Circadian rhythm of melatonin release in pineal gland culture: arg-vasopressin inhibits melatonin release

Brain Research 918 (2001) 67–73 www.elsevier.com / locate / bres

Research report

Circadian rhythm of melatonin release in pineal gland culture: arg-vasopressin inhibits melatonin release Yoshiaki Isobe*, Junko Fujioi, Hitoo Nishino Nagoya City University Medical School, Nagoya 467 -8601, Japan Accepted 6 August 2001

Abstract The mammalian pineal gland is known to receive a noradrenergic sympathetic efferent signal from the suprachiasmatic nucleus (SCN) via the superior cervical ganglion. Arg-vasopressin (AVP) containing neurons in the SCN is one of the output paths of circadian information to the other brain areas. AVP release from the SCN is suppressed by melatonin. In turn, we determined the direct effect of AVP on melatonin release using pineal gland explant culture. AVP (1 mM) suppressed melatonin release. Noradrenaline stimulated melatonin release was attenuated by AVP. In turn, the expression of the melatonin synthesis enzyme arylalkylamine N-acetyltransferase mRNA in the rat SCN was reported. We measured melatonin content in the SCN in rats kept under the light–dark cycle and constant dim light. Melatonin in the SCN was higher during the dark period than that in the light. A similar tendency was also observed in the SCN of animals kept under a constant dim light. It was suggested that the reciprocal regulation of melatonin release and AVP release occurs in the SCN and pineal gland.  2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Circadian rhythm; Explant culture; Pineal gland

1. Introduction Melatonin synthesized and released in a pineal gland is higher during the dark period than during the light period under a light–dark cycle [2,24]. The higher levels of melatonin during the dark period was suppressed by a light pulse [12]. The light signal activates the suprachiasmatic nucleus (SCN), a center of the biological clock, directly through the retinohypothalamic tract [19]. The neurons from the SCN, via the paraventricular nucleus (PVN) of the hypothalamus, project to the intermediolateral column of the spinal cord that innervate the superior cervical ganglion (SCG) [18]. Sympathetic axons from the superior cervical ganglia ascend to the pineal gland [18]. Noradrenaline released from the sympathetic nerve terminals activates the arylalkylamine N-acetyltransferase (AA*Corresponding author. Tel.: 181-52-853-8136; fax: 181-52-8433069. E-mail addresses: [email protected] (Y. Isobe), [email protected] (H. Nishino).

NAT) in the pineal gland and increase the melatonin synthesis following the release [21,25]. Thus, the circadian melatonin production and release are controlled by the SCN via a multisynaptic pathway. The effect of AVP on the synthesis and release of melatonin has been studied little [1,12,21,30]. Some studies reported that AVP potentiated melatonin release during the dark period [1,12]. However, an inhibitory effect of AVP on melatonin synthesis and release was also observed [25,28]. The direct action of AVP on melatonin release is mediated by pinealocyte V1a subtype of vasopressinergic receptors [21,22]. The receptor stimulation induces the phosphoinositide-signaling cascade in parallel with the increase in cAMP [15,21,23,31]. The SCN activities and AVP release are higher during the (subjective) light period [9,16]. AVP containing neurons are one of the output paths of circadian information from the SCN to other brain areas. AVP release from the SCN is suppressed by melatonin [10]. Recently, the expression of the melatonin synthesis enzyme AA-NAT and its coding mRNA in the rat SCN and their rhythmicity

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02936-5

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was reported [7]. The function and role of AA-NAT in the SCN is unknown. In the present study, we examined whether the reciprocal interactions of vasopressin in the SCN with the melatonin release in the pineal gland are functioning, because the major source of AVP in the cerebrospinal fluid (CSF) is depend upon the SCN nerve terminals [24]. Vasopressin containing fibers exist in the pinealocyte and subcommissural organ in the rat [3]. Vasopressin functions at the pineal gland through a V1a receptor to activate the AA-NAT [29,30]. To analyze more precisely the direct actions of AVP on the pineal gland in melatonin release, we adopted the pineal gland organ culture, which conserves the minimal and essential components to produce and release melatonin. In addition, in the present study, we measured melatonin content in the SCN in rats kept under the light–dark cycle and constant dim light.

Before the sampling at 11:00 h, vasopressin, noradrenaline, or both dissolved in culture mediums, was applied in a drop (10 ml) directly onto each of the pineal gland cultures, then left for 30 min. The aspirated medium was collected in sample tubes, then lyophilized and stored at 2808C until the time of RIA.

