ORIGINAL RESEARCH

Increase of c-Fos and c-Jun Expression in Spinal and CranialMotoneurons of the Degenerating Muscle Mouse (Scn8admu)

Hiroyuki Ichikawa • Mitsuhiro Kano • Yoshinaka Shimizu •

Toshihiko Suzuki • Eri Sawada • Wako Ono •

Leona W. G. Chu • Patrice D. Cote

Received: 23 November 2009 / Accepted: 13 January 2010 / Published online: 29 January 2010

� Springer Science+Business Media, LLC 2010

Abstract The degenerating muscle (dmu) mouse harbors

a loss-of-function mutation in the Scn8a gene, which

encodes the a subunit of the voltage-gated sodium channel

(VGSC) NaV1.6. The distribution of c-Fos and c-Jun was

examined in spinal and cranial motoneurons of the dmu

mouse. In the cervical spinal cord, trigeminal motor

nucleus (Vm), facial nucleus (VII), dorsal motor nucleus of

the vagus (X), and hypoglossal nucleus (XII) of wild-type

mice, motoneurons expressed c-Fos and c-Jun-immunore-

activity. The immunoreactivity in wild-type mice was

mostly weak and localized to the nucleus of these neurons

whereas in the spinal cord and brain stem of dmu mice

motoneurons showed intense c-Fos and c-Jun-immunore-

activity. The number of c-Fos-immunoreactive motoneu-

rons was dramatically elevated in the cervical spinal cord

(wild type, 4.8 ± 1.0; dmu, 17.3 ± 1.6), Vm (wild type,

76.2 ± 21.6; dmu, 216.9 ± 30.9), VII (wild type, 162.4 ±

43.3; dmu, 533.3 ± 41.2), and XII (wild type, 58.2 ± 43.3;

dmu, 150.9 ± 25.7). The mutation also increased the

number of c-Jun-immunoreactive motoneurons in the cer-

vical spinal cord (wild type, 1.6 ± 0.8; dmu, 12.1 ± 2.1),

Vm (wild type, 41.4 ± 18.0; dmu, 123.1 ± 11.7), and X

(wild type, 39.1 ± 10.7; dmu, 92.8 ± 17.8). The increase

of these transcription factors may be associated with the

uncoordinated and excessive movement of forelimbs and

degeneration of cardiac muscles in dmu mice.

Keywords Brain stem � c-Fos � dmu Mouse � c-Jun �Motoneuron � Spinal cord � Immunohistochemistry �Voltage-gated sodium channel � NaV1.6 � Scn8a

Introduction

There are many hereditary neuromuscular diseases in

humans. The cause of these diseases can be intrinsic

defects of motoneurons, neuromuscular junctions, or

muscle itself. Several mouse models have been character-

ized for the study of human muscle diseases to elucidate

their etiology. The degenerating muscle (dmu), mutation

arose spontaneously and homozygote individuals for this

autosomal recessive mutation begin to display a progres-

sive paralysis beginning at approximately 11 days after

birth (P11) and die at approximately P21, presumably of

respiratory weakness (De Repentigny et al. 2001). Histo-

pathological and ultrastructural observations reveal that

skeletal and cardiac muscle fibers are atrophied and the

tissues show focal areas of degeneration. However, no

significant morphological abnormalities are detected in

sciatic nerves. The analysis of candidate genes on mouse

chromosome 15 reveals that Scn8a, the gene encoding the

sodium channel 8a subunit (VGSC a subunit NaV1.6)

harbors a loss-of-function mutation and NaV1.6 channels

are absent in dmu mice (De Repentigny et al. 2001; Cote

et al. 2005) Thus as expected, the phenotype of dmu is very

similar to that of med (motor endplate disease) mice, which

H. Ichikawa (&) � M. Kano � Y. Shimizu � T. Suzuki �E. Sawada � W. Ono

Division of Oral and Craniofacial Anatomy, Tohoku University

Graduate School of Dentistry, Sendai, Miyagi 980-8575, Japan

e-mail: hiroichi@anat.dent.tohoku.ac.jp

L. W. G. Chu � P. D. Cote

Department of Biology and Neuroscience Institute Life Sciences

Center, Dalhousie University, Halifax, NS B3H 4J1, Canada

P. D. Cote

Department of Ophthalmology & Visual Sciences, QE II Health

Sciences Centre, Halifax, NS B3H 2Y9, Canada

123

Cell Mol Neurobiol (2010) 30:737–742

DOI 10.1007/s10571-010-9498-8

also harbor a loss-of-function mutation in Scn8a (Searle

1962; Meisler et al. 2004). Scn8a is known to be expressed

in the brain and spinal cord (Alessandri-Haber et al. 2002;

Schaller and Caldwell 2003; Gunasekaran et al. 2009).

