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Am J Physiol Endocrinol Metab 293: E1325-E1334, 2007. First published August 28, 2007; doi:10.1152/ajpendo.00341.2007
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Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats

Maristela O. Poletini, De'Nise T. McKee, Jessica E. Kennett, Jamie Doster, and Marc E. Freeman

Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, Florida

Submitted 31 May 2007 ; accepted in final form 26 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The nature of the circadian signal from the suprachiasmatic nucleus (SCN) required for prolactin (PRL) surges is unknown. Because the SCN neuronal circadian rhythm is determined by a feedback loop of Period (Per) 1, Per2, and circadian locomotor output cycles kaput (Clock) gene expressions, we investigated the effect of SCN rhythmicity on PRL surges by disrupting this loop. Because lesion of the locus coeruleus (LC) abolishes PRL surges and these neurons receive SCN projections, we investigated the role of SCN rhythmicity in the LC neuronal circadian rhythm as a possible component of the circadian mechanism regulating PRL surges. Cycling rats on proestrous day and estradiol-treated ovariectomized rats received injections of antisense or random-sequence deoxyoligonucleotide cocktails for clock genes (Per1, Per2, and Clock) in the SCN, and blood samples were taken for PRL measurements. The percentage of tyrosine hydroxylase-positive neurons immunoreactive to Fos-related antigen (FRA) was determined in ovariectomized rats submitted to the cocktail injections and in a 12:12-h light:dark (LD) or constant dark (DD) environment. The antisense cocktail abolished both the proestrous and the estradiol-induced PRL surges observed in the afternoon and the increase of FRA expression in the LC neurons at Zeitgeber time 14 in LD and at circadian time 14 in DD. Because SCN afferents and efferents were probably preserved, the SCN rhythmicity is essential for the magnitude of daily PRL surges in female rats as well as for LC neuronal circadian rhythm. SCN neurons therefore determine PRL secretory surges, possibly by modulating LC circadian neuronal activity.

norepinephrine; PERIOD1; PERIOD2

IN FEMALE RATS, PROLACTIN (PRL) secretion has been proposed to result from synchronization among a circadian neural signal from the suprachiasmatic nucleus (SCN), both inhibitory and stimulatory neurotransmitter actions on the pituitary gland, and modulating effects of ovarian steroids (19). As a consequence of this synchronization, female rats display cyclical increases of PRL secretion characterized by two surges: the preovulatory surge of proestrous afternoon (64) and the secondary surge of estrous afternoon (12, 70).

The preovulatory surge of PRL depends on the levels of plasma ovarian steroids, because the increase of estradiol titers induces (45) and the increase of progesterone amplifies (76) this surge. Estradiol treatment in ovariectomized (OVX) rats induces daily PRL surges that occur at roughly the same time of day as the proestrous surge (13). Shifting of the light phase results in a coincident shift of the proestrous surge (10), and the estradiol-induced PRL surges of OVX rats free run in constant light (52), but the mechanism regulating the circadian profile of PRL secretion is still unknown.

The SCN coordinates PRL secretion as well as other circadian events (8, 44, 65). SCN neurons have been proposed to provide the circadian signal necessary for the preovulatory surge of PRL that occurs on the afternoon of proestrus. The unequivocal evidence supporting this proposal was that lesion of this nucleus abolishes the PRL surges (11, 37, 48), but no study to date has demonstrated which mechanisms are compromised by the SCN lesion. All attempts to implicate the SCN outputs in this mechanism were unsuccessful; interruption of SCN outputs, i.e., arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP), does not reproduce the results obtained with SCN lesion. AVP effects have been shown to be both suppressive (15, 47, 57) and stimulatory (20), whereas VIP disruption has been shown to have no effect (23), to delay the PRL surge, or to decrease its magnitude (74). Therefore, the mechanism interrupted by SCN lesion remains elusive. Furthermore, experiments with lesions do not exclude the participation of modulatory effects of SCN afferents on the mechanism controlling PRL surges. The molecular basis for the circadian rhythm of SCN neurons consists of positive and negative transcriptional/translational feedback loops. The negative loop involves the regulation of both Period (Per) 1, Per2, and Per3 genes and Cryptochrome (Cry) 1 and Cry2. The rhythmic transcription of these genes is driven by the transcription factors circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocater-like (BMAL). As the translation of PER and CRY occur, these proteins form heterodimers (PER-CRY), which, in the nucleus, interact with CLOCK and/or BMAL1 to inhibit transcription. At the same time, the rhythmic transcription of Bmal1 expresses a phase opposite that of Per and Cry (5, 55). Several studies have demonstrated that the disruption of this core molecular-clock function in the SCN prevents circadian behavior and circadian physiological events (6, 24, 32, 62, 77). Consequently, these clock genes seem to be essential for driving of circadian rhythms in mammals by the SCN neurons. We have shown (59) that an injection of an antisense deoxyoligonucleotide (AS-ODN) cocktail for clock genes (Per1, Per2, and Clock) into the SCN decreases by 60% the expression of the clock genes in these neurons and in turn compromises the circadian release of corticosterone and drinking behavior in OVX rats. Here we describe a specific effect, revealed by this approach, of SCN neuronal circadian rhythm on the mechanism timing PRL surges. We believe the SCN acts as a synchronizer, therefore, determining the time, not the magnitude, of the PRL surges. In addition, with this approach, we were able to identify the relevance of SCN afferents and efferents to this mechanism.

One excitatory component for a PRL surge comes from the locus coeruleus (LC), a pontine nucleus composed of neurons that synthesize norepinephrine (NE) (22, 69). From this nucleus arises the dorsal noradrenergic pathway, which provides NE for almost the entire central nervous system (4, 18). The LC neurons are therefore involved in several physiological processes. Their role in arousal, alertness, and the sleep/wake cycle has received much attention (7), and evidence for their role in hormone secretion has gradually increased. Similar to SCN lesions, lesions of LC neurons abolish the PRL surge that occurs on the afternoon of proestrus (1, 26) and estrus (71) as well as steroid-induced PRL surges in OVX rats (53). Therefore, LC neurons may be one target of SCN neurons, participating in the neural mechanism controlling the timing of PRL surges. Indeed, a circuit originating in the SCN and projecting to the LC neurons has been described (17). In male rats, LC neuronal circadian rhythm of electrical activity (3) has been demonstrated, but these neurons seem not to control PRL secretion, because LC lesioning does not affect secretion of this hormone in male rats (53) and the LC of female and male rats differ morphologically and functionally (36). In the study reported here, we investigated whether LC neurons of female rats display the same circadian profile of activity as those of male rats. Because estradiol exerts strong effects both on NE-biosynthetic enzyme dynamics in LC neurons (35, 60, 61) and on LC neuronal activity (38) in female rats, we characterized the LC neuronal activity in OVX rats and subsequently determined the role of clock-gene expression in the SCN on this rhythm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Adult female Sprague-Dawley rats weighing 250–300 g (Charles River Labs, Wilmington, MA) were housed under a standard 12:12-h light:dark (LD) cycle with lights on at 0600 and constant temperature (25°C) and humidity. Standard rat chow and water were available ad libitum. Vaginal smears were taken daily, and only rats showing at least three consecutive 4-day regular estrous cycles were used. Animals in the OVX groups were bilaterally ovariectomized under halothane vapor. All experimental protocols were approved by the Florida State University Animal Care and Use Committee.

