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School of Biosciences, Cardiff University, Cardiff CF10 3US, Wales, United Kingdom
Address all correspondence and requests for reprints to: Dr. Timothy Wells, School of Biosciences, Cardiff University, P.O. Box 911, Museum Avenue, Cardiff CF10 3US, Wales, United Kingdom. E-mail: wellst{at}cardiff.ac.uk.
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Introduction |
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Photic cues, transmitted via the retinohypothalamic tract, are used by the ventrolateral suprachiasmatic nuclei (SCN) to entrain circadian rhythms to the light/dark cycle through partially defined mechanisms (8, 9). In contrast, the role of photic cues in the entrainment and synchronization of ultradian rhythms is ill defined, although SCN mechanisms are implicated (10). Recently, nocturnal photic stimulation has been shown to induce egr-1 (a transcription factor also known as NGFI-A, Zif-268, Krox-24, and TIS8) (11) mRNA expression in the SCN in a time-dependent manner, maximal expression occurring after 1 h (12). At this time point, egr-1 mRNA (12) and protein expression (13) are also observable in more dorsal locations and, significantly, are colocalized with SRIF in periventricular (PeN) neurons (12, 13). Thus, nocturnal photic stimulation could potentially modulate the pattern of GH secretion via augmented SRIF release.
In this series of experiments, we investigated the effects of nocturnal photic stimulation on the hypothalamopituitary GH axis in conscious rats. This stimulus always coincided with a trough period in spontaneous GH secretory profiles. The potential hypothalamic mechanisms underlying this response were investigated by monitoring the secretory responses to SRIF withdrawal and exogenous GRF treatment in conjunction with nocturnal photic stimulation. Analysis of male GH profiles also revealed that a similar trough period occurred at the commencement of the normal light phase, at a time when Egr-1 expression was also shown to be elevated in the SCN and PeN.
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Materials and Methods |
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Experiment 1: effect of single nocturnal photic stimulation on GH secretion in male rats
Male AS rats (8–9 wk old, weighing 216–248 g; n = 6) were placed in metabolic cages for 3–4 d before the insertion of single-bore jugular vein cannulae under halothane anesthesia. The rats were permitted at least 48 h recovery, during which time body weight and food intake were monitored daily and cannula patency was maintained with an intermittent infusion of sterile heparinized saline (10 U/ml; 20-µl bolus every hour). For 4 nights (commencing on the night of the first blood sampling session), rats were subjected to a single period of nocturnal photic stimulation at 2400 h (light intensity 200–300 lux) for 1 h. A 24-h period of automated serial blood sampling [100-µl samples of 1:5 blood (20 µl blood in 80 µl heparinized saline; 10 U/ml) collected every 10 min] was commenced at 1100 h prior to the first nocturnal photic stimulation. After 3 d recovery, during which time nocturnal photic stimulation and cannula patency were maintained, these rats were subjected to a second identical period of automated blood sampling, after which they were killed by iv injection of Expiral (sodium pentobarbitone; Sanofi Animal Health, Watford, Herts, UK). Circulating rat (r)GH concentrations were determined by RIA in diluted whole blood.
Experiment 2: effect of single nocturnal photic stimulation on GH secretion in female rats
Female AS rats (15–16 wk old, weighing 155–172 g; n = 5) were acclimatized to metabolic cages and prepared with single-bore jugular vein cannulae as above. After at least 48 h recovery, during which time body weight and food intake were monitored daily and cannula patency maintained as previously described, these rats were subjected to automated serial blood sampling (100-µl samples of 1:5 blood every 10 min; commenced at 1100 h). After 3 d recovery, during which time cannula patency was maintained, rats were subjected to a second identical period of automated blood sampling in conjunction with a single period of nocturnal photic stimulation (lights on at 2400 h for 1 h), after which they were killed by iv injection of Expiral. Circulating rGH concentrations were determined by RIA in diluted whole blood.
Experiment 3: effect of dual nocturnal photic stimulation on GH secretion in male rats
The potential waning of this effect was examined in male rats using repeated nocturnal photic stimulation. Male AS rats (10–14 wk old, weighing 167–224 g; n = 7), prepared with single-bore jugular vein cannulae as above, were subjected to automated serial blood sampling (100-µl samples of 1:5 blood every 10 min, commenced at 1100 h) in conjunction with two periods of nocturnal photic stimulation (at 2100 and 0100 h for 1 h), after which they were killed by iv injection of Expiral. Circulating rGH concentrations were determined by RIA in diluted whole blood.
