Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus

Mechanisms of Ageing and Development 123 (2002) 481– 490 www.elsevier.com/locate/mechagedev

Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus Assia Catalani a,*, Maurizio Sabbatini b, Claudia Consoli a, Carlo Cinque a, Daniele Tomassoni b, Efrain Azmitia c, Luciano Angelucci a, Francesco Amenta b a

Dipartimento di Fisiologia Umana e Farmacologia, Uni6ersita` ‘‘La Sapienza’’, P. le. A. Moro 5, 00185 Rome, Italy Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Uni6ersita` di Camerino, 62032 Camerino, Italy c Department of Biology, New York Uni6ersity, 100 Washington Square East, New York, NY 10003 -5181, USA

b

Received 26 March 2001; received in revised form 11 June 2001; accepted 27 July 2001

Abstract The developmental pattern of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes was investigated in the hippocampus (subfields CA1, CA3 and CA4) and in the dentate gyrus of male and female rats aged 11, 16, 30, 90 and 150 days by immunohistochemistry associated with image analysis. Analysis was centred on stratum radiatum, a hippocampal area rich in GFAP-immunoreactive astrocytes. The volume of different portions of hippocampus, the number and the size of astrocytes, the intensity of cell body GFAP immunostaining as well as the extension of astrocyte were assessed. A maturation pattern consisting in higher cellular expression of GFAP, an increase in overall cell size and expanding arborisation from the 11th to the 30th postnatal day, followed by stabilisation of these parameters until the 90th day of life, and a subsequent decrease in the oldest age group studied was found. A sex-related different temporal pattern of astrocytes maturation in size and GFAP content was observed in the CA1 subfield only. The increase of GFAP content during pre-weaning ages was less pronounced in females than in males as well as the decrease between the 90th and the 150th day of age. Moreover, the size of astrocytes was larger in females than in males at the 11th and 150th days of life. These findings suggest that hippocampal astrocytes undergo rapid maturation in the 1st month of postnatal life, followed by a slow consolidation of this process until the 3rd month of life. At 5 months of age, there are still dynamic changes in the mature astrocytes, which become slender and thinner probably as a response to the increased volume of hippocampus noticeable at this age. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Astrocytes; GFAP; Hippocampus; Development; Male; Female; Morphometry; Rat

1. Introduction * Corresponding author. Tel.: + 39-06-499-12513; fax: + 39-06-494-0588. E-mail address: [email protected] (A. Catalani).

Glial fibrillary acidic protein (GFAP) is a specific astroglial protein. Several astrocyte markers have been identified including GFAP, S100

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protein, vimentin and glutamine synthase. GFAP is the principal subunit of cytoplasmic filaments of fibrous astrocytes and to a lesser extent of protoplasmatic astrocytes. It represents the most reliable and widely used marker for in vivo and in vitro identification of astrocytes (Bignami et al., 1972; Bignami and Dahl, 1977). Astrocytes support central nervous system during development, and contribute to the maintenance of brain microenvironment and to the regulation of neural activity and plasticity. Astrocytes are also involved in the synthesis and uptake of some neurotransmitters such as glutamate, GABA (Schousboe, 1982), and serotonin (Inazu et al., 2001), and contribute to brain immune function. Another function of astroglia is the preservation of tissue integrity following injury. In certain conditions reactive astrocytes could provide a permissive substratum for neuritic extension (Hatten and Mason, 1986; Azmitia et al., 1990; Nishi et al., 1997). Moreover, astrocytes express neurotransmitter and hormone receptors such as b-adrenoceptors (Shao and Sutin, 1992), serotonin (Merzak et al., 1996) glucocorticoid and oestrogen receptors (Vielkind et al., 1990; Bohn et al., 1991; Mong and McCarthy, 1999; Mong et al., 1999). Via binding to these receptors, steroid hormones are critical regulators of the gene expression on GFAP. The effects of gonadal steroids on the GFAP are complex and may also be exerted indirectly via changes of the surrounding neurons and/or glial elements (Melcangi et al., 1998). GFAP expression shows cyclic variations in the rat hippocampus during normal oestrous cycle with different regulation of GFAP transcription in various brain regions by 17-b-estradiol (Luquin et al., 1993). Hippocampus is a brain region involved in several important functions including learning and memory. It contains numerous astrocytes that are located in the Ammon’s horn, dentate gyrus and subiculum. Neuronal activity can trigger astrocyte cell-to-cell communication such as modulation of calcium waves (Dani et al., 1992). Neuron –astrocyte interactions play an important role in the development and functional activity of hippocampus including differentiation of hippocampal neurons (Rickmann et al., 1987). It