Immunohistochemical studies have demonstrated that

NaV1.6 is localized to the node of Ranvier in motoneurons

in the spinal cord and Purkinje and granule cells in the

cerebellum. In the heart, the primary VGSC isoform

responsible for action potential conductance between

cardiomyocytes is NaV1.5. However, NaV1.6 along with

the other neuronal VGSCs NaV1.1 and NaV1.3 have been

localized to the transverse tubules of cardiomyocytes

where they have been hypothesized to play a role in elec-

tromechanical coupling based on tetrodotoxin (TTX)

blockade and b-scorpion toxin CssIV activation experi-

ments (Maier et al. 2002, 2004). The specific role of

NaV1.6 has not, however, been assessed. Although neuro-

nal sodium channels have also been implicated in sinoatrial

node pacemaking, NaV1.6 has not been found in sinoatrial

node cells (Maier et al. 2003; Lei et al. 2004; Yoo et al.

2006).

The transcription factor activator protein-1 consists of

a variety of dimers composed of members of the Fos and

Jun families of proteins. c-Fos and c-Jun genes are

induced and activated following physiological stimuli in

the brain and spinal cord (Sakura-Yamashita et al. 1991;

Pennypacker 1995; Tischmeyer and Grimm 1999). Pre-

vious studies have also demonstrated that these proteins

are upregulated after ischemia and stroke, in seizures, or

following axonal injury and during regeneration (Penny-

packer 1995, 1997 Gillardon et al. 1996). Thus, the pro-

teins are considered to be markers for neuronal activity

in physiological and pathological conditions. In addition,

c-Jun is considered to be essential for neuronal cell death

of injured motoneurons in young animals (Gillardon et al.

1996; Garrah et al. 1998). Interestingly TTX changes the

distribution of c-Fos- and c-Jun-positive neurons in the

brain after optical nerve injury (Dai et al. 2009). Differ-

ential induction of c-Fos and c-Jun in the lateral genicu-

late nucleus of rats following unilateral optic nerve injury

with contralateral retinal blockade is observed. Thus,

sodium channels may be associated with expression of

these transcription factors.

In this study, expression of c-Fos and c-Jun was exam-

ined in the spinal cord and brain stem of dmu mice to know

effects of the mutation on spinal and cranial motoneurons.

The behavioral change cannot be observed in dmu mice

within 10 days after birth (De Repentigny et al. 2001). In

addition,the expression of c-Fos and c-Jun is abundant in

the brain of normal animals during perinatal period (Gon-

zalez-Martın et al. 1992; White et al. 1994; Garrah et al.

1998). Thus, wild-type and dmu mice at postnatal day 18

were used for the analysis.

Materials and Methods

Animals

The dmu mutation arose spontaneously within our breeding

colony on a C57BL6XC3H F1 hybrid background

(De Repentigny et al. 2001) and has since been maintained

on a C57BL/6 genetic background. Mice were genotyped

as previously described (Cote et al. 2005). Five wild-type

and five dmu mice were used in this study. Eighteen-day-

old mice were deeply anesthetized with avertin (2,2,2-tribro-

moethanol; 275 mg/kg, i.p.), and transcardially perfused

with 4% paraformaldehyde in 0.1 M phosphate buffer (pH

7.4). The cervical spinal cord, brainstem, and cerebellum

were dissected, and immersion-fixed overnight in the same

fixative at 4�C. Subsequently, the tissues were stored at

4�C in 0.02 M phosphate-buffered saline (PBS) containing

0.1 mM sodium azide until use.