2. Method

The animals were decapitated at the 6th week of age under the LD cycle and dim light (5 lux) condition. The pineal gland was rapidly dissected and homogenized in PBS (20 mM phosphate buffer containing 140 mM NaCl) and centrifuged. The supernatant was lyophilized and stored at 2808C. On the same animal, SCNs were punched out from the coronal brain slices. The SCNs were homogenized in 0.1 M acetic acid containing 0.01 M HCl

2.1. Materials Wistar rats (SLC, Shizudokyo Co. Ltd., Japan) were housed under a 12-h light / dark cycle (LD, light on at 07:00 h; light intensity, 200 lux) or kept under the constant dim light (5 lux) with free access to food and water. Animal care was in accordance with the guidelines of Nagoya City University Medical School for use of animals in research.

2.2.3. Circadian rhythm of melatonin in medium The pineal glands were prepared, at ZT 3 (10:00 h in JST), from pups born and kept under the LD cycle till postnatal days 7–13. The pineal gland culture medium was sampled at 3-h intervals during the fixed corresponding day on postnatal day 17, at 09:00, 12:00, 15:00, 18:00, 21:00, 03:00, 06:00 h, and again at 09:00 h on the following day (Fig. 1). 2.3. Pineal gland and suprachiasmatic nucleus samplings to measure the melatonin and AVP contents

2.2. Organ culture and measurements of melatonin in the medium or tissue extract 2.2.1. Organ culture Pups born under the LD cycle and nursed under a constant dim light (5 lux) were sacrificed in the morning and at night when they were 7–10 days old. The pineal gland were removed and freed of tissue from other areas under a stereomicroscope. The explant organs were placed on meshed inner-plate (40 mm) and cultured in 1.0 ml of medium in 12-well transfer dishes (Transwell Coaster 3506, USA) in a CO 2 (5%) incubator at 378C [26]. The culture medium consisted of DMEM (Gibco BRL, Japan) with 25 mM D-glucose and 44.05 mM NaHCO 3 (90%), and N 2 supplement medium (Gibco BRL) (10%). DMEM was supplemented with 40 mg / 100 ml of streptomycin GK (Meiji, Japan) and 16 000 U of penicillin G sulfate (Meiji). 2.2.2. Medium samplings and drug application Sampling procedures were initiated 3 days after the pineal gland was isolated and prepared. The medium was aspirated and fresh medium was added. To analyze the effects of vasopressin and noradrenaline with vasopressin on the melatonin release, the medium was changed (sampling) four times at 09:00, 11:00, 13:00 and 19:30 h.

Fig. 1. Circadian rhythms of melatonin release from rat pineal gland explant culture. The pineal gland was prepared at ZT 3. Medium samplings were started 10 days after preparation of the pineal gland culture. Each symbol indicates the individual culture wells. The mean6S.E.M. of melatonin release at each time was expressed as the percentage of the 24-h average of three individual wells and is shown as a column. The average melatonin concentration (eight time-points) throughout 24 h is designated as 100% in each well. Acrophase (w )517.1 h, Percent rhythm50.377, P,0.05.

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and centrifuged. The supernatant was lyophilized and stored at 2808C until used for the melatonin and AVP assay. Circadian time (CT) of free-running rhythm of rats was estimated from the locomotor activity rhythm measured for 10 days under constant dim light. The onset time of the actogram was designated as CT 12. The CT obtained from the actogram standardized the time of day, under the free-running condition [9].

2.4. Melatonin assay The aliquot of the lyophilized extracts of the pineal gland and SCN were reconstructed with 450 ml of assay buffer (tricine buffer; 0.1 M tricine, 0. 9% saline, 0.1% gelatin). Melatonin was measured by the RIA procedure, as reported previously [6]. In brief, 100 ml of each sample or the standard were added to the tube containing 100 ml assay buffer. Then, 100 ml of [ 3 H]melatonin (5000 dpm, Amersham Int. Japan) and anti-melatonin (1:6000, Stockland Ltd., UK) were dispensed. After leaving for 22 h at 48C, 500 ml dextran-coated charcoal (DDC) was added. DDC was prepared by suspending activated charcoal (Sigma, Japan) at 0.5% assay buffer with 0.05% dextran T70 (Pharmashia Fine Chemicals, Japan). After incubation for 15 min, the medium was centrifuged at 3000 rpm for 15 min and the supernatant was decanted into vials containing 8 ml scintillation fluid. Radioactivity was then counted. The RIA was done twice to each sample. The mean recovery rate was 90.1%. Intra- and inter-assay variations were 5.8 and 14.8%, respectively.

