Journal of Chemical Neuroanatomy 23 (2002) 187– 198

www.elsevier.com/locate/jchemneu

Expression of c-Fos, ICER, Krox-24 and JunB in the

whisker-to-barrel pathway of rats: time course of induction upon
whisker stimulation by tactile exploration of an enriched
environment
Sebastian Bisler a, Axel Schleicher a, Peter Gass b, Jörg H. Stehle c, Karl Zilles a,d,
Jochen F. Staiger a,*
a
C.&O. Vogt-Institut für Hirnforschung, Heinrich-Heine-Uni6ersität, Uni6ersitätsstr. 1, D-40225 Dusseldorf, Germany
b
Zentralinstitut für seelische Gesundheit, Uni6ersität Mannheim, Germany
c
Institut für Anatomie II, Uni6ersität Frankfurt, Frankfurt, Germany
d
Institut für Medizin, Forschungszentrum Jülich, Jülich, Germany
Received 20 July 2001; received in revised form 24 October 2001; accepted 5 December 2001

Abstract

Modified tactile information has been shown to induce adaptive plasticity in the somatosensory cortex of rat. The cellular
mechanisms resulting in plastic neuronal responses, however, are largely unknown. Inducible transcription factors have been
proposed as one major link in the cascade from modified input to altered neuronal structure and function. We investigated the
spatial and temporal patterns of transcription factor induction in the rat whisker-to-barrel pathway by placing the animals in a
novel, enriched environment while having clipped sets of whiskers on one side of the face. Such stimulation resulted not only in
a specific c-Fos induction in brainstem barrelettes and thalamic barreloids, but also in the barrel-related cortical columns, each
with different time courses. In the barrel cortex, c-Fos and Krox-24 immunostaining showed a rapid induction with peak levels
at 1 h and a return to basal levels after 14 h. JunB was induced after 1 h of exploration, declined at 6 h and returned to basal
levels after this time point. The inducible cyclic AMP early repressor (ICER), a transcription factor of the cAMP signaling
pathway, showed a maximum after 6 h, decreased slowly, but elevated levels were still detectable after 5 days. Our data
demonstrate that upon whisker stimulation by exploration of a novel, enriched environment, (i) subcortical relay stations in the
whisker-to-barrel pathway are able to express elevated levels of c-Fos and (ii) in the barrel cortex c-Fos, JunB, Krox-24 and ICER
are differentially regulated in the temporal domain. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Inducible transcription factors; Barreloids; Primary somatosensory cortex; Stimulus-transcription coupling; Immunocytochemistry

1. Introduction Kaczmarek and Chaudhuri, 1997). ITFs have also been

Inducible transcription factors (ITFs) are considered
to play an important role in adaptive cell responses to
external stimuli. A multitude of studies have investi-
gated the involvement of ITFs in physiological as well
as pathophysiological phenomena (cf. (Angel and
Karin, 1991; Dragunow, 1996; O’Donovan et al., 1999;
* Corresponding author. Tel.: + 49-211-81-12727; fax: + 49-211-
81-12336.
E-mail address: jochen@hirn.uni-duesseldorf.de (J.F. Staiger).
frequently used as markers for neuronal activity
(Chaudhuri, 1997). Here their induction is often linked
to synaptic activity of the expressing neurons (Cole et
al., 1989; Worley et al., 1993). C-Fos, JunB, Krox-24
and inducible cyclic AMP early repressor (ICER) are
ITFs that have received a high level of attention in
recent years. Fos and Jun proteins are able to dimerize
through a leucine zipper motif and, depending on the
particular combination, either activate or repress tran-
scription of genes containing AP-1 sites (Chiu et al.,
1989; Schutte et al., 1989); for review see Angel and
Karin (1991), Morgan and Curran (1991)). Krox-24

0891-0618/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 8 9 1 - 0 6 1 8 ( 0 1 ) 0 0 1 5 5 - 7
188 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198

acts through a zinc finger motif and has, in the cortex,
been implicated in mechanisms leading to learning and
memory (for review see Dragunow (1996)). ICER, a
potent repressor of cAMP-mediated transactivation, is
a member of the cAMP response element modulator
family of transcription factors and has been shown to
be inducible in the visual cortex by exposure of dark-
reared animals to light (Konopka et al., 1998). One of
the most important functions of ITFs, also in the
somatosensory cortex, is their role in stimulus-tran-
scription coupling (Melzer and Steiner, 1997) resulting
in various forms of neuronal plasticity (Lanahan and
Worley, 1998); for general reviews see Kaczmarek and
Chaudhuri (1997), Dragunow (1996)).
A major animal model that was used to investigate
these phenomena is the rat barrel cortex, which receives
its main input from the animal’s vibrissae via the
whisker-to-barrel pathway (Waite and Tracey, 1995).
Sensory input from the whisker follicles is transmitted
by the trigeminal ganglion cells to the ipsilateral brain-
stem (trigeminal nuclei). Next, the information is send
to the contralateral thalamus (ventrobasal complex).
The thalamic projections preferentially terminate in
layer IV of the barrel cortex, the whisker-related part of
the primary somatosensory cortex. Whereas several
studies have shown that the barrel-neurons are capable
of experience-dependent plasticity (Fox, 1994; Kossut,
1998; Polley et al., 1999; Lebedev et al., 2000), there is
still a debate on how far brainstem and thalamus
participate in adaptive neuronal plasticity (Lebedev et
al., 2000).
The enriched environment, a surrounding that pro-
vides new and diverse input to the (somato-) sensory
system, has become an important tool for studying the
processes underlying activity-dependent neuronal plas-
ticity. Keeping animals in such habitats has effects like
the formation of new synapses and dendritic growth
(Wallace et al., 1992; Klintsova and Greenough, 1999).
A study that used an enriched environment to investi-
gate the induction of ITFs in the somatosensory system
of the rat described not only a cortical expression of
c-Fos, but also its induction in the somatic sector of the
thalamic reticular nucleus (TRN) in visually deprived
rats (Montero, 1997) and related this to forms of
neuronal plasticity as mentioned above.
In spite of the frequency of studies that investigated
ITF expression in the somatosensory system, informa-
tion on the temporal profile of their expression re-
mained very scarce. The goal of the present study was
to determine the time course of ITF expression in the
whisker-to-barrel pathway resulting from modified sen-
sory input. We chose clipping of all but two neighbor-
ing whiskers on one side of the face (i.e.
whisker-pairing) as a means to evoke adaptive neuronal
responses, as many physiological studies used this ap-
proach to determine aspects of receptive field plasticity
(Diamond et al., 1994; Polley et al., 1999; Lebedev et
al., 2000). Examining the entire whisker-to-barrel path-
way yielded new insight in ITF expression in brainstem,
thalamus and cortex.
2. Experimental procedures
2.1. Animals and stimulation procedure
Male Wistar rats (body weight 250–350 g) from the
local breeding facility of the Heinrich-Heine-University
were used in the present study (n= 51; Table 1). All
experimental protocols were in accordance with the
German law on animal research. Animals were trans-
ferred to the laboratory 1 or 2 days before the begin-
ning of the experiment, so they could get accustomed to
the room. Rats were anesthetized with Enflurane (3 ml
in a 5 l box) and all but two whiskers (C1 and C2) on
the right side of the face were clipped close to the skin.
Whiskers on the left side remained intact. Next, single
animals were placed in a stimulatory box that was
equipped with maze-like pieces of Styrofoam and cellu-
lose balls as ‘unnatural’ gadgets, as well as piles of
sticks and stones, mixed with seeds of trees (maple tree,
chestnut, oak) as ‘natural’ gadgets, in order to provide
a stimulus-rich environment. Water was freely accessi-
ble and food pellets were distributed among the con-
tents of the box.
Rats were kept in the box for different periods of
time (10 min, 1 h, 6 h, 14 h, 24 h, 48 h and 5 days,
compare Table 1). Light inside the box was constantly
kept dim. Illumination levels at the floor of the box

