Daily Variations in Type II Iodothyronine Deiodinase Activity in the Rat Brain as Controlled by the Biological Clock
http://www.100md.com
内分泌学杂志 2005年第3期
Netherlands Institute for Brain Research (A.K, R.M.B., R.v.S.) and Academic Medical Center (B.Z.D., E.F.), Department of Endocrinology and Metabolism, 1105 AZ Amsterdam, The Netherlands; and Department of Internal Medicine III (E.K., T.J.V.), Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: A. Kalsbeek, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: a.kalsbeek@nih.knaw.nl.
Abstract
Type II deiodinase (D2) plays a key role in regulating thyroid hormone-dependent processes in, among others, the central nervous system (CNS) by accelerating the intracellular conversion of T4 into active T3. Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid axis, D2 activity also appears to show daily variations. However, the mechanisms involved in generating these daily variations, especially in the CNS, are not known. Therefore, we decided to investigate the role the master biological clock, located in the hypothalamus, plays with respect to D2 activity in the rat CNS as well as the role of one of its main hormonal outputs, i.e. plasma corticosterone. D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Ablation of the biological clock abolished the daily variations of D2 activity in all four tissues studied. The main effect of the knockout of the suprachiasmatic nuclei (SCN) was a reduction of nocturnal peak levels in D2 activity. Moreover, contrary to previous observations in SCN-intact animals, in SCN-lesioned animals, the decreased levels of D2 activity are accompanied by decreased plasma levels of the thyroid hormones, suggesting that the SCN separately stimulates D2 activity as well as the hypothalamo-pituitary-thyroid axis.
Introduction
IN THE CENTRAL NERVOUS system, availability of the physiologically active thyroid hormone T3 depends mainly on the cellular uptake and intracellular deiodination of T4, which is in clear contrast to the peripheral tissues, in which most of the nuclear-bound T3 is imported from the plasma pool. Two isoenzymes are known to be involved in the conversion of T4 to T3. Type I iodothyronine deiodinase activity is present in the rat thyroid gland, liver, and kidney, whereas type II iodothyronine deiodinase (D2) activity is mainly present in rat brain, anterior pituitary, brown adipose tissue, and pineal gland. The existence of a separate pathway in the brain for T4 deiodination suggests that the adult central nervous system has the ability to autoregulate thyroid status and maintain T3 availability within physiological levels. Indeed, D2 enzyme activity is increased in hypothyroidism and reduced in hyperthyroidism (1, 2, 3, 4).
Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid (HPT) axis, D2 activity, too, has been reported to have daily variations (5, 6, 7, 8, 9, 10, 11). Although, apart from thyroid status, stress (12, 13), feeding (14, 15), and ?-adrenergic mechanisms (5, 6, 7, 9) also may affect D2 activity, the mechanisms involved in generating the daily variation in D2 activity in the central nervous system are not known. Recently it was shown that in both birds and hamsters, hypothalamic D2 expression can be affected by photoperiod (16, 17). In mammals it is likely that the rhythmic change in pineal D2 activity is controlled by a multisynaptic pathway, emanating from the biological clock that is located in the suprachiasmatic nuclei (SCN) and involving subsequent projections to the hypothalamus, the spinal cord, the superior cervical ganglion, and finally a sympathetic projection to the pineal gland, in a way comparable with that described for pineal arylalkylamine N-acetyltransferase, i.e. the enzyme responsible for the large circadian rhythm in melatonin synthesis (9, 18). In addition, D2 activity in the harderian glands, thymus, and brown adipose tissue shows a daily rhythm and is controlled primarily by the sympathetic nervous system (8, 19, 20, 21, 22, 23, 24, 25). But if and how the biological clock could affect D2 activity in brain areas such as cortex and hypothalamus is not known.
Manic-depressive illness and depression have been associated with alterations in both thyroid function and circadian rhythms. Decreased amplitudes of behavioral, physiological, and neuroendocrine circadian measures and disrupted responses of the circadian pacemaker to the light-dark cycle are frequently observed in depression (26, 27, 28, 29). But apart from those circadian disturbances changes in two major endocrine systems also have been demonstrated in depression, i.e. the HPT axis and hypothalamo-pituitary-adrenal (HPA) axis. Low nocturnal plasma TSH levels and/or a decreased amplitude of the daily plasma TSH rhythm occur in some, but not all, depressed patients (28, 30, 31, 32, 33). Moreover, a blunted TSH response to TRH is a consistent finding in approximately 25% of depressed patients (34), and the antidepressant effect of a total night’s sleep deprivation is accompanied by rises in plasma TSH, T3, and T4 (35). In addition, a slight increase of plasma T4 and a slight decrease of plasma T3 levels in depression have been reported (31, 32).
The hypothesis has been put forward that the changes seen in the HPT axis during depression are explained in part by a reduced cerebral conversion of T4 into T3 due to a reduced activity of the D2 enzyme (36). Indeed, some clinical studies suggest a beneficial effect of addition of T3 to treatment with tricyclic antidepressants (37, 38). Most studies investigating a potential therapeutic effect of thyroid hormone in depression have focused on the daily addition of 12.5–25 μg T3 (i.e. a little less than the daily production rate in humans) to antidepressants (mostly tricyclic antidepressants). The usefulness of thyroid hormone addition to antidepressants has remained controversial. Although increased efficacy through T3 addition was suggested by some studies (39), others were negative. A metaanalysis on this subject identified only four small randomized trials (38). On the other hand, exogenous T4 in TSH-suppressive doses also has been reported to be of potential value in the management of patients with treatment refractory bipolar disorder (40). Reduced activity of the D2 enzyme in depressive patients has been assumed to result from increased plasma cortisol levels (36). A mild hypercortisolism is often noted in depression (28, 41), and CRH-expressing neurons in the paraventricular nucleus of the hypothalamus show an increased activity (42, 43, 44). However, although it has been shown that corticosteroids inhibit D2 activity in mouse mammary gland (45) and human placental tissue (46), it is not known whether this also holds for the central nervous system. In fact, removal of circulating corticosteroids in the rat, by adrenalectomy, did not affect D2 activity in the hypothalamus or pituitary (24, 47), whereas corticosterone treatment during early development did not affect pituitary or cortical D2 activity (48). We therefore decided to investigate the separate roles of the biological clock and plasma corticosterone levels on D2 activity in the rat central nervous system.
Materials and Methods
Animals
Male Wistar rats (Harlan, Horst, The Netherlands), housed at a room temperature of 21 ± 1 C with a 12-h light, 12-h dark (L/D) schedule (lights on at 0700 h), were used for all experiments. Light onset was defined as Zeitgeber time (ZT) = 0 and lights off as ZT12. Animals were allowed to adapt to the new environment for at least 2 wk before the first experiments. Animals were kept with four animals per cage with food and water available ad libitum. At the time of the experiments, animals weighed between 300 and 350 g. Postoperative care was provided after each surgical procedure by a sc injection of Temgesic (Reckitt & Colman, Hull, UK; 0.3 ml/kg) after the animals woke up from anesthesia. All of the following experiments were conducted with the approval of the Animal Care Committee of the Royal Netherlands Academy of Sciences.
Experiment 1.
A total of 36 animals was killed (October 2001) at different times of the L/D cycle. After one-by-one transport to a separate room, animals were weighed, anesthetized, and decapitated within 3 min. Groups of six animals were killed at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22. Mixed arteriovenous trunk blood was collected in heparinized tubes and centrifuged for 15 min, 3000 x g at 4 C, to yield plasma that was decanted and stored at –20 C. After decapitation the brain, pituitary, and pineal gland were rapidly removed. Subsequently three transversal brain cuts were made with the help of a simple blocking device (49). The use of the blocking device prevented asymmetrical or oblique cutting and ensured a standardized and reproducible dissection of all the brains. The first cut was located just rostral to the optic chiasm, and the other two cuts were positioned 2 mm caudal and 4 mm rostral from the first cut, resulting in a thalamic, forebrain, and frontal cortex slice. Only the thalamic and frontal cortex slices were used for further analysis. The thalamic slice was dissected further by two vertical cuts just lateral to the optic chiasm and one horizontal cut just dorsal from the third ventricle, separating the medial part of the hypothalamus adjacent to the third ventricle, thus resulting in a hypothalamic slice. Pineal, pituitary, frontal cortex, and medial hypothalamus tissues were collected in Eppendorf tubes, snap frozen in liquid nitrogen, and stored at –80 C.
Experiment 2.
Bilateral thermic lesions of the SCN were made as described previously (50) when animals weighed 180–200 g. A total of 60 rats was operated on to ensure a sufficient number of effectively lesioned animals. After a 2-wk recovery period, the effectiveness of the SCN lesions was checked by measuring their drinking behavior during a 3-wk period. Intact animals consume only 0–5% of their total daily water intake during the middle 8 h of the light period (i.e. between ZT2 and ZT10). SCN lesions were considered successful when an animal drank more than 30% of its daily water intake during this 8-h period. By this method 24 animals were selected as being completely arrhythmic. Previous experiments have shown that the selection of effectively SCN-lesioned animals by their drinking behavior correlates very well, although not completely, with the selection by histological verification of the SCN lesions (50, 51). In the sixth week after the operation (May 2002), groups of effectively SCN-lesioned animals (n = 6) were killed at four different time points along the L/D cycle (i.e. ZT6, ZT10, ZT18, and ZT22) together with a group of unoperated control animals (n = 6). SCN-lesioned and control animals were killed in an alternating order according to the same protocol as described for experiment 1.
Experiment 3.
A pellet (100 mg) containing 100% cholesterol or a mixture of cholesterol and corticosterone was implanted sc in the interscapular region at the back. Pellets were constructed by gently heating pure cholesterol or a mixture of cholesterol and corticosterone in a small stainless steel spoon over a low gas flame until a liquid was formed, which was then poured into in pill mold (52). In a pilot experiment, groups of animals were provided with 0, 50, and 100% corticosterone pellets together with a jugular vein catheter (53) to determine the long-term effects of the pellets on plasma corticosterone concentrations. Based on the results of the pilot experiments, 48 animals were provided with a pellet containing either 0 or 50% corticosterone. Four weeks later (April 2003), animals were killed at four different time points during the L/D cycle (i.e. ZT6, ZT10, ZT18, and ZT22). At every time point, six animals of the 0 and 50% groups were killed in an alternating order as described for experiment 1.
D2 assay
Tissues were homogenized in 10 volumes of 0.1 M phosphate (pH 7.2), 2 mM EDTA, and 1 mM dithiothreitol (P100E2D1 buffer). D2 activity was assayed by measurement of the release of radioiodide from outer ring-labeled T4 as previously described (54). Appropriate dilutions of homogenates were incubated for 60 min at 37 C with 1 nM (105 cpm) [3',5'-125I]T4 in 0.1 ml P100E2D10 in the presence of 100 nM unlabeled T3 to block inner-ring deiodinase (D3) activity. Blank incubations were carried out in the absence of homogenate. Release of 125IG was determined and corrected for nonenzymatic deiodination. Addition of 100 nM unlabeled T4 resulted in major inhibition of radioiodide production, confirming low-Michaelis constant outer-ring deiodination of T4 by D2.
Hormone measurements
Plasma concentrations of the thyroid hormones T3 and T4 were determined by an in-house RIA (55), with inter and intraassay coefficients of variation of 7–8 and 3–4 and 3–6 and 2–4%, respectively. Detection limits for T3 and T4 were 0.3 nmol/liter and 5 nmol/liter. Plasma TSH concentrations were determined by a chemiluminescent immunoassay (IMMULITE, Diagnostic Products Corp., Los Angeles, CA), using a rat-specific standard. The inter- and intraassay coefficients of variation for TSH were less than 4% and less than 2% at ±3.5 ng/ml, and the detection limit was 0.01 ng/ml. Plasma corticosterone was measured directly without extraction, using a RIA from ICN Biomedicals, Inc. (Costa Mesa, CA) with iodinated corticosterone. The inter- and intraassay coefficients of variation for corticosterone were 6.5 and 7.1% at ±150 ng/ml, and the detection limit was 1 ng/ml.
