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Chronic lithium chloride injection increases glucocorticoid receptor but not mineralocorticoid receptor mRNA expression in rat brain
Neuroscience Research 38 (2000) 313 – 319 www.elsevier.com/locate/neures
Chronic lithium chloride injection increases glucocorticoid receptor but not mineralocorticoid receptor mRNA expression in rat brain Jun’ichi Semba a,c,*, Hideei Watanabe a, Tetsuya Suhara b,c, Nozomi Akanuma d b
a Di6ision of Health Sciences, Uni6ersity of the Air, 2 -11 Wakaba, Mihama-ku, Chiba 261 -8586, Japan Di6ision of Ad6anced Technology for Medical Imaging, National Institute of Radiological Sciences, 9 -1 Anagawa 4 chome, Inage-ku, Chiba 261 -8555, Japan c CREST, Japan Science and Technology Corporation (JST), Tokyo, Japan d Department of Neuropsychiatry, School of Medicine, Tokyo Medical and Dental Uni6ersity, 5 -45 Yushima 1 chome, Bunkyo-ku, Tokyo 113 -8519, Japan
Received 23 May 2000; accepted 3 August 2000
Abstract Lithium has been used clinically for the treatment of bipolar disorders. However, the brain mechanisms, by which lithium acts, are still unclear. An impaired hypothalamic–pituitary – adrenal (HPA) axis has been implicated in the pathogenesis of mood disorders. In this study, we investigated the effects of chronic lithium on the corticosteroid receptors in the brain. Male Wistar rats were injected with LiCl (1.5 mEq/kg) or saline intraperitoneally (i.p.) once a day for 14 days. Twenty-four hours after the last injection, the expressions of mRNA for glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in the brain were determined by non-radioactive in situ hybridization. Chronic administration of LiCl increased the expression of GR mRNA in the hippocampus and paraventricular nucleus of the hypothalamus (PVN). However, no significant changes were observed in the expression of either MR mRNA in the hippocampus or GR mRNA in the locus ceruleus. Since the hippocampus and PVN mediate negative feedback regulation of the HPA axis, an increased expression of GR mRNA in these regions may normalize HPA axis activity in mood disorders. Thus, the effect of chronic lithium on GR function may be involved in its antimanic and/or prophylactic activity in bipolar disorders. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. Keywords: Glucocorticoid receptor; Hippocampus; Hypothalamic– pituitary – adrenal axis; Lithium; Mineralocorticoid receptor
1. Introduction The hyperactivity of the hypothalamic – pituitary– adrenal (HPA) axis, as revealed by hypercortisolemia and non-suppression of the serum cortisol following dexamethasone administration, is widely accepted as one of the major pathophysiological findings in depression (Barden et al., 1995; Holsboer and Barden, 1996). Since corticosteroid receptor in the brain plays an important role in modulating the HPA axis (Barden et al., 1995), altered function of this receptor may be involved in the pathogenesis of mood disorders (Barden et al., 1995; Holsboer and Barden, 1996). * Corresponding author. Tel.: +81-43-2765111, ext. 4144; fax: +81-43-2984379. E-mail address: [email protected] (J. Semba).
There are two types of corticosteroid receptors in the brain — mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), which are characterized by a higher or lower affinity for corticosterone, respectively. Glucocorticoid receptor (GR) is distributed evenly throughout the brain, but is enriched in the hippocampus, paraventricular nucleus of the hypothalamus (PVN) and locus ceruleus (LC), while MR is predominantly localized in the hippocampus (Reul and de Kloet, 1985; Harfstrand et al., 1986; Aronsson et al., 1988). Recent studies (Brady et al., 1991; Peiffer et al., 1991; Seckl and Fink, 1992; Przegalinski and Budziszewska, 1993; Reul et al., 1993; Budziszewska et al., 1994) have shown that chronic antidepressant treatment increases GR and/or MR binding activity or their mRNA expression in the hippocampus, although the extent of the changes varies depending on length of
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treatment and the type and dose of antidepressants used. These animal experiments support the clinical findings that the effective antidepressant treatment normalizes the HPA axis activity in mood disorders (Barden et al., 1995; Holsboer and Barden, 1996). Increased GR and/or MR activity in the hippocampus normalize the hyperactivity of the HPA axis because the hippocampus plays a major role in mediating negative feedback of glucocorticoid (Barden et al., 1995). Lithium is the most commonly used drug for the acute and prophylactic treatment of bipolar disorders (Soares and Gershon, 1998). The precise mechanisms underlying the clinical efficacy of lithium, however, remain unknown. Since the impaired function of HPA axis is also observed in manic patients (Graham et al., 1982; Godwin, 1984; Godwin et al., 1984; Swann et al., 1992), studying the effect of lithium on the corticosteroid receptor function in the brain can be expected to provide useful information. In this paper, we further examine the pattern of the expression of both GR and MR mRNA in three different brain regions (the hippocampus, PVN and LC) after chronic lithium treatment using a non-radioactive in situ hybridization technique. We observed an increase in GR mRNA expression in CA1 and dentate gyrus of the hippocampus and in PVN after chronic lithium treatment. The increased expression of GR mRNA in these regions may reflect the therapeutic effects of lithium in bipolar disorders.
