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Reduction of GFAP induced by long dark rearing is not restricted to visual cortex
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Research Report
Reduction of GFAP induced by long dark rearing is not restricted to visual cortex Luigi Corvetti a,1,2 , Eugenio Aztiria a,2 , Luciano Domenici a,b,⁎ a
International School for Advanced Studies (SISSA), Cognitive Neuroscience Sector, Via Beirut 2-4, 34014 Trieste, Italy Institute of Neuroscience (CNR), Laboratory of Neurophysiology, Via G. Moruzzi 1, 56100 Pisa, Italy
b
A R T I C LE I N FO
AB S T R A C T
Article history:
A key component of the astrocyte cytoskeleton is the glial fibrillary acidic protein (GFAP),
Accepted 22 October 2005
which plays an essential role in neuron/astrocyte interactions. Environmental conditioning,
Available online 15 December 2005
such as visual experience manipulation, can affect neuronal and/or glial plasticity in specific brain areas. Previous work from our laboratory showed that short light deprivation
Theme:
throughout the period of GFAP maturation does not influence the expression profile of GFAP
Development and regeneration
in mouse visual cortex; however, it was strong enough to affect neuronal phenotype. It was
Topic:
suggested that visual experience controls the maturation of the neuronal circuitry in this
Visual system
brain area. Therefore, to see whether the modifications of neuronal activity induced by light deprivation affect the maintenance of normal astrocytic phenotype, the dark rearing
Keywords:
protocol was extended until the adult life. GFAP-immunoreactive cells were dramatically
Glia
affected, showing an 80% decrease in number. In addition, GFAP protein level exhibited a
Glial fibrillary acidic protein
50% reduction, while its mRNA remained unaffected. Besides the visual cortex, two other
Dark rearing
areas of the brain not directly involved in vision, the hippocampus and the motor cortex,
Western blot
were chosen as internal controls. Unexpectedly, also in these areas, astrocytes were affected
Immunohistochemistry
by light deprivation. The present results show that lack of visual experience for long periods
Plasticity
of time deeply affects glial phenotype not only in visual areas but also in brain regions not directly involved in sensory processing. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Postnatal development of GFAP in the visual cortex has been studied in different mammalian species, such as cat (Muller, 1990), rat (Stewart et al., 1986) and mouse (Corvetti et al., 2003). In cat and rat, GFAP develops slowly during the first 2 months of postnatal life, while, in mouse, the acquisition of an adult GFAP phenotype is
faster than in the other mammalian species. In particular, GFAP maturation is complete at the beginning of the fourth postnatal week. Changes of GFAP expression occurring after eye opening raise the interesting question of whether neuronal activity triggered by light represents a key factor in regulating GFAP expression. It is known that visual experience constitutes the driving force for the maturation of neurons and the refinement of neuronal
⁎ Corresponding author. International School for Advanced Studies, Cognitive Neuroscience Sector, Via Beirut 2-4, 34014 Trieste, Italy. E-mail address: [email protected] (L. Domenici). 1 Present address: Rita Levi-Montalcini Center for Brain Repair, Dept. of Neuroscience, Section of Physiology, University of Turin, Corso Raffaello 30, 10125 Turin, Italy. 2 The first 2 authors contributed equally to this work.
0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.072
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connections in the visual cortex (for a review, see Berardi et al., 2000). Sensory-dependent modification of neurons can be considered a component of visual cortex plasticity. Indeed, deprivation of visual input slows down the functional and structural maturation of visual cortical neurons and increases the length of the critical period for monocular deprivation (Fagiolini et al., 1994). Concerning astrocytes, it has been reported that environmental stimuli are able to interfere with GFAP expression. Indeed, after a short period of enriched-environment housing, the density of astrocytes within layer II/III increases in the rat cortex (Jones et al., 1996). In addition, it has been shown that a prolonged period of light deprivation from birth provokes a reduction of GFAP immunostaining in kittens (Muller, 1990) and GFAP level in rats (Stewart et al., 1986). These results suggest that GFAP is altered in dark-reared animals. We have recently re-examined this issue in mouse visual cortex, which is characterized by fast GFAP maturation. We showed that a short period of visual deprivation encompassing the time frame of GFAP maturation was unable to induce appreciable modifications of GFAP expression, thus excluding the possibility that visual input may drive the maturation of GFAP (Corvetti et al., 2003). In the present paper, we used a longer protocol of dark rearing (DR), extended beyond the maturation period of GFAP, to ascertain whether visual input is important to maintain a normal phenotype in visual cortex astrocytes. Mice were reared in the dark from P10, i.e. 4 to 5 days before eye opening, to P40 when maturation of GFAP in visual cortex is already over (Corvetti et al., 2003). As an additional marker, we used the S100 protein, a calcium binding protein well expressed in astrocytes (Cocchia, 1981; Ludwin et al., 1976) and known to be affected by light deprivation in the rat visual cortex (Argandona et al., 2003). The level of endogenous GFAP expression was studied by Western blot and RT-PCR to evaluate the protein and mRNA, respectively. Immunohistochemistry was carried out not only in the primary visual cortex but also in the hippocampus and motor cortex to check whether potential alterations were restricted to visual areas.
2.
Results
2.1. Effects of sensory deprivation on GFAP-immunoreactive cell distribution Mice were kept in the darkness from postnatal day 10 (P10), i.e. before eye opening, until P40. The distribution of GFAPimmunostained astrocytes was investigated in dark-reared and age-matched normally reared mice. In the visual cortex of control mice (Figs. 1A, C, E, G and I), GFAP-IR astrocytes were localized in layers I–III and VI (Figs. 1C and G), and labeled astrocytes were characterized by a star-shaped perikarya with branching cytoplasmic processes (Fig. 1I) similar to what was previously described (Corvetti et al., 2003). In dark-reared animals (Figs. 1B, D, F, H and J), GFAPimmunoreactive cells were reduced in all cortical layers
Fig. 1 – GFAP immunostaining in the visual cortex. Photomicrographs showing brain sections containing the visual cortex of control (A) and DR (B) mice. Low-magnification sections were counterstained with Thionin to identify the different cortical layers. GFAP-IR astrocytes concentrate on layers I–III and on the bottom layer VI. Higher magnification of the different cortical layers of control (C, E and G) and DR (D, F and H). GFAP-positive cells were rarely found in cortex of DR animals, and those few that were detected showed a weak staining with thin and short processes (J). Calibration bars: 100 μm in A and B; 50 μm in C–H; 10 μm in I and J.
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(Fig. 1B), and, at higher magnification, it was possible to observe profound alterations of astrocyte shape (Fig. 1J) that are less intensely stained than in control mice and are characterized by short and thin processes and by a reduced ramification. To quantify the results obtained at a qualitative level, GFAP-immunoreactive cells were counted within a 300 μm wide vertical bar scanning the primary visual cortex from the pial side to the corpus callosum. Cell counts in visual cortex showed that a prolonged light deprivation provoked a reduction in the number of GFAP-IR astrocytes of about 80% (Fig. 4A; n = 8, *P b 0.05, Student's t test).
2.2.
Western blot analysis of GFAP
To establish if the reduction of GFAP-immunostained cells that was observed in the visual cortex after light deprivation corresponds to a reduced level of the protein, the primary visual area was analyzed by Western blot as described in Experimental procedures. Membranes were probed with a polyclonal antibody recognizing GFAP, and a densitometric analysis was carried out on the membranes. Fig. 4B shows the expression profile of GFAP with respect to a house keeping protein such as tubulin, which was used as an internal standard, in the primary visual cortex. The densitometric analysis of the GFAP bands showed that also the protein expression was strongly reduced (∼50%) in dark-reared animals, compared to normal reared mice (n = 8; P b 0.05; Student's t test).
2.3.
Transcriptional expression of GFAP in DR mice
To know whether GFAP protein down-regulation was associated to a reduced expression at the transcriptional level, GFAP mRNA expression was analyzed by a non-saturating RT-PCR in the visual cortex. No difference was found between the control and DR groups, indicating that a prolonged light deprivation does not affect GFAP transcriptional expression (Fig. 4C).
2.4. Effects of sensory deprivation on S100 immunoreactivity To check whether prolonged DR effects were restricted to GFAP expression, the distribution of a second glial marker, the S100 protein, was also investigated by using an antibody recognizing both the A and B isoforms. In the visual cortex of control mice, S100 immunoreactivity is intense and distributed throughout the entire visual cortex (Fig. 2A). After a long DR protocol, S100-IR cells were strongly reduced in all the layers of the visual cortex (Fig. 2B). Furthermore, the shape of S100-immunoreactive astrocytes was compromised. In fact, they normally have a strongly stained, starshaped cell body and well-defined processes (Fig. 2A, insert). After light deprivation, the cell body looked shrunken and undefined and the processes were undefined (Fig. 2B, insert). In addition, the number of S100-IR cells in visual cortex of dark-reared animals was reduced around 75%, when compared to normally reared mice (Fig. 4D; n = 8, *P b 0.05, Student's t test).
