Research Report Internship 2016
Introduction
Megalencephalic leukoencephalopathy with
subcortical cysts (MLC) is a genetic disease that affects the white matter of the brain and is characterized by infantile onset1,2. MLC patients
develop macrocephaly in the first year of life, after which the rate of head growth stabilizes1,2.
Several years later, patients develop slowly progressive cerebellar ataxia, spasticity, mild cognitive deterioration and occasional epileptic seizures1-4. Magnetic resonance imaging (MRI)
scans of MLC patients show diffuse signal abnormality in combination with swelling of the cerebral white matter and subcortical cysts1.
MLC is associated with mutations in the
Mlc1 and Glialcam genes5-7. MLC1 is a plasma
membrane protein expressed in the brain and to a low degree in leukocytes8. In the brain, MLC1 is
exclusively located in astrocytes, at astrocytic endfeet around blood vessels and the CSF-brain barrier, in Bergmann glia and ependymal cells8,9.
GlialCAM is a cell adhesion molecule found predominantly in the brain. A role of GlialCAM is to direct MLC1 to its correct location in
astrocytic endfeet where they co-localize6,10,11.
Several studies have shown that the two proteins are functionally dependent on each other6,10,11.
Glialcam-null mice show a strong decrease in the
expression of MLC1 and a mislocalization of both GlialCAM and MLC110,11. Likewise, loss of
MLC1 changes the localization of GlialCAM and MLC110.
MLC1 and GlialCAM play a crucial role
in regulating ion homeostasis and water balance in the brain6,12,13. Tight regulation of the water-ion
homeostasis is essential for normal brain functioning and highly depends on astrocytes14.
Mutations in MLC1/Mlc1 decrease cell swelling activated volume regulated anion channel (VRAC) currents in astrocytes. This is accompanied by a delayed regulatory volume decrease (RVD)12,13.
MLC patients, and Mlc1-null and Glialcam-null mice, have fluid-filled vacuoles within myelin sheaths and astrocytic endfeet10,13,15. Normal
myelination is important for the fast conduction of action potentials along axons. Myelin vacuolation can result in conduction delays, as seen in mice carrying a null mutation in the
AXONAL ACTION POTENTIAL PROPAGATION IN A
MOUSE MODEL FOR MEGALENCEPHALIC
LEUKOENCEPHALOPATHY WITH SUBCORTICAL
CYSTS (MLC)
Abstract
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an infantile-onset cerebral white matter disease with autosomal recessive inheritance. In the majority of the patients, the disease is caused by mutations in the MLC1 gene. MLC1 is a plasma membrane protein predominantly expressed in astrocytes. It has been demonstrated that MLC1 is involved in the volume regulation of astrocytes. MLC1/Mlc1 mutations are characterized by fluid-filled vacuoles within myelin sheaths and astrocytic endfeet. Intramyelinic vacuolation is associated with a hampered conduction of action potentials along axons. We tested how loss of MLC1 function in a mouse model for MLC may affect the axonal action potential propagation. To test this, we measured compound action potentials (CAPs) in the corpus callosum of acute brain slices upon stimulation and determined the conduction velocity. We found that conduction velocity in myelinated and unmyelinated axons of the corpus callosum is unaffected by the loss of MLC1.
08
Fall
Student: Rosanne Tuip Student number: 10276912 Daily supervisor: Eelke Brouwers1
Principal Investigators: Rogier Min1, Marjo van der Knaap1 & Huib Mansvelder2
Co-assessor: Ilja Boor
1Department of Pediatrics/Child Neurology, Neuroscience Campus Amsterdam, VU University Medical Center 2Department of Integrative
Neurophysiology, Center for Neurogenomics and Cognitive Research, VU University
chloride channel CLC-216,17. However, at this
point it is unknown whether white matter vacuolation in MLC affects the axonal propagation.