is also demonstrated that glial S-100 is involved in neuronal differentiation and maturation (Ueda et al., 1994; Yang et al., 1996). Studies on the development of rat hippocampus have shown that neurogenesis occurs during embryonic periods and is extended beyond birth, continuing postnatally, whereas mature astrocytes develop during postnatal life (Bayer, 1980a,b). The majority of information on GFAP immunoreactive astrocyte development comes from in vitro investigations (Hamprecht, 1986), whereas only a few in vivo studies have analysed the development of hippocampal astrocytes (Nixdorf et al., 1991) or have assessed possible developmental differences of hippocampal astrocytes between males and females. The present study was designed to assess postnatal changes in the number and size of GFAPimmunoreactive astrocytes in different subfields of the hippocampus of male and female rats ranging from 11 to 150 days of age by immunohistochemical techniques associated with image analysis.

2. Materials and methods

2.1. Animals and tissue treatment Female COBS Wistar rats (Charles River, Italy) weighing 280–320 g, were housed in a controlled-temperature room (22–25 °C) and maintained on a 12 h light/dark cycle (light on 08:00 h) with food (Standard Diet Charles River 4RF21) and water available ad libitum. Females were mated with sexually experienced male rats. One female and one male per cage were left undisturbed for 1 week; after this time females were housed individually. The day of birth was counted as day 0, and the next day, litters were culled to eight pups (four males and four females). Weaning was performed at 21 days of age and animals were housed three per cage. Animals were handled according to internationally accepted principles for the care of laboratory animals (EEC Council Directive 86/609, OJ No. L358, 18/12/86).

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Female and male rats were used for the GFAP immunohistochemistry at different ages: two before weaning (11 and 16 days) and three after weaning (30, 90 and 150 days of life). To avoid litter effect, each litter contributed one or maximum two offspring to make the groups. Animals were anaesthetised with Nembutal (60 mg/kg for males and 45 mg/kg for females i.p.) and perfused through the ascending aorta with a 0.9% NaCl solution in 0.1 M phosphate buffer (PBS, pH 7.4) for 5 min, followed by 4% phosphate-buffered paraformaldehyde for 20 min. After perfusion the brains were removed from the skull and post fixed for 48 h in fresh fixative at 4 °C. Brain blocks were cut coronally to obtain hippocampus. Hippocampal formation was dissected out, kept overnight in 20% sucrose– PBS and then frozen in a dry ice– acetone mixture. Hippocampal blocks were then maintained at − 80 °C until used.

2.2. GFAP immunohistochemistry Coronal sections of hippocampal formation were cut on a microtome cryostat at 40 mm, collected in PBS plus 0.3% Triton X 100, left overnight at 4 °C and processed for immunohistochemistry as serial free floating sections. Sections were put in a blocking solution (0.1 M PBS, pH 7.4, 0.1% Triton X-100 and 1% BSA) for 1 h, washed three times with ice-cold 0.1 M PBS (pH 7.4) and then exposed overnight at 4 C° to a primary monoclonal GFAP antibody (Sigma G-3893) diluted 1:10 000 in the above blocking solution. Sections were washed three times with ice-cold PBS and then incubated for 1 h at room temperature in a biotin-labelled (goat anti-mouse) secondary antibody (1:2000), washed in PBS and further incubated for 1 h in a peroxidase-conjugated streptavidin solution. After washing three times in 0.1 M TBS (pH 7.6), sections were reacted in 0.02% 3%,3%-diaminobenzidine in 0.01% H2O2, counterstained with 2% methyl green and dehydrated in ethanol. After clearing in xylene, sections were coverslipped and examined under a Leica DMR light microscope.

