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The Journal of Neuroscience, July 1, 1999, 19(13):5574-5585
Localization of a Suprachiasmatic Nucleus Subregion Regulating
Locomotor Rhythmicity
J.
LeSauter1 and
Rae
Silver1, 2, 3
1 Department of Psychology, Barnard College, New York,
New York 10027, 2 Department of Psychology, Columbia
University, New York, New York 10027, and 3 Department of
Anatomy and Cell Biology, College of Physicians and Surgeons, New York,
New York 10032
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ABSTRACT |
The bilaterally symmetrical suprachiasmatic nuclei (SCN) of the
hypothalamus are the loci of the mammalian clock controlling circadian
rhythms. Previous studies suggested that all regions of the SCN are
equipotential as circadian rhythmicity is sustained after partial
ablation, as long as ~25% of the nuclei are spared. In contrast to
these results, we found that animals bearing partial lesions of the SCN
that spared the subregion delimited by cells containing the
calcium-binding protein calbindin-D28K (CaBP), sustained
circadian locomotor rhythms. Furthermore, there was a correlation
between the strength of the rhythm and the number of spared CaBP cells.
Partial lesions that destroyed this region but spared other
compartments of the SCN resulted in loss of rhythmicity. The next study
indicates that transplants of half-SCN grafts that contain CaBP cells
restore locomotor rhythms in SCN-lesioned host animals, whereas
transplants containing SCN tissue but lacking cells of this subnucleus
fail to restore rhythmicity. Finally, there was a correlation between
the number of CaBP-positive cells in the graft and the strength of the
restored rhythm. Taken together, the results indicate that pacemakers
in the region of the CaBP subnucleus are necessary and sufficient
for the control of locomotor rhythmicity and that the SCN is
functionally heterogeneous.
Key words:
calbindin-D28K; suprachiasmatic
nucleus; hamster; rodent; vasopressin; vasoactive intestinal peptide; circadian rhythms; locomotor activity; pacemaker; oscillator
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INTRODUCTION |
Circadian clocks are ubiquitous
endogenous biological oscillators that measure elapsed and local time,
thereby regulating the temporal organization of organisms. Rapid
progress in the cloning and characterization of clock genes and
clock-controlled genes in several species has brought the promise of
finding a general mechanism for circadian clocks at the molecular level (Dunlap, 1998 ). To relate molecular and genetic mechanisms to the physiology of clocks in multicellular organisms, it must be determined which cells are pacemakers. Properties of pacemakers thought
to be necessary for producing circadian rhythms include endogenous
oscillation, resetting by environmental cues, especially light, and
production of an efferent signal(s) that reaches the rest of the brain.
It is not clear in mammals which combination of these properties reside
within individual cells.
It is well established that in mammals circadian pacemakers that
control the phase of circadian rhythms in the rest of the body lie in
the paired suprachiasmatic nucleus (SCN) of the hypothalamus (Moore and
Silver, 1998). Photic cues from the daily cycle of light and darkness
reach the SCN by means of the retinohypothalamic tract, serving to keep
the organism in phase with its environment. Output signals from the SCN
reach the rest of the brain to ensure the synchronization of circadian
organization throughout the body.
In the rodent the SCN contains ~10,000 cells in each nucleus.
These cells are not homogenous and express a large number of different
peptides and enzymes (van den Pol and Tsujimoto, 1985). Studies using
tract-tracing and fos induction in response to light indicate that the
nucleus is also differentiated on the afferent side, with retinal and
photic input reaching a subset of SCN cells largely restricted to the
ventrolateral aspect in rats and extending more dorsally in hamsters
(Johnson et al., 1988; Rea, 1989; Kornhauser et al., 1990; Rusak et
al., 1990; Morin, 1994). Although it seems that SCN cells are
heterogeneous, lesion studies suggest that many SCN cells function as
pacemakers (see Discussion in van den Pol and Powley, 1979), and
recording of electrical activity in individual SCN neurons (Welsh et
al., 1995) indicates that several SCN cell types are capable of
sustaining circadian rhythms. The present report represents a
multipronged strategy to localize SCN pacemakers that control locomotor rhythmicity.