Experiments

Experiment 1: effect of acute knockdown of clock genes on proestrous PRL surge. On diestrous afternoon, rats were implanted with guide cannulae bilaterally within the dorsal border of the SCN neurons. After 5 days, at 0800 on the next proestrous day, each animal was implanted with a cannula in the jugular vein and then received bilateral injections into the SCN of a cocktail of the AS-ODN or random-sequence deoxyoligonucleotides (RS-ODN) for the clock genes. On the same day, blood samples were collected every 2 h, beginning at 1300 and continuing for 24 h, for measurement of plasma PRL by radioimmunoassay. At the end of the experiment, the animals were perfused with fixative solution and the brains were obtained and processed for immunohistochemistry of CLOCK protein to verify the SCN cannula placements. Results from rats in which the guide cannulae were positioned directly above the SCN were considered to have received effective AS-ODN (n = 6) or RS-ODN (n = 5) treatments. Animals in which injections missed the SCN on one or both sides were included in the experiment as controls (n = 7).

Experiment 2: effect of acute knockdown of clock genes on estradiol-induced PRL surge. Seven days after ovariectomy, animals were implanted with guide cannulae bilaterally within the dorsal border of the SCN neurons. Three days later, at 1000, each received a subcutaneous injection of 20 µg of 17beta-estradiol (Sigma) diluted in 200 µl of corn oil. On the day after the treatment, at 0800, the estradiol-treated OVX animals were submitted to the same protocol described for experiment 1. Results from rats in which the guide cannulae were positioned directly above the SCN were considered to have received effective AS-ODN (n = 6) or RS-ODN (n = 5) treatments. Animals in which injections missed the SCN on one or both sides were included in the experiment as controls (n = 5).

Experiment 3: effect of acute knockdown of clock genes on activity of LC neurons over a 24-h period. Ten days after ovariectomy, rats were placed for 5 days either under LD or in constant darkness (DD) in individual cages. For animals kept in LD, illumination began at Zeitgeber time (ZT) 0 (0600) and ended at ZT 12 (1800). For purposes of comparison, in the last 3 days under both lighting conditions, drinking behavior was recorded for determination of the beginning of the period of rhythmic activity (66), which was designated circadian time (CT) 12, and experiments were performed in DD according to the CT. We counted the number of tyrosine hydroxylase (TH)-positive neurons immunoreactive to FOS-related antigens (FRA) bilaterally in the LC of both OVX rats and OVX rats injected with AS-ODN or RS-ODN cocktail. Groups of animals were perfused with fixative solution from ZT 2 to ZT 22, as well as at ZT 0 and ZT 12 in LD or at the corresponding CT in DD in the OVX group. In a second set of experiments, animals were perfused 6 h after an AS-ODN or RS-ODN cocktail injection into the SCN. In this last experiment, the SCN was also processed for immunohistochemistry for CLOCK so that the position of the guide cannulae could be evaluated, and only results from rats in which the guide cannulae were positioned directly above the SCN were included in the analysis. The results were presented as the mean ± SE of the percentage of TH neurons expressing FRA in the LC calculated from four animals at each time point. In addition, in the animals perfused at ZT 2 and ZT 10, the number of CLOCK-positive cells was determined in a region corresponding to SCN neurons (see General Methods for details). Animals kept under DD lighting conditions were handled and killed in dim red light (<1 lux).

General Methods

Bilateral cannula implantation into the SCN. The animals were anesthetized (100 µl/100 g weight) with a ketamine (49 mg/ml) and xylazine (1.8 mg/ml) cocktail and were implanted stereotaxically with bilateral stainless-steel guide tubes (1.5 mm apart, 9.5 mm in length, 27 gauge) whose tips were placed at the dorsal border of the SCN (0.8 mm posterior to bregma, 7.9 mm ventral to the dorsal surface of the dura mater). Bilateral 33-gauge solid steel mandrels were placed inside the guide tubes, and dust caps were used to secure the apparatus.

Jugular cannulation and blood samples. Polyurethane catheter tubing (Micro-Renathane; Braintree Scientific, Braintree, MA), extended with Tygon tubing (TGY-040-100; Small Parts, Miami Lakes, FL), was inserted into the jugular vein under halothane anesthesia. A tube filled with sterile heparinized saline (30 U/ml) was fitted subcutaneously and was exteriorized at the back of the animal's neck. Blood samples of 200 µl were drawn into plastic heparinized syringes. After removal of each blood sample, the same volume of sterile saline was injected through the catheter to replace the blood volume removed. Plasma was separated by centrifugation at 1,200 g for 15 min at 4°C and was stored at –20°C until it was assayed for PRL.

SCN injection of deoxyoligonucleotides. The oligonucleotide composition of the cocktail is shown in Table 1. Animals were anesthetized with halothane, and the 33-gauge bilateral mandrels were removed. Bilateral internal cannulae (33 gauge, 10.5 mm length, 1 mm extension beyond the guide tube) were inserted into the guide tubes, and 800 nl of AS-ODN or RS-ODN was injected at 200 nl/min with two 1-µl Hamilton syringes attached to an automated microinfusion pump (KD Scientific; Fisher Scientific, Fair Lawn, NJ). AS-ODNs were generated against the 5' transcription start site and 3' cap site of Per1, Per2, and Clock mRNA (Table 1). Control animals were injected with RS-ODNs, which had the same nucleotide content (%AGCT) as the AS-ODN but were not complementary to clock gene mRNA sequences (Table 1). As demonstrated previously in our laboratory, this cocktail leads to a knockdown of PER1, PER2, and CLOCK expression in the SCN by >60% within 6 h of injection at a dose of 3 nmol (2.5 mg/ml, 800 nl injection) (59). In the work reported here, we further confirmed the efficiency of the AS-ODN treatment by demonstrating that the number of CLOCK-positive neurons in the SCN at ZT 2 (0800) and ZT 10 (1600) was lower than that produced by RS-ODN injection.


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  		Table 1. Antisense and random sequence deoxyoligonucleotide sequences

 
RIA. Plasma concentrations of PRL were determined by RIA with specific kits provided by the National Institute of Diabetes and Digestive and Kidney Diseases. The antiserum for PRL was anti-rat PRL-S9, and the reference preparation was PRL-RP3. To prevent interassay variation, we assayed all samples in the same RIA. The lower limit of detection for PRL was 0.10 ng/ml. The intra-assay coefficient of variation was 5%.

