| Human Molecular Genetics | Pages |
Correlation between varying levels of PMP22 expression and the degree of demyelination and reduction in nerve conduction velocity in transgenic mice
Introduction
Results
Transgenic mice expressing different levels of human PMP22
Level of expression of the human PMP22 transgenes
Electrophysiology
Histology
Discussion
Materials And Methods
Generation of transgenic mice
DNA analysis and FISH
RNA analysis
Histology
Electrophysiological recordings
Acknowledgements
References
Correlation between varying levels of PMP22 expression and the degree of demyelination and reduction in nerve conduction velocity in transgenic mice
Charcot-Marie-Tooth disease type 1A is most commonly caused by a duplication of a 1.5 Mb region of chromosome 17 which includes the peripheral myelin protein 22 gene (PMP22). Over-expression of this gene leads to a hypomyelinating/demyelinating neuropathy and to severely reduced nerve conduction velocity. Previous mouse and rat models have had relatively high levels of expression of the mouse or human PMP22 gene leading to severe demyelination. Here we describe five lines of transgenic mice carrying increasing copies of the human PMP22 gene (one to seven) and expressing increasing levels of the transgene. From histological and electrophysiological observations there appears to be a threshold below which expression of PMP22 has virtually no effect; below a ratio of human/mouse mRNA expression of ~0.8, little effect is observed. Between a ratio of 0.8 and 1.5, histological and nerve conduction velocity abnormalities are observed, but there are no behavioural signs of neuropathy. An expression ratio >1.5 leads to a severe neuropathy. A second observation concerns the histology of the different lines; the level of expression does not affect the type of demyelination, but influences the severity of involvement.
INTRODUCTION
The hereditary demyelinating neuropathies are a genetically complex group of disorders of which the commonest form is type 1A Charcot-Marie-Tooth disease (CMT1A) otherwise known as type Ia hereditary motor and sensory neuropathy (HMSN Ia). It is characterized by an onset in childhood and results in widespread demyelination and later axonal loss, associated with severely reduced nerve conduction velocity. It is caused by over-expression or point mutations in the gene for the peripheral myelin protein 22 (PMP22)[for a review, see (1)].
PMP22 is found in compact myelin in peripheral nerve where it makes up ~2-5% of the total myelin proteins (2). Thus PMP22 is not one of the major structural proteins of myelin, such as P0 which makes up ~50% of the myelin proteins, and its role in myelin is not known. It is also expressed in many other cells of the body, though at much lower levels than in Schwann cells, and was actually first identified as a gene which is switched on in growth-arrested fibroblasts. This has led to the suggestion that it has a role in Schwann cell differentiation and division. In vitro, over-expression of PMP22 in Schwann cells reduces cell growth and delays transition from G0/G1 to the S phase of the cell cycle (3), while in NIH-3T3 fibroblasts over-expression causes an apoptotic-like phenotype (4).
The commonest cause of CMT1A is over-expression of PMP22 usually due to a duplication of a 1.5 Mb region on chromosome 17p11.2, including the PMP22 gene [for a review see (1)]. This usually occurs by unequal crossing over between two copies of a repeated sequence (5), most frequently during male meiosis (6) although it can also be of maternal origin (7). The repeated sequence has been determined to be 24 011 bp in length (8,9) and the hotspot of recombination within these repeats occurs near a region with very high homology to the Drosophila Mariner element (10). De novo duplication events have been found in nine out of 10 sporadic cases (11) indicating that it is a relatively common occurrence possibly stimulated by double-stranded breaks at the Mariner-like element. The patients with this over-expression have slowed nerve conduction velocity and a hypertrophic demyelination characterized by abnormally thick myelin on intact nerve fibres, demyelination and remyelination, and numerous onion bulbs characteristic of cycles of demyelination and remyelination [for a review, see (12)].