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2.3. Image analysis Images of slides processed for GFAP immunohistochemistry were transferred via a TV camera from the microscope to the screen of an IAS 2000 image analyser (Delta Sistemi, Rome, Italy). Using an overlap option, CA1, CA3, CA4 subfields and dentate gyrus were delineated. The area of each portion was then calculated by the image analyser in six consecutive sections per animal. The sum of these area values multiplied by section thickness resulted in the volume of each structure examined (West et al., 1978). Numerical density of astrocytes, displaying dark-brown immunoreactive staining, was evaluated according to the dissector method (Sterio, 1984), using a programme of the image analyser. Estimates of the total number of GFAP-immunoreactive astrocytes were calculated by multiplying estimates of astrocytes numerical density with estimates of volume. The morphology of GFAP-immunoreactive astrocytes was further assessed quantitatively at high magnification. For each section, ten astrocytes per subfield were randomly selected and viewed at a final magnification of ×600. The cell body of astrocytes and main branching processes were delineated using an overlap option of the image analyser. The intensity of immune staining was then assessed microdensitometrically taking as ‘‘zero’’ the background of control sections incubated with heat-denatured anti-GFAP antibody or with non-immune serum instead of anti-GFAP antiserum. In these astrocytes the number of primary processes (originating directly from the cell body) and their relative length were assessed. Astrocytes showing at least three primary processes were indicated with ‘‘+ ’’, those displaying four to five primary processes were indicated with ‘‘+ + ’’, whereas astrocytes with more than five primary processes were indicated with ‘‘+ + + ’’. ‘‘L’’ was used to define astrocytes with primary processes two times longer than the cell body, whereas ‘‘LL’’ was indicative of astrocytes with primary processes three or more times longer than the cell body.

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2.4. Data analysis

3. Results

Data of different quantitative parameters investigated were analysed statistically with analysis of variance (ANOVA) followed by Duncan’s multiple range test to evaluate the statistical significance of differences during development. The Student’s t-test was used to assess significance of differences of given parameters between male and female rats.

3.1. General obser6ations Exposure of sections of hippocampus to monoclonal antibody raised against GFAP resulted in a highly selective and reproducible staining pattern. GFAP was localised in the cytoplasm and cell bodies of astrocytes. The morphology and density of GFAP-immunoreactive were similar in stratum

Fig. 1. GFAP immunoreactive astrocytes in stratum radiatum of CA1 subfield of the hippocampus of male (A, C, E) and female (B, D, F) rats. A – B: 11-day old rats; C –D: 30-day-old rats; E – F: 150-day-old rats. Note the changes in GFAP immunoreactivity, astrocyte size, number and characteristics of astrocyte processes throughout development. Calibrations bar: 10 mm.

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Table 1 Volume of stratum radiatum of subfields of hippocampus of rats of different ages Age (days)

11

16

30

90

150

CA1 subfield Males Females

7.50 90.10 7.60 90.06

9.079 0.15a 9.0790.09a

14.13 9 0.12a,b 14.00 90.11a,b

16.73 90.15a,b,c 16.93 90.09a,b,c

17.27 90.07a,b,c,d 17.17 90.15a,b,c,d

CA3 subfield Males Females

3.43 90.09 3.57 90.09

5.109 0.15a 5.07 9 0.12a

5.97 9 0.09a,b 5.87 90.18a,b

7.85 90.06a,b,c 7.67 9 0.20a,b,c

7.90 9 0.06a,b,c 7.90 9 0.12a,b,c

CA4 subfield Males Females

3.13 90.15 3.23 90.12

4.939 0.03a 4.80 90.12a

6.83 90.12a,b 6.90 90.15a,b

7.98 90.09a,b,c 7.97 9 0.15a,b,c

8.00 90.06a,b,c 7.97 9 0.03a,b,c

Dentate gyrus Males Females

4.23 90.07 4.27 90.09

5.539 0.09a 5.6390.03a

9.50 90.10a,b 9.73 9 0.12a,b

11.83 90.10a,b,c 11.73 90.03a,b,c

12.00 90.06a,b,c 11.93 90.19a,b,c

Data are the mean 9S.E. and are expressed in mm3. a PB0.05 vs. 11 days. b PB0.05 vs. 16 days. c PB0.05 vs. 30 days. d PB0.05 vs. 90 days.