Fig. 2 – S100-IR astrocytes in the visual cortex, hippocampus and motor cortex. Visual cortex sections of control and DR mice showing the pattern of expression of S100-IR cells. Positive cells are evenly distributed throughout all cortical layers both in control (A) and DR (B) animals, although S100-stained cells were strongly reduced in DR (B). Inserts show high magnification views of the S100-labeled cells. In control animals, the processes of astrocytes are well visible, and the soma shows a defined and heavily stained contour (A, inset). In DR animals, dramatic morphological changes were apparent at the cellular level as cells showed a reduced number of their processes and the soma display a roughly defined contour and appears weakly stained (B, inset). The effect of DR is apparent also in hippocampus and motor cortex. Panels C and D are representative examples of CA1 area of the hippocampus of control and DR animals, respectively. Panels E and F are representative examples of motor cortex control and DR animals, respectively. Note the strong reduction of the number of S100-IR cells also in these areas non-directly involved in the process of vision. Calibration bars: 100 μm in A, B, C and D; 10 μm in the insets; 20 μm in E and F. CA1, cornu ammonis 1; MC, motor cortex.
2.5. Effects of sensory deprivation on GFAP- and S100-immunoreactive cell distribution in hippocampus and motor cortex Although it was clear that the visual cortex was dramatically affected by GFAP reduction (Figs. 3A and B, primary visual cortex between arrows), we also analyzed two brain areas that do not receive direct visual input, like the hippocampus CA1 subfield and the motor cortex, in order to know whether the
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effects of light deprivation on GFAP and S100 immunoreactivity were restricted to visual areas only. In normal rearing conditions, GFAP-immunoreactive astrocytes are well distributed throughout all CA1 layers (Fig. 3C), exhibiting a well-defined soma and strongly stained branches (Fig. 3D). However, after DR, GFAP immunoreactivity was reduced in the entire hippocampus, especially in the CA1 subfield (Fig. 3E), and the shape of astrocytes was profoundly altered. In fact, when compared to the control group, astrocytes were weakly stained; its soma appeared narrow and stretched, with thinner branches (Fig. 3F). A quantification of
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the GFAP-IR astrocytes in the CA1 area showed a 50% reduction in DR animals with respect to the control group (Fig. 4E; n = 8, *P b 0.05, Student's t test). Interestingly, a similar reduction was found in the motor cortex, an area of the CNS that is far from the visual cortex and not involved in the codification of the visual information. In normal conditions of illumination, GFAP-immunoreactive cells are localized in the subpial layer (Fig. 3G) of the motor cortex. After DR, GFAP immunoreactivity was quite faint (Fig. 3H), and the number of immunoreactive cells was around 50% of that counted in control animals (Fig. 4F; *P b 0.05, Student's t test). In both hippocampus and motor cortex, S100 immunoreactivity was also strongly reduced. In normal light conditions, both the CA1 area of the hippocampus and the motor cortex (Figs. 2C and E respectively) display several S100-IR cells uniformly distributed, while, after the dark rearing protocols, they are dramatically reduced in both these areas (Figs. 2D and F, respectively).
3.
Discussion
In the present paper, we showed that a long period of dark rearing (i) affects GFAP and S100 expression in astrocytes and (ii) alters GFAP and S100 expression not only in the primary visual cortex. The results obtained in the present paper must be coupled to previous data obtained in our laboratory where we showed that the effect of a short period of light deprivation did not influence the maturation of GFAP in mouse visual cortex (Corvetti et al., 2003). However, other authors have reported that, in other species, light deprivation induces an alteration on GFAP expression in visual cortex (Muller, 1990; Stewart et al., 1986). The main differences in the protocol used here relied not only on the species used (mouse instead of rat) but also on the length of the DR protocol used. To establish if there is a different regulation of GFAP in mice and rats, or if chronic sensory deprivation could eventually affect GFAP metabolism in visual cortex, we used a longer visual deprivation protocol in the mouse. We found that a much longer period of DR
Fig. 3 – GFAP expression in the hippocampus. Panels A and B correspond to low-magnification views of the upper part of the brain of control and DR mice, respectively. The effects of sensory deprivation on GFAP expression in the cortex and the hippocampus are apparent (B). The primary visual cortex relies between the arrows. (C–F) Closer views of the GFAP immunostaining pattern in the CA1 subfield of the hippocampus of control (C and D) and DR animals (E and F) show the reduction induced by DR. Higher magnification of astrocytes illustrates that also in the hippocampus the morphology of astrocytes is affected by DR (compare D and F). (G–H) GFAP expression in the motor cortex. Positive cells were mostly localized on the upper cortical layers of motor cortex under both experimental conditions, although, in DR animals (H), the staining was very faint. Calibration bars: 200μm in A and B; 70 μm in C and E; 20 μm in D and F; 20 μm in G and H.
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Fig. 4 – Expression level of GFAP and S100 in the rat brain. (A–C) Quantification of GFAP-IR astrocytes, protein and mRNA level in VC. (A) Bar plot depicting the mean number of GFAP-IR cells per section in the whole VC of control and DR mice. No distinction was made among the different layers. Note the reduction of GFAP-IR (80%) in the DR group. (B) The graph shows the relative amount of GFAP in the visual cortex of control and DR animals, values are given as relative units (RU) as explained in Experimental procedures. A significant reduction in GFAP level (about 50%) can be observed. The panel under the plot reports a representative Western blot showing the expression of GFAP compared to that of tubulin (used as an internal control) under both experimental conditions. (C) Histogram describing the expression level of GFAP mRNA in control and DR mice, values are given as relative units (RU) as explained in Experimental procedures. Differences were not significant. The panel under the plot reports a representative agarose gel showing the expression level of the GFAP mRNA in control and DR animals compared to that of β actin (house keeping gene). (D) Quantification of S100-IR astrocytes in VC. Bar plot showing the mean number of S100-IR cells in the VC of control and DR mice. Note the reduction of S100-IR (75%) in the DR group. (E–F) Quantification of GFAP-IR astrocytes in the CA1 area of the hippocampus (E) and in MC (F). Note the reduction of GFAP-IR astrocytes in DR animals in both hippocampus (50%) and MC (50%). Data are media ± SEM; *P b 0.05, Student's t test, n = 8 per each group. Open bars = control, filled bars = DR; CA1, cornu ammonis 1; MC, motor cortex; VC, visual cortex. IHC = immunohistochemistry, WB = Western blot. RT-PCR = reverse transcriptase-polymerase chain reaction.
strongly reduces GFAP expression, suggesting that normal light exposure is important to maintain the correct astrocyte phenotype rather than being involved in GFAP development. We found a reduction of both GFAP immunoreactivity and GFAP level but not of GFAP mRNA, therefore suggesting an alteration at the protein level with no modifications occurring at the transcriptional level. The fact that GFAP mRNA and protein are regulated in different manner is in accordance with what was shown by other authors, in other brain areas and under different experimental conditions. It is known, for example, that GFAP-IR is reduced in the in the supraoptic nucleus after dehydration in rats (Hawrylak et al., 1998). Conversely, GFAP mRNA is increased in the same area under the same experimental conditions (Lally et al., 2005). Changes in the GFAP synthesis pathway and turnover may account for this phenomenon. One possibility is that the process of synthesis is regulated by visual activity at the post-transcriptional level, for example, through the interaction between the complex S100B-Ca++ and GFAP that hamper the correct assembly of the protein by inhibiting GFAP phosphorylation (Frizzo et al., 2004). To have an overview of the effect of light on the entire astrocyte population, the expression of the S100 protein was
also investigated. In fact, this soluble calcium binding protein is synthesized preferentially in mature astrocytes and expressed in all the layers of the cortex (Cocchia, 1981; Ludwin et al., 1976). Besides its use an astrocyte marker (Cocchia, 1981; Ludwin et al., 1976) and the just mentioned interaction with GFAP (Frizzo et al., 2004), it was suggested that the S100 protein could play an important role in the maturation of the cortical wiring (Muller, 1990). Our data showed that S100 is expressed throughout all the layers of the visual cortex and that a long period of visual deprivation causes a reduction of S100, as shown also by other authors (Argandona et al., 2003). Recent reports (Deloulme et al., 2004) showed that S100 is also expressed by a small class of oligodendrocytes, namely, the oligodendroglial progenitor cells (OPC). OPC accounts only for 5–8% of total glia (Dawson et al., 2000), and, in addition, S100 is strongly reduced in mature oligodendrocytes (Deloulme et al., 2004; Rickmann and Wolff, 1995). These data indicate that in late postnatal development and in adulthood S100 can be normally considered a good marker for the overall population of cortical astrocytes (Ludwin et al., 1976).