Table 1
Table showing the number of animals that were sacrificed per time point/treatment

Number of animals

Basal 14 h sham 10 min 1h 6h 14 h 48 h 5d

Fos 7 6 6 7 6 7 6 6
ICER 6 6 0 7 6 5 5 6
Krox-24 6 6 6 7 6 7 6 6
JunB 7 6 6 6 6 7 6 6

The absolute number of animals that were used in this study is 51. With few exceptions, sections from each animal were stained for each ITF.
 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198
ranged between 0 and 10 Lux. The clipped whiskers of
the animals that were kept in the box for 5 days started
to grow over fur level about two days after the first
clipping and were thus clipped again at day 2 and 4.
The enriched environment was not altered during the
experiment. After the respective period of exploration
time, animals were anesthetized with an overdose of
pentobarbital and perfused. Animals of the basal level
(control) group were taken out of their home cages and
were then immediately anesthetized and perfused.
2.2. Fixation and tissue processing
Animals were perfused through the aorta, first with
: 30 ml of physiological saline and second, with 500 ml
of freshly depolymerized paraformaldehyde (in 0.1 M
phosphate buffer (PB), pH 7.4) by hydrostatic pressure.
Subsequently the brain and the trigeminal ganglion
were dissected out of the skull and two blocks were
trimmed: one from the forebrain and one from the
hindbrain. After a postfixation of 2 h in the same
fixative, the forebrain was further trimmed to contain
the primary somatosensory cortex and the ventrobasal
thalamic complex. The forebrain blocks were glued to
the stage of a vibratome (Series 1000, TPI; Polyscience
Europe, Eppelheim, Germany) with cyanoacrylate and
8 – 10 parallel series of 50 mm thickness were cut. The
blocks of the hindbrain containing the trigeminal nuclei
were immersed in cryoprotectant (25% saccharose, 10%
glycerol in 0.01 M PB) until sunk and sectioned on a
cryomicrotome (Leica, Bensheim, Germany) at 50 mm.
Four series of sections were collected. From this stage
on, all tissue was treated identically. After cutting, the
sections were washed extensively in 0.1 M PB (4× 30
min). Between the second and third rinse, sections were
incubated in 1% H2O2 in PB for 45 min. Several rinses
with 0.1 M PB (2× 30 min) and 0.05 M TRIS-buffered
saline with 0.1% Triton X-100 (TBS-Tx; 2× 15 min),
both at pH 7.4, followed. Slices were incubated in 10%
normal goat serum (in TBS-Tx) for 45 min. Immunos-
taining was done in three steps. First step: incubation of
primary polyclonal antisera (diluted in TBS-Tx) for 60
h (at 6 °C) against: (i) c-Fos (raised in rabbit, 1:5000
(sc-52, Santa Cruz, Heidelberg, Germany); (ii) JunB
(raised in rabbit, 1:20 000; (Herdegen et al., 1993); (iii)
Krox-24 (raised in rabbit, 1:20 000; (Herdegen et al.,
1993); (iv) ICER, raised in rabbit, 1:30 000; (Maronde
et al., 1999). Subsequently, it was rinsed with TBS-Tx
(4× 15 min). Second step: incubation of biotinylated
anti-rabbit serum (1:100, in TBS-Tx; Vector,
Burlingame, CA) for 2 h at room temperature followed
by rinsing with TBS-Tx (4× 15 min). Third step: incu-
bation of peroxidase coupled to an avidin-biotin com-
plex (1:200, in TBS-Tx; ABC, Vector, Burlingame, CA)
for 2 h at room temperature. Sections were then rinsed
(2 × 15 min each) in TBS (without Triton X-100) and
189
0.05 M TRIS-buffer (TB, pH 7.6). Subsequently, the
buffer was exchanged by a chromogen solution consist-
ing of 0.015% 3,3%-diaminobenzidine (Sigma, Deisen-
hofen, Germany) and 0.4% ammonium nickel sulfate
(Fluka, Buchs, Switzerland). After a preincubation of
10 min, the reaction was started by adding 0.006%
hydrogen peroxide. Progress of the reaction was con-
trolled by frequently inspecting sections under the mi-
croscope. The reaction was stopped by rinsing with TB
when visual inspection confirmed that no further in-
crease in the intensity of blue-staining in nuclei oc-
curred. Sections were mounted on slides, air-dried and
coverslipped with DePeX (Merck, Darmstadt, Ger-
many) after immersion in xylene.
Since the specificity of the antisera was extensively
tested and approved by the manufacturers, in the
present study, immunohistochemical controls only in-
cluded a replacement of the primary antiserum by
appropriately diluted normal serum. This always re-
sulted in the absence of any staining. In this report the
nomenclature of Paxinos and Watson for the rat brain
is used (Paxinos and Watson, 1998).
2.3. Quantification
The reaction products of c-Fos, JunB, Krox-24 and
ICER in the barrel cortex were quantified by calculat-
ing a gray-level-index (GLI), as described (Zilles et al.,
1993). In our application, GLI values correspond to the
area fraction of immunoreactive cell nuclei in relation
to the entire reference area. One histological section per
animal at the level of the largest columnar diameter in
layer IV (referring to the concept that a barrel is the
morphological correlate in layer IV of a functional
column spanning all cortical layers) was digitized to
high-resolution gray value images (2048× 2048 pixel, 8
bit, 1.34×1.34 mm2 per pixel). Columnar regions were
drawn interactively to define the area of the corre-
sponding columns of the stimulated whiskers in the
barrel cortex as reference area. The size of the reference
area was set by the largest diameter of the ITF-response
in layer IV of the stimulated column and reached from
the layer I/II border to that of layer VI with the white
matter. In sections from basal level- or sham-groups,
regions were drawn that corresponded in location and
size to the barrel-related columns C1 and C2. Grey
value thresholds were optimized using an automated
procedure to differentiate immunoreactive nuclei from
occasional patches of high background staining. The
amount of nuclear staining in the reference areas was
quantified by GLI and linearly scaled to the range
0–100%. Background level, which was automatically
determined by an algorithm, was set to 0% while the
highest GLI value of the respective time point was set
to 100%. Data are presented as means9 SEM. GLI
values of each experimental group were compared with
190 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198