Analysis of drinking rhythms. Drinking was measured with an automated device (Dilog Instruments, Tallahassee, FL) that counted individual licks in 30-s bins over 24 h. Drinking behavior was used as an index of activity of the animal (65). The beginning of the 12-h activity period, identified as CT 12 in DD, was determined from the data obtained by the automated device counting licks for 2 days before experiments for each animal. The two times were averaged to predict the onset of activity on the following day. The EPS 500 plotting program (version 5.3.10, developed by Ross Henderson, Florida State University) was used to process these data, in which a 12-h moving average around a central peak of activity was imposed by means of a filter of 20 bins/10 min. CT 12 was used as a reference time for experimental procedures in the DD condition, and light entrainment was used in LD. ZT 12 (0600) is when the lights went off, and the activity period of animals began at 1730 ± 0.3 h under LD in our colony. Using this approach, we could make direct comparisons between the rhythmic activities of LC neurons observed under LD and DD lighting conditions. In a separate group of animals, in which drinking behavior was also recorded, as described in experiment 3, recordings were obtained several days after injections of AS-ODN or RS-ODN into the SCN. When the cannulae were positioned outside the SCN, the data were grouped as misplaced injections. Therefore, the effects of AS-ODN on drinking behavior were evaluated both under entrainment and under free-running conditions.

Immunohistochemistry and cannula replacement. Animals were deeply anesthetized with 83 mg/kg body wt of pentobarbital sodium solution (Nembutal, Abbott Laboratories, Chicago, IL) and were transcardially perfused with 40 ml of sterile saline containing heparin (5 IU/ml), followed by 200 ml of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in 0.1 M PBS (pH 7.2). After perfusion, brains were removed, postfixed in 4% paraformaldehyde for 12 h, and cryoprotected in 20% sucrose in 0.1 M PBS at 4°C for at least 24 h. For evaluation of the LC neuronal activity, frontal sections of 30 µm were cut in a sliding microtome (Richard-Allan Scientific, Kalamazoo, MI) through the entire rostrocaudal extension of the LC, between 9.16 and 10.52 mm postbregma (50). To evaluate the guide-cannula replacement, we also cut sections of 30 µm between 0.80 and 1.60 mm postbregma, where the SCN neurons are located (50). Adjacent sections of both nuclei were stored in one of three adjacent series at –20°C in culture dishes containing cryoprotectant solution (75). Each of the three series therefore comprised adjacent sections separated by 60 µm.

We rinsed one series of sections of both LC and SCN neurons five times for 10 min in 0.1 M PBS (pH 7.35) containing 0.1% Triton X-100 and 0.1% sodium azide to remove cryoprotectant (Sigma-Aldrich). Nonspecific binding was blocked in 10% normal donkey serum (Chemicon, Temecula, CA) in rinsing solution. All primary and secondary antibodies were diluted in 0.1 M PBS (pH 7.35) containing 0.4% Triton X-100 and 0.1% sodium azide. Sections of LC neurons were incubated with primary antibodies against TH (mouse host, 1:10,000, monoclonal; Chemicon) and against FRA (K-25, goat host, 1:5,000, polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) for 24 h at 4°C. Sections of SCN neurons were incubated with anti-CLOCK (S-19, goat host, 1:10,000, polyclonal; Santa Cruz Biotechnology). Sections were rinsed three times for 10 min each. In the LC sections, donkey anti-goat Cy3-conjugated and anti-mouse Cy2-conjugated secondary antibodies were added (1:600; Jackson Immunochemicals, West Grove, PA), in the SCN sections, the anti-goat Cy3 (1:400; Jackson Immunochemicals) was added, and sections were again incubated at 4°C for 24 h. After being rinsed, the sections were mounted and cover slips were applied with diluted Aqua-Poly/Mount (Polysciences, Warrington, PA). After 24 h, the edges of the coverslips were sealed with nail polish. The specificities of both antibodies, anti-FRA and anti-CLOCK, were tested in sections incubated with 0.4% Triton X-100 in PBS without the primary antibodies, and no stains were observed (data not shown). The specificity for anti-CLOCK was also demonstrated elsewhere (34, 46). Images were taken with a Leica DM LB microscope fitted with short-pass filters (Cy2, 488 nm; Cy3, 596 nm) and a SPOT RT monochrome camera (Diagnostic Instruments, Sterling Heights, MI) attached to a microcomputer. Image acquisition and analysis were conducted with MetaMorph software (Universal Imaging, Downingtown, PA). Grayscale images of LC neurons immunoreactive to both TH and FRA were overlaid and pseudocolored in MetaMorph. The number of TH neurons expressing FRA was determined bilaterally in LC neurons of one series. The results are presented as the percentage of TH neurons expressing FRA. The grayscale of CLOCK-positive SCN neurons was obtained, and the position of the guide cannulae as well as the track of AS-ODN and RS-ODN injections was evaluated. In the OVX rats perfused at ZT 2 and ZT 10 in experiment 3, we counted the number of CLOCK-positive neurons in an area corresponding to the SCN, which was determined by immunohistochemistry in both the RS-ODN- and the AS-ODN-injected group. A circle 500 µm in diameter was drawn bilaterally adjacent to the ventricle and slightly above the optic chiasm for counting of the neurons, as shown in Fig. 1. The circle was drawn in the same position in all SCN sections, and the CLOCK-positive neurons were counted in six sections per animal. The results were summed and then averaged for each experimental group (AS-ODN and RS-ODN). The results are presented as the number of CLOCK-positive cells in the SCN. The efficiency of the treatment could therefore be tested. The counting for both the percentage of TH neurons expressing FRA in the LC and CLOCK-positive cells in the SCN was performed blindly.


Figure 1
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  		Fig. 1. Photomicrograph of 3 representative coronal sections of suprachiasmatic nucleus (SCN) neurons [A: guide cannula misplaced; B: injected with random-sequence oligonucleotides (RS-ODN) for clock genes; C: injected with antisense deoxyoligonucleotides (AS-ODN) for clock genes] incubated with anti-circadian locomotor output cycles kaput (CLOCK) and with donkey anti-goat secondary antibody conjugated to Cy3 (red stain). Arrow indicates track of guide cannula. Diagram modified from Paxinos's atlas (50) shows localization in SCN, –0.96 mm from bregma. Scale bar = 500 µm. OC, optic chiasm; 3V, 3rd ventricle.