oriens, radiatum and lacunosum-molecolare of CA1 and CA3 subfields (data nor shown). In view of this, quantitative data were related to stratum radiatum of CA1 and CA3 only. In rats of different ages astrocytes revealed a maturation pattern in the arborisation number (Fig. 1). Astrocytes at 11 postnatal days (P) appeared with a rather large cell body and short branches both in the CA1 (Fig. 1A and B) and CA3 subfields of hippocampus and in the dentate gyrus. In older age groups, astrocytes showed a smaller cell body, with longer processes from which originated thin and ramified branches (Fig. 1C –F).

3.2. Volume of hippocampus Morphometric analysis revealed in the CA1 subfield a progressive increase of the volume of stratum radiatum from P11 to P150 (Table 1) and from P11 to P90 in the remaining portions of hippocampus investigated (Table 1).

3.3. Number of GFAP-immunoreacti6e astrocytes An increase in the number of GFAP-positive

astrocytes was observed both in male and female rats from 11 to 30 days of life (Table 2). No further increase was noticeable between P30 and P150 (Table 2). The numerical increase of astrocytes was more remarkable between P11 and P16 and less pronounced between P16 and P30 days (Table 2). The numerical increase of GFAPimmunoreactive astrocytes was similar in the different portions of hippocampus investigated (Table 2).

3.4. Morphometry of GFAP-immunoreacti6e astrocytes and GFAP expression Data on the size of GFAP-immunoreactive astrocytes in the different subfields of hippocampus of male and female rats of different ages are summarised in Fig. 2. In the CA1 subfield the size of astrocytes increased from P11 to P30 followed by a decrease until P150 (Fig. 2). At P11 and P150 GFAP-immunoreactive astrocytes were larger in female than in male rats (Fig. 2A). In the CA3 subfield a decrease in size of GFAP-immunoreactive astrocytes was observed both in male and female rats from P11 to P150 (Fig. 2B). In the CA4 subfield and in the dentate gyrus of

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Table 2 Number of GFAP immunoreactive astrocytes in the different subfields of rat hippocampus Age (days)

11

16

30

90

150

CA1 subfield Males Females

148 95 151 93

358 9 7a 3619 4a

44596a,b 44997a,b

458 93a,b 460 97a,b

434 94a,b 431 93a,b

CA3 subfield Males Females

76 95 76 93

19294a 1899 5a

23993a,b 24192a,b

241 9 2a,b 244 9 2a,b

239 9 2a,b 238 9 2a,b

CA4 subfield Males Females

81 93 83 92

20993a 210 9 4a

257 9 1a,b 26092a,b

262 9 2a,b 265 93a,b

262 93a,b 261 92a,b

Dentate gyrus Males Females

116 93 112 94

3139 4a 3159 6a

39593a,b 39396a,b

398 9 4a,b 394 94a,b

385 94a,b 387 94a,b

Data are the mean 9S.E. and expressed as number of cells×103. a PB0.05 vs. 11 days. b PB0.05 vs. 16 days.

both male and female rats, an increased size of astrocytes from P11 to P30 followed by a subsequent decrease until P150 was observed (Fig. 2C and D). Increased number of branches and of length of astrocyte processes were found in the CA1

subfield from P11 to P90. The subsequent decrease of these parameters was more evident in female rats and by P150 male astrocytes displayed wider and longer processes than female rats (Table 3 and Fig. 1E and F). In the CA3 subfield of male and female astrocyte arborisa-

Table 3 Morphometric evaluation of number and length of processes of GFAP-immunoreactive astrocytes in the hippocampus of male and female rats of different age groups Age (days)

11

16

30

90

150

CA1 subfield Males Females

+ ++

++ ++L

++L ++L

+++LL +++LL

++LL +LL

CA3 subfield Males Females

+++ +++

++ ++

++L ++L

++LL ++LL

++L ++L

CA4 subfield Males Females

+LL +LL

+LL +LL

+LL +LL

+LL +LL

+LL +LL

Dentate gyrus Males Females

++ ++

++ ++

++ ++

+++ +++

++ ++

Details on morphometric analysis protocols are reported in Section 2.3. +: Astrocytes showing until three primary processes; ++: astrocytes displaying until five primary processes; +++: astrocytes with more than five primary processes; L: astrocytes with primary processes longer two times more than the cell body; LL: astrocytes with primary processes three or more times longer than the cell body.

