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Dexamethasone induces different wheel running activity than corticosterone through vasopressin release from the suprachiasmatic nucleus
Brain Research 1028 (2004) 219 – 224 www.elsevier.com/locate/brainres
Research report
Dexamethasone induces different wheel running activity than corticosterone through vasopressin release from the suprachiasmatic nucleus Yoshiaki Isobe*, Takako Torii, Takatsune Kawaguchi, Hitoo Nishino Department of Neuro-physiology and Brain Sciences, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan Accepted 21 September 2004
Abstract During the analysis of wheel running activity, we found that corticosterone (1 mg/100 g BW) injection decreased wheel activity, while dexamethasone (0.1 mg/100 g) increased the activity. To clarify the functional differences between corticosterone and dexamethasone, we measured Arg-vasopressin (AVP) release from the suprachiasmatic nucleus (SCN) slice culture in vitro and AVP coding mRNA in the SCN in vivo. The corticosterone (0.2 and 2 Ag/ml, final concentration in medium) decreased the AVP release, while it increased by dexamethasone (0.2 and 2 Ag/ml). An AVP mRNA in the SCN was decreased by both corticosterone (1 mg/100 g) and dexamethasone (0.1 mg/100 g). The differences in wheel activity by corticosterone and dexamethasone are discussed from the changes of AVP in the SCN. D 2004 Elsevier B.V. All rights reserved. Theme: Neuronal basis of behavior Topic: Biological rhythms and sleep Keywords: Corticosterone; Dexamethasone; Wheel activity; Arg-vasopressin; Suprachiasmatic nucleus
1. Introduction Wheel running activity is widely used in analyzing the mechanisms of drug effects, defects in motor functions in the CNS and circadian rhythm. An animal’s motivation to start, continue and stop wheel running activity is not yet clear; neurotransmitters, neuropeptides and many factors in the CNS should be involved. If the nature of the running wheel activity is elucidated, it will be an advantage to help clarify the mechanisms of circadian rhythm, stress and motor functions. Stress-induced behavioral sensitivity is augmented by corticosterone and dexamethasone [26,28]. Stressed animals allowed to run showed lower corticosterone levels than those not allowed to run [27]. In blinded
* Corresponding author. Tel.: +81 52 853 8136; fax: +81 52 843 3069. E-mail address: [email protected] (Y. Isobe). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.09.021
rats, the corticosterone levels were high at the beginning of the running phase and low at its end [6]. From the circadian rhythm point of view, it was reported that rodents allowed access to wheel or not affected the phase shift in the circadian rhythm [24,29]. Higher doses of dexamethasone phase-shift the body temperature rhythm [8]; however, progress in this field of study is difficult to define. With respect to stress, there is some evidence that Argvasopressin (AVP) enhances or reduces the locomotor activity [3,5]. In response to stress, a glucocorticoid negative feedback to AVP and corticotropin-releasing factor (CRF) gene transcription at the paraventricular nucleus (PVN) had been advocated [16]. AVP containing neurons is one of the output paths from the suprachiasmatic nucleus (SCN), which is the center of biological rhythm in mammals. AVP containing neurons in the SCN connects with the PVN. Recently, it was reported that glucocorticoid
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negatively regulated the AVP mRNA transcription at the promoter region in glucocorticoid responsible elements (GRE) of the AVP gene [13,15]. In addition, mesolimbic dopaminergic and serotonergic systems coordinate well with glucocorticoid in modulating the locomotor activity [3,11,22,30,31]. In the present research, we found different responses of corticosterone and dexamethasone in the wheel running activity and AVP release from the SCN slice culture. An action and the feedback mechanism were reported to be different for corticosterone and dexamethasone in their functions [17,20]. Based on the present results, we discussed the effects of corticosterone and dexamethasone on the behavioral effects in relation to the vasopressin functions in the SCN.