2. Methods and materials
2.1. Drug administration Male Wistar rats weighing 200 – 210 g were used. They were housed in groups of 3 – 4 in a temperaturecontrolled room under light/dark cycle (lights on from 7:00 to 19:00 h) with food and water ad libitum. In a chronic experiment, the animals were administered with LiCl (1.5 mEq/kg) or saline intraperitoneally (i.p.) once a day for 14 days. In an acute experiment, rats were injected i.p. with LiCl (1.5 and 3 mEq/kg). Twenty-four hours after the last injection, the rats were killed by decapitation. Their brains were removed promptly, frozen in powdered dry ice and stored at − 80°C. Trunk blood was collected in ice-cold heparin-coated tubes and centrifuged (2000 ×g, 15 min, 4°C). Plasma was stored at −30°C until assayed.
2.2. Plasma corticosterone assay Plasma corticosterone levels were determined by radioimmunoassay (ICN Biochemicals, USA) and expressed in ng/ml.
2.3. Non-radioacti6e in situ hybridization Coronal brain sections of 12-mm thickness were cut in a cryostat kept at −20°C and mounted on salinecoated glass slides. The sections were dried on a hot plate at 40°C and then kept frozen until used. The RNA probe for rat GR (plasmid generously donated by M.A. Smith, NIMH, Bethesda, MD, USA; Miesfeld et al., 1986) consisting of an 1155-bp PstI fragment encoding amino acids 140–525 was subcloned into pBluescript (II). The RNA probe for rat MR (plasmid generously donated by R.M. Evans, Salk Institute, San Diego, CA, USA; Arriza et al., 1988) consisted of a 513-bp EcoRI fragment encoding the 25 amino acid carboxy-terminal (steroid-binding domain) and a portion of the 3%-untranslated region was subcloned into pGEM4. Antisense digoxigenin-labeled RNA probe was prepared using digoxigenin-11-UTP (Boehringer Mannheim, Germany) and T7 or SP6 polymerase to transcribe the GR or MR insert cDNA, respectively. Sense strand RNA probes were generated using T3 or T7 RNA polymerase, respectively. Dot blotting was performed to confirm that the labeling reaction was successful and that the product yield was sufficient for use. Non-radioactive in situ hybridization was performed as described earlier (Hashimoto and Obata, 1991; Semba et al., 1999). Sections were post-fixed in 4% para-formaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 5 min. They were then rinsed three times in PBS for 3 min each and acetylated in 0.1 M triethanolamine (pH 8.0)/0.25% acetic anhydride. Hybridization was undertaken with 10 mg of a digoxigenin-labeled RNA probe diluted in 1 ml of hybridization buffer for 16 h at 42°C. After hybridization, the slides were washed twice in 2× SSC, 50% formamide at 55°C for 15 min and treated with 20 mg/ml RNase A for 40 min at 37°C. They were rinsed in 2×SSC, 50% formamide for 15 min at 55°C and twice in 1×SSC, 50% formamide for 15 min at room temperature, then in 2× SSC for 10 min, and finally in Tris-buffered saline (TBS, pH 7.4) for 10 min. For immunological detection of digoxigenin, the slides were pre-blocked in 1% blocking reagent (Boehringer Mannheim) in TBS for 30 min and then incubated with anti-digoxigenin conjugated to alkaline phosphatase (1:1000 dilution) for 30 min at 4°C. After incubation, the slides were washed in TBS and developed in a solution of NBT/BCIP (Boehringer Mannheim) in the presence of levamisole for 24 h. The color reaction was stopped using 1-mM ethylene diamine tetra aceticacid (EDTA), 10 mM Tris–HCl. After a wash in distilled water, the slides were dehydrated and cover-slipped. Sections of each brain region were processed simultaneously to allow direct comparison between groups using the same digoxigenin-labeled probes. Only back-
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Table 1 Effect of chronic lithium on weight gain in ratsa Treatment
Saline Lithium
Body weight (g) Day 1
Day 15
213.39 3.4 217.39 1.9
290.5 9 4.5 280.59 4.1
Data are represented as mean 9 S.E.M. (n = 7–8). There is no significant difference in the bodyweight at day 15 (Student’s t-test, P\0.05). a
ground labeling is observed when hybridization is conducted with the corresponding sense probes.