Staining Procedure

The materials were cryoprotected by immersing until

sunken in PBS containing 20% sucrose (pH 7.3), and

frontally and serially frozen-sectioned at 40 lm. Complete

series of free floating sections were divided into three

subsets. Each subseries of every third section was pro-

cessed for Nissl stain or immunohistochemistry. For c-Fos-

and c-Jun-immunoreactivity, the sections were incubated

overnight with rabbit antiserum against c-Fos protein

(Santa Cruz, Biotechnology, Inc., USA) and phosphory-

lated c-Jun protein (Cell Signaling Technology, Inc., USA),

followed by the incubation with biotinylated goat anti-

rabbit IgG and avidin–biotin-horseradish peroxidase com-

plex (Vector Laboratories). Immunoreaction products were

visualized with diaminobenzidine and nickel ammonium

sulfate. The specificity of the primary antisera used in this

study has been described elsewhere (Sugimoto et al. 1993;

de Ruiter et al. 2000).

Morphometric Analysis

Lamina IX in the spinal cord and sensory and motor nuclei

in the brain stem as well as molecular, granule cell, and

Purkinje cell layers in the cerebellum was identified in

Nissl-stained sections under a light microscope and in

immuno-stained sections under a dark-field microscope.

For analysis of spinal motoneurons, the number of c-Fos-

and c-Jun-immunoreactive motoneurons was counted in

each section of the cervical spinal cord and their average

number was recorded for each lamina IX on the right and/

or left sides. For analysis of cranial motoneurons, the total

number of c-Fos- and c-Jun-immunoreactive motoneurons

in the trigeminal motor nucleus (Vm), facial nucleus (VII),

738 Cell Mol Neurobiol (2010) 30:737–742

123

dorsal motor nucleus of the vagus (X), and hypoglossal

nucleus (XII) was obtained from every third section of the

serial sections of the brain stem, and recorded for each

nucleus on the right and/or left sides. Neuron counts were

performed by a person who was unaware of the nature of

the samples. All differences were analyzed by Student’s

t-test.

The experiments were carried out under the control of

the Animal Research Control Committee in accordance

with the guidelines of the Canadian Council on Animal

Care and with The Guidelines for Animal Experiments of

Okayama University Medical School, Government Animal

Protection and Management Law (No. 105), and Japanese

Government Notification on Feeding and Safekeeping of

Animals (No. 6). All efforts were made to minimize the

number of animals used and their suffering.

Results

In Nissl-stained sections, spinal, brain stem, and cerebellar

neurons were abundant and their distribution was similar in

wild-type and dmu mice.

c-Fos

c-Fos-immunoreactive cells were occasionally detected in

the spinal cord and brain stem of wild-type mice (Figs. 1a,

2a, c, e, g). The immunoreactivity was weak and localized

to the nucleus but not the cytoplasm of these cells (Fig. 3a,

c, e, g). In dmu mice, however, many cells with intense

immunoreactivity appeared and the number of c-Fos-

immunoreactive cells dramatically increased (Figs. 1b, 2b,

d, f, h). In the spinal cord, the number of c-Fos-immuno-

reactive cells in the dorsal and ventral horns was elevated

by the mutation (Fig. 1b). c-Fos-immunoreactive moto-

neurons increased in the lamina IX of the spinal cord of

dmu mice (Fig. 3a, b, Table 1). In the brain stem, the

mutation also increased the number of c-Fos-immunore-

active motoneurons; such neurons in the Vm, VII, and XII

of dmu mice were more numerous compared to wild-type

mice (Fig. 3c–h, Table 1). However, the number of c-Fos-

immunoreactive motoneurons was quite variable in the X

of dmu mice; in some dmu mice the positive neurons were

far more numerous while in others they were comparable to

those in wild-type mice. In the cerebellum of wild-type and

dmu mice, c-Fos-immunoreactive cells in the granular layer

were abundant and their immunoreactive intensity was

variable (Fig. 2e, f). Purkinje cells in some dmu mice

contained weak c-Fos-immunoreactiviy, whereas those

cells in other dmu mice as well as wild-type mice were

devoid of the immunorecactivity. c-Fos-immunoreactive

cells in the molecular layer were relatively rare in wild-

type and dmu mice.