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Fig. 2. Area of GFAP immunoreactive astrocytes during development of hippocampus in male (“) and female () rats. Data are expressed in mm2 and are the mean 9S.E. A: CA1 subfield; B: CA3 subfield; C: CA4 subfield; D: dentate gyrus. Figures in the axis of abscissa indicate the age (days) of animal groups investigated. * P B 0.05 between male and female rats; + PB0.05 vs. previous age group.

tion was well developed at P11 (Table 3). A decreased arborisation accompanied by elongation of processes was noticeable after P30 (Table 3). In the CA4 subfield, GFAP-immunoreactive astrocytes showed a similar pattern of arborisa-

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Fig. 3. Values of microdensitometric analysis of GFAP immunoreactivity of astrocytes during development of hippocampus in male (“) and female () rats. Data are expressed in arbitrary units proportional to the intensity of the immunostaining and were assessed as indicated in Section 2.3. Values are the mean 9S.E. A: CA1 subfield; B: CA3 subfield; C: CA4 subfield; D: dentate gyrus. Figures in the axis of abscissa indicate the age (days) of animal groups investigated. * P B0.05 between male and female rats; + P B0.05 vs. previous age group.

tion in male and female rats of different age groups investigated (Table 3). In the dentate gyrus, at P90 the arborisation of GFAP-immunoreactive astrocytes was greater in male and

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female rats compared with younger cohorts or P150 rats (Table 3). Microdensitometric analysis of the expression of astrocyte GFAP immunoreactivity in the CA1 subfield of 11-day-old rats revealed a lower immunoreactivity in male than in female rats (Fig. 3A). A subsequent gradual increase of GFAP immunoreactivity was observed until 30 days of age, followed by a stabilisation until 90P (Fig. 3A) and a significant decrease in the oldest age group investigated (Fig. 3A). The increase of GFAP immunoreactivity between P11 and P16 was more pronounced in male than in female rats (Fig. 3A), as well as the decrease of immunoreactivity between P90 and P150 (Fig. 3A). In the CA3 subfield a progressive decrease of GFAP immunoreactivity similar in male and female rats throughout the different ages investigated from P11 to P150 was found (Fig. 3B), whereas the expression of GFAP immunoreactivity was similar in astrocytes of the CA4 subfield of male and female rats of different ages (Fig. 3C). In the dentate gyrus GFAP immunoreactivity increased from P11 to P16, remained constant until P90 and decreased at P150 (Fig. 3D). This pattern of immunoreactivity was similar in the two sexes with the exception of a more pronounced decrease at P150 in male than in female rats (Fig. 3D).

4. Discussion The above results provide direct evidence that the morphology of GFAP-immunoreactive astrocytes undergoes significant changes during hippocampal postnatal development and that both the CA1 subfield and the dentate gyrus show large changes in the volume of the stratum radiatum between 16 and 30 days of life. The area showing the most pronounced changes was the CA1 subfield, in which the astrocyte cell body became smaller, but developed longer processes with thin and ramified branches. This area also displayed the most pronounced sexual dimorphism during astrocyte development. The present study represents an extension of former investigations (Ling and Leblond, 1973; Parnavelas et al., 1983; Nixdorf-Bergweiler et al., 1994), since in

