Brain Research Bulletin, Vol. 57, No. 5, pp. 631– 638, 2002
Copyright © 2002 Elsevier Science Inc.
Printed in the USA. All rights reserved
0361-9230/02/$–see front matter
PII S0361-9230(01)00757-2
Fos induction in cortical interneurons during
spontaneous wakefulness of rats in a familiar or
enriched environment
Giuseppe Bertini,1* Ze-Chun Peng,1† Paolo F. Fabene,1 Gigliola Grassi-Zucconi2 and Marina Bentivoglio1
1
Department of Morphological and Biomedical Sciences, University of Verona, Verona, Italy; and 2Department
of Cell Biology, University of Perugia, Perugia, Italy
[Received 28 August 2001; Revised 23 October 2001; Accepted 25 October 2001]
ABSTRACT: It has been repeatedly reported that Fos is spontaneously induced in several brain structures, including the
cerebral cortex, during wakefulness. To ascertain whether cortical interneurons are involved in this state-dependent oscillation of gene regulation, we combined Fos immunocytochemistry with immunostaining of either parvalbumin or calbindin,
known markers of cortical interneurons. Immunopositive neurons were examined in the sensorimotor and cingulate cortex.
In rats perfused in basal conditions, a minor proportion (around
8%) of Fos-immunoreactive neurons in the parietal cortex were
also parvalbumin- or calbindin-immunoreactive; these double
immunostained cells accounted for 13% of the parvalbuminand 34% of the calbindin-labeled neurons. Colocalization of Fos
with either calcium-binding protein was instead not observed in
the cingulate cortex. In rats stimulated by novel environmental
cues during the period of wakefulness preceding perfusion,
Fos-positive neurons increased markedly relative to unstimulated animals, and involved the majority of the calbindin- or
parvalbumin-labeled cell populations (60 –75% and over 95%,
respectively). In the neuronal populations in which Fos was
induced by exposure to the enriched environment, the proportion of calbindin- and parvalbumin-labeled cells was larger than
in the unstimulated cases, and the increment was statistically
significant in the cingulate cortex. The results demonstrate that
Fos induction occurring in the cortex during undisturbed wakefulness in a familiar environment involves a minor proportion of
interneurons. Furthermore, the findings indicate that the addition of novel environmental stimuli results in an increase of
Fos-expressing neurons whose recruitment, at least in the cingulate cortex, involves a higher proportion of interneurons than
of projection neurons. © 2002 Elsevier Science Inc.
its protein product Fos are induced during wakefulness in the rat
cerebral cortex [7,8,18]. These and other studies have provided
evidence that gene expression fluctuates considerably in relation to
the circadian variation of functional states, as well as to the type of
activity that the animal is engaged in [15,19]. However, it remains
to be determined whether certain cortical cell populations are
preferentially involved in such oscillation.
In the present study, we aimed at a characterization of cortical
neurons involved in behavioral state-dependent Fos induction. In
particular, we sought to ascertain whether Fos is induced during
wakefulness in interneurons of the cingulate and parietal cortex,
and we examined this issue with a double immunocytochemical
approach.
In the cerebral cortex, short-axon neurons comprise heterogeneous subsets of inhibitory GABAergic cells, which represent
approximately 15–30% of the total neuronal cell population (see
[6] for review). On the other hand, cortical projection neurons,
which are excitatory and utilize amino acids as neurotransmitters,
constitute the largest component (approximately 70 – 85%) of the
total population of cortical neurons [6]. A number of different
neuroactive molecules, including neuropeptides and calcium-binding proteins of the EF-hand family, have been shown to be colocalized with GABA in cortical interneurons. In particular, cells
immunostained by the calcium-binding proteins parvalbumin (Pv)
or calbindin D-28k (Cb) represent, taken together, more than 90%
of cortical GABAergic interneurons, although with areal and species variations [6]. In the rat cortex, 70% of the GABAergic cells
contain Pv [4], while Cb-positive neurons include a much lower
percentage of cortical nonpyramidal cells [5,6]. Pv- and Cb-containing cells mostly represent discrete subpopulations of cortical
interneurons, and co-localization of the two proteins in the rat
somatosensory cortex can be demonstrated in a very limited subset
of neurons [20]. In the present study, we selected Pv and Cb
immunoreactivity, which is fully compatible with Fos immunostaining (at variance with GABA immunocytochemistry, which
requires a different perfusion recipe), to characterize populations
of interneurons in the cerebral cortex of rats perfused during
KEY WORDS: Calbindin, Parvalbumin, Exploratory behavior, Immediate early genes, Immunocytochemistry.