2. Materials and method 2.1. Materials Wistar rats (SLC, Shizudokyo, Japan) were housed under a 12-h light/12-h dark cycle (LD, light on at 7:00; light intensity, 200 lux) with free access to food and water. A minimum number of animals were used to perform our strategy. Animal care was in accordance with the guideline of Nagoya City University, Graduate School of Medical Sciences, for use of animals in research. 2.2. Drugs Corticosterone (Sigma, Japan) and dexamethasone (Sigma) were used. Corticosterone and dexamethasone was dissolved in ethanol then resolvated (suspended) in saline. Drug injection methods are shown later. 2.3. Locomotor activity Six rats were used. Running wheel activity was measured as turns using wheel cages with an attached resting room. The length of the wheel circles was 100 cm. The activities were counted for 20 min just after the treatment (saline and drug injection or no injection) using a cumulative event recorder, and data were stored on a computer disk [11]. 2.4. Drug injection Corticosterone (1 mg/100 g BW) was dissolved in ethanol then resolvated (suspended) in saline and mixed vigorously just before the injection. Dexamethasone (0.1 mg/100 g BW) was dissolved in ethanol then resolvated in saline. The drugs and saline were injected intraperitoneally, at the morning (08:30–09:30) and at night (20:30–21:30), once in a 24-h period, respectively. Each group of six animals was treated eight times as follows: none for the control (no injection) or injection of saline and the drug
(corticosterone and dexamethasone) during the light and dark periods. The treatment was given on a 3-day interval. The sequence of treatments with no injection and the drugs was randomized. For the control group, the average measurements in the morning and at night were defined as 100% in each rat. On the basis of this value, the number of wheel turns was expressed as percent values at the treatment. 2.5. Slice culture and measurement of AVP SCN slice culture was performed according to our previous method [10]. Briefly, rats maintained under the same conditions as described above were sacrificed at 22 to 30 days old. The brains were rapidly removed and cut coronally into 500-Am slices, using a tissue cutter (Brain Matrix, Activational Systems, USA), and the SCN regions were freed of tissue from other areas under a stereomicroscope. Two independent series of preparations, each consists of five wells of SCN culture, were analyzed. 2.5.1. Slice culture The culture medium consisted of Hank’s balanced solution (40%, Invitrogen, USA), DMEM (50%, Invitrogen, Japan) and N2 supplement medium (10%, Invitrogen, USA) with sodiumbicarbonate and glucose. AVP release from isolated SCN slice was recorded after the corticosterone and dexamethasone administration at a final concentrations of 0.2 and 2 Ag/ml, respectively. The drugs were applied directly onto each of the paired SCN slice cultures in drops of 10 Al for 30 min. The drug application and sampling procedures were performed between 10:00–11:00 h. The medium was aspirated, and fresh medium was added with or without drugs after 0 and 30 min. The aspirated medium was collected in sample tubes filled with 50 Al of 10 N acetic acid containing 0.2 N HCl. Aspirated media were heated at 100 8C for 10 min, then chilled on ice and centrifuged at 10,000 g for 10 min. Aliquots of the supernatant were lyophilized and stored at 80 8C until enzyme immunoassay (EIA). 2.5.2. EIA The concentrations of AVP in the medium were measured by the double antibody solid-phase method, as described previously [10]. Briefly, the lyophilized samples were reconstituted in 110 Al of assay buffer. One hundred microliters of assay buffer, 50 Al of sample or standard and the first antibody to AVP (1:2500, Peptide Inst., Japan) were added to each well of the microplates coated with the second antibody (1:800, Shibayagi, Japan) and incubated overnight at 4 8C. Fifty microliters of HRP–AVP conjugate prepared by the peroxidase oxidation method was added to the microplate and incubated for an additional 3 h. Each well was then washed and incubated with 250 Al of the substrate (OPD) at room temperature for 30 min. The reaction was terminated by the addition of 50 Al of 0.25 M H2SO4 to each
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well. The optical absorbance of the resultant reaction products was then measured at 492 nm. The sensitivity of the present EIA system for AVP was 10 fmol/well. Two independent series of samples were collected for each drug analyzed. Values were calculated as meansFSEM of 10 wells (5 wells2) from two series. Control levels of AVP release before the drug application ranged from 0.98F0.29 to 1.52F032 pg/ml depending on the SCN culture well. The changes of AVP release were expressed as the percentage against the control levels. 2.6. AVP mRNA measurement after the corticosterone and dexamethasone injection Corticosterone or dexamethasone was injected during light (10:00) and dark (22:00) periods for doses of 1 and 0.1 mg/100 g BW, respectively. Corticosterone was dissolved in ethanol then resolvated (suspended) in saline and mixed vigorously just before injection. 2.6.1. mRNA preparation and RT–PCR Animals were killed by decapitation at time 0, 30 and 180 min after the corticosterone or dexamethasone injection. The number of animals was three rats at each time point. The brain was quickly isolated, semifrozen and cut into coronal sections with a brain slicer (Activational Systems) with a thickness of 700 Am. A small tissue block containing the bilateral SCNs were punched out using a 20-gauge needle. Total RNA was isolated from each tissue using TRIzol (Invitrogen, USA) [12]. 2.6.2. RT–PCR Relative mRNA levels in the tissue blocks were quantified using a quantitative RT–PCR analysis, as described previously [12]. Total RNA (0.2 Ag) after DNase treatment was reverse transcribed into cDNAs, using oligo(dT)-primer (Thermoscript, Invitrogen). In the rAVP cDNA (Gene bank accession no. M25646) amplification, the forward primer 5V-CGCAGTGCCCACCTATGCTCGCCA (7–31) and reverse primer 5V-TCGGCCACGCAGCTCTCATCGCTG (371–347) were used for 32 cycles of 94 8C for 1 min, 60 8C for 1 min and 72 8C for 1 min. The 365-bp fragment was amplified [19]. For the controls and normalizing the quantities of each tube, rath-actin mRNA was adopted. Theh-actin cDNA forward primer 5V-TTGTAACCAACTGGGACGATATGG and reverse primer, 5VGATCTTGATCTTCATGGTGCTAGG were used for 28 cycles of 94 8C for 1 min, 60 8C for 1 min and 72 8C for 1 min. The 765-bp fragment was amplified (Clontech, #55061, USA). The conditions were set to produce liner relation between the amounts of template cDNA and the amplified products. 2.6.3. Electrophoresis The PCR products of rAVP cDNAs with rh-actin cDNA were subjected to 2% NuSieve agarose (FMC
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Bioproducts, USA) gel electrophoresis, stained with ethidium bromide and then detected with an image analyzer; Multi-Imager Fluor-S (BioRad, USA). The amounts of the products with the predicted size were quantified using Multi-Analyst (BioRad). The maximum intensity in each electrophoresis throughout the light and dark values was designated as 100%. The relative density of each Et-Br staining from the imager at each time was normalized against the density of the h-actin cDNA product. Group analyzed data were expressed against the percentage at time 0 (no injection of drug). 2.7. Statistics To analyze the differences in the wheel running activity and AVP peptide levels, the Mann-Whitney U test was used. Value of pb0.05 was taken to indicate significance. To analyze the differences in the mean values of OD in the RT– PCR, the results at time 0 were adjusted to 100%. The effects of the drugs at each time point were compared with value at time 0.