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Due to the fact that the S100 protein is expressed by other cells besides the astrocytes, we cannot exclude that prolonged DR also affect other cellular types in addition to astrocytes, nevertheless, due the poor amount of the other cells expressing S100, in CNS, we conclude that the reduction observed in the number of S100-immunoreactive cells is mainly due to S100 expressed by astrocytes. Since both GFAP and S100 are reduced, the possibility has to be considered that the process of astrocyte cell death might occur in DR mice. The data showing that GFAP mRNA was not changed after DR indirectly suggest that astrocytes expressing the GFAP mRNA were not reduced in number. However, since (i) in the neocortex only a subpopulation of astrocytes express GFAP (Cocchia, 1981; Ludwin et al., 1976) and (ii) in DR mice also S100 which stains a larger population of cortical astrocytes is affected, we cannot completely exclude the possibility that visual deprivation induces astrocytes death. In our opinion, long dark rearing affects the cytoskeletal properties of astrocytes by acting on the synthesis and/or the turnover of the protein rather than on astrocyte survival. Considering the mutual interaction between GFAP and S100, it might be possible that, in mice, our DR protocols affect the calcium buffering properties of astrocytes. Indeed, the morphological alteration observed might be a consequence of the altered intracellular distribution of GFAP, maybe due to an alteration in the conformation of the protein. Ultrastructural studies will be required to verify whether there is really a morphological change. However, these experiments are far from the goal of this paper. We also checked if, under the present experimental conditions, GFAP and S100 alterations were confined to visual cortex or if areas not directly involved in visual processing were affected as well. Indeed, we found a reduction of both GFAP and S100 also in hippocampus and motor cortex, two areas that do not take part in the elaboration of the visual signal. A first explanation for this phenomenon could be a non-homogenous penetration of the antibody, however, tissue from control and DR animals was processed simultaneously by using the same amount and batch of the two antibodies. To further support the specificity of results obtained in DR mice, we run a second group of immunohistochemical experiments that confirmed the previous results. Another hypothesis, for GFAP reduction, takes into account the sexual dimorphism. It has been shown that the number of GFAP-IR astrocytes, in hippocampus, is reduced in females with respect to male mice (Mouton et al., 2002), this reduction is around 20% in young adult mice showing a trend to increase at late postnatal ages. Since long DR induced a much dramatic reduction of GFAP-stained cells, in VC (80%) and hippocampus (20%), gender differences cannot completely account for the alterations reported. Indeed, though we found that the motor cortex and the hippocampus are less affected by visual deprivation, compared to VC, this result suggests that the influences of light deprivation on astrocyte phenotype, rather than being direct, could be mediated by a wider phenomenon involving the entire CNS. What are the mechanisms responsible for astrocyte alterations?
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It is well known that neuronal activity can influence astrocytes and vice versa. In addition, light deprivation induces several alterations on neuronal phenotype (reviewed in Berardi et al., 2000) and on the expression of several proteins including neurotrophins (Mouton et al., 2002) and their receptors (Castren et al., 1992; Tropea et al., 2001) and neurotransmitters (Viegi et al., 2002). On the other hand, some tyrosine kinase receptors (TrkB and C) are expressed in astroglial and oligodendroglial cells in culture (Morales et al., 2002). Prolonged sensory deprivation might modify several physiological responses including the perception of the circadian period. Nevertheless, animals reared under constant light show an over-expression of GFAP, not only in the visual cortex but also in the cerebellum and the hippocampus (Condorelli et al., 1995). Indeed, GFAP immunoreactivity in the suprachiasmatic nucleus, the area in charge of regulating behavioral and physiological circadian rhythm in mammals, appears to be highly sensitive to circadian time and light conditions (Baydas et al., 2002). Elegant experiments by Lavialle et al. (2001) demonstrated that disruption of the retino-hypothalamic projections promotes a dramatic decrease in GFAP expression and GFAP-immunoreactive redistribution. The lack of cyclic information input affecting the suprachiasmatic nucleus of the hypothalamus might in turn influence the whole CNS metabolism of GFAP. The present results confirm our previous hypothesis that the development of GFAP in mouse is not regulated by light deprivation, but prolonged deprivation of visual experience influences the maintenance of the correct astrocytic phenotype. Nonetheless, they suggest that long DR affects the entire CNS. Thus, the demonstration that astrocyte alterations are not restricted to visual cortical areas imposes a caveat in the use of prolonged periods of DR as a physiological tool to study the role of light in maturation and maintenance of visual cortical circuitry in mouse.
4.
Experimental procedures
4.1.
Animals
Experiments were conducted on C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) in accordance to the European Community Council Directive for animal treatment. In total, we used 16 animals per each group, brain extracts from 8 animals were processed for PCR and Western blot, while the brain from the other 8 animals was processed for immunohistochemistry. 4.2.
Dark rearing
Two different litters were used in these experiments, and, for each litter, pups were randomly assigned to the DR or to the control group (6 animals per group). DR mice were kept in complete darkness for 1 month, from P10, i.e. 4–5 days before eye opening, to P40, when the maturation period is nearly over, while the control group was placed in an artificial 12 h/12 h light/dark cycle. At P40, each experimental group was further divided into two groups: one was perfused and processed for immunohistochemistry, while Western blot and RT-PCR were carried out on the other group. All the analyses described in this paper were performed blindly with respect to the experimental treatment. Furthermore, to avoid spurious results and to exclude the possibility that results
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obtained were due to potential artifacts, attributable to the dark rearing protocol, two more groups of animals (5 animals per group) were placed in DR and the tissue was processed as above. Results were compared with two different groups of control. 4.3.
Immunohistochemistry
Immunohistochemistry was assessed as described elsewhere (Corvetti et al., 2003). Briefly, mice were deeply anesthetized with 20% urethane (Sigma, St. Louis, MO, USA) and then transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). Brains were removed, post-fixed for 1 h at 4 °C in the same solution and then cryoprotected by immersion in 30% sucrose/PBS. Forty-micron thick coronal sections, containing the primary visual cortex, (approximately −2.70 to −5.00 from Bregma, according to Paxinos and Franklin, 1997) were obtained using a freezing sliding microtome, and serial sections were collected in PBS. Sections were first incubated in 0.3% hydrogen peroxide in Tris-buffered saline (TBS) to quench endogenous peroxidase and then processed with the different antibodies. After a preincubation (45 min at room temperature) in mouse Ig blocking reagent (Vector Labs Inc., Burlingame, CA, USA), sections were processed for detection of GFAP or S100. All the primary and secondary antibodies were diluted in phosphate-buffered saline plus 0.1% Triton X-100 (PBST) containing 5% fetal calf serum. Tissue sections were incubated with mouse monoclonal antibodies anti-GFAP (1:100, clone G-A-5, Sigma) or anti-S100 (1:100; clone 4C4.9, Neo Markers, Fremont, CA, USA) for 18 h at 4 °C. The day after, sections were incubated with a goat anti-mouse biotinylated antibody (1:200; Vector) for 3 h at room temperature and then with horseradish-peroxidase-conjugated avidin–biotin complex (1:100; Elite Standard kit, Vector) for 1 h. The peroxidase complex was visualized using 3,3-diaminobenzidine tetrachloride (Sigma) dissolved in Tris/HCl, pH 7.5. Tissue from the DR and the control group were processed in parallel. GFAP-immunostained sections were counterstained with Thionin (1%; Merck, Darmstadt, Germany) to identify the different cortical layers. 4.4.
