The MEPP amplitude histograms were also more skewed to the right in the control diaphragms Ž1.38 " 0.03. than the dy 2 J r dy 2 J diaphragms Ž0.64 " 0.04. in contrast to the soleus and EDL. The MEPP amplitude distributions for all muscle types are shown in Fig. 1. The MEPP amplitudes recorded in the control diaphragm are in agreement to those reported previously w15,29x. Likewise the MEPP amplitudes recorded in control soleus and EDL are similar to those recorded by Edwards et al. w8x. The mean MEPP rise times were also increased in the dy 2 J r dy 2 J soleus and EDL but not in the diaphragm when compared to the controls. Table 2 Transmitter release in dy 2 J r dy 2 J muscle Muscle
EPP latency Žms. Ž n)90, 5. Quantal content Ž n) 75, 5.
Diaphragm Control 1.75"0.03 dy 2 J r dy 2 J 2.27"0.06 p- 0.0001
15.83"0.71 17.31"0.74 NS
Soleus Control 2.01"0.04 dy 2 J r dy 2 J 1.93"0.05 NS
19.71"0.89 30.48"1.21 p- 0.0001
EDL Control 1.54"0.03 dy 2 J r dy 2 J 1.63"0.03 p- 0.05
19.74"1.00 20.21"1.02 NS
The values are means"S.E.M. The figures in parentheses are the number of observations on the left Ž n for the statistical analysis. and number of animals on the right. The results obtained from the dy 2 J r dy 2 J muscles were compared with their respective controls using Student’s t-test for normally distributed data and the Mann–Whitney U-test for non-normally distributed data, the significance of any differences between the means are shown, NSs not significant.
266
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
Higher MEPP frequencies were recorded from the diaphragm, soleus and EDL of dy 2 J r dy 2 J mice, although only in the soleus did this increase reach statistical significance. In the diaphragm and soleus the mean MEPP frequencies in the dy 2 J r dy 2 J were over 30% higher than their respective controls. 3.4. EÕoked transmitter release EPPs were recorded from over 90% of both control and dy 2 J r dy 2 J muscles fibres impaled, confirming that few, if any, fibres were denervated ŽTable 2.. EPP latencies were found to be significantly prolonged in the dy 2 J r dy 2 J diaphragm and EDL. In soleus, however, there was no significant difference in EPP latency between dy 2 J r dy 2 J and controls. The mean EPP latency in the control soleus and EDL were very similar to those previously published w8x. The length of nerve aspirated into the suction electrode was of approximately the same in both dystrophic and control preparations to prevent any apparent increases in latency due to using a longer length of nerve. In soleus preparations the length of nerve used was always far shorter than that of the EDL and diaphragm. Quantal contents of EPPs were very significantly increased Žby 55%. in the dy 2 J r dy 2 J soleus. In the diaphragm and EDL the quantal contents appeared to be slightly higher in the dy 2 J r dy 2 J muscles, but these differences were not statistically significant. The quantal contents recorded in the control diaphragm are in close agreement with those calculated by the direct method in the mouse diaphragm paralysed using 2–4 m grml m-conotoxin w15x.
Fig. 2. Rundown in diaphragm EPPs evoked by indirect stimulation at 2 Žsquare., 10 Žtriangle., 20 Žhexagon., 40 Ždiamond., and 60 Žstar. Hz. The results for the control fibres are shown by a complete line and closed symbols whereas the dystrophics have a dashed line and open symbols. Muscle contractions were blocked using m-conotoxin 2–4 m grml. The results are the means"S.E.M. of 18 fibres from four control preparations and 14 fibres from three dystrophic muscles. Each point represents the EPP amplitude expressed as a percentage of the EPP amplitude at time 0 Žstandardised to 100%..
