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The Journal of Neuroscience, August 13, 2003, 23(19):7412-7414
 Previous Article | Next Article 
BRIEF COMMUNICATION
Lateralization of Circadian Pacemaker Output: Activation of Left- and Right-Sided Luteinizing Hormone-Releasing Hormone Neurons Involves a Neural Rather Than a Humoral Pathway
Horacio O. de la Iglesia,
Jennifer Meyer, and
William J. Schwartz
Department of Neurology, University of Massachusetts Medical School,
Worcester, Massachusetts 01655
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Abstract
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Locomotor activity and luteinizing hormone (LH) secretion in golden
hamsters share a common circadian pacemaker in the suprachiasmatic nucleus
(SCN), but the rhythms do not seem to share a common output pathway from the
SCN. Locomotion is believed to be driven by humoral factor(s), whereas LH
secretion may depend on specific ipsilateral neural efferents from the SCN to
LH releasing hormone (LHRH)-containing neurons in the preoptic area. In this
paper we provide the first functional evidence for such efferents in
neurologically intact hamsters by exploiting a phenomenon known as
"splitting" in constant light, in which circa-12 hr (approximately
12 hr) locomotor activity bouts reflect an antiphase oscillation of the left
and right sides of the bilaterally paired SCN. In ovariectomized,
estrogen-treated (OVX + E2) female hamsters, splitting is also
known to include circa-12 hr LH secretory surges. Here we show that
behaviorally "split" OVX + E2 females exhibit a marked
left-right asymmetry in immunoreactive c-Fos expression in both SCN and
activated LHRH neurons, with the percentage of
LHRH+/c-Fos+ double-labeled cells approximately fivefold
higher on the side corresponding to the side of the SCN with higher c-Fos
immunoreactivity. Our results suggest that splitting involves alternating
left- and right-sided stimulation of LHRH neurons; under such circumstances,
the functional activity of the neuroendocrine hypothalamus mirrors intrinsic
side-to-side differences in SCN gene expression. The circadian regulation of
reproductive activity depends on lateralized, point-to-point axonal
projections rather than on diffusible factors.
Key words: gonadotropin; hypothalamus; luteinizing hormone; preoptic; suprachiasmatic; fos
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Introduction
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The master circadian pacemaker in mammals, located in the suprachiasmatic
nucleus (SCN) of the hypothalamus, governs a diverse array of behavioral,
physiological, and hormonal rhythms, all of which express different phases and
waveforms (for review, see Herzog and
Schwartz, 2002 ). How the SCN organizes this temporal program is
not well understood and appears to be complex. In golden hamsters, circadian
regulation of both locomotor (wheel-running) activity and luteinizing hormone
(LH) secretion is abolished by SCN lesions
(Turek and Van Cauter, 1988),
and both rhythms are altered in parallel in the short-period phenotype
(tau) caused by mutation of the casein kinase 1 gene
(Lucas et al., 1999). Despite
sharing a common pacemaker in the SCN, however, the rhythms do not seem to
share a common output pathway from the SCN. Circadian wheel running is
believed to be driven by humoral factor(s) released by SCN cells. The rhythm
persists after surgical isolation of the SCN as a hypothalamic island in
situ (Hakim et al., 1991),
and it is restored in arrhythmic, SCN-lesioned animals by transplantation of
SCN tissue encased in semipermeable capsules
(Silver et al., 1996). On the
other hand, circadian LH secretion and reproductive cycles are abolished by
discrete knife cuts dorsocaudal to the SCN in rats
(Watts et al., 1989), and they
are not restored by transplants in hamsters
(Meyer-Bernstein et al., 1999).
These data have raised the possibility that specific neural efferents from the
SCN carry the output signal for this function, presumably via a direct,
predominantly ipsilateral projection to LH releasing hormone (LHRH)-containing
neurons in the preoptic area (de la Iglesia
et al., 1995; van der Beek et
al., 1997), but interpretation of arrhythmicity in these
experiments is compromised by nonspecific surgical effects. In this report, we
exploit an unusual property of the circadian system to investigate LHRH
neuronal activation in neurologically intact animals and to implicate a neural
rather than humoral mechanism for the control of this neuroendocrine output by
the SCN.