2.5. Arg-vasopressin assay The measurements of AVP were performed by EIA twice for each material. In the measurements of the AVP, aliquots of lyophilized SCN extracts were dissolved in assay buffer of EIA. The contents of immunoreactive AVP were measured by a double antibody solid-phase method [9]. Synthetic AVP were purchased from the Peptide Research Foundation (Japan). The first antibody of AVP were purchased from ICN (USA).

2.6. Statistics To evaluate the effects of drugs on the melatonin release, the melatonin concentration was expressed as the percentage before the drug application. The significance of the differences was analyzed by ANOVA. All values were expressed as the mean6S.E.M. To determine significant differences between values from the light and dark periods, a Student’s t-test was performed. Rhythmicity of melatonin release was calculated by least-square spectrum analysis [9,20].

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3. Results Spontaneous release of melatonin from the pineal gland to culture medium was analyzed throughout 24 h on the same pineal gland. Typical examples of the circadian melatonin release are shown in Fig. 1. Medium samplings were started 10 days after preparation of the pineal gland culture. Pineal gland culture was prepared at ZT 3 (10:00 h in JST). Circadian rhythms were obvious in each well. The peak time presented during the light period (subjective day; that was out of phase with that in vivo), indicated that the circadian melatonin releasing rhythm was free-running. The statistically analyzed rhythm index of the mean values of three wells was: Mesor (mean)518.27 pg / ml, Amp (amplitude)55.8, Acrophase (peak time) w 517.1 h (o’clock) and Percent rhythm50.377, P,0.05. In a separate series of melatonin releasing rhythm experiments using another pineal gland cultures, medium samplings were started 15 days after the preparation. The preparation time was the same at ZT 3. The peak time of the melatonin rhythm presented at 20.5 h (o’clock) (data not shown). The suppression of melatonin release by vasopressin is shown in Fig. 2. The melatonin concentration varied wellby-well depending on the individual pineal conditions [2]. The melatonin concentration was normalized and expressed as the percentage before the vasopressin application at 09:00 h. The repeated medium exchanges did not induce a fluctuation in the melatonin concentration (Fig. 2A). Although, the sampling time was the same, the spontaneous release of melatonin in controls (no drugs) were at different times of the day (Fig. 2A and B). Compared with the melatonin levels at 09:00 and 11:00 h, there were no significant differences, as shown in Fig. 2A (top), while the spontaneous melatonin release was elevated at 11:00 h (145% increase, P,0.01) and at 13:00 h (82% increase, P,0.05) in Fig. 2B (bottom). The disagreements in the melatonin concentrations in the spontaneous releasing levels were caused by the temporal culture conditions, because the medium samplings were begun on day 3 in A and day 10 in B from the start of cultures. These findings indicated that the spontaneous release of melatonin in organ culture show circadian variations, as shown in Fig. 1. After the vasopressin administration, the melatonin release was reduced to 45625% (P,0.01, compared with the value at 09:00 h) in Fig. 1A. The circadian morning increase of melatonin release was suppressed to 105625% (P,0.01, compared with the value in control at 11:00 h, while no differences with that before the vasopressin apply at 09:00 h) in Fig. 2B. The effects of vasopressin did not continued until the following sampling at 13:00 h. The inhibitory effects of vasopressin was dose-dependent, because with vasopressin doses at 0.1, 1 and 10 mM, the melatonin release was suppressed compared with before vasopressin administration by 15631.9, 34632.4 and 35624%, respectively. The stimulated release of melatonin by noradrenaline

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Fig. 3. Noradrenaline stimulated release of melatonin was suppressed by vasopressin. The melatonin concentration is expressed as the percentage before the drug application (100% at 09:00 h). Noradrenaline (1 mM, open circle) or vasopressin (1 mM, closed circle) with noradrenaline (1 mM) was applied for 30 min as indicated by the horizontal dark rectangular column. [[ Significant difference compared with the control value of noradrenaline alone. ** P,0.01 and * P,0.05; Significant differences compared with before the noradrenaline application (n56).