4.6. GFAP reverse transcription-polymerase chain reaction (RT-PCR)
Cell counts
In both control and dark-reared mice, sections containing the visual cortex were divided in 12 consecutive series (four–five slices/series) (Howard et al., 1998). One every 12 sections was chosen following the rostro-caudal axis, choosing the first section randomly. In this way, 12 series of 4–5 sections each were sampled uniformly at random. A single random series (1/12) of coronal sections/animal was analyzed, and thereby enabling all visual cortical sections to be sampled with the same probability. Following immunohistochemistry, GFAP-IR cells in the visual cortex, motor cortex and hippocampus and S100-positive cells in the visual cortex were counted using a 40× objective. A 300-μm wide grid was superimposed on the section to delimit the area of sampling. Cells within each frame and in the different focal planes were manually counted using the “optical dissector” method (Gundersen, 1986). GFAP-IR cells, within an entire cortical stack going from the pial side to the white matter (i.e. including all the cortical layers) of both the motor and the visual cortex, were counted. The final thickness of the sections, coming from either the dark rearing or the control group, is 34 ± 5 μm. 4.5.
same stereotaxic coordinates as for immunohistochemistry. Only one of the two hemispheres was used for the Western blot, while the other one was immediately frozen in dry ice for RNA analysis. Visual cortices were homogenized in an extracting buffer (1 g tissue/5 ml buffer) containing 50 mM Tris/HCl pH 8, 150 mM EDTA, 0.1% deoxycholic acid, 1% igepal, 1 mM aprotinine, 5 mg/ml phenylmethylsulfonylfluoride, 1 mM iodoacetamide, 5 mg/ml leupeptin, 4 mg/ml soybean trypsin inhibitor and 10 mg/ml turkey egg white inhibitor. Protein concentration was assessed by Lowry colorimetric assay (Lowry et al., 1951), and 20 μg of total protein, extracted from a single brain, was loaded for each lane and resolved in a 12% SDS polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech; Little Chalfont, UK), blocked for 1 h at 37 °C in 5% nonfat milk/PBS (pH 7.4) and then incubated overnight at 4 °C with the proper primary antibody dissolved in 0.05% Tween-20/PBS, pH 7.4 (PBST). A goat anti-mouse antibody against GFAP at a 1:300 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a rat anti-mouse antibody against tubulin at a 1:250 dilution (mAb YOL1, kindly provided by Dr. Cesar Milstein, MRC, Cambridge, UK) were used. After washing in PBS, membranes were incubated for 2 h at 37 °C with the secondary anti-goat (1:1000; Vector) or anti-rat (1:500; Dako Laboratories, Glostrup, Denmark) biotinylated antibody, washed and incubated for 30 min at room temperature with alkaline-phosphatase-conjugated ABC kit (1:1000; Vector). Reaction was revealed using p-Nitro Blue Tetrazolium chloride (NBT; Sigma, 0.5 mg/ml) and 5-Bromo-4-Chloro-3-Indoyl Phosphate ptoluidine salt (BCIP; Sigma, 0.25 mg/ml) in developing buffer (0.1 M Tris, 0.5 mM MgCl2, pH 9.5). Each Western blot was run in duplicate. For a semi-quantitative analysis, after blotting, membranes were scanned, and the density of the single bands was determined using the NIH Image software (Rasband and Bright, 1995). For each band, optical density (OD) was calculated multiplying the mean optical density by the area of the band, and values were given as relative units (RU) according to the following formula: (GFAP OD / tubulin OD) × 100.
Western blot analysis
To evaluate the level of GFAP expression, proteins were extracted from visual cortex, as described previously (Corvetti et al., 2003), and the amount of GFAP was analyzed. Mice were deeply anesthetized with 20% urethane, the brain was removed, and visual cortex was separated from the rest of the brain, using the
The relative amount of GFAP mRNA was evaluated in the visual cortex by RT-PCR. To detect the protein-specific message, the total RNA was extracted from the visual cortex using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) then treated with DNAse I (Ambion, Austin, TX, USA) in order to ensure the absence of genomic DNA in the sample. Two micrograms of total RNA was reverse transcribed (RT) into cDNA using Moloney-Murine leukemia virus (M-MLV) reverse transcriptase (Gibco BRL, Milan, Italy) using random primers. After RT, 1 μl of cDNA was used as template in 25 μl of PCR mixture containing: 0.5 mM final concentration of proteinspecific primers, 0.2 mM deoxinucleotides, GeneAmp 10× buffer and 0.5 U of Platinum Taq DNA Polymerase (Gibco BRL). Primers for GFAP and for β-actin GFAP were designed from published sequence data (Ghazanfari and Stewart, 2001) as follows: β-actinU: 5′-AAC CCT AAG GCC AAC CGT GAA AAG-3′; β-actin-D: 5′-CTA GGA GCC AGG GCA GTA ATC T-3′; GFAP-U: 5′-AGT CCC TCC GCG GCA CGA ACG A-3′; GFAP-D: 5′-ACC ATC CCG CAT CTC CAC AGT CTT TAC CAC-3′. Each PCR reaction was carried out following a temperature protocol consisting of a warm up period of 5 min at 95 °C, 26 cycles of PCR (95 °C for 1 min, 55 °C for 1 min, 72 °C for 5 min) and a final elongation period of 10 min at 75 °C. All samples were tested simultaneously for the two different primer sets, and all PCRs were run in duplicate to eliminate the possibility of spurious results. PCR products were analyzed on 1.5% agarose gel in Tris– borate EDTA buffer, stained with ethidium bromide. To assess semi-quantitatively the expression of the GFAP gene, the agarose gel pictures were digitized and analyzed using the NIH Image program mentioned before (Rasband and Bright, 1995). The optical
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density (OD) of the GFAP bands was normalized by comparison to that of β-actin, which was used as an internal standard and expressing the obtained values as RU, as previously described. The expression level of mRNA between control and treated was studied by using non-saturating PCR conditions. The identity of the PCR products was determined by sequencing the gel-purified bands. 4.7.
Statistics
Data are expressed as mean ± SEM of eight animals per group. Statistical analysis of data was performed by using Student's t test. The values were considered significant with P b 0.05.
Acknowledgment Thanks to Mr. Ian Martin Williams for carefully reading the manuscript.
REFERENCES
Argandona, E., Rossi, M.L., Lafuente, J.V., 2003. Visual deprivation effects on the S-100β positive astrocytic population in the developing rat visual cortex: a quantitative study. Brain Res. Dev. Brain Res. 141, 63–69. Baydas, G., Reiter, R.J., Nedzvetskii, V.S., Nerush, P.A., Kirichenko, S. V., 2002. Altered glial fibrillary acidic protein content and in the hippocampus, cortex and cerebellum of rats to constant light: reversal by melatonin. J. Pineal Res. 33, 134–139. Berardi, N., Pizzorusso, T., Maffei, L., 2000. Critical periods during sensory development. Curr. Opin. Neurobiol. 10, 138–145. Castren, E., Zafra, F., Thoenen, H., Lindholm, D., 1992. Light regulates the expression of brain-derived neurotrophic factor in rat visual cortex. Proc. Natl. Acad. Sci. U. S. A. 19, 9444–9448. Cocchia, D., 1981. Immunocytochemical localization of S-100 protein in the brain of adult rat. An ultrastructural study. Cell Tissue Res. 214, 529–540. Condorelli, D.F., Salin, T., Dell' Albani, P., Mudo, G., Corsaro, M., Timmusk, T., Metsis, M., Belluardo, N., 1995. Neurotrophins and their trk receptors in cultured cells of the glial lineage and in white matter of the central nervous system. J. Mol. Neurosi. 6, 237–248. Corvetti, L., Capsoni, S., Cattaneo, A., Domenici, L., 2003. Postnatal development of GFAP in mouse visual cortex is not affected by light deprivation. Glia 41, 404–414. Dawson, M.R., Levine, J.M., Reynolds, R., 2000. NG2-expressing cells in the central nervous system: are they oligodendroglial progenitors? J. Neurosci. Res. 61, 471–479. Deloulme, J.C., Raponi, E., Gentil, B.J., Bertacchi, N., Marks, A., Labourdette, G., Baudier, J., 2004. Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation. Mol. Cell. Neurosci. 27, 453–465. Fagiolini, M., Pizzorusso, T., Berardi, N., Domenica, L., Maffei, L., 1994. Functional postnatal development of the rat primary visual cortex and the role of visual experience: dark rearing and monocular deprivation. Vision Res. 34, 709–720.