Statistics
The time dependency (i.e. the existence of a daily variation) of D2 activity and hormone concentrations in experiment 1 was analyzed by one-way ANOVA. A two-way ANOVA was used to analyze the differences between experimental groups in experiments 2 (i.e. SCN-lesioned vs. intact) and 3 (i.e. corticosterone-treated vs. cholesterol-treated). The two-way ANOVA used the factors time (four levels) and group (two levels). When significant differences were found, post hoc tests were performed to determine which time points differed significantly from each other (Bonferroni) and at which time points treatment groups differed significantly (unpaired Student’s t test with Bonferroni correction). All data are given as means ± SEM. Results yielding a P < 0.05 were considered significant.
Results
Experiment 1
Although their mean levels showed the expected daily fluctuations, i.e. highest plasma levels of TSH, T3, and T4 during the light period (Fig. 1), none of these three parameters showed significant differences, depending on the time of day (TSH: P = 0.413; T3: P = 0.092; and T4: P = 0.141). Only plasma corticosterone concentrations (Fig. 1C) showed a significant diurnal variation (P < 0.001), with peak concentrations attained at ZT10. On the other hand, D2 activity showed clear effects of time of day in all four tissues studied. Peak activities were attained during the dark period, although most tissues had their own specific peak and trough times (Fig. 2). The most pronounced rhythms were found in the pineal gland and frontal cortex, i.e. P = 0.008 and P < 0.001, respectively. Pineal D2 activity was very low during all of the light period and even during the first part of the dark period (i.e. ZT14). Peak levels were attained at ZT18, and at ZT22, D2 activity was still significantly increased. Cortical D2 activity showed more gradual changes over the L/D cycle, with peak and trough times found at ZT22 and ZT10. The daily peaks of D2 activity rhythms in the pituitary and hypothalamus were located at the beginning and end of the night, respectively. However, the diurnal fluctuations of D2 activity were less pronounced in the pituitary and hypothalamus, i.e. P = 0.037 and P = 0.033, respectively, than in the frontal cortex and pineal gland.
FIG. 1. Twenty-four-hour plasma concentrations of TSH, T4, corticosterone, and T3 in male Wistar rats entrained to a regular 12/12 = L/D cycle before their killing (experiment 1). Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. *, P < 0.05; ***, P < 0.005.
FIG. 2. Twenty-four-hour rhythms of D2 activity in cortical, pineal, pituitary, and medial hypothalamus tissue of male Wistar rats entrained to a regular 12/12 = L/D cycle (experiment 1). Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. Despite a significant time effect for the hypothalamic D2 activity, post hoc testing did not reveal two specific time points that differed significantly. *, P < 0.05; ***, P < 0.005.
Experiment 2
The drinking scores (percentage of daily water intake between ZT2 and ZT10) of the SCN-lesioned animals were 33.6 ± 1.3, 33.0 ± 1.1, 34.7 ± 1.2, and 36.3 ± 1.6% for the groups killed at, respectively, ZT6, ZT10, ZT18, and ZT22. One-way ANOVA indicated the existence of a diurnal variation in SCN-intact animals in plasma corticosterone (P = 0.018) and plasma TSH (P = 0.016) but not for thyroid hormone concentrations in plasma (P > 0.1). In SCN-lesioned animals, none of these parameters showed a significant diurnal variation (all P > 0.05). Two-way ANOVA indicated significant effects of both group and group x time for all four hormonal parameters (Fig. 3 and Table 1). Despite the reduction from six to four sampling points, a significant time dependency in D2 activity could still be recovered in three of the four brain areas of the SCN-intact animals, i.e. pineal, cortex, and hypothalamus (all P < 0.001). In the pituitary, however, the significant rhythm as detected in experiment 1 was lost (P = 0.148), probably because the time of peak activity (i.e. ZT14) was not included in the sampling points of experiment 2. Two-way ANOVA revealed very significant effects of the SCN removal on D2 activity in all three brain areas showing a significant diurnal variation (i.e. for all group and group x time, P < 0.005; see also Table 1), the main effect of the SCN-lesion being a disappearance of the normal nocturnal increase of D2 activity (Fig. 4).
FIG. 3. Plasma concentrations of TSH, T4, corticosterone, and T3 in SCN-lesioned male Wistar rats (light bars) and control rats (dark bars) killed at the same time (experiment 2). All animals were housed in a regular 12/12 = L/D cycle before their killing. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. A bracket with asterisks indicates a time point in which the two experimental groups differ significantly from each other. *, P < 0.05.
TABLE 1. Analysis of the diurnal variations of plasma corticosterone, TSH, T3, and T4 concentrations and D2 activity in cortical, pineal, pituitary, and hypothalamic tissue of SCN-lesioned and intact control animals
FIG. 4. Diurnal rhythms of D2 activity in cortical, pineal, pituitary, and medial hypothalamus tissue of SCN-lesioned male Wistar rats (light bars) and control rats (dark bars) killed at the same time (experiment 2). All animals were housed in a regular 12/12 = L/D cycle. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. A bracket with asterisks indicates a time point in which the two experimental groups differ significantly from each other. *, P < 0.05; **, P < 0.01; ***, P < 0.005.
Experiment 3
In the pilot experiment, animals were provided with jugular vein catheters to allow for repeated blood sampling in conscious and undisturbed animals. Stress-free blood samples were taken at ZT2 and ZT10 twice a week. Results from these pilot experiments showed that the plasma corticosterone concentrations produced by the different pellets were constant for at least 5 wk. During these 5 wk, mean plasma corticosterone concentrations at ZT2 reached 14 ± 1, 47 ± 14, and 132 ± 6 ng/ml for the 0, 50, and 100% pellet groups (n = 4–5 animals), respectively. At ZT10 all three groups had mean plasma corticosterone concentrations greater than 100 ng/ml, i.e. 130 ± 8, 101 ± 12, and 124 ± 12 ng/ml for the 0, 50, and 100% corticosterone groups, respectively. The lack of a dose-dependent effect of the 50 and 100% pellets on the plasma corticosterone concentrations at ZT10 is probably due to a negative feedback on the HPA axis at the level of the hypothalamus and pituitary. In addition, the 100% pellet group showed an almost complete arrest of its body weight development after implantation of the pellet. Five weeks after implantation of the pellet, the body weight increment was 76 ± 7, 56 ± 11, and 6 ± 6 g for the 0, 50, and 100% groups, respectively, despite a similar body weight at the start of the experiment, i.e. 333 ± 2, 335 ± 5, and 337 ± 10 g. Due to the negative effects of the 100% pellets on body weight, in the subsequent experiment, only the 0 and the 50% corticosterone pellets were used.
Neither body weight nor the plasma concentrations of one of the hormones (corticosterone, TSH, T3, and T4) showed clear effects of the corticosterone treatment, i.e. no significant effects of group or group x time (Fig. 5 and Table 2). Significant diurnal variations were observed for plasma corticosterone and T3 concentrations but not plasma T4 (P = 0.077) or TSH (P = 0.117). However, whereas daily fluctuations in plasma corticosterone and T3 concentrations were significant or almost significant in both corticosterone- and cholesterol-treated animals (Table 2), daily fluctuations in plasma T4 concentrations approached significance in only the cholesterol- but not the corticosterone-treated animals (P = 0.066 and P = 0.804, respectively). D2 activity again showed a significant diurnal fluctuation in all four brain areas studied (Fig. 6), but no significant effects of group or group x time were detected. However, the 4-wk corticosterone treatment did cause a disappearance of the daily variation in pituitary D2 activity, as shown by the absence of a significant effect (P = 0.091) of time in the one-way ANOVA.
FIG. 5. Plasma concentrations of TSH, T4, corticosterone, and T3 in corticosterone-treated male Wistar rats (light bars) and sham-treated rats (dark bars) killed at the same time (experiment 3). The ZT2 data in the corticosterone figure are derived from the pilot experiment. All animals were housed in a regular 12/12 = L/D cycle before their killing. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value.*, P < 0.05; **, P < 0.01.
TABLE 2. Analysis of the diurnal variations of plasma corticosterone, TSH, T3, and T4 concentrations and D2 activity in cortical, pineal, pituitary, and hypothalamic tissue of corticosterone- and cholesterol-treated animals
FIG. 6. Diurnal rhythms of D2 activity in cortical, pineal, pituitary, and medial hypothalamus tissue of corticosterone-treated male Wistar rats (light bars) and sham-treated rats (dark bars) killed at the same time (experiment 3). All animals were housed in a regular 12/12 = L/D cycle. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. *, P < 0.05; **, P < 0.01; ***, P < 0.005.
Experiments 2 and 3 were performed in spring (April/May), whereas experiment 1 was performed in late autumn (October/November). Previously significant seasonal variations of both the HPA axis (56, 57, 58) and HPT axis (56, 59, 60, 61) as well as D2 activity have been reported (17). Nevertheless, in the present experiment, only plasma T3 concentrations showed a consistent seasonal variation, with higher concentrations in autumn than spring. Also, in the studies just cited, the most pronounced effects of season (or photoperiod) were found for plasma T3 concentrations. However, the changes reported by Wong et al. (60), i.e. higher plasma T3 concentrations in spring than in winter, were exactly the opposite of our results. On the other hand, Ahlersova et al. (61) found no differences between long and short photoperiods (i.e. L/D = 16/8 and L/D = 8/16, respectively) but found the highest plasma T3 concentrations during the L/D = 12/12 photoperiod. Moreover, because in our study the samples from the different experiments were not analyzed in one assay, we cannot be completely sure the variations are really due to seasonal changes.
Discussion
D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as in the hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Removal of the master biological clock, located in the hypothalamus, abolished the daily fluctuations of D2 activity in all four tissues studied, with the main effect of the SCN knockout being a reduction of nocturnal peak levels. Remarkably, previously (but also in the present study), we found that the main effect of SCN lesions on plasma TSH and thyroid hormone levels is also a disappearance of the diurnal peak and a reduction of the daily mean (62). By contrast, reducing the amplitude of the daily corticosterone rhythm during 4 wk through an elevation of its daily trough levels only had minor effects on the daily rhythm of D2 activity in the different tissues. Only in the pituitary and medial hypothalamus did the corticosterone treatment tend to reduce the amplitude of the daily rhythm in D2 activity. The present results thus show that a removal of the biological clock has pronounced effects on the activity of the D2 enzyme, whereas a slight elevation of the basal plasma corticosterone concentrations affects D2 activity only to a minor degree.
The presently reported daily rhythms of D2 activity in general confirm the results of previous studies on the pituitary, the hypothalamus, cortex, and pineal gland (5, 6, 8, 9, 10, 11, 18, 20, 63). Our results on the SCN-lesioned animals show that the daily rhythms in D2 activity were abolished in all four tissues investigated, as would be expected from a circadian rhythm controlled by the SCN. Moreover, these results confirm the previously indicated circadian nature of the D2 rhythms in the pineal gland and pituitary. However, with the exception of sympathetic tissues, such as the pineal and harderian glands, there is no indication to date how the circadian rhythms in pituitary and brain D2 activity are controlled by the central pacemaker. Apart from the autonomic nervous system, hormones, such as the thyroid hormones and glucocorticoids, also have been proposed as mediators of SCN output. However, because these hormonal rhythms are also abolished in SCN-lesioned animals, the SCN-lesion experiment gives no indication as to the importance of these hormonal factors.
Due to its pronounced circadian rhythmicity, we decided to investigate the importance of corticosterone as a mediator of SCN output. Thus far, in a number of animal tissues, no clear effects of corticosterone on D2 activity could be found (7, 12, 13, 24, 48), with the notable exception of D2 in the adrenal gland (64). On the other hand, two studies (45, 46) using cultured human placental and mouse mammary epithelium cells showed inhibitory effects of glucocorticoids on D2 activity. Our pilot experiments indicated a clear and sustained elevation of the daily trough levels of plasma corticosterone by the implantation of a sc pellet containing 50% corticosterone. Because the pellets containing 100% corticosterone resulted in an almost complete arrest of the normal increase in body weight, we provided our experimental animals with pellets containing only 50% corticosterone. The moderate increase during 4 wk, of basal corticosterone levels appeared to cause only minor changes in the daily rhythms of D2 activity. Unfortunately, the time points selected for killing the animals (i.e. the trough and acrophase of the daily rhythm in D2 activity) did not include the daily trough of the corticosterone rhythm (i.e. ZT2), and the significant effect of the corticosterone treatment on basal plasma corticosterone concentrations, as found in the pilot experiment, could thus not be demonstrated in the killed animals. Moreover, despite the well-known inhibitory effect of high plasma corticosterone levels on the HPT axis (65, 66), plasma TSH and thyroid hormone concentrations were not affected by the corticosterone treatment. Therefore, it is not clear yet whether the absence of a clear effect of our corticosterone treatment on the D2 activity rhythms indicates that corticosterone is not involved in the propagation of the SCN rhythm or whether the lack of effect is due to the minor disturbance in the corticosterone rhythm introduced by our treatment. In addition, a pilot experiment investigating D3 activity in the cortical tissue of the corticosterone- and cholesterol-treated animals did not reveal any significant effects of either time or group either (data not shown).