Patients heterozygous for point mutations in the PMP22 gene have been reported with slow nerve conduction and demyelination (13-18). Some of these mutations cause CMT1A with later onset and intermediate nerve conduction velocities (13,15) while others cause a more severe demyelination with earlier onset and very slow nerve conduction sometimes referred to as the Dejerine-Sottas syndrome (14,16-18). There are two mouse mutants which have dominantly acting point mutations in the Pmp22 gene. Trembler (Tr) is a dominant mutation causing severe hypomyelination and continuing Schwann cell proliferation throughout life (19). Trembler-J (TrJ) has a mutation which corresponds to one found in a human CMT1A family (13). It is semidominant with some gene dosage effect. Homozygotes are very severely affected with peripheral myelin deficiency and early death at postnatal day 17 or 18 (20).
DNA changes which cause null mutations in the PMP22 gene, such as a 2 bp frameshifting deletion (21) or the complete deletions due to the reciprocal of the CMT duplication event (22,23), in the heterozygous condition give the human disease hereditary neuropathy with liability to pressure palsies (HNPP). This is characterized by a less severe reduction in nerve conduction velocity and by focal myelin thickenings termed tomacula. Clearly the point mutations in PMP22 which cause prominent demyelination (see above), as opposed to the less severe demyelination with tomacula seen in HNPP, are not acting as haplo-insufficient null mutations but as dominantly acting mutations. Humans homozygous for null mutations have not been reported.
Three animal models with over-expression have been reported. A rat carrying about three copies of the mouse Pmp22 gene on a cosmid has quite severe demyelination and slowed nerve conduction. The homozygote is more severely affected with virtually no myelin in the peripheral nervous system (24). Mice with seven copies of the human gene have a severe demyelination (25) while mice with 16 or more copies of the mouse gene have very severe demyelination (26). In this paper we report a series of transgenic mouse lines with different copy numbers of the PMP22 gene with correlation to the levels of expression. We have related the level of expression in the different lines with the observed histology and electrophysiology.
RESULTS
Transgenic mice expressing different levels of human PMP22
The YAC 49G7 (27,25) contains the 40 kb human PMP22 gene as well as ~300 kb of upstream and ~100 kb of downstream sequences (Fig. 1A). This YAC DNA was gel purified and used for pronuclear injection giving rise to six transgenic lines called C1, C2, C16, C22, C58 and C61. One of the lines, C22, showed a dominant phenotype of weakness and progressive paralysis of the hind legs which we have already described (25). Heterozygotes of the other lines do not show an abnormal phenotype. However, C61 homozygotes have a clear weakness and paralysis similar to C22. Homozygotes of C1 and C58 homozygotes look normal but do not breed well while C2 and C16 homozygotes do not show an abnormal phenotype. C22 mice have never bred to give homozygotes for this line although four pairs have been together for 6 months. The mouse lines and phenotypes are summarized in Table 1.
Figure
The lines were analysed for six markers located in the YAC including the left and right arms of the YAC, the markers D17S122, FVG11 and D17S261 and the PMP22 gene itself (Fig. 1A, Table 2). Only C22 and C61 contain all six markers. Intactness of the region around the transgene was further analysed by digestion with SfiI which gives an ~100 kb fragment spanning the PMP22 gene in the YAC (Fig. 1A and B). All the lines except C16 showed the intact SfiI fragment indicating that they contain the intact gene and surrounding DNA. C16 only showed a smaller fragment indicating that the YAC is deleted and this line was not analysed further.
Table 1.
Table 2.
The copy numbers of the YAC transgenes were determined by digestion with HindIII, followed by hybridization with the PMP22 exon 2 probe. An example of this is shown for the line C58 which clearly has a copy number of two (Fig. 1C). The copy numbers in the lines are; one in C2, two in C1 and C58, four in C61 and seven in C22 (Table 1). Interestingly, the line with the highest copy number, C22 (seven copies), is the only one showing a dominant phenotype while C61 with four copies only shows the phenotype in the homozygous state (eight copies) (see Table 1).
The lines were also analysed by FISH and found to have different integrations of YAC DNA into a mouse chromosome as shown for C1, C2, C61 and C22 in Figure 2. The C1 integration is into chromosome 7, C2 into chromosome 1, C22 into chromosome 12, C58 into chromosome 15 and C61 into chromosome 10. Thus the lines have different integration sites and integration does not seem to occur preferentially in a particular region of the chromosomes such as centromeres, telomeres, heterochromatin or euchromatin. . FISH analysis of the transgenic lines C1, C2, C61 and C22 showing the different integration sites.
