M . Maruyama et al. Brain Research 887 2000 350 –358
351
microglias [24]. Moreover analysis performed in ATF2 chased from CLEA Japan Inc. Under deep anesthesia with
null mice has indicated that ATF2 plays a critical role in an intraperitoneal injection of sodium pentobarbital, mice
the hippocampal CA3 [20]. These results suggest that p38 were perfused through the left cardiac ventricle with saline
may play a critical part in maintenance of central nervous followed by ice-cold 4 paraformaldehyde in 0.1 M
system CNS through the determination of survival and phosphate buffered saline PBS, pH 7.4. The brain was
or death in response to extracellular stimuli. then removed and immersion fixed in the same fixative for
Here we report the localization of p38 in the adult 12 h at 48C. For paraffin sections, the brain was dehydrated
mouse brain as a first step to understanding its physiologi- in graded ethanol, embedded in paraffin, and sectioned at 4
cal functions in the CNS. mm thickness. For cryostat sections, the brain was im-
mersed in the series of 5, 15 and 30 sucrose solutions in PBS for cryoprotection, frozen in Tissue Tek O.C.T.
2. Material and methods compound Miles Inc., and sectioned at 10 mm thickness.
For immunoperoxidase labeling, sections were blocked 2.1. Antibodies
by incubation with 10 normal horse serum in PBS for 30 min followed by several washes with PBS. They were then
A polyclonal anti-p38 antibody was raised by immuniz- incubated with a primary antibody in PBS for 1 h followed
ing rabbits
with an
affinity purified
recombinant by several washes with PBS containing 0.05 Triton
glutathione-S-transferase-p38a GST-p38 [22]. The anti- X-100
PBST. Then
sections were
incubated in
GST-p38 serum was repeatedly applied to GST-c-Jun [11] biotinylated secondary antibody for 1 h followed by
column to remove antibodies against GST and bacterial several washes with PBS. They were then incubated in
proteins. Then, partially purified serum was multi-step ABC reagent followed by several washes with PBS. Bound
affinity purified by binding to GST-p38 column. Mono- peroxidase was visualized by incubating sections with
clonal antibodies against neurofilament 68, 160, 200 and 0.05 diaminobenzidine and 0.01 H O
in 50 mM
2 2
CNPase were purchased from Sigma-Aldrich, Japan K. K. Tris–HCl, pH 7.4. Control sections were incubated with
Alexa 488– and Alexa 594–conjugated second antibodies pre-immune serum. All incubations were performed at
were purchased from Molecular Probes. Biotinylated sec- room temperature.
ondary antibodies and ABC reagents were purchased from For immunofluorescence double labeling, sections were
Vector Laboratories. blocked by incubating with 10 normal goat serum in
PBS for 30 min followed by several washes with PBS. 2.2. Immunoblot analysis
Then they were incubated with a primary antibody in PBS with 2 normal goat serum for 1 or 3 h followed by
Dissected brain tissues for immunoblotting were ob- several washes with PBST. Alexa 488– and Alexa 594–
tained from adult mouse and sonicated in buffer containing conjugated second antibodies were diluted 2000-fold and
50 mM Hepes–KOH pH 7.4, 150 mM NaCl, 5 mM incubated for 1 h in PBS containing 2 goat serum. After
EDTA, 5 mM EGTA, 20 mM NaF, 1 mM dithiothreitol, 1 extensive washing with PBS, sections were examined by
mM phenylmethylsulfonylfluoride, 1 mM sodium ortho- using fluorescent microscopy Olympus. Control sections
vanadate, 1 mM p-nitrophenylphosphate, 20 mM b- were incubated either with pre-immune serum or antibody
glycerophosphate and 1 Triton X-100. Then 100 mg of preabsorbed with GST-p38.
protein was loaded in each lane after boiling for 3 min in The terminology of brain areas was mainly after Paxinos
sample buffer containing sodium dodecyl sulfate SDS. and Watson [18] and Franklin and Paxinos [6]
The tissue samples were resolved by 12 SDS–poly- acrylamide gel electrophoresis and transferred onto Im-
mobilon Millipore. The filters were blocked for 1 h in 5 non-fat dried milk in buffer containing 10 mM Tris–HCl
3. Results
pH 7.8 and 144 mM NaCl TBS supplemented with 0.05 Tween-20 TBST. Then filters were incubated for
3.1. Specificity of the anti-p38 antibody 2 h in 1 non-fat dried milk in TBST containing anti-p38
antibody or pre-immune serum. After several washes, the To study the localization of p38 MAP kinase at the
blots were incubated with horseradish–peroxidase conju- protein level, we used a rabbit polyclonal antibody against
gated anti-rabbit IgG Santa Cruz in 1 non-fat dried recombinant
GST-p38. Immunoblotting
experiments milk containing TBST for 1 h followed by several washes
showed that the anti-p38 antibody but not preimmune with TBST, and developed using SuperSignal Pierce.
serum specifically recognized a single band of about 42 kD in mouse brain extracts and extracts of murine embryonic
1 2
2.3. Immunohistochemistry fibroblasts prepared from E10.5 of progeny of p38
intercrosses [23] Fig. 1. Moreover, the immunoreactivity Eight-week-old C57BL 6J male mice n56 were pur-
recognized by the anti-p38 antibody disappeared when
352 M
Fig. 1. Specificity of anti-p38 antibody against brain extracts and extracts of murine embryonic fibroblasts MEF prepared from E10.5 progeny of
1 2
p38 intercrosses. 100 mg of brain homogenate lanes 1, 2 and 10 mg
of MEF extracts lanes 3–8 were applied for immunoblotting experi- ments. Lanes: 1, 3–5 pre-immune serum was used as 1st antibody
negative control: Lanes: 2, 6–8 anti-p38 antibody recognizes a single
2 2
band of 42 kD except for p38 MEF lane 8. The positions of
molecular mass markers are indicated on the left.
coronal sections were stained either with preimmune serum or with antibody pre-absorbed with GST-p38 Fig. 2.