2.4. Analysis of hybridization signal NIH Image 1.6 software (Wayne Rasband, NIMH) was used to collect images for semi-quantitative examination of GR and MR mRNA expression. Sampled areas were digitized through a microscope with a CCD camera equipped with a Macintosh computer (PowerMac 9500). The NIH image outline tool was used to circumscribe the subregions of the hippocampus (CA1, CA2, CA3 and DG), PVN and LC. The mean gray level of the sampled area was corrected for background by subtracting the value of an unlabeled brain region within the same tissue section (e.g. corpus callosum). The resulting corrected gray levels for each brain region were analyzed statistically by Mann – Whitney U-test.
3. Results In an acute experiment, a single injection of LiCl (1.5 and 3.0 mEq/kg, i.p.) did not change the expression of either GR or MR mRNA in all brain regions examined (data not shown). Chronic lithium administration was not associated with any significant effects on weight gain (Table 1). All rats appeared healthy and no abnormal behavior was observed. Also, there were no significant changes in plasma corticosterone levels between saline- and lithium-treated rats (Table 2). Table 2 Effect of chronic lithium treatment on plasma corticosterone level in ratsa Treatment
Plasma corticosterone (ng/dl)
Saline Lithium
38.0 95.0 34.3 9 5.2
Data are represented as mean 9 S.E.M. (n = 7–8). There is no significant difference in plasma corticosterone level (Student’s t-test, P\0.05). a
Fig. 1. Effect of chronic lithium on GR mRNA expression in rat hippocampus. Top, photomicrographs of GR mRNA expression in the hippocampus; bottom, densitometic analysis of alteration in GR mRNA expression in the hippocampus. Data are represented as mean 9S.E.M. (n =7 – 8). *, PB 0.05 or **, PB 0.01 vs. saline-injected controls (Mann – Whitney U-test).
As shown in Fig. 1, chronic lithium treatment increased GR mRNA expression in CA1 and dentate gyrus (DG) subregions of the hippocampus. In CA2 and CA3 subregions, GR mRNA expression was relatively low and no difference was observed between chronic lithium- and saline-injected rats. Although MR mRNA is relatively highly expressed in CA2 and CA3 subregions of the hippocampus, there was no difference in the expression of this gene (Fig. 2). In the hypothalamus, strong expression of GR mRNA was observed in PVN (Fig. 3). Chronic treatment with lithium increased the GR mRNA expression in this region significantly. Although there were dense GR mRNA signals in LC, chronic lithium did not change the intensity (Fig. 4).
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4. Discussion Our main finding is that chronic lithium treatment increased GR mRNA in the hippocampus and PVN but not LC. MR mRNA in the hippocampus, however, did not change following chronic treatment with lithium In a previous northern hybridization study (Peiffer et al., 1991), significant increases in GR mRNA content were observed in the hippocampus following i.p. injection with a high dose of LiCl (6 mEq/kg) daily for 10 days. Our in situ hybridization study confirmed their findings in hippocampal GR. However, in our preliminary experiment, rats injected with 6 mEq/kg daily could not survive more than 7 days. We chose the dose of lithium 1.5 mEq/kg in our experiment, since signifi-
Fig. 2. Effect of chronic lithium on MR mRNA expression in rat hippocampus. Top, photomicrographs of MR mRNA expression in the hippocampus; bottom, densitometic analysis of alteration in MR mRNA expression in the hippocampus. Data are represented as mean9 S.E.M. (n = 7 – 8).