As documented before, effective SCN lesions result in the disappearance of all daily rhythms. In general, SCN lesions disturb only the temporal distribution of a certain parameter but not its overall level. For instance the mean 24-h food intake, water consumption, plasma corticosterone, and plasma glucose concentrations are quite similar in SCN-lesioned and control animals (51, 67). On the other hand, for some parameters SCN lesions may cause a clear increase or decrease of the 24-h means, as we recently demonstrated for plasma leptin (50) and plasma melatonin (51) concentrations. Apart from the disappearance of the daily rhythms of D2 activity, the SCN lesions also resulted in an overall decrease of daily D2 activity, not only in the pineal gland but also in the hypothalamic and cortical tissue. In agreement with our previous study (62), plasma TSH and especially thyroid hormone concentrations also were decreased in the SCN-lesioned animals. However, a direct control of the daily D2 activity rhythm in brain tissue by thyroid hormones seems unlikely. Indeed, hypothyroid rats show a pronounced increase, instead of a decrease, of hypothalamic and cortical D2 activity (1, 3). On the other hand, a primary and sole effect of the SCN lesion to decrease D2 activity also seems unlikely because then an increased release of TSH, and subsequently the thyroid hormones, would have been expected due to a diminished inhibitory feedback of the thyroid hormones at the level of the pituitary and hypothalamus. Indeed, D2 knockout mice show elevated plasma levels of T4 and TSH (68, 69). Apparently SCN activity provides a stimulatory input to both the HPT axis and the activity of the D2 enzyme. The absence of a stimulatory SCN input to the TRH neurons prevents an up-regulation of TSH release as a result of a diminished T3 feedback to the hypothalamus and pituitary (62). On the other hand, an altered D3 activity also could contribute to the changes observed. Although we do not know whether, and if yes, how the SCN (lesions) affected D3 activity, pilot experiments (as mentioned before) did not show daily fluctuations in cortical D3 activity.
Our results, as well as most of the previous results, were obtained in regular L/D conditions and therefore as such do not indicate whether the daily rhythms in D2 activity are endogenous or an (in)direct consequence of the L/D cycle. Apart from the pineal gland, thus far the endogenous nature of the daily D2 fluctuation has been demonstrated clearly only for the pituitary (20). Nocturnal light exposure or extension of the light phase into the dark period of the L/D cycle prevents the normal nocturnal increase of D2 activity in sympathetic tissues such as the pineal, thymus, and harderian glands (5, 6, 9, 20, 22, 24, 25) but also in the pituitary (20). On the other hand, in the frontal cortex, extension of the light phase into the dark period of the L/D cycle significantly enhanced D2 activity (5), whereas continuous darkness abolished the daily rhythm of D2 activity in both the frontal cortex and hypothalamus (5, 63). Moreover, recently it was found that D2 expression in the quail hypothalamus can be induced by light exposure (16), indicating that daily rhythms in D2 activity might indeed not be endogenous (i.e. circadian) but light driven. However, in the Djungarian hamster, the photoperiodic D2 expression was not driven directly by light exposure (17). In addition, because our SCN-lesioned animals were housed in L/D = 12/12 conditions, the disappearance of the daily rhythms of D2 activity in these animals strongly suggest that they are not light driven but endogenous and originating from the SCN. On the other hand, for the pineal rhythms of D2 (but also arylalkylamine N-acetyltransferase) expression, it is well known that they are both circadian and sensitive to light (9).
A direct effect of light on D2 activity mediated by the SCN or involving direct projections of the retina to the hypothalamus and cortex that are damaged by the SCN lesion can thus not be excluded completely. However, in view of the differential timing of the acrophase in D2 activity in the different brain structures, this possibility does not seem very likely either. Moreover, if SCN lesions are blocking a direct effect of light, we would expect increased, instead of decreased, 24-h means in SCN-lesioned animals because in intact animals the lowest levels of D2 activity are in general found during the light period. Therefore, with the exception of sympathetic tissues, such as the pineal and harderian glands, there is no indication to date how the circadian rhythms in pituitary and brain D2 activity are generated. On the other hand, the recent discovery that the SCN is not required for all 24-h timekeeping functions in individual tissues (70) indicates the possibility that daily rhythms of D2 activity in the cortex, hypothalamus, and pituitary are mainly sustained by tissue-specific clock gene expression and are only loosely connected to the SCN. Interestingly, the expression of the thyroid hormone receptor (TR) as well as that of the transcription factor Rev-erb in the central nervous system is modulated by antipsychotic drugs (71). Moreover, in mammals Rev-erb is an integral component of the biological clock (72, 73, 74) and is involved in the splicing of the TR1 and TR2 isoforms (75). However, at present there is no evidence linking the (rhythmic) activity of the Rev-erb gene to the control of D2 activity.
Thyroid hormone comedication with antidepressants may be an effective method to increase response rate in treatment-resistant depressive patients. It was hypothesized earlier that the beneficial effect of T3 treatment is due to a mild T3 deficiency in the central nervous system as a consequence of reduced D2 activity. The proposed inhibition of D2 activity has been assumed to result from elevated plasma cortisol concentrations (36, 37, 38). The plasma corticosterone concentrations produced by our treatment protocol nicely reflected the daily corticosterone rhythm as often observed in depressed patients, i.e. increased basal trough concentrations and a diminished amplitude (28, 41). Thus, according to the present results, the disturbed corticosterone rhythm in depressed patients probably presents only a minor contribution to the putatively reduced T3 bioavailability in these patients. Therefore, with regard to the often observed low plasma T3 concentrations in depressed patients and the proposed decreased D2 activity, it seems more likely that this is due to a lesser activity of the SCN (76, 77, 78) than to a slight hypercortisolism. In fact, the reduced SCN activity in depression might even be responsible for the decreased TRH gene expression in the paraventricular nucleus of the hypothalamus (79).
In conclusion, the present study provides further evidence for the endogenous nature of the daily rhythms of D2 activity in the cortex, hypothalamus, and pituitary. However, it is not yet clear how the SCN controls these rhythms because no clear effects of light and plasma corticosterone on D2 activity could be detected.
Acknowledgments
We thank Wilma Verweij for correcting our English and Henk Stoffels for making the figures. Eric Endert and the staff of the endocrinology laboratory are gratefully acknowledged for performing the TSH and thyroid hormone assays, as is the expert assistance of Jilles Timmer in animal husbandry.
References
Burmeister LA, Pachucki J, St. Germain DL 1997 Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138:5231–5237
Riskind PN, Kolodny JM, Larsen PR 1987 The regional hypothalamic distribution of type II 5'-monodeiodinase in euthyroid and hypothyroid rats. Brain Res 420:194–198
Tu HM, Kim SW, Salvatore D, Bartha T, Legradi G, Larsen PR, Lechan RM 1997 Regional distribution of type 2 thyroxine deiodinase messenger ribonucleic acid in rat hypothalamus and pituitary and its regulation by thyroid hormone. Endocrinology 138:3359–3368
Visser TJ, Leonard JL, Kaplan MM, Larsen PR 1982 Kinetic evidence suggesting two mechanisms for iodothyronine 5'-deiodination in rat cerebral cortex. Proc Natl Acad Sci USA 79:5080–5084
Guerrero JM, Puig-Domingo M, Reiter RJ 1988 Thyroxine 5'-deiodinase activity in pineal gland and frontal cortex: nighttime increase and the effect of either continuous light exposure or superior cervical ganglionectomy. Endocrinology 122:236–241
Murakami M, Greer MA, Greer SE, Hjulstad S, Tanaka K 1989 Comparison of the nocturnal temporal profiles of N-acetyl-transferase and thyroxine 5'-deiodinase in rat pineal. Neuroendocrinology 50:88–92
Jimenez J, Osuna C, Reiter RJ, Rubio A, Guerrero JM 1993 Adrenalectomy or superior cervical ganglionectomy modifies the nocturnal increase in rat pineal type-II thyroxine 5'-deiodinase. Chronobiol Int 10:87–93
Soutto M, Guerrero JM, Osuna C, Molinero P 1998 Nocturnal increases in the triiodothyronine/thyroxine ratio in the rat thymus and pineal gland follow increases of type II 5'-deiodinase activity. Int J Biochem Cell Biol 30:235–241
Kamiya Y, Murakami M, Araki O, Hosoi Y, Ogiwara T, Mizuma H, Mori M 1999 Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by ?-adrenergic mechanism in the rat pineal gland. Endocrinology 140:1272–1278
Murakami M, Tanaka K, Greer MA 1988 There is a nyctohemeral rhythm of type II iodothyronine 5'-deiodinase activity in rat anterior pituitary. Endocrinology 123:1631–1635
Campos-Barros A, Musa A, Flechner A, Hessenius C, Gaio U, Meinhold H, Baumgartner A 1997 Evidence for circadian variations of thyroid hormone concentrations and type II 5'-iodothyronine deiodinase activity in the rat central nervous system. J Neurochem 68:795–803
Baumgartner A, Hiedra L, Pinna G, Eravci M, Prengel H, Meinhold H 1998 Rat brain type II 5'-iodothyronine deiodinase activity is extremely sensitive to stress. J Neurochem 71:817–826
Bianco AC, Nunes MT, Hell NS, Maciel RM 1987 The role of glucocorticoids in the stress-induced reduction of extrathyroidal 3,5,3'-triiodothyronine generation in rats. Endocrinology 120:1033–1038
Diano S, Naftolin F, Goglia F, Csernus V, Horvath TL 1998 Monosynaptic pathway between the arcuate nucleus expressing glial type II iodothyronine 5'-deiodinase mRNA and the median eminence projective TRH cells of the rat paraventricular nucleus. J Neuroendocrinol 10:731–742
Glick Z, Wu SY, Lupien J, Reggio R, Bray GA, Fisher DA 1985 Meal-induced brown fat thermogenesis and thyroid hormone metabolism in rats. Am J Physiol 249:E519–E524
Yoshimura T, Yasuo S, Watanabe M, Iigo M, Yamamura T, Hirunagi K, Ebihara S 2003 Light-induced hormone conversion of T4 to T3 regulates photoperiodic response of gonads in birds. Nature 426:178–181
Watanabe M, Yasuo S, Watanabe T, Yamamura T, Nakao N, Ebihara S, Yoshimura T 2004 Photoperiodic regulation of type 2 deiodinase gene in Djungarian hamster: possible homologies between avian and mammalian photoperiodic regulation of reproduction. Endocrinology 145:1546–1549
Murakami M, Greer MA, Hjulstad S, Greer SE, Tanaka K 1988 The role of the superior cervical ganglia in the nocturnal rise of pineal type-II thyroxine 5'-deiodinase activity. Brain Res 438:366–368
Silva JE, Larsen PR 1983 Adrenergic activation of triiodothyronine production in brown adipose tissue. Nature 305:712–713