The number of c-Fos-immunoreactive neurons also

increased in the trigeminal sensory nuclei [subnuclei cau-

dalis, interpolaris (figure not shown), oralis, and princip-

alis], solitary tract nucleus, lateral reticular nucleus,

parvicellular reticular formation, cochlear nucleus, locus

coeruleus, subcoeruleus nucleus, dorsal raphe nucleus,

pontine nuclei as well as in the inferior colliculi and

periaqueductal gray of dmu mice (Fig. 2a–h).

c-Jun

In wild-type mice, c-Jun-immunoreactive cells were rarely

seen in the spinal cord whereas such cells were abundantly

detected throughout the brain stem (Fig. 4a, c, e). In these

cells, the immunoreactivity was mostly weak, and localized

to the nucleus. In the spinal cord and brain stem of dmu

mice, c-Jun- immunoreactive motoneurons increased

(Fig. 4a–f, Table 1). In the lamina IX of the spinal cord as

well as the Vm and X of dmu mice, motoneurons with

strong c-Jun-immunoreactivity increased and the number

of c-Jun-immunoreactive motoneurons was elevated

(Fig. 4a–f, Table 1). However, difference in the numbers

of c-Jun-immunoreactive motoneurons between wild-type

and dmu mice in the VII, and XII was not found to be

statistically significant (Table 1). The distribution of c-Jun-

immunoreactive cells in other regions of the spinal cord

and brain stem was also similar in wild-type and dmu mice.

In the cerebellum of wild-type and dmu mice, cell bodies

and processes of glial cells in the Purkinje cell layer

showed weak c-Jun-immunoreactivity (figure not shown).

However, c-Jun-immunoreactive neurons could not be

detected in the cerebellum of these mice.

Fig. 1 Microphotographs of c-Fos in the cervical spinal cord of wild-

type (a) and dmu mice (b). In the cervical spinal cord of wild-type

mice, the dorsal (DH) and ventral horns (VH) contain few c-Fos-ir

cells (a). In dmu mice, however, the number of Fos-ir cells

dramatically increased. The number of c-Fos-ir cells in the VH was

more numerous than in the DH of dmu mice (b). Bar = 500 lm (a).

Panels a and b are at the same magnification

Cell Mol Neurobiol (2010) 30:737–742 739

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Discussion

This study describes c-Fos and c-Jun expression in P18

wild-type and dmu mice. In the spinal cord, brain stem, and

cerebellum of wild-type mice, c-Fos-positive neurons were

detected and the immunoreactivity was weak. These find-

ings are consistent with previous observations that c-Fos

protein is expressed by developing neurons in the postnatal

central nervous system (Sakura-Yamashita et al. 1991;

Pennypacker 1995). It is considered that c-Fos expression

is the sign of the attained maturity of signal transduction

mechanisms in developing neurons. In dmu mice, however,

the expression of c-Fos was elevated and the number of

c-Fos-positive neurons increased in various regions which

were associated with the sensory (the dorsal and ventral

horn of the spinal cord, the solitary tract, trigeminal

sensory and reticular and cochlear nuclei, and reticular

formation), motor (the lamina IX of the spinal cord, Vm,

VII and XII) and autonomic functions (the locus coeruleus

and the subcoeruleus and dorsal raphe nuclei). The func-

tional significance and mechanism of c-Fos increase still

remains unclear in this study. However, it is unlikely that

the increase is correlated to degeneration of cardiac mus-

cle, because the statistical difference of the number of

c-Fos-immunoreactive motoneurons in the X could not be

detected between wild-type and dmu mice. The dmu mice

progressively lose mobility in their hindlimbs, and cannot

run or jump by the end of second week after birth

(De Repentigny et al. 2001). Instead, it crawls using its

forelimbs. Increase of c-Fos expression in motoneurons

within the cervical spinal cord of dmu mice may be asso-

ciated with the uncoordinated and excessive movement.

This is supported by a previous finding that the excessive

exercise, walking on the rotating rod for 1 h, induced c-Fos

Fig. 2 Microphotographs of c-Fos in the brain stem of wild-type

(a, c, e, g) and dmu mice (b, d, f, h). In wild-type mice, the brain stem

has a few c-Fos-ir cells (a, c, e, g). In dmu mice, the number of c-Fos-

ir cells increases in the XII (a, b), VII (c, d), and Vm (e, f) as well as

in the trigeminal sensory nuclei [subnuclei caudalis (Vc in a, b), oralis

(Vo in c, d), and principalis (Vp in e, f)], solitary tract nucleus (NTS

in a, b), lateral reticular nucleus (LRN in a, b), parvicellular reticular

formation (RFp in c, d), cochlear nucleus (CN in c, d), locus

coeruleus (LC in e, f), subcoeruleus nucleus (SN in e, f), dorsal raphe

nucleus (DRN in g, h), periaqueductal gray (PAG in g, h), pontine

nuclei (PN in g, h), and inferior colliculi (IC in g, h). Bars = 1 mm

(a, c, e, g). Panels a and b, c and d, e and f, and g and h are at the

same magnification, respectively

740 Cell Mol Neurobiol (2010) 30:737–742

123

expression in rat spinal motoneurons (Jasmin et al. 1994).

