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 Previous Article | Next Article 
The Journal of Neuroscience, July 1, 2001, 21(13):4864-4874
Contrasting Effects of Ibotenate Lesions of the Paraventricular
Nucleus and Subparaventricular Zone on Sleep-Wake Cycle and
Temperature Regulation
J.
Lu1,
Y.-H.
Zhang1,
T. C.
Chou1,
S. E.
Gaus1,
J. K.
Elmquist1,
P.
Shiromani2, and
C.
B.
Saper1
1 Department of Neurology and Program in Neuroscience,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts 02115, and 2 Department of Psychiatry,
Brockton Veterans Administration Hospital, Harvard Medical School,
Boston, Massachusetts 02115
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ABSTRACT |
The suprachiasmatic nucleus (SCN), the circadian pacemaker for the
brain, provides a massive projection to the subparaventricular zone
(SPZ), but the role of the SPZ in circadian processes has received
little attention. We examined the effects on circadian rhythms of
sleep, body temperature, and activity in rats of restricted ibotenic
acid lesions of the ventral or dorsal SPZ that spared the immediately
adjacent paraventricular hypothalamic nucleus (PVH) and the SCN.
Ventral SPZ lesions caused profound reduction of measures of circadian
index of sleep (by 90%) and locomotor activity (75% reduction) but
had less effect on body temperature (50% reduction); dorsal SPZ
lesions caused greater reduction of circadian index of body temperature
(by 70%) but had less effect on circadian index of locomotor activity
(45% reduction) or sleep (<5% reduction). The loss of circadian
regulation of body temperature or sleep was replaced by a strong
ultradian rhythm (period ~3 hr). Lesions of the PVH, immediately
dorsal to the SPZ, had no significant effect on any circadian rhythms
that we measured, nor did the lesions affect the baseline body
temperature. However, the fever response after intravenous injection of
lipopolysaccharide (5 µg/kg) was markedly decreased in the rats with
PVH lesions (66.6%) but not dorsal SPZ lesions. These results indicate
that circadian rhythms of sleep and body temperatures are regulated by
separate neuronal populations in the SPZ, and different aspects of
thermoregulation (circadian rhythm and fever response) are controlled
by distinct anatomical substrates.
Key words:
circadian rhythm; ultradian rhythm; ibotenic acid; suprachiasmatic nucleus; c-Fos; fever
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INTRODUCTION |
The suprachiasmatic nucleus (SCN) of
the hypothalamus controls the circadian rhythms of behavioral and
physiological states, including sleep-wake cycles and body temperature
in mammals (Ibulka and Kawamura, 1975 ; Mouret et al., 1978; Eastman et
al., 1984; for review, see Harrington et al., 1994). Although
remarkable progress has been made in revealing molecular mechanisms of
circadian rhythm within the mammalian SCN in the last few years (for
review, see Dunlap, 1998), the output pathways by which the SCN exerts control over circadian rhythms are not well understood.
By far the densest SCN projections are to the subparaventricular zone
(SPZ) (Watts and Swanson, 1987; Watts et al., 1987; Watts, 1991). The
SPZ is defined by the SCN terminal field, beginning at the dorsal
border of the SCN [the ventral SPZ (vSPZ)] and continuing dorsally
and caudally into the region ventral to the paraventricular nucleus
[the dorsal SPZ (dSPZ)]. Many SCN fibers extend farther caudally into
the medial part of the dorsomedial hypothalamic nucleus (DMH). Because
of its relationship with the SCN, this output pathway has been proposed
as a major component of the outflow for circadian regulation (Watts,
1991). This hypothesis is supported by the observation that
electrolytic lesions including the DMH and the dorsal SPZ abolish
circadian rhythms of feeding, drinking, and corticosterone secretion
(Bernardis, 1973; Bellinger et al., 1976; Cipolla-Neto et al.,
1988). These studies do not distinguish between the effects of
injuring cell bodies versus fibers of passage, but suggest that the SPZ
plays an important role in circadian regulation.
The parvicellular divisions of the paraventricular nucleus of the
hypothalamus (PVH) also receive a small part of the output of the SCN
(Vrang et al., 1995a). Dorsal parvicellular PVH neurons project to the
sympathetic preganglionic column in the spinal cord (for review, see
Saper, 1995), where they regulate the nocturnal secretion of melatonin
(Klein et al., 1983). Medial parvicellular neurons in the PVH secrete
corticotropin-releasing hormone into the median eminence, thereby
regulating the diurnal secretion of adrenal corticosteroids. It is not
known, however, whether the circadian control of these functions is
exerted by direct SCN inputs to the PVH or by relays through the SPZ,
for example to the caudal DMH, which supplies a major projection back
to the PVH (Elmquist et al., 1998). In addition, it is not known to
what extent the cycles of melatonin and adrenal corticosteroid
secretion mediated by the PVH influence other circadian functions, such as sleep or body temperature.
To determine and differentiate the roles of the SPZ and the PVH in the
circadian regulation of sleep, activity, and body temperature cycles,
we have placed small ibotenic acid lesions in the SPZ (ventral and
dorsal subdivisions) and PVH and examined circadian rhythms of sleep,
activity, and body temperature. We also tested the effects of these
lesions on fever response after intravenous injection of
lipopolysaccharide (LPS).