cant changes in g-amino butyricacid (GABA) receptors in rat brain have been reported after chronic injection at this dose of lithium (Motohashi, 1992; Motohashi et al., 1989). Even at this dosage, increased GR mRNA in the hippocampus was observed without a significant impairment of growth, suggesting that our results may not be due to a toxic effect of lithium. Increased expression of GR gene in our study cannot be explained by a down-regulation of GR following decreased secretion of glucocorticoid. In our experiment, there were no significant changes in plasma corticosterone levels between saline- and lithium-treated rats. This is supported by the previous studies, in which chronic administration of lithium resulted in no change (Aulakh et al., 1991) or rather an increase (Vatal and Aiyar, 1983; Storlien et al., 1985; Sugawara et al., 1988) in plasma corticosterone levels. Moreover, the increase in GR mRNA after chronic lithium treatment may not be due to a non-specific stress such as handling or injection, since chronic stress paradigms usually decrease (Herman et al., 1995; Makino et al., 1995) or has no effect (Mamalaki et al., 1992; Helmreich et al., 1997) on MR and/or GR mRNA levels within the hippocampus or PVN. Long-term secretion of glucocorticoids stimulated by repeated stress may down-regulate GR and finally decrease GR activity in the brain. The most conservative explanation for the increase in GR mRNA after chronic lithium can be made by the interaction between serotonin function and glucocorticoid. Many previous studies have shown that chronic lithium enhances serotonergic transmission at a variety of levels, including synthesis, turnover, release and uptake (Price et al., 1990). Serotonin is also important for the maintenance of corticosteroid receptor gene expression in the hippocampus. Central depletion of serotonin leads to a reduction of corticosteroid receptor mRNA expression in hippocampus (Seckl and Fink, 1991; Yau et al., 1994; Novotney and Lowy, 1995), while the application of 5-HT increases corticosteroid receptor sites in hippocampal cell culture (Mitchell et al., 1992; Hery et al., 2000). Since long-term stimulation of 5-HT or its agonist was required to increase GR binding in the hippocampus (Mitchell et al., 1992; Hery et al., 2000), it is suggested that the 5-HT-induced increase in GR binding may involve the synthesis of new GR receptors. Thus, the increased serotonin function induced by chronic lithium may induce the increase in GR mRNA in the hippocampus. However, since MR mRNA in the hippocampus did not simultaneously increase together with GR mRNA, other mechanisms must be considered. Increased GR mRNA expression cannot be explained by altered NE function after chronic lithium, in spite of the fact that lithium is shown to enhance the uptake of norepinephrine (NE) into nerve terminals and reduce stimulated release of NE from synapse (Slotkin et al.,
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Fig. 3. Effect of chronic lithium on GR mRNA expression in rat paraventricular nucleus of the hypothalamus (PVN). Top, photomicrographs of GR mRNA expression in the PVN; bottom, densitometic analysis of alteration in GR mRNA expression in the hippocampus. Data represented as mean9S.E.M. (n =7 –8). **, P B0.01 vs. saline-injected controls (Mann – Whitney U-test).
1980; Gross and Hanft, 1990). Central lesions with 6-hydroxydopamine decreased MR mRNA and increased MR binding activity but had no effect on GR in the rat hippocampus (Maccari et al., 1992; Yau and Seckl, 1992). Recent studies (Rossby et al., 1995; Eiring and Sulser, 1997) showed that an increase in the synaptic availability of NE is not responsible for the action of tricyclic antidepressants on GR gene expression in the hippocampus. However, there remains a possibility that alterations of other neurotransmitter function may underlie the changes of GR gene expression (Lenox and Manji, 1998). The biological roles of the cells with dense GR mRNA signals in the LC are not known. However, since NE cells in the LC contain GR-immunoreactivity (Harfstrand et al., 1986; Kitayama et al., 1988), it appears that the noradrenergic neurons in the LC may be target cells for glucocorticoids (Kitayama et al., 1988). In our experiment, chronic lithium did not change GR mRNA in the LC. This may be explained by the fact that lithium has only a limited effect on noradrenergic systems as described above (Lenox and Manji, 1998). Much of the research concerning lithium has focused on changes of the activities of cellular transduction
systems, especially the phosphoinositide and cyclic AMP second messenger systems (Jope and Williams, 1994; Manji and Lenox, 1994). It is reported that protein kinase C modulates the expression of GR (Sheppard, 1994) and lithium mimics dexamethasone in stimulating DNA synthesis by WI-38 cells (Sorger and Cristofalo, 1992). Most recently, a novel gene, which is modulated by lithium, has been identified using a differential display technique (Wang et al., 1999). Such lithium-induced genes might regulate GR function in the brain. Otherwise, lithium might increase the GR gene promoter activity at the genome level. This can be supported partially by a previous experiment that desipramine increases the GR gene promoter activity in LTK− cells, which do not contain any catecholamines (Pepin et al., 1992). The hippocampus and PVN are the major brain regions, which mediate negative feedback regulation of the HPA axis. Thus, our results suggest that increased expression of GR mRNA in the hippocampus and PVN may normalize the hyperactivity of HPA axis, which manifests itself as non-suppression of the serum cortisol following a dexamethasone suppression test in mania. We supposed that this altered GR function might mediate antimanic and/or prophylactic effects of lithium in the treatment of bipolar disorders.
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Fig. 4. Effect of chronic lithium on GR mRNA expression in rat locus ceruleus (LC). Top, photomicrographs of GR mRNA expression in the LC; bottom, densitometic analysis of alteration in GR mRNA expression in the LC. Data are represented as mean 9 S.E.M. (n=7– 8).
