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Endocrinology Vol. 143, No. 11 4165-4171
Copyright © 2002 by The Endocrine Society

ARTICLE

Neuronal Activity Is Required for the Circadian Rhythm of Vasopressin Gene Transcription in the Suprachiasmatic Nucleus in Vitro

Hiroshi Arima, Shirley B. House, Harold Gainer and Greti Aguilera

Section on Endocrine Physiology (H.A., G.A.), Developmental Endocrinology Brach, National Institute of Child Health and Human Development, and Laboratory of Neurochemistry (H.G.), National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Hiroshi Arima, First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Syowa-ko, Nagoya 466-8550, Japan. E-mail: arima105{at}med.nagoya-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Arginine vasopressin (AVP) is synthesized in and secreted by the suprachiasmatic nucleus (SCN) in a circadian pattern. Transcription of the AVP gene in organotypic cultures of rat SCN was studied by using an intronic in situ hybridization. AVP gene transcription in the cultured SCN maintained a daily rhythm with a peak in the daytime. Inhibition of spontaneous activity by the sodium channel blocker, tetrodotoxin (TTX), dramatically decreased AVP heteronuclear RNA levels and suppressed rhythmicity, indicating that ongoing neural activity was required for the AVP gene transcription. In the presence of TTX, the adenylate cyclase stimulator, forskolin, increased AVP transcription in the SCN. In contrast, the protein kinase C activator, phorbol 12-myristate 13-acetate, greatly increased AVP transcription in the absence of TTX, but this effect was blocked by TTX, indicating that the phorbol 12-myristate 13-acetate acted indirectly via synaptic input. Neither protein kinase A nor protein kinase C pathways appear to be involved in the rhythmicity of AVP transcription in the SCN because selective inhibitors of these protein kinases were without effect. In contrast, the MAPK pathway inhibitor, PD98059, profoundly decreased AVP transcription and abolished its daily rhythm. Hence, a functional MAPK signaling pathway appears to be critical for AVP gene expression in the SCN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SUPRACHIASMATIC NUCLEUS (SCN) of the hypothalamus is the principal pacemaker involved in the generation of circadian rhythms in mammals (1, 2, 3, 4, 5). The nucleus is composed of multiple single-cell circadian oscillators (6, 7, 8, 9, 10), which act in synchrony to generate a coherent rhythmic output. Synaptic transmission and a number of neurotransmitters have been implicated in the synchronization and amplification of circadian signals in the SCN, integration of the photic input, and transmission of the rhythm from the SCN to other regions of the brain (2, 11, 12, 13, 14, 15, 16).

One of the major neuropeptide neurotransmitters in the SCN is arginine vasopressin (AVP). In contrast to the regulation of AVP gene expression in other hypothalamic regions, the gene expression and secretion of AVP in the SCN have an intrinsic circadian rhythm (12, 17, 18, 19, 20). AVP mRNA levels in the SCN have been shown to depend on changes in mRNA stability associated with changes in length of the poly-A tail (20, 21), as well as regulation at the transcriptional level (22). The AVP gene promoter contains an E-box (23), an element responsive to the clock proteins, CLOCK and brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1), which are known to be involved in the generation of circadian rhythms in the SCN (24, 25). The elimination of the circadian patterns in AVP mRNA expression in the SCN in CLOCK gene knockout mice suggests that the E-box enhancer in the AVP gene is involved in the intrinsic rhythmicity of AVP expression in the AVP neuron (23). However, whether the circadian pattern of AVP gene transcription in the SCN is regulated only by the E-box and clock genes within the AVP neuron, or requires additional signals and transcriptional factors remains to be elucidated.