These results demonstrated the specificity of the anti-p38 antibody.
3.2. Localization of p38 in mouse brain To determine the localization of p38 in adult mouse
brain, the anti-p38 antibody was applied to paraffin-em- bedded sections and cryostat sections, which resulted in
the similar patterns Figs. 3 and 4. Strong p38 immuno- reactivity was observed in fiber bundles, but not in cell
bodies or nuclei.
In the telencephalon, strong p38 immunoreactivity was observed in the lateral olfactory tract, anterior commissure,
corpus callosum, cingulum, internal capsule, external capsule, stria terminalis, fimbria and alveus hippocampi. In
the deeper layers of the neocortex, many fibers running in various directions were also immunoreactive for p38 Fig.
3B. In the diencephalon, strong immunoreactivity was observed in the fornix, mammilothalamic tract, stria
Fig. 2. Specific recognition by anti-p38 antibody in brain sections.
medullaris, fasciculus retroflexus, optic chiasm, and optic
Coronal sections were incubated with either anti-p38 antibody A,
tract. In the brainstem, many fiber bundles were strongly
pre-immune serum B or preabsorption of anti-p38 antibody with GST-
immunoreactive for p38, including cerebral peduncles,
p38 C. dlo, dorsolateral olfactory tract; lo, lateral olfactory tract. Scale
longitudinal fasciculus and transverse fibers of the pons.
bar5100 mm.
In the cerebellum, the white matter corpus medullare was strongly immunoreactive for p38 Fig. 3D.
It is noteworthy that no fibers in CA1 through dentate were immunoreactive for neurofilaments Fig. 5A, B.
gyrus in the hippocampus were immunoreacitve for p38, Dendrites of Purkinje cells in the molecular layer were not
whereas many fibers including mossy fibers in CA3 region immunoreactive for p38 in the cerebellum Fig. 5C, D.
M . Maruyama et al. Brain Research 887 2000 350 –358
353
Fig. 3. Distribution of p38 immunoreactivity in the forebrain and the cerebellum in sagittal sections. Intense p38 immunoreactivity is observed in ac, anterior commissure; f, fornix; cc, corpus callosum; cg, cingulum; sm, stria medullaris; fr, fasciculus retroflexus; ox, optic chiasm; lfp, longitudinal
fasciculus; tfp, transverse fibers of the pons; wm, white matter in the cerebellum. gcc, genu corpus callosum; MHb, medial habenular nucleus; fr, fasciculus retroflexus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer. A to F were paraffin-embedded sections and G was a cryostat section. Scale
bars530 mm for A,E,G, 60 mm for B,C,D and 150 mm for F.
354 M
Fig. 4. Distribution of p38 immunoreactivity in horizontal and coronal sections. Dense p38 immunoreactivity is observed in dlo, dorsolateral olfactory tract; lo, lateral olfactory tract A; sm, stria medullaris; cc, corpus callosum B; alv, alveus hippocampi; bsc, brachium superior colliculus; ec, external
capsule; fi, fimbria hippocampus; st, stria terminalis C in horizontal sections and cg, cingulum; fr, fasciculus retroflexus D; fr; ic, internal capsule; opt, optic tract; mt, mammillothalamic tract; cp, cerebral peduncles E in coronal section. CPu, caudate putamen; Ld, lambdoid septal zone; MHb, medial
habenular nucleus; Rt, reticular thalamic nucleus. Scale bar5150 mm.
3.3. Cellular localization of p38 immunoreactivity fiber bundles, it is possible that axons and or myelin
sheaths express p38. To identify p38-immunoreactive Since strong p38 immunoreactivity was observed only in
structures at the cellular level, sections were first double-
M . Maruyama et al. Brain Research 887 2000 350 –358
355
Fig. 5. Absence of p38 immunoreactivity in dentate gyrus and parallel fibers. Horizontal A,B and sagittal sections C,D were labeled with either anti-p38 A,C or anti-neurofilament antibodies B,D. CA3, area CA3; DG, dentate gyrus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer; wm,
white matter. Scale bar5150 mm.
labeled with anti-p38 and anti-neurofilament 68, 160 and adult mouse brain. Previous studies concerning p38 ex-
180 antibodies. At higher resolution, p38 immunoreactivity pression in the brain are very limited. In the hippocampus
was often observed in myelin sheath-like structures sur- CA1, phosphorylated p38 is increased in microglia but not
rounding axons Fig. 6A, C, D, F, but not in axons, which neurons after ischemia [24]. Phosphorylated p38 is also
were immunoreactive for neurofilaments Fig. 6B, C, E, increased in cultured cerebellar granule cells under gluta-
F. Localization of p38 in myelin sheath structures was mate-induced apoptosis [14]. This is the first report
confirmed by double labeling with anti-p38 and anti- regarding the expression of p38 in the central nervous
CNPase antibodies Fig. 7C, F. These results suggest that system under physiological conditions. Our results demon-
in some thick fibers, p38 is predominantly expressed in the strated that p38 MAP kinase is highly expressed in fiber
myelin sheath but not axons. bundles including the olfactory tract, anterior commissure,
corpus callosum, cingulum, internal capsule, stria ter- minalis, fimbria and alveus hippocampi, fornix, stria
4. Discussions medullaris, optic chiasm, and optic tract. Although similar