Murakami M, Greer MA, Greer SE, Hjulstad S, Tanaka K 1988 Effect of short-term constant light or constant darkness on the nyctohemeral rhythm of type-II iodothyronine 5'-deiodinase activity in rat anterior pituitary and pineal. Life Sci 42:1875–1879
Eley J, Himms-Hagen J 1989 Brown adipose tissue of mice with GTG-induced obesity: altered circadian control. Am J Physiol 256:773–779
Guerrero JM, Gonzalez-Brito A, Santana C, Reiter RJ 1989 Nocturnal increase of type II thyroxine 5'-deiodinase activity in the Syrian hamster harderian gland is abolished by light exposure and induced by isoproterenol. Proc Soc Exp Biol Med 190:186–189
Kates AL, Himms-Hagen J 1990 Defective regulation of thyroxine 5'-deiodinase in brown adipose tissue of ob/ob mice. Am J Physiol 258:E7–E15
Osuna C, Jimenez J, Reiter RJ, Rubio A, Guerrero JM 1992 Adrenergic regulation of type II 5'-deiodinase circadian rhythm in rat harderian gland. Am J Physiol 263:E884–E889
Soutto M, Molinero P, Guerrero JM 1997 Continuous light exposure modifies the nocturnal increase in rat thymus type II thyroxine 5'-deiodinase. Cell Mol Life Sci 53:697–699
McEachron DL, Schull J 1993 Hormones, rhythms, and the blues. In: Schulkin J, ed. Hormonally induced changes in mind and brain. San Diego: Academic Pres Inc.; 287–355
Healy D, Waterhouse JM 1995 The circadian system and the therapeutics of the affective disorders. Pharmacol Therapeut 65:241–263
Duncan WC 1996 Circadian rhythms and the pharmacology of affective illness. Pharmacol Therapeut 71:253–312
Van Cauter E, Turek FW 1986 Depression: a disorder of timekeeping? Perspect Biol Med 29:510–519
Weeke A, Weeke J 1978 Disturbed circadian variation of serum thyrotropin in patients with endogenous depression. Acta Psychiatr Scand 57:281–289
Wilson DA, Mulder RT, Joyce PR 1992 Diurnal profiles of thyroid hormones are altered in depression. J Affect Disord 24:11–16
Maes M, Meltzer HY, Cosyns P, Suy E, Schotte C 1993 An evaluation of basal hypothalamic-pituitary-thyroid axis function in depression: results of a large-scaled and controlled study. Psychoneuroendocrinology 18:607–620
Duval F, Mokrani MC, Crocq MA, Jautz M, Bailey P, Diep TS, Macher JP 1996 Effect of antidepressant medication on morning and evening thyroid function tests during a major depressive episode. Arch Gen Psychiatry 53:833–840
Lenzinger E, Meszaros K, Hornik K, Parzer P, Hollerer E, Langer G, Resch F, Legros JJ 1996 Correlation between vasopressin baseline and TSH-blunting in depressives. Biol Psychiatry 39:341–345
Baumgartner A, Riemann D, Berger M 1990 Neuroendocrinological investigations during sleep deprivation in depression. II. Longitudinal measurement of thyrotropin, TH, cortisol, prolactin, GH, and LH during sleep and sleep deprivation. Biol Psychiatry 28:569–587
Jackson IM 1996 Does thyroid hormone have a role as adjunctive therapy in depression? Thyroid 6:63–67
Kirkegaard C, Faber J 1998 The role of thyroid hormones in depression. Eur J Endocrinol 138:1–9
Aronson R, Offman HJ, Joffe RT, Naylor CD 1996 Triiodothyronine augmentation in the treatment of refractory depression. A meta-analysis. Arch Gen Psychiatry 53:842–848
Altshuler LL, Bauer M, Frye MA, Gitlin MJ, Mintz J, Szuba MP, Leight KL, Whybrow PC 2001 Does thyroid supplementation accelerate tricyclic antidepressant response? A review and meta-analysis of the literature. Am J Psychiatry 158:1617–1622
Bauer MS, Whybrow PC 1990 Rapid cycling bipolar affective disorder. II. Treatment of refractory rapid cycling with high-dose levothyroxine: a preliminary study. Arch Gen Psychiatry 47:435–440
Dahl RE, Ryan ND, Puig-Antich J, Nguyen NA, Al-Shabbout M, Meyer VA, Perel J 1991 24-Hour cortisol measures in adolescents with major depression: a controlled study. Biol Psychiatry 30:25–36
Raadsheer FC, Hoogendijk WJ, Stam FC, Tilders FJ, Swaab DF 1994 Increased numbers of corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus of depressed patients. Neuroendocrinology 60:436–444
Raadsheer FC, Van Heerikhuize JJ, Lucassen PJ, Hoogendijk WJG, Tilders FJH, Swaab DF 1995 Corticotropin-releasing hormone mRNA levels in the paraventricular nucleus of patients with Alzheimer’s disease and depression. Am J Psychiatry 152:1372–1376
Wong ML, Kling MA, Munson PJ, Listwak S, Licinio J, Prolo P, Karp B, McCutcheon IE, Geracioti Jr TD, DeBellis MD, Rice KC, Goldstein DS, Veldhuis JD, Chrousos GP, Oldfield EH, McCann SM, Gold PW 2000 Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: relation to hypercortisolism and corticotropin-releasing hormone. Proc Natl Acad Sci USA 97:325–330
Song S, Oka T 2003 Regulation of type II deiodinase expression by EGF and glucocorticoid in HC11 mouse mammary epithelium. Am J Physiol 284:E1119–E1124
Hidal JT, Kaplan MM 1988 Inhibition of thyroxine 5'-deiodination type II in cultured human placental cells by cortisol, insulin, 3',5'-cyclic adenosine monophosphate, and butyrate. Metabolism 37:664–668
Anguiano B, Valverde C 2001 Cold-induced increment in rat adrenal gland type II deiodinase is corticosterone dependent. Endocrine 15:87–91
McCann UD, Shaw EA, Kaplan MM 1984 Iodothyronine deiodination reaction types in several rat tissues: effects of age, thyroid status, and glucocorticoid treatment. Endocrinology 114:1513–1521
Kalsbeek A, Matthijssen MA, Uylings HB 1989 Morphometric analysis of prefrontal cortical development following neonatal lesioning of the dopaminergic mesocortical projection. Exp Brain Res 78:279–289
Kalsbeek A, Fliers E, Romijn JA, La Fleur SE, Wortel J, Bakker O, Endert E, Buijs RM 2001 The suprachiasmatic nucleus generates the diurnal changes in plasma leptin levels. Endocrinology 142:2677–2685
Perreau-Lenz S, Kalsbeek A, Garidou ML, Wortel J, Van Der Vliet J, Van Heijningen C, Simonneaux V, Pévet P, Buijs RM 2003 Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms. Eur J Neurosci 17:221–228
Akana SF, Scribner KA, Bradbury MJ, Strack AM, Walker CD, Dallman MF 1992 Feedback sensitivity of the rat hypothalamo-pituitary-adrenal axis and its capacity to adjust to exogenous corticosterone. Endocrinology 131:585–594
Steffens AB 1969 A method for frequent sampling blood and continuous infusion of fluids in the rat without disturbing the animal. Physiol Behav 4:833–836
Richard K, Hume R, Kaptein E, Sanders JP, van Toor H, De Herder WW, den Hollander JC, Krenning EP, Visser TJ 1998 Ontogeny of iodothyronine deiodinases in human liver. J Clin Endocrinol Metab 83:2868–2874
Wiersinga WM, Chopra IJ 1982 Radioimmunoassays of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'diiodothyronine (T2). Methods Enzymol 84:272–303
Wong CC, Dohler KD, Atkinson MJ, Geerlings H, Hesch RD, Von Zur Mühlen A 1983 Circannual variations in serum concentrations of pituitary, thyroid, parathyroid, gonadal and adrenal hormones in male laboratory rats. J Endocrinol 97:179–185
Ahlersova E, Ahlers I, Smajda B 1992 Influence of light regimen and time of year on circadian oscillations of insulin and corticosterone in rats. Physiol Res 41:307–314
Ahlersova E, Ahlers I, Smajda B, Kassayova M 1992 The effect of various photoperiods on daily oscillations of serum corticosterone and insulin in rats. Physiol Res 41:315–321
Ahlersova E, Ahlers I, Smajda B 1991 Influence of light regimen and the time of year on circadian oscillations of thyroid hormones in rats. Physiol Rev 40:305–315
Wong CC, D?hler KD, Atkinson MJ, Geerlings H, Hesch RD, Von Zur Mühlen A 1983 Influence of age, strain and season on diurnal periodicity of thyroid stimulating hormone, thyroxine, triiodothyronine and parathyroid hormone in the serum of male laboratory rats. Acta Endocrinol (Copenh) 102:377–385
Ahlersova E, Ahlers I, Kassayova M, Smajda B 1997 Circadian oscillations of serum thyroid hormones in the laboratory rat: the effect of photoperiods. Physiol Res 46:443–449
Kalsbeek A, Fliers E, Franke AN, Wortel J, Buijs RM 2000 Functional connections between the suprachiasmatic nucleus and the thyroid gland as revealed by lesioning and viral tracing techniques in the rat. Endocrinology 141:3832–3841
Luna M, Guzman G, Navarro L, Delapena SS, Valverde C 1995 Circadian rhythm of type II 5'deiodinase activity in the rat hypothalamic-pituitary-adrenal axis. Endocrine 3:597–601
Anguiano B, Quintanar A, Luna M, Navarro L, Ramirez del Angel A, Pacheco P, Valverde C 1995 Neuroendocrine regulation of adrenal gland and hypothalamus 5'deiodinase activity. II. Effects of splanchnicotomy and hypophysectomy. Endocrinology 136:3346–3352
Van Haasteren GAC, Linkels E, Klootwijk W, Van Toor H, Rondeel JMM, Themmen APN, De Jong FH, Valentijn K, Vaudry H, Bauer K, Visser TJ, De Greef WJ 1995 Starvation-induced changes in the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA and the hypothalamic release of proTRH-derived peptides: role of the adrenal gland. J Endocrinol 145:143–153
Samuels MH 2000 Effects of metyrapone administration on thyrotropin secretion in healthy subjects—a clinical research center study. J Clin Endocrinol Metab 85:3049–3052
La Fleur SE, Kalsbeek A, Wortel J, Buijs RM 1999 An SCN generated rhythm in basal glucose levels. J Neuroendocrinol 11:643–652
de Jesus LA, Carvalho SD, Ribeiro MO, Schneider M, Kim SW, Harney JW, Larsen PR, Bianco AC 2001 The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue. J Clin Invest 108:1379–1385
Schneider MJ, Fiering SN, Pallud SE, Parlow AF, St. Germain DL, Galton VA 2001 Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol 15:2137–2148
Granados-Fuentes D, Prolo LM, Abraham U, Herzog ED 2004 The suprachiasmatic nucleus entrains, but does not sustain, circadian rhythmicity in the olfactory bulb. J Neurosci 24:615–619
Langlois MC, Beaudry G, Zekki H, Rouillard C, Levesque D 2001 Impact of antipsychotic drug administration on the expression of nuclear receptors in the neocortex and striatum of the rat brain. Neuroscience 106:117–128
Torra IP, Tsibulsky V, Delaunay F, Saladin R, Laudet V, Fruchart JC, Kosykh V, Staels B 2000 Circadian and glucocorticoid regulation of Rev-erb expression in liver. Endocrinology 141:3799–3806
Preitner N, Damiola F, Molina LL, Zakany J, Duboule D, Albrecht U, Schibler U 2002 The orphan nuclear receptor REV-ERB controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110:251–260
Sato TK, Panda S, Miraglia LJ, Reyes TM, Rudic RD, McNamara P, Naik KA, FitzGerald GA, Kay SA, Hogenesch JB 2004 A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43:527–537
Chawla A, Lazar MA 1993 Induction of Rev-ErbA, an orphan receptor encoded on the opposite strand of the -thyroid hormone receptor gene, during adipocyte differentiation. J Biol Chem 268:16265–16269
Zhou JN, Riemersma RF, Unmehopa UA, Hoogendijk WJ, van Heerikhuize JJ, Hofman MA, Swaab DF 2001 Alterations in arginine vasopressin neurons in the suprachiasmatic nucleus in depression. Arch Gen Psychiatry 58:655–662
Bernstein HG, Heinemann A, Krell D, Mawrin C, Bielau H, Danos P, Diekmann S, Keilhoff G, Bogerts B, Baumann B 2002 Further immunohistochemical evidence for impaired NO signaling in the hypothalamus of depressed patients. Ann NY Acad Sci 973:91–93
Overeem S, van Vliet JA, Lammers GJ, Zitman FG, Swaab DF, Ferrari MD 2002 The hypothalamus in episodic brain disorders. Lancet Neurol 1:437–444
Alkemade A, Unmehopa UA, Brouwer JP, Hoogendijk WJ, Wiersinga WM, Swaab DF, Fliers E 2003 Decreased thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus of patients with major depression. Mol Psychiatry 8:838–839(Andries Kalsbeek, Ruud M.)