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Frizzo, J.K., Tramontina, F., Bortoli, E., Gottfried, C., Leal, R.B., Lengyel, I., Donato, R., Dunkley, P.R., Goncalves, C.A., 2004. S100B-mediated inhibition of the phosphorylation of GFAP is prevented by TRTK-12. Neurochem. Res. 29, 735–740. Ghazanfari, F.A., Stewart, R.R., 2001. Characteristics of endothelial cells derived from the blood–brain barrier and of astrocytes in culture. Brain Res. 890, 49–65. Gundersen, H.J., 1986. Stereology of arbitrary particles, a review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson. J. Microsc. 143, 3–45. Hawrylak, N., Fleming, J.C., Salm, A.K., 1998. Dehydration and rehydration selectively and reversibly alter glial fibrillary acidic protein immunoreactivity in the rat supraoptic nucleus and subjacent glial limitans. Glia 22, 260–271. Howard, C.V., Reed, M.G., 1998. Unbiased Stereology. Springer-Verlag, New York, NY. Jones, T.A., Hawrylak, N., Greenough, W.T., 1996. Rapid laminar-dependent changes in GFAP immunoreactive astrocytes in the visual cortex of rats reared in a complex environment. Psychoneuroendocrinology 21, 189–201. Lally, B.E., Albrecht, P.J., Levison, S.W., Salm, A.K., 2005. Divergent glial fibrillary acidic protein and its mRNA in the activated supraoptic nucleus. Neurosci. Lett. 380, 295–299. Lavialle, M., Begue, A., Papillon, C., Vilaplana, J., 2001. Modifications of retinal afferent activity induce changes in astroglial plasticity in the hamster circadian clock. Glia 34, 88–100. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Pholin phenol reagent. J. Biol. Chem. 193, 265–275. Ludwin, S.K., Kosek, J.C., Eng, L.F., 1976. The topographical distribution of S-100 and GFA proteins in the adult rat brain: an immunohistochemical study using horseradish peroxidase-labelled antibodies. J. Comp. Neurol. 165, 197–207. Morales, B., Choi, S.Y., Kirkwood, A., 2002. Dark rearing alters the development of GABAergic transmission in visual cortex. J. Neurosci. 22, 8084–8090. Mouton, P.R., Long, J.M., Lei, D.L., Howard, V., Jucker, M., Calhoun, M.E., Ingram, D.K., 2002. Age and gender effects on microglia and astrocyte numbers in brains of mice. Brain Res. 956, 30–35. Muller, C.M., 1990. Dark-rearing retards the maturation of astrocytes in restricted layers of cat visual cortex. Glia 3, 487–494. Paxinos, J., Franklin, K.B.J., 1997. The Mouse Brain in Stereotaxic Coordinates. Academic Press, London. Rasband, W.S., Bright, D.S., 1995. NIH Image: a public domain image processing program for the Macintosh. Microbeam, Anal. Soc. J. 4, 37–149. Rickmann, M., Wolff, J.R., 1995. S100 immunoreactivity in a subpopulation of oligodendrocytes and Ranvier's nodes of adult rat brain. Neurosci. Lett. 67, 991–997. Stewart, M.G., Bourne, R.C., Gabbott, P.L., 1986. Decreased levels of an astrocytic marker, glial fibrillary acidic protein, in the visual cortex of dark-reared rats: measurement by enzyme-linked immunosorbent assay. Neurosci. Lett. 63, 147–152. Tropea, D., Capsoni, S., Tongiorgi, E., Giannotta, S., Cattaneo, A., Domenica, L., 2001. Mismatch between BDNF mRNA and protein expression in the developing visual cortex: the role of visual experience. Eur. J. Neurosci. 13, 709–721. Viegi, A., Cotrufo, T., Berardi, N., Mascia, L., Maffei, L., 2002. Effects of dark rearing on phosphorylation of neurotrophin Trk receptors. Eur. J. Neurosci. 16, 1925–1930.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Reduction of GFAP induced by long dark rearing is not restricted to visual cortex Luigi Corvetti a,1,2 , Eugenio Aztiria a,2 , Luciano Domenici a,b,⁎ a
International School for Advanced Studies (SISSA), Cognitive Neuroscience Sector, Via Beirut 2-4, 34014 Trieste, Italy Institute of Neuroscience (CNR), Laboratory of Neurophysiology, Via G. Moruzzi 1, 56100 Pisa, Italy
b
A R T I C LE I N FO
AB S T R A C T
Article history:
A key component of the astrocyte cytoskeleton is the glial fibrillary acidic protein (GFAP),
Accepted 22 October 2005
which plays an essential role in neuron/astrocyte interactions. Environmental conditioning,
Available online 15 December 2005
such as visual experience manipulation, can affect neuronal and/or glial plasticity in specific brain areas. Previous work from our laboratory showed that short light deprivation
Theme:
throughout the period of GFAP maturation does not influence the expression profile of GFAP
Development and regeneration
in mouse visual cortex; however, it was strong enough to affect neuronal phenotype. It was
Topic:
suggested that visual experience controls the maturation of the neuronal circuitry in this
Visual system
brain area. Therefore, to see whether the modifications of neuronal activity induced by light deprivation affect the maintenance of normal astrocytic phenotype, the dark rearing
Keywords:
protocol was extended until the adult life. GFAP-immunoreactive cells were dramatically
Glia
affected, showing an 80% decrease in number. In addition, GFAP protein level exhibited a
Glial fibrillary acidic protein
50% reduction, while its mRNA remained unaffected. Besides the visual cortex, two other
Dark rearing
areas of the brain not directly involved in vision, the hippocampus and the motor cortex,
Western blot
were chosen as internal controls. Unexpectedly, also in these areas, astrocytes were affected
Immunohistochemistry
by light deprivation. The present results show that lack of visual experience for long periods
Plasticity
of time deeply affects glial phenotype not only in visual areas but also in brain regions not directly involved in sensory processing. © 2005 Elsevier B.V. All rights reserved.
1.
Introduction
Postnatal development of GFAP in the visual cortex has been studied in different mammalian species, such as cat (Muller, 1990), rat (Stewart et al., 1986) and mouse (Corvetti et al., 2003). In cat and rat, GFAP develops slowly during the first 2 months of postnatal life, while, in mouse, the acquisition of an adult GFAP phenotype is
faster than in the other mammalian species. In particular, GFAP maturation is complete at the beginning of the fourth postnatal week. Changes of GFAP expression occurring after eye opening raise the interesting question of whether neuronal activity triggered by light represents a key factor in regulating GFAP expression. It is known that visual experience constitutes the driving force for the maturation of neurons and the refinement of neuronal
⁎ Corresponding author. International School for Advanced Studies, Cognitive Neuroscience Sector, Via Beirut 2-4, 34014 Trieste, Italy. E-mail address: [email protected] (L. Domenici). 1 Present address: Rita Levi-Montalcini Center for Brain Repair, Dept. of Neuroscience, Section of Physiology, University of Turin, Corso Raffaello 30, 10125 Turin, Italy. 2 The first 2 authors contributed equally to this work.
0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.072
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connections in the visual cortex (for a review, see Berardi et al., 2000). Sensory-dependent modification of neurons can be considered a component of visual cortex plasticity. Indeed, deprivation of visual input slows down the functional and structural maturation of visual cortical neurons and increases the length of the critical period for monocular deprivation (Fagiolini et al., 1994). Concerning astrocytes, it has been reported that environmental stimuli are able to interfere with GFAP expression. Indeed, after a short period of enriched-environment housing, the density of astrocytes within layer II/III increases in the rat cortex (Jones et al., 1996). In addition, it has been shown that a prolonged period of light deprivation from birth provokes a reduction of GFAP immunostaining in kittens (Muller, 1990) and GFAP level in rats (Stewart et al., 1986). These results suggest that GFAP is altered in dark-reared animals. We have recently re-examined this issue in mouse visual cortex, which is characterized by fast GFAP maturation. We showed that a short period of visual deprivation encompassing the time frame of GFAP maturation was unable to induce appreciable modifications of GFAP expression, thus excluding the possibility that visual input may drive the maturation of GFAP (Corvetti et al., 2003). In the present paper, we used a longer protocol of dark rearing (DR), extended beyond the maturation period of GFAP, to ascertain whether visual input is important to maintain a normal phenotype in visual cortex astrocytes. Mice were reared in the dark from P10, i.e. 4 to 5 days before eye opening, to P40 when maturation of GFAP in visual cortex is already over (Corvetti et al., 2003). As an additional marker, we used the S100 protein, a calcium binding protein well expressed in astrocytes (Cocchia, 1981; Ludwin et al., 1976) and known to be affected by light deprivation in the rat visual cortex (Argandona et al., 2003). The level of endogenous GFAP expression was studied by Western blot and RT-PCR to evaluate the protein and mRNA, respectively. Immunohistochemistry was carried out not only in the primary visual cortex but also in the hippocampus and motor cortex to check whether potential alterations were restricted to visual areas.
2.