On the other hand, previous immunohistochemical studies

have demonstrated that NaV1.6 is expressed by motoneu-

rons in the spinal cord (Alessandri-Haber et al. 2002;

Gunasekaran et al. 2009). Therefore, it might be possible

that absence of NaV1.6 in dmu mice is directly associated

with increase of c-Fos expression in spinal motoneurons.

Previous studies have demonstrated that axotomy indu-

ces a prolonged expression of c-Jun transcription factor in

neurons, and suggested that c-Jun is involved in neurore-

generation in vivo (Gillardon et al. 1996). In contrast,

recent in vitro studies have indicated that induction of

c-Jun is necessary for neuronal cell death induced by

growth factor deprivation (Palmada et al. 2002; Ricart et al.

2006). According to in vivo experiments using young rats,

intense c-Jun-immunoreactivity is considered to be a

marker for fragmented DNA in the nucleus of axotomized

motoneurons (Gillardon et al. 1996). In this study, weak

c-Jun-immunoreactivity was detected in brain stem neu-

rons as well as spinal and cerebellar neurons in wild-type

and dmu mice. The expression may be associated with

Fig. 3 Microphotographs of c-Fos in the cervical spinal cord (a, b)

and brain stem (c–h) of wild-type (a, c, e, g) and dmu mice (b, d, f, h).

The number of c-Fos-ir motoneurons dramatically increases in the

spinal cord (b), Vm (d), VII (f), and XII (h) of dmu mice compared to

wild-type mice (a, c, e, g). Bars = 50 lm (a) and 100 lm (c). Panels

a and b, and c–h are at the same magnification, respectively

Table 1 The number of immunoreactive spinal and cranial motoneurons in wild-type and dmu mice

c-Fos c-Jun

Wild type dmu Wild type dmu

Spinal 4.8 ± 1.0 (n = 5) 17.3 ± 1.6** (n = 4) 1.6 ± 0.8 (n = 4) 12.1 ± 2.1** (n = 4)

Vm 76.2 ± 21.6 (n = 10) 216.9 ± 30.9** (n = 7) 41.4 ± 18.0 (n = 8) 123.1 ± 11.7** (n = 8

VII 162.4 ± 43.3 (n = 8) 533.3 ± 41.2** (n = 7) 179.3 ± 106.4 (n = 4) 214.8 ± 21.5 (n = 4)

X 23 ± 4.7 (n = 10) 60.9 ± 21.0 (n = 8) 39.1 ± 10.7 (n = 8) 92.8 ± 17.8* (n = 8)

XII 58.2 ± 43.3 (n = 10) 150.9 ± 25.7** (n = 8) 48.8 ± 31.3 (n = 8) 110 ± 24.1 (n = 8)

Value represents mean ± SEM. Difference between wild-type and dmu mice was significant (* P \ 0.05 and ** P \ 0.01, Student’s t-test)

Fig. 4 Microphotographs of c-Jun in the cervical spinal cord (a, b)

and brain stem (c–f) of wild-type (a, c, e) and dmu mice (b, d, f). The

number of c-Jun-ir motoneurons in the spinal cord (b), Vm (d), and X

(f) of dmu mice is more numerous than in those of wild-type mice

(a, c, e). Bars = 50 lm (a) and 100 lm (c). Panels a, b, e and f, and

c and d are at the same magnification, respectively

Cell Mol Neurobiol (2010) 30:737–742 741

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development and maturation of these neurons. In the cer-

vical spinal cord, Vm and X of dmu mice, however, c-Jun

expression was increased. In these regions, motoneurons

which contained intense c-Jun-immunoreactivity appeared.

Together with the degeneration of cardiac muscles in dmu

mice (De Repentigny et al. 2001), these findings suggest

that the mutation causes cell death of motoneurons in the

X. In dmu mice, however, muscle degeneration has been

also reported in hindlimbs but not forelimbs or mandibles

(De Repentigny et al. 2001). Therefore, the functional

significance of increase in c-Jun expression in the cervical

spinal cord and Vm of dmu mice is unclear. Further studies

will be necessary to determine the relationship between

the mutation and increase of c-Jun in cervical spinal and

trigeminal motoneurons.

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