Department of Legal Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan,
1 URC for Neuroendocrinology, University of Bristol, BRI, Marlborough Street, Bristol BS2 8HW,
2 School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 1TS, UK and
3 Department of Forensic Medicine, Kagawa Medical University, 1750-1, Miki, Kita, Kagawa, 761-0793, Japan
Received 8 November 2001; in revised form 5 February 2002; accepted 20 February 2002
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ABSTRACT |
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INTRODUCTION |
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The expression of the immediate early genes (IEGs), such as c-fos, has been widely used as a marker of neuronal activity in the central nervous system (Sagar et al., 1988). Following ethanol (3 g/kg) administration, the levels of c-fos mRNA in PVN are markedly increased (Zoeller and Fletcher, 1994
; Ogilvie et al., 1998
). However, there have been no reports on the relationship between acetaldehyde and c-fos expression in the PVN. Our previous observations (Kinoshita et al., 2001a
), prompted us to hypothesize that c-fos mRNA may be induced in the PVN by the accumulation of acetaldehyde in ethanol- and cyanamide-treated rats. In the present study, we examined the acute effects of acetaldehyde combined with ethanol on c-fos expression in the PVN using in situ hybridization histochemistry (ISHH).
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MATERIALS AND METHODS |
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Six experimental groups were used as follows: saline (as control), cyanamide (50 mg/kg) alone, low dose of ethanol (1 g/kg body weight) alone, high dose of ethanol (3 g/kg body weight) alone, low dose of ethanol with cyanamide and high dose of ethanol with cyanamide. Cyanamide (Sigma Chemical Co., Dorset, Poole, UK) or saline in a volume of 0.1 ml/100 g body weight was injected intraperitoneally (i.p.) 60 min before ethanol administration. Ethanol for i.p. injection was diluted with saline. Animals were killed by decapitation 30 min following ethanol administration and trunk blood was collected. This timepoint has previously been shown to be a suitable one for demonstrating a significant increase in c-fos mRNA after stress or ethanol administration (Imaki et al., 1992; Harbuz et al., 1993
; Ogilvie et al., 1998
). Brains were rapidly removed, frozen on dry-ice and stored at 80°C until sectioning. Sections 12 µm thick, containing the medial parvocellular region of PVN, were cut and thaw-mounted on gelatin-coated slides and stored at 80°C before hybridization. Trunk blood was collected for the measurement of ethanol and acetaldehyde concentrations. Experimental procedures were performed on a project licence granted by the Home Secretary under the terms of the Animal (Scientific Procedures) Act 1986.
In situ hybridization histochemistry
ISHH was performed as described previously (Young et al., 1986; Harbuz and Lightman, 1989
; Harbuz et al., 1991
; Kinoshita et al., 2001a
). In brief, the sections were warmed at room temperature, allowed to dry for 10 min, and then fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 5 min. Then the sections were washed twice in PBS, and incubated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min. The sections were passed through 70 (1 min), 80 (1 min), 95 (2 min) and 100% (1 min) ethanol, 100% chloroform (5 min), and 100 (1 min) and 95% (1 min) ethanol for dehydration, delipidation and partial rehydration.
The probe used for c-fos was a 48-mer oligonucleotide complementary to part of the exonic mRNA sequence (Perkin Elmer, Warrington, UK). The specificity of the probe has been determined previously (Harbuz et al., 1993). The probe was 3'-end-labelled with [35S]deoxy-ATP (1000 µCi/mmol; NEN, Boston, MA, USA) by terminal deoxyribonucleotidyl transferase (BoehringerMannheim, Lewes, Sussex, UK) and column-purified by QIA quick nucleotide removal kit (Qiagen Ltd, Crawley, West Sussex, UK). The specific activity of the probe was 6.70 x 1018 d.p.m./mol. Approximately 100 000 c.p.m. probe (per 45 µl) were applied to each slide. Hybridization was performed overnight at 37°C. All the sections were processed at the same time. The sections were washed in four 15-min rinses of 1 x SSC (1 x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) at 55°C, followed by two 30-min washes in 1 x SSC at room temperature to remove non-specific binding before two short water rinses and then air-dried. The sections, together with 14C-labelled standard (ARC146C; American Radiolabeled Chemicals Inc., St Louis, MO, USA) were exposed to Hyperfilm MP autoradiography film (Amersham International plc, Amersham, Bucks, UK). The autoradiographic image was measured as previously described (Harbuz et al., 1994
) using a computer-assisted image analysis system (Image 1.22, developed by W. Rasband, NIH, Bethesda, MD, USA) run on Apple Macintosh.
Gas chromatography
The concentration of ethanol and acetaldehyde was measured simultaneously by head-space gas chromatography (Okada and Mizoi, 1982).
