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Netherlands Institute for Brain Research (A.K., A.N.F., J.W., R.M.B.), Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands; Academic Medical Center (E.F.), Department of Endocrinology and Metabolism, F5–171, Meibergdreef 9, 1105 AZ Amsterdam
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{at}nih.knaw.nl
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Introduction |
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While not as readily apparent as the behavioral changes, also the internal environment of the organism adapts to the 24-h light/dark cycle and undergoes pronounced daily fluctuations. It now appears that the release of probably all hormones, both pituitary-dependent ones and hormones that are not directly related to the hypothalamic-pituitary axis, is affected by the circadian clock system one way or another. In a number of cases, the effect of removal of the biological clock (i.e. SCN), on these rhythms is clearly documented (1, 2). Because it has been impossible to reinstate hormonal rhythms by SCN transplantation (3), the neural projections of the SCN seem to be a pivotal controlling factor. In the case of pituitary hormones such as LH and ACTH, detailed information is available showing the involvement of specific SCN transmitters, projections, and target areas in the circadian control of these hormones (4, 5). Next to a direct SCN control of the releasing factor-containing neuroendocrine "motorneurons" (6, 7), several studies now have demonstrated that the SCN affects peripheral organs also by the autonomic nervous system. This neuronal influence not only holds for nonpituitary-dependent hormones such as melatonin and insulin (8, 9, 10), but also for the adrenal cortical hormone corticosterone (4, 11, 12).
Surprisingly little is known, still, about the daily rhythmicity of the thyroidal axis. In humans, a daily TSH rhythm, with higher levels during the night, is well known (13). Despite the pronounced rhythm in plasma TSH, no rhythms have been reported in thyroid hormones (14). Comparing data across different species indicates that the major period of thyroid activity aligns with the sleep period. Indeed, in rats most studies show that daytime concentrations of TSH are usually higher than nighttime levels, but animal studies showing a clear daily pattern of thyroid hormone plasma concentrations are scarce (15, 16, 17, 18, 19). In fact, the definitive proof for the circadian nature of thyroid activity is still lacking because, as far as we know, no studies have been reported yet showing a rhythm in plasma TSH or thyroid hormones during constant conditions. The effect of SCN ablation on TSH levels, but not on thyroid hormones, has been described in two studies (20, 21).
The present study was initiated to describe in more detail: 1) the daily rhythmicity of plasma TSH and thyroid hormones, and 2) the effects of SCN removal on plasma TSH and thyroid hormone levels. However, the minor effects of SCN-lesions on TSH release indicated that apart from the neuroendocrine pathway, the SCN might use additional (neuronal) pathways to control circadian thyroid activity. Despite the fact that in many species a dense innervation of the thyroid gland has been shown, by nerves originating from both sympathetic and parasympathetic ganglia, virtually nothing is known about the central nuclei that control the autonomic innervation or its functional significance (22). Therefore, we extended the study to describe also 3) the central origin of the autonomic innervation of the thyroid gland using transsynaptic virus tracing.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Operations
For SCN-lesions, 30 animals of 180–200 g, anesthetized with
Hypnorm (Duphar, The Netherlands; 0.6 ml/kg, im) and Dormicum
(Roche, The Netherlands; 0.4 ml/kg sc), were mounted with
their heads in a David Kopf stereotact with the toothbar set at +5.0
mm, and sustained a bilateral lesion of the SCN (coordinates: 1.4 mm
rostral to bregma; 1.1 lateral to the midline; 8.3 mm below the brain
surface) using bilateral lesion electrodes, 0.2 mm in diameter, with
temperature set at 85 C for 1 min (lesion generator, Radiotronics).