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MATERIALS AND METHODS |
Animals
Pathogen-free adult male Sprague Dawley rats (275-300 gm;
n = 69) purchased from Taconic (Germantown, NY) were
used. The rats were individually housed and had ad libitum
access to food and water. All animals were housed under controlled
conditions (12 hr light starting at 7:00 A.M.; 200 lux) in an isolated
ventilated chamber maintained at 20-22°C. All protocols were
approved by the Institutional Animal Care and Use Committees of Beth
Israel Deaconess Medical Center and Harvard Medical School.
Venous catheterization and thermal telemetry transmitter
All surgeries were performed under chloral hydrate anesthesia
(7% in saline, 0.35 gm/kg) using aseptic techniques. During the
preliminary surgical session, a temperature/activity transmitter (type
TA10-F40, Data Science International) was implanted in each animal via
a ventral midline incision into the peritoneal cavity. In some animals
who needed venous access for later injections of LPS, a 2 cm incision
was made along the medial thigh, and the femoral vein was exposed. A
SILASTIC catheter containing heparinized saline (10 U/ml of
saline) was inserted into the vein and sutured in place. The free end
of the catheter was passed under the skin, exteriorized between the
scapulas, and plugged with a sterile wire stylet. All wounds were
closed with wound clips, and animals were allowed to recover for 3 d, after which body temperature and activity data were recorded every 5 min to provide a prelesion baseline.
Ibotenic acid injection and EEG/EMG implantation
It was necessary to have a second surgical session to place a
lesion in the SPZ or PVH. The scalp was incised, and a burr hole was
made at the injection site. A fine glass pipette containing ibotenic
acid was lowered into the SPZ or PVH stereotaxically. The coordinates
used for the PVH (Paxinos and Watson, 1986) were anteroposterior (AP)
 1.8 mm, dorsoventral (DV) 7.2 mm, mediolateral (ML) ±0.5 mm
relative to bregma. For the dorsal SPZ, we used AP 1.8 mm, DV 7.6
mm, ML ±0.5 mm; for ventral SPZ, we used AP 1.3 mm, DV 8.0 mm, ML
±0.4 mm. The tooth bar was 3.3 mm below the ear bar. Twenty nanoliters
containing 10 nmol ibotenic acid (Sigma, St. Louis, MO) in saline was
injected by air pressure through a glass pipette. After 2 min, the
pipette was slowly withdrawn.
EEG/EMG electrodes could be implanted only after the ibotenic acid
lesions were made, because the implantation procedure obscured skull
landmarks necessary for accurate placement of ibotenic acid injections.
Four screw electrodes were implanted into the skull, in the frontal
(two screws) and parietal bones (two screws) of each side, and two
flexible wire electrodes were placed in the nuchal muscles. The
electrodes were soldered in sockets that were connected via flexible
recording cables and a commutator to a Grass polygraph and computer.
Analyses of physiological data
The EEG/EMG signals were amplified by a polygraph (Grass) and
digitized by an Apple Macintosh computer running ICELUS (G Systems, Inc). Wake-sleep states were manually scored in 12 sec epochs of the
digitized EEG/EMG. Wakefulness was identified by the presence of
desynchronized EEG and phasic EMG activity. Non-rapid eye movement (NREM) sleep consisted of high amplitude slow wave EEG together with
low EMG tone relative to waking. REM sleep was identified by the
presence of regular theta EEG activity coupled with low EMG tone
relative to NREM sleep. The amount of time spent in wake, NREM sleep,
and REM sleep was determined for each hour. Body temperature and
activity were monitored with Minimitter telemetry antennas placed under
the cages of individual animals. Activity recordings with this
apparatus reflect movement of the implanted transmitter with respect to
the antenna. Signals were averaged over 5 min and recorded on a
Macintosh computer running Minimitter software.
Perfusion and immunohistochemistry
The rats were deeply anesthetized with chloral hydrate (350 mg/kg, i.p) and perfused with saline (100 ml) and then 10% neutral buffered formalin (500 ml) transcardially. The brains were cut coronally at 40 µm in four series. The sections were washed in PBS
and incubated with primary antiserum in PBS containing 0.25% Triton
X-100 [c-Fos, Ab-5, 1:150,000 (Oncogene); arginine vasopressin (AVP)
1:10,000 (Peninsula Laboratories, Belmont, CA); vasoactive intestinal
polypeptide (VIP) 1:100,000 (Chemicon, Temecula, CA)] for 1 d at
room temperature. The sections were washed again in PBS and incubated
in biotinylated secondary antiserum (1:1000; Jackson ImmunoResearch,
West Grove, PA) for 1 hr, washed, and incubated in ABC regents (1:1000;
Vector Laboratories, Burlingame, CA) for 1 hr, then washed again and
incubated in a 0.06% solution of 3,3-diaminobenzidine
tetrahydrochloride (DAB; Sigma) and 0.05% CoCl and 0.01%
NiSO4 (NH4) in PBS plus 0.02%
H202 for ~5 min. Finally,
the sections were mounted on slides, dehydrated, cleared, and coverslipped.
Nissl staining
A series of adjacent sections was mounted on gelatin-coated
slides, washed, and incubated in 0.25% thionin solution for 2 min,
then washed and dehydrated in gradient ethanols, and cleared in xylene
before being coverslipped.