There is a compact subnucleus of calcium-binding protein
calbindin-D28K (CaBP)-positive cells in the core of
the hamster SCN (Fig. 1; Silver et al.,
1996). Within this region is a population of substance P cells (Morin
et al., 1992), forming a subset of the CaBP cells with most substance P
cells expressing CaBP (78%) and only 8% of the CaBP cells expressing
substance P (Silver et al., 1996). Approximately 79% of the CaBP cells
are fos-positive in response to a light pulse (Silver et al., 1996). We
now report that circadian rhythmicity is retained after partial lesions
of the SCN if this region is spared but not if this region is ablated. Furthermore, transplantation of parts of the SCN into SCN-lesioned animals rescues circadian rhythms if the CaBP subnucleus is present but
not when it is absent.
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Figure 1.
Photomicrograph of a sagittal brain section
showing the location of the CaBP subnucleus (arrow).
This subregion is highly localized in the caudal SCN, making it
possible to ablate this part of the nucleus but to leave other
compartments intact. Scale bar, 200 µm.
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MATERIALS AND METHODS |
Subjects and housing. Subjects were wild-type
(free-running period of ~24 hr) adult male or female LVG hamsters
(Mesocricetus auratus) and male mutant hamsters heterozygous
and homozygous for the tau mutation (free-running periods of
~22 and 20 hr, respectively). Animals were housed individually in
translucent polypropylene cages (48 × 27 × 20 cm) and
provided with access to food and water ad libitum. The room
was kept at ~23°C and was equipped with a white noise generator (91 dB sound pressure level) to mask environmental noise. At ~7
weeks of age, animals were transferred from a light/dark cycle (14 hr
light, 10 hr dark) to constant darkness (DD) in cages equipped with a
running wheel (17 cm diameter). A dim red light (<1 lux; Delta 1, Dallas, TX) allowed for maintenance. All experimental protocols
conformed to the Institutional Animal Care and Use Committee guidelines of Columbia University.
Experiment 1: lesion study. After 1-2 weeks in DD, hamsters
(110-120 gm) were anesthetized (100 mg/kg sodium pentobarbital) and
placed in a stereotaxic instrument, and bilateral electrolytic lesions
were made using a Grass Instruments (Quincy, MA) LM-5 lesion maker and
stainless steel 00 electrodes, insulated with Epoxylite (The Epoxylite
Corp., Irvine, CA) except at the tip (0.25 mm). Current was passed for
either 10 (n = 24) or 20 (n = 45)
sec at 0.55 mA. Lesions were placed 0.8 mm anterior to bregma, 0.1 mm
lateral to the midline, and 7.9 mm below the dura. Activity was
monitored for 4 weeks in animals that sustained rhythmicity and for 12 weeks in animals that became arrhythmic after surgery.
Experiment 2: transplantation study. Locomotor activity of
tau mutant hamsters was monitored for 1-3 weeks. Animals were then given SCN lesions as described above, using a 20 sec current (see above). Hamsters (n = 36) that had become arrhythmic
after SCN lesions were used as host animals for transplants.
Timed pregnant female hamsters (Charles River Laboratories, Wilmington,
MA) were received on gestation day 12 and housed in DD. Hypothalamic
grafts were taken from pups on the day of birth. Brains were dissected
out and placed on a sterile Petri dish. Vibratome slices were cut at
500 µm in either the coronal or sagittal plane. The sections were
placed in sterile saline on a cold sterile microscope slide for
visualization of the SCN under an inverted light microscope (LeSauter
et al., 1996). On coronal sections, the SCN was cut with a scalpel
blade at an angle of ~45° (right side; 315° left side) to
separate the dorsomedial and ventrolateral parts of the nucleus. Next,
two additional cuts were made at the outer limit of the SCN. On
sagittal slices, the SCN was cut in half, separating the rostral and
caudal SCN. Then, the other cuts were made to separate the SCN from the
rest of the brain. Tissue from three donors was pooled in a drop of
saline and implanted via a modified 20 gauge needle, which was lowered
7.6 mm below dura, using the opening in the skull made during the lesion.