this work the observation has assessed male and female rats independently and it was extended until the 5th month of age. In this work we have observed that the number of astrocytes increases in the rat hippocampus during the 1st month of age. The greatest increase in the number of GFAP positive cells was observed before weaning between 11 and 16 days of life. A further, but smaller increase occurred between 16 days and 1 month of age. At this time adult number of astrocytes was reached. The above changes were observed both in males and females in all the subfields of the hippocampus examined. Only a few quantitative morphological studies on changes in number of astrocytes during development and adult life are available. Our data on early stabilisation of astrocyte number are consistent with the more general observation on the topic performed in different brain areas (Ling and Leblond, 1973; Parnavelas et al., 1983) including hippocampus (Nixdorf-Bergweiler et al., 1994). Analysis of the morphology and GFAP content in astrocytes revealed a maturation pattern of these cells, reaching its maximal expression at P30–P90 and then decreasing at P150. Throughout development, astrocytes change their morphology undergoing thinning of their cell body accompanied by a different expression of GFAP content. These data are consistent with those of another group that used different morphological techniques (Nixdorf-Bergweiler et al., 1994). Comparative analysis of different aspects of development of GFAP-immunoreactive astrocytes suggests that the mature form of astrocytes is that observed at 5 months of age. Based on our data it is probable to hypothesise that GFAP-immunoreactive astrocytes adapt their morphology to cover progressively with their processes the increased tissue volume and then retract their own processes between P90 and P150. This behaviour may be related to nervous tissue maturation occurring during first postnatal period. The uneven pattern of astrocyte maturation in various subfields of hippocampus may be related to a different function of neuronal field of hippocampus. An interrelationship between astrocyte morphology and synaptic function has been

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hypothesised (Bjo¨ rklund et al., 1985; Mu¨ ller, 1992; Barres, 1991; Mong and McCarthy, 1999). It is probable that astrocyte changes observed in our work may be related to maturation and synaptic status of hippocampal neurons. An interesting finding is the different behaviour between male and female in astrocytes maturation that may be pointed out as a different regulation pattern present in females compared to male rats, perhaps due to the particular hormonal influence present in females. Throughout development, as well as in postpubertal animals, gonadal steroid exert an important influence over the central nervous system resulting in sexual dimorphism in some areas at both morphological and physiological levels. Moreover, the perinatal developmental period is characterised by dramatic sex differences in circulating gonadal steroids (Garcia-Segura et al., 1994a; Maclusky and Naftolin, 1981). This may explain the gender differences we found in GFAP expression and astrocyte morphology. Studies in the adult animal show that GFAP levels are regulated by gonadal steroids: GFAP may vary depending on the steroid under investigation and the brain regions (Nichols, 1999; Garcia-Segura et al., 1994b; Laping et al., 1994; Kohama et al., 1995). Oestrogen-induced synaptic sprouting in female rats influences GFAP expression in the dentate gyrus of the hippocampus (Stone et al., 2000). In male rats GFAP expression in the hippocampus is enhanced by castration and inhibited by sex steroids (Day et al., 1993; Hajos et al., 1999). In certain brain areas a clear cut sexual dimorphism exists for GFAP. In the hypothalamus, GFAP m-RNA levels and GFAP immunoreactivity were found to be higher in males than in females (Chowen et al., 1995). It has been demonstrated that gonadal steroids promote glial differentiation in the developing hypothalamus in a regionally specific manner (Mong et al., 1999). Our data contribute to insight into the sex differences in the maturation of astrocytes and GFAP expression in the hippocampus suggesting an influence of gonadal hormones on these cells also during development. Future studies will look at the effects of different types of steroids on hippocampal glial development.