3. Results 3.1. Corticosterone and dexamethasone on wheel running activity The injection of corticosterone (1 mg/100 g BW) attenuated the wheel activity in the morning (Fig. 1). A similar tendency of a suppressing effect of corticosterone was observed at the darkness but was not significantly
Fig. 1. The effects of corticosterone (1 mg/100 g BW) and dexamethasone (0.1 mg/100 g BW) injections on wheel running activities. Each rectangle with vertical bar represents the meanFS.E.M. of six animals. Values are expressed as a percentage of the turns in control animals (none). The 100% level indicates the average counts in the morning and at night in the control group. Empty and dark columns represent the responses in the morning (08:30–09:30) and at night (20:30–21:30), respectively. None—no injection. #Significant differences between control animals and those injected with drugs, analyzed by Mann-Whitney U test. *Significantly small counts compared with rats injected with dexamethasone.
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different from controls ( pb0.07). The injection of dexamethasone (0.1 mg/100 g BW) enhanced the wheel activity at the dark period while dexamethasone did not show significant changes in the number of wheel turns during the light period. The effects of corticosterone and dexamethasone on wheel activity (responses to the wheel) showed reversed responses at night ( pb0.01). Both in none and saline-injected group showed a higher responses (wheel turn) at the dark period than at light but was not significant. 3.2. AVP peptide release from SCN slice culture after the corticosterone and dexamethasone applications in vitro After the corticosterone was applied to the SCN culture medium, AVP peptide release was decreased at the dose of 2 Ag/ml (for a final concentration). Similar results were observed at a lower dose (0.2 Ag/ml), but it was not significantly different from controls (Fig. 2). Through the dexamethasone administration (0.2 and 2 Ag/ml), the AVP release was significantly increased with both concentrations. The effects of corticosterone and dexamethasone on AVP release were reciprocal. 3.3. AVP mRNA changes after the corticosterone and dexamethasone injection in vivo Typical examples of electrophoresed band of AVP cDNA of PCR products and grouped analyzed data are shown in Fig. 3A and B, respectively. AVP coding mRNA indexed by AVP cDNA from RT–PCR products was decreased both by the corticosterone and dexamethasone injections (Fig. 3). During the light period, AVP mRNA decreased at 30 and 60 min after the corticosterone injection. With dexamethasone administration, AVP mRNA decreased at 60 min after the
Fig. 3. AVP mRNA decrease after corticosterone and dexamethasone injection. (A) Band intensity of PCR products of cDNA of AVP. Comp. B—corticosterone (1 mg/100 g BW), Dex—dexamethasone (0.1 mg/100 g BW). (B) At the arrowhead, corticosterone or dexamethasone was injected at 10:00 during the light period and 22:00 during the dark period, in each. Values were collected from three independent RT–PCR studies of three animals at each time point, meanFS.E.M. Significant differences compared with the values at time 0, analyzed by t test. **pb0.01 and ***pb0.005. There was no significant difference in the no-injection groups (control) at 3 h later compared with the values at time 0 in both the light and dark period, indicated by black circle with the standard error. #Significantly lower compared with that at time 0 during the light period.
injection. During dark period, AVP mRNA significantly decreased from 30 to 60 min after both corticosterone and dexamethasone injection. The AVP mRNA levels during darkness at time 0 (ZT 20) was significantly lower compared with those during the light period (time 0, ZT 10).
4. Discussion
Fig. 2. AVP release from the suprachiasmatic nucleus slice culture. Corticosterone (Cort) or dexamethasone (Dex) was applied in each culture well at a final concentration to 0.2 or 2 Ag/ml. After 30 min, the AVP concentration in the medium was measured. Each rectangle with small vertical bars represents the meansFS.E.M. from 10 slice culture dishes. Values were obtained from the two independent experiments. Significant differences compared with the values of saline, analyzed by Mann-Whitney U test. *pb0.05, **pb0.01 and ***pb0.005.