 
Statistical Analysis

Data from experiments 1 and 2 are presented as means ± SE of plasma PRL concentrations. Statistical differences between AS-ODN and RS-ODN were determined by two-way ANOVA followed by Bonferroni's post hoc test. Temporal differences within the same group were determined by one-way ANOVA followed by Bonferroni's post hoc test. Data from experiment 3 are presented as means ± SE of the four animals perfused at each ZT or CT from different treatment groups (AS-ODN-injected and RS-ODN-injected groups). Both statistical differences between lighting schemes and those between treatment groups in experiment 3 were determined by two-way ANOVA followed by Bonferroni's post hoc test. Differences among ZTs or CTs in the same experimental group were analyzed by one-way ANOVA followed by Newman-Keuls multiple-comparison tests. Statistical differences in the number of CLOCK-positive neurons in the SCN between AS-ODN and RS-ODN were determined by two-way ANOVA followed by Bonferroni's post hoc analysis. All analyses were performed with GraphPad Prism (GraphPad Software, San Diego, CA), and P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1 illustrates the position of the guide cannulae on one side of the SCN. Figure 1A shows a guide cannula that missed the SCN; Fig. 1, B and C show cannulae successfully positioned above the SCN. The track of the injected cocktail (AS-ODN or RS-ODN) is marked by the tissue damage toward the SCN neurons (Fig. 1, B and C). As demonstrated by the number of CLOCK-positive neurons in the SCN of rats perfused at ZT 2 and ZT 10, the AS-ODN treatment significantly decreased CLOCK expression in the SCN (Fig. 2). Analysis of drinking behavior showed that the activity period started at ~1730 in animals kept under LD (mean ± SE of 1730 ± 0.3 h). Five days after the transition to DD, the beginning of the activity period (CT 12) was delayed ~2 h (mean ± SE of 19:30 ± 0.3 h; data not shown). The antisense cocktail clearly disrupted circadian drinking behavior. Figure 3 shows lick recordings of one representative rat from each experimental group (AS-ODN and RS-ODN) and from rats in which the SCN guide cannulae were misplaced. The AS-ODN decreased the licking and acutely abolished the rhythmicity, which was restored on the third day after the treatment (Fig. 3). This effect was observed under both lighting conditions (Fig. 3). Drinking behavior decreased to a minimum during this period, marked by an average of 10 licks per 10-min bin. These data suggest that AS-ODN cocktail transiently and specifically disrupted the function of the central molecular oscillator within the SCN.


Figure 2
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  		Fig. 2. Effects of knockdown of PERIOD (PER)1, PER2, and CLOCK expression in SCN on number of CLOCK-positive cells in SCN. A cocktail of either AS-ODN or RS-ODN for clock genes was injected into SCN of ovariectomized rats, and animals under a 12:12-h light:dark scheme were perfused at Zeitgeber time (ZT) 2 (2 h after lights on) and ZT 10. ***P < 0.001 vs. RS-ODN, determined by 2-way ANOVA followed by Bonferroni's post hoc test. Number of animals used at each ZT is in parentheses.

 

Figure 3
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  		Fig. 3. Effects of knockdown of PER1, PER2, and CLOCK expression in SCN on drinking behavior under entrainment (A: 12:12-h light:dark) or free-running (B: constant darkness) conditions. Lick recordings are presented from 1 representative rat in each experimental group (AS-ODN or RS-ODN for clock genes) and from rats in which SCN guide cannulae were misplaced. Recordings were done 24 h before and several days after injections into SCN. AS-ODN decreased licking and acutely abolished rhythmicity, which was restored by 3rd day after treatment. Arrows indicate times of SCN injections.

 
Experiments 1 and 2: Knockdown of Clock Genes in the SCN Neurons Abolishes Both the Proestrous and Estradiol-Induced PRL Surges

A broad surge of PRL characteristic of proestrous afternoon was seen in the RS-ODN group. In this group, plasma PRL concentrations started to increase at 1500 and remained higher until 2100 (Fig. 4A). Because of lack of progesterone effects (2, 76), the estradiol-induced PRL surge in OVX rats is sharp and smaller than that of proestrus. This typical surge of PRL was observed in the RS-ODN estradiol-treated group (Fig. 4B), which showed a peak of PRL secretion at 1700. In both proestrous and estradiol-treated OVX rats, these same secretory PRL surges were observed in animals in which injections missed the SCN on one or both sides (Fig. 4). The injection of AS-ODN cocktail for clock genes into the SCN abolished both proestrous day and estradiol-induced PRL surges (Fig. 4).


Figure 4
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  		Fig. 4. Effects of knockdown of PER1, PER2, and CLOCK expression in SCN on profile of plasma prolactin (PRL) levels on day of proestrus (A) and in ovariectomized rats treated with 20 µg estradiol at 1000 (B). A cocktail of either AS-ODN or RS-ODN for clock genes was injected into SCN, and blood samples were taken. AS-ODN injection abolished both surges of PRL on day of proestrus and in estradiol-treated animals. Black bar on x-axis indicates period of darkness in animal quarters. aP < 0.01 vs. 1300 (1-way ANOVA followed by Bonferroni's post hoc test); *P < 0.05 vs. RS-ODN and group with misplaced injection (2-way ANOVA followed by Bonferroni's post hoc test). Numbers of animals used in experimental groups are in parentheses.

 
Experiment 3: Knockdown of Clock Genes in the SCN Neurons Alters the Circadian Rhythm of LC Neuronal Activity in OVX Rats

Figure 5 shows a photomicrograph of neurons expressing TH and FRA in a representative section of the LC. Double-labeled cells are those exhibiting a green cytoplasmic stain (neurons positive for TH) and a red nucleus (positive for FRA).


Figure 5
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  		Fig. 5. Photomicrograph of locus coeruleus (LC) neurons incubated with anti-tyrosine hydroxylase (TH) and donkey anti-mouse secondary antibody conjugated to Cy2 (green stain) and incubated with anti-Fos-related antigen (FRA) and donkey anti-goat secondary antibody conjugated to Cy3 (red stain). Arrow indicates overlay of a TH-positive cell expressing FRA. Inset: overlay in higher magnification. Scale bar = 500 µm. 4V, 4th ventricle.

 
In LD, the percentage of TH neurons expressing FRA was significantly lower at ZT 10 than at any other ZT and reached the highest levels at ZT 14 (Fig. 6A). In DD, a higher percentage of TH neurons expressing FRA was also observed at CT 14 and a lower percentage was observed at CT 6 than at any other CT (Fig. 6B). No differences were found between percentages at any ZT observed in LD and the corresponding CT in DD.


Figure 6
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  		Fig. 6. Endogenous circadian rhythm of FRA expression in TH-positive neurons of LC. Percentage of LC neurons immunoreactive to TH expressing FRA was determined in groups of animals perfused every 4 h from ZT 2 to ZT 22 as well as at ZT 0 and ZT 12 under a light:dark cycle of 12:12 h (A) and at their respective circadian times (CT) under continuous darkness (B). LC neuronal activities under different lighting conditions did not differ (2-way ANOVA followed by Bonferroni's post hoc test). aP and bP < 0.05 vs. all other times (1-way ANOVA followed by Newman-Keuls test). Average percentage TH neurons expressing FRA was calculated from 4 animals at each time point.