Address all correspondence and requests for reprints to: A. Kalsbeek, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: a.kalsbeek@nih.knaw.nl.
Abstract
Type II deiodinase (D2) plays a key role in regulating thyroid hormone-dependent processes in, among others, the central nervous system (CNS) by accelerating the intracellular conversion of T4 into active T3. Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid axis, D2 activity also appears to show daily variations. However, the mechanisms involved in generating these daily variations, especially in the CNS, are not known. Therefore, we decided to investigate the role the master biological clock, located in the hypothalamus, plays with respect to D2 activity in the rat CNS as well as the role of one of its main hormonal outputs, i.e. plasma corticosterone. D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Ablation of the biological clock abolished the daily variations of D2 activity in all four tissues studied. The main effect of the knockout of the suprachiasmatic nuclei (SCN) was a reduction of nocturnal peak levels in D2 activity. Moreover, contrary to previous observations in SCN-intact animals, in SCN-lesioned animals, the decreased levels of D2 activity are accompanied by decreased plasma levels of the thyroid hormones, suggesting that the SCN separately stimulates D2 activity as well as the hypothalamo-pituitary-thyroid axis.
Introduction
IN THE CENTRAL NERVOUS system, availability of the physiologically active thyroid hormone T3 depends mainly on the cellular uptake and intracellular deiodination of T4, which is in clear contrast to the peripheral tissues, in which most of the nuclear-bound T3 is imported from the plasma pool. Two isoenzymes are known to be involved in the conversion of T4 to T3. Type I iodothyronine deiodinase activity is present in the rat thyroid gland, liver, and kidney, whereas type II iodothyronine deiodinase (D2) activity is mainly present in rat brain, anterior pituitary, brown adipose tissue, and pineal gland. The existence of a separate pathway in the brain for T4 deiodination suggests that the adult central nervous system has the ability to autoregulate thyroid status and maintain T3 availability within physiological levels. Indeed, D2 enzyme activity is increased in hypothyroidism and reduced in hyperthyroidism (1, 2, 3, 4).
Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid (HPT) axis, D2 activity, too, has been reported to have daily variations (5, 6, 7, 8, 9, 10, 11). Although, apart from thyroid status, stress (12, 13), feeding (14, 15), and ?-adrenergic mechanisms (5, 6, 7, 9) also may affect D2 activity, the mechanisms involved in generating the daily variation in D2 activity in the central nervous system are not known. Recently it was shown that in both birds and hamsters, hypothalamic D2 expression can be affected by photoperiod (16, 17). In mammals it is likely that the rhythmic change in pineal D2 activity is controlled by a multisynaptic pathway, emanating from the biological clock that is located in the suprachiasmatic nuclei (SCN) and involving subsequent projections to the hypothalamus, the spinal cord, the superior cervical ganglion, and finally a sympathetic projection to the pineal gland, in a way comparable with that described for pineal arylalkylamine N-acetyltransferase, i.e. the enzyme responsible for the large circadian rhythm in melatonin synthesis (9, 18). In addition, D2 activity in the harderian glands, thymus, and brown adipose tissue shows a daily rhythm and is controlled primarily by the sympathetic nervous system (8, 19, 20, 21, 22, 23, 24, 25). But if and how the biological clock could affect D2 activity in brain areas such as cortex and hypothalamus is not known.
Manic-depressive illness and depression have been associated with alterations in both thyroid function and circadian rhythms. Decreased amplitudes of behavioral, physiological, and neuroendocrine circadian measures and disrupted responses of the circadian pacemaker to the light-dark cycle are frequently observed in depression (26, 27, 28, 29). But apart from those circadian disturbances changes in two major endocrine systems also have been demonstrated in depression, i.e. the HPT axis and hypothalamo-pituitary-adrenal (HPA) axis. Low nocturnal plasma TSH levels and/or a decreased amplitude of the daily plasma TSH rhythm occur in some, but not all, depressed patients (28, 30, 31, 32, 33). Moreover, a blunted TSH response to TRH is a consistent finding in approximately 25% of depressed patients (34), and the antidepressant effect of a total night’s sleep deprivation is accompanied by rises in plasma TSH, T3, and T4 (35). In addition, a slight increase of plasma T4 and a slight decrease of plasma T3 levels in depression have been reported (31, 32).
The hypothesis has been put forward that the changes seen in the HPT axis during depression are explained in part by a reduced cerebral conversion of T4 into T3 due to a reduced activity of the D2 enzyme (36). Indeed, some clinical studies suggest a beneficial effect of addition of T3 to treatment with tricyclic antidepressants (37, 38). Most studies investigating a potential therapeutic effect of thyroid hormone in depression have focused on the daily addition of 12.5–25 μg T3 (i.e. a little less than the daily production rate in humans) to antidepressants (mostly tricyclic antidepressants). The usefulness of thyroid hormone addition to antidepressants has remained controversial. Although increased efficacy through T3 addition was suggested by some studies (39), others were negative. A metaanalysis on this subject identified only four small randomized trials (38). On the other hand, exogenous T4 in TSH-suppressive doses also has been reported to be of potential value in the management of patients with treatment refractory bipolar disorder (40). Reduced activity of the D2 enzyme in depressive patients has been assumed to result from increased plasma cortisol levels (36). A mild hypercortisolism is often noted in depression (28, 41), and CRH-expressing neurons in the paraventricular nucleus of the hypothalamus show an increased activity (42, 43, 44). However, although it has been shown that corticosteroids inhibit D2 activity in mouse mammary gland (45) and human placental tissue (46), it is not known whether this also holds for the central nervous system. In fact, removal of circulating corticosteroids in the rat, by adrenalectomy, did not affect D2 activity in the hypothalamus or pituitary (24, 47), whereas corticosterone treatment during early development did not affect pituitary or cortical D2 activity (48). We therefore decided to investigate the separate roles of the biological clock and plasma corticosterone levels on D2 activity in the rat central nervous system.
Materials and Methods
Animals
Male Wistar rats (Harlan, Horst, The Netherlands), housed at a room temperature of 21 ± 1 C with a 12-h light, 12-h dark (L/D) schedule (lights on at 0700 h), were used for all experiments. Light onset was defined as Zeitgeber time (ZT) = 0 and lights off as ZT12. Animals were allowed to adapt to the new environment for at least 2 wk before the first experiments. Animals were kept with four animals per cage with food and water available ad libitum. At the time of the experiments, animals weighed between 300 and 350 g. Postoperative care was provided after each surgical procedure by a sc injection of Temgesic (Reckitt & Colman, Hull, UK; 0.3 ml/kg) after the animals woke up from anesthesia. All of the following experiments were conducted with the approval of the Animal Care Committee of the Royal Netherlands Academy of Sciences.
Experiment 1.
A total of 36 animals was killed (October 2001) at different times of the L/D cycle. After one-by-one transport to a separate room, animals were weighed, anesthetized, and decapitated within 3 min. Groups of six animals were killed at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22. Mixed arteriovenous trunk blood was collected in heparinized tubes and centrifuged for 15 min, 3000 x g at 4 C, to yield plasma that was decanted and stored at –20 C. After decapitation the brain, pituitary, and pineal gland were rapidly removed. Subsequently three transversal brain cuts were made with the help of a simple blocking device (49). The use of the blocking device prevented asymmetrical or oblique cutting and ensured a standardized and reproducible dissection of all the brains. The first cut was located just rostral to the optic chiasm, and the other two cuts were positioned 2 mm caudal and 4 mm rostral from the first cut, resulting in a thalamic, forebrain, and frontal cortex slice. Only the thalamic and frontal cortex slices were used for further analysis. The thalamic slice was dissected further by two vertical cuts just lateral to the optic chiasm and one horizontal cut just dorsal from the third ventricle, separating the medial part of the hypothalamus adjacent to the third ventricle, thus resulting in a hypothalamic slice. Pineal, pituitary, frontal cortex, and medial hypothalamus tissues were collected in Eppendorf tubes, snap frozen in liquid nitrogen, and stored at –80 C.
Experiment 2.
Bilateral thermic lesions of the SCN were made as described previously (50) when animals weighed 180–200 g. A total of 60 rats was operated on to ensure a sufficient number of effectively lesioned animals. After a 2-wk recovery period, the effectiveness of the SCN lesions was checked by measuring their drinking behavior during a 3-wk period. Intact animals consume only 0–5% of their total daily water intake during the middle 8 h of the light period (i.e. between ZT2 and ZT10). SCN lesions were considered successful when an animal drank more than 30% of its daily water intake during this 8-h period. By this method 24 animals were selected as being completely arrhythmic. Previous experiments have shown that the selection of effectively SCN-lesioned animals by their drinking behavior correlates very well, although not completely, with the selection by histological verification of the SCN lesions (50, 51). In the sixth week after the operation (May 2002), groups of effectively SCN-lesioned animals (n = 6) were killed at four different time points along the L/D cycle (i.e. ZT6, ZT10, ZT18, and ZT22) together with a group of unoperated control animals (n = 6). SCN-lesioned and control animals were killed in an alternating order according to the same protocol as described for experiment 1.
Experiment 3.
A pellet (100 mg) containing 100% cholesterol or a mixture of cholesterol and corticosterone was implanted sc in the interscapular region at the back. Pellets were constructed by gently heating pure cholesterol or a mixture of cholesterol and corticosterone in a small stainless steel spoon over a low gas flame until a liquid was formed, which was then poured into in pill mold (52). In a pilot experiment, groups of animals were provided with 0, 50, and 100% corticosterone pellets together with a jugular vein catheter (53) to determine the long-term effects of the pellets on plasma corticosterone concentrations. Based on the results of the pilot experiments, 48 animals were provided with a pellet containing either 0 or 50% corticosterone. Four weeks later (April 2003), animals were killed at four different time points during the L/D cycle (i.e. ZT6, ZT10, ZT18, and ZT22). At every time point, six animals of the 0 and 50% groups were killed in an alternating order as described for experiment 1.
D2 assay
Tissues were homogenized in 10 volumes of 0.1 M phosphate (pH 7.2), 2 mM EDTA, and 1 mM dithiothreitol (P100E2D1 buffer). D2 activity was assayed by measurement of the release of radioiodide from outer ring-labeled T4 as previously described (54). Appropriate dilutions of homogenates were incubated for 60 min at 37 C with 1 nM (105 cpm) [3',5'-125I]T4 in 0.1 ml P100E2D10 in the presence of 100 nM unlabeled T3 to block inner-ring deiodinase (D3) activity. Blank incubations were carried out in the absence of homogenate. Release of 125IG was determined and corrected for nonenzymatic deiodination. Addition of 100 nM unlabeled T4 resulted in major inhibition of radioiodide production, confirming low-Michaelis constant outer-ring deiodination of T4 by D2.
Hormone measurements
Plasma concentrations of the thyroid hormones T3 and T4 were determined by an in-house RIA (55), with inter and intraassay coefficients of variation of 7–8 and 3–4 and 3–6 and 2–4%, respectively. Detection limits for T3 and T4 were 0.3 nmol/liter and 5 nmol/liter. Plasma TSH concentrations were determined by a chemiluminescent immunoassay (IMMULITE, Diagnostic Products Corp., Los Angeles, CA), using a rat-specific standard. The inter- and intraassay coefficients of variation for TSH were less than 4% and less than 2% at ±3.5 ng/ml, and the detection limit was 0.01 ng/ml. Plasma corticosterone was measured directly without extraction, using a RIA from ICN Biomedicals, Inc. (Costa Mesa, CA) with iodinated corticosterone. The inter- and intraassay coefficients of variation for corticosterone were 6.5 and 7.1% at ±150 ng/ml, and the detection limit was 1 ng/ml.
Statistics
The time dependency (i.e. the existence of a daily variation) of D2 activity and hormone concentrations in experiment 1 was analyzed by one-way ANOVA. A two-way ANOVA was used to analyze the differences between experimental groups in experiments 2 (i.e. SCN-lesioned vs. intact) and 3 (i.e. corticosterone-treated vs. cholesterol-treated). The two-way ANOVA used the factors time (four levels) and group (two levels). When significant differences were found, post hoc tests were performed to determine which time points differed significantly from each other (Bonferroni) and at which time points treatment groups differed significantly (unpaired Student’s t test with Bonferroni correction). All data are given as means ± SEM. Results yielding a P < 0.05 were considered significant.