Hamsters maintained in constant light (LL) can exhibit
"splitting" of their locomotor activity rhythm, in which the
single daily bout of activity dissociates into two components stably coupled
180° ( 12 hr) apart. In ovariectomized, estrogen-treated (OVX +
E2) female hamsters, splitting also includes circa-12 hr
(approximately 12 hr) LH secretory surges
(Swann and Turek, 1985);
behaviorally "unsplit" OVX + E2 females exhibit only a
single daily surge in LL. The splitting phenomenon is believed to represent
the activity of two circadian oscillators cycling oppositely in antiphase, and
we have shown recently that these two oscillators correspond to the left and
right sides of the bilaterally paired SCN
(de la Iglesia et al., 2000).
We reasoned that if the ipsilateral innervation of LHRH neurons by the SCN
mediates circadian gating of the LH surge, then each surge in a
"split" female might reflect the activation of either left- or
right-sided LHRH neurons. We tested this prediction, identifying activated
LHRH neurons by their expression of immunoreactive c-Fos 1 hr before locomotor
activity onset (Doan and Urbanski,
1994), just after the start of the expected LH surge.
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Materials and Methods
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Female golden hamsters (Charles River, Kingston, NY), 21 d old at time of
delivery, were used in accordance with the regulations of the University of
Massachusetts Institutional Animal Care and Use Committee. They were housed
individually in LL (300-400 lux), and wheel-running activity was recorded
continuously to monitor for split activity rhythms (see
Fig. 1). Behaviorally split and
nonsplit control females were ovariectomized under ketamine-xylazine
anesthesia, returned to their cages for an additional 14 d in LL, and then
each was anesthetized and implanted subcutaneously with two 1-cm-long SILASTIC
capsules filled with powdered estradiol benzoate (EB). Two days after EB
implantation, behaviorally split animals were killed 1 hr before the onset of
either of their two split activity bouts, and nonsplit animals were killed
either 1 hr (1 hr controls) or 13 hr (13 hr controls) before the onset of
their single daily activity bout. After anesthetic overdose, animals were
perfused with PBS followed by 4% paraformaldehyde in 0.1 M
phosphate buffer, brains were removed and postfixed overnight at 4°C, and
50 µm coronal sections were cut from the lateral septum and diagonal band
through the preoptic area to the SCN. Double-label immunohistochemistry was
performed by simultaneous incubation with rabbit anti-c-Fos (1:10,000; SC52,
Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-LHRH (1:1000; HU4H,
generous gift of Dr. H. F. Urbanski, Oregon Health and Science University,
Portland, OR) for 72 hr at 4°C, followed by treatment with biotinylated
goat anti-rabbit-diaminobenzidine and goat anti-mouse-SG kit (Vector
Laboratories, Burlingame, CA), respectively. Alternate sections were prepared
for immunofluorescence and confocal microscopy using Alexa 594 goat
anti-rabbit and Alexa 488 goat anti-mouse secondary antibodies (1:200;
Molecular Probes, Eugene, OR). Excitation wavelengths for Alexa 594 and 488
were 568 and 488 nm, respectively. For each animal, the total number of
LHRH+ single-labeled and LHRH+/c-Fos+
double-labeled cells was counted on a set of sections spanning the rostral
lateral septum to the caudal preoptic area [15.1 ± 0.5 sections (mean
± SE) counted for each animal], and the percentage of double-labeled
cells was calculated separately for the left and right sides of the brain.
Also counted was the number of c-Fos+ cells in the section of the
SCN that represented its largest mediolateral and ventrodorsal extent.
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Figure 1. Behavioral splitting and immunoreactive c-Fos expression in the SCN. Left,
Wheel-running activity of a representative female hamster maintained in LL,
with the number of wheel revolutions over the course of each 24 hr period
charted horizontally from left to right and succeeding days stacked vertically
from top to bottom. At the white arrow, the animal's single daily bout of
activity dissociated into two split components stably coupled 12 hr apart
(30% of females exposed to LL exhibited splitting within 90 d). The
animal was then ovariectomized (OVX), subcutaneously implanted with an
estradiol benzoate capsule 14 d later (E2), and killed after 2 d (asterisk);
20% of the females, whether split or nonsplit, had disrupted wheel-running
rhythms after OVX and were discarded from the study. Right, Coronal brain
section through the SCN of a behaviorally split female hamster, killed 1 hr
before the onset of a split activity bout and processed for c-Fos
immunohistochemistry. 3V, Third ventricle; OC, optic chiasm. Scale bar, 500
µm.