Neuromuscular rundown is shown in Fig. 2. Statistical analysis accounted for three independent variables: group Žnormal vs. dystrophic., stimulation frequency and time. Overall the effect of increasing stimulation frequency, the effect of time and the interaction between the two Ži.e., increasing frequency over time. were highly significant Ž p - 0.001.. The overall effect of dystrophy was not significant, highlighting the fact that there is very little separation between the datasets as a whole ŽFig. 2.. However, the three-way interaction between group, stimulation frequency and time was significant Ž p s 0.01., indicating differences between controls and dystrophics in the time course of the response to increasing frequency Ži.e., the effect of increasing frequency over time was different between controls and dystrophics.. For example, it can be seen that at the lower stimulation frequencies Ž2, 10 and 20 Hz. the dy 2 J r dy 2 J neuromuscular junctions showed a slightly increased rate of ‘rundown’ to the controls. Also, at the higher stimulation frequencies Ž40 and 60 Hz., the dy 2 J r dy 2 J neuromuscular junctions showed a much faster initial rate of ‘rundown’ than was seen in the controls, although by 30-s rundown was similar. Failures in neurotransmission were also seen, prior to complete ‘rundown’ in 4 out of 14 dy 2 J r dy 2 J muscle fibres at stimulation frequencies above 20 Hz a feature never seen in any of the 18 control muscle fibres sampled.
4. Discussion The clinical observations of the affected dy 2 J r dy 2 J mice concur with previous descriptions of this form of murine dystrophy w12,26,33x. The smaller values for RMP found in the dy 2 J r dy 2 J diaphragm and EDL are in agreement with previous work on the mdx mouse w20,29x, the REJ 129 dy r dy mouse w13x, and the C57 BLr6J dy 2 J r dy 2 J mouse w12,33x. The reasons for the increased RMP in dy 2 J r dy 2 J soleus compared to the control are unclear, particularly as the soleus is histologically more severely affected w5x. It is possible that small regenerating fibres were more frequently impaled in soleus, which shows a more generalised distribution of affected fibres than the EDL w1x. The smaller RMP in dystrophic muscle is thought to be caused by changes in the sarcolemmal permeability to sodium or potassium ions brought about by membrane defects w29x. The larger MEPP amplitudes in dy 2 J r dy 2 J soleus and EDL probably reflect a higher fibre input resistance of affected muscle fibres. Dystrophic murine muscle fibres have previously been shown to have a higher mean input resistance w13,21x. The MEPP amplitude distribution for the dy 2 J r dy 2 J diaphragm shows a very different pattern from the control diaphragm indicating MEPP amplitudes are more variable in the dy 2 J r dy 2 J diaphragm. It should be noted, however, that pooling of data from five muscles could have contributed to an apparent
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
skew distribution. Smaller MEPP amplitudes have also been recorded the diaphragm of the mdx mouse w29x which might suggest that this is a characteristic of murine dystrophy. Developing muscle fibres and reinnervated fibres have a large proportion of small-skewed MEPPs w28x and it would have been interesting to determine if these were present in the dy 2 J r dy 2 J diaphragm. However, since an anticholinesterase was not used in the present experiments and our sampling method excluded events - 0.1 mV, the small-skewed MEPPs described in normal mouse muscle by Kriebel et al. w17x would not have been detected. The MEPP amplitudes quoted in this paper have not been corrected to a predetermined RMP. If the values for the dy 2 J r dy 2 J fibres are corrected w16x to the RMP of their respective controls, the value for the dy 2 J r dy 2 J diaphragm increases to 1.37 mV Žnow significantly larger than the control., whereas for the EDL the corrected value is 1.17 mV, and the soleus value is 1.25 mV Žboth still significantly larger than their controls.. It would appear from the results that dystrophic muscle fibres are found in all three of the muscles examined Žthe slow twitch, oxidative soleus, fast twitch, glycolytic, EDL and fast, oxidative, diaphragm. in 6- to 8-week-old mice and not just in the slow twitch, oxidative muscles, such as the soleus, as reported to occur early in the condition w5x. Agbenyega et al. w1x have recently reported that in this particular colony of mice dystrophic