Experiment 4: effect of single nocturnal photic stimulation on the rebound GH response to SRIF withdrawal in male rats
The possible contribution of reduced GRF secretion to the reduction in GH release after nocturnal photic stimulation was examined in male rats subjected to repeated SRIF withdrawal. Male AS rats (10 wk old, weighing 165–179 g; n = 4), prepared with double-bore jugular vein cannulae as above, were subjected to automated serial blood sampling (100-µl samples of 1:5 blood every 10 min, commenced at 1100 h) in conjunction with five 2.5-h periods of exogenous iv SRIF infusion (30 µg/100 µl/h; Peninsula Laboratories Europe Ltd., St. Helens, UK; catalog no. 8001), separated by 30 min. The fourth SRIF infusion was timed to terminate 30 min into a 1-h period of nocturnal photic stimulation (which commenced at 2400 h). After sampling, the rats were killed by iv injection of Expiral. Circulating rGH concentrations were determined by RIA in diluted whole blood.
Experiment 5: effect of single nocturnal photic stimulation on the pituitary GH response to GRF treatment in male rats
The possible contribution of reduced sensitivity of the pituitary to GRF after nocturnal photic stimulation was examined in male rats in which GH secretion was entrained by repeated exogenous GRF injection. Male AS rats (10–14 wk old, weighing 167–220 g; n = 6), prepared with single-bore jugular vein cannulae as above, were subjected to automated serial blood sampling (100-µl samples of 1:5 blood every 10 min, commenced at 1100 h) in conjunction with four 100-µl bolus injections of rat GRF [1 µg/100 µl rGRF(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)NH2; Bachem, Bubendorf, Switzerland; generously donated by Novo Nordisk A/S, Bagsværd, Denmark] separated by 3 h. The fourth bolus injection of GRF was timed to occur 30 min into a 1-h period of nocturnal photic stimulation (which commenced at 2400 h). Circulating rGH concentrations were determined in diluted whole blood.
Experiment 6: effect of the commencement of the normal light phase on hypothalamic Egr-1 expression in male rats
The possibility that induction of egr-1 expression might occur in the SCN and PeN after the commencement of the normal light phase was investigated in male rats. Groups of male Sprague Dawley rats (13–14 wk old; n = 3 for each group) were killed by cervical dislocation at 0500, 0700, and 0800 h (lights on at 0600 h), with an additional group killed in the dark at 0800 h after the dark phase was extended by 2 h. Male AS rats were also killed at 0500 h (n = 1) and 1 h after the commencement of the light phase (0700 h; n = 1) for interstrain comparison. Coronal blocks containing the hypothalamus were cut from partially frozen brains and snap frozen in isopentane at –20 C for subsequent determination of Egr-1 expression.
Tissue analyses
Circulating rGH concentrations were determined immediately after collection in whole blood by RIA, with the results expressed in terms of the reference preparation RP-2 (rGH), using the reagents generously supplied by National Institute of Diabetes and Digestive and Kidney Diseases (intraassay variation 1.2%; sensitivity 0.25 ng/ml).
Measurement of hypothalamic Egr-1 expression
Coronal brain sections (10 µm) cut through the PeN and SCN of the hypothalamus were taken at –20 C and thaw mounted onto polylysine-coated slides (BDH, Poole, UK) for subsequent determination of Egr-1 expression by immunohistochemistry. Brain sections were postfixed in 4% paraformaldehyde in PBS for 5 min and permeabilized in methanol at –20 C for 2 min. Tissue sections were blocked in 10% normal goat serum (Vector Laboratories, Burlingame, CA) in PBS containing 0.1% Triton X-100 (Sigma, St. Louis, MO; PBS-T) for 20 min at room temperature. Subsequently excess serum was washed away and the sections were incubated with the primary antibody [1:400 dilution of rabbit anti-Egr-1 antibody (catalog no. C19; Santa Cruz Biotechnology Inc., Santa Cruz, CA) in PBS-T] for 60 min at room temperature. Sections were then washed in PBS for 5 min and incubated in the dark for 30 min with the secondary antibody [1:500 dilution of Alexa Fluor 488 goat antirabbit IgG (Molecular Probes Inc., Eugene, OR) in PBS-T]. Sections were washed twice in PBS before being mounted in 50 µl/section Vectashield mounting medium with DAPI (4',6-diamidino-2-phenylindole; for counterstaining nucleic acid; 1.5 µg/ml; Vector Laboratories). In control studies, we have shown that omission of the primary, Egr-1, antiserum results in a loss of detectable immunoreactivity (13). Digital images were captured using a Leica DM-RD fluorescence microscope and Spot Advanced Image capture system (Spot software 2.2; Diagnostic Instruments Inc., Sterling Heights, MI). Egr-1-positive neurons were counted in unilateral hypothalamic nuclei, identified in matched sections according to the brain atlas coordinates of Paxinos and Watson (14), and the data presented as Egr-1-positive cells/section (with a minimum of eight sections/brain and three rats/group).