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Results
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As we reported previously for male hamsters
(de la Iglesia et al., 2000),
behaviorally split OVX + E2 female hamsters exhibited a dramatic
left-right asymmetry in immunoreactive c-Fos expression in the SCN
(Fig. 1), with 323 ± 18
labeled cells on the "high" side (whether left or right) and 59
± 9 labeled cells on the "low" side (n = 10). In
the septal-preoptic area of these animals, LHRH+ single-labeled and
LHRH+/c-Fos+ double-labeled cells were present on both
sides of the brain, but the percentage of double-labeled cells was
approximately fivefold higher on the side corresponding to the side of the SCN
with higher c-Fos immunoreactivity (Fig.
2). In behaviorally split OVX + E2 females, 21.2
± 4.0% of LHRH+ cells were also c-Fos+ on the
side ipsilateral to high SCN c-Fos expression (64 ± 7 total
LHRH+ cells per side per animal), but only 4.4 ± 1.2% were
double labeled on the side contralateral to high SCN c-Fos expression (64
± 6 total LHRH+ cells per side per animal). In nonsplit OVX
+ E2 1 hr controls (n = 7), there was no such side-to-side
asymmetry (12.3 ± 3.4 and 11.4 ± 4.4% double-labeled cells, with
69 ± 10 and 68 ± 10 total LHRH+ cells per side per
animal; the high SCN side in nonsplit animals was defined as the side with
marginally higher cell counts). The side-to-side difference in the percentage
of double-labeled cells in the behaviorally split group, but not in the
nonsplit group, was statistically significant (p = 0.002; two-tailed
t test). The absolute number of double-labeled cells per animal
(regardless of side) was not significantly different between the split and
nonsplit groups (17.1 ± 5.4 and 16.9 ± 3.8), raising the
possibility that this might represent the minimum number of activated LHRH
cells necessary for an LH surge in OVX + E2 animals. As expected,
no double-labeled cells were observed in nonsplit 13-hr controls (n =
2).
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Figure 2. Asymmetric activation of LHRH-containing neurons during splitting.
LHRH+ (blue) single-labeled cells (white arrows, left) and
LHRH+ (blue)/c-Fos+ (brown) double-labeled cells (black
arrows, right) from the left and right sides (red squares in insets) of the
brain of the hamster whose SCN is shown in
Figure 1. Similar results were
observed using confocal microscopy (0.5-µm-thick optical sections) and
immunofluorescence (data not shown). Scale bar, 50 µm.
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Of note, one of the seven nonsplit 1 hr controls included in the above
analysis exhibited a marked left-right asymmetry in the percentage of
LHRH+/c-Fos+ double-labeled cells (24.1 vs 2.0%), along
with a correspondingly asymmetrical pattern of SCN c-Fos expression (268 vs 93
labeled cells, compared with 241 ± 29 vs 200 ± 30 labeled cells
in the remaining 1 hr controls). Although this was the only such animal in the
control group, this case hints that a time delay might exist between the onset
of splitting within the SCN oscillatory apparatus and its subsequent
reflection as split bouts of wheel-running activity.
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Discussion
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Asymmetric activation of LHRH neurons in behaviorally split hamsters
provides a unique functional demonstration of the ipsilateral innervation of
these neurons by each side of the SCN, heretofore identified solely by lesion,
histochemical, and tract-tracing methods
(de la Iglesia et al., 1995;
van der Beek et al., 1997).
During splitting, we know that the SCN is reorganized as two oppositely
phased, left- and right-sided circadian pacemakers, and the present data
suggest that such a split SCN also activates bilateral LHRH neurons in
antiphase. Alternating left- and right-sided stimulation of these neurons
likely accounts for the generation of the circa-12 hr LH secretory surge in
behaviorally split females. Thus, at least under these circumstances, the
functional activity of neuroendocrine hypothalamus mirrors an intrinsic
side-to-side difference in SCN gene expression.
Our results point to an organization of SCN output pathways that is
multimodal. Although locomotor activity may be regulated by diffusible factors
released by SCN cells (Kramer et al.,
2001; Cheng et al.,
2002), reproductive activity depends instead on lateralized,
point-to-point axonal projections to specific regional targets. The
multiplicity of SCN output mechanisms could provide a substrate for
independent adjustment of the temporal sequencing of disparate
clock-controlled events.
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Footnotes
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Received Apr. 28, 2003;
revised Jun. 12, 2003;
accepted Jun. 25, 2003.
This research was supported by the National Institute of Neurological
Disorders and Stroke. Correspondence should be addressed to Horacio O. de la
Iglesia, Department of Biology, University of Washington, Box 351800, Seattle,
WA 98195-1800. E-mail:
horaciod{at}u.washington.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/237412-03$15.00/0
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Top
Abstract
Introduction