This temperature was found empirically to result in lesions large
enough to eliminate the SCN bilaterally, but small enough to leave the
surrounding tissue and PVN intact. After a 2-week recovery period, the
effectiveness of the lesions was checked by measuring water intake
during the middle 8 h of the light period (0900–1700 h) during a
3-week period. Animals showing a water intake of >30% during the
middle part of the light period were considered to have complete
lesions of the SCN. After the experiments, SCN-lesions were checked
histologically by immunocytochemical staining of hypothalamic sections
for the presence of VP and/or vasoactive intestinal polypeptide (VIP)
containing cell bodies or fibers in the SCN area or its target areas,
respectively (for details see Ref. 5). For undisturbed
blood sampling, both SCN-lesioned animals showing >30% daytime
drinking, and SCN-intact animals were provided with a permanent silicon
heart cannula (id 0.5 mm; od 1.0 mm) into the entrance of the right
atrium (vena cava) via an external jugular venotomy according to the
method of Steffens (23). The cannula was externalized and
fixed on top of the skull with three screws and dental cement. Animals
were operated when their body weight had reached 300–325 g.
Twenty-four hour profiles
After the operation, a 7-day recovery period was included to
allow complete reinstatement of circadian rhythms in activity, body
temperature, and plasma corticosterone. During this period, animals
became accustomed to the experimental conditions in Plexiglass cages,
designed to allow blood sampling under unrestrained conditions. All
experiments were performed in the animals’ own home cage. Tubings were
threaded through a stainless steel support spring that was attached to
the dental cement on the skull. The entire assembly was suspended from
the animal by a counter-balanced beam and did not interfere with the
animals’ behavior. Daily changes in the plasma levels of
corticosterone, TSH, T3, and
T4 were assessed in two experimental sessions of
12 h each by taking a 0.6 ml blood sample every 2 h. Blood
samples were collected in heparinized tubes placed on ice and
centrifuged, and plasma was stored at -20 C until assay.
Transneuronal tracing
Male animals (n = 23) were anesthetized with a mixture of
Hypnorm (Duphar, The Netherlands; 0.6 ml/kg, im) and Dormicum
(Roche, The Netherlands; 0.4 ml/kg sc). Two to 5 µl
of the viral suspension of pseudo-rabies virus (PRV), Bartha strain
(containing 1 x 106 plague-forming units),
a generous gift of Dr. C. E. Jacobse (Institute for Animal Science
and Health, Agricultural Research Department, Lelystad, The
Netherlands), was pressure-injected into the left thyroid gland with a
30-gauge needle attached to a Hamilton syringe. Evans Blue was added to
the viral suspension to allow visual inspection of the injection site.
To evaluate possible effects of leakage, control animals received a
similar amount of virus on top of the thyroid gland without the needle
entering it (n = 3) and survived for 4 days. All animals were
allowed to survive exactly 2 (n = 5), 21/2 (n = 6), 3
(n = 5) 31/2 (n = 2), or 4 days (n = 2), after
which they were killed by deep anesthesia with sodium pentobarbital and
perfused through the left ventricle of the heart with saline followed
by a solution of 4% paraformaldehyde and 0.15% glutaraldehyde in PBS
(pH 7.2). The brains and spinal cord were removed and kept in 4%
paraformaldehyde in PBS for overnight postfixation.
Immunocytochemistry
Vibratome 50 µm transverse brain sections were extensively
washed in Tris-buffered saline (TBS, pH 7.4). For confocal laser
scanning microscopy analysis, a monoclonal mouse anti-PRV [a generous
donation of Dr. H. Pol (Institute for Animal Science and Health,
Agricultural Research Department, Lelystad, The Netherlands) 1:6000]
was used and the secondary antibodies were conjugated to FITC for PRV
and CY3 (Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) for either VP [Truus, Netherlands Institute for Brain
Research (NIBR), Amsterdam, 1:4000], VIP (Viper, NIBR, Amsterdam,
1:4000) or TRH (no. 8880, kindly donated by Dr. T. J. Visser,
Dept. of Internal Medicine, Erasmus University Medical School,
Rotterdam, The Netherlands, 1:6000) staining. VP, VIP and TRH
antibodies were raised in rabbits. The filters used did not allow
cross-talk between these fluorophores [for further details on this
procedure see (24)].