 
After administration of RS-ODN to OVX rats, a higher percentage of TH neurons expressing FRA was observed in the LC at ZT 14 in LD and at CT 14 in DD. In the RS-ODN group under LD, lower LC neuronal activity was observed at ZT 10 (Fig. 7, A and B). The administration of AS-ODN abolished the increase in percentage of TH neurons expressing FRA at ZT 14 and CT 14, observed at LD and DD, respectively (Fig. 7, B and D). In addition, in LD, this treatment caused a premature decrease, at ZT 6 rather than ZT 10 (Fig. 7B). Therefore, the knockdown of clock genes in the SCN neurons changed the profile of LC activity under both lighting conditions.


Figure 7
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  		Fig. 7. Effects of knockdown of PER1, PER2, and CLOCK expression in SCN on endogenous circadian rhythm of neurons in LC. Injection of AS-ODN or RS-ODN for clock genes was made into SCN. After 6 h, groups of animals were perfused every 4 h from ZT 2 to ZT 18 as well as at ZT 0 and ZT 12 under a light:dark cycle of 12:12 h (A and B) and at their respective CT under continuous darkness (C and D). aP and bP < 0.05 vs. all other times (1-way ANOVA followed by Newman-Keuls test). *P < 0.05 vs. RS-ODN group (2-way ANOVA followed by Bonferroni's post hoc test). Average percentage TH neurons expressing FRA was calculated from 4 animals at each time point.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the study reported here, for the first time, the effect of the SCN clocks regulating neuronal circadian rhythms on the PRL surges of proestrus was revealed. Interestingly, when SCN rhythmicity, the synchronizing factor in the mechanism controlling PRL secretion, was taken away, both the proestrous afternoon and the estradiol-induced PRL surges were abolished. Because SCN afferents and efferents might have been preserved, in contrast to the case of a lesion, we can now further suggest that the SCN rhythmicity is essential for the magnitude of PRL surges in female rats. We also have demonstrated that LC neurons are one of the possible targets of the SCN. The SCN neuronal rhythmicity was disrupted by knockdown of clock gene (Per1, Per2, and Clock) expression within the SCN. This procedure compromised the functional circadian clock, as confirmed by disruption of circadian drinking behavior. Clock genes in the SCN therefore control the estradiol-induced circadian PRL surges, possibly by modulating LC neuronal activity.

We have shown here that an injection of AS-ODN for clock genes into the SCN abolishes both the proestrous and estradiol-induced PRL surges in OVX rats, whereas injection with RS-ODN, or with AS-ODN that missed the SCN, did not do so. These results suggest a specific role for SCN rhythmicity in the mechanism controlling PRL secretion.

Because the transcriptional/translational feedback loops of these genes have been proposed to determine the circadian rhythm in the SCN (55), the AS-ODN cocktail injection may disrupt the rhythmicity of the SCN neurons. Indeed, we have previously demonstrated (59) that this treatment disrupts the two well-known events that depend on the circadian signal from the SCN, i.e., circadian drinking behavior and circadian corticosterone secretion in OVX rats. The drinking-behavior disruption was confirmed in the present study. Furthermore, here we have shown that this treatment decreases CLOCK expression in the SCN, but because CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although their responses to light are altered (14), we do not interpret the decreased CLOCK expression in the SCN as a proof of lost rhythmicity but rather as indicating that expression of the clock-gene feedback loop might be compromised. Because we used a cocktail in the present study, we obviously cannot identify the specific gene(s) involved in the PRL rhythm. Clock-mutant mice, however, display a disrupted neuroendocrine system marked by decline in progesterone levels at midpregnancy together with a shortened duration of pseudopregnancy, suggesting that PRL release is compromised (41). In addition, CLOCK-BMAL1 seems to regulate PRL gene expression at least at the level of the pituitary (34). Because a diurnal increase of Per1 and Per2 expression in the rat SCN starts before the time of the surge of PRL (63, 68), these genes most probably drive the circadian signal involved in the mechanism controlling PRL secretion.

We cannot disregard the possibility that the lack of SCN rhythmicity affects the SCN projections. VIP and AVP might be compromised after interruption of the feedback loop of clock-gene expression. CLOCK-BMAL1 heterodimer accelerates AVP transcription (30), and clock-mutant mice show a decrease of AVP specifically in the SCN (27, 40). VIP and AVP expression peak at the same time as Per1 and Per2 gene expression, suggesting that their expressions are causally related (56).

The circadian signal from the SCN is always present in the rat, but a PRL surge is only observed after the increase of estradiol titers on diestrus or estradiol treatment in OVX rats. A secretory preovulatory PRL surge is therefore a consequence of the modulatory effects of ovarian steroids on an endogenous rhythm controlling PRL secretion. In the absence of estradiol, the rhythmic inhibitory and stimulatory inputs governing PRL secretion are extant, but a PRL surge only occurs after estradiol priming (19). Estradiol treatment in OVX rats promotes an advance in the acrophase of Per2 RNA expression in the SCN, but this hormone is unlikely to act at the level of the SCN to affect PRL secretion, because injection of estradiol into the SCN does not induce a PRL surge (49) and these neurons are not an estradiol-concentrating site (51). We therefore suggest that, although a circadian signal is given every day by the SCN, it does not modify PRL secretion unless the endogenous rhythm controlling PRL secretion has been affected by estradiol.

The AS-ODN for Clock, Per1, and Per2 mRNA injected into the SCN abolished the increase of LC neuron activity observed at CT 14 under both lighting conditions and altered the decrease in activity observed at ZT 10 in LD and at CT 6 in DD lighting conditions. LC neurons might be involved in the daily signal from the SCN that controls PRL secretion, but the present findings do not reveal whether the role of the LC is permissive or whether the regulation of the LC by the SCN is critical to the surge. Moreover, although our sampling times were rigorous, they do not exclude the possibility that the treatments merely phase shifted the rhythm in the LC.