Studies in both healthy and transgenic rodents have revealed that the manifestation of vacuoles is activity dependent16-18. For example,
upon evoked seizures, Mlc1-null mice show an increase in myelin vacuoles throughout the brain18. It would be interesting to see whether this
effect, observed in vivo, can be reproduced by long duration stimulation in brain slices.
In the current study we aimed to investigate the axonal propagation of action potentials in a mouse model for the white matter disease MLC13. For this aim, extracellular
recordings were performed in the corpus callosum of acute brain slices from 3-5 month-old mice. The speed of action potential propagation and the action potential amplitudes were measured and calculated after single stimulating pulses and upon long duration stimulation. Subsequently, the sections were processed for the quantification of vacuoles.
Based on findings of previous
research16,19,21, we expected that the speed of
action potential propagation is decreased in the
Mlc1-null mice compared to wild-type (WT)
littermates in response to single stimulation pulses. Moreover, we expected the conduction velocity to decrease and a reduction in signal amplitude at earlier time points in Mlc1-null mice during long duration stimulation. Additionally, we predicted that the amount of vacuoles is increased to a greater extent in Mlc1-null mice compared to wild-type mice upon long duration stimulation.
This study will help us to gain understanding on the influence of white matter vacuolization, as seen in MLC and several other
leukodystrophies, on action potential
propagation. Another objective of the study was to better understand how neural activity relates to the development of vacuoles.
Materials and methods
Animals
All experiments were conducted with the approval of the Animals Ethical Committee at the VU University in Amsterdam and in accordance with Dutch regulations. Experiments were performed on 9 acute brain slices of 3-5 month-old C57BL/6-Tg(Mlc1-Egfp) transgenic mice,
‘Mlc1-null mice’ (n=4), and 7 acute brain slices of wild-type (WT) C57BL/6 littermates (n=5) of matching age. The generation of
C57BL/6-Tg(Mlc1-Egfp) transgenic mice was previously
described by Dubey et al., (2015). These mice express enhanced green fluorescent protein GFP (eGFP) instead of MLC1.
Brain slice preparation
The mice were rapidly decapitated and their brains dissected in sucrose-based slicing solution,
consisting of 25 mM D-glucose, 25 mM
NaHCO3, 70 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 5 mM MgSO4, 1 mM CaCl2, 70 mM
sucrose, 1 mM sodium ascorbate and 3 mM sodium pyruvate, and adjusted to 300 ± 5 mOsm. The brains were cut with a vibrating microtome (Thermo Scientific Microm HM 650 V) to obtain 400 μm-thick coronal sections and from this moment on, the sections were constantly oxygenated with carbogen gas (95% O2, 5%
CO2). Directly after slicing, the sections were
kept in 32 °C sucrose-based slicing solution for 20 minutes. After this recovery period, the sections were transferred to a holding artificial cerebrospinal fluid (aCSF) solution, containing
10 mM D-glucose, 26 mM NaHCO3, 125 mM NaCl, 3 mM KCl, 1.2 mM NaH2PO4, 2 mM
MgCl2, 2 mM CaCl2, 1 mM sodium ascorbate and
3 mM sodium pyruvate (300-305 mOsm), and kept at room temperature. Sections containing an intact corpus callosum were selected for electrophysiological recordings.
Electrodes
Clark borosilicate theta glass capillaries (2.0 OD x 0.3 wall x 0.22 septum x 150 L mm, Warner Instruments, LLC, Hamden, CT) were pulled with a single stage glass microelectrode puller (model PP-830, Narishige, Tokyo, Japan), after which a piece of the tip was cut off to create glass capillaries with a tip of 50 μm in diameter. These capillaries consist of 2 compartments separated by a thin membrane, each 25 μm in diameter at the tip after cutting. 2 silver chloride electrodes were connected to the output poles of the stimulus isolator, one to the positive pole and one to the negative pole. The glass capillaries were filled with aCSF and the silver chloride electrodes were inserted in either one of the compartments.