Analysis of hormone data
Plasma concentrations of the thyroid hormones
T3 and T4 were determined
by in-house RIA (25), and TSH by a chemiluminescent
immunoassay (IMMULITE, Diagnostic Products Corp., Los
Angeles, CA; detection limit 0.4 ng/ml). Plasma corticosterone was
measured directly without extraction using a RIA from ICN Biomedicals, Inc. (Costa Mesa, CA) with iodinated
corticosterone. The interassay coefficient of variation for
corticosterone was <4%, and the detection limit 1 ng/ml. All
quantitative results are expressed as mean ± SEM. The
significance of diurnal variations in plasma TSH,
T3, and T4 values (time
dependence) was assessed using a one-way ANOVA with repeated measures.
ANOVA was followed by a Student’s t test (paired) to
establish which time points differed significantly from trough values.
Multivariate ANOVA (MANOVA) with factors Time and Group (repeated
measures) was used to determine whether the daily release patterns of
female or SCN-lesioned animals differed significantly from those of the
control males, followed by a Student’s t test to establish
at which timepoints the two groups differed significantly. However,
when used repeatedly to compare various time points in one experiment,
the Student’s t test has a real probability threshold much
higher than its nominal threshold (i.e. 0.05). Therefore,
the Student’s t test was applied using the Bonferroni
correction. The results were considered significant if the probability
of error was less than 5%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). None of the SCN-lesions caused damage
to more distant hypothalamic areas such as the supraoptic nuclei,
lateral hypothalamus, or PVN (Fig. 1). Inspection of brain sections for
the presence of VP and VIP cells or fibers revealed remnants of VIP and
VP cells at the lesion side in only one of these SCN-lesioned animals.
In addition, this animal also showed remaining VIP fibers in the PVN.
Complete daily profiles (i.e. 12 samples) for TSH,
T3, and T4 were obtained
from 8 unoperated control male animals, 4 control female animals, and
10 (i.e. 11 minus 1) SCN-lesioned male animals. The
T3 profile of one male control animal is missing
due to an insufficient volume of plasma. Daily profiles of TSH,
T3, and T4 of SCN-intact
animals are displayed in Fig. 2. In male
animals, ANOVA showed significant variations during the light/dark
cycle for both T3 and T4.
Daily fluctuations of TSH just missed significance (P =
0.056) in SCN-intact males (Table 1). In
female animals, all three hormonal parameters showed pronounced daily
fluctuations. A clear sex-difference was apparent in the 24-h means of
the three hormones. Female 24-h means for TSH and
T4 were approximately 40–50% lower compared
with those of males, whereas their T3 levels were
approximately 20% higher. In general, intact males and females showed
peak levels for all three hormones during (the first half of) the light
period and trough levels in the early dark period. A second period of
peak levels usually occurred during the middle of the dark period,
although it only reached significance for TSH release. In females, the
acrophase of the diurnal peaks in TSH, T3, and
T4 seemed delayed, and the nocturnal peak was
less obvious. Both in males and females, the secondary or nocturnal
peak was most clearly visible in circulating TSH levels.
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). Daily fluctuations in thyroid hormones were
significant but at a much lower level of significance than in control
males. SCN lesions significantly affected daily
T3 and T4, but not TSH,
profiles (Fig. 3; Table 1). MANOVA
detected no significant effects of either Group or Group x Time
on TSH levels due to the ablation of the SCN, although the interaction
effect was close to significance. Neither the daily mean nor the daily
fluctuations were affected by SCN lesions. As is clear from Fig. 3, the
only time to expect an effect of the SCN lesion is a narrow time window
around ZT6, when control animals have their peak values. In contrast to
its effect on TSH, removal of the SCN had a profound effect on thyroid
hormone levels. SCN lesions induced a considerable flattening of the
daily variations in circulating T3 and
T4 levels (Fig. 3). This change in
T3 and T4 release patterns
was also recognized by the MANOVA as evidenced by the significant
interaction effects (Table 1). Differences between SCN-intact and
SCN-lesioned animals were concentrated in the light period. In addition
to a disappearance of the rhythmic T3 and
T4 release, ablation of the SCN also caused a
22% decrease of mean T4 levels.