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Chronic lithium chloride injection increases glucocorticoid receptor but not mineralocorticoid receptor mRNA expression in rat brain Jun’ichi Semba a,c,*, Hideei Watanabe a, Tetsuya Suhara b,c, Nozomi Akanuma d b
a Di6ision of Health Sciences, Uni6ersity of the Air, 2 -11 Wakaba, Mihama-ku, Chiba 261 -8586, Japan Di6ision of Ad6anced Technology for Medical Imaging, National Institute of Radiological Sciences, 9 -1 Anagawa 4 chome, Inage-ku, Chiba 261 -8555, Japan c CREST, Japan Science and Technology Corporation (JST), Tokyo, Japan d Department of Neuropsychiatry, School of Medicine, Tokyo Medical and Dental Uni6ersity, 5 -45 Yushima 1 chome, Bunkyo-ku, Tokyo 113 -8519, Japan
Received 23 May 2000; accepted 3 August 2000
Abstract Lithium has been used clinically for the treatment of bipolar disorders. However, the brain mechanisms, by which lithium acts, are still unclear. An impaired hypothalamic–pituitary – adrenal (HPA) axis has been implicated in the pathogenesis of mood disorders. In this study, we investigated the effects of chronic lithium on the corticosteroid receptors in the brain. Male Wistar rats were injected with LiCl (1.5 mEq/kg) or saline intraperitoneally (i.p.) once a day for 14 days. Twenty-four hours after the last injection, the expressions of mRNA for glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in the brain were determined by non-radioactive in situ hybridization. Chronic administration of LiCl increased the expression of GR mRNA in the hippocampus and paraventricular nucleus of the hypothalamus (PVN). However, no significant changes were observed in the expression of either MR mRNA in the hippocampus or GR mRNA in the locus ceruleus. Since the hippocampus and PVN mediate negative feedback regulation of the HPA axis, an increased expression of GR mRNA in these regions may normalize HPA axis activity in mood disorders. Thus, the effect of chronic lithium on GR function may be involved in its antimanic and/or prophylactic activity in bipolar disorders. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. Keywords: Glucocorticoid receptor; Hippocampus; Hypothalamic– pituitary – adrenal axis; Lithium; Mineralocorticoid receptor
1. Introduction The hyperactivity of the hypothalamic – pituitary– adrenal (HPA) axis, as revealed by hypercortisolemia and non-suppression of the serum cortisol following dexamethasone administration, is widely accepted as one of the major pathophysiological findings in depression (Barden et al., 1995; Holsboer and Barden, 1996). Since corticosteroid receptor in the brain plays an important role in modulating the HPA axis (Barden et al., 1995), altered function of this receptor may be involved in the pathogenesis of mood disorders (Barden et al., 1995; Holsboer and Barden, 1996). * Corresponding author. Tel.: +81-43-2765111, ext. 4144; fax: +81-43-2984379. E-mail address: [email protected] (J. Semba).
There are two types of corticosteroid receptors in the brain — mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), which are characterized by a higher or lower affinity for corticosterone, respectively. Glucocorticoid receptor (GR) is distributed evenly throughout the brain, but is enriched in the hippocampus, paraventricular nucleus of the hypothalamus (PVN) and locus ceruleus (LC), while MR is predominantly localized in the hippocampus (Reul and de Kloet, 1985; Harfstrand et al., 1986; Aronsson et al., 1988). Recent studies (Brady et al., 1991; Peiffer et al., 1991; Seckl and Fink, 1992; Przegalinski and Budziszewska, 1993; Reul et al., 1993; Budziszewska et al., 1994) have shown that chronic antidepressant treatment increases GR and/or MR binding activity or their mRNA expression in the hippocampus, although the extent of the changes varies depending on length of
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treatment and the type and dose of antidepressants used. These animal experiments support the clinical findings that the effective antidepressant treatment normalizes the HPA axis activity in mood disorders (Barden et al., 1995; Holsboer and Barden, 1996). Increased GR and/or MR activity in the hippocampus normalize the hyperactivity of the HPA axis because the hippocampus plays a major role in mediating negative feedback of glucocorticoid (Barden et al., 1995). Lithium is the most commonly used drug for the acute and prophylactic treatment of bipolar disorders (Soares and Gershon, 1998). The precise mechanisms underlying the clinical efficacy of lithium, however, remain unknown. Since the impaired function of HPA axis is also observed in manic patients (Graham et al., 1982; Godwin, 1984; Godwin et al., 1984; Swann et al., 1992), studying the effect of lithium on the corticosteroid receptor function in the brain can be expected to provide useful information. In this paper, we further examine the pattern of the expression of both GR and MR mRNA in three different brain regions (the hippocampus, PVN and LC) after chronic lithium treatment using a non-radioactive in situ hybridization technique. We observed an increase in GR mRNA expression in CA1 and dentate gyrus of the hippocampus and in PVN after chronic lithium treatment. The increased expression of GR mRNA in these regions may reflect the therapeutic effects of lithium in bipolar disorders.