To address this issue whether additional signaling pathways are involved in the mechanism of rhythmic AVP transcription in the SCN, we employed intronic in situ hybridization to measure AVP transcription in organotypic cultures of rat hypothalamic sections. This made it possible to examine changes in the AVP neurons in the SCN under rigorously controlled experimental conditions. Probes directed against intronic sequences allow the detection of nascent AVP gene transcripts (heteronuclear RNA, hnRNA) and provide a sensitive indicator for AVP transcription (26). The results of this study show that AVP transcription in the SCN in long-term organotypic cultures exhibits a circadian rhythm, which is dependent on ongoing electrical activity and synaptic transmission in the cultures, as well as an intact MAPK-dependent pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hypothalamic slice cultures
Hypothalamic slice-explant cultures were performed as described previously (27, 28). In brief, 7-d-old Sprague Dawley rats (Taconic Farms, Inc., Germantown, NY; lights on 0600–1800 h) were killed by decapitation between 0900 and 1200 h, and hypothalamic tissues were sectioned at 300-µm thickness on a Mcllwain tissue chopper. Three coronal slices containing SCN were separated and placed in Gey’s balanced salt solution (Life Technologies, Inc., Grand Island, NY) enriched with glucose (5 mg/ml). Selected sections were trimmed dorsally at the level of anterior commissure and laterally at the level of optic chiasma. Those sections were kept in Gey’s solution until completion of tissue collection, for 30 min to 3 h at 4 C. Explants from individual rat were then placed on 0.4-µm Millicell-CM filter inserts (Millipore Corp., Bedford, MA; pore size 0.4 mm, diameter 30 mm), and each filter insert was placed in a Petri dish (35 mm), containing 1.1 ml of culture medium. All cultures were performed at 35 C in 5% CO2-enriched air, under stationary conditions. All procedures were carried out in accordance with the NIH guidelines on the Care and Use of Animals and an animal study protocol approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee.

Medium and incubation
The standard culture medium was composed of 50% Eagle basal medium (Sigma, St. Louis, MO), 25% heat-inactivated serum (Life Technologies, Inc.), 25% Hanks’ balanced salt solution (Life Technologies, Inc.), 25 U/ml penicillin/streptomycin (Life Technologies, Inc.), 2 mM L-glutamine (Life Technologies, Inc.), and 0.5% glucose. The serum-free medium was composed of Neurobasal A medium (Life Technologies, Inc.) supplemented with 2% B27 (Life Technologies, Inc.), 1 mM sodium pyruvate (Biofluids, Rockville, MD), 2 mM glutamax (Life Technologies, Inc.), 10 mM HEPES (Biofluids), 0.075% sodium bicarbonate (Biofluids), 0.5% glucose, and 100 U/ml penicillin/streptomycin (Life Technologies, Inc.). Cultures were maintained in the standard medium for 11 or 12 d, and the medium was changed to defined serum-free medium for an additional 5 d before subjecting slices to different experimental conditions. The medium was changed three times a week.

Circadian pattern of AVP gene transcription in the SCN
To determine whether AVP gene transcription shows a circadian rhythm in the SCN in the organotypic cultures, slices containing the SCN were fixed with 4% formaldehyde in PBS for 30 min at 0600, 1200, 1800, and 2400 h through 2 d consecutively (d 16 and 17). Slices were then washed twice in PBS, mounted onto polylysine-coated slides, dried at room temperature, and kept at -80 C until processed for in situ hybridization. The role of neural transmission in the generation of circadian variations of AVP expression was studied at 1200 and 2400 h by incubating the sections in the presence and in the absence of the sodium channel blocker, tetrodotoxin (TTX, Sigma), at a concentration of 1 µM for 3 h before fixation. Under these experimental conditions, TTX has been shown to completely block action potentials (29).

Effects of forskolin and phorbolesters on AVP hnRNA expression
To investigate the signal transduction systems involved in the regulation of AVP gene transcription in the SCN, slices were incubated with the adenylyl cyclase stimulator, forskolin (10 µM, Sigma), the protein kinase C (PKC) stimulator, phorbol 12-myristate 13-acetate (PMA, 1 µM, Sigma) or 0.1% dimethylsulfoxide (DMSO) (Sigma) used as vehicle, for 2 h before fixation at 1200 or 2400 h. To determine whether the effects of forskolin and PMA on AVP hnRNA expression were dependent on action potentials, additional groups of sections were studied in presence of TTX, at a concentration of 1 µM. Forskolin, PMA, or DMSO (vehicle control) was added to the cultures after 30-min preincubation with TTX, and slices were incubated for an additional 2 h.

