Elsevier

Acta Histochemica

Volume 112, Issue 2, March 2010, Pages 147-160
Acta Histochemica

Single-dose and chronic corticosterone treatment alters c-Fos or FosB immunoreactivity in the rat cerebral cortex

https://doi.org/10.1016/j.acthis.2008.10.001Get rights and content

Summary

The aim of this study was to examine the effects of single-dose and chronic corticosterone treatment on the inducible transcription factor c-Fos and FosB, and thereby to estimate the effects of high-doses of corticosterone on calcium-dependent neuronal responses in the rat cerebral cortex. At the same time we investigated the distribution of interneurons containing calretinin (CR), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in chronically treated animals in order to collect data on the involvement of inhibitory neurons in this process. Adult male rats were injected subcutaneously with 10 mg corticosterone, whereas controls received the vehicle (sesame oil). The animals were fixed by transcardial perfusion 12 and 24 h following single corticosterone injection, and the brains were processed for c-Fos and FosB immunohistochemistry. To investigate the effects of repeated corticosterone administration, rats were daily treated with the same amount of corticosterone (10 mg/animal, subcutaneously) for 21 days. Controls were injected with vehicle. At the end of the experiment, the rats were perfused and immunohistochemistry was used to detect the presence of the FosB protein, CR, VIP and NPY. Quantitative evaluation of immunolabelled cells was performed in the neocortex and the hippocampus. The number of immunoreactive nuclei per unit area was used as a quantitative measure of the effects of corticosterone. It was found that a single-dose administration of corticosterone resulted in a significant, time-dependent increase of c-Fos protein immunoreactivity in the granule cell layer of the dentate gyrus, as well as in regions CA1 and CA3 of the hippocampus 12 and 24 h post-injection with respect to control animals. Significant enhancement of c-Fos immunoreactivity was also observed in the neocortex at 12 and 24 h post-injection. Single-dose treatment did not significantly alter FosB immunolabelling. Repeated administration of corticosterone produced a complex pattern of changes in FosB immunolabelling: significant increase in FosB immunoreactivity was detected in the granule cell layer of the dentate gyrus, with no significant changes in the CA1 and CA3 layers of the hippocampus and in the neocortex. However, a significant decrease of FosB induction in the neocortex was observed in chronically treated rats in comparison to single-dose injected animals (12 h before immunohistochemistry). Analysis of immunohistochemical detection of interneuronal markers revealed a significant reduction of the CR immunolabelling in the CA3 area of the hippocampus. No changes in VIP or NPY immunoreactivity were found in the Ammon's horn 3 weeks following daily corticosterone treatment. NPY immunoreactivity was significantly attenuated in the neocortex.

The present data suggest that single-dose corticosterone treatment increases immunoreactivity of c-Fos protein in a time-dependent manner, 12 and 24 h post-injection in the rat hippocampus and the neocortex, whereas chronic corticosterone treatment influences FosB immunoreactivity, primarily in the dentate gyrus. Chronic corticosterone administration seems to affect CR levels in the CA3 area of the hippocampus.

Introduction

Several lines of evidence indicate that glucocorticoids influence activity of brain structures and that excess glucocorticoid may have deleterious consequences in the central nervous system, predominantly in the hippocampus (Sapolsky et al., 1995; Joëls, 2001). Chronic exposure to excess glucocorticoid could endanger hippocampal cell function and morphology, leading to decreased neurogenesis and increased dendritic atrophy in the hippocampus and prefrontal cortex, which might be involved in memory deficits and ensuing psychopathology (Gould and Tanapat, 1999; Sapolsky, 2000; Wellmann, 2001). Apart from the chronic effects of glucocorticoids, they may have acute, short-term neuronal effects, the molecular mechanisms of which still remain to be clarified (Haller et al., 1998). The mechanisms of action of glucocorticoids in the central nervous system comprise both genomic and non-genomic effects (Wolkowitz et al., 2001; Joëls, 2001). Most of the effects of glucocorticoids are mediated by modulation of gene transcription, through interaction with corticosteroid receptors: the activated receptors translocate to the nucleus and affect gene expression either directly by binding to DNA as homodimers or indirectly, interacting with other transcription factors (Ayroldi et al., 2002). The hippocampus is a primary target site for glucocorticoids, the receptors for glucocorticoids being densely located in the Ammon's horn, as well as in the septum and amygdala, regions of the brain thought to be intimately involved in behavior, learning and memory function (McEwen et al., 1986; De Kloet, 1991; Wolkowitz et al., 2001). The prefrontal cortex is similarly a behaviorally relevant target for glucocorticoids since stress alters prefrontal cortical functions and glucocorticoid receptors (Rajkowska, 2000; Wolkowitz et al., 2001).