Results
2.1. Effects of sensory deprivation on GFAP-immunoreactive cell distribution Mice were kept in the darkness from postnatal day 10 (P10), i.e. before eye opening, until P40. The distribution of GFAPimmunostained astrocytes was investigated in dark-reared and age-matched normally reared mice. In the visual cortex of control mice (Figs. 1A, C, E, G and I), GFAP-IR astrocytes were localized in layers I–III and VI (Figs. 1C and G), and labeled astrocytes were characterized by a star-shaped perikarya with branching cytoplasmic processes (Fig. 1I) similar to what was previously described (Corvetti et al., 2003). In dark-reared animals (Figs. 1B, D, F, H and J), GFAPimmunoreactive cells were reduced in all cortical layers
Fig. 1 – GFAP immunostaining in the visual cortex. Photomicrographs showing brain sections containing the visual cortex of control (A) and DR (B) mice. Low-magnification sections were counterstained with Thionin to identify the different cortical layers. GFAP-IR astrocytes concentrate on layers I–III and on the bottom layer VI. Higher magnification of the different cortical layers of control (C, E and G) and DR (D, F and H). GFAP-positive cells were rarely found in cortex of DR animals, and those few that were detected showed a weak staining with thin and short processes (J). Calibration bars: 100 μm in A and B; 50 μm in C–H; 10 μm in I and J.
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(Fig. 1B), and, at higher magnification, it was possible to observe profound alterations of astrocyte shape (Fig. 1J) that are less intensely stained than in control mice and are characterized by short and thin processes and by a reduced ramification. To quantify the results obtained at a qualitative level, GFAP-immunoreactive cells were counted within a 300 μm wide vertical bar scanning the primary visual cortex from the pial side to the corpus callosum. Cell counts in visual cortex showed that a prolonged light deprivation provoked a reduction in the number of GFAP-IR astrocytes of about 80% (Fig. 4A; n = 8, *P b 0.05, Student's t test).
2.2.
Western blot analysis of GFAP
To establish if the reduction of GFAP-immunostained cells that was observed in the visual cortex after light deprivation corresponds to a reduced level of the protein, the primary visual area was analyzed by Western blot as described in Experimental procedures. Membranes were probed with a polyclonal antibody recognizing GFAP, and a densitometric analysis was carried out on the membranes. Fig. 4B shows the expression profile of GFAP with respect to a house keeping protein such as tubulin, which was used as an internal standard, in the primary visual cortex. The densitometric analysis of the GFAP bands showed that also the protein expression was strongly reduced (∼50%) in dark-reared animals, compared to normal reared mice (n = 8; P b 0.05; Student's t test).
2.3.
Transcriptional expression of GFAP in DR mice
To know whether GFAP protein down-regulation was associated to a reduced expression at the transcriptional level, GFAP mRNA expression was analyzed by a non-saturating RT-PCR in the visual cortex. No difference was found between the control and DR groups, indicating that a prolonged light deprivation does not affect GFAP transcriptional expression (Fig. 4C).
2.4. Effects of sensory deprivation on S100 immunoreactivity To check whether prolonged DR effects were restricted to GFAP expression, the distribution of a second glial marker, the S100 protein, was also investigated by using an antibody recognizing both the A and B isoforms. In the visual cortex of control mice, S100 immunoreactivity is intense and distributed throughout the entire visual cortex (Fig. 2A). After a long DR protocol, S100-IR cells were strongly reduced in all the layers of the visual cortex (Fig. 2B). Furthermore, the shape of S100-immunoreactive astrocytes was compromised. In fact, they normally have a strongly stained, starshaped cell body and well-defined processes (Fig. 2A, insert). After light deprivation, the cell body looked shrunken and undefined and the processes were undefined (Fig. 2B, insert). In addition, the number of S100-IR cells in visual cortex of dark-reared animals was reduced around 75%, when compared to normally reared mice (Fig. 4D; n = 8, *P b 0.05, Student's t test).
Fig. 2 – S100-IR astrocytes in the visual cortex, hippocampus and motor cortex. Visual cortex sections of control and DR mice showing the pattern of expression of S100-IR cells. Positive cells are evenly distributed throughout all cortical layers both in control (A) and DR (B) animals, although S100-stained cells were strongly reduced in DR (B). Inserts show high magnification views of the S100-labeled cells. In control animals, the processes of astrocytes are well visible, and the soma shows a defined and heavily stained contour (A, inset). In DR animals, dramatic morphological changes were apparent at the cellular level as cells showed a reduced number of their processes and the soma display a roughly defined contour and appears weakly stained (B, inset). The effect of DR is apparent also in hippocampus and motor cortex. Panels C and D are representative examples of CA1 area of the hippocampus of control and DR animals, respectively. Panels E and F are representative examples of motor cortex control and DR animals, respectively. Note the strong reduction of the number of S100-IR cells also in these areas non-directly involved in the process of vision. Calibration bars: 100 μm in A, B, C and D; 10 μm in the insets; 20 μm in E and F. CA1, cornu ammonis 1; MC, motor cortex.
2.5. Effects of sensory deprivation on GFAP- and S100-immunoreactive cell distribution in hippocampus and motor cortex Although it was clear that the visual cortex was dramatically affected by GFAP reduction (Figs. 3A and B, primary visual cortex between arrows), we also analyzed two brain areas that do not receive direct visual input, like the hippocampus CA1 subfield and the motor cortex, in order to know whether the
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effects of light deprivation on GFAP and S100 immunoreactivity were restricted to visual areas only. In normal rearing conditions, GFAP-immunoreactive astrocytes are well distributed throughout all CA1 layers (Fig. 3C), exhibiting a well-defined soma and strongly stained branches (Fig. 3D). However, after DR, GFAP immunoreactivity was reduced in the entire hippocampus, especially in the CA1 subfield (Fig. 3E), and the shape of astrocytes was profoundly altered. In fact, when compared to the control group, astrocytes were weakly stained; its soma appeared narrow and stretched, with thinner branches (Fig. 3F). A quantification of
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the GFAP-IR astrocytes in the CA1 area showed a 50% reduction in DR animals with respect to the control group (Fig. 4E; n = 8, *P b 0.05, Student's t test). Interestingly, a similar reduction was found in the motor cortex, an area of the CNS that is far from the visual cortex and not involved in the codification of the visual information. In normal conditions of illumination, GFAP-immunoreactive cells are localized in the subpial layer (Fig. 3G) of the motor cortex. After DR, GFAP immunoreactivity was quite faint (Fig. 3H), and the number of immunoreactive cells was around 50% of that counted in control animals (Fig. 4F; *P b 0.05, Student's t test). In both hippocampus and motor cortex, S100 immunoreactivity was also strongly reduced. In normal light conditions, both the CA1 area of the hippocampus and the motor cortex (Figs. 2C and E respectively) display several S100-IR cells uniformly distributed, while, after the dark rearing protocols, they are dramatically reduced in both these areas (Figs. 2D and F, respectively).
3.
Discussion
In the present paper, we showed that a long period of dark rearing (i) affects GFAP and S100 expression in astrocytes and (ii) alters GFAP and S100 expression not only in the primary visual cortex. The results obtained in the present paper must be coupled to previous data obtained in our laboratory where we showed that the effect of a short period of light deprivation did not influence the maturation of GFAP in mouse visual cortex (Corvetti et al., 2003). However, other authors have reported that, in other species, light deprivation induces an alteration on GFAP expression in visual cortex (Muller, 1990; Stewart et al., 1986). The main differences in the protocol used here relied not only on the species used (mouse instead of rat) but also on the length of the DR protocol used. To establish if there is a different regulation of GFAP in mice and rats, or if chronic sensory deprivation could eventually affect GFAP metabolism in visual cortex, we used a longer visual deprivation protocol in the mouse. We found that a much longer period of DR
Fig. 3 – GFAP expression in the hippocampus. Panels A and B correspond to low-magnification views of the upper part of the brain of control and DR mice, respectively. The effects of sensory deprivation on GFAP expression in the cortex and the hippocampus are apparent (B). The primary visual cortex relies between the arrows. (C–F) Closer views of the GFAP immunostaining pattern in the CA1 subfield of the hippocampus of control (C and D) and DR animals (E and F) show the reduction induced by DR. Higher magnification of astrocytes illustrates that also in the hippocampus the morphology of astrocytes is affected by DR (compare D and F). (G–H) GFAP expression in the motor cortex. Positive cells were mostly localized on the upper cortical layers of motor cortex under both experimental conditions, although, in DR animals (H), the staining was very faint. Calibration bars: 200μm in A and B; 70 μm in C and E; 20 μm in D and F; 20 μm in G and H.