Mouse
genotype Copy
no.Ratio human/
mouse mRNAMCVa
(m/s)Histology
Visible phenotype
Wild type
0
-
38
normal
none
C2 het
1
0.4
nd
normal
none
C2 hom
2
0.8
41
normal
none
C1 het
2
0.5
46
normal
none
C58 het
2
0.6
49
normal
none
C1 hom
4
1.3
26
mild demyelination
don't mate well
C58 hom
4
nd
21
mild demyelination
don't mate well
C61 het
4
1.0
25
mild demyelination
none
C22 het
7
1.6
4
demyelination
peripheral neuropathy
C61 hom
8
nd
4
demyelination
peripheral neuropathy
C22 hom
14
-
-
-
never produced
Line
Left arm
PMP22
D17S122
FVG11
D17S261
Right arm
C1
+
+
+
+
+
-
C2
+
+
+
+
-
+
C16
+
+
-
+
-
+
C22
+
+
+
+
+
+
C58
+
+
+
+
+
-
C61
+
+
+
+
+
+
Level of expression of the human PMP22 transgenes
The level of expression of the human PMP22 transgenes was determined relative to the level of mouse Pmp22 mRNA in sciatic nerves of the mice. Two primers were used which amplify both the mouse and human cDNAs equally. The product was then cut with either TaqI, which cuts the human cDNA, or AluI, which cuts the mouse cDNA, and the products quantified (25). RNA from a single mouse was analysed four times starting with different cDNA reactions and the average taken. One mouse was analysed for each genotype except C58 homozygote and C61 homozygote and the results are shown in Table 1.
The level of human transgene expression relative to the mouse mRNA is plotted against the transgene copy number in Figure 3A. The level of expression is roughly proportional to the copy number of the YAC transgene irrespective of the different positions of integration in the different lines. However, the expression of each transgene copy is only about half that of each mouse gene and the mice with four copies of the transgene have approximately the same amount of human as mouse mRNA.
Figure
Electrophysiology
Nerve conduction was examined under terminal anaesthesia. Conduction velocity was measured in the tibial division of the sciatic nerve, together with the distal motor and F wave latencies. The results are given in Tables 1 and 3 and Figures 3 and 4. Seven wild-type mice were found to have an average motor nerve conduction velocity (MCV) of 38.2 ± 6.3 m/s and an average distal motor latency (DML) of 0.82 ± 0.1 ms (Table 3, Fig. 4A).
Figure
Nine C22 heterozygotes were measured (Table 3, Fig. 4E). In three, no response to nerve stimulation could be recorded. The others had an average MCV of 3.7 ± 2.2 m/s which is very slow and an average DML of 4.3 ± 1.6 ms which is much greater than normal. The mice analysed ranged from 30 to 336 days of age but the MCVs and DMLs did not seem to correlate with age and the 30-day-old mouse had one of the slowest MCV recordings (Table 3). Similarly the three males did not appear to be different from the six females analysed (Table 3). In these animals with severely reduced motor conduction velocity, or in which no response to nerve stimulation could be obtained, denervation of the muscle was confirmed by the recording of spontaneous fibrillation potentials and myotonic runs on needle movement.
Table 3.
For the other lines, a single mouse of each genotype was investigated and measurements were taken on both sides (Tables 1 and 3, Figs 3 and 4). A C2 homozygous mouse, a C1 heterozygote and a C58 heterozygote all had motor conduction velocities and distal motor latencies in the normal range (MCV > 35 m/s, DML < 1 ms). A C1 homozygote, a C58 homozygote and a C61 heterozygote all had intermediate values (MCV 20-30 m/s, DML 1-2 ms). Finally, a C61 homozygote had a MCV of 4.3 m/s which is as slow as the C22 mice. The DML for this mouse was 1.7 ms which is surprisingly low for the slow MCV and may be an artefact.