Statistical analysis
Because the spontaneous episodes of GH secretion are not completely synchronized among rats over the 24-h period, the effects of nocturnal photic stimulation on secretory profiles were visualized initially by superimposing multiple individual profiles (experiments 1, 2, and 3). Total secretory output for 1-h time periods before and during either nocturnal photic stimulation or normal lights on were determined by calculating the mean area under curve (AUC), statistical comparisons for the dark-light interface being made using paired Student’s t tests. In experiments 4 and 5, in which GH secretion was triggered by an exogenous stimulus, data shown are mean ± SEM, with 30-min AUC values determined and statistical comparisons made with paired Student’s t tests. AUC values were calculated using GraphPad Prism (GraphPad Inc., San Diego, CA). Statistical analysis of Egr-1 expression was performed by one-way ANOVA and Bonferroni post hoc test (experiment 6).
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), with mean AUC for the 1-h period of photic stimulation being less than half that in the 1-h period immediately before stimulation [Fig. 1B; P = 0.226 (t = 1.43; n = 5)]. In one rat in which a 3-h episode of GH secretion was due to occur during the period of nocturnal photic stimulation, the GH peak occurred immediately after the resumption of darkness (individual data not shown separately). The effect of nocturnal photic stimulation was not diminished by 4 d of exposure to the stimulus (Fig. 1D), with mean AUC during photic stimulation being 5% of that in the preceding 1-h period [Fig. 1E; P = 0.072 (t = 2.734; n = 4)].
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). Three days later, nocturnal photic stimulation elicited a marked suppression in both the peak and baseline secretion of GH (Fig. 2D), with mean AUC during photic stimulation reduced to 30% of that in the preceding 1-h period [Fig. 2E; P = 0.004 (t = 8.11; n = 4)].
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), with mean AUC for the 1-h period of photic stimulation being less than half that of the preceding 1-h period of darkness (Fig. 3B; P = 0.118 (t = 1.825; n = 7)]. However, there was no suppression of GH secretion associated with a second period of photic stimulation (commenced 3 h after the resumption of darkness) (Fig. 3, A and C).
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, A and B). Nocturnal photic stimulation suppressed the effect of SRIF withdrawal on GH secretion, peak height being less than 7% of that in the preceding (third) withdrawal (Fig. 4C, fourth rebound; P < 0.0001; t = 732.2; n = 3) and mean AUC for the 30-min period of fourth SRIF withdrawal being less than 4% of that in the preceding (third) withdrawal (Fig. 4D; P = 0.017; t = 7.509; n = 3). One rat, in which GH secretion was not entrained by SRIF withdrawal (data not shown), was not included in the statistical analysis.
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, A and B). Nocturnal photic stimulation abolished the effect of exogenous GRF on GH secretion, peak height being reduced to 3% of that in the preceding (third injection) response (Fig. 5C; P = 0.006; t = 4.665; n = 6) and mean AUC for the 30-min period after GRF treatment being reduced to less than 3% of that in the preceding (third injection) response (P = 0.024; t = 3.193; n = 6). A small increase in circulating GH was observed after the resumption of the dark phase. Additionally, some rats displayed a spontaneous episode of GH secretion 3 h after the last GRF injection (Fig. 5A). One rat, in which GH secretion was not entrained by GRF injection (data not shown) was not included in the statistical analysis.
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, A and D) and female rats (Fig. 2, A and D). AUC values for the first 1-h period of the light phase were less than those in the last hour of the dark phase [males: Fig. 1C; P = 0.036 (t = 3.12; n = 5) and Fig. 1F; P = 0.045 (t = 3.34; n = 4); females: Fig. 2C; P = 0.019 (t = 3.84; n = 5); and Fig. 2F; P = 0.058; (t = 3.00; n = 4)]. This phenomenon was unaffected by prior exposure to a single period of nocturnal photic stimulation (e.g. Figs. 1, C and F, and 2F) but was completely abolished after exposure to dual nocturnal photic stimulation (Fig. 3, A and D).