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Anatomy
To evaluate to what extent these pronounced daily rhythms in
thyroid hormones could be explained by a neuronal influence on the
thyroid, PRV was injected in the thyroid gland. The distribution and
order of appearance of PRV infected CNS neurons is summarized in Table 2. Animals surviving for only 2 days had
an infection limited to neurons in the thoracic part of the spinal
cord, mainly in the intermediolateral column (IML) with some neurons in
the lateral funiculus and intercalated nucleus ipsilateral to the
injection. In addition to these sympathetic motorneurons,
parasympathetic motorneurons also became visible, especially in the
rostral part of the dorsal motor nucleus of the vagus (DMV; Fig. 4G).
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) and ventromedial
parvocellular subdivision (mvPVN). Also in the anterior PVN (aPVN),
labeled neurons and labeled dendrites were observed. In addition to
these hypothalamic sites, PRV-labeled neurons were also seen in the
prefrontal cortex.
After 4 days of survival, third order neurons became visible, in
addition to the labeled neurons of the second order. In the thoracic
spinal cord, numerous infected neurons were observed bilaterally in the
IML, intercalated nucleus and lateral funiculus, and in lamina VII and
X. In the brain stem, in addition to the first and second order
neurons, infected neurons were seen in the C1and C3 noradrenergic
groups, the magnocellular reticular nucleus and paragiganto cellular
nucleus. In the hypothalamus, labeled neurons were found in areas known
to project to the PVN, e.g. the medial preoptic area (MPO),
anterior hypothalamic area (AHA), subfornical organ (SFO), bed nucleus
of the stria terminalis (BNST), arcuate nucleus, and SCN. Relatively
few labeled neurons were found in the dorsomedial and ventromedial
hypothalamic nuclei. In the SCN, labeling was present particularly in
the rostral part of the nucleus. Labeled neurons were found in all
parts of the medial, mediodorsal, and medioventral aspects of the
nucleus (Fig. 4A).
In principle, it is feasible that when PRV is injected into the thyroid gland it can leak out, spread to neighboring tissues, and subsequently enter the bloodstream. In order, to check for possible infection of the brain through tissues neighboring the thyroid gland or through the general circulation and subsequent passage through one of the circumventricular organs, in 3 animals we applied the same amount of virus on top of the thyroid gland. Animals receiving an injection on top of the thyroid did not show any viral labeling up to 4 days after tracer application. The lack of central infection by way of the general circulation is in agreement with the large number of control experiments reported previously by us (11, 26) and others (27, 28, 29). In fact, the differential timing of labeling in AP (day 3) vs. OVLT and SFO (day 4) already indicates that there is not a common route (i.e. the general circulation) of labeling. Labeling in OVLT and SFO is only found after a long survival time, i.e. when already a large number of other brain areas not open to the blood-brain barrier have been labeled. Therefore, labeling in OVLT and SFO represents transsynaptic labeling from the PVN and the labeling in the AP is due to transsynaptic labeling from the DMV. Moreover, labeling in the ME was never observed.