To produce the recording electrodes, Clark borosilicate standard wall glass capillaries (1.5 OD x 0.86 x 100 L mm, Warner Instruments,
LLC, Hamden, CT) were pulled with a
micropipette puller (model P-87, Sutter
Instrument, Novato, CA). Glass electrodes were pulled to obtain a tip resistance of 2.5- 3 MΩ. The electrodes were filled with aCSF and fixed on the silver chloride electrodes on the headstages.
Compound action potential (CAP)
measurements
A compound action potential (CAP) is the summation of all action potentials generated by all axons in a fiber tract19. A CAP consists of a
fast component, N1, which reflects the fast depolarization of the myelinated axons, followed by a slow component, N2, representing the slower depolarization of the unmyelinated axons.
All electrophysiological recordings were
performed at room temperature.
Brain sections were transferred to a recording chamber containing standard aCSF, consisting of 10 mM D-glucose, 26 mM NaHCO3, 125 mM NaCl, 3 mM KCl, 1.2 mM NaH2PO4, 1 mM MgCl2, 2 mM CaCl2 (300-305 mOsm). The flowing speed of the aCSF was set at 2 mL/min via a gravity perfusion system (exodrop, B Braun, Melsingen, Germany). A stimulating electrode was lowered into the corpus callosum 80–160 μm from the midline. Two recording electrodes were placed in the corpus callosum of the contralateral hemisphere at a distance of 780 ± 73,2 μm and 1,2 mm ± 87,2 μm away from the stimulation electrode. The CAPs were recorded in voltage clamp mode set at a membrane potential of 0 mV.
Before investigating the conduction speed and CAP amplitude, the maximum stimulus intensity was specified for each section19. During this process, every 6 seconds
(0.16 Hz) a monophasic current pulse was applied while the intensity of the stimulus pulses was varied from threshold spiking level until the maximum amplitude was reached. The maximum amplitudes of the N1 and N2 components of the CAPs were subjectively determined during the recordings.
During the next step, CAPs were generated by presenting monophasic single stimulating current pulses with an intensity that induced CAPs with maximum N1 and N2 amplitudes (figure1, left). During this low frequency single stimulation protocol the current pulses were 6 seconds apart. The peak latencies of the N1 and N2 components were determined at
both recording sites and used to calculate the conduction velocity19.
Subsequently, current pulses were
applied with a frequency of 5 HZ for a period of 1 hour (figure 1, middle). The development of mean conduction velocity and mean N1 and N2 amplitudes will be determined during this stimulation period.
During the recovery period, the sections were presented with current pulses every 10 seconds (0.1 Hz) for 5 minutes (figure 1, right).
All electrophysiological recordings were
performed blind for the genotype.
Vacuolation quantification
The brain sections were postfixed in 1.5 % paraformaldehyde (PFA) for 4-8 weeks after each CAP measurement. Subsequently, the sections were fixed in 4% PFA for at least 6 hours and
embedded in paraffin. Paraffin-embedded
sections were cut to obtain 5 μm-think coronal sections. Sections were histochemically stained with hematoxylin and eosin (H&E)21. Vacuoles
were visualized under a light microscope and quantified using ImageJ.
Statistical Analysis
Electrophysiology data were analyzed with Matlab and differences between Mlc1-null and wildtype mice were evaluated using an unpaired t-test or a Mann-Whitney U test with GraphPad Prism. The staining data will be tested for significant differences in number of vacuoles with the unpaired t-test or the Mann-Whitney U with GraphPad Prism. Results are presented as mean ± SD. A p-value of < 0.05 was considered significant.
Figure 1. Stimulation protocol. For maximum single stimulation pulses, current pulses were generated every 6 seconds (left). During the long duration stimulation protocol, current pulses were applied at 5 Hz for 1 hour (middle). The recovery period consisted of 5 minutes current pulses separated by 10 seconds (right).