Effects of endogenous protein kinase inhibition on AVP hnRNA expression
To determine the role of endogenous signaling pathways on AVP gene transcription in the SCN, slices were exposed to the protein kinase A (PKA) inhibitor, H89 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA; 10 µM), the PKC inhibitor, calphostin C (BIOMOL, 1 µM), the MAPK inhibitor PD98059 (BIOMOL, 75 µM), or DMSO used as vehicle for 3 h before fixation at 1200 or 2400 h. The doses of inhibitors used here were determined based on previous studies in which similar doses were effective in brain slices (30, 31, 32).

In situ hybridization and quantification
The rat AVP intronic probe (kindly provided by Dr. Thomas Sherman, Georgetown University, Washington, DC) was a 735-bp fragment of intron 1 of the rat AVP gene subcloned into pGEM-3 and linearized by HindIII.

Prehybridization and hybridization procedures were performed as previously described (33). Briefly, before hybridization, fixed sections were thawed at room temperature and acetylated for 10 min at room temperature in 0.25% acetic anhydride in 0.1 M trietanolamine/0.9% NaCl (pH 8.0). Sections were dehydrated and delipidated by sequential transfers through ethanol and chloroform, and air-dried before hybridization. Sections were hybridized at 55 C, overnight, with 2 x 106 cpm 35S-labeled probes, and then nonspecifically bound probes were removed by washing in 50% formamide/250 mM NaCl at 60 C for 15 min, followed by ribonuclease A treatment for 30 min at 37 C and washes in saline sodium citrate. Sections were air-dried and exposed to x-ray films for 3–6 d, and the OD were measured using a computerized system and the public domain NIH Image (developed at the U.S. NIH, and available at http://rsb.info.nih.gov/nih-image).

Analysis and quantification
AVP hnRNA levels were quantified by the measurement of the integrated OD (OD x area) of the SCN in the sections from each rat. Statistical significance of differences between groups was calculated by Student’s t test or one-way ANOVA followed by Fisher’s protected least significant difference (PLSD) test. Differences between groups were considered statistically significant when P values were less than 0.05. Experiments were repeated and results are expressed as the mean ± SEMs of pooled data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Circadian rhythms of AVP gene expression in the SCN
In situ hybridization of hypothalamic slices on d 16 and 17 of culture with AVP intronic probes revealed AVP hnRNA expression in the SCN (Fig. 1Go). Analysis of AVP hnRNA levels in these sections showed a clear circadian rhythm with a peak at 1200 h and a nadir at 2400 h (Fig. 1Go). Levels of AVP hnRNA were significantly higher at 1200 h than at 2400 h (e.g. 100 ± 11 vs. 47 ± 5 arbitrary units, F (3, 76) = 28.47, P < 0.01, in Fig. 2Go, left).



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  		Figure 1. Daily rhythm of AVP gene transcription in SCNs after 2 wk in vitro. A, Quantitative in situ hybridization analysis of the levels of AVP hnRNA was made in hypothalamic slices fixed at 0600, 1200, 1800, and 2400 h over 2 d consecutively (d 16 and 17 in vitro). Bars are the mean and SEMs of the pooled data obtained from four independent experiments. The total numbers of rats used at each time point are 14 (0600, 1200, and 2400 h) and 5 (1800 h). The times shown in the abscissa of (A) and in the other figures correspond to the times of the light-dark cycle in the animal facility before the day of culture. Statistical significance of the differences between groups was calculated by one-way ANOVA followed by Fisher’s PLSD test. *, P < 0.05 as compared with AVP hnRNA levels at 2400 h on each day. B, Representative film autoradiographs of in situ hybridization for AVP hnRNA at 1200 and 2400 h in hypothalamic organotypic cultures containing the SCN.