To investigate the nature of the first order IML and second order PVN
neurons infected by the virus, spinal cord and hypothalamic sections
were stained for both PRV and TRH. Because we did not use
colchicine, only a few TRHimmunoreactivity (ir)
neurons were observed in the parvocellular parts of the PVN. On the
other hand, dense networks of TRH-labeled fibers were observed in the
dorsomedial and ventromedial hypothalamic nuclei and in the
parvocellular part of the PVN. High concentrations of intensely labeled
TRH-ir fibers were also observed in the median eminence. No TRH-ir
neurons could be observed in the brainstem but a pronounced
TRH-containing innervation was observed in the nucleus of the solitary
tract and the surroundings of the DMV. Infected DMV neurons, however,
only showed occasional contacts with TRH-ir fibers (Fig. 4E). In the
spinal cord, a dense network of TRH-containing fibers virtually
outlined the preganglionic sympathetic neurons in the IML. A large
number of the viral positive neurons in the IML were seen to be
contacted by TRH containing fibers (Fig. 4, F and H). In agreement with
this observation, we occasionally observed in the dorsal and
ventrolateral part of the PVN neurons that combined fluorescence for
PRV with that for TRH (Fig. 4D). After 4 days of survival, the SCN
input to the second order neurons in the PVN was confirmed by
demonstrating the colocalization of VP with PRV in cell bodies of the
SCN (Fig. 4B), and the presence of either VIP- or VP-positive contacts
on PRV infected PVN neurons. In addition, the double staining
experiments revealed VIP-positive contacts on TRH-containing PVN
neurons.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observed daily rhythmicity of TSH is in agreement with previous rodent studies using less frequent sampling protocols and different sampling techniques, i.e. jugular vein puncture, cardiac puncture, or decapitation (15, 16, 17, 19). In general a diurnal TSH peak is found in all studies, although factors such as age and strain of the rats, light, and season may modify its appearance (18). The description of a daily rhythm in thyroid hormones has been more equivocal. Some studies did not detect such a rhythm (16), whereas others did report a significant daily rhythm in T3 and/or T4 (17, 18, 19). In general, the pattern of TSH and thyroid hormone secretion is similar in both sexes. Clear sex-differences, however, are present in mean 24-h plasma concentrations. The sex-differences found in the present study are in the same direction as described previously by Fukuda et al. (1975), although in that study the T3 and T4 differences did not reach statistical significance. The lower T4 levels found in females are in line with their lower TSH levels. It is not clear, however, what causes the higher T3 levels in female rats. It has been reported that the turnover and degradation rate of T4 are greater in female than in male rats (16). A higher peripheral conversion of T4 to T3 in females may indeed result in higher T3 plasma levels in females compared with males.
The second TSH peak has also been recognized in previous animal, but
not human, studies (see figures in Refs. 15, 18, 33). Thus
far, no explanations have been offered for its occurrence. It is
tempting to speculate on a correlation between the increased feeding
activity at night in rats and the increased TSH release,
e.g. it has been hypothesized that (feeding-induced
nocturnal) insulin release may up-regulate thyroid activity
(34). Indeed, it is well known that fasting will decrease
thyroid activity (35, 36). In addition, it has been shown
that restricted feeding will abolish the nocturnal rise in plasma TSH
(37). Another intriguing point is that the nocturnal peak
is especially observed in plasma TSH, but not thyroid hormone, levels
(Fig. 2). The absence of significant increases in plasma
T3 and T4 in response to
the nocturnal TSH surge might be due to an inhibitory effect of the
increased nocturnal sympathetic input to the thyroid gland
(38, 39, 40, 41). It is not clear if an increased TRH release is
also involved in the regulation of the nocturnal TSH peak. The
aforementioned TRH data in general only indicate one period of
increased TRH synthesis and release each 24-h cycle.
Since, as yet, no studies have been performed examining TSH or thyroid hormone rhythms during constant dark (DD) or constant light (LL) conditions, there is no proof yet for a circadian rhythm in thyroid activity in the rat. On the other hand, in humans the circadian nature of TSH release has been clearly proven (13). Together the human data and the present data on the effect of SCN ablation provide a strong case for a circadian regulation of the hypothalamo-pituitary-thyroid axis as well as for instance the hypothalamo-pituitary-adrenal axis. In addition, the fact that in our studies both SCN-intact and SCN-lesioned animals were housed in L/D conditions provides additional evidence for the fact that daily rhythms in thyroid hormones are not controlled by environmental lighting, i.e. if environmental lighting conditions were to be important also SCN-lesioned animals should have displayed daily rhythms in thyroid hormones.
It is probable that the daily rhythm in plasma TSH levels is secondary to an increased release of hypothalamic TRH. Daily changes in the hypothalamic content of TRH peptide and mRNA have been reported (42, 43, 44), as well as daily changes in its hypothalamic release (45). Although TRH neurons are distributed widely throughout the brain, the major population of TRH neurons dedicated to regulating pituitary-thyroid function appears to be the one residing in the PVN (30, 31, 46). In addition, the PVN is a prominent target area of SCN efferents (47, 48, 49, 50). Indeed, the present study has evidenced direct SCN projections to TRH-containing PVN neurons and a daily rhythm in the amount of TRH mRNA in the PVN has been described previously (51).