INTRODUCTION
The expression of the immediate early gene c-fos has been reported to oscillate during the spontaneous sleep/wakefulness cycle
in neuronal populations of the brain (see [1] for review). In
particular, it has been repeatedly described that c-fos mRNA and
* Address for correspondence: Dr. G. Bertini, Department of Morphological and Biomedical Sciences, Section of Anatomy and Histology, Medical
Faculty, Strada Le Grazie 8, 37134 Verona, Italy. Fax: ⫹39-045-802-7163; E-mail: giuseppe.bertini@univr.it
†
Present address: Brain Research Institute, University of California at Los Angeles, Los Angeles, CA, USA.
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BERTINI ET AL.
wakefulness, both in the absence of specific stimulation and after
introducing novel environmental cues that trigger exploratory behavior.
MATERIALS AND METHODS
Animals and Perfusion
The study was based on six adult male Wistar rats (250 –300 g)
that were housed under a 12 h/12 h light/dark cycle (lights on at
0700h), with controlled temperature and food and water ad libitum, for at least 2 weeks before the experiment. The experimental
protocols received institutional approval and authorization by the
Italian Ministry of Health. All efforts were made to avoid animal
suffering and minimize the number of animals used.
In the first three animals (rats 1–3; group F, maintained in a
familiar environment during the experiment), epidural electrodes
were implanted under barbiturate anesthesia (Nembutal 50 mg/kg,
i.p.), and the animals were familiarized with electroencephalogram
(EEG) recording for at least 2 h every day for 1 week. On the day
of the experiment, EEG was continuously recorded for 3– 4 h
starting at lights-off time. The other three animals (rats 4 – 6; group
E, exposed to an enriched environment) were not implanted with
electrodes and EEG was not recorded. On the day of the experiment, at lights off, environment of the latter animals was enriched
by introducing novel objects, such as stones and leaves, as well as
oddly shaped pieces of plastic in their home cages. All animals
(rats 1– 6) were rapidly anesthetized between 2300h and 2400h and
transcardially perfused with saline followed by 4% phosphatebuffered paraformaldehyde. In addition to the EEG monitoring of
rats of group F, all animals were individually observed and their
behavior manually recorded for the whole 3– 4 h preceding perfusion.
Histology and Immunohistochemistry
After a brief postfixation and overnight cryoprotection in 30%
phosphate-buffered sucrose, brains were cut into 30-m-thick
serial sections with a freezing microtome. In the rats of group F,
every 10th section was processed for Fos immunocytochemistry,
whereas the adjacent series was processed for double immunocytochemistry with anti-Fos antibodies, and either anti-Cb (rats 1 and
2) or anti-Pv (rats 2 and 3) antibodies. In the rats of group E, every
10th section was processed for Fos and Cb double immunocytochemistry, and the adjacent series of sections was processed for
Fos and Pv double immunocytochemistry. In all cases, one series
of sections was stained with cresyl violet for cytoarchitectonic
control.