 


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  		Figure 2. Effects of inhibition of action potentials by TTX on the daily rhythm of AVP hnRNA expression in the SCN in organotypic cultures. On d 17 of culture, sections were incubated with 1 µM TTX 3 h before fixation at 1200 and 2400 h. Bars are the mean and SEMs of the pooled data (n = 20) obtained from two independent experiments. Statistical significance of the differences between groups was calculated by one-way ANOVA followed by Fisher’s PLSD test. Significant differences between 1200 and 2400 h values are indicated in the graph. TTX decreased AVP hnRNA levels significantly at 1200 h (a, P < 0.01) and at 2400 h (b, P < 0.05). Although AVP hnRNA levels are significantly (P < 0.01) higher at 1200 h than at 2400 h in the absence of TTX, there were no significant differences in the levels between 1200 and 2400 h in the presence of TTX. ns, Not significant.

 
To determine whether the circadian behavior of AVP transcription was independent of electrical activity and synaptic input to the AVP neurons in the SCN, the cultured SCNs were incubated with TTX for 3 h before fixation. TTX produced significant decreases in AVP hnRNA levels at both 1200 and 2400 h (Fig. 2Go), suggesting that, although the clock genes may be necessary for the rhythmic AVP gene expression in the SCN, they are clearly not sufficient. There was no significant difference in the level of AVP hnRNA between 1200 and 2400 h in the presence of TTX (Fig. 2Go, right).

Effect of forskolin and PMA on AVP hnRNA expression
To investigate the signaling mechanisms that might be involved in the regulation of AVP transcription in the SCN, culture slices were incubated with forskolin or PMA for 2 h before fixation at 1200 or at 2400 h. In the absence of TTX incubation of SCN slices with forskolin had no significant effect on AVP hnRNA levels at 1200 h when levels were already high, but this treatment produced a significant increase at 2400 h (Fig. 3Go, left). In the presence of TTX, incubation with forskolin significantly increased AVP hnRNA levels both at 1200 and 2400 h, but the increase was significantly higher at 1200 h than at 2400 h (100 ± 19 vs. 46 ± 13 arbitrary units, P < 0.05; Fig. 3Go, right). The latter data suggested that the responsiveness of the AVP neurons in the SCN to forskolin also had a daily rhythm.



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  		Figure 3. Effects of forskolin on AVP hnRNA expression in the SCN at 1200 and 2400 h in the presence or absence of 1 µM TTX. Sections were incubated with 10 µM forskolin 2 h before fixation at 1200 and 2400 h. In the TTX experiments, forskolin or DMSO (vehicle control) was added to the cultures 30 min after preincubation with TTX, and then incubation continued for two additional hours. Bars show the means and SEMs of the pooled data (n = 20) obtained from two independent experiments. Statistical significance of the differences between groups was calculated by Student’s t test. In the absence of TTX, forskolin had no significant effect on AVP hnRNA levels at 1200 h but caused a significant increase at 2400 h. In the presence of TTX, forskolin increased AVP hnRNA levels significantly both at 1200 and at 2400 h. The absolute values after forskolin treatment are significantly (P < 0.05) higher at 1200 than at 2400 h in the presence of TTX, which was confirmed in independent experiments. *, P < 0.05; **, P < 0.01 as compared with AVP hnRNA levels of control at each time point; ns, not significant.

 
In an interesting contrast to the results with forskolin, incubation of the cultures in PMA greatly increased AVP hnRNA levels both at 1200 and 2400 h in the absence of TTX (Fig. 4Go, left). However, these AVP hnRNA responses to PMA were completely abolished by TTX both at 1200 and 2400 h (Fig. 4Go, right), suggesting an indirect, presynaptic action of the PMA.



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  		Figure 4. Effects of the phorbol ester, PMA, on AVP hnRNA expression in the SCN at 1200 and 2400 h in the presence or absence of 1 µM TTX. Sections were incubated with 1 µM PMA 2 h before fixation at 1200 and 2400 h. In the TTX experiments, PMA or DMSO (vehicle control) was added to the cultures 30 min after preincubation with TTX, and then incubation continued for an additional 2 h. Bars show the means and SEMs of the pooled data (n = 20) obtained from two independent experiments. Statistical significance of the differences between groups was calculated by Student’s t test. In the absence of TTX, PMA increased AVP hnRNA levels significantly both at 1200 and 2400 h. In the presence of TTX, however, PMA had no significant effects on AVP hnRNA levels at either 1200 or 2400 h. **, P < 0.01 as compared with AVP hnRNA levels of control at each time point; ns, not significant.