Results
Normal conduction velocity in
Mlc1-null mice
To evaluate the consequence of MLC1 loss on the action potential propagation in the corpus callosum, CAPs were recorded in coronal brain slices at two different sites (figure 2A). Typical CAP traces are shown in figure 2B.
In response to single stimulation pulses of maximum stimulus intensity, the average conduction velocity along myelinated axons (N1) in Mlc1-null mice (n = 9) was 2.22 ± 2.08 m/s. This was not significantly different compared to a conduction velocity of 1.15 ± 0.19 m/s for wild-type mice (n = 7) (figure 3A; p > 0.05). Three
Mlc1-null mice showed notably high N1
conduction velocities. The conduction velocities of these animals, 3.06 m/s, 3.5 m/s and 7.1 m/s, are depicted in the upper part of figure 3A. These conduction velocities are from a biological perspective not possible19,32. In addition, a N1
conduction velocity of 7.1 m/s is greater than twice the standard deviation (2.1 m/s), significantly deviating from the mean. Thus, the analysis was repeated while excluding these animals. Excluding these animals slightly reduced the average N1 conduction velocity to 1.05 ± 0.17 m/s for Mlc1-null mice. This did not change the statistical outcome (figure 3B; p > 0.05).
The average conduction velocity along unmyelinated axons (N2) was 0.51 ± 0.2 m/s in the Mlc1-null group. This was not significantly different in comparison to 0.48 ± 0.1 m/s for wild-type mice (figure 4A; p > 0.05). For one
Mlc1-null mouse a conduction velocity of 1 m/s
was observed. This value is biologically not possible. Moreover, it is outside the error margin of the mean, as it is greater that twice the standard deviation of 0.2 m/s. Exclusion of this animal changed the average N2 conduction velocity of the Mlc1-null group to 0.45 ± 0.07 m/s. This did not change the statistical outcome for (figure 4B; p > 0.05).
To ensure that observed conduction velocities of the ‘outliers’ were not unreliably determined by the presence of an outlier within the CAP recordings, we compared the conduction velocities after each individual stimulation pulse for these animals. The conduction velocities were increased in response to the majority of the stimulation pulses for all three animals when
compared to the corresponding group mean (Supplementary figure S1).
The development of conduction
velocity during single stimulation
pulses
The conduction velocity could change over the course of the 10 single stimulation pulses. To test this, we compared the conduction velocity of N1 and N2 in response to the first stimulation pulse with the conduction velocity after the last stimulation pulse. The Mlc1-null mouse that showed a N1 conduction velocity of 7.1 m/s had a time difference of the N1 peak recorded at the proximal and distal electrode of 0 s after the last stimulation pulse. As a result, the conduction velocity could not be calculated and this mouse was excluded from the analysis.
For wild-type mice, the conduction velocity of N1 was 1.06 ± 0.29 m/s upon the first stimulation pulse. This did not significantly differ from a conduction velocity of 1.22 ± 0.22 m/s found after the last stimulation pulse (figure 5A; p > 0.05). In Mlc1-null mice, a N1 conduction velocity of 1.51 ± 0.8 m/s after the first and 1.57 + 1 m/s after the last stimulation pulse did also not significantly differ (figure 5B;
p > 0.05). The conduction velocity of N2 was
0.47 + 0.15 m/s after the first stimulation pulse in wild-type mice. No significant difference was observed when compared to the conduction velocity of 0.41 + 0.11 m/s after the last stimulation pulse (figure 6A; p > 0.05). Similarly, no significant difference for N2 was found between a conduction velocity of 0.58 ± 0.32 m/s after the first and 0.51 + 0.17 m/s upon the last pulse in Mlc1-null mice (figure 6B; p > 0.05).