Experiment 6: effect of the commencement of the normal light phase on hypothalamic Egr-1 expression in male rats
Following our observation that the initiation of the light phase was accompanied by a suppression of GH secretion, we investigated the possibility that hypothalamic Egr-1 expression might be induced during the same period. Egr-1 expression was not observed in either the SCN or PeN of rats killed in the dark phase (1 h before lights on) (Figs. 6A and 7, A and B), although a small, diffuse population of Egr-1-positive cells was identified in the anterior hypothalamic area. One hour after the commencement of the light phase nuclear expression of Egr-1 could be seen in the SCN [Figs. 6, B, F, and G, and 7A; F = 13.22; P < 0.01 (t = 5.14; n = 3)], predominantly in the venterolateral portion (Fig. 6B), and, to a lesser extent, in the dorsomedial region. Egr-1-positive neurons were also observable in the PeN [Figs. 6, F and H, and 7B; F = 12.88; P < 0.05 (t = 3.84; n = 3)] and medial preoptic area (MPoA; Fig. 6, F and H). After 2 h light exposure (0600–0800 h), Egr-1 expression was not as robust in the SCN (Figs. 6C and 7A) but remained significantly higher than that observed before the commencement of the light phase [Fig. 7A; P < 0.05 (t = 3.50; n = 3)]. In contrast, the expression of Egr-1 was sustained in the PeN (Figs. 6C and 7B) and increased in the MPoA [Fig. 7C; F = 6.18; P < 0.05 (t = 4.02; n = 3)] at 0800 h. A similar pattern of Egr-1 expression was also observed in male AS rats (n = 2) killed 2 h after the commencement of the light phase (data not shown). This induction of hypothalamic Egr-1 expression was not seen in a cohort of rats maintained in the dark until being killed at 0800 h (Figs. 6D and 7, A–C). As expected, constitutive nuclear expression of Egr-1 was observed in the cingulate cortex in samples from both light and dark periods (Fig. 6I).
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Discussion |
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The induction of a 1-h period of photic stimulation during the dark phase was associated with a suppression of the episodic and baseline secretion of GH in both male (Fig. 1, A and B) and female rats (Fig. 2, D and E). This effect was sustained after 4 d of exposure to the photic stimulus (Fig. 1, D and E) but rapidly repeated periods of photic stimulation did not appear to be as effective (Fig. 3, A and C). A number of hypothalamic mechanisms may contribute to the generation of this phenomenon.
First, the amplitude of the spontaneous episodes of GH secretion is thought to be regulated by the quantity of GRF secreted from arcuate neurons (4). To investigate the possible suppression of GRF neurons by photic stimulation in vivo, we studied the GRF-dependent rebound release of GH after SRIF withdrawal (4, 16). The near-complete inhibition of the GH response by nocturnal photic stimulation (Fig. 4) implies that GRF release is abolished by this manipulation. Although monosodium glutamate treatment abolishes the entrainment of GH pulsatility to the light/dark cycle (17) and a transient increase in hypothalamic GRF content occurs 1 h after normal light onset (18), modulation of GRF release via a direct projection from the SCN to the arcuate nuclei is not supported by anatomical studies. It has been suggested, therefore, that the SCN may influence the arcuate nuclei indirectly via synaptic connections in the preoptic and retrochiasmatic areas (19).
Second, GH secretion may be inhibited during photic stimulation via increased release of SRIF. It has previously been reported that nocturnal photic stimulation induces Egr-1 expression in PeN SRIF neurons without affecting arcuate expression (13). Thus, it is possible that during photic stimulation, endogenous GRF release may continue, but its efficacy may be masked by a large induction of SRIF secretion. This is supported by the profound inhibition of the GH response to exogenous GRF injection by nocturnal photic stimulation (Fig. 5) and may account for the inhibition of basal GH secretion in female rats (Fig. 2, D and E).