values of the basal level using Dunnett’s test for multi-
ple comparisons. Significance levels indicated are:
***P B 0.001; **P B 0.01; *P B 0.05 (Fig. 5). GLI val-
ues in the sham group were tested against basal level
using one way analysis of variance (ANOVA) Systat®
for Windows, Version 9; SPSS, Chicago, IL).
2.4. Documentation of results
Sections were photographed with a CCD-camera
(Photometrics CoolSNAP; Visitron, Puchheim, Ger-
many) attached to an Axioplan 2 microscope (Zeiss,
Oberkochen, Germany). Files were imported into Pho-
toshop 5.0 (Adobe Systems, San Jose, CA). In order to
optimize gray values, brightness and contrast, the ap-
propriate commands were executed. No other manipu-
lations of the files were performed. Figures were
assembled and lettered with Illustrator 8.0 (Adobe
Systems).
barreloids C1 and C2 (Fig. 1B and D) could constantly
be observed, virtually exclusively at 1 h in the group of
experimental animals, contralateral to the paired
whiskers. Neither ICER (Fig. 1E), nor the other investi-
gated transcription factors (Krox-24, JunB; not shown)
displayed an up-regulation in VPM at any of the time
points investigated.
3.2. Basal le6els of ITFs in barrel cortex
One group of animals was used to determine the
basal levels of the investigated transcription factors.
C-Fos and JunB showed a very low level of basal
expression in the cortex (Fig. 2A and D), whereas
Krox-24 and ICER displayed a moderate level of basal
expression (Fig. 2B and C). In contrast to the other
transcription factors, ICER was also constitutively ex-
pressed in the ventrobasal thalamus. This expression
was at least partly due to nuclear labeling in glial cells
(Fig. 1F).

3. Results
3.1. ITF induction in subcortical relay stations of the
whisker-to-barrel pathway
In the trigeminal ganglion, no immunocytochemical
signal related to the experimental paradigm was ob-
served. C-Fos and Krox-24 did not show any nuclear
staining at all. ICER showed low basal level staining.
In the brainstem, induction of c-Fos could be observed
in the nucleus principalis and nucleus spinalis nervi
trigemini, ipsilateral to the paired whiskers. The nucleus
principalis constantly displayed clusters of stained nu-
clei (Fig. 1A and C) that resembled barrelettes of the
C-row. These clusters were observed at time points 10
min, 1 h, 6 h and 48 h (14 h and 5 d here not
determined; n.d.). ICER and Krox-24 did not show any
specific induction (JunB n.d.).
The ventral posteromedial thalamic nucleus ventral
posteromedial (VPM) also showed specific induction of
c-Fos. Clusters of stained nuclei located in the area of
3.3. ITF induction in barrel cortex
3.3.1. C-Fos
After 10 min exploration time, already a weak
columnar induction was detected in the barrel cortex
(Fig. 3A, Fig. 5A). After 1 h, the c-Fos– IR reached
peak-levels presenting a strong columnar induction pat-
tern (Fig. 3B, Fig. 5A). The strongest focus of induc-
tion was in layer IV and lower layer III. This laminar
maximum of induction was present at all time points
examined, not only for c-Fos, but also for every other
transcription factor that showed an induction (ICER,
Krox-24, JunB).The induction of c-Fos then decreased
considerably until 14 h, but was still significant after 6
and 14 h (Fig. 3C and D; Fig. 5A). After 48 h and 5 d
of exploration time, the c-Fos expression was not sig-
nificantly increased in comparison to basal levels (Fig.
5A). However, barrel-related columns with a very weak
signal could still be observed qualitatively (Fig. 3E and
F).