Thus far, the effect of SCN-ablation on daily TSH rhythms has been investigated in only two studies (20, 21), whereas to our knowledge no studies have yet investigated its effect on thyroid hormones. The study of Abe et al. (1979) compared three timepoints and indicated a small increase of TSH levels in SCN-lesioned animals at one timepoint. Peschke et al. (1989) detected no effect of their SCN lesions on mean TSH levels, but in this case only four time points were compared. The present study investigated the effect of SCN lesions on both TSH and thyroid hormones during a complete 24-h light/dark cycle using a 2-h sampling protocol. Statistical analysis showed that the effects of SCN-lesions on the TSH release pattern approached significance (group vs. time, P = 0.085). The main effect of the SCN lesion was a more erratic distribution of the highest TSH values, compared with the daytime preference in control animals. The dual peak pattern and the small amplitude of the daily TSH rhythm (±10%) in control males, together with the lack of effect of SCN lesions on mean 24-h TSH levels, hindered the detection of a significant effect of SCN lesions on the TSH rhythm. In fact, the pronounced circadian TSH rhythm in humans only became apparent after the inhibitory effect of sleep was discovered (13, 52). As of now, no data are available indicating a similar effect of sleep deprivation on the rat TSH rhythm. Ablation of the SCN, however, did result in a significant flattening of the daily T3 and T4 release patterns, and a significant decrease of the 24-h mean T4 levels. The more pronounced effect of SCN lesions on T3 and T4 rhythms than on TSH rhythms indicates that the SCN may stimulate thyroid hormone secretion from the thyroid in a diurnal pattern via a neuronal control of thyroid. Although, at present, it cannot be excluded that the significant lower daytime TSH levels in SCN-lesioned animals are responsible for the disappearance of the daytime T3 and T4 peaks. A neuronal input to the thyroid may also explain the presence of clear-cut daily T3 and T4 rhythms in the absence of pronounced daily fluctuations in TSH release. The lower plasma levels of T4, together with similar levels of TSH, suggest that in SCN-lesioned animals thyroid sensitivity to TSH is decreased. The autonomic nervous system may interfere with plasma T3 and T4 concentrations by affecting either thyroid hormone release, thyroid TSH responsiveness, or extrathyroidal deiodination. Previously, we have shown that SCN lesions modulate adrenal sensitivity to ACTH (53). Next we demonstrated labeled SCN neurons after transneuronal tracing from the adrenal gland and provided evidence for a physiological SCN-adrenal cortex link (11). We concluded that SCN control of the daily corticosterone rhythm involves two mechanisms: 1) an SCN control of ACTH release via its input to CRH neurons, and 2) an SCN control of adrenal sensitivity via its input to autonomic PVN neurons innervating the sympathetic preganglionic neurons in the spinal cord (11, 54). We now propose that the SCN may control thyroid activity in a similar way by affecting both TSH release and thyroid sensitivity. In support of such a role, it has been shown that increased sympathetic activity will inhibit the response of the thyroid gland to TSH (38, 39, 40) and that the sympathetic input to the thyroid gland shows a peak of activity during the dark period (41). In addition, it has been proposed that some of the thyroidal changes observed with nonthyroidal illness (NTI), i.e. a fall in T4 and reduced thyroidal responsiveness to TSH, are caused by a change in the sympathetic input to the thyroid gland (39).
In principal, ablation of the SCN may also affect the release of TSH by inducing changes in other (neuroendocrine) inputs to the TRH neuron. Well-known examples are the effects of glucocorticoids on the HPT axis (55) and the effect of somatostatin and dopamine on TSH release (32, 56, 57). However, in view of the limited effect of SCN-lesions on 24-h mean TSH levels, no major changes in these systems are to be expected. Indeed, although highly irregular, mean 24-h plasma corticosterone concentrations in SCN-lesioned animals are only slightly increased compared with those of control animals (i.e. 65 ± 8 vs. 50 ± 8 ng/ml) see also (58, 59, 60). In our experience, corticosterone will only inhibit TSH release when plasma corticosterone concentrations are >300 ng/ml (our unpublished observations). Also, hypothalamic dopamine concentrations do not show major changes after SCN-lesions (61). Additional in situ hybridization experiments are planned to investigate the effect of SCN-lesions on preproTRH mRNA and to see if indeed the early morning increase in TRH mRNA (51) is abolished by SCN-lesions.