Determination of lesion region
The area of the lesion was determined by loss of neurons and
presence of gliosis in Nissl-stained sections. To further verify the
lesion in specific areas, we combined the immunostaining for c-Fos,
VIP, and AVP. In the SCN, the ventrolateral division contains VIP-immunoreactive (ir) neurons, whereas the dorsomedial division contains AVP-ir neurons. In the PVH, AVP-ir neurons are found mainly in
the magnocellular subdivision. By using immunostaining for these
peptides, we could determine whether the lesions included the SCN or
the PVH and whether the lesions affected the fibers of passage through
the SPZ. Because LPS induces Fos expression in the PVH, we also used
loss of Fos-ir neurons in the PVH to assess the integrity of the PVH
after LPS administration. Because most of the cells in the SPZ do not
contain any single known neurotransmitters, we used VIP-ir axons (see
Fig. 1) to delineate the SPZ and used counting of Nissl-stained cells
within the VIP axonal field of the SPZ to determine loss of cells.
Cell counting in the SPZ and PVH
For the dorsal SPZ, we diagonally placed a box (300 × 200 µm) along the ventral border of the PVH at a level corresponding to
1.7 mm posterior to bregma in the atlas of Paxinos and Watson (1986).
For the ventral SPZ, we counted cells in a vertical box (100 × 300 µm) 100 µm lateral to the third ventricle, with the bottom edge
directly above the dorsal border of the SCN, at a level corresponding
to 1.5 mm posterior to bregma in the atlas of Paxinos and Watson
(1986). For the PVH, we placed a box (500 × 300 µm)
horizontally, 100 µm lateral to the third ventricle with its
upper border at the same level as the most dorsal point of the third
ventricle at the level equivalent to 1.8 mm posterior to bregma in the
atlas of Paxinos and Watson (1986).
Animals with bilateral cell loss >70% in the counting areas were
considered to have substantial lesions, and animals with no lesion (for
example, injections into the third ventricle) were used as the
controls. We used the entire series of animals to determine whether the
degree of cell loss in the specific target area correlated with the
physiological response.
Data analyses of circadian rhythm: circadian index and
cosinor amplitude
The circadian index (CI) for a physiological variable was
calculated as CI = (meannight meanday)/mean24hr, scaled
to an index of 100 for the mean for the control animals. For sleep, the
mean percentage of the 12 hr period (night and day) spent in total
sleep was calculated and used to determine the circadian index. For
body temperature, the mean of temperature recorded every 10 min across
the 12 hr period was used. Total activity was summated over the entire
12 hr period. The "night" cycle corresponded to the 7:00 P.M.-7:00
A.M. period, and "day" corresponded to 7:00 A.M.-7:00 P.M., even
when the animals were kept in a completely dark environment. Animals
kept in a dark environment for 6 d showed minimal drift in their
circadian phase. Using cosinor analysis, there was a phase delay of
23.7 ± 14.6 min for body temperature peak in the control animals
on day 6 in constant darkness (DD), and 20.4 ± 16.8 min for the
animals with vSPZ lesions. Animals with dSPZ lesions had a phase delay
in sleep peak by 34.3 ± 20.7 min. Because the phase delays were
so small, and because they had to be measured on different variables
(because of differential reduction of sleep vs body temperature
circadian amplitude with different lesions), we did not correct for the
circadian phase in determining circadian index.
As a further control for the circadian index as a measure of circadian
amplitude, we therefore also used cosinor analysis to determine the
amplitudes of circadian rhythms of sleep, body temperature, and
activity, independent of circadian phase, for the last 48 hr of the
continuously dark period. Briefly, the cosinor amplitude
(A) was determined by:
when:
and then was scaled so that the mean for the control animals was
100. We found that the amplitude derived from cosinor analysis was
consistent with the circadian index (see Results).
In addition to the circadian index and cosinor analysis, we also used a
t test to determine whether there was a significant difference between the means of physiological variables during the day
and the night. Values are expressed throughout this text as mean ± SEM.
Experimental protocol
The effects of lesions in the SPZ and PVH on circadian
rhythmicity. During the prelesion control period, we continuously
recorded body temperature and activity for 3 d. Beginning 1 d
after the ibotenic acid lesions were placed, we recorded EEG/EMG, body
temperature, and activity continuously in both lesion and control
animals for the next 6 d under a 12 hr light/dark schedule (LD)
and then for 6 more days under DD. Data for the cosinor analysis
of amplitude of circadian rhythms of sleep, body temperature, and
locomotor activity were collected during the last 48 hr of the DD
period; the circadian index was measured on data from the last 24 hr of the DD period.
We used two groups of control animals (controls for vSPZ lesion = 7; controls for dSPZ lesion = 10) because the experiments were
performed in two series a few months apart. The control animals in each
series were animals drawn from the series in which the injections of
ibotenic acid had been made into the third ventricle, and no lesions in
the hypothalamus were found. Rats that had >70% loss of cells in the
vSPZ (n = 8), dSPZ (n = 7), or PVH
(n = 7) bilaterally were identified as having
substantial lesions. The animals with 20-70% cell loss bilaterally in
the vSPZ (n = 10) or dSPZ (n = 10) were
compared with them by correlation analyses to assess whether the
observed physiological effects correlated with cell loss in the SPZ.
This is an important component of the analysis, because the lesions
always extend beyond the intended target, including variable amounts of
adjacent tissue. We found in our earlier work that some effects seen in
animals with substantial lesions (e.g., in the ventrolateral preoptic
nucleus) were not correlated with cell loss in that structure, thus
allowing us to determine that the effects arose from adjacent
structures (Lu et al., 2000).