After recovery from anesthesia, the animals were returned to their
cages. Activity was monitored for 16-20 weeks after transplantation.
Perfusion and histology. Hamsters were heavily anesthetized
(pentobarbital, 200 mg/kg) and perfused intracardially with 150 ml of
0.9% saline followed by 300-400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Brains were post-fixed for 18-24 hr at 4°C and cryoprotected in 20% sucrose in 0.1 M phosphate buffer overnight. Coronal sections were cut on
a freezing microtome and processed as free-floating sections.
For the lesion and transplant studies, every fourth brain section was
immunostained for neurophysin-associated vasopressin (NP), as a marker
for vasopressin in the SCN, and vasoactive intestinal polypeptide
(VIP), and every other section was immunostained for CaBP. Polyclonal
antisera against NP and VIP (Incstar, Stillwater, MN) were used at
dilutions of 1:10,000. Preabsorption of each milliliter of diluted
antiserum for 24 hr at 4°C with the purified peptides NP and VIP
(Peninsula Laboratories, Belmont, CA) completely eliminated all
immunoreaction product. Monoclonal CaBP antibody (Sigma, St. Louis, MO)
was used at 1:20,000. Antisera were detected using appropriate
biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) and an avidin-biotin-HRP conjugate (Vector
Laboratories) with 3,3'-diaminobenzidine (Polysciences, Warrington, PA)
as the chromogen. Sections were mounted on glass slides, dehydrated, cleared, and coverslipped.
Analysis of the SCN. To assess the SCN in lesions and
grafts, we took advantage of the fact that the SCN has several unique features when compared with the adjacent hypothalamus. VIP and VP cells
are present in a characteristic topographical arrangement (also see
LeSauter et al., 1996; Moore and Silver, 1998) and are absent in the
adjacent hypothalamus. Densely packed CaBP-IR cells with very
fine efferents in the SCN lie within the NP-VIP region, in an
"island" surrounded by a region lacking this peptide (Fig. 1;
Silver et al., 1996). Dispersed CaBP-IR cells are seen in the adjacent
hypothalamus. CaBP cells of SCN and extra-SCN origin were distinguished
by their size: it is known that "... SCN cells are the smallest
neurons in the hypothalamus, and among the smallest in the brain"
(van den Pol, 1991). CaBP cells of the SCN were significantly smaller
than those of adjacent hypothalamic regions (overall
F(4,195) = 107.5; p < 0.0001). The perimeter of CaBP cells of the SCN was 33.6 ± 0.4 µm (mean ± SEM; n = 40 cells). CaBP cells in
extra-SCN sites in adjacent hypothalamus were significantly smaller, as follows: rostral (42.1 ± 5.5 µm;
n = 40; p < 0.0001), lateral
(42.4 ± 0.5 µm; n = 40; p < 0.0001), and caudal (47.8 ± 5.6 µm; n = 40;
p < 0.0001). The SCN CaBP cells within the grafts were
slightly smaller than in the intact SCN (31.0 ± 0.5 µm,
n = 40; p = 0.005).
Based on these features, we used the following criteria to identify the
SCN in transplants and at the lesion site. The presence of overlapping
regions of VIP and VP-IR in adjacent sections definitively denoted the
presence of SCN tissue at the lesion site or in grafts. The
determination of whether CaBP cells originated in the SCN or adjacent
hypothalamus was determined by measuring cell size. Thus, the presence
of these peptides in their characteristic distribution and
distinguishing cell size, taken together, were used to analyze the SCN
lesion and graft.
Brain sections were captured using a CCD video camera (XC77; Sony,
Tokyo, Japan) attached to an Olympus (Tokyo, Japan) BH2 microscope
using NIH Image (version 1.61). In grafted animals, the size of
the transplanted SCN was measured by taking the overall area of the
section with the largest NP- or VIP-IR plexus (used for Fig. 5). This
system was also used to measure size of CaBP cells (described above)
and SCN volume after partial ablation. The size of the cells in these
different regions was compared by ANOVA with post hoc Fisher's PLSD
for comparison between groups.