For Fos immunohistochemistry, the sections were repeatedly
washed in 0.01 M phosphate-buffered saline, pH 7.4 (PBS),
soaked in 0.75% H2O2 in PBS and preincubated in 5% normal
rabbit serum and 0.2% Triton X-100 in PBS for 1 h. The sections
were then incubated for 36 – 40 h in sheep polyclonal antibodies
that recognize Fos (Cambridge Research Biochemicals, Cambridge, UK), diluted 1:4000 in the preincubation solution to which
0.1% bovine serum albumin and 0.05% sodium azide had been
added. The sections were then incubated for 2 h in biotinylated
rabbit anti-sheep secondary antibodies (Vector, Burlingame, CA,
USA), diluted 1:200 in 2% normal rabbit serum and 0.2% Triton
X-100 in PBS. The sections were finally reacted with avidin-biotin
peroxidase (ABC kit, Vector) using a heavy metal intensification
of 3-3⬘-diaminobenzidine (DAB; Sigma, St. Louis, MO, USA):
0.025% DAB, 0.1% nickel ammonium sulfate, and 0.03% H2O2 in
PBS.
Following Fos immunostaining, sections destined to double
immunohistochemistry were repeatedly washed in PBS, and then
treated with mouse monoclonal antibodies that recognize either Pv
(Sigma; diluted 1:10,000) or Cb (Swant, Bellinzona, Switzerland;
diluted 1:5000). The same procedure outlined above was followed,
except that normal horse serum and biotinylated horse anti-mouse
IgG (Vector) as secondary antibodies were used. A previously
described protocol [17] was adopted in the last step of the procedure. Briefly, sections were reacted in a freshly prepared solution
of 0.05% ␣-naphtol, 0.1% ammonium carbonate, and 0.003%
H2O2, and then incubated in 0.1% pyronin B in 0.1 M phosphate
buffer, pH 7.4. This recipe results in a pink-purple color of the
reaction products.
All sections were then mounted on gelatinized slides, dehydrated through the alcohol series, cleared in xylene, and coverslipped with Entellan.
Data Analysis
The material was studied at the microscope under bright-field
illumination with the aid of an X-Y digital plotting system connected to the microscope stage by means of transducers, and of a
JVC CCD KY-F58 digital camera connected to the microscope
and the image analysis software ImagePro Plus 4.1 (Media Cybernetics, Silver Spring, MD, USA). Analysis of the areal and laminar
distribution of labeled cells in the cases of group F was performed
by charting double and single Fos-immunoreactive (ir) and Cb- or
Pv-ir neurons in the cerebral cortex, using sample sections at the
level of the frontoparietal cortex. Due to the very high number of
Fos-ir cells in the rats of group E, only double and single Cb- or
Pv-ir neurons were instead charted in sample sections through the
frontoparietal cortex of these animals.
Quantitative analysis of the material was performed by counting single-immunostained and double-immunostained neurons in
selected areas of the parietal and cingulate cortex. A strategy of
unbiased section and field sampling was adopted in order to obtain
samples at consistent locations across animals. First, using a 25⫻
objective we acquired digitized images of two entire coronal
sections for each experimental animal (Fig. 1). With the aid of the
image analysis software, we then measured for each section the
perimeter of the cortex on the coronal plane between the rhinal
sulcus and the indusium griseum (Fig. 1, arrowheads), which
served as reference points. We then placed 300 ⫻ 1500 m frames
spanning all cortical layers (from the pial surface to the border
with the white matter) in the parietal and in the cingulate cortex of
one hemisphere (Fig. 1), at the same relative distance from the two
reference points across animals. Based on anatomical landmarks
and cytoarchitecture of adjacent cresyl violet-stained sections, all
the frames were located within the Par1 and Cg1 fields, as defined
by Zilles [21]. Immunostained cells found within the frames’
boundaries were directly observed with 25⫻ and 40⫻ objectives
(at final magnifications of 250⫻ and 400⫻) and individually
counted through all focal planes.
For statistical evaluation, data were summarized by averaging
together cell counts for the two rostro-caudal levels and for the
different animals in each group. The effects of exploratory behavior on Fos induction in different classes of neurons were analyzed
on the basis of 2 ⫻ 2 contingency tables of Fos-labeled neurons.
In particular, we compared the mean number of Fos-positive cells
that contained either calcium-binding protein to those that did not
(dependent variable), in the sections obtained from groups F and E
(independent variable). Tables were obtained for each of the two
studied calcium-binding proteins and for each of the two cortical
areas analyzed, and were statistically evaluated with chi-square
(2) tests.