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Fig. 4 – Expression level of GFAP and S100 in the rat brain. (A–C) Quantification of GFAP-IR astrocytes, protein and mRNA level in VC. (A) Bar plot depicting the mean number of GFAP-IR cells per section in the whole VC of control and DR mice. No distinction was made among the different layers. Note the reduction of GFAP-IR (80%) in the DR group. (B) The graph shows the relative amount of GFAP in the visual cortex of control and DR animals, values are given as relative units (RU) as explained in Experimental procedures. A significant reduction in GFAP level (about 50%) can be observed. The panel under the plot reports a representative Western blot showing the expression of GFAP compared to that of tubulin (used as an internal control) under both experimental conditions. (C) Histogram describing the expression level of GFAP mRNA in control and DR mice, values are given as relative units (RU) as explained in Experimental procedures. Differences were not significant. The panel under the plot reports a representative agarose gel showing the expression level of the GFAP mRNA in control and DR animals compared to that of β actin (house keeping gene). (D) Quantification of S100-IR astrocytes in VC. Bar plot showing the mean number of S100-IR cells in the VC of control and DR mice. Note the reduction of S100-IR (75%) in the DR group. (E–F) Quantification of GFAP-IR astrocytes in the CA1 area of the hippocampus (E) and in MC (F). Note the reduction of GFAP-IR astrocytes in DR animals in both hippocampus (50%) and MC (50%). Data are media ± SEM; *P b 0.05, Student's t test, n = 8 per each group. Open bars = control, filled bars = DR; CA1, cornu ammonis 1; MC, motor cortex; VC, visual cortex. IHC = immunohistochemistry, WB = Western blot. RT-PCR = reverse transcriptase-polymerase chain reaction.
strongly reduces GFAP expression, suggesting that normal light exposure is important to maintain the correct astrocyte phenotype rather than being involved in GFAP development. We found a reduction of both GFAP immunoreactivity and GFAP level but not of GFAP mRNA, therefore suggesting an alteration at the protein level with no modifications occurring at the transcriptional level. The fact that GFAP mRNA and protein are regulated in different manner is in accordance with what was shown by other authors, in other brain areas and under different experimental conditions. It is known, for example, that GFAP-IR is reduced in the in the supraoptic nucleus after dehydration in rats (Hawrylak et al., 1998). Conversely, GFAP mRNA is increased in the same area under the same experimental conditions (Lally et al., 2005). Changes in the GFAP synthesis pathway and turnover may account for this phenomenon. One possibility is that the process of synthesis is regulated by visual activity at the post-transcriptional level, for example, through the interaction between the complex S100B-Ca++ and GFAP that hamper the correct assembly of the protein by inhibiting GFAP phosphorylation (Frizzo et al., 2004). To have an overview of the effect of light on the entire astrocyte population, the expression of the S100 protein was
also investigated. In fact, this soluble calcium binding protein is synthesized preferentially in mature astrocytes and expressed in all the layers of the cortex (Cocchia, 1981; Ludwin et al., 1976). Besides its use an astrocyte marker (Cocchia, 1981; Ludwin et al., 1976) and the just mentioned interaction with GFAP (Frizzo et al., 2004), it was suggested that the S100 protein could play an important role in the maturation of the cortical wiring (Muller, 1990). Our data showed that S100 is expressed throughout all the layers of the visual cortex and that a long period of visual deprivation causes a reduction of S100, as shown also by other authors (Argandona et al., 2003). Recent reports (Deloulme et al., 2004) showed that S100 is also expressed by a small class of oligodendrocytes, namely, the oligodendroglial progenitor cells (OPC). OPC accounts only for 5–8% of total glia (Dawson et al., 2000), and, in addition, S100 is strongly reduced in mature oligodendrocytes (Deloulme et al., 2004; Rickmann and Wolff, 1995). These data indicate that in late postnatal development and in adulthood S100 can be normally considered a good marker for the overall population of cortical astrocytes (Ludwin et al., 1976).
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Due to the fact that the S100 protein is expressed by other cells besides the astrocytes, we cannot exclude that prolonged DR also affect other cellular types in addition to astrocytes, nevertheless, due the poor amount of the other cells expressing S100, in CNS, we conclude that the reduction observed in the number of S100-immunoreactive cells is mainly due to S100 expressed by astrocytes. Since both GFAP and S100 are reduced, the possibility has to be considered that the process of astrocyte cell death might occur in DR mice. The data showing that GFAP mRNA was not changed after DR indirectly suggest that astrocytes expressing the GFAP mRNA were not reduced in number. However, since (i) in the neocortex only a subpopulation of astrocytes express GFAP (Cocchia, 1981; Ludwin et al., 1976) and (ii) in DR mice also S100 which stains a larger population of cortical astrocytes is affected, we cannot completely exclude the possibility that visual deprivation induces astrocytes death. In our opinion, long dark rearing affects the cytoskeletal properties of astrocytes by acting on the synthesis and/or the turnover of the protein rather than on astrocyte survival. Considering the mutual interaction between GFAP and S100, it might be possible that, in mice, our DR protocols affect the calcium buffering properties of astrocytes. Indeed, the morphological alteration observed might be a consequence of the altered intracellular distribution of GFAP, maybe due to an alteration in the conformation of the protein. Ultrastructural studies will be required to verify whether there is really a morphological change. However, these experiments are far from the goal of this paper. We also checked if, under the present experimental conditions, GFAP and S100 alterations were confined to visual cortex or if areas not directly involved in visual processing were affected as well. Indeed, we found a reduction of both GFAP and S100 also in hippocampus and motor cortex, two areas that do not take part in the elaboration of the visual signal. A first explanation for this phenomenon could be a non-homogenous penetration of the antibody, however, tissue from control and DR animals was processed simultaneously by using the same amount and batch of the two antibodies. To further support the specificity of results obtained in DR mice, we run a second group of immunohistochemical experiments that confirmed the previous results. Another hypothesis, for GFAP reduction, takes into account the sexual dimorphism. It has been shown that the number of GFAP-IR astrocytes, in hippocampus, is reduced in females with respect to male mice (Mouton et al., 2002), this reduction is around 20% in young adult mice showing a trend to increase at late postnatal ages. Since long DR induced a much dramatic reduction of GFAP-stained cells, in VC (80%) and hippocampus (20%), gender differences cannot completely account for the alterations reported. Indeed, though we found that the motor cortex and the hippocampus are less affected by visual deprivation, compared to VC, this result suggests that the influences of light deprivation on astrocyte phenotype, rather than being direct, could be mediated by a wider phenomenon involving the entire CNS. What are the mechanisms responsible for astrocyte alterations?
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It is well known that neuronal activity can influence astrocytes and vice versa. In addition, light deprivation induces several alterations on neuronal phenotype (reviewed in Berardi et al., 2000) and on the expression of several proteins including neurotrophins (Mouton et al., 2002) and their receptors (Castren et al., 1992; Tropea et al., 2001) and neurotransmitters (Viegi et al., 2002). On the other hand, some tyrosine kinase receptors (TrkB and C) are expressed in astroglial and oligodendroglial cells in culture (Morales et al., 2002). Prolonged sensory deprivation might modify several physiological responses including the perception of the circadian period. Nevertheless, animals reared under constant light show an over-expression of GFAP, not only in the visual cortex but also in the cerebellum and the hippocampus (Condorelli et al., 1995). Indeed, GFAP immunoreactivity in the suprachiasmatic nucleus, the area in charge of regulating behavioral and physiological circadian rhythm in mammals, appears to be highly sensitive to circadian time and light conditions (Baydas et al., 2002). Elegant experiments by Lavialle et al. (2001) demonstrated that disruption of the retino-hypothalamic projections promotes a dramatic decrease in GFAP expression and GFAP-immunoreactive redistribution. The lack of cyclic information input affecting the suprachiasmatic nucleus of the hypothalamus might in turn influence the whole CNS metabolism of GFAP. The present results confirm our previous hypothesis that the development of GFAP in mouse is not regulated by light deprivation, but prolonged deprivation of visual experience influences the maintenance of the correct astrocytic phenotype. Nonetheless, they suggest that long DR affects the entire CNS. Thus, the demonstration that astrocyte alterations are not restricted to visual cortical areas imposes a caveat in the use of prolonged periods of DR as a physiological tool to study the role of light in maturation and maintenance of visual cortical circuitry in mouse.
4.
Experimental procedures
4.1.
Animals
Experiments were conducted on C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) in accordance to the European Community Council Directive for animal treatment. In total, we used 16 animals per each group, brain extracts from 8 animals were processed for PCR and Western blot, while the brain from the other 8 animals was processed for immunohistochemistry. 4.2.
Dark rearing
Two different litters were used in these experiments, and, for each litter, pups were randomly assigned to the DR or to the control group (6 animals per group). DR mice were kept in complete darkness for 1 month, from P10, i.e. 4–5 days before eye opening, to P40, when the maturation period is nearly over, while the control group was placed in an artificial 12 h/12 h light/dark cycle. At P40, each experimental group was further divided into two groups: one was perfused and processed for immunohistochemistry, while Western blot and RT-PCR were carried out on the other group. All the analyses described in this paper were performed blindly with respect to the experimental treatment. Furthermore, to avoid spurious results and to exclude the possibility that results
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obtained were due to potential artifacts, attributable to the dark rearing protocol, two more groups of animals (5 animals per group) were placed in DR and the tissue was processed as above. Results were compared with two different groups of control. 4.3.
