Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus

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.

Introduction

Glial fibrillary acidic protein (GFAP) is a specific astroglial protein. Several astrocyte markers have been identified including GFAP, S100 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 β-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-β-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, Bayer, 1980b). 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 GFAP-immunoreactive 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.