FRA expression in the TH neurons has been demonstrated as a reliable index of neuronal activity (28, 29). We chose this technique to evaluate LC activity, although FRA expression and neuronal activity are not always interchangeable. FRA expression lags behind neuronal activation by 2 h (31). In view of this delay, the increase in FRA expression observed in TH-positive neurons at ZT 14 in LD or at CT 14 in DD reflects changes in the LC neurons' activity that occurred at the beginning of the animals' active period, corresponding to the onset of darkness, ZT 12. This result agrees with the circadian profile of LC electrical activity described in male rats (3). Because the phenotype of the LC neurons was identified in our study, our results further indicate that the noradrenergic neurons of the LC display circadian rhythm of activity in OVX rats. This circadian increase in the NE neuronal activity of LC coincided with the time of the PRL surges, but it did not precede the estradiol-induced PRL surge. As previously demonstrated, however, LC neuronal activity does increase before the PRL surge on proestrous afternoon (38). Because this surge results from an increase in ovarian steroid levels (64), we believe that ovarian steroids exert a stimulatory effect on LC neurons, not only amplifying their activity but precipitating it. Indeed, LC neurons have been shown to have estrogen and progesterone receptors (25, 39). Also, LC neurons concentrate [3H]estradiol (58), and the mRNA for TH in the LC neurons is increased by estradiol (60). We believe that this effect of estradiol on LC neuronal activity might predict an NE increase before the surges. Such an increase is observed in target areas controlling PRL secretion, such as the ventromedial hypothalamus and medial preoptic area (43, 54, 72). Because electrochemical stimulation of the LC induces release of NE in these areas (21), whereas lesion of this nucleus leads to a decrease (1, 71), LC neurons are candidates for providing NE for these areas. We therefore suggest that the action of estradiol in the LC neurons promotes an increase in their activity; these neurons, once activated, release NE to areas controlling PRL secretion. Further experiments are required to determine the effects of ovarian steroids on the circadian rhythm of LC neuron activity. Because lesions of LC neurons (1, 53) reproduce the results of SCN lesion (11, 37, 48), i.e., abolition of both the preovulatory surge of proestrous afternoon and the estradiol-induced surge of OVX rats, we hypothesized that these neurons convert the increase of estradiol levels (which is required for PRL surges) to the circadian daily signal provided by the SCN.

In a previous study (59), we have shown that the expression of clock genes in the SCN is reduced by 60% after AS-ODN cocktail injection, so the remaining clock protein could still be present in the SCN controlling the LC neuronal activity. Its presence might explain why the abolition of LC circadian rhythm has only been seen in DD, when both clock-gene circadian inputs from the SCN and the light cue were missing. Because the effects of SCN on LC activity have been proposed to be indirect, through dorsomedial hypothalamus (3), this area might be responsible for the circadian LC activity observed in the present study.

The technique we used (antisense cocktail for clock genes) allows the identification of some of the specified components involved in the complex interconnection between circadian rhythmicity and reproduction. In addition, this technique has the advantage of transiently affecting the rhythmicity. One of the immediate implications of a better understanding of the role of the circadian timing system in reproductive hormone profiles is to open a prospective basis for therapeutic intervention in abnormal reproductive function. Women with circadian rhythm abnormalities, caused, for example, by shift work, winter depression, or sleep disturbances, present low PRL secretion (16, 42, 67, 73). These abnormalities have been associated with an increased risk of subfecundity and changes in menstrual function (9, 33). In the present study, we have demonstrated that clock-gene knockdown in the SCN abolished estradiol-induced PRL surges in cycling and OVX rats. Because this treatment also desynchronizes the rhythmicity of LC neurons in OVX rats, we suggest that SCN neurons time PRL secretory surges by modulating the circadian rhythm of LC neurons.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Health Grants DA-19356 to R. Bertram, M. Egli, and M. E. Freeman and DK-43200 to M. E. Freeman.


   ACKNOWLEDGMENTS
 
We thank C. Pye for technical support, Dr. M. T. Sellix and Dr. J. Tabak for helpful comments and suggestions, C. Badland for image production, and Dr. A. B. Thistle for editing the text. Dr. A. Parlow is recognized for his provision of reagents for RIA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. O. Poletini, Dept. of Biological Science, Florida State Univ., Tallahassee, FL 32306-4340 (e-mail: poletini{at}neuro.fsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
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 REFERENCES
 