changes are seen in both the EDL and SOL of 12-week-old mice. Increased MEPP frequencies, compared with the controls, have previously been recorded from dy 2 J r dy 2 J soleus and EDL w33x. High MEPP frequencies, and increased quantal content are indicative of presynaptic abnormalities which could be the functional correlates of morphological changes. Reduced numbers of synaptic vesicles and retraction of the axolemma away from the postsynaptic specialisations of the sarcolemma have been observed w9x. It seems unlikely that a reduction in synaptic vesicles and retraction of the axolemma would lead to an increase in MEPP frequency and quantal content, however. The fact that the largest increases in MEPP frequency and quantal content are in the soleus which is histologically the most severely affected muscle suggests these changes are directly related to the dystrophy in the muscle. Increases in mean MEPP frequency and quantal content could also be caused by the sampling of the large neuromuscular junctions of hypertrophied fibres, which is more likely to occur in SOL which has a more generalised distribution of spherical hypertrophied fibres than the EDL w1x. It has previously been demonstrated that MEPP frequency and quantal content increase with endplate size w17,18x and endplates increase in size as the muscle fibre grows w2x. Sampling of the large endplates of hypertrophied fibres would increase the mean MEPP frequency and quantal content. We were surprised that the dy 2 J r dy 2 J muscle were as susceptible to m-conotoxin as the controls and could be
267
completely paralysed by a concentrations as low as 2 m grml Žthe same concentrations as controls.. Previous work had shown that distal hind limb muscles of C57 BLr6J dy 2 J r dy 2 J mice show tetrodotoxin ŽTTX. resistant action potentials w12x indicative of the widespread distribution of the embryonic form of sodium channel along the sarcolemma. It has been demonstrated that mconotoxin ŽGeographutoxin II. distinguishes more clearly between TTX-sensitive Žadult. and TTX-insensitive Žembryonic. sodium channels than TTX itself and may have no action at all at TTX-insensitive sodium channels w10x. We therefore expected that the dystrophic muscle would be difficult to paralyse with m-conotoxin, as we find in developing rat muscle Žunpublished observations., where embryonic sodium channels predominate w20x. This finding suggests that the TTX-insensitive sodium channels in dystrophic muscle differ from those found in the developing rat muscle. The increased latency of the EPP seen in the dy 2 J r dy 2 J EDL and diaphragm and the increased rate of rundown and incidence of failures in evoked neurotransmission at the dystrophic diaphragm are indicative of neuronal abnormalities. Extensive widening of the nodes of Ranvier, retraction of Schwann cell cytoplasm, and paranodal thinning of the myelin sheaths of peripheral nerves have been reported w4x. It is unclear why the latency of EPP in the dy 2 J r dy 2 J soleus did not increase. These results demonstrate that neuromuscular transmission is largely intact in the C57 BLr6J dy 2 J r dy 2 J mouse since no evidence of denervation Žmuscles failing to show MEPPs or EPPs and fibrillation potentials. were seen. Some of the abnormalities in neurotransmission observed, notably, the changes in mean MEPP amplitude and MEPP amplitude distribution, reflect muscle fibre changes. Increases in MEPP frequency, EPP quantal content, and the increased latency of the EPP are indicative of neuronal abnormalities. It appears that laminin-2 Žmerosin. is not of major importance for neuromuscular differentiation, unlike b 2-containing laminins Žs-laminin. w30x. This is perhaps not surprising as laminin-2 Žmerosin. is expressed late in muscle development w19x making it an extremely unlikely candidate for a primary role in regulating the development of the neuromuscular junction. However, the inability of the truncated form of the a 2 subunit of the dy 2 J r dy 2 J mice to form a stable link with membrane associated glycoproteins such as a-dystroglycan does causes a breakdown in the structural integrity of the muscle and affects the properties of the neuromuscular junction. Acknowledgements We wish to thank Mr. Alan Nevitt of the Department of Medical Computation for his help with the statistical analysis. J.P. Edwards is supported by the Medical Research Council. P.A. Hatton holds a Wellcome Trust Prize Studentship.