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References Azmitia, E.C., Dolan, K., Whitaker-Azmitia, P.M., 1990. S100B but not NGF, EGF, insulin or calmodulin is a CNS serotonergic growth factor. Brain Res. 516, 354 – 356. Barres, B.A., 1991. New roles for glia. J. Neurosci. 11, 3685 – 3694. Bayer, S.A., 1980a. Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography. J. Comp. Neurol. 190, 87 – 114. Bayer, S.A., 1980b. Development of the hippocampal region in the rat. II. Morphogenesis during embryonic and early postnatal life. J. Comp. Neurol. 190, 115 – 134. Bignami, A., Dahl, D., 1977. Specificity of the glial fibrillary acidic protein for astroglia. J. Histochem. Cytochem. 25, 466 – 469. Bignami, B.A., Eng, L.F., Dahl, D., Uyeda, C.T., 1972. Localization of the glial fibrillary acidic protein isolated from fibrous astrocytes. Brain Res. 43, 429 – 435. Bjo¨ rklund, H., Eriksdotter-Nilsson, M., Dahl, D., Rose, G., Hoffer, B., Olson, L., 1985. Image analysis of GFA-positive astrocytes from adolescence to senescence. Exp. Brain Res. 58, 163 – 170. Bohn, M.C., Howard, E., Vielkind, U., Krozowski, Z., 1991. Glial cells express both mineralocorticoid and glucocorticoid receptors. J. Steroid Biochem. Mol. Biol. 40, 105 – 111. Chowen, J.A., Busiguina, S., Garcia-Segura, L.M., 1995. Sexual dimorphism and sex steroid modulation of glial fibrillary acidic protein messenger RNA and immunoreactivity levels in the rat hypothalamus. Neuroscience 69, 519 – 532. Dani, J.W., Chernjavsky, A., Smith, S.J., 1992. Neuronal activity triggers calcium waves in hippocampal astrocyte networks. Neuron 8, 429 –440. Day, J.R., Laping, N.J., Lampert-Etchells, M., Brown, S.A., O’Callaghan, J.P., McNeill, T.H., Finch, C.E., 1993. Gonadal steroids regulate the expression of glial fibrillary acidic protein in the adult male rat hippocampus. Neuroscience 55, 435 – 443. Garcia-Segura, L.M., Luquin, S., Parducz, A., Naftolin, F., 1994a. Gonadal hormone regulation of glial fibrillary acidic protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus. Glia 10, 59 – 69. Garcia-Segura, L.M., Chowen, J.A.:, Parducz, A., Naftolin, F., 1994b. Gonadal hormones as promoter of structural synaptic plasticity: cellular mechanisms. Prog. Neurobiol. 44, 279 – 307. Hajos, F., Halasy, K., Gerics, B., Szalay, F., 1999. Glial fibrillary acidic protein (GFAP)-immunoreactivity is reduced by castration in the interpeduncular nucleus of male rats. Neuroreport 10, 2229 –2233. Hamprecht, B., 1986. Astroglia cells in culture: Receptors and cyclic nucleotides. In: Fedoroff, S., Vernadakis, A. (Eds.), Astrocytes, In: Biochemistry, Physiology and Pharmacology of Astrocytes, vol. 2. Academic Press, New York, pp. 77 – 106. Hatten, M.E., Mason, C.A., 1986. Neuron-astroglia interactions in vitro and in vivo. Trends Neurosci. 9, 168 – 174.