Results
Experiment 1
Although their mean levels showed the expected daily fluctuations, i.e. highest plasma levels of TSH, T3, and T4 during the light period (Fig. 1), none of these three parameters showed significant differences, depending on the time of day (TSH: P = 0.413; T3: P = 0.092; and T4: P = 0.141). Only plasma corticosterone concentrations (Fig. 1C) showed a significant diurnal variation (P < 0.001), with peak concentrations attained at ZT10. On the other hand, D2 activity showed clear effects of time of day in all four tissues studied. Peak activities were attained during the dark period, although most tissues had their own specific peak and trough times (Fig. 2). The most pronounced rhythms were found in the pineal gland and frontal cortex, i.e. P = 0.008 and P < 0.001, respectively. Pineal D2 activity was very low during all of the light period and even during the first part of the dark period (i.e. ZT14). Peak levels were attained at ZT18, and at ZT22, D2 activity was still significantly increased. Cortical D2 activity showed more gradual changes over the L/D cycle, with peak and trough times found at ZT22 and ZT10. The daily peaks of D2 activity rhythms in the pituitary and hypothalamus were located at the beginning and end of the night, respectively. However, the diurnal fluctuations of D2 activity were less pronounced in the pituitary and hypothalamus, i.e. P = 0.037 and P = 0.033, respectively, than in the frontal cortex and pineal gland.
FIG. 1. Twenty-four-hour plasma concentrations of TSH, T4, corticosterone, and T3 in male Wistar rats entrained to a regular 12/12 = L/D cycle before their killing (experiment 1). Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. *, P < 0.05; ***, P < 0.005.
FIG. 2. Twenty-four-hour rhythms of D2 activity in cortical, pineal, pituitary, and medial hypothalamus tissue of male Wistar rats entrained to a regular 12/12 = L/D cycle (experiment 1). Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. Despite a significant time effect for the hypothalamic D2 activity, post hoc testing did not reveal two specific time points that differed significantly. *, P < 0.05; ***, P < 0.005.
Experiment 2
The drinking scores (percentage of daily water intake between ZT2 and ZT10) of the SCN-lesioned animals were 33.6 ± 1.3, 33.0 ± 1.1, 34.7 ± 1.2, and 36.3 ± 1.6% for the groups killed at, respectively, ZT6, ZT10, ZT18, and ZT22. One-way ANOVA indicated the existence of a diurnal variation in SCN-intact animals in plasma corticosterone (P = 0.018) and plasma TSH (P = 0.016) but not for thyroid hormone concentrations in plasma (P > 0.1). In SCN-lesioned animals, none of these parameters showed a significant diurnal variation (all P > 0.05). Two-way ANOVA indicated significant effects of both group and group x time for all four hormonal parameters (Fig. 3 and Table 1). Despite the reduction from six to four sampling points, a significant time dependency in D2 activity could still be recovered in three of the four brain areas of the SCN-intact animals, i.e. pineal, cortex, and hypothalamus (all P < 0.001). In the pituitary, however, the significant rhythm as detected in experiment 1 was lost (P = 0.148), probably because the time of peak activity (i.e. ZT14) was not included in the sampling points of experiment 2. Two-way ANOVA revealed very significant effects of the SCN removal on D2 activity in all three brain areas showing a significant diurnal variation (i.e. for all group and group x time, P < 0.005; see also Table 1), the main effect of the SCN-lesion being a disappearance of the normal nocturnal increase of D2 activity (Fig. 4).
FIG. 3. Plasma concentrations of TSH, T4, corticosterone, and T3 in SCN-lesioned male Wistar rats (light bars) and control rats (dark bars) killed at the same time (experiment 2). All animals were housed in a regular 12/12 = L/D cycle before their killing. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. A bracket with asterisks indicates a time point in which the two experimental groups differ significantly from each other. *, P < 0.05.
TABLE 1. Analysis of the diurnal variations of plasma corticosterone, TSH, T3, and T4 concentrations and D2 activity in cortical, pineal, pituitary, and hypothalamic tissue of SCN-lesioned and intact control animals
FIG. 4. Diurnal rhythms of D2 activity in cortical, pineal, pituitary, and medial hypothalamus tissue of SCN-lesioned male Wistar rats (light bars) and control rats (dark bars) killed at the same time (experiment 2). All animals were housed in a regular 12/12 = L/D cycle. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. A bracket with asterisks indicates a time point in which the two experimental groups differ significantly from each other. *, P < 0.05; **, P < 0.01; ***, P < 0.005.
Experiment 3
In the pilot experiment, animals were provided with jugular vein catheters to allow for repeated blood sampling in conscious and undisturbed animals. Stress-free blood samples were taken at ZT2 and ZT10 twice a week. Results from these pilot experiments showed that the plasma corticosterone concentrations produced by the different pellets were constant for at least 5 wk. During these 5 wk, mean plasma corticosterone concentrations at ZT2 reached 14 ± 1, 47 ± 14, and 132 ± 6 ng/ml for the 0, 50, and 100% pellet groups (n = 4–5 animals), respectively. At ZT10 all three groups had mean plasma corticosterone concentrations greater than 100 ng/ml, i.e. 130 ± 8, 101 ± 12, and 124 ± 12 ng/ml for the 0, 50, and 100% corticosterone groups, respectively. The lack of a dose-dependent effect of the 50 and 100% pellets on the plasma corticosterone concentrations at ZT10 is probably due to a negative feedback on the HPA axis at the level of the hypothalamus and pituitary. In addition, the 100% pellet group showed an almost complete arrest of its body weight development after implantation of the pellet. Five weeks after implantation of the pellet, the body weight increment was 76 ± 7, 56 ± 11, and 6 ± 6 g for the 0, 50, and 100% groups, respectively, despite a similar body weight at the start of the experiment, i.e. 333 ± 2, 335 ± 5, and 337 ± 10 g. Due to the negative effects of the 100% pellets on body weight, in the subsequent experiment, only the 0 and the 50% corticosterone pellets were used.
Neither body weight nor the plasma concentrations of one of the hormones (corticosterone, TSH, T3, and T4) showed clear effects of the corticosterone treatment, i.e. no significant effects of group or group x time (Fig. 5 and Table 2). Significant diurnal variations were observed for plasma corticosterone and T3 concentrations but not plasma T4 (P = 0.077) or TSH (P = 0.117). However, whereas daily fluctuations in plasma corticosterone and T3 concentrations were significant or almost significant in both corticosterone- and cholesterol-treated animals (Table 2), daily fluctuations in plasma T4 concentrations approached significance in only the cholesterol- but not the corticosterone-treated animals (P = 0.066 and P = 0.804, respectively). D2 activity again showed a significant diurnal fluctuation in all four brain areas studied (Fig. 6), but no significant effects of group or group x time were detected. However, the 4-wk corticosterone treatment did cause a disappearance of the daily variation in pituitary D2 activity, as shown by the absence of a significant effect (P = 0.091) of time in the one-way ANOVA.
FIG. 5. Plasma concentrations of TSH, T4, corticosterone, and T3 in corticosterone-treated male Wistar rats (light bars) and sham-treated rats (dark bars) killed at the same time (experiment 3). The ZT2 data in the corticosterone figure are derived from the pilot experiment. All animals were housed in a regular 12/12 = L/D cycle before their killing. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value.*, P < 0.05; **, P < 0.01.
TABLE 2. Analysis of the diurnal variations of plasma corticosterone, TSH, T3, and T4 concentrations and D2 activity in cortical, pineal, pituitary, and hypothalamic tissue of corticosterone- and cholesterol-treated animals
FIG. 6. Diurnal rhythms of D2 activity in cortical, pineal, pituitary, and medial hypothalamus tissue of corticosterone-treated male Wistar rats (light bars) and sham-treated rats (dark bars) killed at the same time (experiment 3). All animals were housed in a regular 12/12 = L/D cycle. Horizontal black bars indicate the dark phase of the L/D cycle. Data are mean ± SEM (bars) values of six animals. P values in the upper left corner indicate the significance of the one-way ANOVA. Asterisks indicate time points that differ significantly from the peak value. *, P < 0.05; **, P < 0.01; ***, P < 0.005.
Experiments 2 and 3 were performed in spring (April/May), whereas experiment 1 was performed in late autumn (October/November). Previously significant seasonal variations of both the HPA axis (56, 57, 58) and HPT axis (56, 59, 60, 61) as well as D2 activity have been reported (17). Nevertheless, in the present experiment, only plasma T3 concentrations showed a consistent seasonal variation, with higher concentrations in autumn than spring. Also, in the studies just cited, the most pronounced effects of season (or photoperiod) were found for plasma T3 concentrations. However, the changes reported by Wong et al. (60), i.e. higher plasma T3 concentrations in spring than in winter, were exactly the opposite of our results. On the other hand, Ahlersova et al. (61) found no differences between long and short photoperiods (i.e. L/D = 16/8 and L/D = 8/16, respectively) but found the highest plasma T3 concentrations during the L/D = 12/12 photoperiod. Moreover, because in our study the samples from the different experiments were not analyzed in one assay, we cannot be completely sure the variations are really due to seasonal changes.
Discussion
D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as in the hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Removal of the master biological clock, located in the hypothalamus, abolished the daily fluctuations of D2 activity in all four tissues studied, with the main effect of the SCN knockout being a reduction of nocturnal peak levels. Remarkably, previously (but also in the present study), we found that the main effect of SCN lesions on plasma TSH and thyroid hormone levels is also a disappearance of the diurnal peak and a reduction of the daily mean (62). By contrast, reducing the amplitude of the daily corticosterone rhythm during 4 wk through an elevation of its daily trough levels only had minor effects on the daily rhythm of D2 activity in the different tissues. Only in the pituitary and medial hypothalamus did the corticosterone treatment tend to reduce the amplitude of the daily rhythm in D2 activity. The present results thus show that a removal of the biological clock has pronounced effects on the activity of the D2 enzyme, whereas a slight elevation of the basal plasma corticosterone concentrations affects D2 activity only to a minor degree.
The presently reported daily rhythms of D2 activity in general confirm the results of previous studies on the pituitary, the hypothalamus, cortex, and pineal gland (5, 6, 8, 9, 10, 11, 18, 20, 63). Our results on the SCN-lesioned animals show that the daily rhythms in D2 activity were abolished in all four tissues investigated, as would be expected from a circadian rhythm controlled by the SCN. Moreover, these results confirm the previously indicated circadian nature of the D2 rhythms in the pineal gland and pituitary. However, with the exception of sympathetic tissues, such as the pineal and harderian glands, there is no indication to date how the circadian rhythms in pituitary and brain D2 activity are controlled by the central pacemaker. Apart from the autonomic nervous system, hormones, such as the thyroid hormones and glucocorticoids, also have been proposed as mediators of SCN output. However, because these hormonal rhythms are also abolished in SCN-lesioned animals, the SCN-lesion experiment gives no indication as to the importance of these hormonal factors.
Due to its pronounced circadian rhythmicity, we decided to investigate the importance of corticosterone as a mediator of SCN output. Thus far, in a number of animal tissues, no clear effects of corticosterone on D2 activity could be found (7, 12, 13, 24, 48), with the notable exception of D2 in the adrenal gland (64). On the other hand, two studies (45, 46) using cultured human placental and mouse mammary epithelium cells showed inhibitory effects of glucocorticoids on D2 activity. Our pilot experiments indicated a clear and sustained elevation of the daily trough levels of plasma corticosterone by the implantation of a sc pellet containing 50% corticosterone. Because the pellets containing 100% corticosterone resulted in an almost complete arrest of the normal increase in body weight, we provided our experimental animals with pellets containing only 50% corticosterone. The moderate increase during 4 wk, of basal corticosterone levels appeared to cause only minor changes in the daily rhythms of D2 activity. Unfortunately, the time points selected for killing the animals (i.e. the trough and acrophase of the daily rhythm in D2 activity) did not include the daily trough of the corticosterone rhythm (i.e. ZT2), and the significant effect of the corticosterone treatment on basal plasma corticosterone concentrations, as found in the pilot experiment, could thus not be demonstrated in the killed animals. Moreover, despite the well-known inhibitory effect of high plasma corticosterone levels on the HPT axis (65, 66), plasma TSH and thyroid hormone concentrations were not affected by the corticosterone treatment. Therefore, it is not clear yet whether the absence of a clear effect of our corticosterone treatment on the D2 activity rhythms indicates that corticosterone is not involved in the propagation of the SCN rhythm or whether the lack of effect is due to the minor disturbance in the corticosterone rhythm introduced by our treatment. In addition, a pilot experiment investigating D3 activity in the cortical tissue of the corticosterone- and cholesterol-treated animals did not reveal any significant effects of either time or group either (data not shown).