To count the number of CaBP profiles (what is seen of a cell in a
histological section; Coggeshall and Lekan, 1996) after a partial SCN
lesion or a transplant cells were counted within the area stained for
VIP and VP on adjacent sections. The presence or absence of a rhythm
and the absolute power of the restored rhythm were correlated with the
number of SCN CaBP profiles, the number of SCN clusters, and the size
of the clusters.
Acquisition of behavioral data. Locomotor activity was
monitored continuously using a computer-based data acquisition
system (Dataquest; Data Sciences, St. Paul, MN). The power of the
rhythm was assessed using Fourier analysis (Dataquest). An animal
was considered rhythmic when the highest peak occurred at ~1
cycle/d, with absolute power of at least 0.005 mV/Hz.
Brain sections were analyzed by an investigator blind to the
experimental groups and to the behavioral aspects of the results.
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RESULTS |
Experiment 1: partial SCN lesions
In 51 of 69 subjects, the SCN were partially (n = 47) or fully (n = 4) ablated on both sides (summarized
in Table 1). The remaining 18 animals
were not used in the study, because the lesion either missed the SCN
completely (n = 14) or ablated the SCN on one side
partially (n = 3) or totally (n = 1),
leaving the other side completely intact. These animals remained
rhythmic.
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Table 1.
The peptidergic analysis of animals with partial lesions of
the SCN shows that the presence of locomotor rhythmicity is dependent
on the presence of the CaBP subnucleus
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Twenty-four animals with sparing of one (n = 18) or
both CaBP (n = 6) subnuclei remained rhythmic. In
marked contrast, animals with lesions that bilaterally destroyed the
subregion of CaBP cells were arrhythmic, although sparing of other SCN
tissue occurred (n = 23).
The extent of SCN damage is shown for six animals with partial SCN
lesions. Animals with a lesion ablating the CaBP cells and sparing of
NP and VIP cells and fibers are shown in Figures 2 (B26-V34) and
3 (B56-CA29). The behavioral analysis for
these animals (Fig. 4) shows that both
are arrhythmic after the lesion. Figure 5
shows schematics from images captured on the computer of the area of
SCN damage in adjacent sections stained for VIP and CaBP for four
animals. The first animal (top left panel, B54-CA23) has
sparing of VIP cells and fibers. A few CaBP cells are seen unilaterally
in the CaBP subnucleus. This animal retained locomotor rhythmicity. The
other three animals depicted in Figure 5 all had sparing of VIP cells,
but no CaBP immunoreactivity could be detected in the core of the SCN.
These three animals were arrhythmic.
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Figure 2.
Photomicrographs of coronal brain sections showing
a partial SCN lesion in animal B26-V34. Much of the SCN is spared in
this animal, although circadian rhythmicity is lost after the lesion
(behavior shown in Fig. 4A).
A-C, Adjacent low-power sections stained
for NP, VIP, and CaBP, respectively. A. Low-power view
of a section through the SCN stained for NP (arrows
indicate NP cells in the dorsomedial SCN). B. The
lateral aspect of the SCN is damaged (asterisks). The
arrow points to the region shown in high power in
E. C, No immunostained cells could be
detected in the SCN, although sparse CaBP cells can be seen in the
adjacent hypothalamus (arrow). The box
denotes the region that is shown in high power in F.
D-F, High-power photomicrographs of
sections stained for NP, VIP, and CaBP, respectively. No CaBP cells
could be detected in the SCN. Scale bars:
A-C, 100 µm; D,
E, 10 µm; F, 20 µm.
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Figure 3.
Photomicrographs of coronal brain sections
showing a partial SCN lesion in animal B56-CA29 (behavior shown in Fig.
4B). A-C, Adjacent
low-power sections stained for NP, VIP, and CaBP, respectively.
A, B, Asterisks indicates
partial SCN ablation at the ventral aspect. The arrows
in A and B point to the region shown in
high power in D and E, respectively.