  1. Anselmo-Franci JA, Franci CR, Krulich L, Antunes-Rodrigues J, McCann SM. Locus coeruleus lesions decrease norepinephrine input into the medial preoptic area and medial basal hypothalamus and block the LH, FSH and prolactin preovulatory surge. Brain Res 767: 289–296, 1997.[CrossRef][Web of Science][Medline]
  2. Arbogast LA, Ben-Jonathan N. The preovulatory prolactin surge is prolonged by a progesterone-dependent dopaminergic mechanism. Endocrinology 126: 246–252, 1990.[Abstract/Free Full Text]
  3. Aston-Jones G, Chen S, Zhu Y, Oshinsky ML. A neural circuit for circadian regulation of arousal. Nat Neurosci 4: 732–738, 2001.[CrossRef][Web of Science][Medline]
  4. Aston-Jones G, Shipley MT, Grzanna R. The locus coeruleus A5 and A7 noradrenergic cell groups. In: The rat nervous system. edited by Paxinos G. New York: Academic, 1995, p. 183–214.
  5. Aton SJ, Herzog ED. Come together right...now: synchronization of rhythms in a mammalian circadian clock. Neuron 48: 531–534, 2005.[CrossRef][Web of Science][Medline]
  6. Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR. Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30: 525–536, 2001.[CrossRef][Web of Science][Medline]
  7. Berridge CW, Waterhouse BD. The locus coeruleus-noradrenergic system: modulation of behavioral state and state-dependent cognitive processes. Brain Res Brain Res Rev 42: 33–84, 2003.[CrossRef][Medline]
  8. Bethea CL, Neill JD. Lesions of the suprachiasmatic nuclei abolish the cervically stimulated prolactin surges in the rat. Endocrinology 107: 1–5, 1980.[Abstract/Free Full Text]
  9. Bisanti L, Olsen J, Basso O, Thonneau P, Karmaus W. Shift work and subfecundity: a European multicenter study. European Study Group on Infertility and Subfecundity. J Occup Environ Med 38: 352–358, 1996.[CrossRef][Web of Science][Medline]
  10. Blake CA. Effects of pinealectomy on the rat oestrous cycle and pituitary gonadotrophin release. J Endocrinol 69: 67–75, 1976.[Abstract/Free Full Text]
  11. Brown-Grant K, Raisman G. Abnormalities in reproductive function associated with the destruction of the suprachiasmatic nuclei in female rats. Proc R Soc Lond B Biol Sci 198: 279–296, 1977.[Medline]
  12. Butcher RL, Collins WE, Fugo NW. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17beta throughout the 4-day estrous cycle of the rat. Endocrinology 94: 1704–1708, 1974.[Abstract/Free Full Text]
  13. Caligaris L, Astrada JJ, Taleisnik S. Oestrogen and progesterone influence on the release of prolactin in ovariectomized rats. J Endocrinol 60: 205–215, 1974.[Abstract/Free Full Text]
  14. DeBruyne JP, Noton E, Lambert CM, Maywood ES, Weaver DR, Reppert SM. A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron 50: 465–477, 2006.[CrossRef][Web of Science][Medline]
  15. DePaolo LV, Berardo PV, Carrillo AJ. Intraventricular administration of arginine vasopressin suppresses prolactin release via a dopaminergic mechanism. Peptides 7: 541–544, 1986.[CrossRef][Web of Science][Medline]
  16. Depue RA, Arbisi P, Spoont MR, Krauss S, Leon A, Ainsworth B. Seasonal and mood independence of low basal prolactin secretion in premenopausal women with seasonal affective disorder. Am J Psychiatry 146: 989–995, 1989.[Abstract/Free Full Text]
  17. Deurveilher S, Semba K. Indirect projections from the suprachiasmatic nucleus to major arousal-promoting cell groups in rat: implications for the circadian control of behavioural state. Neuroscience 130: 165–183, 2005.[CrossRef][Web of Science][Medline]
  18. Foote SL, Bloom FE, Aston-Jones G. Nucleus locus ceruleus: new evidence of anatomical and physiological specificity. Physiol Rev 63: 844–914, 1983.[Free Full Text]
  19. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev 80: 1523–1631, 2000.[Abstract/Free Full Text]
  20. Funabashi T, Aiba S, Sano A, Shinohara K, Kimura F. Intracerebroventricular injection of arginine-vasopressin V1 receptor antagonist attenuates the surge of luteinizing hormone and prolactin secretion in proestrous rats. Neurosci Lett 260: 37–40, 1999.[CrossRef][Web of Science][Medline]
  21. Gitler MS, Barraclough CA. Effects of drugs which modify catecholamine activity on amplification of LH release induced by locus coeruleus electrical stimulation. Brain Res 437: 332–338, 1987.[CrossRef][Web of Science][Medline]
  22. Grzanna R, Molliver ME. The locus coeruleus in the rat: an immunohistochemical delineation. Neuroscience 5: 21–40, 1980.[CrossRef][Web of Science][Medline]
  23. Harney JP, Scarbrough K, Rosewell KL, Wise PM. In vivo antisense antagonism of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in the estradiol-induced luteinizing hormone and prolactin surges. Endocrinology 137: 3696–3701, 1996.[Abstract]
  24. Hastings MH, Field MD, Maywood ES, Weaver DR, Reppert SM. Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism. J Neurosci 19: RC11, 1999.[Abstract/Free Full Text]
  25. Helena CV, de Oliveira PM, Sanvitto GL, Hayashi S, Franci CR, Anselmo-Franci JA. Changes in alpha-estradiol receptor and progesterone receptor expression in the locus coeruleus and preoptic area throughout the rat estrous cycle. J Endocrinol 188: 155–165, 2006.[Abstract/Free Full Text]
  26. Helena CV, Franci CR, Anselmo-Franci JA. Luteinizing hormone and luteinizing hormone-releasing hormone secretion is under locus coeruleus control in female rats. Brain Res 955: 245–252, 2002.[CrossRef][Web of Science][Medline]
  27. Herzog ED, Grace MS, Harrer C, Williamson J, Shinohara K, Block GD. The role of Clock in the developmental expression of neuropeptides in the suprachiasmatic nucleus. J Comp Neurol 424: 86–98, 2000.[CrossRef][Web of Science][Medline]
  28. Hoffman GE, Le WW, Abbud R, Lee WS, Smith MS. Use of Fos-related antigens (FRAs) as markers of neuronal activity: FRA changes in dopamine neurons during proestrus, pregnancy and lactation. Brain Res 654: 207–215, 1994.[CrossRef][Web of Science][Medline]
  29. Hoffman GE, Smith MS, Verbalis JG. c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front Neuroendocrinol 14: 173–213, 1993.[CrossRef][Web of Science][Medline]
  30. Jin X, Shearman LP, Weaver DR, Zylka MJ, De Vries GJ, Reppert SM. A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96: 57–68, 1999.[CrossRef][Web of Science][Medline]
  31. Kovacs KJ. Invited review c-Fos as a transcription factor: a stressful (re)view from a functional map. Neurochem Int 33: 287–297, 1998.[CrossRef][Web of Science][Medline]
  32. Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, Hastings MH, Reppert SM. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98: 193–205, 1999.[CrossRef][Web of Science][Medline]
  33. Labyak S, Lava S, Turek F, Zee P. Effects of shiftwork on sleep and menstrual function in nurses. Health Care Women Int 23: 703–714, 2002.[CrossRef][Medline]
  34. Leclerc GM, Boockfor FR. Pulses of prolactin promoter activity depend on a noncanonical E-box that can bind the circadian proteins CLOCK and BMAL1. Endocrinology 146: 2782–2790, 2005.[Abstract/Free Full Text]
  35. Liaw JJ, He JR, Hartman RD, Barraclough CA. Changes in tyrosine hydroxylase mRNA levels in medullary A1 and A2 neurons and locus coeruleus following castration and estrogen replacement in rats. Brain Res Mol Brain Res 13: 231–238, 1992.[Medline]
  36. Luque JM, de Blas MR, Segovia S, Guillamon A. Sexual dimorphism of the dopamine-beta-hydroxylase-immunoreactive neurons in the rat locus ceruleus. Brain Res Dev Brain Res 67: 211–215, 1992.[CrossRef][Medline]
  37. Mai LM, Shieh KR, Pan JT. Circadian changes of serum prolactin levels and tuberoinfundibular dopaminergic neuron activities in ovariectomized rats treated with or without estrogen: the role of the suprachiasmatic nuclei. Neuroendocrinology 60: 520–526, 1994.[Web of Science][Medline]