Fig. 1. Expression of ITFs in subcortical relay stations of the whisker-to-barrel pathway. (A) and (B) Diagrams of coronal sections showing the
localisation of nucleus principalis nervi trigemini (PrV, Bregma − 10.10 mm) and ventral posteromedial nucleus of the thalamus (VPM, Bregma
−3.25 mm). The diagrams were taken from the stereotaxic atlas of Swanson (Swanson, 1998). PO, posterior thalamic nucleus; RT, reticular
thalamic nucleus; SI, primary somatosensory cortex; sptV, tractus spinalis nervi trigemini; VIIn, tractus nervi facialis; VPL, ventral posterolateral
nucleus of the thalamus. Frame in A indicates the region of the brainstem that is shown in (C). Frame in (B) indicates the thalamic region that
is shown in (D), (E) (left hemisphere) and (G) (right hemisphere). Insets in (A) and (B) indicate the location (vertical black line) of the coronal
sections in relation to the whole rat brain. (C) Cluster of c-Fos immunoreactive nuclei in barrelettes C1 and C2 (arrowheads) after whisker-pairing
and 1 h of exploration of an enriched environment. Scale bar: 300 mm. (D) Expression of c-Fos in the VPM. The arrowhead indicates the cluster
of c-Fos stained nuclei in barreloids C1 and C2. Treatment of animal as in (C). Scale bar: 300 mm. (E) Basal level-like pattern of
ICER-immunoreactivity in the somatosensory thalamus after 6 h of exploration. ICER did not show any specific up-regulation in the barreloids
corresponding to the paired whiskers. Scale bar: 300 mm. (F) Double labeling with antisera directed against ICER and GFAP (glial fibrillary acidic
protein, an astrocyte marker) shows an example of an ICER-immunoreactive nucleus of a glial cell, the perikaryon of which has been stained by
GFAP. Scale bar: 20 mm. (G) Ventrobasal thalamus (contra-l. =contralateral to the experimental side) of an animal that displayed clusters of
stained nuclei on the experimental side (compare (D)). The ventral posteromedial thalamus displays no specific cluster of Fos-stained nuclei. Scale
bar: 300 mm.
 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198 191

Fig. 1.

3.3.2. Inducible cAMP early repressor
A small induction of ICER protein was already
detected in the barrel cortex at 1 h. At 6 h, a clear
columnar pattern emerged corresponding with peak
levels of ICER expression (Fig. 4B, Fig. 5B). Beyond 6
h, expression decreased constantly, but was still de-
tectable after 5 d (Fig. 5B). The laminar pattern of
ICER in the experimental barrel-related columns was
very similar to c-Fos.
3.3.3. Krox-24
At 10 min, an induction of Krox-24, which showed
no columnar pattern, preceded the specific columnar
induction at 1 h (Fig. 4C). At that time point, Krox-24
192 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198

Fig. 2. Coronal sections of the barrel field, showing basal levels of ITFs. (A) Staining for c-Fos visualizes a very low constitutive expression of
this ITF in untreated animals. Only few immunoreactive nuclei, mainly in layers II and VI, can be detected. (B) ICER, in contrast, shows a high
level of basal expression, especially in layers IV, lower V and VI. (C) Krox-24 was also expressed throughout the barrel cortex, with intense
staining in layers IV and VI. (D) Staining for JunB in the barrel field of untreated animals revealed only sparse basal expression, mainly in layer
II and upper layer III. Scale bars: 300 mm.

reached peak levels (Fig. 5C). Beyond 1 h, staining 3.4. Controls

intensity decreased and—based on GLI-measure-
ments— ceased to be significantly elevated at 14 h,
although, like c-Fos, barrel-related columns could be
found at later time points (not shown). The quantifica-
tion showed that at least up to 6 h, Krox-24 displayed
a statistically significant induction (Fig. 5C). Beyond 6
h, the columnar pattern was likely to be caused by
depression of Krox-24 in the neighboring areas, corre-
sponding to the clipped whiskers, which were deprived
of synaptic input. The laminar pattern was similar to
c-Fos and ICER.
The control group was a sham group that served to
evaluate the effect of the anesthetic procedure on the
ITF induction (n=6) after short stimulation times (01
h). The animals were anesthetized with Enflurane and
put back into their home cage for 1 h before perfusion.
C-Fos showed a slight induction that was statistically
not significantly different from basal level (P\0.05).
The other transcription factors also did not show a
significant increase in staining following this treatment
(not shown).

3.3.4. JunB
Following an early up-regulation after 10 min, JunB
peaked after 1 h (Fig. 5D), showing a clear columnar
pattern of expression (Fig. 4D), however, at low inten-
sity. JunB expression had decreased after 6 h and
remained close to basal level beyond that time point.
The expression was most prominent in layer IV and
lower layer III.
4. Discussion
The present results demonstrate in the rat whisker-to-
barrel pathway that of stimulating sets of whiskers by
exposure to an enriched environment results in distinct
temporal patterns of transcription factor induction. The
investigated ITFs are differentially regulated at the
 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198 193

Fig. 3. Time course of c-Fos expression in the barrel cortex following whisker-pairing and exploration of a novel environment. (A) Ten minutes
of exploration already induced a weak columnar pattern of c-Fos expression in the barrel cortex. (B) After 1 h, levels of c-Fos staining peaked
and formed a strong barrel-related column. (C, D) At 6 h (note also the supragranular induction) and 14 h, the expression of c-Fos started to
decrease, but still displayed a clear columnar pattern. (E, F) At 48 h and 5 d, expression slowly returned to basal levels, though a weak columnar
pattern was still visible. Width of the barrels in layer IV varies due to variable location of the section in center or side of the barrel. Scale bars:
300 mm.
194 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198

Fig. 4. Maximal induction of four different transcription factors in the barrel cortex of adult animals related to the experimental paradigm. (A)
C-Fos reached peak-levels at 1 h and was induced in a strong and layer-specific fashion. (B) and (C) Because of the high constitutive level of ICER
and Krox-24, the supra- and infragranular induction is not as readily visible as in the case of c-Fos. (D) Immunostaining against JunB did not
yield very intensively stained nuclei, yet a closer inspection revealed an induction, at least in layer IV and layer III. Scale bars: 300 mm.