After the corticosterone injection, wheel activity was significantly suppressed during the light period and showed same tendency to be suppressed during the dark period (Fig. 1). The suppression of wheel activity by the corticosterone might be thought to be curious because stress-induced wheel running activity is well correlated with plasma corticosterone levels [6], and behavioral sensitivity is augmented by corticosterone [28]. In contrast, the dexamethasone on wheel activity was different by the time of day in the present study (Fig. 3). Dexamethasone facilitated the activity at night, although no obvious changes were shown during the light period. The reason for the differences in the effects of corticosterone and dexamethasone (synthetic strong corticoid) is not clear. There is little evidence of functional
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differences between corticosterone and dexamethasone. The dose responses of glucocorticoid on the behavioral reactivity toward an object in a novel environment showed U-shape; this phenomenon is hypothesized to be due to the balance of receptor occupation by mineralocorticoid receptors (MR, Type I) and glucocorticoid receptors (GR, Type II; in review [4]). In addition to the glucocorticoid function, neurotransmitter-mediated locomotor activity changes have been reported. The injection of dexamethasone increases central dopamine activity [25], while corticosterone depletion by adrenalectomy reduces it [30]. The site of action in the brain area was different in corticosterone with dexamethasone on adrenal-ectomized animals [17]. The reciprocal responses in AVP release against corticosterone (decrease) and dexamethasone (increase) were found using a SCN slice culture (Fig. 2). The results might also be explained due to the U-shape glucocorticoid responses [4]. However, the rapid inhibitory effect of corticosterone on AVP release from the hypothalamus slice culture was strongly suggested to be mediated by membrane-associated receptor and not through the genomic (classical) responses [20]. The contents of AVP coding mRNA in the SCN decreased after the injection of both corticosterone and dexamethasone (Fig. 3). These results suggest that both steroids down-regulate the transcription of AVP mRNA. These findings indicate that corticosterone and dexamethasone are acting at the transcription level in addition to functioning at the membrane receptor level. There is increasing evidence that glucocorticoids act inside the nucleus at the gene transcription level. The effects of glucocorticoids on SCN gene expression are likely to be mediated via glucocorticoid receptors because the effects were also observed with the selective synthetic glucocorticoid dexamethasone [18,21]. After injection of corticosterone in adrenal-ectomized rats, AVP mRNA levels in the parvicellular PVN remained elevated [17,21]. Actually, the AVP gene promoter contains a glucocorticoid responsible element (GRE); the dexamethasone inhibited AVP promoter activity [13]. The partial deletions of 5V flanking region of the AVP gene increased the transcription indexed by luciferase activity [13,15]. These previous results and the present findings strongly suggest that the altered pattern of AVP gene expression (mRNA changes) in the SCN is mediated by transcription level rather than by alteration of overall metabolic state by glucocorticoid treatment. It is easy to understand that corticosterone depresses the AVP mRNA transcription accompanying the decrease in AVP release. However, it is intriguing that the decease in AVP mRNA by dexamethasone followed the increase in AVP release (Fig. 2). This discrepancy might possibly be explained, as the posttranscription levels for (i) special AVP releasing mechanisms and (ii) its dependence on the time of day, as follows. For the special AVP releasing mechanisms, (i) neurosecretory vesicles and/or the transport system might be differentially functioning in corticosterone and dexame-
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thasone in the AVP containing neurons at cytosole levels [23]. The AVP release might be altered by unclarified mechanisms. For possibility, (ii) AVP in the SCN plays inhibitory and facilitatory effects on the release of corticosterone depending on the time of day [14]. The effects of dexamethasone on the AVP contents in the SCN and circulating corticosterone were different by the administration times [9]. Dexamethasone when injected early during the light period decreased the AVP levels, while the corticosterone level was elevated at 1 and 3 h after injection. Conversely, following injection early during the dark period, dexamethasone increased the AVP levels accompanying the corticosterone rise [9]. It is interesting that the functional differences in the AVP release evoked by corticosterone and dexamethasone that developed within 30 min and were not mediated by nuclear transcription factor activation were reciprocal and were similar to the results of wheel running activity [9,20,21]. However, the AVP release was measured at a fixed time of day between 10:00–11:00 (subjective daytime). The almost reciprocal AVP release is only considered to be caused by the differences in the glucocorticoids. It was also reported that acute dexamethasone injection did not prevent induction of neuropeptide or transcription factor responses to ether stress [16]. It is suggested that the functional differences of corticosterone and dexamethasone on AVP release should be clarified in relation to the receptor and/or the transcription and translation mechanisms. To clarify the mechanisms of glucocorticoid function from the locomotor activity, AVP release and to their molecular levels in relation to the psychological incidence to motivate the running wheel activity also require clarification. As another possibility for the differences to AVP release and motor function by corticosterone and dexamethasone, dopaminergic and serotonergic systems would be considered. The physiologically secreted corticosterone facilitates dopamine-mediated locomotor enhancement [22]. As mentioned in the introduction, the serotonergic (5-HT) system is well coupled with the adrenocortical functions. The 5-HT terminals in the suprachiasmatic nucleus are responsible for the circadian variations in corticosterone secretion [31]. As reported previously, the locomotor suppressive effects of corticosterone were increased by buspirone (5-HT1a agonist) [7]. Also, the AVP affects the locomotor activity via the dopaminergic and serotonergic systems (in review [2]). The AVP release is suggested to be modified by dopamine and serotonin. However, the AVP release was measured in SCN slice culture in the present study, the changes in the AVP release were a direct function of corticosterone and dexamethasone. The AVP release from the SCN stimulated by dexamethasone is a physiological range in doses (b20 pmol/CSF, estimated from the results of Fig. 2). However, the intrinsic nature might not be the same in the locomotor activity, barrel rotation and wheel activity were reduced by the higher dose of AVP and were increased by the lower dose
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(Isobe et al., unpublished data, [1,3]). The lower dose of AVP corresponds to the present results of AVP release (Fig. 2) in dose. Therefore, it might be suggested that the effects of attenuation by corticosterone and promotion by dexamethasone on running wheel activity might depend on the decrease and increase of AVP release, respectively, at least in part. In conclusion, the present results showed that the wheel running activity was suppressed by corticosterone and was facilitated by dexamethasone. We measured the AVP peptide release from SCN slice cultures and AVP mRNA in the SCN. The AVP mRNA was decreased both by the corticosterone and dexamethasone; however, AVP release was decreased by corticosterone and dexamethasone.