Statistics
The data are expressed as mean ± SEM. All groups within each data set were compared by one-way analysis of variance (ANOVA) followed by Fishers protected least-significant difference test for multiple comparisons. A value of P < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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The expression of c-fos is rapidly induced in various brain regions in response to a variety of physiological manipulations, such as immobilization, hypertonic saline injection, irradiation, cocaine or morphine administration and high dose ethanol administration (Ceccatelli et al., 1989; Imaki et al., 1992
; Moratalla et al., 1993
; Liu et al., 1994
; Zoeller and Fletcher, 1994
; Usenius et al., 1996
; Ogilvie et al., 1998
). However, the role of c-fos in the central nervous system is unclear; it has been proposed that IEGs, including c-fos, have a function as mediators of the response to external stimuli (Sheng and Greenberg, 1990
). Acute stress results in an increase in c-fos mRNA in CRF-containing neurons in the PVN, suggesting a close relationship (Ceccatelli et al., 1989
; Imaki et al., 1992
). The activation of c-fos and CRF gene expression have been linked following ethanol administration (Ogilvie et al., 1998
). However, the precise relationship between CRF and c-fos in the PVN remains obscure and has been the subject of much investigation, resulting in a number of studies that have questioned this relationship. The induction of heteronuclear CRF mRNA has been observed prior to an increase in c-fos mRNA, suggesting that Fos protein may not mediate the activation of CRF gene transcription (Kovacs and Sawchenko, 1996
). It has also been noted that c-fos mRNA in the PVN can be dissociated from the activation of CRF mRNA in the rat model of adjuvant-induced arthritis (Harbuz and Jessop, 1999
). In addition, the CRF promoter lacks an activator protein (AP)-1 site (Ogilvie et al., 1998
). In our previous study (Kinoshita et al., 2000
), we noted a significant and sustained increase in plasma corticosterone that was maintained for 4 h and that was reflected by an increase in CRF mRNA in the parvocellular cells of the PVN following this dose of cyanamide alone. The ability to increase CRF mRNA (Kinoshita et al., 2000
) in the absence of a change in c-fos mRNA (the present study) provides further evidence for a dissociation between the activation of c-fos and CRF mRNA. Taken together, these data question the relationship between CRF and c-fos. However, although accumulating evidence suggests c-fos mRNA does not appear to regulate directly CRF mRNA expression, it has been suggested that Fos protein may interact with other transcription factors to modulate CRF gene expression (Imaki et al., 1996
), and fos remains a useful marker of neuronal activation.
In the present study, we could not determine whether acetaldehyde activates PVN neurons in a direct or indirect manner. The c-fos mRNA activation in fat-storing cell cultures may be mediated by protein kinase C activation (Casini et al., 1994). However, as both CRF and c-fos promoters have a functional cyclic AMP (cAMP) response element, it has been speculated that a cAMP-dependent system might mediate this activation (Kovacs and Sawchenko, 1996
; Ogilvie et al., 1998
). The relationship between CRF neurons and c-fos in the PVN remains to be determined, but may involve other neurotransmitter systems. It has been reported that glutamate is a major neurotransmitter in the region of the PVN (Brann, 1995
), and glutamate, catecholamine and acetylcholine are candidate neurotransmitters for mediation of the induction of a number of IEGs including c-fos in the PVN (Imaki et al., 1996
). Acetaldehyde modulates monoamine metabolism in brain (Hashimoto et al., 1989
) and can induce catecholamine release from peripheral adrenergic nerve terminals (Brien and Loomis, 1983
; Chiba and Tsukada, 1988
). Also in the periphery, high concentrations of acetaldehyde have been noted to stimulate cholinergic neurons in intestine (Kinoshita et al., 2001b
). However, there are no reports describing the effects of acetaldehyde on the release of neurotransmitters in PVN and the involvement of these requires further investigation. A number of studies have investigated the interaction of the cAMP response element and the glucocorticoid response element in the CRF gene. Clearly, glucocorticoid feedback (via the type II glucocorticoid receptor present in the PVN), is a potent regulator of CRF mRNA. Future studies could elucidate the interplay of these elements and neurotransmitters following treatment with acetaldehyde.
In conclusion, we have demonstrated that acetaldehyde significantly and maximally increased c-fos mRNA in the PVN in the presence of a low dose of ethanol. These results suggest that acetaldehyde exerts central actions inducing neuronal activation, resulting in activation of the HPA axis. These data also provide further evidence for a dissociation between the activation of c-fos mRNA and CRF mRNA in the parvocellular cells of the PVN. It remains to be determined whether this activation is due to a direct action of acetaldehyde on CRF neurons, or due to modulation of other neurotransmitter systems activated in response to elevated blood concentrations of acetaldehyde.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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