The effects of lesions in the PVH and SPZ on fever response.
To examine fever responses in animals with either PVH
(n = 7) or dSPZ lesions (n = 7), dSPZ
controls (n = 10) were returned after recording in a DD
cycle for 6 d to a 12 hr LD schedule (light on 7:00 A.M.). After a
further 3 d for adaptation, during which they were handled daily,
the animals received an intravenous injection of 5 µg/kg LPS
(Salmonella typhimurium, Sigma) at 10:00 A.M. and then were
perfused at 3 P.M. The 5 hr (10:00 A.M. - 3 P.M.) delay after LPS
injections allowed us to measure dynamic changes in body temperature
and then to determine expression of Fos protein in the brains by
immunohistochemistry (Elmquist et al., 1996).
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RESULTS |
Defining lesions of the SPZ and PVH
Although Watts and colleagues (1987) originally described the SPZ
based on the tendency for SCN axons to terminate ventral to the PVH
rather than within it, the name SPZ does not fully describe the region.
As defined by the continuous column occupied by dense SCN axonal
arborization and termination, the SPZ begins just dorsal to the
SCN (Fig.
1A,B)
where it overlaps with the peri-SCN region. As the SCN axons progress
caudally, the terminal zone becomes progressively more dorsal to occupy
the region just ventral to the PVH (Fig. 1C). It is possible
in Nissl-stained sections to identify the SPZ by the presence of
bundles of dorsoventrally oriented axons piercing the medial part of
the anterior hypothalamic area. At the level of the PVH, the SCN
termination zone turns laterally, running just ventral to the PVH.
However, relatively few SCN axons further penetrate the PVH capsule;
instead, the remaining SCN axons continue caudally to terminate
diffusely throughout the rostromedial DMH. Even more caudally, a few
axons terminate in the caudal DMH (Fig. 1D) but avoid
its compact subnucleus. We defined the vSPZ as the region of SCN
terminals in the medial part of the anterior hypothalamic area for ~1
mm dorsal and caudal to the SCN and the dSPZ as the more caudal part of
the SPZ centered on the area ventral to the PVH. The SCN terminal field
continues back into the DMH, which is continuous with the SPZ, and
receives substantial SPZ inputs (Elmquist et al., 1998).
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Figure 1.
Photomicrographs showing VIP-ir axons and
terminals that define the subparaventricular zone (SPZ)
from rostral to caudal levels. A, B, The
ventral SPZ demonstrated by a column of VIP-ir axons leaving the dorsal
margin of the suprachiasmatic nucleus (SCN).
C, The dorsal SPZ, ventral to the paraventricular
nucleus (PVH). Notice the VIP-ir terminals in the
medial PVH. D, The continuation of VIP-ir terminals from
the SCN into the rostral DMH.
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We defined the cell loss in our animals by using a combination of
Nissl, c-Fos, AVP, and VIP staining. The extent of vSPZ lesions was
defined by Nissl cell counts within the region identified by VIP fibers
at the level of the SCN, and the extent of dSPZ lesions was defined by
counts at the level of the PVH (see Materials and Methods). To
determine whether the SCN was injured in our vSPZ lesions, we used
immunostaining for AVP, VIP, and Fos staining in addition to Nissl
staining. In most cases, we found that there was no morphological
damage to neurons in the SCN, which generally appeared resistant to
ibotenic acid (Fig.
2A-C); if
the SCN was damaged, the case was excluded from the analysis. Dorsal
SPZ lesions in general did not injure the PVH as judged by loss of
Nissl- or AVP-stained neurons within the nucleus, or by loss of Fos-ir neurons after LPS stimulation (Fig.
2D-F). The PVH lesions usually included a small but variable number of cells in the dSPZ as determined by counts of Nissl-stained neurons in the region marked by VIP fibers
(Fig. 2G-I). The locations of lesions in
selected animals of all three groups are illustrated in Figure
3.
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Figure 2.
Photomicrographs showing lesions (outlined by
light dashed line) in the ventral SPZ
(A-C, vSPZ), dorsal SPZ
(D-F, dSPZ), and PVH
(G-I). Lesions of the vSPZ
typically spared the SCN as confirmed by immunostaining for VIP and AVP
(A-C). Lesions of the dSPZ
(D) did not affect the PVH, as shown by
immunostaining for Fos (E, induced by LPS) and AVP
(F). Lesions of the PVH (G, PVH
border outlined in heavy dashed line) were verified by
loss of Fos-ir neurons after LPS injection and by loss of AVP-ir
neurons (H, I).
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Figure 3.
A series of camera lucida drawings illustrating
lesion areas in the vSPZ (A), dSPZ
(B), and PVH (C).
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The effects of SPZ lesions on circadian rhythms of body
temperature, sleep, and activity
Ventral SPZ
We obtained 8 rats with >70% cell loss bilaterally in the vSPZ
and 10 rats that had cell loss ranging from 20 to 70%. None of our
lesions appeared to affect neuronal morphology in the SCN as judged by
AVP, VIP, Nissl, and Fos staining (Fig.
2A-C). We did not analyze animals in
which the vSPZ was damaged only unilaterally. For controls, we used
animals (n = 7) that had injections into the third
ventricle without apparent damage to the SCN or the SPZ.