2. Methods and materials
2.1. Drug administration Male Wistar rats weighing 200 – 210 g were used. They were housed in groups of 3 – 4 in a temperaturecontrolled room under light/dark cycle (lights on from 7:00 to 19:00 h) with food and water ad libitum. In a chronic experiment, the animals were administered with LiCl (1.5 mEq/kg) or saline intraperitoneally (i.p.) once a day for 14 days. In an acute experiment, rats were injected i.p. with LiCl (1.5 and 3 mEq/kg). Twenty-four hours after the last injection, the rats were killed by decapitation. Their brains were removed promptly, frozen in powdered dry ice and stored at − 80°C. Trunk blood was collected in ice-cold heparin-coated tubes and centrifuged (2000 ×g, 15 min, 4°C). Plasma was stored at −30°C until assayed.
2.2. Plasma corticosterone assay Plasma corticosterone levels were determined by radioimmunoassay (ICN Biochemicals, USA) and expressed in ng/ml.
2.3. Non-radioacti6e in situ hybridization Coronal brain sections of 12-mm thickness were cut in a cryostat kept at −20°C and mounted on salinecoated glass slides. The sections were dried on a hot plate at 40°C and then kept frozen until used. The RNA probe for rat GR (plasmid generously donated by M.A. Smith, NIMH, Bethesda, MD, USA; Miesfeld et al., 1986) consisting of an 1155-bp PstI fragment encoding amino acids 140–525 was subcloned into pBluescript (II). The RNA probe for rat MR (plasmid generously donated by R.M. Evans, Salk Institute, San Diego, CA, USA; Arriza et al., 1988) consisted of a 513-bp EcoRI fragment encoding the 25 amino acid carboxy-terminal (steroid-binding domain) and a portion of the 3%-untranslated region was subcloned into pGEM4. Antisense digoxigenin-labeled RNA probe was prepared using digoxigenin-11-UTP (Boehringer Mannheim, Germany) and T7 or SP6 polymerase to transcribe the GR or MR insert cDNA, respectively. Sense strand RNA probes were generated using T3 or T7 RNA polymerase, respectively. Dot blotting was performed to confirm that the labeling reaction was successful and that the product yield was sufficient for use. Non-radioactive in situ hybridization was performed as described earlier (Hashimoto and Obata, 1991; Semba et al., 1999). Sections were post-fixed in 4% para-formaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 5 min. They were then rinsed three times in PBS for 3 min each and acetylated in 0.1 M triethanolamine (pH 8.0)/0.25% acetic anhydride. Hybridization was undertaken with 10 mg of a digoxigenin-labeled RNA probe diluted in 1 ml of hybridization buffer for 16 h at 42°C. After hybridization, the slides were washed twice in 2× SSC, 50% formamide at 55°C for 15 min and treated with 20 mg/ml RNase A for 40 min at 37°C. They were rinsed in 2×SSC, 50% formamide for 15 min at 55°C and twice in 1×SSC, 50% formamide for 15 min at room temperature, then in 2× SSC for 10 min, and finally in Tris-buffered saline (TBS, pH 7.4) for 10 min. For immunological detection of digoxigenin, the slides were pre-blocked in 1% blocking reagent (Boehringer Mannheim) in TBS for 30 min and then incubated with anti-digoxigenin conjugated to alkaline phosphatase (1:1000 dilution) for 30 min at 4°C. After incubation, the slides were washed in TBS and developed in a solution of NBT/BCIP (Boehringer Mannheim) in the presence of levamisole for 24 h. The color reaction was stopped using 1-mM ethylene diamine tetra aceticacid (EDTA), 10 mM Tris–HCl. After a wash in distilled water, the slides were dehydrated and cover-slipped. Sections of each brain region were processed simultaneously to allow direct comparison between groups using the same digoxigenin-labeled probes. Only back-
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Table 1 Effect of chronic lithium on weight gain in ratsa Treatment
Saline Lithium
Body weight (g) Day 1
Day 15
213.39 3.4 217.39 1.9
290.5 9 4.5 280.59 4.1
Data are represented as mean 9 S.E.M. (n = 7–8). There is no significant difference in the bodyweight at day 15 (Student’s t-test, P\0.05). a
ground labeling is observed when hybridization is conducted with the corresponding sense probes.