subcortical relay stations and the barrel cortex. Only
c-Fos shows a specific up-regulation in trigeminal
brainstem nuclei and the VPM at particular time
points. In the barrel cortex, c-Fos, Krox-24 and JunB
display an early induction with peak levels around 1 h,
followed by a steady decline. The induction of Krox-24
and c-Fos ceased to be significantly elevated after 14
and 48 h, respectively, although qualitatively, barrel-re-
lated columns could still be detected at later time
points. Interestingly, ICER remained significantly ele-
vated for a much longer period and was still elevated
after the longest period tested.
4.1. Controls
Own previous studies addressed the question in how
far c-Fos and ICER induction depended either solely
on the effects of whisker-pairing or on the effects of the
enriched environment, in comparison to a combination
of both procedures. We found that whisker-clipping
alone can produce barrel-related columns, but this ef-
fect is much more robust and strong when the animals
are placed in the enriched environment as well (Staiger
et al., 2000). We have also shown that on the side of the
barrel cortex where all related whiskers had been left
unclipped, an ‘ubiquitous’ up-regulation of the ITFs
takes place whereas clipping of all whiskers suppresses
the ITF up-regulation completely.
4.2. Brainstem
In the past, studies that used physiological stimuli to
induce transcription factors in the somatosensory
pathways did not examine or did not report changes of
ITF expression in the brainstem. In our present study,
an induction of c-Fos in the nucleus principalis nervi
trigemini could be observed at time points ranging from
10 min up to 48 h. A study by Takemura et al. (2000)
reported an induction of c-Fos in trigeminal brainstem
nuclei 3 h after electrical stimulation of the trigeminal
ganglion. These results suggest that already at the
brainstem level, the somatosensory system displays
adaptive molecular responses upon altered sensory
stimulation.
 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198 195

Fig. 5. Time course of transcription factor induction in barrel cortex following whisker-pairing and exploration of novel environment. Exploration
time (abscissa) drawn as categories. (A) C-Fos, (C) Krox-24 and (D) JunB show similar dynamics with an early up-regulation after 10 min in the
enriched environment and peak levels around 1 h when also the strongest columnar expression was observed (compare Fig. 4A – D). (B) ICER,
in contrast, displays its maximum around 6 h (compare Fig. 4B) with a much slighter decrease and still significant induction after 5 days under
experimental conditions.