Acknowledgement This work was supported by MEXT in Japan, Grant-inAid for Scientific Research (C) No. 10670060.
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Research report
Dexamethasone induces different wheel running activity than corticosterone through vasopressin release from the suprachiasmatic nucleus Yoshiaki Isobe*, Takako Torii, Takatsune Kawaguchi, Hitoo Nishino Department of Neuro-physiology and Brain Sciences, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan Accepted 21 September 2004
Abstract During the analysis of wheel running activity, we found that corticosterone (1 mg/100 g BW) injection decreased wheel activity, while dexamethasone (0.1 mg/100 g) increased the activity. To clarify the functional differences between corticosterone and dexamethasone, we measured Arg-vasopressin (AVP) release from the suprachiasmatic nucleus (SCN) slice culture in vitro and AVP coding mRNA in the SCN in vivo. The corticosterone (0.2 and 2 Ag/ml, final concentration in medium) decreased the AVP release, while it increased by dexamethasone (0.2 and 2 Ag/ml). An AVP mRNA in the SCN was decreased by both corticosterone (1 mg/100 g) and dexamethasone (0.1 mg/100 g). The differences in wheel activity by corticosterone and dexamethasone are discussed from the changes of AVP in the SCN. D 2004 Elsevier B.V. All rights reserved. Theme: Neuronal basis of behavior Topic: Biological rhythms and sleep Keywords: Corticosterone; Dexamethasone; Wheel activity; Arg-vasopressin; Suprachiasmatic nucleus
1. Introduction Wheel running activity is widely used in analyzing the mechanisms of drug effects, defects in motor functions in the CNS and circadian rhythm. An animal’s motivation to start, continue and stop wheel running activity is not yet clear; neurotransmitters, neuropeptides and many factors in the CNS should be involved. If the nature of the running wheel activity is elucidated, it will be an advantage to help clarify the mechanisms of circadian rhythm, stress and motor functions. Stress-induced behavioral sensitivity is augmented by corticosterone and dexamethasone [26,28]. Stressed animals allowed to run showed lower corticosterone levels than those not allowed to run [27]. In blinded
* Corresponding author. Tel.: +81 52 853 8136; fax: +81 52 843 3069. E-mail address: [email protected] (Y. Isobe). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.09.021
rats, the corticosterone levels were high at the beginning of the running phase and low at its end [6]. From the circadian rhythm point of view, it was reported that rodents allowed access to wheel or not affected the phase shift in the circadian rhythm [24,29]. Higher doses of dexamethasone phase-shift the body temperature rhythm [8]; however, progress in this field of study is difficult to define. With respect to stress, there is some evidence that Argvasopressin (AVP) enhances or reduces the locomotor activity [3,5]. In response to stress, a glucocorticoid negative feedback to AVP and corticotropin-releasing factor (CRF) gene transcription at the paraventricular nucleus (PVN) had been advocated [16]. AVP containing neurons is one of the output paths from the suprachiasmatic nucleus (SCN), which is the center of biological rhythm in mammals. AVP containing neurons in the SCN connects with the PVN. Recently, it was reported that glucocorticoid
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negatively regulated the AVP mRNA transcription at the promoter region in glucocorticoid responsible elements (GRE) of the AVP gene [13,15]. In addition, mesolimbic dopaminergic and serotonergic systems coordinate well with glucocorticoid in modulating the locomotor activity [3,11,22,30,31]. In the present research, we found different responses of corticosterone and dexamethasone in the wheel running activity and AVP release from the SCN slice culture. An action and the feedback mechanism were reported to be different for corticosterone and dexamethasone in their functions [17,20]. Based on the present results, we discussed the effects of corticosterone and dexamethasone on the behavioral effects in relation to the vasopressin functions in the SCN.
2. Materials and method 2.1. Materials Wistar rats (SLC, Shizudokyo, Japan) were housed under a 12-h light/12-h dark cycle (LD, light on at 7:00; light intensity, 200 lux) with free access to food and water. A minimum number of animals were used to perform our strategy. Animal care was in accordance with the guideline of Nagoya City University, Graduate School of Medical Sciences, for use of animals in research. 2.2. Drugs Corticosterone (Sigma, Japan) and dexamethasone (Sigma) were used. Corticosterone and dexamethasone was dissolved in ethanol then resolvated (suspended) in saline. Drug injection methods are shown later. 2.3. Locomotor activity Six rats were used. Running wheel activity was measured as turns using wheel cages with an attached resting room. The length of the wheel circles was 100 cm. The activities were counted for 20 min just after the treatment (saline and drug injection or no injection) using a cumulative event recorder, and data were stored on a computer disk [11]. 2.4. Drug injection Corticosterone (1 mg/100 g BW) was dissolved in ethanol then resolvated (suspended) in saline and mixed vigorously just before the injection. Dexamethasone (0.1 mg/100 g BW) was dissolved in ethanol then resolvated in saline. The drugs and saline were injected intraperitoneally, at the morning (08:30–09:30) and at night (20:30–21:30), once in a 24-h period, respectively. Each group of six animals was treated eight times as follows: none for the control (no injection) or injection of saline and the drug