In the later experiments, F wave latency with ankle stimulation was also recorded. Mean F wave latency for five normal animals was 3.80 ms (Fig. 4B). For animals with moderately reduced motor conduction velocity it was 11.25 ms (mean for three animals)(Fig. 4D); responses were undetectable for the severely affected animals.
Genotype
Copy
no.Age
(days)Sex
Right MCV
(m/s)Left MCV
(m/s)Right DML
(ms)Left DML
(ms)
Wild type
0
30
F
34.1
25.0
0.66
0.85
Wild type
0
83
F
35.9
38.2
0.80
0.95
Wild type
0
83
F
34.4
34.7
0.74
0.75
Wild type
0
140
F
40.0
39.0
0.73
0.90
Wild type
0
320
F
47.0
49.0
0.88
1.04
Wild type
0
52
M
37.3
-
0.77
-
Wild type
0
211
M
36.2
46.0
0.68
0.96
C22 het
7
30
F
NR
1.7
NR
5.6
C22 het
7
30
F
NR
NR
NR
NR
C22 het
7
83
F
1.4
3.4
5.02
3.36
C22 het
7
83
F
NR
NR
NR
NR
C22 het
7
118
F
7.3
7.0
2.81
3.56
C22 het
7
336
F
NR
NR
8.0
2.74
C22 het
7
52
M
3.0
2.6
4.40
4.50
C22 het
7
140
M
NR
NR
NR
NR
C22 het
7
211
M
3.4
NR
3.4
NR
C61 hom
8
299
F
5.1
3.4
1.57
1.74
C61 het
4
214
F
24.0
26.0
1.19
1.08
C1 hom
4
305
F
28.0
24.0
0.84
1.50
C1 het
2
233
F
45.0
47.0
0.73
1.00
C58 hom
4
305
M
21.0
21.0
1.21
1.74
C58 het
2
221
F
46.0
51.0
0.84
0.94
C2 hom
2
139
M
44.0
37.0
0.89
0.88
Histology
Comparison of sections of sciatic nerves from the same mice as used for the nerve conduction studies showed different degrees of severity of demyelination depending on the genotype. C22 heterozygous and C61 homozygous mice showed severe demyelination affecting most of the large axons (Fig. 5A). C1 homozygotes, C58 homozygotes and C61 heterozygotes all had intermediate degrees of demyelination (Fig. 5B), while C2 homozygotes, C1 heterozygotes and C58 heterozygotes showed only occasional demyelination of degenerating fibres (Fig. 5C).
Figure
Examination of a variety of nerves by light microscopy showed different levels of demyelination. When different fascicles of a nerve and plexus were considered, such as the median nerve and brachial plexus, they could have quite different levels of demyelination (Fig. 6A-D). In other nerve specimens, for instance the sciatic nerve, there were patches of demyelinated fibres and patches of myelinated fibres (Fig. 6E).
Figure
Electronmicroscopic examination of the nerves of mice of different genotypes showed that they all had a similar pattern of demyelination although different numbers of fibres were affected. Features included uncompacted myelin characteristically on the outsides of the myelin sheaths of medium sized axons (Fig. 7A), demyelination of large axons with reduplicated basal lamina (basal lamina onion bulbs)(Fig. 7B), completely demyelinated large axons (Fig. 7C), and macrophages indicating the occurrence of active demyelination (Fig. 7D).
Figure
DISCUSSION
In this paper we report the generation of transgenic mice carrying one, two, four, seven or eight copies of the human PMP22 gene carried on a YAC, and their associated phenotypes. In lines with different integration sites but the same number of copies, the level of expression of human versus mouse mRNA is very similar indicating that the position of integration of the gene is not affecting the level of expression. The level of expression of human mRNA is also roughly proportional to the copy number of the transgene. Thus the expression appears to be position independent and copy number dependent as expected for a gene surrounded by a large region of genomic DNA. However, each transgene only gives about half as much expression as each single mouse gene which could be due to the transgenes being of human origin and thus not functioning completely efficiently in the mouse environment. The integrations at different loci also indicate that the observed phenotypes are not due to disruptions of genes on integration.