4.3. Thalamus rats exploring (2 h) an enriched environment with two

In untreated animals, VPM is lacking c-Fos ex-
pression (Herdegen et al., 1995 and in this study). In
the VPM contralateral to the paired whiskers, a robust
barreloid-shaped expression of c-Fos was observed at 1
h. Interestingly, in contrast to the cortex, the VPM does
not show an induction on the side where input from all
whiskers had been intact. To our knowledge an induc-
tion of c-Fos in VPM resulting from physiological
stimuli has not been reported before. With passive
whisker stimulation, own preliminary studies in the
mouse showed a comparable c-Fos induction in the
VPM (Staiger et al., 1996). A number of investigators
found the VPM to be spared of IEG induction. Mon-
tero (1997) observed c-Fos induction in the TRN of
intact sets of vibrissae, but found the VPM devoid of
stained nuclei. We also found c-Fos induction in the
TRN (not shown), but could not detect any differences
between the experimental and the control side of the
animals that would indicate an effect of the stimulation
procedure.
Mechanical or thermal noxious stimulation,
applied for a longer period of time (up to 4 h) resulted
in a slight induction of c-Fos in ventral thalamic nuclei
of rat (Bullitt, 1990). Application of formalin into the
plantar skin of one hindpaw has been shown not only
to induce c-Fos, but also Krox-24 and JunB expression
in medial thalamic nuclei (Pertovaara et al., 1993).
Zimmer et al., (1997) showed that soman strongly
induces c-Fos in the somatosensory cortex at 1–8 h,
196 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198
but spares the VPM. Despite this relative unresponsive-
ness of the IEG response in VPM, different authors
reported induction of a functional reorganization on
the electrophysiological level in VPM following lesions,
at least in young animals (Nicolelis et al., 1991; Gar-
raghty and Kaas, 1991). The functional relevance of the
reported c-Fos induction in VPM remains to be shown.
A possible partner for dimerization with c-Fos in VPM
would be JunD as this transcription factor has been
reported to be expressed in the VPM (Herdegen et al.,
1995).
4.4. Cortex
4.4.1. C-Fos shows an early induction and a prolonged
time course of expression
C-Fos expression in the rat cortex has been investi-
gated in a number of studies. However, not much focus
was put on its temporal pattern of expression caused by
physiological stimuli. Melzer and Steiner (1997) re-
ported an increase of c-Fos (and Krox-24) mRNA in a
barrel-related column after 5 min of stimulating vibris-
sae with the help of a magnetic device plus 5 min
survival time. A study by Montero (1997) found an
up-regulation of c-Fos in the barrel cortex resulting
from 2 h of exploration of an enriched environment.
C-Fos up-regulation was also observed after brushing
of vibrissae on one side of the snout for 20 min and 2
h survival time (Filipkowski et al., 2000). Chaudhuri et
al. (2000) traced the time course of c-Fos expression in
the rat visual cortex after exposing dark reared animals
to light. A first increase was shown at 30 min with peak
levels around 1.5 h and return to basal level after 6 h.
The protracted time course in our study is likely to be
related to a more enduring novelty effect in the en-
riched environment setting.
Studies that used pharmacological agents or lesions
to induce c-Fos induction looked at the time course of
induction more often. It was reported that soman in-
duced c-Fos expression in rat neocortex lasted from 1
to 8 h after the exposition to the poison with intense
staining at 2 and 4 h (Zimmer et al., 1997). Also
induced by soman was an induction of c-Fos mRNA in
rat hippocampus and piriform cortex that was detected
already 5 min after administration (Baille-Le Crom et
al., 1996). C-Fos mRNA reached maximum levels at
2– 4 h and stayed sustained up to 24 h. Actually, most
authors found maximum levels of c-Fos expression
around 2 h, resulting from treatments like bicuculline
administration (Gass et al., 1992; Herdegen et al., 1993)
and photochemical stroke (Comelli et al., 1993).
4.4.2. ICER, Krox-24 and JunB show differential time
courses of induction
Very few studies exist on ICER induction in the
cerebral cortex or elsewhere in the brain. Two studies
using light exposure to the lateral eyes of animals as the
stimulating cue showed a rather delayed increase in
ICER mRNA in the suprachiasmatic nucleus after 2 h
(Stehle et al., 1996), and after 6 h in the visual
cortex, respectively (Konopka et al., 1998). In contrast,
under several in vitro conditions ICER mRNA induc-
tion was shown to appear rapidly already after 15– 60
min (Stehle et al., 1993; Luckman and Cox, 1995;
Fitzgerald et al., 1996a,b; Molina et al., 1993;
Lamas and Sassone-Corsi, 1997). Previous studies on
the expression of ICER protein in the parietal cortex
upon glutamatergic stimulation (Storvik et al.,
2000) and in the pineal gland upon adrenergic stimula-
tion (Pfeffer et al., 1999; Maronde et al., 1999) found a
late increase after about 6 h. The consistent fact that
elevated levels of this TF are still present 24 h after the
stimulation predicts its function as part of a sensitive
cellular protection mechanism of self-restriction in
cAMP-inducible gene transcription. This interpretation
goes along with our finding of a later peak and long-
lasting up-regulation of ICER in the rat barrel
cortex, contrasting the early peaks and more restricted
time courses of the other investigated TFs. Thus,
ICER may act as an inhibitor on gene transcription of
activating TFs and/or late-response genes after having
reached a sufficient concentration in the nucleus.
It was shown, for example, that ICER is able to
attenuate c-Fos expression resulting from cAMP- or
NGF-activated pathways in PC 12 cells (Monaco and
Sassone-Corsi, 1997). On the other hand, it is
not known, in how far the cell populations that
express ICER and c-Fos overlap in the barrel-related
columns.
An induction of Krox-24 mRNA in the barrel cortex
of rats was reported after 5–15 min of passive whisker
stimulation (Melzer and Steiner, 1997). Other authors
who used bicuculline-induced seizures found increased
Krox-24 protein expression after 0.5– 1 h with peak
levels after 2 h and return to basal level after 4–8 h
(Gass et al., 1992; Herdegen et al., 1993). Lanteri et al.
(1993) induced a continuous noxious stimulation by
causing a monoarthritic inflammation and reported
Krox-24 expression in superficial layers of dorsal horn
for up to 4 weeks. The quantification of the present
study did not show a significant increase at time points
later than 6 h, which leads us to the conclusion that the
columnar pattern in a previous study (Staiger et al.,
2000) was caused by a down-regulation of Krox-24 in
the area of the clipped whiskers. It has been shown
frequently that suppressing neuronal input can lead to a