 
Endogenous signaling pathways and AVP gene transcription in the SCN
To determine which specific kinase pathways might be involved in generating the endogenous circadian pattern of AVP gene transcription in the SCN, the cultured slices were exposed to various kinase inhibitors. These included the PKA inhibitor, H89 (10 µM), the PKC inhibitor, calphostin C (1 µM), or the MAPK pathway inhibitor PD98059 (75 µM), each of which was applied 3 h before fixation either at 1200 or 2400 h. Incubation of slices with H89 had no significant effect on AVP hnRNA levels at 1200 or at 2400 h (Fig. 5AGo). Similarly, AVP hnRNA levels in the SCN were unaffected after incubation with calphostin C at 1200 or at 2400 h (Fig. 5BGo). However, PD 98059 markedly decreased AVP hnRNA levels in the SCN (F (3, 76) = 13.7, P < 0.01) at both 1200 (100 ± 19 vs. 23 ± 6 arbitrary units, P < 0.01; Fig. 5CGo) and 2400 h (47 ± 6 vs. 18 ± 4 arbitrary units, P < 0.01; Fig. 5CGo). In presence of the PD 98059, there was no significant difference between AVP hnRNA levels at 1200 and 2400 h.



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  		Figure 5. Effects of the PKA inhibitor H89 (10 µM; A), the PKC inhibitor, calphostin C (1 µ M; B) and the MAPK pathway inhibitor PD 98059 (75 µM; C) on AVP hnRNA levels in the SCN at 1200 and 2400 h. Slices were incubated with the inhibitors for 3 h before fixation at 1200 or at 2400 h. Bars are the mean and SEMs of the pooled data (n = 20) obtained from two independent experiments. Statistical significance of the differences between groups was calculated by one-way ANOVA followed by Fisher’s PLSD test. Note that the PD 98059 treatment decreased AVP hnRNA levels significantly at both 1200 and 2400 h, whereas the H89 and calphostin C treatments did not affect the levels significantly at either time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The SCN is the central pacemaker that regulates circadian rhythms in mammals (3, 5). There are multiple neurotransmitters that operate in the SCN (12). Among these, AVP appears to play an important excitatory role by activating V1a receptors (12) to increase the amplitude of firing rates in the SCN during subjective day (34, 35) and enhancing the SCN's output (11, 12). In addition to its intranuclear function, AVP also serves as an important neurotransmitter secreted by SCN efferents in other hypothalamic centers orchestrating the corticosterone and GnRH rhythms in the organism (16, 36, 37).

The biosynthesis and release of AVP in the SCN is regulated by an endogenous circadian mechanism. AVP mRNA peaks during the subjective day and is at a nadir during subjective night (21, 38, 39), with corresponding daily changes in AVP peptide levels in the SCN (40, 41). Nuclear run-on experiments suggested that the daily rhythm was due to regulation at the level of transcription (22), and the secretion of AVP by the SCN has also been shown to exhibit a circadian pattern in vitro (6, 42, 43).

Jin et al. (23) proposed and tested the hypothesis that AVP gene transcription is positively regulated by CLOCK-BMAL1 heterodimers binding to the E-box in the AVP gene’s 5'upstream region, as shown for the Period (Per) genes (24). These authors showed that homozygous CLOCK/CLOCK knockout mice had greatly reduced Per gene rhythms as well as abolition of the AVP mRNA rhythm in their SCNs. They also showed in in vitro transcription studies, using mouse NIH 3T3 cells, that an E-box (CACGTC) element in the AVP gene, located about 150 residues upstream of the transcription start site, is necessary for CLOCK-BMAL1-mediated transcriptional activation, and that this was under negative feedback regulation by Per proteins (23). Silver et al. (44) confirmed the finding that CLOCK mutant mice exhibited no significant day/night differences in SCN AVP mRNA levels, and also demonstrated that this effect was specific for the AVP gene. The cholecystokinin, vasoactive intestinal peptide, and substance P genes also have upstream consensus E-box sequences; however, these do not show any deficiencies in peptide gene expression in CLOCK mutant mice. The latter observations (44) are also consistent with a recent report showing that the circadian rhythms of Per1, Per2, and Per3 expression occur specifically in the shell compartment of the SCN where AVP is synthesized, in contrast to the location of vasoactive intestinal peptide expression in the core compartment of the SCN that is not under endogenous circadian regulation (45).