As documented before, effective SCN lesions result in the disappearance of all daily rhythms. In general, SCN lesions disturb only the temporal distribution of a certain parameter but not its overall level. For instance the mean 24-h food intake, water consumption, plasma corticosterone, and plasma glucose concentrations are quite similar in SCN-lesioned and control animals (51, 67). On the other hand, for some parameters SCN lesions may cause a clear increase or decrease of the 24-h means, as we recently demonstrated for plasma leptin (50) and plasma melatonin (51) concentrations. Apart from the disappearance of the daily rhythms of D2 activity, the SCN lesions also resulted in an overall decrease of daily D2 activity, not only in the pineal gland but also in the hypothalamic and cortical tissue. In agreement with our previous study (62), plasma TSH and especially thyroid hormone concentrations also were decreased in the SCN-lesioned animals. However, a direct control of the daily D2 activity rhythm in brain tissue by thyroid hormones seems unlikely. Indeed, hypothyroid rats show a pronounced increase, instead of a decrease, of hypothalamic and cortical D2 activity (1, 3). On the other hand, a primary and sole effect of the SCN lesion to decrease D2 activity also seems unlikely because then an increased release of TSH, and subsequently the thyroid hormones, would have been expected due to a diminished inhibitory feedback of the thyroid hormones at the level of the pituitary and hypothalamus. Indeed, D2 knockout mice show elevated plasma levels of T4 and TSH (68, 69). Apparently SCN activity provides a stimulatory input to both the HPT axis and the activity of the D2 enzyme. The absence of a stimulatory SCN input to the TRH neurons prevents an up-regulation of TSH release as a result of a diminished T3 feedback to the hypothalamus and pituitary (62). On the other hand, an altered D3 activity also could contribute to the changes observed. Although we do not know whether, and if yes, how the SCN (lesions) affected D3 activity, pilot experiments (as mentioned before) did not show daily fluctuations in cortical D3 activity.
Our results, as well as most of the previous results, were obtained in regular L/D conditions and therefore as such do not indicate whether the daily rhythms in D2 activity are endogenous or an (in)direct consequence of the L/D cycle. Apart from the pineal gland, thus far the endogenous nature of the daily D2 fluctuation has been demonstrated clearly only for the pituitary (20). Nocturnal light exposure or extension of the light phase into the dark period of the L/D cycle prevents the normal nocturnal increase of D2 activity in sympathetic tissues such as the pineal, thymus, and harderian glands (5, 6, 9, 20, 22, 24, 25) but also in the pituitary (20). On the other hand, in the frontal cortex, extension of the light phase into the dark period of the L/D cycle significantly enhanced D2 activity (5), whereas continuous darkness abolished the daily rhythm of D2 activity in both the frontal cortex and hypothalamus (5, 63). Moreover, recently it was found that D2 expression in the quail hypothalamus can be induced by light exposure (16), indicating that daily rhythms in D2 activity might indeed not be endogenous (i.e. circadian) but light driven. However, in the Djungarian hamster, the photoperiodic D2 expression was not driven directly by light exposure (17). In addition, because our SCN-lesioned animals were housed in L/D = 12/12 conditions, the disappearance of the daily rhythms of D2 activity in these animals strongly suggest that they are not light driven but endogenous and originating from the SCN. On the other hand, for the pineal rhythms of D2 (but also arylalkylamine N-acetyltransferase) expression, it is well known that they are both circadian and sensitive to light (9).
A direct effect of light on D2 activity mediated by the SCN or involving direct projections of the retina to the hypothalamus and cortex that are damaged by the SCN lesion can thus not be excluded completely. However, in view of the differential timing of the acrophase in D2 activity in the different brain structures, this possibility does not seem very likely either. Moreover, if SCN lesions are blocking a direct effect of light, we would expect increased, instead of decreased, 24-h means in SCN-lesioned animals because in intact animals the lowest levels of D2 activity are in general found during the light period. Therefore, with the exception of sympathetic tissues, such as the pineal and harderian glands, there is no indication to date how the circadian rhythms in pituitary and brain D2 activity are generated. On the other hand, the recent discovery that the SCN is not required for all 24-h timekeeping functions in individual tissues (70) indicates the possibility that daily rhythms of D2 activity in the cortex, hypothalamus, and pituitary are mainly sustained by tissue-specific clock gene expression and are only loosely connected to the SCN. Interestingly, the expression of the thyroid hormone receptor (TR) as well as that of the transcription factor Rev-erb in the central nervous system is modulated by antipsychotic drugs (71). Moreover, in mammals Rev-erb is an integral component of the biological clock (72, 73, 74) and is involved in the splicing of the TR1 and TR2 isoforms (75). However, at present there is no evidence linking the (rhythmic) activity of the Rev-erb gene to the control of D2 activity.
Thyroid hormone comedication with antidepressants may be an effective method to increase response rate in treatment-resistant depressive patients. It was hypothesized earlier that the beneficial effect of T3 treatment is due to a mild T3 deficiency in the central nervous system as a consequence of reduced D2 activity. The proposed inhibition of D2 activity has been assumed to result from elevated plasma cortisol concentrations (36, 37, 38). The plasma corticosterone concentrations produced by our treatment protocol nicely reflected the daily corticosterone rhythm as often observed in depressed patients, i.e. increased basal trough concentrations and a diminished amplitude (28, 41). Thus, according to the present results, the disturbed corticosterone rhythm in depressed patients probably presents only a minor contribution to the putatively reduced T3 bioavailability in these patients. Therefore, with regard to the often observed low plasma T3 concentrations in depressed patients and the proposed decreased D2 activity, it seems more likely that this is due to a lesser activity of the SCN (76, 77, 78) than to a slight hypercortisolism. In fact, the reduced SCN activity in depression might even be responsible for the decreased TRH gene expression in the paraventricular nucleus of the hypothalamus (79).
In conclusion, the present study provides further evidence for the endogenous nature of the daily rhythms of D2 activity in the cortex, hypothalamus, and pituitary. However, it is not yet clear how the SCN controls these rhythms because no clear effects of light and plasma corticosterone on D2 activity could be detected.
Acknowledgments
We thank Wilma Verweij for correcting our English and Henk Stoffels for making the figures. Eric Endert and the staff of the endocrinology laboratory are gratefully acknowledged for performing the TSH and thyroid hormone assays, as is the expert assistance of Jilles Timmer in animal husbandry.
References
Burmeister LA, Pachucki J, St. Germain DL 1997 Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138:5231–5237
Riskind PN, Kolodny JM, Larsen PR 1987 The regional hypothalamic distribution of type II 5'-monodeiodinase in euthyroid and hypothyroid rats. Brain Res 420:194–198
Tu HM, Kim SW, Salvatore D, Bartha T, Legradi G, Larsen PR, Lechan RM 1997 Regional distribution of type 2 thyroxine deiodinase messenger ribonucleic acid in rat hypothalamus and pituitary and its regulation by thyroid hormone. Endocrinology 138:3359–3368
Visser TJ, Leonard JL, Kaplan MM, Larsen PR 1982 Kinetic evidence suggesting two mechanisms for iodothyronine 5'-deiodination in rat cerebral cortex. Proc Natl Acad Sci USA 79:5080–5084
Guerrero JM, Puig-Domingo M, Reiter RJ 1988 Thyroxine 5'-deiodinase activity in pineal gland and frontal cortex: nighttime increase and the effect of either continuous light exposure or superior cervical ganglionectomy. Endocrinology 122:236–241
Murakami M, Greer MA, Greer SE, Hjulstad S, Tanaka K 1989 Comparison of the nocturnal temporal profiles of N-acetyl-transferase and thyroxine 5'-deiodinase in rat pineal. Neuroendocrinology 50:88–92
Jimenez J, Osuna C, Reiter RJ, Rubio A, Guerrero JM 1993 Adrenalectomy or superior cervical ganglionectomy modifies the nocturnal increase in rat pineal type-II thyroxine 5'-deiodinase. Chronobiol Int 10:87–93
Soutto M, Guerrero JM, Osuna C, Molinero P 1998 Nocturnal increases in the triiodothyronine/thyroxine ratio in the rat thymus and pineal gland follow increases of type II 5'-deiodinase activity. Int J Biochem Cell Biol 30:235–241
Kamiya Y, Murakami M, Araki O, Hosoi Y, Ogiwara T, Mizuma H, Mori M 1999 Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by ?-adrenergic mechanism in the rat pineal gland. Endocrinology 140:1272–1278
Murakami M, Tanaka K, Greer MA 1988 There is a nyctohemeral rhythm of type II iodothyronine 5'-deiodinase activity in rat anterior pituitary. Endocrinology 123:1631–1635
Campos-Barros A, Musa A, Flechner A, Hessenius C, Gaio U, Meinhold H, Baumgartner A 1997 Evidence for circadian variations of thyroid hormone concentrations and type II 5'-iodothyronine deiodinase activity in the rat central nervous system. J Neurochem 68:795–803
Baumgartner A, Hiedra L, Pinna G, Eravci M, Prengel H, Meinhold H 1998 Rat brain type II 5'-iodothyronine deiodinase activity is extremely sensitive to stress. J Neurochem 71:817–826
Bianco AC, Nunes MT, Hell NS, Maciel RM 1987 The role of glucocorticoids in the stress-induced reduction of extrathyroidal 3,5,3'-triiodothyronine generation in rats. Endocrinology 120:1033–1038
Diano S, Naftolin F, Goglia F, Csernus V, Horvath TL 1998 Monosynaptic pathway between the arcuate nucleus expressing glial type II iodothyronine 5'-deiodinase mRNA and the median eminence projective TRH cells of the rat paraventricular nucleus. J Neuroendocrinol 10:731–742
Glick Z, Wu SY, Lupien J, Reggio R, Bray GA, Fisher DA 1985 Meal-induced brown fat thermogenesis and thyroid hormone metabolism in rats. Am J Physiol 249:E519–E524
Yoshimura T, Yasuo S, Watanabe M, Iigo M, Yamamura T, Hirunagi K, Ebihara S 2003 Light-induced hormone conversion of T4 to T3 regulates photoperiodic response of gonads in birds. Nature 426:178–181
Watanabe M, Yasuo S, Watanabe T, Yamamura T, Nakao N, Ebihara S, Yoshimura T 2004 Photoperiodic regulation of type 2 deiodinase gene in Djungarian hamster: possible homologies between avian and mammalian photoperiodic regulation of reproduction. Endocrinology 145:1546–1549
Murakami M, Greer MA, Hjulstad S, Greer SE, Tanaka K 1988 The role of the superior cervical ganglia in the nocturnal rise of pineal type-II thyroxine 5'-deiodinase activity. Brain Res 438:366–368
Silva JE, Larsen PR 1983 Adrenergic activation of triiodothyronine production in brown adipose tissue. Nature 305:712–713