FOS EXPRESSION IN THE AWAKE RAT CORTEX
633
Immunocytochemistry
FIG. 1. The drawing illustrates the location of the cortical regions of
interest (shaded rectangles) for quantitative analysis; sections were located
⫺0.3 mm (A) and ⫺2.8 mm (B) from bregma [21]; reference landmarks
for positioning of the regions of interest are indicated by the arrowheads;
the empty arrowheads point to the rhinal sulcus, and the filled ones to the
indusium griseum.
RESULTS
Behavior
Records of behavioral observation indicated that the rats of
group F were active (grooming, eating, walking, and rearing) for
most of the time preceding perfusion, with short periods of rest
lasting few minutes. Systematic EEG scoring in these rats confirmed that the animals were almost always awake during the 3– 4
hours preceding perfusion, with short episodes of slow-wave sleep,
and a single episode of paradoxical sleep, lasting less than 2 min,
observed in case 3 (Table 1).
The rats of group E showed substantially more intense activity
than those of group F, and displayed patterns of exploratory
behavior consisting of systematic inspection, accompanied by constant whisking, of each of the objects recently added to the cage.
These systematic exploratory routines were spontaneously repeated several times.
TABLE 1
PROPORTION OF TIME SPENT IN WAKEFULNESS (W), SLOW-WAVE
SLEEP (SS), AND PARADOXICAL SLEEP (PS) DURING THE HOURS
PRECEDING PERFUSION BY EACH OF THE 3 ANIMALS OF GROUP F,
STUDIED IN THE FAMILIAR ENVIRONMENT
Rat #
W
SS
PS
1
2
3
99.6
95.8
90.7
0.4
4.2
8.5
0.0
0.0
0.8
The patterns of Pv and Cb immunostaining in all examined
cases were consistent with those previously reported in the literature. Briefly, Pv-positive cells were found throughout the neocortex, and exhibited a relatively uniform distribution in layers II–VI.
Neurons containing Cb were overall less numerous and, although
present in all layers, more irregularly distributed.
In all rats of group F, numerous Fos-immunostained cells were
seen in the cortex, concentrated in the cingulate and sensorimotor
areas. In terms of laminar distribution, Fos-ir cells were mainly
located in a more superficial band (layers II–III) and in a deeper
band (mostly layer V) (Fig. 2). Fos immunoreactivity was consistently bilateral, and labeled neurons were often densely clustered.
Foci of dense labeling were observed in several different cortical
fields (including Cg1, Par1, Fr1, Pir, Te1, Oc2 [21]) and exhibited
a mirror distribution in the two hemispheres.
In the rats of group E, Fos-ir cells were extremely numerous
throughout the cerebral cortex and in all layers. The bilaminar
distribution of labeled cells, observed in the previous cases, was
still evident but less marked, and many Fos-ir neurons were also
seen in layers IV and VI.
As previously described [17], in the sections processed for
double immunostaining the different colors of the reaction products and their subcellular distribution enabled a clear distinction
between single Fos-ir neurons and single Pv-ir or Cb-ir ones. Thus,
dark brown-black Fos immunoreactivity was confined to the neuronal nuclei, whereas bright pink-purple immunostaining filled the
perikarya and proximal dendrites of Cb-ir or Pv-ir neurons (Fig. 3).
Double immunostained neurons displayed Fos immunoreactivity
in the nucleus and calcium-binding protein immunoreactivity in
the cytoplasm (Fig. 3B, C). Neuropil staining was rather high in
the sections processed for Pv immunocytochemistry, but not in
those processed with anti-Cb antibodies (Fig. 3D). In the latter
sections, however, a relatively high background was observed in
the superficial cortical layers, as reported in previous studies on the
distribution of Cb in the rat cortex [5].