Corticosterone, the endogenous glucocorticoid of most rodents, binds to central corticosteroid receptors, the mineralocorticoid and glucocorticoid receptors, which are co-expressed in the neurons of limbic regions, such as the CA1 pyramidal and dentate granule cells of the hippocampal formation (Joëls, 2001). Low levels of corticosterone are sufficient to activate mineralocorticoid receptors (which are mostly restricted to limbic brain regions), whereas activation of hippocampal glucocorticoid receptors only occurs at high corticosterone levels, as seen after stressful events or following peripheral corticosterone administration (Reul et al., 1987; Joëls, 2001). Potential targets for corticosteroid actions that could alter neuronal membrane properties are the voltage-gated ion channels (Kerr et al., 1992; Karst et al., 1994) and neurotransmitter-mediated receptors, including ligand-gated ion channels and G-protein-coupled receptors (Joëls, 2001; Czyrak et al., 2002; Wiegert et al., 2005). Exposure to high levels of corticosterone has been reported to increase Ca2+ influx into hippocampal cells through voltage-gated Ca2+ channels by glucocorticoid receptor activation (Kerr et al., 1992), resulting in enhanced cell firing accommodation. However, prolonged exposure to high concentrations of corticosterone and glucocorticoid receptor activation may lead to excessive and uncontrolled Ca2+ influx with enhanced vulnerability and cell death (Joëls, 2001). In addition to the marked sensitivity of the voltage-gated Ca2+ conductances to corticosteroids, high concentrations of the hormone modulate glutamate and/or GABA-mediated fast synaptic transmission, including the development of long-term potentiation (LTP) related to N-methyl-d-aspartate (NMDA)-receptor-mediated Ca2+ influx in the CA1 region of the hippocampus within 1 h (Vidal et al., 1986; Kawato et al., 2001; Shibuya et al., 2003). High concentrations of corticosterone administered acutely (within 30 min) decrease NMDA-receptor-mediated Ca2+ elevation in the CA1 region in hippocampal slices (Sato et al., 2004). Nevertheless, these rapid modulatory effects of corticosteroids are not only fast in onset, but often also reversible within minutes (for a review, see: Joëls, 2001), revealing the possibility that corticosteroids may drive, apart from their genomic effects, acute non-genomic pathways (Joëls, 2001; Sato et al., 2004).

Members of the activator protein 1 (AP-1) family, c-Fos and FosB belong to the inducible proto-oncogenes, which exert various regulatory actions in the cell nucleus, including late-response gene transcription that may be critical for neuronal adaptive responses (Morgan and Curran, 1995; Herdegen and Leah, 1998; Greenberg and Ziff, 2001). Among the transcription factors encoded by these immediate early genes (IEGs) are members of the Fos family of proteins, such as Fos, FosB, Fra-1 and Fra-2, all possessing leucine zippers. These IEGs are induced by different stimuli, including membrane depolarisation and Ca2+ influx. Activation of c-Fos transcription requires Ca2+ influx, mediated mainly by neurotransmitters acting on ionotropic receptors, such as NMDA and AMPA types of glutamate receptors and through voltage-sensitive Ca2+-channels (Greenberg and Ziff, 2001). Fos immunohistochemistry has widely been used in neuropharmacological investigations to determine neuronal activation patterns. Accordingly, previous studies from our laboratory indicated that immunohistochemical detection of Fos protein serves as a marker of neuronal activation in forebrain structures (Mihály et al., 2001; Szakács et al., 2003; Fazekas et al., 2006). In the experiments reported here, we investigated the changes in c-Fos and FosB immunolabelling following a single injection of corticosterone and we tested the alterations of FosB immunoreactivity after chronic corticosterone treatment in order to estimate the effects of high-doses of corticosterone on calcium-dependent neuronal responses. Several investigations have revealed the fundamental role of inhibitory neurons in the control of (calcium-dependent) synaptic mechanisms, synaptic plasticity and network activity in the hippocampus (Acsády et al., 1996; Freund and Buzsáki, 1996). We therefore used immunolabelling to identify calcium-binding protein, calretinin (CR)-containing interneurons, as well as interneurons containing neuropeptides, such as vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), which label specific populations of interneurons.

Section snippets

Animals and treatments

Adult male Wistar rats weighing 200–220 g were used in the study. The animals were housed in standard laboratory conditions at constant temperature and humidity, under a 12 h light–dark cycle. The rats had free access to water and food. The experiments were conducted in accordance with prevailing laws and ethical considerations. Written permission was granted from the Faculty Ethical Committee on Animal Experiments, University of Szeged.

Corticosterone and sesame oil were purchased from

Effects of single-dose corticosterone administration on c-Fos immunoreactivity

c-Fos-immunoreactive cell nuclei were present in areas CA1-3 of the hippocampus, the granule cell layer of the DG, as well as in every layer of the CX 12 and 24 h following corticosterone administration. Most of the labelled nuclei in the Ammon's horn were detected in the pyramidal cell layer, which was strongly immunoreactive, whilst the Strata oriens, radiatum and lacunosum-moleculare contained a few, scattered c-Fos immunopositive nuclei. c-Fos immunopositive cell nuclei were observed in the

Discussion

Our previous results were in accord with data in the literature concerning the detection and evaluation of Fos immunoreactivity as a marker of neuronal activation (Mihály et al., 2001; Szakács et al., 2003; Fazekas et al., 2006). Expression of c-Fos is induced mainly by transmitter-mediated input and voltage-dependent Ca2+ currents (Greenberg and Ziff, 2001), and thereby Fos protein detection is a recognized marker of rapid changes in neuronal activity, reliable and widely used for

Acknowledgements

This study was supported by the Hungarian National Research Fund and by the Ministry of Education (OTKA T 046152/2004).

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