Discussion
Patients with MLC develop macrocephaly in infancy1,2. Brain MRI reveals diffuse white
matter signal abnormality related to elevated water content of the white matter1,22. In brain
biopsies of MLC patients, fluid-filled vacuoles were observed within myelin sheaths and the endfeet of astrocytes15. These vacuoles were also
found in brain tissue of Mlc1-null mice10,13.
White matter vacuolation has been associated with a reduced axonal conduction velocity in mice lacking CLCN216. In the present study we
Figure 2. Experimental set-up CAP recording. A. Coronal brain section including a stimulation electrode (stim) lowered into the corpus callosum and two recording electrodes (rec) in the contralateral hemisphere. B. Two representative CAP traces recorded at the proximal (red) and distal (black) recording site. The two CAP peaks correspond to N1 and N2 respectively.
Figure 3. The conduction velocity in myelinated axons of the corpus callosum in Mlc1-null mice and wild-type mice. A. Plot showing the conduction velocity in myelinated axons (N1) in Mlc1-null mice (green) and wild-type mice (blue) (n = 7 brain slices for wild-type and n = 9 brain slices for Mlc1-null). p > 0.05. The average conduction velocity is 2.22 m/s in Mlc1-null and 1.15 m/s in wild-type mice. Three Mlc1-null mice show biologically unexplainable conduction velocities of 3.06 m/s, 3.5 m/s and 7.1 m/s B. Plot showing the N1 conduction velocity in Mlc1-null mice (green) and wild-type mice (blue) after excluding the outliers (n = 7 brain slices for wild-type and n = 6 brain slices for Mlc1-null). p > 0.05. The new average conduction velocity for Mlc1-null mice is 1.05 m/s.
Figure 4. The conduction velocity in unmyelinated axon of the corpus callosum in Mlc1-null mice and wild-type mice. A. Plot
B
A
B
A
B
showing the conduction velocity in unmyelinated axons (N2) in Mlc1-null mice (green) and wild-type mice (blue) (n = 7 brain slices for wild-type and n = 9 brain slices for Mlc1-null). p > 0.05. The average conduction velocity is 0.48 m/s for Mlc1-null and 0.51 m/s for wild-type mice. B. Plot showing the conduction velocity in unmyelinated axons (N2) in Mlc1-null mice (green) and wild-type mice (blue) after excluding the outlier (n = 7 brain slices for wild-type and n = 8 brain slices for Mlc1-null). p > 0.05. The new average conduction velocity for Mlc1-null mice is 0.45 m/s.
Figure 5. The conduction velocity of N1 after the first and last single stimulation pulse. A. The conduction velocity along myelinated axons for wild-type mice was 1.06 m/s after the first and 1.22 m/s after the last stimulation pulse for (n = 7 brain slices). p > 0.05. B. In Mlc1-null mice a N1 conduction velocity of 1.51 m/s was observed upon the first pulse and 1.57 m/s upon the last pulse (n = 8 brain slices). p > 0.05.
Figure 6. The conduction velocity of N2 after the first and last single stimulation pulse. A. For wild-type mice, the conduction velocity along unmyelinated axons was 0.47 m/s after the first and 0.41 m/s after the last stimulation pulse (n = 7 brain slices). p > 0.05. B. In Mlc1-null, the N2 conduction velocity upon the first pulse was 0.58 m/s and upon the last pulse 0.51 m/s (n = 8 brain slices). p > 0.05.
propagation is altered in Mlc1-null mice and how prolonged increased neural activity affects
myelin vacuolation. Based on previous
observations we hypothesized that the axonal action potential propagation is altered in
Mlc1-null mice and the amount of vacuoles to be increased after high neural activity. For that reason, we structured our experimental setup in such a way as to determine the conduction velocity and amplitude of CAPs after single
A
B
stimulation pulses and during a long duration stimulation protocol.