In the cases of group F, Fos was found to be colocalized with
either calcium-binding protein in a relatively small subset of
neurons of the sensorimotor cortex. In particular, both Fos/Pvlabeled neurons and Fos/Cb-labeled ones were observed in the
parietal cortex, with a slight prevalence for layers II/III (Fig. 2),
and the findings appeared consistent across animals. At the quantitative evaluation, these double-labeled cells were found to represent a minor proportion of the Fos-ir cells in the parietal cortex,
i.e. approximately 8% for both Pv and Cb (Table 2 and Fig. 4).
With respect to the cell population expressing either calciumbinding protein, the double-labeled cells accounted for approximately 13% of the total Pv-ir neurons and for 34% of the Cb-ir
neurons in the parietal cortex. No double-labeled cells were instead
observed in the cingulate cortex (Table 2 and Fig. 4).
In the rats of group E, which had been stimulated by novel
environmental cues, double-labeled cell populations were instead
commonly observed in both the parietal and cingulate cortex. The
distribution of double-labeled neurons in these rats was consistent
with that of calcium-binding proteins in the cases of group F, with
a prevalence in layers II/III and layer V, especially for the Pvlabeled cells (Fig. 2). At the quantitative analysis, over 95% of the
Pv-ir neurons were found to express also Fos in both the examined
cortical fields, while the proportion of Fos/Cb-positive cells accounted for 60% of the Cb-ir cell population in the cingulate cortex
and for 75% of the same cell population in the parietal cortex
(Table 2). The quantitative analysis also revealed that the Fos/Pv-ir
neurons accounted for an average proportion of about 14% of the
total Fos-ir cell population in the parietal cortex, and the Fos/Cb-ir
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BERTINI ET AL.
FIG. 2. Graphic representation of the distribution of immunoreactive neurons in the parietal cortex. Panels represent the regions of interest from
sections processed with Fos/Pv ((Fos ⫹ Pv, left-side panels) and Fos/Cb (Fos ⫹ Cb, right-side panels) double immunocytochemistry. Comparison of
the immunoreactivity in the samples obtained from rats exposed to familiar vs. enriched environment demonstrates the markedly larger population of
Fos-ir neurons, as well as the substantial increase of double-immunostained neurons, in the latter cases. Fos-ir cells are represented by small black
dots, whereas neurons containing either calcium-binding protein are shown in red and double-stained (Fos/Pv or Fos/Cb) cells are blue. For clarity
of the graphic representation, each dot represents two labeled neurons of each cell population.
FIG. 3. (Next page) The microphotographs illustrate double Fos/Pv immunostaining (A–C) and Fos/Cb immunostaining (D–F) in the somatosensory cortex
of a rat of the first group (rat 3), perfused in basal conditions after 3 h of wakefulness documented by electroencephalographic recording. Fosimmunopositive neurons exhibit brown labeling of the cell nuclei, whereas Pv or Cb immunostaining is revealed by the purple-pink labeling. (A)
Low-power view, illustrating the density of both labels and their laminar prevalence. (B) and (C) show at higher power Pv/Fos double-labeled neurons
(arrowheads) intermingled with single labeled ones. (D) Low-power view of layers II/III showing Cb-positive and Fos-positive cells, the intermingling of
which is illustrated at higher power in (E) and (F); note that the labels are evident in distinct neuronal populations. Scale bars: A, 140 m; C, 25 m (applies
also to B); D, 80 m; F, 45 m (applies also to E).
FOS EXPRESSION IN THE AWAKE RAT CORTEX
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BERTINI ET AL.