(corticosterone and dexamethasone) during the light and dark periods. The treatment was given on a 3-day interval. The sequence of treatments with no injection and the drugs was randomized. For the control group, the average measurements in the morning and at night were defined as 100% in each rat. On the basis of this value, the number of wheel turns was expressed as percent values at the treatment. 2.5. Slice culture and measurement of AVP SCN slice culture was performed according to our previous method [10]. Briefly, rats maintained under the same conditions as described above were sacrificed at 22 to 30 days old. The brains were rapidly removed and cut coronally into 500-Am slices, using a tissue cutter (Brain Matrix, Activational Systems, USA), and the SCN regions were freed of tissue from other areas under a stereomicroscope. Two independent series of preparations, each consists of five wells of SCN culture, were analyzed. 2.5.1. Slice culture The culture medium consisted of Hank’s balanced solution (40%, Invitrogen, USA), DMEM (50%, Invitrogen, Japan) and N2 supplement medium (10%, Invitrogen, USA) with sodiumbicarbonate and glucose. AVP release from isolated SCN slice was recorded after the corticosterone and dexamethasone administration at a final concentrations of 0.2 and 2 Ag/ml, respectively. The drugs were applied directly onto each of the paired SCN slice cultures in drops of 10 Al for 30 min. The drug application and sampling procedures were performed between 10:00–11:00 h. The medium was aspirated, and fresh medium was added with or without drugs after 0 and 30 min. The aspirated medium was collected in sample tubes filled with 50 Al of 10 N acetic acid containing 0.2 N HCl. Aspirated media were heated at 100 8C for 10 min, then chilled on ice and centrifuged at 10,000 g for 10 min. Aliquots of the supernatant were lyophilized and stored at 80 8C until enzyme immunoassay (EIA). 2.5.2. EIA The concentrations of AVP in the medium were measured by the double antibody solid-phase method, as described previously [10]. Briefly, the lyophilized samples were reconstituted in 110 Al of assay buffer. One hundred microliters of assay buffer, 50 Al of sample or standard and the first antibody to AVP (1:2500, Peptide Inst., Japan) were added to each well of the microplates coated with the second antibody (1:800, Shibayagi, Japan) and incubated overnight at 4 8C. Fifty microliters of HRP–AVP conjugate prepared by the peroxidase oxidation method was added to the microplate and incubated for an additional 3 h. Each well was then washed and incubated with 250 Al of the substrate (OPD) at room temperature for 30 min. The reaction was terminated by the addition of 50 Al of 0.25 M H2SO4 to each
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well. The optical absorbance of the resultant reaction products was then measured at 492 nm. The sensitivity of the present EIA system for AVP was 10 fmol/well. Two independent series of samples were collected for each drug analyzed. Values were calculated as meansFSEM of 10 wells (5 wells2) from two series. Control levels of AVP release before the drug application ranged from 0.98F0.29 to 1.52F032 pg/ml depending on the SCN culture well. The changes of AVP release were expressed as the percentage against the control levels. 2.6. AVP mRNA measurement after the corticosterone and dexamethasone injection Corticosterone or dexamethasone was injected during light (10:00) and dark (22:00) periods for doses of 1 and 0.1 mg/100 g BW, respectively. Corticosterone was dissolved in ethanol then resolvated (suspended) in saline and mixed vigorously just before injection. 2.6.1. mRNA preparation and RT–PCR Animals were killed by decapitation at time 0, 30 and 180 min after the corticosterone or dexamethasone injection. The number of animals was three rats at each time point. The brain was quickly isolated, semifrozen and cut into coronal sections with a brain slicer (Activational Systems) with a thickness of 700 Am. A small tissue block containing the bilateral SCNs were punched out using a 20-gauge needle. Total RNA was isolated from each tissue using TRIzol (Invitrogen, USA) [12]. 2.6.2. RT–PCR Relative mRNA levels in the tissue blocks were quantified using a quantitative RT–PCR analysis, as described previously [12]. Total RNA (0.2 Ag) after DNase treatment was reverse transcribed into cDNAs, using oligo(dT)-primer (Thermoscript, Invitrogen). In the rAVP cDNA (Gene bank accession no. M25646) amplification, the forward primer 5V-CGCAGTGCCCACCTATGCTCGCCA (7–31) and reverse primer 5V-TCGGCCACGCAGCTCTCATCGCTG (371–347) were used for 32 cycles of 94 8C for 1 min, 60 8C for 1 min and 72 8C for 1 min. The 365-bp fragment was amplified [19]. For the controls and normalizing the quantities of each tube, rath-actin mRNA was adopted. Theh-actin cDNA forward primer 5V-TTGTAACCAACTGGGACGATATGG and reverse primer, 5VGATCTTGATCTTCATGGTGCTAGG were used for 28 cycles of 94 8C for 1 min, 60 8C for 1 min and 72 8C for 1 min. The 765-bp fragment was amplified (Clontech, #55061, USA). The conditions were set to produce liner relation between the amounts of template cDNA and the amplified products. 2.6.3. Electrophoresis The PCR products of rAVP cDNAs with rh-actin cDNA were subjected to 2% NuSieve agarose (FMC
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Bioproducts, USA) gel electrophoresis, stained with ethidium bromide and then detected with an image analyzer; Multi-Imager Fluor-S (BioRad, USA). The amounts of the products with the predicted size were quantified using Multi-Analyst (BioRad). The maximum intensity in each electrophoresis throughout the light and dark values was designated as 100%. The relative density of each Et-Br staining from the imager at each time was normalized against the density of the h-actin cDNA product. Group analyzed data were expressed against the percentage at time 0 (no injection of drug). 2.7. Statistics To analyze the differences in the wheel running activity and AVP peptide levels, the Mann-Whitney U test was used. Value of pb0.05 was taken to indicate significance. To analyze the differences in the mean values of OD in the RT– PCR, the results at time 0 were adjusted to 100%. The effects of the drugs at each time point were compared with value at time 0.