Exploration of the different lines, heterozygotes as well as homozygotes, has led to the following observations. In mice with one or two copies of the transgene, there was no detectable phenotype abnormality, the mice bred well, only minor histological abnormalities were present and motor nerve conduction was also normal. These mice expressed about half as much human as mouse messenger RNA which is equivalent to the mice having one extra copy of the PMP22 gene. One might therefore expect to see a phenotype comparable with human CMT1A whereas in fact an abnormal phenotype is not detectable. Possibly, over-expression of the human gene is not affecting the mice to the same extent as over-expression of the human gene affects humans. Alternatively, clinical signs appear during the first and second decades of life in humans, mainly related to loss of axons. This aspect would make the comparison of relatively young mice with older humans inappropriate.
In mice with four copies of the gene there was slightly more human than mouse mRNA, equivalent in level of expression to two extra copies of the mouse gene. There is no overt weakness or paralysis, although they do not move as smoothly as wild-type and the C1 and C58 homozygotes did not mate well. However, there was considerable demyelination in the peripheral nerves of these mice. Also the motor nerve conduction velocities, distal motor latencies and F wave latencies were intermediate between the normal and the severely affected mice. The phenotype is roughly equivalent to early CMT1A in terms of slowed nerve conduction, the degree of demyelination and slight physical disability, but the hypermyelination (28) is not reproduced.
In mice with seven or eight copies there was ~1.5 times as much human as mouse mRNA. There is a severe phenotype of weakness and progressive paralysis of the rear legs as described previously for the C22 line (25). These mice also had severe demyelination of the peripheral nerves and very slow nerve conduction velocities and prolonged distal motor latencies.
One of the most striking features is the high degree of correlation between the number of YAC copies, the level of expression and the phenotypes in different lines. Two examples are particularly illustrative. C1 heterozygotes (two copies) have no abnormal phenotype. C1 homozygotes (four copies) have a mild demyelination and intermediate nerve conduction velocity. C61 heterozygotes (four copies) present the same mild demyelination and intermediate nerve conduction velocity as C1 homozygotes (same number of YAC copies and same level of transgene expression). However, these lines do not present any overt disability. Finally, C61 bred to homozygosity (eight copies) has a severe peripheral neuropathy similar to C22 heterozygotes (seven copies).
One question about our mice is whether the human transgene is acting in the same way that over-expression of the mouse gene would, or whether it is acting more like a point mutation in the PMP22 gene due to differences between the mouse and human proteins. The published rat model with three copies of the mouse gene has a visibly abnormal phenotype and corresponds to a condition somewhat milder than our severe class (24). The nerve histology is quite different with the rats having extensive onion bulbs at 6 months of age while our mice have rare basal lamina onion bulbs. This could either be due to the fact that we have a mouse rather than a rat model or that we have over-expressed the human rather than the mouse gene. There is also a published mouse model with 16 or more copies of the mouse Pmp22 gene which has almost no peripheral myelin (26). This is clearly more severe than our mice with seven or eight copies of the human gene which have an appreciable amount of myelin. Unfortunately, as the mice with the mouse Pmp22 transgene are much more severely affected than our mice with the human transgene, a comparison of the histologies does not address the question of whether the mouse and human transgenes are acting by the same mechanism.
An important observation is that there appears to be a threshold level of expression under which there is almost no effect on the histology and nerve conduction velocity. Thus, mice expressing up to 0.8 times as much human as mouse mRNA are barely affected while as soon as the expression is >~1:1 the demyelination gets rapidly worse. CMT1A patients with a 17p11.2 duplication present with variable degrees of severity, including asymptomatic carriers. This has led authors to suggest the existence of modifier genes (29). However, a report about twins with variable degrees of expression of the disorder (30), makes the hypothesis of modifier genes, strongly affecting the phenotype, unlikely. From our data, we would suggest that the over-expression in humans is close to the threshold, and that variations in severity among patients could be due to individual variations in the expression of PMP22, which place the patient on one or other side of the threshold. As the relationship between over-expression and phenotypes seems not to be linear, small variations of expression of PMP22 can have a strong effect on the phenotype. If this holds true for humans, it suggests that a partial correction of the over-expression in patients could have a strong effect on the pathology, and that it will not be necessary to totally suppress the over-expression to benefit patients.