Fig. 2. Melatonin release was suppressed by vasopressin. The melatonin concentration was expressed as the percentage before the vasopressin application (100% at 09:00 h). On the 3rd (A) and 10th day (B, different culture dish with A) after the beginning of the pineal organ culture medium samplings were started. Vasopressin (1 mM) was applied for 30 min as indicated by the horizontal dark rectangular column. [,[[ Significant suppression (closed circle) compared with control value (open circle). ** P,0.01 and * P,0.05, Significant differences compared with before the drug application (n56).

was attenuated by vasopressin (Fig. 3). Noradrenaline (1 mM) alone or vasopressin (1 mM) with noradrenaline (1 mM) was applied for 30 min before the medium sampling at 11:00 h. Noradrenaline stimulated melatonin release to 252670% at 11:00 h and 180640% at 13:00 h (2 h later after the normal medium). While, after the simultaneously administration of noradrenaline with vasopressin, the stimulated release of melatonin was attenuated. The melatonin release was significantly suppressed by 122665% (from 252670 to 130673%, P,0.01), compared with the noradrenaline alone at 09:00 h. The day–night differences in vasopressin content in the SCN, and melatonin content in the SCN and in the pineal gland which were measured in the same rat are shown in Fig. 4A. Not only the light–dark entrained condition, but also under free-running conditions in the constant dim light, the AVP content in the SCN was higher during the light period or subjective day than during the dark period or subjective night. Melatonin concentrations both in the SCN and the pineal gland was higher during the (subjective) night than during (subjective) day, while the signifi-

cant differences were found only in the pineal grand. The immunogenical cross reactivity with vasopressin was not found in melatonin assay (RIA). Individual relationships between the AVP concentration in the SCN with melatonin in the SCN and melatonin in the pineal gland are shown in Fig. 5. Original findings were the same as shown in Fig. 4. Correlation coefficients at ZT 3 of the AVP content with melatonin in the SCN and the AVP content with melatonin in the pineal gland were 20.172 and 0.070, respectively. The correlation coefficients at ZT 15 of the AVP content with melatonin in the SCN and the AVP content with melatonin in the pineal gland were 0.307 and 20.070, respectively. Although, these correlation coefficients were not significant, a tendency towards a negative correlation was observed (Fig. 5).

4. Discussion In the circadian melatonin releasing rhythm, the peak time (acrophase) was different depending on the culture well (Fig. 1). This indicates that the melatonin released in the rat pineal gland organ culture showed a free-running circadian rhythm. The melatonin releasing rhythm was free-running when liberated from the in situ environment of the brain. From the shifts of the peak time of the circadian melatonin rhythm, the free-running period was calculated to 25.53 h [2411.53 h; the peak time of melatonin rhythm at post natal day 7 is expected to 1.00 h, after 10 days in culture, the peak shifts to 16.3 h; (16.32 1.0 h) / 1051.53 h]. In general, a pineal organ culture in mammals could be

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Fig. 5. Individual relationships between the AVP concentration in the SCN with melatonin in the SCN and melatonin in the pineal gland. Rats were sacrificed at ZT 3 or ZT 15, the SCN and pineal gland were removed from the same rat. Original findings were the same data in Fig. 4. Correlation coefficients at ZT 3 of AVP–Mel (SCN) and AVP–Mel (pineal) were 20.172 and 0.070, respectively, and at ZT 15 of AVP–Mel (SCN) and AVP–Mel (pineal) were 0.307 and 20.070, respectively.

Fig. 4. Day (ZT 3) and night (ZT 15) or subjective day (CT 8) and subjective night (CT 20) differences in arg-vasopressin (AVP) content in the SCN (A), and melatonin contents in the SCN and the pineal gland (B). Amounts of vasopressin in the SCN and melatonin in the SCN and the pineal gland were measured in the same rat. The length of the column and attached vertical bar indicates the mean and S.E.M., respectively. ** P,0.01 and P,0.05; significant differences between the (subjective) day and (subjective) night period. [,[[ Significant large content compared with ZT3 and CT8, respectively. The numbers of rats were 12 kept in LD entrained group and six animals kept in constant dim light. ZT 12 is designated as light off under the LD cycle. CT 12 is designated as locomotor activity onset which was estimated from the actogram in each rat (see Method).

difficult to perform, because it is unclear whether the pineal gland in rat is light sensitive or insensitive. However, it was reported that pinealocytes are not light sensitive, and no rod-opsin immunoreactivity is found in the pineal organs of adult albino rats [5,13]. Several studies analyzed the pineal gland activity in mammals using a pineal gland organ- and dispersed cell-culture system [5,22,26,30,31]. Recently, it was reported that the photosensitivity of cultured pineal gland developed in melatonin synthesis that was prepared from pups of neonatal day 1 [32]. However, in the present study, we prepared the pineal organ culture from pups at post-natal days 7–10. The induction of photosensitivity would not be