Our viral tracing experiments provide clear evidence for both a
sympathetic and parasympathetic input to the thyroid gland,
i.e. first order neurons are found in the IML and DMV, in
agreement with previous morphological studies (22). The
SCN may affect both branches of the autonomic input to the thyroid
gland through its projection to the PVN. Several experiments now have
provided evidence for an SCN control of the sympathetic input to a
number of peripheral organs via its input to the PVN > IML
> sympathetic ganglia pathway (11, 26, 62, 63). PVN
neurons also project heavily to the brain stem regions containing the
parasympathetic motorneurons; therefore, second order labeling of PVN
neurons could also occur via this parasympathetic route
(i.e. similar to the second order labeling found in the
central nucleus of the amygdala). Additional experiments are necessary
to know if the SCN only contacts "sympathetic PVN" neurons or also
"parasympathetic PVN" neurons. Several reports have indicated that
central administration of TRH enhances both sympathetic and
parasympathetic efferent activity (64, 65, 66). Indeed LM
studies, using colchicine to enhance neuronal TRH staining, indicated
that TRH containing neurons in the raphe and ventral medulla project
both to the IML and DMV/nts area (67, 68). Interestingly,
with help of the high-resolution confocal laser scanning microscope,
our double-labeling experiments now show that following PRV injections
in the thyroid gland only the sympathetic preganglionic neurons show an
extensive TRH input. Virus-infected preganglionic IML neurons are
heavily innervated by TRH-ir fibers, whereas in the brainstem there is
only an occasional TRH-positive nerve ending found on first order
parasympathetic neurons in the DMV. On the other hand, respiratory- and
gastric-related parasympathetic motorneurons do seem to be contacted by
a TRH-containing innervation (64, 65, 69). Anatomical
tracing and lesion studies have indicated that the major part of the
TRH innervation of autonomic preganglionic neurons is not derived from
the PVN, but instead from the raphe nucleus and ventral medulla
(67, 68, 70, 71). Because we did not use a colchine
pretreatment, we could not visualize the brain stem TRH neurons, and
only a few TRH-containing neurons in the PVN (Fig. 4, C and D).
Nevertheless, the colocalization of TRH and virus particles in PVN
neurons provides evidence for the (indirect) involvement of
hypothalamic TRH neurons in the control of automomic activity as well.
Indeed, recently Westerhaus and Loewy (29) found
colocalization of TRH and PRV in preoptic neurons after application of
PRV to the stellate ganglion.
In conclusion, the present study shows clear effects of SCN ablation on thyroid activity. SCN lesions abolish daily fluctuations of T3 and T4 release and cause a reduction of mean 24-h T4 concentrations. The minor effects of SCN-lesions on TSH release indicate that besides to the neuroendocrine pathway, the SCN recruits additional pathways to control thyroid activity. Our viral tracing experiments show that indeed the SCN might use neuronal pathways to set the sensitivity of the thyroid gland to TSH. In addition, through its connections with multiple parts of the autonomic system (54, 63), the SCN may be able to control the peripheral conversion of T4 to T3 (72, 73). It will be clear, however, that additional experiments are needed to elucidate the functional significance of the previously (11, 63, 74) and presently described neuronal pathways connecting the biological clock with peripheral (endocrine) organs.
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Acknowledgments |
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Footnotes |
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Received April 3, 2000.