2.4. Analysis of hybridization signal NIH Image 1.6 software (Wayne Rasband, NIMH) was used to collect images for semi-quantitative examination of GR and MR mRNA expression. Sampled areas were digitized through a microscope with a CCD camera equipped with a Macintosh computer (PowerMac 9500). The NIH image outline tool was used to circumscribe the subregions of the hippocampus (CA1, CA2, CA3 and DG), PVN and LC. The mean gray level of the sampled area was corrected for background by subtracting the value of an unlabeled brain region within the same tissue section (e.g. corpus callosum). The resulting corrected gray levels for each brain region were analyzed statistically by Mann – Whitney U-test.
3. Results In an acute experiment, a single injection of LiCl (1.5 and 3.0 mEq/kg, i.p.) did not change the expression of either GR or MR mRNA in all brain regions examined (data not shown). Chronic lithium administration was not associated with any significant effects on weight gain (Table 1). All rats appeared healthy and no abnormal behavior was observed. Also, there were no significant changes in plasma corticosterone levels between saline- and lithium-treated rats (Table 2). Table 2 Effect of chronic lithium treatment on plasma corticosterone level in ratsa Treatment
Plasma corticosterone (ng/dl)
Saline Lithium
38.0 95.0 34.3 9 5.2
Data are represented as mean 9 S.E.M. (n = 7–8). There is no significant difference in plasma corticosterone level (Student’s t-test, P\0.05). a
Fig. 1. Effect of chronic lithium on GR mRNA expression in rat hippocampus. Top, photomicrographs of GR mRNA expression in the hippocampus; bottom, densitometic analysis of alteration in GR mRNA expression in the hippocampus. Data are represented as mean 9S.E.M. (n =7 – 8). *, PB 0.05 or **, PB 0.01 vs. saline-injected controls (Mann – Whitney U-test).
As shown in Fig. 1, chronic lithium treatment increased GR mRNA expression in CA1 and dentate gyrus (DG) subregions of the hippocampus. In CA2 and CA3 subregions, GR mRNA expression was relatively low and no difference was observed between chronic lithium- and saline-injected rats. Although MR mRNA is relatively highly expressed in CA2 and CA3 subregions of the hippocampus, there was no difference in the expression of this gene (Fig. 2). In the hypothalamus, strong expression of GR mRNA was observed in PVN (Fig. 3). Chronic treatment with lithium increased the GR mRNA expression in this region significantly. Although there were dense GR mRNA signals in LC, chronic lithium did not change the intensity (Fig. 4).
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4. Discussion Our main finding is that chronic lithium treatment increased GR mRNA in the hippocampus and PVN but not LC. MR mRNA in the hippocampus, however, did not change following chronic treatment with lithium In a previous northern hybridization study (Peiffer et al., 1991), significant increases in GR mRNA content were observed in the hippocampus following i.p. injection with a high dose of LiCl (6 mEq/kg) daily for 10 days. Our in situ hybridization study confirmed their findings in hippocampal GR. However, in our preliminary experiment, rats injected with 6 mEq/kg daily could not survive more than 7 days. We chose the dose of lithium 1.5 mEq/kg in our experiment, since signifi-
Fig. 2. Effect of chronic lithium on MR mRNA expression in rat hippocampus. Top, photomicrographs of MR mRNA expression in the hippocampus; bottom, densitometic analysis of alteration in MR mRNA expression in the hippocampus. Data are represented as mean9 S.E.M. (n = 7 – 8).