TABLE 2
MEAN NUMBER AND STANDARD DEVIATION OF IMMUNOREACTIVE NEURONS IN SAMPLES PROCESSED WITH FOS/PV AND FOS/CB DOUBLE
IMMUNOCYTOCHEMISTRY IN THE TWO GROUPS OF CASES (GROUP F, FAMILIAR ENVIRONMENT AND GROUP E, ENRICHED ENVIRONMENT)
Parietal cortex
Cingulate cortex
Familiar
Enriched
Familiar
Enriched
Pv
Fos/Pv
Fos
85.50 ⫾ 7.78
12.50 ⫾ 0.71
138.00 ⫾ 4.24
3.75 ⫾ 3.10
84.00 ⫾ 6.06
525.75 ⫾ 27.29
84.00 ⫾ 4.24
0.00 ⫾ 0.00
132.00 ⫾ 9.90
3.25 ⫾ 1.89
90.25 ⫾ 6.70
499.50 ⫾ 26.25
Cb
Fos/Cb
Fos
26.00 ⫾ 8.52
13.25 ⫾ 2.63
159.50 ⫾ 20.17
13.25 ⫾ 2.50
38.25 ⫾ 3.86
590.50 ⫾ 36.86
26.75 ⫾ 14.24
0.00 ⫾ 0.00
141.50 ⫾ 13.92
11.50 ⫾ 8.54
17.25 ⫾ 4.79
553.00 ⫾ 47.25
neurons accounted for 6% of the total Fos-ir cells in the same area
(Table 2 and Fig. 4). In addition, as mentioned above, in the rats
of group E double-immunolabeled cells were also seen in the
cingulate cortex (Table 2). In the latter area, Fos/Pv-ir neurons
accounted for an average proportion of 15%, and Fos/Cb-ir cells
for 3%, of the total Fos-ir cell population (Fig. 4).
The above data indicate that the increment of calcium-binding
protein-containing neurons that expressed Fos after exposure to the
enriched environment was larger than the total increment of Fos-ir
cells. Thus, while the cases of group E showed on average four
times as many Fos-ir neurons in the parietal cortex than those of
group F (Table 2), the proportion of Pv-ir cells among Fosexpressing neurons in group E was 7.5 times larger than in group
F. The latter finding raised the question as to whether recruitment
of Fos-expressing neurons as a consequence of exploratory behavior preferentially involved calcium-binding protein-containing interneurons rather than being equally distributed among neuronal
populations. This issue was addressed with chi-square tests of the
already described contingency tables.
In the cingulate cortex, the analyses indicated that both the
Pv-ir and Cb-ir cell populations were involved by the environ-
ment-dependent increase of Fos expression significantly more than
neurons that did not contain either calcium-binding protein, and
that this effect was highly significant for Pv-expressing neurons
(Pv: 2 ⫽ 23.09, p ⬍ 0.0001; Cb: 2 ⫽ 4.38, p ⬍ 0.05). In the
parietal cortex, a similar (although nonsignificant) trend was revealed for the Pv-ir cell population, while the effect was absent for
Cb-ir neurons (Pv: 2 ⫽ 3.26; Cb: 2 ⫽ 0.57).
DISCUSSION
Consistent with previous reports of circadian oscillations in the
pattern of c-fos expression in the brain (reviewed in [1]), our
results show that the Fos protein is spontaneously induced in
neuronal subsets of the cerebral cortex during wakefulness even in
the absence of any specific stimulation. Previous studies have also
shown that Fos expression in the cerebral cortex during sleep is
instead very low, especially in relation to the relative amount of
slow-wave sleep [7,8]. Altogether these findings indicate that the
induction of the c-fos gene in cortical neurons is a specific feature
of wakefulness-dependent behavior.
A variety of studies of stimulus-induced Fos expression have
FIG. 4. The charts illustrate the differences in the average proportion of calbindin- or parvalbumin-immunoreactive (ir) neurons relative to the total Fos-ir cell populations (i.e., Fos/parvalbumin and Fos/calbindin
double-labeled neurons) in the two groups of cases, examined after wakefulness in a familiar environment and
in an enriched environment, respectively.
FOS EXPRESSION IN THE AWAKE RAT CORTEX
demonstrated that the protein becomes detectable in the neuronal
nucleus 1–2 h following an effective stimulus and peaks shortly
afterwards (reviewed in [9]). Naturally, a reliable assessment of
“spontaneous” expression requires particular methodological care.