The other important points raised by our work concern the histology of the different lines. The histological findings indicate that over-expression of the PMP22 gene does not prevent the association of Schwann cells with axons and initiation of myelination but that with increased expression of the gene the Schwann cells become incapable of maintaining myelination, in particular on larger axons. The variation in the degree of demyelination both within a nerve fascicle and between fascicles is of interest, perhaps suggesting differential vulnerability of different functional classes of nerve fibres. This requires further study. As the level of expression increases, the number of axons affected and the amount of demyelination increases but the process of demyelination (thin or absent myelin on the larger axons, decompaction of the outside of the myelin sheath and some basal lamina onion bulbs) remains the same. This indicates that it is the same mechanism acting in all the mice but with different severity.
The different lines we have constructed represent a unique tool to test different approaches to correct the phenotype. This is particularly true as the different lines have been bred now up to the eighth generation, without any variation in the expression of the transgene, or expression of the various phenotypes.
MATERIALS AND METHODS
Generation of transgenic mice
The intact YAC DNA was prepared as described previously (31,25). Transgenic mice were generated by pronuclear injection using standard techniques (32). Mice used for the generation of transgenics and for all subsequent crosses were C57BL/6J × CBA/Ca F1. Homozygotes were identified by crossing to wild-type mice.
DNA analysis and FISH
Mouse DNA was extracted from cultured fibroblasts using standard procedures both as low-molecular-weight DNA for analysis of copy number and as high-molecular-weight DNA in agarose blocks. Blocks were digested overnight with 20 U of SfiI. Pulsed field gel electrophoresis was performed on a BioRad CHEF DRII with a pulse time of 10-45 s over 33 h at 160 V. Gels were blotted on PALL membranes in 10 × SSC overnight. The probe was labelled using random priming and stringent washes were performed at 0.1 × SSC at 65°C. The signal was imaged and quantitated with a phosphorimager. The PMP22 exon 2 probe does cross-hybridize with the mouse gene but the sizes of the HindIII and SfiI fragments differ between mouse and human DNA.
For FISH analysis, Alu-PCR products from the YAC 49G7 were synthesized and labelled as described elsewhere (33). One hundred ng of labelled probe was pre-annealed to 20-fold excess of Cot1 DNA for 20 min at 37°C and then precipitated in ethanol and centrifuged. The pellet was resuspended in 10 µl and hybridized to denatured chromosome spreads at 37°C overnight in 50% formamide. The probe sequences were detected with avidin-FITC (Sigma). Slides were examined on a Zeiss Axiophot microscope equipped with a 3 CCD camera.
RNA analysis
Sciatic nerves were dissected from both legs of a mouse, snap frozen in liquid nitrogen and stored at -80°C for up to 3 weeks. Poly(A)+ RNA was extracted using the Invitrogen Micro-Fast Track kit according to the manufacturer's instructions. cDNA synthesis was carried out using the Invitrogen cDNA cycle kit. In order to quantify the level of human transgene mRNA in relation to mouse mRNA, the cDNA was amplified using two primers; 5[prime]-GTC TCC ACC/G ATC GTC AGC CAA TG-3[prime] (starts at position 249 of accession D11428) and 5[prime]-CTC ATC ACG CAC AGA CCA GCA AG-3[prime] (starts at position 523 of accession D11428) which are homologous to both the mouse and the human cDNAs and amplify across an intron. The 275 bp human product cuts with TaqI to give two fragments of 154 and 121 bp while the mouse product cuts with AluI to give fragments of 198 and 77 bp. Amplification for 24-32 cycles of PCR was found to give the same ratio of human to mouse product, so 30 cycles was used for quantification. 33P was incorporated during the PCR and the 2% agarose gel was dried and quantitated using a Phosphorimager (Molecular Dynamics, `Imagequant' program).