268
J.P. Edwards et al.r Brain Research 788 (1998) 262–268
References w1x E.T. Agbenyega, R.H. Morton, P.A. Hatton, A.C. Wareham, Effect of the b 2-adrenergic agonist clenbuterol on the growth of fast- and slow-twitch skeletal muscle of the dystrophic ŽC57Bl6J dy 2 J r dy 2 J . mouse, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 112 Ž1995. 397–403. w2x R.J. Balice-Gordon, S.M. Breedlove, S. Bernstein, J.W. Lichtman, Neuromuscular junctions shrink and expand as muscle fiber size is manipulated: in vivo observations in the androgen-sensitive bulbocavernosus muscle of mice, J. Neurosci. 10 Ž1990. 2660–2671. w3x W.G. Bradley, M. Jenkinson, Neural abnormalities in the dystrophic mouse, J. Neurol. Sci. 25 Ž2. Ž1975. 249–255. w4x W.G. Bradley, E. Jaros, M. Jenkinson, The nodes of Ranvier in the nerves of mice with muscular dystrophy, J. Neuropathol. Exp. Neurol. 36 Ž5. Ž1977. 797–806. w5x J. Butler, E. Cosmos, Histochemical and structural analyses of the phenotypic expression of the dystrophic gene in the 129rReJ dyr dy and the C57BLr6J dy 2 J r dy 2 J mice, Exp. Neurol. 57 Ž1977. 661– 681. w6x S. Carbonetto, M. Lindenbaum, The basement membrane at the neuromuscular junction: a synaptic mediatrix, Curr. Opin. Neurobiol. 5 Ž1995. 596–605. w7x L.R. Dribin, S.B. Simpson, Histochemical and morphological study of dystrophic ŽC57BLr6J dy 2 J r dy 2 J . and normal muscle, Exp. Neurol. 56 Ž1977. 480–497. w8x J.P. Edwards, C.-C. Lee, L.W. Duchen, The evolution of an experimental distal motor axonopathy, physiological studies of changes in neuromuscular transmission caused by cycloleucine, an inhibitor of methionine adenosyltransferase, Brain 117 Ž1994. 959–974. w9x J.J. Gilbert, M.C. Steinberg, B.Q. Banker, Ultrastructural alterations of the motor endplate in myotonic dystrophy of the mouse Ž dy 2 J r dy 2 J ., J. Neuropathol. Exp. Neurol. 32 Ž3. Ž1973. 345–362. w10x T. Gonoi, Y. Ohizumi, H. Nakamura, J. Kobayashi, W.A. Catterall, The Conus toxin geographutoxin II distinguishes two functional sodium channel subtypes in rat muscle cells developing in vitro, J. Neurosci. 7 Ž6. Ž1987. 1728–1731. w11x J.B. Harris, The resting membrane potential of fibres of fast and slow twitch muscles in normal and dystrophic mice, J. Neurol. Sci. 12 Ž1971. 45–52. w12x J.B. Harris, A. Montgomery, Some mechanical and electrical properties of distal hind limb muscles of genetically dystrophic mice ŽC57BLr6J dy 2 J r dy 2 J ., Exp. Neurol. 48 Ž1975. 569–585. w13x J.B. Harris, R.R. Ribchester, Muscular dystrophy in the mouse: neuromuscular transmission and the concept of functional denervation, Ann. New York Acad. Sci. 317 Ž1979. 152–170. w14x Y.K. Hayashi, E. Engvall, E. Arikawa-Hiraswa, K. Goto, R. Koga, I. Nonaka, H. Sugita, K. Arahata, Abnormal localization of laminin subunits in muscular dystrophies, J. Neurol. Sci. 119 Ž1993. 53–64. w15x S.J. Hong, C.C. Chang, Use of geographutoxin II Ž m-conotoxin. for the study of neuromuscular transmission in mouse, Br. J. Pharmacol. 97 Ž1989. 934–940. w16x B. Katz, S. Thesleff, On factors which determine the amplitude of the ‘miniature endplate potentials’, J. Physiol. ŽLondon. 137 Ž1957. 267–278. w17x M.E. Kriebel, R. Hanna, C. Muniak, Synaptic vesicle diameters and synaptic cleft widths at the mouse diaphragm in neonates and adults, Dev. Brain Res. 27 Ž1986. 19–29. w18x M. Kuno, S.A. Turkanis, J.N. Weakley, Correlation between nerve