3. Results 3.1. Corticosterone and dexamethasone on wheel running activity The injection of corticosterone (1 mg/100 g BW) attenuated the wheel activity in the morning (Fig. 1). A similar tendency of a suppressing effect of corticosterone was observed at the darkness but was not significantly
Fig. 1. The effects of corticosterone (1 mg/100 g BW) and dexamethasone (0.1 mg/100 g BW) injections on wheel running activities. Each rectangle with vertical bar represents the meanFS.E.M. of six animals. Values are expressed as a percentage of the turns in control animals (none). The 100% level indicates the average counts in the morning and at night in the control group. Empty and dark columns represent the responses in the morning (08:30–09:30) and at night (20:30–21:30), respectively. None—no injection. #Significant differences between control animals and those injected with drugs, analyzed by Mann-Whitney U test. *Significantly small counts compared with rats injected with dexamethasone.
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different from controls ( pb0.07). The injection of dexamethasone (0.1 mg/100 g BW) enhanced the wheel activity at the dark period while dexamethasone did not show significant changes in the number of wheel turns during the light period. The effects of corticosterone and dexamethasone on wheel activity (responses to the wheel) showed reversed responses at night ( pb0.01). Both in none and saline-injected group showed a higher responses (wheel turn) at the dark period than at light but was not significant. 3.2. AVP peptide release from SCN slice culture after the corticosterone and dexamethasone applications in vitro After the corticosterone was applied to the SCN culture medium, AVP peptide release was decreased at the dose of 2 Ag/ml (for a final concentration). Similar results were observed at a lower dose (0.2 Ag/ml), but it was not significantly different from controls (Fig. 2). Through the dexamethasone administration (0.2 and 2 Ag/ml), the AVP release was significantly increased with both concentrations. The effects of corticosterone and dexamethasone on AVP release were reciprocal. 3.3. AVP mRNA changes after the corticosterone and dexamethasone injection in vivo Typical examples of electrophoresed band of AVP cDNA of PCR products and grouped analyzed data are shown in Fig. 3A and B, respectively. AVP coding mRNA indexed by AVP cDNA from RT–PCR products was decreased both by the corticosterone and dexamethasone injections (Fig. 3). During the light period, AVP mRNA decreased at 30 and 60 min after the corticosterone injection. With dexamethasone administration, AVP mRNA decreased at 60 min after the
Fig. 3. AVP mRNA decrease after corticosterone and dexamethasone injection. (A) Band intensity of PCR products of cDNA of AVP. Comp. B—corticosterone (1 mg/100 g BW), Dex—dexamethasone (0.1 mg/100 g BW). (B) At the arrowhead, corticosterone or dexamethasone was injected at 10:00 during the light period and 22:00 during the dark period, in each. Values were collected from three independent RT–PCR studies of three animals at each time point, meanFS.E.M. Significant differences compared with the values at time 0, analyzed by t test. **pb0.01 and ***pb0.005. There was no significant difference in the no-injection groups (control) at 3 h later compared with the values at time 0 in both the light and dark period, indicated by black circle with the standard error. #Significantly lower compared with that at time 0 during the light period.
injection. During dark period, AVP mRNA significantly decreased from 30 to 60 min after both corticosterone and dexamethasone injection. The AVP mRNA levels during darkness at time 0 (ZT 20) was significantly lower compared with those during the light period (time 0, ZT 10).
4. Discussion
Fig. 2. AVP release from the suprachiasmatic nucleus slice culture. Corticosterone (Cort) or dexamethasone (Dex) was applied in each culture well at a final concentration to 0.2 or 2 Ag/ml. After 30 min, the AVP concentration in the medium was measured. Each rectangle with small vertical bars represents the meansFS.E.M. from 10 slice culture dishes. Values were obtained from the two independent experiments. Significant differences compared with the values of saline, analyzed by Mann-Whitney U test. *pb0.05, **pb0.01 and ***pb0.005.