Histology
The nerves were fixed either with 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide (Fig. 7A and B) or in 1% paraformaldehyde/1% glutaraldehyde in 0.1 M PIPES [piperazine-N,N[prime]-bis(2-ethanesulfonic acid)] buffer at pH 7.4 with postfixation in 1% osmium tetroxide in the same buffer plus 2% sucrose, 3% sodium iodate and 1.5% potassium ferricyanide (Figs 5 and 7C and D). The specimens were embedded in resin after dehydration through graded concentrations of ethanol. Semithin sections were stained with haematoxylin-phloxin-saffron and paraphenylene diamine (PDD) (Fig. 6). Ultrathin sections for electron microscopy were contrasted with lead citrate and methanolic uranyl acetate (Figs 5 and 7).
Electrophysiological recordings
Electromyographic measurements were carried out under terminal anaesthesia. Terminal anaesthesia was achieved by intraperitoneal injection of between 0.15 and 0.5 ml of a 1 in 10 dilution of Sagatal. The severely demyelinated mice (C22 line) were more sensitive to Sagatal; they became anaesthetized with less and occasionally died rapidly. Recordings were obtained via a fine concentric needle electrode inserted into the muscles of the first interosseous space of the hind foot employing a Medelec Saphire IL electromyograph. The sciatic nerve at the sciatic notch and the tibial nerve at the ankle were stimulated via fine stainless steel electrodes, the anode being inserted in the midline sacral region. Latency measurements were made by an electronic cursor on the oscilloscope screen. The tracings were also stored photographically. For the calculation of motor nerve conduction velocity between the proximal and distal recording sites, measurements of interelectrode distance were made on the skin with calipers with the leg in the fully extended position used for the recordings. In initial experiments, the accuracy of the surface measurement was verified by the injection of India ink through needles inserted at the sites of the stimulating electrodes. Throughout the recording the temperature of the limb was maintained at 38 ± 0.5°C using an electrically heated plate to which the animal was strapped by adhesive tape, and by a lamp above the animal.
ACKNOWLEDGEMENTS
We thank Dr Feuerstein for his help in mouse dissection and Gail Baker in genotyping. This work has been supported by AFM (Association Française contre les Myopathies) and the Wellcome, Leverhulme and Philip and Barbara Attenborough Trusts.
REFERENCES
*To whom correspondence should be addressed. M. Fontés: Tel: +33 91 78 44 77; Fax: +33 91 80 43 19; C. Huxley: Tel: +44 171 594 3771; Fax: +44 171 706 3272; Email: c.huxley@ic.ac.uk
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 14 Feb 1998
Copyright© Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Robaglia-Schlupp, J. Pizant, J.-C. Norreel, E. Passage, D. Saberan-Djoneidi, J.-L. Ansaldi, L. Vinay, D. Figarella-Branger, N. Levy, F. Clarac, et al. PMP22 overexpression causes dysmyelination in mice Brain, October 1, 2002; 125(10): 2213 - 2221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Perea, A. Robertson, T. Tolmachova, J. Muddle, R. H. M. King, S. Ponsford, P. K. Thomas, and C. Huxley Induced myelination and demyelination in a conditional mouse model of Charcot-Marie-Tooth disease type 1A Hum. Mol. Genet., May 1, 2001; 10(10): 1007 - 1018. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Niemann, M. W. Sereda, U. Suter, I. R. Griffiths, and K.-A. Nave Uncoupling of Myelin Assembly and Schwann Cell Differentiation by Transgenic Overexpression of Peripheral Myelin Protein 22 J. Neurosci., June 1, 2000; 20(11): 4120 - 4128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wrabetz, M. L. Feltri, A. Quattrini, D. Imperiale, S. Previtali, M. D'Antonio, R. Martini, X. Yin, B. D. Trapp, L. Zhou, et al. P0 Glycoprotein Overexpression Causes Congenital Hypomyelination of Peripheral Nerves J. Cell Biol., March 6, 2000; 148(5): 1021 - 1034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sancho, J. P. Magyar, A. Aguzzi, and U. Suter1 Distal axonopathy in peripheral nerves of PMP22-mutant mice Brain, August 1, 1999; 122(8): 1563 - 1577. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||