Murakami M, Greer MA, Greer SE, Hjulstad S, Tanaka K 1988 Effect of short-term constant light or constant darkness on the nyctohemeral rhythm of type-II iodothyronine 5'-deiodinase activity in rat anterior pituitary and pineal. Life Sci 42:1875–1879
Eley J, Himms-Hagen J 1989 Brown adipose tissue of mice with GTG-induced obesity: altered circadian control. Am J Physiol 256:773–779
Guerrero JM, Gonzalez-Brito A, Santana C, Reiter RJ 1989 Nocturnal increase of type II thyroxine 5'-deiodinase activity in the Syrian hamster harderian gland is abolished by light exposure and induced by isoproterenol. Proc Soc Exp Biol Med 190:186–189
Kates AL, Himms-Hagen J 1990 Defective regulation of thyroxine 5'-deiodinase in brown adipose tissue of ob/ob mice. Am J Physiol 258:E7–E15
Osuna C, Jimenez J, Reiter RJ, Rubio A, Guerrero JM 1992 Adrenergic regulation of type II 5'-deiodinase circadian rhythm in rat harderian gland. Am J Physiol 263:E884–E889
Soutto M, Molinero P, Guerrero JM 1997 Continuous light exposure modifies the nocturnal increase in rat thymus type II thyroxine 5'-deiodinase. Cell Mol Life Sci 53:697–699
McEachron DL, Schull J 1993 Hormones, rhythms, and the blues. In: Schulkin J, ed. Hormonally induced changes in mind and brain. San Diego: Academic Pres Inc.; 287–355
Healy D, Waterhouse JM 1995 The circadian system and the therapeutics of the affective disorders. Pharmacol Therapeut 65:241–263
Duncan WC 1996 Circadian rhythms and the pharmacology of affective illness. Pharmacol Therapeut 71:253–312
Van Cauter E, Turek FW 1986 Depression: a disorder of timekeeping? Perspect Biol Med 29:510–519
Weeke A, Weeke J 1978 Disturbed circadian variation of serum thyrotropin in patients with endogenous depression. Acta Psychiatr Scand 57:281–289
Wilson DA, Mulder RT, Joyce PR 1992 Diurnal profiles of thyroid hormones are altered in depression. J Affect Disord 24:11–16
Maes M, Meltzer HY, Cosyns P, Suy E, Schotte C 1993 An evaluation of basal hypothalamic-pituitary-thyroid axis function in depression: results of a large-scaled and controlled study. Psychoneuroendocrinology 18:607–620
Duval F, Mokrani MC, Crocq MA, Jautz M, Bailey P, Diep TS, Macher JP 1996 Effect of antidepressant medication on morning and evening thyroid function tests during a major depressive episode. Arch Gen Psychiatry 53:833–840
Lenzinger E, Meszaros K, Hornik K, Parzer P, Hollerer E, Langer G, Resch F, Legros JJ 1996 Correlation between vasopressin baseline and TSH-blunting in depressives. Biol Psychiatry 39:341–345
Baumgartner A, Riemann D, Berger M 1990 Neuroendocrinological investigations during sleep deprivation in depression. II. Longitudinal measurement of thyrotropin, TH, cortisol, prolactin, GH, and LH during sleep and sleep deprivation. Biol Psychiatry 28:569–587
Jackson IM 1996 Does thyroid hormone have a role as adjunctive therapy in depression? Thyroid 6:63–67
Kirkegaard C, Faber J 1998 The role of thyroid hormones in depression. Eur J Endocrinol 138:1–9
Aronson R, Offman HJ, Joffe RT, Naylor CD 1996 Triiodothyronine augmentation in the treatment of refractory depression. A meta-analysis. Arch Gen Psychiatry 53:842–848
Altshuler LL, Bauer M, Frye MA, Gitlin MJ, Mintz J, Szuba MP, Leight KL, Whybrow PC 2001 Does thyroid supplementation accelerate tricyclic antidepressant response? A review and meta-analysis of the literature. Am J Psychiatry 158:1617–1622
Bauer MS, Whybrow PC 1990 Rapid cycling bipolar affective disorder. II. Treatment of refractory rapid cycling with high-dose levothyroxine: a preliminary study. Arch Gen Psychiatry 47:435–440
Dahl RE, Ryan ND, Puig-Antich J, Nguyen NA, Al-Shabbout M, Meyer VA, Perel J 1991 24-Hour cortisol measures in adolescents with major depression: a controlled study. Biol Psychiatry 30:25–36
Raadsheer FC, Hoogendijk WJ, Stam FC, Tilders FJ, Swaab DF 1994 Increased numbers of corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus of depressed patients. Neuroendocrinology 60:436–444
Raadsheer FC, Van Heerikhuize JJ, Lucassen PJ, Hoogendijk WJG, Tilders FJH, Swaab DF 1995 Corticotropin-releasing hormone mRNA levels in the paraventricular nucleus of patients with Alzheimer’s disease and depression. Am J Psychiatry 152:1372–1376
Wong ML, Kling MA, Munson PJ, Listwak S, Licinio J, Prolo P, Karp B, McCutcheon IE, Geracioti Jr TD, DeBellis MD, Rice KC, Goldstein DS, Veldhuis JD, Chrousos GP, Oldfield EH, McCann SM, Gold PW 2000 Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: relation to hypercortisolism and corticotropin-releasing hormone. Proc Natl Acad Sci USA 97:325–330
Song S, Oka T 2003 Regulation of type II deiodinase expression by EGF and glucocorticoid in HC11 mouse mammary epithelium. Am J Physiol 284:E1119–E1124
Hidal JT, Kaplan MM 1988 Inhibition of thyroxine 5'-deiodination type II in cultured human placental cells by cortisol, insulin, 3',5'-cyclic adenosine monophosphate, and butyrate. Metabolism 37:664–668
Anguiano B, Valverde C 2001 Cold-induced increment in rat adrenal gland type II deiodinase is corticosterone dependent. Endocrine 15:87–91
McCann UD, Shaw EA, Kaplan MM 1984 Iodothyronine deiodination reaction types in several rat tissues: effects of age, thyroid status, and glucocorticoid treatment. Endocrinology 114:1513–1521
Kalsbeek A, Matthijssen MA, Uylings HB 1989 Morphometric analysis of prefrontal cortical development following neonatal lesioning of the dopaminergic mesocortical projection. Exp Brain Res 78:279–289
Kalsbeek A, Fliers E, Romijn JA, La Fleur SE, Wortel J, Bakker O, Endert E, Buijs RM 2001 The suprachiasmatic nucleus generates the diurnal changes in plasma leptin levels. Endocrinology 142:2677–2685
Perreau-Lenz S, Kalsbeek A, Garidou ML, Wortel J, Van Der Vliet J, Van Heijningen C, Simonneaux V, Pévet P, Buijs RM 2003 Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms. Eur J Neurosci 17:221–228
Akana SF, Scribner KA, Bradbury MJ, Strack AM, Walker CD, Dallman MF 1992 Feedback sensitivity of the rat hypothalamo-pituitary-adrenal axis and its capacity to adjust to exogenous corticosterone. Endocrinology 131:585–594
Steffens AB 1969 A method for frequent sampling blood and continuous infusion of fluids in the rat without disturbing the animal. Physiol Behav 4:833–836
Richard K, Hume R, Kaptein E, Sanders JP, van Toor H, De Herder WW, den Hollander JC, Krenning EP, Visser TJ 1998 Ontogeny of iodothyronine deiodinases in human liver. J Clin Endocrinol Metab 83:2868–2874
Wiersinga WM, Chopra IJ 1982 Radioimmunoassays of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'diiodothyronine (T2). Methods Enzymol 84:272–303
Wong CC, Dohler KD, Atkinson MJ, Geerlings H, Hesch RD, Von Zur Mühlen A 1983 Circannual variations in serum concentrations of pituitary, thyroid, parathyroid, gonadal and adrenal hormones in male laboratory rats. J Endocrinol 97:179–185
Ahlersova E, Ahlers I, Smajda B 1992 Influence of light regimen and time of year on circadian oscillations of insulin and corticosterone in rats. Physiol Res 41:307–314
Ahlersova E, Ahlers I, Smajda B, Kassayova M 1992 The effect of various photoperiods on daily oscillations of serum corticosterone and insulin in rats. Physiol Res 41:315–321
Ahlersova E, Ahlers I, Smajda B 1991 Influence of light regimen and the time of year on circadian oscillations of thyroid hormones in rats. Physiol Rev 40:305–315
Wong CC, D?hler KD, Atkinson MJ, Geerlings H, Hesch RD, Von Zur Mühlen A 1983 Influence of age, strain and season on diurnal periodicity of thyroid stimulating hormone, thyroxine, triiodothyronine and parathyroid hormone in the serum of male laboratory rats. Acta Endocrinol (Copenh) 102:377–385
Ahlersova E, Ahlers I, Kassayova M, Smajda B 1997 Circadian oscillations of serum thyroid hormones in the laboratory rat: the effect of photoperiods. Physiol Res 46:443–449
Kalsbeek A, Fliers E, Franke AN, Wortel J, Buijs RM 2000 Functional connections between the suprachiasmatic nucleus and the thyroid gland as revealed by lesioning and viral tracing techniques in the rat. Endocrinology 141:3832–3841
Luna M, Guzman G, Navarro L, Delapena SS, Valverde C 1995 Circadian rhythm of type II 5'deiodinase activity in the rat hypothalamic-pituitary-adrenal axis. Endocrine 3:597–601
Anguiano B, Quintanar A, Luna M, Navarro L, Ramirez del Angel A, Pacheco P, Valverde C 1995 Neuroendocrine regulation of adrenal gland and hypothalamus 5'deiodinase activity. II. Effects of splanchnicotomy and hypophysectomy. Endocrinology 136:3346–3352
Van Haasteren GAC, Linkels E, Klootwijk W, Van Toor H, Rondeel JMM, Themmen APN, De Jong FH, Valentijn K, Vaudry H, Bauer K, Visser TJ, De Greef WJ 1995 Starvation-induced changes in the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA and the hypothalamic release of proTRH-derived peptides: role of the adrenal gland. J Endocrinol 145:143–153
Samuels MH 2000 Effects of metyrapone administration on thyrotropin secretion in healthy subjects—a clinical research center study. J Clin Endocrinol Metab 85:3049–3052
La Fleur SE, Kalsbeek A, Wortel J, Buijs RM 1999 An SCN generated rhythm in basal glucose levels. J Neuroendocrinol 11:643–652
de Jesus LA, Carvalho SD, Ribeiro MO, Schneider M, Kim SW, Harney JW, Larsen PR, Bianco AC 2001 The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue. J Clin Invest 108:1379–1385
Schneider MJ, Fiering SN, Pallud SE, Parlow AF, St. Germain DL, Galton VA 2001 Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol 15:2137–2148
Granados-Fuentes D, Prolo LM, Abraham U, Herzog ED 2004 The suprachiasmatic nucleus entrains, but does not sustain, circadian rhythmicity in the olfactory bulb. J Neurosci 24:615–619
Langlois MC, Beaudry G, Zekki H, Rouillard C, Levesque D 2001 Impact of antipsychotic drug administration on the expression of nuclear receptors in the neocortex and striatum of the rat brain. Neuroscience 106:117–128
Torra IP, Tsibulsky V, Delaunay F, Saladin R, Laudet V, Fruchart JC, Kosykh V, Staels B 2000 Circadian and glucocorticoid regulation of Rev-erb expression in liver. Endocrinology 141:3799–3806
Preitner N, Damiola F, Molina LL, Zakany J, Duboule D, Albrecht U, Schibler U 2002 The orphan nuclear receptor REV-ERB controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110:251–260
Sato TK, Panda S, Miraglia LJ, Reyes TM, Rudic RD, McNamara P, Naik KA, FitzGerald GA, Kay SA, Hogenesch JB 2004 A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43:527–537
Chawla A, Lazar MA 1993 Induction of Rev-ErbA, an orphan receptor encoded on the opposite strand of the -thyroid hormone receptor gene, during adipocyte differentiation. J Biol Chem 268:16265–16269
Zhou JN, Riemersma RF, Unmehopa UA, Hoogendijk WJ, van Heerikhuize JJ, Hofman MA, Swaab DF 2001 Alterations in arginine vasopressin neurons in the suprachiasmatic nucleus in depression. Arch Gen Psychiatry 58:655–662
Bernstein HG, Heinemann A, Krell D, Mawrin C, Bielau H, Danos P, Diekmann S, Keilhoff G, Bogerts B, Baumann B 2002 Further immunohistochemical evidence for impaired NO signaling in the hypothalamus of depressed patients. Ann NY Acad Sci 973:91–93
Overeem S, van Vliet JA, Lammers GJ, Zitman FG, Swaab DF, Ferrari MD 2002 The hypothalamus in episodic brain disorders. Lancet Neurol 1:437–444
Alkemade A, Unmehopa UA, Brouwer JP, Hoogendijk WJ, Wiersinga WM, Swaab DF, Fliers E 2003 Decreased thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus of patients with major depression. Mol Psychiatry 8:838–839(Andries Kalsbeek, Ruud M.)