After the corticosterone injection, wheel activity was significantly suppressed during the light period and showed same tendency to be suppressed during the dark period (Fig. 1). The suppression of wheel activity by the corticosterone might be thought to be curious because stress-induced wheel running activity is well correlated with plasma corticosterone levels [6], and behavioral sensitivity is augmented by corticosterone [28]. In contrast, the dexamethasone on wheel activity was different by the time of day in the present study (Fig. 3). Dexamethasone facilitated the activity at night, although no obvious changes were shown during the light period. The reason for the differences in the effects of corticosterone and dexamethasone (synthetic strong corticoid) is not clear. There is little evidence of functional
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differences between corticosterone and dexamethasone. The dose responses of glucocorticoid on the behavioral reactivity toward an object in a novel environment showed U-shape; this phenomenon is hypothesized to be due to the balance of receptor occupation by mineralocorticoid receptors (MR, Type I) and glucocorticoid receptors (GR, Type II; in review [4]). In addition to the glucocorticoid function, neurotransmitter-mediated locomotor activity changes have been reported. The injection of dexamethasone increases central dopamine activity [25], while corticosterone depletion by adrenalectomy reduces it [30]. The site of action in the brain area was different in corticosterone with dexamethasone on adrenal-ectomized animals [17]. The reciprocal responses in AVP release against corticosterone (decrease) and dexamethasone (increase) were found using a SCN slice culture (Fig. 2). The results might also be explained due to the U-shape glucocorticoid responses [4]. However, the rapid inhibitory effect of corticosterone on AVP release from the hypothalamus slice culture was strongly suggested to be mediated by membrane-associated receptor and not through the genomic (classical) responses [20]. The contents of AVP coding mRNA in the SCN decreased after the injection of both corticosterone and dexamethasone (Fig. 3). These results suggest that both steroids down-regulate the transcription of AVP mRNA. These findings indicate that corticosterone and dexamethasone are acting at the transcription level in addition to functioning at the membrane receptor level. There is increasing evidence that glucocorticoids act inside the nucleus at the gene transcription level. The effects of glucocorticoids on SCN gene expression are likely to be mediated via glucocorticoid receptors because the effects were also observed with the selective synthetic glucocorticoid dexamethasone [18,21]. After injection of corticosterone in adrenal-ectomized rats, AVP mRNA levels in the parvicellular PVN remained elevated [17,21]. Actually, the AVP gene promoter contains a glucocorticoid responsible element (GRE); the dexamethasone inhibited AVP promoter activity [13]. The partial deletions of 5V flanking region of the AVP gene increased the transcription indexed by luciferase activity [13,15]. These previous results and the present findings strongly suggest that the altered pattern of AVP gene expression (mRNA changes) in the SCN is mediated by transcription level rather than by alteration of overall metabolic state by glucocorticoid treatment. It is easy to understand that corticosterone depresses the AVP mRNA transcription accompanying the decrease in AVP release. However, it is intriguing that the decease in AVP mRNA by dexamethasone followed the increase in AVP release (Fig. 2). This discrepancy might possibly be explained, as the posttranscription levels for (i) special AVP releasing mechanisms and (ii) its dependence on the time of day, as follows. For the special AVP releasing mechanisms, (i) neurosecretory vesicles and/or the transport system might be differentially functioning in corticosterone and dexame-
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thasone in the AVP containing neurons at cytosole levels [23]. The AVP release might be altered by unclarified mechanisms. For possibility, (ii) AVP in the SCN plays inhibitory and facilitatory effects on the release of corticosterone depending on the time of day [14]. The effects of dexamethasone on the AVP contents in the SCN and circulating corticosterone were different by the administration times [9]. Dexamethasone when injected early during the light period decreased the AVP levels, while the corticosterone level was elevated at 1 and 3 h after injection. Conversely, following injection early during the dark period, dexamethasone increased the AVP levels accompanying the corticosterone rise [9]. It is interesting that the functional differences in the AVP release evoked by corticosterone and dexamethasone that developed within 30 min and were not mediated by nuclear transcription factor activation were reciprocal and were similar to the results of wheel running activity [9,20,21]. However, the AVP release was measured at a fixed time of day between 10:00–11:00 (subjective daytime). The almost reciprocal AVP release is only considered to be caused by the differences in the glucocorticoids. It was also reported that acute dexamethasone injection did not prevent induction of neuropeptide or transcription factor responses to ether stress [16]. It is suggested that the functional differences of corticosterone and dexamethasone on AVP release should be clarified in relation to the receptor and/or the transcription and translation mechanisms. To clarify the mechanisms of glucocorticoid function from the locomotor activity, AVP release and to their molecular levels in relation to the psychological incidence to motivate the running wheel activity also require clarification. As another possibility for the differences to AVP release and motor function by corticosterone and dexamethasone, dopaminergic and serotonergic systems would be considered. The physiologically secreted corticosterone facilitates dopamine-mediated locomotor enhancement [22]. As mentioned in the introduction, the serotonergic (5-HT) system is well coupled with the adrenocortical functions. The 5-HT terminals in the suprachiasmatic nucleus are responsible for the circadian variations in corticosterone secretion [31]. As reported previously, the locomotor suppressive effects of corticosterone were increased by buspirone (5-HT1a agonist) [7]. Also, the AVP affects the locomotor activity via the dopaminergic and serotonergic systems (in review [2]). The AVP release is suggested to be modified by dopamine and serotonin. However, the AVP release was measured in SCN slice culture in the present study, the changes in the AVP release were a direct function of corticosterone and dexamethasone. The AVP release from the SCN stimulated by dexamethasone is a physiological range in doses (b20 pmol/CSF, estimated from the results of Fig. 2). However, the intrinsic nature might not be the same in the locomotor activity, barrel rotation and wheel activity were reduced by the higher dose of AVP and were increased by the lower dose
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(Isobe et al., unpublished data, [1,3]). The lower dose of AVP corresponds to the present results of AVP release (Fig. 2) in dose. Therefore, it might be suggested that the effects of attenuation by corticosterone and promotion by dexamethasone on running wheel activity might depend on the decrease and increase of AVP release, respectively, at least in part. In conclusion, the present results showed that the wheel running activity was suppressed by corticosterone and was facilitated by dexamethasone. We measured the AVP peptide release from SCN slice cultures and AVP mRNA in the SCN. The AVP mRNA was decreased both by the corticosterone and dexamethasone; however, AVP release was decreased by corticosterone and dexamethasone.
Acknowledgement This work was supported by MEXT in Japan, Grant-inAid for Scientific Research (C) No. 10670060.
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