The above-described studies provide compelling evidence that the E-box enhancer is necessary for the AVP gene’s day/night rhythm of expression. However, is it sufficient? To address this question, we turned to long-term organotypic cultures of the SCN, and measurements of AVP gene transcription (hnRNA) by the use of an AVP intronic probe and quantitative in situ hybridization histochemistry (26). Although the neurochemical signaling of these long-term cultures from neonatal rats might differ from the properties of the mature SCN in vivo (46), the fundamental circadian mechanisms in the SCN have been found to be similar at all ages (3), and the use of these culture systems have proven invaluable to study the regulation of neuronal function in the SCN (7, 9, 42, 43, 47).

We first examined whether the rhythmicity of AVP gene transcription is maintained in vitro. As shown by the SEMs in Fig. 1Go, there was variability in AVP expression in the SCN of different rats at the same time points. This variability could be due, in part, to variations in the phases of the circadian rhythms between the individual rat SCNs after free-running in vitro. However, despite this variability, our data showed a clear day/night difference of AVP gene transcription after 17 d in vitro. One of the key aspects of this study was the use of the voltage-dependent sodium channel blocker, TTX, to directly block the spontaneous and evoked action potentials and indirectly the synaptic activity in the cultures. Much to our surprise, we found that, in the presence of TTX, AVP gene transcription decreased both at 1200 and 2400 h and that rhythmicity of expression was lost (Fig. 2Go). Whereas the E-box and CLOCK genes appear to be involved in the rhythmic expression of AVP gene transcription in the SCN (23, 44, 45), the present data indicate that action potentials and the synaptic activity in the SCN are also essential, presumably through signaling mechanisms required for the regulation of transcription in the AVP neurons. Because measurements were performed only at 1200 and 2400 h, the times at which diurnal variations of AVP hnRNA were at their peak and nadir, respectively, the possibility exists that TTX treatment shifted the time of phase. However, we view this as highly unlikely because it has been shown that inhibition of spontaneous activity by TTX does not affect the fundamental circadian control mechanisms in the SCN either in vitro (47, 48) or in vivo (49).

With respect to other signaling mechanisms, the rat AVP promoter contains putative cAMP response element (CRE) sites (19, 50), and several lines of evidence have pointed to roles for cAMP and CRE-mediated gene expression in the SCN. There have been reports of diurnal fluctuations of cAMP and PKA (51, 52, 53), circadian rhythms of type II adenylate cyclase mRNA in the SCN (54), and circadian variations in levels of the phospho-active forms the transcription factor, CRE binding protein (CREB) (32). Our experiments using forskolin suggested that cAMP was capable of activating AVP transcription in AVP cells of the SCN (Fig. 3Go). In the absence of TTX, forskolin stimulated AVP gene transcription greatly during the night (2400 h), but it was totally ineffective during the day (1200 h), when presumably the cAMP-driven mechanism was already fully active. When the AVP hnRNA levels were reduced by TTX, forskolin stimulated AVP gene transcription both during the day and the night showing 1) that forskolin acts on AVP neurons independently from action potentials and 2) that it could substitute for the inhibited neuronal activity. These data indicate that forskolin stimulates AVP gene transcription directly in the AVP neurons, though it is not possible to rule out an action through interneuronal communication independent of sodium channel-dependent action potentials (55, 56). In the presence of TTX, the stimulatory effect of forskolin was greater in the day than the night (P < 0.05), suggesting that there is also a circadian regulation of the response in the AVP neuron to forskolin.