w19x
w20x
w21x w22x w23x w24x
w25x
w26x w27x
w28x
w29x w30x
w31x
w32x
w33x
w34x w35x
w36x
w37x
w38x
terminal size and transmitter release at the neuromuscular junction of the frog, J. Physiol. ŽLondon. 213 Ž1971. 545–556. I. Leiro, E. Engvall, Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblasts, is expressed late in nerve and muscle development, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 1544–1548. M.T. Lupa, D.M. Krzemien, K.L. Schaller, J.H. Caldwell, Aggregation of sodium channels during development of the neuromuscular junction, J. Neurosci. 13 Ž3. Ž1993. 1326–1336. P.R. Lyons, C.R. Slater, Structure and function of the neuromuscular junction in young adult mdx mice, J. Neurocytol. 20 Ž1991. 969–981. A.R. Martin, A further study of the statistical composition of the end-plate potential, J. Physiol. ŽLondon. 130 Ž1955. 114–122. A.R. Martin, Quantal nature of synaptic transmission, Physiol. Rev. 46 Ž1966. 51–66. E.M. McLachlan, A.R. Martin, Non-linear summation of end-plate potentials in the frog and mouse, J. Physiol. ŽLondon. 311 Ž1981. 307–324. G. McIsaac, J.A. Kiernan, Complete staining of neuromuscular innervation with bromoindigo and silver, Stain. Technol. 49 Ž4. Ž1974. 211–214. H. Meier, J.L. Southard, Muscular dystrophy in the mouse caused by an allele at the dy-locus, Life Sci. 9 Ž1970. 137–144. A.M. Michelson, E.S. Russell, P.J. Harman, Dystrophia muscularis: a hereditary primary myopathy in the house mouse, Proc. Natl. Acad. Sci. U.S.A. 41 Ž1955. 1079–1084. C.G. Muniak, M.E. Kriebel, C.G. Carlsom, Changes in MEPP and EPP amplitude distributions in the mouse diaphragm during synapse formation and degeneration, Brain Res. Ž1982. 123–138. A. Nagel, F. Lehmann-Horn, A.G. Engel, Neuromuscular transmission in the mdx mouse, Muscle Nerve 13 Ž1990. 742–749. P.G. Noakes, M. Gautam, J. Mudd, J.R. Sanes, J.P. Merlie, Aberrant differentiation of neuromuscular junctions in mice lacking slamininrlaminin Beta2, Nature ŽLondon. 374 Ž1995. 258–262. E. Ozawa, M. Yoshida, A. Suzuki, Y. Mizuno, Y. Hagiwara, S. Noguchi, Dystrophin-associated proteins in muscular dystrophy, Hum. Mol. Genet. 4 Ž1995. 1711–1716. C.A. Sewry, J. Philpot, D. Mahony, L.A. Wilson, F. Muntoni, V. Dubowitz, Expression of laminin subunits in congenital muscular dystrophy, Neuromuscular Disord. 5 Ž4. Ž1995. 307–316. P.M. Shalton, A.C. Wareham, Some factors affecting spontaneous transmitter release in dystrophic mice, Muscle Nerve 3 Ž1980. 120–127. C.A. Stirling, Abnormalities in Schwann cell sheaths in spinal nerve roots of dystrophic mice, J. Anat. 119 Ž1975. 169–180. Y. Sunuda, S.M. Bernier, C.A. Kozak, Y. Yamada, K.P. Cambell, Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin m chain gene to dy locus, J. Biol. Chem. 269 Ž19. Ž1994. 13729–13732. F.M.S. Tome, T. Evangelista, A. Leclerc, Y. Sunada, E. Manole, B. Estournet, A. Bardis, K.P. Campbell, M. Fardeau, Congenital muscular dystrophy with merosin deficiency, C. R. Acad. Sci. Ser. III 317 Ž1994. 351–357. H. Xu, P. Christmas, X.R. Wu, U.M. Wewer, E. Engvall, Defective muscle basement and lack of M-laminin in the dystrophic dyr dy mouse, Proc. Natl. Acad. Sci. U.S.A. 91 Ž12. Ž1994. 5572–5576. H. Xu, X.R. Wu, U.M. Wewer, E. Engvall, Murine muscular dystrophy caused by a mutation in the laminin a 2 Ž Lama2 . gene, Nat. Genet. 8 Ž1994. 297–302.