Typical lesions in this group included the vSPZ and often infringed on
the rostroventral edge of the dSPZ (Fig. 3). VIP and AVP fibers in the
vSPZ were intact (Fig. 2B,C). In
the animals with vSPZ lesions, sleep, body temperature, and activity
were synchronized to day/night cycles while on an LD 12 hr cycle (Fig. 4A). In constant
darkness, the animals with vSPZ lesions lost their circadian rhythms of
sleep (CI = 9.8 ± 4.7, mean ± SEM; compared with
controls, p < 0.01; cosinor amplitude = 14.8 ± 4.1, compared with controls, p < 0.01) (Figs.
5, 6).
Daytime total sleep averaged 56.8 ± 2.9%, which was not
significantly different from night sleep time, at 50.1 ± 3.3%,
p > 0.05. These animals also lost circadian rhythm of
activity [CI = 27.0 ± 6.2, p < 0.05; cosinor amplitude 22.1 ± 3.7, p < 0.01 (Fig.
7); daytime activity 2.2 ± 0.3 U, night-time activity 2.8 ± 0.4, p > 0.05 (Fig. 5)]. Body temperature retained a statistically
significant circadian rhythm as determined by comparison of body
temperature during the day (37.3 ± 0.04°C) and night (37.7 ± 0.05°C; p < 0.05) (Fig. 4A),
but the amplitude of the rhythm was about half as great as in controls
(CI = 48.7 ± 11.5; cosinor amplitude = 68.1 ± 4.2; both different from controls at p < 0.05) (Fig.
7). A strong ultradian component of sleep and body temperature emerged
after vSPZ lesions, with a period of ~3-3.4 hr corresponding to
seven to eight cycles across 24 hr. This ultradian rhythm was
superimposed on the remaining underlying circadian rhythm of body
temperature (Fig. 5B). Sleep tended to occur during the
troughs between body temperature circadian peaks, whereas locomotor
activity corresponded to the body temperature peaks. Alternating
ultradian peaks of sleep and locomotor activity were also noted during
the night (waking) cycle and may have been associated with minor
variations in body temperature (Fig. 5A).
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Figure 4.
Plots showing long-term effects of representative
lesions of the vSPZ (A), dSPZ
(B), and PVH (C) on body
temperature. After the vSPZ lesion (A), body
temperature showed a decreased circadian amplitude but still retained a
circadian rhythm even under constant darkness (subjective day vs night
mean value different at p < 0.05), which could be
recognized visually in the long-term recording of body temperature. The
dSPZ lesion (B) initially disrupted body
temperature rhythm, which slowly recovered and showed a low amplitude
during light/dark cycle conditions. However, in constant darkness the
circadian rhythm of body temperature was lost almost completely. In
contrast, the rat with PVH lesions (C) showed a
normal circadian rhythm in body temperature under both light/dark and
constant darkness conditions. The arrow indicates the
time of placing the lesions. Black bars represent the
periods of darkness.
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Figure 5.
Combined graphs showing EEG delta power
(gray line), body temperature (light black
line), and locomotor activity (heavy black line)
under constant darkness in a representative control animal
(A) and in an animal with a vSPZ lesion
(B). The left y-axis
demarcates delta power, the right y-axis
shows body temperature, and the x-axis is time = 24 hr. Locomotor activity is in arbitrary units. The control rat
(A) showed robust circadian rhythms of EEG delta
power, body temperature, and activity. Sleep was increased
predominantly during the subjective day, whereas body temperature and
activity were increased during the subjective night. After a vSPZ
lesion (B), there was a loss of circadian
variation in delta power and activity.
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Figure 6.
Percentage rapid eye movement
(REM) sleep per hour and non-REM
(NREM) sleep per hour across 24 hr (constant
darkness) in controls (A, B) and vSPZ
lesioned rats (C, D). There was loss of
circadian variation in both REM and NREM sleep after lesions in the
vSPZ, whereas both REM and NREM sleep showed a robust circadian rhythm
in the control group.
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Figure 7.
The effects of lesions in the dSPZ, vSPZ, and PVH
on circadian index (A) and cosinor amplitude
(B) of NREM sleep, body temperature
(Tb), and activity under constant darkness. The
circadian index and cosinor amplitude were normalized to the mean
circadian amplitude of the control animals (100). The dSPZ lesions
significantly reduced the circadian index of body temperature but did
not significantly affect circadian index or cosinor amplitude of NREM
sleep or activity. The vSPZ lesions dramatically reduced circadian
index and cosinor amplitude for sleep and activity and had somewhat
less intense, although still significant, effects on body temperature.
*p < 0.05; **p < 0.01.
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Furthermore, across the entire group of animals with 20-70% loss of
neurons bilaterally in the SPZ, we found that the number of
Nissl-stained neurons in the vSPZ was significantly correlated with the
circadian indices of sleep (r = 0.80; p < 0.01) and activity (r = 0.63; p < 0.05) but not that of body temperature (r = 0.51; p > 0.05) (Fig. 8).
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Figure 8.
Correlation of the number of surviving neurons in
the vSPZ with the circadian index of body temperature, sleep, and
locomotor activity. For sleep and activity, there was a significant
(p < 0.05) linear correlation of circadian
index with the number of surviving neurons in the vSPZ, whereas the
circadian index of body temperature did not show a significant
correlation (p > 0.05), suggesting that
alterations of body temperature rhythm may have been caused by the
lesions including tissue beyond the vSPZ counting box.