Due to unforeseen complications
concerning data-analyses of the long duration
stimulation CAP measurements and the
vacuolation quantification, these data could not be included in this paper. Therefore, the conclusions we draw here are based on the outcome of the single stimulation recordings in combination with the findings from previous studies. We demonstrate that the conduction velocity of CAPs in the corpus callosum is not
significantly different in Mlc1-null mice
compared to wild-type mice upon single stimulating pulses.
MLC1 is involved in controlling the
ion-water homeostasis7. This strengthens the
suggestion that the formation of intramyelinic vacuoles is caused by the accumulation of potassium and water as a result of a disturbed potassium and water redistribution23.
Well-regulated ion-water homeostasis is essential for action potentials to propagate along axons. The repolarization of the membrane potential after the depolarizing effect of the initial sodium influx, depends on an efflux of potassium24. The excess
potassium ions are transported through the panglial syncytium, a network of interconnected oligodendrocytes, astrocytes and ependymal cells, linked by gap junctions25.
Over the years, the notion that astrocytes act as spatial buffers of potassium has been a widely accepted theory26,27. While the precise
mechanisms remain unknown, the theory holds that astrocytes take up the excess potassium and thereby restore the extracellular concentration of potassium28. This process causes the astrocytes to
swell. After initial swelling, the astrocytes regain their original cell volume. The astrocytes release potassium into the extracellular space at sites with reduced potassium levels and the potassium ions are supposedly eventually transported to the peri-capillary space around blood vessels or the
cerebrospinal fluid28,29. The movement of
potassium through the panglial syncytium is accompanied by the diffusion of obligatory-associated osmotic water28. In MLC, swelling
induced RVD is slowed down in astrocytes12.
Several disorders that are associated with the disruption of the panglial syncytium are characterized with reduced or blocked action potential conduction caused by vacuoles within myelin sheaths16,21,28. Examples are
Charcot-Marie-Tooth (CMT) neuropathy, caused by
mutations in connexin32, and
leukoencephalopathies caused by mutations in the chloride channel CLC-216. However, in MLC
we did not find conduction delays. This contradiction may be clarified by a less severe and slower vacuolation in Mlc1-null mice compared to Clcn2-null mice10. Moreover, in
Mlc1-null mice, the corpus callosum contains
smaller vacuoles compared to other white matter areas, and is even mostly spared in MLC patients1,13. The minor presence or absence of
intramyelinic vacuoles could explain the unaffected axonal action potential propagation. These postulations need to be verified by the anticipated data pertaining to myelin vacuolation.
A second possible explanation for the observed unaltered conduction velocity is that single stimulation pulses do not challenge the network enough. Early recordings on Amphibian astrocytes showed that higher stimulation frequencies of 5 Hz, as opposed to low-frequency stimulation pulses of 0.5 Hz, results in a stronger depolarization of astrocytes26. This indicates a
larger astrocytic take up of potassium28. We
speculate that during the long duration stimulation protocol (5 Hz), extracellular potassium will accumulate in both groups. Additionally, the slowed RVD of astrocytes in
Mlc1-null mice will lead to an increased
accumulation of extracellular potassium
compared to wild-type mice. A high extracellular
potassium concentration depolarizes the
membrane potential of the axons. As a result, the sodium channels open up and allow an influx of sodium. During the following repolarization phase, the membrane potential cannot fully reach the normal resting membrane potential and stays depolarized. Some sodium channels will stay in their inactivated state24,30. This would be reflected
by the reduction of conduction velocity and eventually conduction block. Ten stimulation pulses, separated by 6 seconds possibly gives the network enough time to recover and do not result in these physiological changes. This idea is supported by the finding that the conduction velocity between the first and the last stimulation pulse is similar. This was observed in both Mlc1-null and wild-type mice. We would expect to find conduction delays during the long duration stimulation recordings in both groups. In addition, we expect this to occur at earlier time points in Mlc1-null mice compared to the wild-type mice. Moreover, we expect an earlier reduction in N1 and N2 amplitudes Mlc1-null
mice as the number of axons able to fire action potentials would decrease faster. During a recovery period of single stimulation pulses, the decrease of extracellular potassium is expected to occur slower in Mlc1-null mice due to the disturbed RVD. This indicates that N1 and N2 amplitudes increase at a lower rate in this group. The data from the long duration stimulation and subsequent recovery measurements have to shed some light on these speculations.