The PKC-dependent regulatory pathway was also studied by the application of the stimulatory phorbol ester, PMA. Evidence exists for the presence of multiple isoforms of PKC in the SCN (57, 58), with the calcium-dependent forms, PKC{alpha} and PKC ß1, found in the AVP neurons. Interestingly, these isoforms also exhibit circadian rhythms in the SCN (57, 59). The application of PMA to the organotypic cultures produced a very large increase in AVP gene transcription in the SCN in the absence of TTX both in the day and night (Fig. 4Go). However, unlike the effect of forskolin, PMA in the presence of TTX was completely inactive (Fig. 4Go), indicating that PMA is acting on neurons that communicate with AVP cells via sodium-dependent action potentials. In this regard, it is interesting to note that a major known role for PKC is to presynaptically modulate neurotransmitter release (60). Which presynaptic system in the SCN is being regulated by PMA is not yet known, but it may involve blockade of an inhibitory pathway, or/and release of a neurotransmitter exceptionally potent in activating AVP gene transcription (note the more than 2-fold increase over the control hnRNA levels at 1200 h in Fig. 4Go). The greater effect of the PMA-induced activation vs. stimulation by forskolin suggests that a cAMP-independent pathway exists in the SCN that could stimulate AVP gene transcription.

CREB/CRE-mediated gene transcription need not be activated by cAMP/PKA only. It is well known that the key transcription factor CREB can be activated by phosphorylation through a variety of protein kinases, including PKA, calcium-calmodulin protein kinase, and MAPK (61, 62). In view of this, we then studied the effects of various protein kinase inhibitors on the endogenous circadian variations of AVP gene transcription in the SCN. Inhibitors of PKA and PKC did not affect AVP gene transcription in the SCN either in the day or at night (Fig. 5Go, A and B). However, the MAPK inhibitor, PD98059, dramatically decreased AVP hnRNA levels in the SCN in both day and night (Fig. 5CGo), suggesting that this kinase pathway is a key regulator of AVP gene expression in the SCN. This observation is consistent with other reports of the circadian rhythm of MAPK in the rat SCN (32) and its involvement in the circadian function of various other biological systems (63, 64, 65, 66, 67). The lack of effect of forskolin in the morning, when AVP hnRNA levels were at their peak, suggested to us that the peak was due to the action of endogenous cAMP. Thus, the inability of the PKA inhibitor, at doses shown to be effective in organotypic cultures (30), to reduce morning levels of AVP hnRNA was unexpected, and suggested that cAMP was acting through a PKA-independent pathway. In this regard, it has been shown that cAMP can activate transcription in a PKA-independent manner by interacting with cAMP binding proteins capable to directly activate the Ras signaling pathways, including MAPK (68, 69). It is unclear from this study whether or not MAPK-dependent pathways regulate AVP gene transcription in the AVP neurons. Given that forskolin could increase AVP gene transcription in the absence of action potentials, it is possible that MAPK-dependent pathways regulate AVP gene transcription downstream of cAMP in the AVP cells. Alternatively, MAPK-dependent pathways might regulate the presynaptic release of neurotransmitters, which in turn could regulate AVP gene transcription through other signaling mechanisms.

In conclusion, this study demonstrates that AVP gene transcription in the rat SCN exhibits a circadian rhythm in long-term, organotypic culture, which is dependent upon ongoing synaptic transmission. Investigation of the neurotransmitters involved and analysis of the upstream and downstream components of the MAPK pathways regulating AVP gene transcription remain important directions for future research.


   Acknowledgments
 


   Footnotes
 
This research was partially supported by the Japan Society for Promotion in Science.

Abbreviations: AVP, Arginine vasopressin; BMAL1, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1; CRE, cAMP response element; CREB, CRE binding protein; DMSO, dimethylsulfoxide; hnRNA, heteronuclear RNA; Per, period; PLSD, protected least significant difference; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SCN, suprachiasmatic nucleus; TTX, tetrodotoxin.

Received April 10, 2002.

Accepted for publication July 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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