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Dorsal SPZ
We obtained 7 rats that had >70% cell loss bilaterally in the
dSPZ (Fig. 2D-F) and 10 rats that
had cell loss ranging from 20 to 70% that were used for correlation
analysis. In these rats, we avoided damaging the vSPZ region by placing
the lesion center more dorsally and caudally. As a result, dSPZ lesions
usually included the rostral edge of the DMH. We did not use any animal with a unilateral dSPZ lesion for correlation analysis. Ten rats that
had injections in the third ventricle were used as controls.
Immediately after bilateral lesions in the dSPZ, body temperature
showed greatly reduced circadian rhythmicity even under light/dark
conditions (CI = 30.1 ± 5.3 on days 1-3; cosinor = 40.5 ± 3.9; p < 0.05) (Fig.
4B). Instead, a robust ultradian rhythm (with a
period of ~3 hr, similar to that found after vSPZ lesions) clearly
emerged, and its amplitude reached as high as 1.0°C (Figs. 4B, 9A). By
6-7 d after the lesion, body temperature began to synchronize to the
light/dark cycle with a low circadian amplitude (daytime body
temperature 37.6 ± 0.05°C, nighttime 37.3 ± 0.04°C; p < 0.01). When the same rats were placed in constant
darkness, the circadian temperature rhythm was reduced further (daytime 37.5 ± 0.04°C; nighttime 37.3 ± 0.04°C) (Fig.
4B) to the point where there was no statistically
significant difference between phases (p > 0.05), and there was a reduction of CI to 32.5 ± 7.4 and cosinor
amplitude to 44.6 ± 4.2 (both different from controls at
p < 0.05) (Fig. 7). The same lesions had no
significant effects on circadian rhythms of sleep in either the
light/dark cycle or constant darkness [in constant darkness, CI = 96.6 ± 4.9; cosinor amplitude = 95.6 ± 2.8, not
different from control at p > 0.05 (Fig. 7); daytime
sleep 63.8 ± 5.8% of total time, nighttime 43.5 ± 5.4%,
day and night significantly different at p < 0.05 (Figs. 9, 10)]. When we plotted delta
power (0.5-4 Hz) and body temperature in the animals with the dSPZ
lesions on the same axes, we found that although the circadian rhythm
of body temperature was abolished, sleep episodes still usually
occurred at the trough of ultradian body temperature variations,
whereas bouts of locomotor activity tended to occur during the
ultradian body temperature peaks (Fig. 9A).
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Figure 9.
Combined graphs showing the effects on EEG delta
power (light black line), locomotor activity
(heavy black line), and body temperature
(gray line) of lesions in the dSPZ
(A) and the PVH (B). The
left y-axis demarcates delta power, the
right y-axis shows body temperature, and the
x-axis is time = 24 hr. Locomotor activity is in
arbitrary units. After a dSPZ lesion (A), there
was loss of circadian rhythm in body temperature, but delta power and
activity from the same animal showed a clear circadian rhythm. In
B (PVH lesion), body temperature, EEG delta power, and
locomotor activity all showed robust circadian rhythms.
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Figure 10.
Circadian rhythms of the percentage NREM sleep
during a 12 hr light/dark cycle (A, C,
E) and under constant darkness (B,
D, F), in controls
(A, B), and after lesions of the dSPZ
(C, D) or PVH (E,
F). Neither lesion significantly affected
circadian rhythms of NREM sleep compared with controls.
|
|
Although the circadian rhythm of locomotor activity was decreased in
the animals with dSPZ lesions (CI = 55.5 ± 16.5; cosinor amplitude = 69.4 ± 11.9), it was not statistically
significantly different from the controls (p > 0.05) (Fig. 7).
The correlation analysis showed a significant linear correlation of the
number of surviving neurons in the dSPZ with the CI of body temperature
(r = 0.71; p < 0.05) but not sleep
(r = 0.24; p > 0.05) or activity
(r = 0.58; p > 0.05) (Fig.
11). Taken together, dSPZ lesions
significantly reduced circadian rhythms of body temperature by
~60-70% but had much smaller effects on locomotor activity that
were not statistically significant. Sleep showed nearly normal circadian patterning, but a strong ultradian pattern of both body temperature and sleep emerged.
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Figure 11.
Correlation of the numbers of surviving neurons
in the dSPZ plotted against the circadian index of sleep
(A), locomotor activity
(B), and body temperature
(C). The circadian index of body temperature but
not sleep or activity showed a strong correlation with the numbers of
surviving neurons (per side) in the dSPZ.
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The effects of lesions of the PVH on circadian rhythms
We obtained 7 rats that had substantial bilateral (>70%) lesions
of the PVH and 10 rats with <50% cell loss in the PVH or with both
injections into the third ventricle that were used as controls. Lesions
typically destroyed most cells in the dorsal and medial parvicellular
PVH that show Fos expression during fever (Figs.
2G,I), but some cells in the
periventricular PVH were often spared. The lesions were verified by
Fos, Nissl, and AVP staining (Figs.
2G-I).
Bilateral PVH lesions did not affect the circadian rhythms of body
temperature, sleep, or activity in the light/dark condition or in
constant darkness (Figs. 4B, 7, 9, 10). The circadian
index of body temperature, sleep, and locomotor activity was not
different from that of the controls (CI of body temperature = 102.5 ± 33.3; sleep = 98.3 ± 8.6; activity = 98.6 ± 21.3; p > 0.05 for all three) (Fig. 7).