Myelin vacuolation is activity
dependent16-18. For instance, induced epileptic
seizures in healthy rats lead to the development of vacuoles16. The long duration stimulation data
could reveal whether prolonged activity
increases, or induces, myelin vacuolation in
Mlc1-null mice and to what extent compared to
wild-type mice. We predict that the long duration stimulation induced accumulation of potassium and water causes the formation of vacuoles in the corpus callosum in both groups. The combination of high activity and a delayed RVD is expected to result in more severe vacuolation in the corpus callosum of Mlc1-null mice compared to wild-type mice. A different approach to study the formation of vacuoles is by incubating the brain slices in aCSF containing neural activity increasing compounds instead of electrophysiological stimulation. This might be an interesting experiment for the future.
In addition to a tight control of the ion-water homeostasis, optimal myelin conditions are essential for facilitating action potentials. Myelin sheaths around the internodal regions of axons act as electrical insulators, increasing the membrane resistance and reducing the membrane capacitance31. The high membrane resistance
prevents ions from flowing through the membrane. The low membrane capacitance reduces the amount of ions lined up along the membrane. These passive myelin properties increase the conduction velocity of propagating action potentials. While it has been proposed that demyelination decreases the membrane resistance and increases the membrane capacitance31, it is
unclear if and how intramyelinic vacuolation affects these myelin properties. It could be possible that only severe vacuolation, as observed in Clcn2-null mice, influences the membrane resistance and/or capacitance. The unaltered conduction velocity along unmyelinated axons is in line with our expectations. Logically, myelin
vacuoles in surrounding myelinated axons may
not alter the membrane properties of
unmyelinated axons.
Besides the finding that the conduction velocity is unchanged, we did another interesting observation. Three Mlc1-null mice showed increased conduction velocities along the myelinated axons (N1) of 3.1 m/s, 3.5 m/s and 7.1 m/s. Based on previous work that demonstrate a callosal N1 conduction velocity in mice of 1.1 – 1.7 m/s and a comparable average in this study, we assume that values of 3.1 m/s, 3.5 m/s and 7.1 m/s are not in a biologically explainable range19,32. The mice that showed a
conduction velocity for N1 of 3.1 m/s and 3.5 m/s
had normal conduction velocities along
unmyelinated axons (N2). Contrarily, the mouse with a N1 conduction velocity of 7.1 m/s also showed a (non-significant) higher conduction velocity of 1 m/s. The average conduction velocity in the corpus callosum of mice is 0.4-0.5 m/s, similar to what we found. It is known that with rising temperatures, the conduction velocity of action potentials increases33. We can affirm
that during these recordings, there were no
apparent methodological aberrations. One
possible theory holds that the stimulus artifact temporally overlapped the recorded signal. Significant distortion to the first peak, N1, could have occurred, decreasing the difference in latency between the two electrodes. Whereas this clarifies the results of the two mice with increased N1 conduction velocities and average values for N2, it does not explain the increased N2 conduction velocity in the third mouse. Importantly, it should be noted that these are merely theories and that currently we have no plausible explanation for these observations.
In summary, our findings indicate that during a period of low frequency activity, the speed of action potential propagation along both myelinated and unmyelinated axons of the corpus callosum is unaffected by loss of MLC1. Data of the long duration stimulation recordings should reveal how the conduction of action potentials develops during periods of prolonged activity. The quantification of vacuolation will help us gain more knowledge on the formation of intramyelinic vacuoles. As there is no known treatment for MLC, the results we provide here yield important information and guide the field of
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