Immunohistochemistry
Immunohistochemistry was assessed as described elsewhere (Corvetti et al., 2003). Briefly, mice were deeply anesthetized with 20% urethane (Sigma, St. Louis, MO, USA) and then transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). Brains were removed, post-fixed for 1 h at 4 °C in the same solution and then cryoprotected by immersion in 30% sucrose/PBS. Forty-micron thick coronal sections, containing the primary visual cortex, (approximately −2.70 to −5.00 from Bregma, according to Paxinos and Franklin, 1997) were obtained using a freezing sliding microtome, and serial sections were collected in PBS. Sections were first incubated in 0.3% hydrogen peroxide in Tris-buffered saline (TBS) to quench endogenous peroxidase and then processed with the different antibodies. After a preincubation (45 min at room temperature) in mouse Ig blocking reagent (Vector Labs Inc., Burlingame, CA, USA), sections were processed for detection of GFAP or S100. All the primary and secondary antibodies were diluted in phosphate-buffered saline plus 0.1% Triton X-100 (PBST) containing 5% fetal calf serum. Tissue sections were incubated with mouse monoclonal antibodies anti-GFAP (1:100, clone G-A-5, Sigma) or anti-S100 (1:100; clone 4C4.9, Neo Markers, Fremont, CA, USA) for 18 h at 4 °C. The day after, sections were incubated with a goat anti-mouse biotinylated antibody (1:200; Vector) for 3 h at room temperature and then with horseradish-peroxidase-conjugated avidin–biotin complex (1:100; Elite Standard kit, Vector) for 1 h. The peroxidase complex was visualized using 3,3-diaminobenzidine tetrachloride (Sigma) dissolved in Tris/HCl, pH 7.5. Tissue from the DR and the control group were processed in parallel. GFAP-immunostained sections were counterstained with Thionin (1%; Merck, Darmstadt, Germany) to identify the different cortical layers. 4.4.
4.6. GFAP reverse transcription-polymerase chain reaction (RT-PCR)
Cell counts
In both control and dark-reared mice, sections containing the visual cortex were divided in 12 consecutive series (four–five slices/series) (Howard et al., 1998). One every 12 sections was chosen following the rostro-caudal axis, choosing the first section randomly. In this way, 12 series of 4–5 sections each were sampled uniformly at random. A single random series (1/12) of coronal sections/animal was analyzed, and thereby enabling all visual cortical sections to be sampled with the same probability. Following immunohistochemistry, GFAP-IR cells in the visual cortex, motor cortex and hippocampus and S100-positive cells in the visual cortex were counted using a 40× objective. A 300-μm wide grid was superimposed on the section to delimit the area of sampling. Cells within each frame and in the different focal planes were manually counted using the “optical dissector” method (Gundersen, 1986). GFAP-IR cells, within an entire cortical stack going from the pial side to the white matter (i.e. including all the cortical layers) of both the motor and the visual cortex, were counted. The final thickness of the sections, coming from either the dark rearing or the control group, is 34 ± 5 μm. 4.5.
same stereotaxic coordinates as for immunohistochemistry. Only one of the two hemispheres was used for the Western blot, while the other one was immediately frozen in dry ice for RNA analysis. Visual cortices were homogenized in an extracting buffer (1 g tissue/5 ml buffer) containing 50 mM Tris/HCl pH 8, 150 mM EDTA, 0.1% deoxycholic acid, 1% igepal, 1 mM aprotinine, 5 mg/ml phenylmethylsulfonylfluoride, 1 mM iodoacetamide, 5 mg/ml leupeptin, 4 mg/ml soybean trypsin inhibitor and 10 mg/ml turkey egg white inhibitor. Protein concentration was assessed by Lowry colorimetric assay (Lowry et al., 1951), and 20 μg of total protein, extracted from a single brain, was loaded for each lane and resolved in a 12% SDS polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech; Little Chalfont, UK), blocked for 1 h at 37 °C in 5% nonfat milk/PBS (pH 7.4) and then incubated overnight at 4 °C with the proper primary antibody dissolved in 0.05% Tween-20/PBS, pH 7.4 (PBST). A goat anti-mouse antibody against GFAP at a 1:300 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a rat anti-mouse antibody against tubulin at a 1:250 dilution (mAb YOL1, kindly provided by Dr. Cesar Milstein, MRC, Cambridge, UK) were used. After washing in PBS, membranes were incubated for 2 h at 37 °C with the secondary anti-goat (1:1000; Vector) or anti-rat (1:500; Dako Laboratories, Glostrup, Denmark) biotinylated antibody, washed and incubated for 30 min at room temperature with alkaline-phosphatase-conjugated ABC kit (1:1000; Vector). Reaction was revealed using p-Nitro Blue Tetrazolium chloride (NBT; Sigma, 0.5 mg/ml) and 5-Bromo-4-Chloro-3-Indoyl Phosphate ptoluidine salt (BCIP; Sigma, 0.25 mg/ml) in developing buffer (0.1 M Tris, 0.5 mM MgCl2, pH 9.5). Each Western blot was run in duplicate. For a semi-quantitative analysis, after blotting, membranes were scanned, and the density of the single bands was determined using the NIH Image software (Rasband and Bright, 1995). For each band, optical density (OD) was calculated multiplying the mean optical density by the area of the band, and values were given as relative units (RU) according to the following formula: (GFAP OD / tubulin OD) × 100.
Western blot analysis
To evaluate the level of GFAP expression, proteins were extracted from visual cortex, as described previously (Corvetti et al., 2003), and the amount of GFAP was analyzed. Mice were deeply anesthetized with 20% urethane, the brain was removed, and visual cortex was separated from the rest of the brain, using the
The relative amount of GFAP mRNA was evaluated in the visual cortex by RT-PCR. To detect the protein-specific message, the total RNA was extracted from the visual cortex using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) then treated with DNAse I (Ambion, Austin, TX, USA) in order to ensure the absence of genomic DNA in the sample. Two micrograms of total RNA was reverse transcribed (RT) into cDNA using Moloney-Murine leukemia virus (M-MLV) reverse transcriptase (Gibco BRL, Milan, Italy) using random primers. After RT, 1 μl of cDNA was used as template in 25 μl of PCR mixture containing: 0.5 mM final concentration of proteinspecific primers, 0.2 mM deoxinucleotides, GeneAmp 10× buffer and 0.5 U of Platinum Taq DNA Polymerase (Gibco BRL). Primers for GFAP and for β-actin GFAP were designed from published sequence data (Ghazanfari and Stewart, 2001) as follows: β-actinU: 5′-AAC CCT AAG GCC AAC CGT GAA AAG-3′; β-actin-D: 5′-CTA GGA GCC AGG GCA GTA ATC T-3′; GFAP-U: 5′-AGT CCC TCC GCG GCA CGA ACG A-3′; GFAP-D: 5′-ACC ATC CCG CAT CTC CAC AGT CTT TAC CAC-3′. Each PCR reaction was carried out following a temperature protocol consisting of a warm up period of 5 min at 95 °C, 26 cycles of PCR (95 °C for 1 min, 55 °C for 1 min, 72 °C for 5 min) and a final elongation period of 10 min at 75 °C. All samples were tested simultaneously for the two different primer sets, and all PCRs were run in duplicate to eliminate the possibility of spurious results. PCR products were analyzed on 1.5% agarose gel in Tris– borate EDTA buffer, stained with ethidium bromide. To assess semi-quantitatively the expression of the GFAP gene, the agarose gel pictures were digitized and analyzed using the NIH Image program mentioned before (Rasband and Bright, 1995). The optical
BR A I N R ES E A RC H 1 0 6 7 ( 2 00 6 ) 1 4 6 –1 53
density (OD) of the GFAP bands was normalized by comparison to that of β-actin, which was used as an internal standard and expressing the obtained values as RU, as previously described. The expression level of mRNA between control and treated was studied by using non-saturating PCR conditions. The identity of the PCR products was determined by sequencing the gel-purified bands. 4.7.
Statistics
Data are expressed as mean ± SEM of eight animals per group. Statistical analysis of data was performed by using Student's t test. The values were considered significant with P b 0.05.
Acknowledgment Thanks to Mr. Ian Martin Williams for carefully reading the manuscript.
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