Our experimental procedure ensured that the observed pattern of
Fos expression reflected brain activity during a period of undisturbed wakefulness, physiologically triggered by the onset of darkness, and spanning approximately 3 to 1 h prior to perfusion. The
high number of Fos-ir cells observed when the animals had been
stimulated by novel environmental cues also indicated that Fos
induction during wakefulness parallels the animal’s activity and
exploratory behavior. Our experiment, however, does not allow a
differentiation between sensorimotor activity-related Fos expression and gene induction concurrent with intrinsically generated
activation states.
The present study demonstrates that in animals examined at the
end of a period of wakefulness in a familiar environment Fos is
induced in a minor proportion of local circuit neurons that express
Pv or Cb in the parietal cortex, whereas no Fos induction was
found in Cb- or Pv-positive interneurons of the cingulate cortex.
Our data on Fos induction in cortical inhibitory cells during
relatively “quiet” wakefulness are in line with previous evidence,
obtained in different experimental paradigms of cortical activation
such as epilepsy [10,14] and thalamic stimulation [2], demonstrating c-fos induction in relatively small proportions of cortical
interneurons, as defined by their colocalization with calciumbinding proteins.
In the animals that had been exposed to an enriched environment, however, Fos induction was detected in a much higher
number of cortical neurons than in the behaviorally unstimulated
cases. This finding is in agreement with the recent report [19] of a
significant increase of the protein products of immediate early
genes, including c-fos, in the barrel cortex of rats placed overnight
in an enriched environment. Interestingly, in the present study we
could determine that the general increase of cortical cells in which
Fos was induced by the exploratory behavior was also accompanied by a substantial increment of Fos-ir interneurons, which did
not simply parallel an overall increase in Fos expression in the
corresponding cortical areas. Rather, cortical interneurons, at least
in the cingulate cortex, were significantly more recruited during
behaviorally relevant sensorimotor activity than projection neurons. It is also interesting to note that such relative increase of
Fos-ir cortical interneurons in actively exploring rats, compared to
animals habituated to their environment, was especially marked in
the Pv-containing cell population. Taken together, these results
indicate that meaningful activity, such as exploration of environmental novelty, is accompanied by selective recruitment of functionally different types of interneurons involved in regulatory
actions on pyramidal cells of distinct cortical areas.
In this respect, it should be pointed out that Pv- or Cb-containing cortical interneurons have different physiological properties. In
particular, nonpyramidal cells that express Pv have been identified
as fast spiking cells in layers II–III and V of the rat frontal cortex
[3,11–13]. On the other hand, Cb-containing cells were characterized as low-threshold spike cells in layer V of the frontal cortex
[12], but the physiological characteristics of neocortical nonpyramidal neurons that express Cb were also found to be more heterogeneous and complex [3].
On the other hand, the very high proportion of Fos-positive
cells that did not express Pv or Cb in our material provides indirect
evidence that the majority of cells expressing Fos in basal conditions are represented by projection neurons. In addition, the laminar distribution of Fos-positive cortical cells during spontaneous
wakefulness indicated that the oscillation of gene expression involved both corticocortical projection neurons and neurons of the
637
deep layers that innervate subcortical targets. It should be pointed
out, however, that Fos induction was observed also in layer IV
neurons of the animals exposed to novel environmental stimuli.
The latter findings support previous data on marked Fos induction
in neurons of layer IV and supragranular layers of the primary
visual and somatosensory cortices of rats exploring a novel environment, suggesting that the activation of these layers may be
specifically related to the attention paid to environmental stimuli
[15].
c-fos is a transcription factor involved in the signal transduction
cascade that links extracellular events to long-term intracellular
adaptations, and has been reported to be induced in neurons by a
variety of stimuli [9,16]. The significance of the spontaneous
fluctuation of gene expression in wake/sleep conditions is still
unknown, but the present findings point out that distinct subsets of
interneurons are involved in the cortex in intrinsic regulatory
mechanisms that occur in state-dependent behavior. Our data also
indicate that the neural correlates of the processing of novel
environmental stimuli involve a transcriptional response of select
populations of cortical interneurons.
ACKNOWLEDGEMENT
The study was supported by grants of the Italian Ministry of University
and Research.
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