expected, as reported previously by Tosini et al. [32] when after exposure to noradrenaline, melatonin release was higher during the dark period than in the light period, even in the pineal explant culture. The melatonin rhythms in the present study, without a supply of noradrenaline, are considered to represent an intrinsic phenomenon, but not a variation against the light. The role of g-aminobutyric acid (GABA) in the PVN for the control of melatonin release was reported; the light induced melatonin suppression was inhibited by the locally applied GABA antagonist (bicuculline) to the PVN [1,12]. They also hypothesized that the AVP released from the SCN to the PVN nerve terminals promoted the release of melatonin during the night time. However, these experiment were carried out in vivo, so the possibility that the other signals from other brain areas were affecting to the control of melatonin release were not excluded [12]. Including the differences in the experimental situations, to explain the discrepancies between previous studies that the AVP promotes melatonin release [1,12] with our present results that the AVP inhibits melatonin release, three explanations could be considered, at least. That the (i) isolated pineal gland shows a different profile with the in vivo condition, (ii) a difference in the source of the AVP, and / or (iii) AVP has dual effect on the melatonin release to promote and inhibit. For the first possibility, for the neuronal connection controlling the release of melatonin, a cholinergic input to pinealocytes was found [27]. The pineal gland functions in mammals are influenced by sympathetic innervation that serves as the major regulatory system [13,27]. In anatomical studies, the neural connections from the SCN to

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the pineal gland to control melatonin release is not clarified other pathways, except for the sympathetic nerves through the PVN [27]. The output signal from the SCG is noradrenaline, not vasopressin. So, the surrounding conditions of the experimental conditions should be similar with those in vivo. The differences in the experimental conditions are not a main reason to explain the discrepancy. The second possibility concerns the source of AVP from the vicinity of the pineal gland; the AVP in the pineal gland except for derived from the hypothalamus was reported [3,14]. Vasopressin-immunoreactive fibers extend via a subcommissural organ or habenular commissure into the pineal stalk and terminate in the anterior part of the pineal gland [3,17]. However, the function of AVP in the vicinity of the pineal gland is not clear, the AVP might also contribute to control the melatonin synthesis and release. For the third possibility, the stimulatory roles of AVP in melatonin release, activation of sympathetic neurons, via the AVP released to the PVN from the SCN, was reported to be the main pathway [1,12]. In the hypothalamus slice culture, bath applied AVP showed excitatory responses on PVN neuron electrical activity [8]. These stimulatory actions of AVP at the PVN might be overridden by the direct inhibitory actions of AVP to the pineal gland in the present study. Intra-arterially administered AVP inhibits the nocturnal melatonin synthesis [28]. The pineal gland is exposed to the CSF at any time in vivo. Both the contents of AVP in the SCN and the concentration in the CSF were higher during the (subjective) light period [9,24]. A source of vasopressin in the CSF relies on the AVP at the AVP nerve terminals from the SCN. If the higher levels of AVP stimulate the melatonin release from the pineal gland, the melatonin levels should be higher during the light period than that under the dark period. Intra-ventricularly injected AVP (1 mg) in rats decreased the serum melatonin levels (unpublished data). The SCN lesions increase the melatonin levels during the daytime, while they decrease during the night [11,12]. The SCN is active during the light period, when AVP is high. These findings that a disinhibition of the SCN output pathway to the PVN caused the increase of melatonin release, indicate that AVP in the SCN shows the inhibitory function against melatonin release during the daytime. The vasopressin V1a receptor and its coding mRNA were found in pineal gland [22,29]. The AVP released from the nerve terminals of the SCN at the PVN, the subventricular zone and the dorsomedial hypothalamus etc. [34] to the CSF might be directly functioning to the pineal gland, and might inhibit the melatonin release through the V1a receptor. The content of immuno-reactive melatonin detected by RIA in the SCN was unexpectedly high (Fig. 4). The contents of melatonin corresponded to one-third of the pineal gland, and 100-fold of the CSF (calculated from Ref. [24]). However, the function of melatonin in the SCN is unknown, although melatonin receptors were recognized

in the SCN [4]. Melatonin inhibits the spontaneous and stimulated release of AVP from the SCN organ- and dispersed cell-culture [9,33]. A tendency towards a negative reciprocal relation were found between the AVP content with melatonin both in the SCN and in the pineal gland melatonin, although it was not significant (Figs. 4 and 5). A reciprocal connection between AVP in the SCN and melatonin in the pineal gland will contribute to coordinate control of the circadian rhythm. Moreover, melatonin in the SCN might regulate activity levels of AVP-containing neurons to establish a circadian clock in a self-regulating manner. In conclusion, melatonin released from the pineal gland showed a free-running rhythm under the pineal gland explant culture. The spontaneous and noradrenaline stimulated release of melatonin was inhibited by bath applied vasopressin. The inhibitory effect of vasopressin on melatonin release might be different from AVP released to the PVN from SCN nerve terminals which promote the release of melatonin [1,12].

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