Previous studies have reported that peak levels of SRIF expression occur in the hypothalamus (18), including the SCN (20), immediately after the normal onset of the light phase. However, somatostatinergic neurons in the SCN are unlikely to mediate direct suppression of GH secretion because the majority appear to function as intranuclear interneurons (21, 22). Our demonstration of the induction of Egr-1 expression in the PeN, after the commencement of the normal light phase (Figs. 6 and 7B), coupled with our previous finding that approximately 50% of SRIF-positive neurons in this nucleus coexpress Egr-1 after nocturnal photic stimulation (13), suggests that the activation of PeN SRIF neurons may underlie the photic suppression of GH secretion. Although further studies are required to quantify the proportion of Egr-1-positive neurons in the PeN that coexpress SRIF, the inhibition of the GH response to exogenous GRF during nocturnal photic stimulation implies significant activation of hypophysiotrophic SRIF neurons during this stimulus. Light-induced SRIF secretion could also explain our consistent observation of trough periods in GH secretion after the commencement of the normal light phase (Figs. 1, C and F, and 2, C and F) and a previous report of the suppression of GH pulses at the commencement of the light phase. The parallel suppression of GH secretion seen at the normal dark-light interface and after nocturnal photic stimulation implies that the latter manipulation could represent a powerful paradigm for studying the hypothalamic mechanisms of light-induced entrainment.
Whereas this study clearly indicates a direct inhibitory action of SRIF on GH release after nocturnal photic stimulation, our data do not exclude the possibility that elevated SRIF release may also inhibit arcuate GRF secretion. In support of this alternative mechanism, it has been shown that somatostatinergic neurons project from the periventricular to the arcuate nuclei (23) and GRF neurons express binding sites for somatostatin (24), and there is also growing circumstantial evidence that SRIF may contribute to the regulation of GH pulsatility via inhibition of GRF release (25). SRIF release from arcuate neurons does not appear to be significant in this context because the nocturnal light stimulus does not induce gene expression in this nucleus (13).
Light-induced elevation of SRIF release from PeN neurons may represent a powerful mechanism for entrainment of GH secretion to the dark-light interface. However, it is clear that this entrainment is only transient. Although trough periods in GH secretion were observed approximately 3 h after that induced by normal lights-on, by the end of the light phase, individual profiles were no longer synchronized (Figs. 1D and 3A), probably resulting from individual variation in hypothalamic periodicity. The transient nature of this entrainment has implications for the assumptions underlying the measurement of spontaneous GH secretion. It is interesting to note that the suppression of GH secretion at the normal dark-light interface is not seen after dual nocturnal photic stimulation (Fig. 3, A and D). This may be related to the fact that the start of the second period of photic stimulation is not in (3-hourly) phase with either the commencement of normal lights-on or the initiation of the preceding nocturnal photic stimulus.
Given the pleiotropic properties of SRIF in the hypothalamus and adenohypophysis, the phenomenon we have described may have a wider endocrine significance. Because, under certain circumstances, elevated SRIF secretion also inhibits ACTH (26), TSH (27, 28, 29), and prolactin (PRL) (29, 30) release, the commencement of the light phase may entrain multiple hormone systems to the dark-light interface. Indeed, this mechanism could account for the reported suppression of GH and vasopressin secretion and the delay in the nocturnal PRL peak after bright light exposure in humans (31). However, our attempts to determine the effects of nocturnal photic stimulation on PRL secretion in female rats were more equivocal than those reported here for the GH axis (Davies, J. S., unpublished data).
The observation of elevated Egr-1 expression in the somatostatinergic neurons in the PeN is interesting in its own right. Although Egr-1 binding sites are not found within the regulatory regions of the prepro-SRIF gene (32), Egr-1 may, nevertheless, function as a transcriptional regulator of other genes in these neurons. The observation that Egr-1 expression is induced in PeN neurons after both artificial (13) and normal photic stimuli suggests that this transcription factor may be the most appropriate marker of activation in this population of hypothalamic neurons.
In conclusion, we have demonstrated that nocturnal photic stimulation suppresses the GRF-GH axis, probably via the activation of SRIF neurons in the PeN. Suppression of GH secretion also occurs at the commencement of the light phase and may lead to transient daily entrainment of endocrine rhythms to the photic environment. The nocturnal photic stimulation paradigm employed in this study may represent a powerful new manipulation to dissect the molecular and neuronal events mediating the hypothalamic responses to dark-light transition.
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Acknowledgments |
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Footnotes |
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Abbreviations: AS, Albino Swiss; AUC, area under curve; GRF, GH-releasing factor; MPoA, medial preoptic area; PeN, periventricular nucleus; PRL, prolactin; r, rat; SCN, suprachiasmatic nucleus; SRIF, somatostatin.
Received September 17, 2003.
Accepted for publication February 10, 2004.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, P < 0.05,