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References |
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 L. P. Klieverik, S. F. Janssen, A. v. Riel, E. Foppen, P. H. Bisschop, M. J. Serlie, A. Boelen, M. T. Ackermans, H. P. Sauerwein, E. Fliers, et al. Thyroid hormone modulates glucose production via a sympathetic pathway from the hypothalamic paraventricular nucleus to the liver PNAS, April 7, 2009; 106(14): 5966 - 5971. [Abstract] [Full Text] [PDF]
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 L. P. Klieverik, H. P. Sauerwein, M. T. Ackermans, A. Boelen, A. Kalsbeek, and E. Fliers Effects of thyrotoxicosis and selective hepatic autonomic denervation on hepatic glucose metabolism in rats Am J Physiol Endocrinol Metab, March 1, 2008; 294(3): E513 - E520. [Abstract] [Full Text] [PDF]
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 M. J Boden and D. J Kennaway Circadian rhythms and reproduction. Reproduction, September 1, 2006; 132(3): 379 - 392. [Abstract] [Full Text] [PDF]
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P. Kok, F. Roelfsema, M. Frolich, A. E. Meinders, and H. Pijl Spontaneous Diurnal Thyrotropin Secretion Is Enhanced in Proportion to Circulating Leptin in Obese Premenopausal Women J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6185 - 6191. [Abstract] [Full Text] [PDF]
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J. B. Young, M. E. Burgi-Saville, U. Burgi, and L. Landsberg Sympathetic nervous system activity in rat thyroid: potential role in goitrogenesis Am J Physiol Endocrinol Metab, May 1, 2005; 288(5): E861 - E867. [Abstract] [Full Text] [PDF]
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 A. Kalsbeek, R. M. Buijs, R. van Schaik, E. Kaptein, T. J. Visser, B. Z. Doulabi, and E. Fliers Daily Variations in Type II Iodothyronine Deiodinase Activity in the Rat Brain as Controlled by the Biological Clock Endocrinology, March 1, 2005; 146(3): 1418 - 1427. [Abstract] [Full Text] [PDF]
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B. Z. Doulabi, M. P.-T. Schiphorst, A. Kalsbeek, E. Fliers, O. Bakker, and W. M. Wiersinga Diurnal Variation in Rat Liver Thyroid Hormone Receptor (TR)-{alpha} Messenger Ribonucleic Acid (mRNA) Is Dependent on the Biological Clock in the Suprachiasmatic Nucleus, whereas Diurnal Variation of TR{beta}1 mRNA Is Modified by Food Intake Endocrinology, March 1, 2004; 145(3): 1284 - 1289. [Abstract] [Full Text] [PDF]
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 N. S. Bradley and D. Y. Jahng Selective Effects of Light Exposure on Distribution of Motility in the Chick Embryo at E18 J Neurophysiol, September 1, 2003; 90(3): 1408 - 1417. [Abstract] [Full Text] [PDF]
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 E. L. Bittman, L. Doherty, L. Huang, and A. Paroskie Period gene expression in mouse endocrine tissues Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2003; 285(3): R561 - R569. [Abstract] [Full Text] [PDF]
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 V. Simonneaux and C. Ribelayga Generation of the Melatonin Endocrine Message in Mammals: A Review of the Complex Regulation of Melatonin Synthesis by Norepinephrine, Peptides, and Other Pineal Transmitters Pharmacol. Rev., June 1, 2003; 55(2): 325 - 395. [Abstract] [Full Text] [PDF]
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S. Takahashi, S.-i. Yokota, R. Hara, T. Kobayashi, M. Akiyama, T. Moriya, and S. Shibata Physical and Inflammatory Stressors Elevate Circadian Clock Gene mPer1 mRNA Levels in the Paraventricular Nucleus of the Mouse Endocrinology, November 1, 2001; 142(11): 4910 - 4917. [Abstract] [Full Text] [PDF]
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 T. J. Bartness, C. K. Song, and G. E. Demas SCN Efferents to Peripheral Tissues: Implications for Biological Rhythms J Biol Rhythms, June 1, 2001; 16(3): 196 - 204. [Abstract] [PDF]
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) rats.
Asterisks and diamonds indicate time
points significantly different from the lowest time point (marked by an
underscore), for males and females, respectively.
). The
shaded area indicates mean ± SEM for
the control males. Asterisks indicate time points that
differ significantly between SCN-lesioned and SCN-intact males.