  38. Martins-Afferri MP, Ferreira-Silva IA, Franci CR, Anselmo-Franci JA. LHRH release depends on Locus Coeruleus noradrenergic inputs to the medial preoptic area and median eminence. Brain Res Bull 61: 521–527, 2003.[CrossRef][Web of Science][Medline]
  39. Merchenthaler I, Shugrue PJ. Estrogen receptor-beta: a novel mediator of estrogen action in brain and reproductive tissues. Morphological considerations. J Endocrinol Invest 22: 10–12, 1999.[Medline]
  40. Miller BH, Olson SL, Levine JE, Turek FW, Horton TH, Takahashi JS. Vasopressin regulation of the proestrous luteinizing hormone surge in wild-type and Clock mutant mice. Biol Reprod 75: 778–784, 2006.[Abstract/Free Full Text]
  41. Miller BH, Olson SL, Turek FW, Levine JE, Horton TH, Takahashi JS. Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy. Curr Biol 14: 1367–1373, 2004.[CrossRef][Web of Science][Medline]
  42. Miyauchi F, Nanjo K, Otsuka K. Effects of night shift on plasma concentrations of melatonin, LH, FSH and prolactin, and menstrual irregularity. Sangyo Igaku 34: 545–550, 1992.[Medline]
  43. MohanKumar PS, ThyagaRajan S, Quadri SK. Correlations of catecholamine release in the medial preoptic area with proestrous surges of luteinizing hormone and prolactin: effects of aging. Endocrinology 135: 119–126, 1994.[Abstract]
  44. Moore RY, Eichler VB. Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res 42: 201–206, 1972.[CrossRef][Web of Science][Medline]
  45. Neill JD, Freeman ME, Tillson SA. Control of the proestrus surge of prolactin and luteinizing hormone secretion by estrogens in the rat. Endocrinology 89: 1448–1453, 1971.[Abstract/Free Full Text]
  46. Oishi K, Shirai H, Ishida N. CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor alpha (PPARalpha) in mice. Biochem J 386: 575–581, 2005.[CrossRef][Web of Science][Medline]
  47. Palm IF, van der Beek EM, Swarts HJ, van d V, Wiegant VM, Buijs RM, Kalsbeek A. Control of the estradiol-induced prolactin surge by the suprachiasmatic nucleus. Endocrinology 142: 2296–2302, 2001.[Abstract/Free Full Text]
  48. Pan JT, Gala RR. Central nervous system regions involved in the estrogen-induced afternoon prolactin surge. I. Lesion studies. Endocrinology 117: 382–387, 1985.[Abstract/Free Full Text]
  49. Pan JT, Gala RR. Central nervous system regions involved in the estrogen-induced afternoon prolactin surge. II. Implantation studies. Endocrinology 117: 388–395, 1985.[Abstract/Free Full Text]
  50. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic, 1998.
  51. Pfaff D, Keiner M. Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J Comp Neurol 151: 121–158, 1973.[CrossRef][Web of Science][Medline]
  52. Pieper DR, Gala RR. The effect of light on the prolactin surges of pseudopregnant and ovariectomized, estrogenized rats. Biol Reprod 20: 727–732, 1979.[Abstract]
  53. Poletini MO, Szawka RE, Franci CR, Anselmo-Franci JA. Role of the locus coeruleus in the prolactin secretion of female rats. Brain Res Bull 63: 331–338, 2004.[CrossRef][Web of Science][Medline]
  54. Rance N, Wise PM, Selmanoff MK, Barraclough CA. Catecholamine turnover rates in discrete hypothalamic areas and associated changes in median eminence luteinizing hormone-releasing hormone and serum gonadotropins on proestrus and diestrous day 1. Endocrinology 108: 1795–1802, 1981.[Abstract/Free Full Text]
  55. Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature 418: 935–941, 2002.[CrossRef][Medline]
  56. Reppert SM, Weaver DR. Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 63: 647–676, 2001.[CrossRef][Web of Science][Medline]
  57. Salisbury RL, Krieg RJ Jr, Seibel HR. Effects of arginine vasotocin, oxytocin, and arginine vasopressin on steroid-induced surges of luteinizing hormone and prolactin in ovariectomized rats. Acta Endocrinol 94: 166–173, 1980.
  58. Sar M, Stumpf WE. Central noradrenergic neurones concentrate 3H-oestradiol. Nature 289: 500–502, 1981.[CrossRef][Medline]
  59. Sellix MT, Egli M, Poletini MO, McKee DT, Bosworth MD, Fitch CA, Freeman ME. Anatomical and functional characterization of clock gene expression in neuroendocrine dopaminergic neurons. Am J Physiol Regul Integr Comp Physiol 290: R1309–R1323, 2006.[Abstract/Free Full Text]
  60. Serova L, Rivkin M, Nakashima A, Sabban EL. Estradiol stimulates gene expression of norepinephrine biosynthetic enzymes in rat locus coeruleus. Neuroendocrinology 75: 193–200, 2002.[CrossRef][Web of Science][Medline]
  61. Serova LI, Maharjan S, Sabban EL. Estrogen modifies stress response of catecholamine biosynthetic enzyme genes and cardiovascular system in ovariectomized female rats. Neuroscience 132: 249–259, 2005.[CrossRef][Web of Science][Medline]
  62. Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee CC, van der Horst GT, Hastings MH, Reppert SM. Interacting molecular loops in the mammalian circadian clock. Science 288: 1013–1019, 2000.[Abstract/Free Full Text]
  63. Shieh KR. Distribution of the rhythm-related genes rPERIOD1, rPERIOD2, and rCLOCK, in the rat brain. Neuroscience 118: 831–843, 2003.[CrossRef][Web of Science][Medline]
  64. Smith MS, Freeman ME, Neill JD. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96: 219–226, 1975.[Abstract/Free Full Text]
  65. Stephan FK, Zucker I. Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc Natl Acad Sci USA 69: 1583–1586, 1972.[Abstract/Free Full Text]
  66. Stephan FK, Zucker I. Rat drinking rhythms: central visual pathways and endocrine factors mediating responsiveness to environmental illumination. Physiol Behav 8: 315–326, 1972.[CrossRef][Medline]
  67. Stojek A, Kasprzak B, Slabikowski A. Intraocular pressure and prolactin measures in seasonal affective disorder. Psychiatr Pol 25: 8–12, 1991.[Medline]
  68. Sumova A, Jac M, Sladek M, Sauman I, Illnerova H. Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod. J Biol Rhythms 18: 134–144, 2003.[Abstract/Free Full Text]
  69. Swanson LW. The locus coeruleus: a cytoarchitectonic, Golgi and immunohistochemical study in the albino rat. Brain Res 110: 39–56, 1976.[CrossRef][Web of Science][Medline]
  70. Szawka RE, Anselmo-Franci JA. A secondary surge of prolactin on the estrus afternoon. Life Sci 75: 911–922, 2004.[CrossRef][Web of Science][Medline]
  71. Szawka RE, Helena CV, Rodovalho GV, Monteiro PM, Franci CR, Anselmo-Franci JA. Locus coeruleus norepinephrine regulates the surge of prolactin during oestrus. J Neuroendocrinol 17: 639–648, 2005.[CrossRef][Web of Science][Medline]
  72. ThyagaRajan S, MohanKumar PS, Quadri SK. Cyclic changes in the release of norepinephrine and dopamine in the medial basal hypothalamus: effects of aging. Brain Res 689: 122–128, 1995.[CrossRef][Web of Science][Medline]
  73. Touitou Y, Motohashi Y, Reinberg A, Touitou C, Bourdeleau P, Bogdan A, Auzeby A. Effect of shift work on the night-time secretory patterns of melatonin, prolactin, cortisol and testosterone. Eur J Appl Physiol Occup Physiol 60: 288–292, 1990.[CrossRef][Web of Science][Medline]
  74. van der Beek EM, Swarts HJ, Wiegant VM. Central administration of antiserum to vasoactive intestinal peptide delays and reduces luteinizing hormone and prolactin surges in ovariectomized, estrogen-treated rats. Neuroendocrinology 69: 227–237, 1999.[CrossRef][Web of Science][Medline]
  75. Watson RE, Wiegand SJ, Clough RW, Hoffman GE. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 7: 155–159, 1986.[Web of Science][Medline]
  76. Yen SH, Pan JT. Progesterone advances the diurnal rhythm of tuberoinfundibular dopaminergic neuronal activity and the prolactin surge in ovariectomized, estrogen-primed rats and in intact proestrous rats. Endocrinology 139: 1602–1609, 1998.[Abstract/Free Full Text]
  77. Zheng B, Albrecht U, Kaasik K, Sage M, Lu W, Vaishnav S, Li Q, Sun ZS, Eichele G, Bradley A, Lee CC. Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell 105: 683–694, 2001.[CrossRef][Web of Science][Medline]




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