In parallel, the cosinor amplitudes of sleep, body temperature, and
activity showed no difference from the controls (sleep = 86.3 ± 3.2, body temperature = 106.3 ± 8.5, activity = 88.5 ± 12.5) (Fig. 7).
The effects of lesions of the dorsal SPZ and the PVH on
fever responses
After administration of LPS to the animals with dSPZ lesions, the
fever response was identical to control animals, rising 1.1 ± 0.2°C in animals with dSPZ lesions 5 hr after LPS injection, compared
with 1.2 ± 0.2°C in control animals (p > 0.05) (Fig. 12). The expression of
Fos protein in the PVH, the ventromedial preoptic nucleus (VMPO), and
the nucleus of solitary tract (NTS) was likewise identical in the two
groups (Table 1).
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Figure 12.
Fever responses in rats with lesions in the PVH
(gray diamonds), dSPZ (black
squares), or controls (gray triangles)
after intravenous LPS injection. The PVH lesion significantly
attenuated fever (66.6%; p < 0.01) compared with
the controls. In contrast, the rats with dSPZ lesions showed fever
responses that did not differ significantly from the controls
(p > 0.05). The injection time is at zero
on the x-axis.
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In the rats with PVH lesions, after intravenous LPS injection, fever
was reduced by 66.6% (Fig. 12), and few Fos-ir cells were found in the
PVH (Table 1; Fig. 2H), although Fos expression in
the VMPO and NTS was identical to that of normal controls.
| |
DISCUSSION |
The principal findings of this study were that lesions of the vSPZ
profoundly disrupted the circadian rhythms in sleep and locomotor
activity but had smaller effects on body temperature that did not
correlate significantly with vSPZ cell loss. By contrast, lesions of
the dSPZ disrupted circadian rhythms in body temperature but had less
of an effect on locomotor activity and virtually no effect on sleep.
PVH lesions did not affect circadian rhythms of sleep, body
temperature, or activity; however, the loss of neurons in the PVH
significantly attenuated LPS-induced fever. The dSPZ lesions did not
affect fever responses. These results suggest that the SPZ is a complex
region consisting of neuronal subsystems that may differentially
regulate circadian rhythms of different physiological responses.
Conversely, the PVH appears to be an autonomic and endocrine effector
site but does not play a major role in organizing circadian rhythms of
sleep, activity, or body temperature.
Technical considerations
One potential problem was the anatomical proximity of the PVH to
the dSPZ, raising the question of whether lesions of the dSPZ would
also damage the PVH. Because the fibrous capsule of the PVH acts as a
diffusion barrier to ibotenic acid, we were able to make cell-specific
lesions in the dSPZ without damaging the PVH. We were also concerned
that the close proximity of the vSPZ to the SCN might make it difficult
to damage the vSPZ without affecting the SCN, lesions of which would
disrupt all circadian rhythms. On the basis of histology of Nissl-,
Fos-, AVP-, and VIP-stained sections through the SCN region, we found
that SCN neurons were resistant to ibotenic acid [see also Peterson
and Moore (1980)]. Moreover, after vSPZ lesions, body temperature retained a significant circadian rhythm, whereas sleep did not, which
would not be expected if the effects were caused by SCN damage.
To define the border of the SPZ, we had to rely on the SCN terminal
field, particularly the VIP-ir fiber zone. In our quantitative studies,
we counted Nissl-stained neurons within the fields determined by these
fibers. We recognize that other SCN outputs contain different neurotransmitters, but in our preliminary studies the course of the
VIP-ir terminal field closely approximated the SCN output as judged in
anterograde labeling from the SCN using biotinylated dextran (S. E. Gaus and C. B. Saper, unpublished observations).
The role of the subparaventricular zone in
circadian regulation
The SPZ has traditionally been identified by tracing the efferent
terminal field from the SCN, particularly its ventrolateral component
(Watts and Swanson, 1987; Watts et al., 1987; Watts, 1991; Vrang et
al., 1995). However, anatomical studies suggest dorsal and ventral
regions within the SPZ that receive somewhat different inputs. For
example, the vSPZ receives direct retinal afferents (Johnson et al.,
1988; Mikkelsen, 1990), which may be important in reconstituting
circadian cycles in a light/dark environment, even after SCN lesions.
The dSPZ, by contrast, receives a substantial input from the
dorsomedial part of the ventromedial hypothalamic nucleus (VMH)
(Elmquist et al., 1998), which may be important in synchronizing
circadian regulation with food availability (Challet et al., 1997; Choi
et al., 1998). Our physiological observations support the separation of
the SPZ into ventral and dorsal regions that differentially regulate
circadian rhythms of sleep and body temperature.
The vSPZ lesions profoundly diminished the circadian rhythm of sleep
and locomotor activity. Although these same lesions caused a
statistically significant reduction in the circadian rhythm of body
temperature, this function retained a significant degree of
rhythmicity. More importantly, the loss of cells in the vSPZ correlated
closely with the loss of sleep circadian rhythm but did not reach
statistical significance for correlating with the loss of body
temperature rhythm. These findings suggest that the changes in
circadian rhythm of body temperature with the vSPZ lesions may have
been caused by injury outside the vSPZ counting area, perhaps in the
rostral edge of the dSPZ, which was included in some cases. Lesions of
the dSPZ, by contrast, disrupted circadian control of body temperature
(as judged by cosinor analysis or comparing day/night mean
temperatures) to a significantly greater degree than did the vSPZ
lesions (p < 0.05) but had no discernable effect on circadian rhythm of sleep or locomotor activity.