490

A. Catalani et al. / Mechanisms of Ageing and De6elopment 123 (2002) 481–490

Inazu, M., Takeda, H., Ikoshi, H., Sugisawa, M., Uchida, Y., Matsumiya, T., 2001. Pharmacological characterization and visualization of the glial serotonin transporter. Neurochem. Int. 39, 39 – 49. Kohama, S.G., Goss, J.R., McNeill, T.H., Finch, C.E., 1995. Glial fibrillary acidic protein mRNA increased at proestrous in the arcuate nucleus of mice. Neurosci. Lett. 183, 164 –166. Laping, N.J., Teter, B., Nichols, N.R., Rozovsky, I., Finch, C.E., 1994. Glial fibrillary acidic protein: regulation by hormones, cytokines, and growth factors. Brain Pathol. 4, 259– 275. Ling, E.A., Leblond, C.P., 1973. Investigation of glial cells in semithin sections. II. Variations with age in the numbers of the numbers of the various glial cell types in rat cortex and corpus callosum. J. Comp. Neurol. 149, 73 –82. Luquin, S., Naftolin, F., Garcia-Segura, L.M., 1993. Natural fluctuation and gonadal hormone regulation of astrocyte immunoreactivity in dentate gyrus. J. Neurobiol. 24, 913 – 924. Maclusky, N.J., Naftolin, F., 1981. Sexual differentiation of the central nervous system. Science 211, 1294 – 1302. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Riva, M.A., Piva, F., Martini, L., 1998. Effects of steroid hormones on gene expression of glial markers in the central and peripheral nervous system: variations induced by aging. Exp. Gerontol. 33, 827 – 836. Merzak, A., Koochekpour, S., Fillion, M.P., Fillion, G., Pilkington, G.J., 1996. Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration. Brain Res. Mol. Brain Res. 41, 1 – 7. Mong, J.A., Glaser, E., McCarthy, M.M., 1999. Gonadal steroids promote glial differentiation and alter neuronal morphology in the developing hypothalamus in a regionally specific manner. J. Neurosci. 19, 1464 –1472. Mong, J.A., McCarthy, M.M., 1999. Steroid-induced developmental plasticity in hypothalamic astrocytes: implications for synaptic patterning. J. Neurobiol. 40, 602 –619. Mu¨ ller, C.M., 1992. A role for glial cells in activity-dependent central nervous plasticity? Review and hypothesis. Int. Rev. Neurobiol. 34, 215 – 281. Nichols, N.R., 1999. Glial responses to steroids as markers of brain aging. J. Neurobiol. 15, 585 –601. Nishi, M., Kawata, M., Azmitia, E.C., 1997. S100beta promotes the extension of microtubule associated protein2 (MAP2)-immunoreactive neurites retracted after colchicine

treatment in rat spinal cord culture. Neurosci. Lett. 229, 212 – 214. Nixdorf, B.E., Albrecht, D., Heinemann, U., 1991. Development of GFAP-positive cells in the CA1 region of the rat hippocampus. Soc. Neurosci. Abstr. 17, 734. Nixdorf-Bergweiler, B.E., Albrecht, D., Heinemann, U., 1994. Developmental changes in the number, size, and orientation of GFAP-positive cells in the CA1 region of rat hippocampus. Glia 12, 180 – 195. Parnavelas, J.G., Luder, R., Pollard, S.G., Sullivan, K., Lieberman, A.R., 1983. A qualitative and quantitative ultrastructural study of glial cells in the developing visual cortex of the rat. Philos. Trans. R. Soc. (Lond.) (Biol.) 301, 55 – 84. Rickmann, M., Amaral, D.G., Cowan, W.M., 1987. The organization of radial glial cells during the formation of the dentate gyrus of the rat. J. Comp. Neurol. 264, 449 – 479. Schousboe, A., 1982. Transport and metabolism of glutamate and GABA in neurons and glial cells. Int. Rev. Neurobiol. 22, 1 – 45. Shao, Y., Sutin, J., 1992. Expression of adrenergic receptors in individual astrocytes and motor neurons isolated from the adult rat brain. Glia 6, 108 – 117. Sterio, D.C., 1984. The unbiased estimation of number and sizes of arbitrary particles using the dissector. J. Microsc. 134, 127 – 136. Stone, D.J., Rozovsky, I., Morgan, T.E., Anderson, C.P., Lopez, L.M., Shick, J., Finch, C.E., 2000. Effects of age on gene expression during estrogen-induced synaptic sprouting in the female rat. Exp. Neurol. 165, 46 – 57. Ueda, S., Gu, X.F., Whitaker-Azmitia, P.M., Naruse, I., Azmitia, E.C., 1994. Neuro-glial neurotrophic interaction in the S-100 beta retarded mutant mouse (Polydactyly Nagoya). I. Immunocytochemical and neurochemical studies. Brain Res. 633, 275 – 283. Vielkind, U., Walencewicz, A., Levine, J.M., Bohn, M.C., 1990. Type II glucocorticoid receptors are expressed in oligodendrocytes and astrocytes. J. Neurosci. Res. 27, 360 – 373. West, M.J., Danscher, G., Gydesen, H., 1978. A determination of volumes of the layers of the rat hippocampal region. Cell Tissue Res. 188, 345 – 359. Yang, Q., Hamberger, A., Wang, S., Haglid, K.G., 1996. Appearance of neuronal S-100 beta during development of the rat brain. Brain Res. Dev. Brain Res. 91, 181 – 189.