cant changes in g-amino butyricacid (GABA) receptors in rat brain have been reported after chronic injection at this dose of lithium (Motohashi, 1992; Motohashi et al., 1989). Even at this dosage, increased GR mRNA in the hippocampus was observed without a significant impairment of growth, suggesting that our results may not be due to a toxic effect of lithium. Increased expression of GR gene in our study cannot be explained by a down-regulation of GR following decreased secretion of glucocorticoid. In our experiment, there were no significant changes in plasma corticosterone levels between saline- and lithium-treated rats. This is supported by the previous studies, in which chronic administration of lithium resulted in no change (Aulakh et al., 1991) or rather an increase (Vatal and Aiyar, 1983; Storlien et al., 1985; Sugawara et al., 1988) in plasma corticosterone levels. Moreover, the increase in GR mRNA after chronic lithium treatment may not be due to a non-specific stress such as handling or injection, since chronic stress paradigms usually decrease (Herman et al., 1995; Makino et al., 1995) or has no effect (Mamalaki et al., 1992; Helmreich et al., 1997) on MR and/or GR mRNA levels within the hippocampus or PVN. Long-term secretion of glucocorticoids stimulated by repeated stress may down-regulate GR and finally decrease GR activity in the brain. The most conservative explanation for the increase in GR mRNA after chronic lithium can be made by the interaction between serotonin function and glucocorticoid. Many previous studies have shown that chronic lithium enhances serotonergic transmission at a variety of levels, including synthesis, turnover, release and uptake (Price et al., 1990). Serotonin is also important for the maintenance of corticosteroid receptor gene expression in the hippocampus. Central depletion of serotonin leads to a reduction of corticosteroid receptor mRNA expression in hippocampus (Seckl and Fink, 1991; Yau et al., 1994; Novotney and Lowy, 1995), while the application of 5-HT increases corticosteroid receptor sites in hippocampal cell culture (Mitchell et al., 1992; Hery et al., 2000). Since long-term stimulation of 5-HT or its agonist was required to increase GR binding in the hippocampus (Mitchell et al., 1992; Hery et al., 2000), it is suggested that the 5-HT-induced increase in GR binding may involve the synthesis of new GR receptors. Thus, the increased serotonin function induced by chronic lithium may induce the increase in GR mRNA in the hippocampus. However, since MR mRNA in the hippocampus did not simultaneously increase together with GR mRNA, other mechanisms must be considered. Increased GR mRNA expression cannot be explained by altered NE function after chronic lithium, in spite of the fact that lithium is shown to enhance the uptake of norepinephrine (NE) into nerve terminals and reduce stimulated release of NE from synapse (Slotkin et al.,
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Fig. 3. Effect of chronic lithium on GR mRNA expression in rat paraventricular nucleus of the hypothalamus (PVN). Top, photomicrographs of GR mRNA expression in the PVN; bottom, densitometic analysis of alteration in GR mRNA expression in the hippocampus. Data represented as mean9S.E.M. (n =7 –8). **, P B0.01 vs. saline-injected controls (Mann – Whitney U-test).
1980; Gross and Hanft, 1990). Central lesions with 6-hydroxydopamine decreased MR mRNA and increased MR binding activity but had no effect on GR in the rat hippocampus (Maccari et al., 1992; Yau and Seckl, 1992). Recent studies (Rossby et al., 1995; Eiring and Sulser, 1997) showed that an increase in the synaptic availability of NE is not responsible for the action of tricyclic antidepressants on GR gene expression in the hippocampus. However, there remains a possibility that alterations of other neurotransmitter function may underlie the changes of GR gene expression (Lenox and Manji, 1998). The biological roles of the cells with dense GR mRNA signals in the LC are not known. However, since NE cells in the LC contain GR-immunoreactivity (Harfstrand et al., 1986; Kitayama et al., 1988), it appears that the noradrenergic neurons in the LC may be target cells for glucocorticoids (Kitayama et al., 1988). In our experiment, chronic lithium did not change GR mRNA in the LC. This may be explained by the fact that lithium has only a limited effect on noradrenergic systems as described above (Lenox and Manji, 1998). Much of the research concerning lithium has focused on changes of the activities of cellular transduction
systems, especially the phosphoinositide and cyclic AMP second messenger systems (Jope and Williams, 1994; Manji and Lenox, 1994). It is reported that protein kinase C modulates the expression of GR (Sheppard, 1994) and lithium mimics dexamethasone in stimulating DNA synthesis by WI-38 cells (Sorger and Cristofalo, 1992). Most recently, a novel gene, which is modulated by lithium, has been identified using a differential display technique (Wang et al., 1999). Such lithium-induced genes might regulate GR function in the brain. Otherwise, lithium might increase the GR gene promoter activity at the genome level. This can be supported partially by a previous experiment that desipramine increases the GR gene promoter activity in LTK− cells, which do not contain any catecholamines (Pepin et al., 1992). The hippocampus and PVN are the major brain regions, which mediate negative feedback regulation of the HPA axis. Thus, our results suggest that increased expression of GR mRNA in the hippocampus and PVN may normalize the hyperactivity of HPA axis, which manifests itself as non-suppression of the serum cortisol following a dexamethasone suppression test in mania. We supposed that this altered GR function might mediate antimanic and/or prophylactic effects of lithium in the treatment of bipolar disorders.
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Fig. 4. Effect of chronic lithium on GR mRNA expression in rat locus ceruleus (LC). Top, photomicrographs of GR mRNA expression in the LC; bottom, densitometic analysis of alteration in GR mRNA expression in the LC. Data are represented as mean 9 S.E.M. (n=7– 8).
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