The differential role of the two SPZ regions in circadian control of
sleep versus body temperature may be related to their connections. One
prominent target of the SPZ is the preoptic area (Watts, 1991).
Although relatively small in size, the preoptic area contains complex
populations of cells that control body temperature, sleep, and
reproduction (for review, see Satinoff, 1978; McGinty and Szymusiak,
1990; Argiolas, 1999). The medial preoptic area, including the recently
identified VMPO, plays an important role in thermoregulation (Satinoff
et al., 1982; Elmquist et al., 1996, Lu et al., 2000), whereas the
ventrolateral preoptic nucleus (VLPO) plays a key role in the
regulation of sleep (Sherin et al., 1996, 1998; Szymusiak et al., 1998;
Lu et al., 2000). It will be important to determine whether projection
from the dSPZ and vSPZ to the VMPO and VLPO may relay critical
information for the differential circadian regulation of body
temperature versus sleep.
The SPZ also projects to the DMH (Elmquist et al., 1998), which
projects to many of the same preoptic targets as the SPZ. Previous studies have implicated the DMH in the circadian regulation of
feeding, drinking, and corticosterone secretion (Bernardis, 1973;
Bellinger et al., 1976; Cipolla-Neto et al., 1988; Kalsbeek et
al., 1996). Our preliminary studies suggest that DMH lesions also have
profound effects on circadian rhythms of sleep, locomotor activity, and
body temperature (Chou et al., 2000). Hence the SPZ may act primarily
as a link, integrating circadian input from the SCN and retina with
behavioral inputs, e.g., from the VMH, and projecting to the DMH and
preoptic targets to organize circadian responses.
The role of the paraventricular nucleus in
circadian regulation
Compared with the SPZ, the PVH receives relatively small amounts
of SCN inputs, mainly to its parvicellular subdivisions. Neurons in the
PVH that are innervated by the SCN are thought to control sympathetic
preganglionic cells that regulate melatonin secretion (Klein et al.,
1983; Larsen et al., 1998; Teclemariam-Mesbah et al., 1999). Similarly,
direct SCN projections to the PVH may regulate secretions of
corticotrophin-releasing factor (Vrang et al., 1995b). The patterns of
secretion of these hormones could potentially affect circadian rhythms
of body temperature or sleep. To control for the possible effects of
even minor damage to the PVH in our study, we analyzed the effect of
ibotenic acid lesions of the nucleus on sleep, activity, and body
temperature. Our results were consistent with earlier studies using
electrolytic lesions of the PVH that had reported no major effects on
body temperature (Horn et al., 1994) or the circadian regulation of
sleep (Piepenbrock et al., 1985).
Our observations are consistent with the PVH being a target, rather
than a pacesetter, for the circadian control system. The SPZ may
influence the PVH via its projections to the DMH, which in turn
provides substantial PVH input (Watts et al., 1987; Elmquist et al.,
1998).
Regulation of ultradian rhythms
The emergence of ultradian rhythms after lesions in the SCN and
SPZ suggests that ultradian rhythms are generated by different mechanisms and suppressed by circadian rhythms generated by the SCN and
SPZ. Large electrolytic lesions in the SCN region and the adjacent
retrochiasmatic area or lesions in the retrochiasmatic area-arcuate
nucleus eliminate ultradian rhythms of sleep (Wollnik and Turek, 1989;
Gerkema et al., 1990), suggesting that the retrochiasmatic area may
play a role in ultradian oscillation. Although ultradian rhythms are
relatively weak in the presence of circadian control, they may still be
recognized, especially during the night (wake period), as shown by the
ultradian rhythm in delta power (Fig. 5A).
The paraventricular nucleus and the febrile response
Several lines of circumstantial evidence suggest that the PVH
plays an important role in the fever response. Large number of
Fos-active cells are seen in the PVH during fever in rats produced either by intravenous or intraperitoneal LPS (Elmquist et al., 1996;
Konsman et al., 1999) or by interleukin-1 (Rivest et al., 1992;
Ericsson et al., 1994; Day and Akil, 1996). Parvicellular neurons in
the dorsal PVH are Fos positive during fever and project directly to
the sympathetic preganglionic column in the spinal cord (Zhang et al.,
2000). This pathway may play an important role in fever response by
stimulating brown adipose tissue to produce heat by activating the
uncoupling protein UCP1, causing the adrenal glands to secrete
adrenaline, and constricting the tail artery to reduce cutaneous heat
loss (for review, see Zhang et al., 2000). Electrolytic lesions of the
PVH decrease fever responses without affecting responses to a cold or
hot environment (Horn et al., 1994). Our studies using excitotoxic
lesions further support the role of the PVH as a key site for
regulating fever response but not circadian rhythm of body temperature.
| |
FOOTNOTES |
Received Jan. 2, 2001; revised March 19, 2001; accepted April 11, 2001.
This research was supported by National Institutes of Health
Grants NS 33987 and MH55772. We thank Quan Hue Ha, Joseph F. Kelly, and Charlotte Lee for excellent technical help.
Correspondence should be addressed to Dr. C. B. Saper, Department
of Neurology, Beth Israel Deaconess Medical Center, 330 Brookline
Avenue, Boston, MA 02215. E-mail:
csaper{at}caregroup.harvard.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