rapid down-regulation of Krox-24 (Melzer and Steiner,
1997; Steiner and Gerfen, 1994).
JunB protein induction in rat neocortex has been
described to occur upon physiological as well as patho-
physiological stimuli. Own studies found JunB expres-
 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198
sion in rat neocortex after clipping of whiskers and
exploration of a novel environment (Staiger et al.,
2000). Bicuculline-induced seizures resulted in JunB
expression at 0.5 h, reaching peak levels at 2 h and
lasting up to 8 h (Herdegen et al., 1993). Intraperitoneal
administration of MK-801 first increased JunB levels in
rat neocortex after 1 h with peak levels between 3 and
24 h (Gass et al., 1993).
4.4.3. Possible target genes
In order to establish a link between the induction of
transcription factors and structural or physiological
changes, more information on the effective regulation
of possible target genes is needed. An interesting
candidate gene is cpg15, coding for a membrane-bound
signaling molecule that has been shown to promote
dendritic growth. Its expression is increased in layer IV
of barrel cortex after 12 h of whisker stimulation
(Nedivi et al., 1998). JunB, in the presence of c-Fos, has
been shown to inhibit activation mediated by JunD,
such as the transcription of the proenkephalin
gene (Kobierski et al., 1991). This transcriptional
regulation of the proenkephalin gene is likely to be
mediated by binding to the CRE-2 site in an enhancer
of the gene.
5. Conclusion
This study has shown that all subcortical relay
stations in the whisker-to-barrel pathway are able to
express the transcription factor c-Fos in response to
physiological whisker stimulation and thus display signs
for adaptive plasticity or increased ‘house-keeping’
demands. We also report differential temporal patterns
of ITF induction in the barrel cortex with early rise and
peak of c-Fos, Krox-24 and JunB. In contrast to these,
ICER peaks later and declines much slower. The
knowledge of these time courses should help to
understand the complex interplay of transcription
factors that regulate the cell’s genomic response to
sensory stimuli. In order to elucidate the functional
significance of the investigated ITFs, an intensified
search for the target genes is clearly warranted. It is to
be expected that among these, proteins necessary for
the assembly of new spines or synapses as well as
enzymes regulating the metabolic cellular demands in
an adaptive manner will be found.
Acknowledgements
The authors like to thank U. Opfermann-Emmerich
for skilled technical assistance. This study was
supported by the Deutsche Forschungsgemeinschaft
(Sta 431/2-2, 431/2-3).
197
References
Angel, P., Karin, M., 1991. The role of Jun, Fos and the AP-1
complex in cell-proliferation and transformation. Biochem. Bio-
phys. Acta 1072, 129 – 157.
Baille-Le Crom, V., Collombet, J.M., Burckhart, M.F., Foquin, A.,
Pernot-Marino, I., Rondouin, G., Lallement, G., 1996. Time
course and regional expression of C-FOS and HSP70 in
hippocampus and piriform cortex following soman-induced
seizures. J. Neurosci. Res. 45, 513 – 524.
Bullitt, E., 1990. Expression of c-fos-like protein as a marker for
neuronal activity following noxious stimulation in the rat. J.
Comp. Neurol. 296, 517 – 530.
Chaudhuri, A., 1997. Dual activity maps in primate visual cortex
produced by different temporal patterns of zif268 mRNA and
protein expression. Proc. Natl. Acad. Sci. USA 94, 2671 –2675.
Chaudhuri, A., Zangenehpour, S., Rahbar-Dehgan, F., Ye, F., 2000.
Molecular maps of neural activity and quiescence. Acta Neuro-
biol. Exp. 60, 403 – 410.
Chiu, R., Angel, P., Karin, M., 1989. Jun-B differs in its biological
properties from, and is a negative regulator of, c-Jun. Cell 59,
979 – 986.
Cole, A.J., Saffen, D.W., Baraban, J.M., Worley, P.F., 1989. Rapid
increase of an immediate early gene messenger RNA in hippocam-
pal neurons by synaptic NMDA receptor activation. Nature 340,
474 – 476.
Comelli, M.C., Guidolin, D., Seren, M.S., Zanoni, R., Canella, R.,
Rubini, R., Manev, H., 1993. Time course, localization and
pharmacological modulation of immediate early inducible genes,
brain-derived neurotrophic factor and trkB messenger RNAs in
the rat brain following photochemical stroke. Neuroscience 55,
473 – 490.
Diamond, M.E., Huang, W., Ebner, F.F., 1994. Laminar comparison
of somatosensory cortical plasticity. Science 265, 1885 – 1888.
Dragunow, M., 1996. A role for immediate-early transcription factors
in learning and memory. Behav. Genet. 26, 293 – 299.
Filipkowski, R.K., Rydz, M., Berdel, B., Morys, J., Kaczmarek, L.,
2000. Tactile experience induces c-fos expression in rat barrel
cortex. Learn. Mem. 7, 116 – 122.
Fitzgerald, L.R., Li, Z., Machida, C.A., Fishman, P.H., Duman,
R.S., 1996a. Adrenergic regulation of ICER (inducible cyclic
AMP early repressor) and beta1-adrenergic receptor gene expres-
sion in C6 glioma cells. J. Neurochem. 67, 490 – 497.
Fitzgerald, L.R., Vaidya, V.A., Terwilliger, R.Z., Duman, R.S.,
1996b. Electroconvulsive seizure increases the expression of
CREM (cyclic AMP response element modulator) and ICER
(inducible cyclic AMP early repressor) in rat brain. J. Neurochem.
66, 429 – 432.
Fox, K., 1994. The cortical component of experience-dependent
synaptic plasticity in the rat barrel cortex. J. Neurosci. 14, 7665 –
7679.
Garraghty, P.E., Kaas, J.H., 1991. Functional reorganization in adult
monkey thalamus after peripheral nerve injury. Neuroreport 2,
747 – 750.
Gass, P., Herdegen, T., Bravo, R., Kiessling, M., 1992. Induction of
immediate early gene encoded proteins in the rat hippocampus
after bicuculline-induced seizures: differential expression of
KROX-24, FOS and JUN proteins. Neuroscience 48, 315 –324.
Gass, P., Herdegen, T., Bravo, R., Kiessling, M., 1993. Induction and
suppression of immediate early genes in specific rat brain regions
by the non-competitive N-methyl-D-aspartate receptor antagonist
MK-801. Neuroscience 53, 749 – 758.
Herdegen, T., Kovary, K., Buhl, A., Bravo, R., Zimmermann, M.,
Gass, P., 1995. Basal expression of the inducible transcription
factors c-Jun, JunB, JunD, c-Fos, FosB, and Krox-24 in the adult
rat brain. J. Comp. Neurol. 354, 39 – 56.
198 S. Bisler et al. / Journal of Chemical Neuroanatomy 23 (2002) 187–198
Herdegen, T., Sandkuhler, J., Gass, P., Kiessling, M., Bravo, R.,
Zimmermann, M., 1993. JUN, FOS, KROX, and CREB transcrip-
tion factor proteins in the rat cortex: basal expression and induction
by spreading depression and epileptic seizures. J. Comp. Neurol.
333, 271 – 288.
Kaczmarek, L., Chaudhuri, A., 1997. Sensory regulation of immediate-
early gene expression in mammalian visual cortex: implications for
functional mapping and neural plasticity. Brain Res. Rev. 23,
237 – 256.
Klintsova, A.Y., Greenough, W.T., 1999. Synaptic plasticity in cortical
systems. Curr. Opin. Neurobiol. 9, 203 – 208.
Kobierski, L.A., Chu, H.M., Tan, Y., Comb, M.J., 1991. cAMP-depen-
dent regulation of proenkephalin by JunD and JunB: positive and
negative effects of AP-1 proteins. Proc. Natl. Acad. Sci. USA 88,
10222 – 10226.
Konopka, D., Szklarczyk, A.W., Filipkowski, R.K., Trauzold, A.,
Nowicka, D., Hetman, M., Kaczmarek, L., 1998. Plasticity- and
neurodegeneration-linked cyclic-AMP responsive element modula-
tor/inducible cyclic-AMP early repressor messenger RNA expres-
sion in the rat brain. Neuroscience 86, 499 – 510.
Kossut, M., 1998. Experience-dependent changes in function and
anatomy of adult barrel cortex. Exp. Brain Res. 123, 110 – 116.
Lamas, M., Sassone-Corsi, P., 1997. The dynamics of the transcrip-
tional response to cyclic adenosine 3%,5%- monophosphate: recurrent
inducibility and refractory phase. Mol. Endocrinol. 11, 1415 – 1424.
Lanahan, A., Worley, P., 1998. Immediate-early genes and synaptic
function. Neurobiol. Learn. Mem. 70, 37 –43.
Lanteri, M., de Pommery, J., Herdegen, T., Weil, F.J., Bravo, R.,
Menetrey, D., 1993. Differential time course and spatial expression
of Fos, Jun, and Krox-24 proteins in spinal cord of rats undergoing
subacute or chronic somatic inflammation. J. Comp. Neurol. 333,
223 – 235.
Lebedev, M.A., Mirabella, G., Erchova, I., Diamond, M.E., 2000.
Experience-dependent plasticity of rat barrel cortex: redistribution
of activity across barrel-columns. Cereb. Cortex 10, 23 –31.
Luckman, S.M., Cox, H.J., 1995. Expression of inducible cAMP early
repressor (ICER) in hypothalamic magnocellular neurons. Mol.
Brain Res. 34, 231 – 238.
Maronde, E., Pfeffer, M., Olcese, J., Molina, C.A., Schlotter, F.,
Dehghani, F., Korf, H.W., Stehle, J.H., 1999. Transcription factors
in neuroendocrine regulation: rhythmic changes in pCREB and
ICER levels frame melatonin synthesis. J. Neurosci. 19, 3326 – 3336.
Melzer, P., Steiner, H., 1997. Stimulus-dependent expression of imme-
diate-early genes in rat somatosensory cortex. J. Comp. Neurol.
380, 145 – 153.
Molina, C.A., Foulkes, N.S., Lalli, E., Sassone-Corsi, P., 1993.
Inducibility and negative autoregulation of CREM: an alternative
promoter directs the expression of ICER, an early response
repressor. Cell 75, 875 –886.
Monaco, L., Sassone-Corsi, P., 1997. Cross-talk in signal transduction:
Ras-dependent induction of cAMP-responsive transcriptional re-
pressor ICER by nerve growth factor. Oncogene 15, 2493 – 2500.
Montero, V.M., 1997. c-fos induction in sensory pathways of rats
exploring a novel complex environment: shifts of active thalamic
reticular sectors by predominant sensory cues. Neuroscience 76,
1069 – 1081.
Morgan, J.I., Curran, T., 1991. Stimulus-transcription coupling in the
nervous system: involvement of the inducible proto-oncogenes fos
and jun. Ann. Rev. Neurosci. 14, 421 – 451.
Nedivi, E., Burbach, B., Svoboda, K., 1998. Regulation of cpg15
expression in barrel cortex during experience-dependent receptive
field plasticity. Soc. Neurosci. Abstr. 24, 634.
Nicolelis, M.A., Chapin, J.K., Lin, R.C., 1991. Thalamic plasticity
induced by early whisker removal in rats. Brain Res. 561, 344 – 349.
O’Donovan, K.J., Tourtellotte, W.G., Millbrandt, J., Baraban, J.M.,
1999. The EGR family of transcription-regulatory factors: progress
at the interface of molecular and systems neuroscience. Trends
Neurosci. 22, 167 – 173.
Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordi-
nates. Academic Press, San Diego, CA.
Pertovaara, A., Bravo, R., Herdegen, T., 1993. Induction and suppres-
sion of immediate-early genes in the rat brain by selective alpha-2-
adrenoceptor agonist and antagonist following noxious peripheral
stimulation. Neuroscience 54, 117 – 126.
Pfeffer, M., Maronde, E., Molina, C.A., Korf, H.W., Stehle, J.H., 1999.
Inducible cyclic AMP early repressor protein in rat pinealocytes:
a highly sensitive natural reporter for regulated gene transcription.
Mol. Pharmacol. 56, 279 – 289.
Polley, D.B., Chen-Bee, C.H., Frostig, R.D., 1999. Two directions of
plasticity in the sensory-deprived adult cortex. Neuron 24, 623 –637.
Schutte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., Minna, J., 1989.
Jun-B inhibits and c-fos stimulates the transforming and trans-ac-
tivating activities of c-jun. Cell 59, 987 – 997.
Staiger, J.F., Bisler, S., Schleicher, A., Gass, P., Stehle, J.H., Zilles, K.,
2000. Exploration of a novel environment leads to the expression
of inducible transcription factors in barrel-related columns. Neuro-
science 99, 7 – 16.
Staiger, J.F., Zilles, K., Gass, P., Welker, E., 1996. Immediate early
gene expression upon whisker stimulation in the adult mouse: an
immunocytochemical study. Eur. J. Neurosci. (Suppl. 9), 52.04.
Stehle, J.H., Foulkes, N.S., Molina, C.A., Simonneaux, V., Pevet, P.,
Sassone, C.P., 1993. Adrenergic signals direct rhythmic expression
of transcriptional repressor CREM in the pineal gland. Nature 365,
314 – 320.
Stehle, J.H., Pfeffer, M., Kuhn, R., Korf, H.W., 1996. Light-induced
expression of transcription factor ICER (inducible cAMP early
repressor) in rat suprachiasmatic nucleus is phase-restricted. Neu-
rosci. Lett. 217, 169 – 172.
Steiner, H., Gerfen, C.R., 1994. Tactile sensory input regulates basal
and apomorphine-induced immediate-early gene expression in rat
barrel cortex. J. Comp. Neurol. 344, 297 – 304.
Storvik, M., Linden, A.M., Kontkanen, O., Lakso, M., Castren, E.,
Wong, G., 2000. Induction of cAMP response element modulator
(CREM) and inducible cAMP early repressor (ICER) expression
in rat brain by uncompetitive N-methyl-D-aspartate receptor antag-
onists. J. Pharmacol. Exp. Ther. 294, 52 – 60.
Swanson, L.W., 1998. Brain Maps, Structure of the Rat Brain. Elsevier,
Amsterdam, Netherlands.
Takemura, M., Shimada, T., Sugiyo, S., Nokubi, T., Shigenaga, Y.,
2000. Mapping of c-Fos in the trigeminal sensory nucleus following.
Exp. Brain Res. 130, 113 – 123.
Waite, P.M.E., Tracey, D.J., 1995. Trigeminal Sensory System. In:
Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, San
Diego, pp. 705 – 724.
Wallace, C.S., Kilman, V.L., Withers, G.S., Greenough, W.T., 1992.
Increases in dendritic length in occipital cortex after 4 days of
differential housing in weanling rats. Behav. Neural Biol. 58, 64 –68.
Worley, P.F., Bhat, R.V., Baraban, J.M., Erickson, C.A., Mc-
Naughton, B.L., Barnes, C.A., 1993. Thresholds for synaptic
activation of transcription factors in hippocampus: correlation with
long-term enhancement. J. Neurosci. 13, 4776 – 4786.
Zilles, K., Hajos, F., Csillag, A., Kalman, M., Sotonyi, P., Schleicher,
A., 1993. Vasoactive intestinal polypeptide immunoreactive struc-
tures in the mouse barrel field. Brain Res. 618, 149 – 154.
Zimmer, L.A., Ennis, M., el Etri, M., Shipley, M.T., 1997. Anatomical
localization and time course of Fos expression following soman-in-
duced seizures. J. Comp. Neurol. 378, 468 – 481.