C. No CaBP cells could be detected in the SCN. The
box denotes the region that is shown in high power in
F. D-F, High-power photomicrographs. The
animal lost locomotor rhythmicity (Fig. 4B) after
the lesion, although NP (D) and VIP
(E) cells (arrows) and fibers were
spared. F, No CaBP cells could be detected in the SCN.
Scale bars: A-C, 200 µm; D, F, 20 µm; E, 10 µm.
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Figure 4.
Wheel running rhythm of the animals for which
anatomy are presented in Figures 2 and 3.
To facilitate inspection of the rhythms, the daily activity is plotted
twice on a 48 hr time scale. The animals received an SCN lesion
(SCN-X) at the point indicated on the
left of the actogram. Spectral analyses of the data are
shown on the right of the actograms. The black
vertical bars on the right of the actograms
indicate the days (intact and lesioned) for which the analyses were
done. A, The intact heterozygote tau mutant hamster
B26-V34 had a period of 22.6 hr (days 1-12). After the partial SCN
lesion, it became arrhythmic (analyses shown for days 34-74).
B, The intact wild-type hamster B56-CA29 had a period of
24 hr (days 1-12). The animal became arrhythmic after a partial lesion
of the SCN ablating the CaBP subnucleus (analysis shown for days
42-67).
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Figure 5.
Schematics of the caudal aspect of the
SCN depicting the area of damage (gray) in
hamsters with partial SCN lesions in one animal with sparing and three
animals with ablation of the CaBP subregion. The approximate outline of
the SCN is indicated by broken lines. For each animal,
the drawings show sections stained for VIP and CaBP. For each peptide,
the schematics are shown at three levels, separated by 200 µm through
areas rostral to, centered in, and caudal to the CaBP subnucleus.
Animal B54-CA23 (top left panel) had a few CaBP
cells remaining on one side (arrow). This animal was
rhythmic after the lesion. For the other three animals, much of the
SCN is spared, and VIP cells and fibers were seen, but CaBP-IR cells
were not detected. These three animals were arrhythmic after the
lesion.
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In 21 of 23 arrhythmic animals, both VIP and NP could be seen in
the SCN. Dramatically, one animal (B26-V34; Fig. 2) with sparing of
~67% of the SCN but with no detectable CaBP cells within the SCN was
arrhythmic. In 3 of the foregoing 21 animals, CaBP cells overlapped
with sparse NP fibers but not with VIP. These CaBP cells were from the
outside of the SCN, because they were much larger than the SCN CaBP
cells (B52-Z27: 39.4 ± 1.2 µm; n = 20 cells;
p < 0.0001; B41-27T: 41.5 ± 1.7 µm;
n = 20 cells; p < 0.0001).
Additionally, 1 animal had NP, but no VIP cells or fibers could be
detected in the SCN, although VIP-IR could be seen in other parts of
the brain. Finally, one animal with sparing of a few CaBP cells was
arrhythmic. No area of the SCN other than the CaBP subnucleus was
consistently damaged in arrhythmic animals. Not surprisingly, animals
(n = 4) with complete SCN lesions expressed no
circadian rhythms. All animals lacking SCN CaBP-positive cells remained arrhythmic.
Experiment 2: SCN transplant
Transplants were scored in two ways. The first analysis was based
on staining for VIP, NP, and CaBP peptides in the graft (Table
2). The results reveal that nine half-SCN
grafts restored donor-specific rhythmicity. Of these, five contained
NP, VIP, and CaBP, whereas VIP was not detected in four animals. In
summary, of the 10 animals with CaBP-positive cells in the graft, 9 expressed circadian rhythmicity, whereas of the six animals lacking
CaBP cells, none was rhythmic.
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Table 2.
Transplant of half-SCN punches indicates that the
restoration of rhythmicity is dependent on the presence of tissue from
the CaBP subregion
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For the second analysis, grafts were analyzed in terms of type of donor
tissue (for coronal sections, dorsomedial vs ventrolateral SCN; for
sagittal sections, rostral or caudal SCN). Although some grafts within
each of these categories restored rhythmicity, this analysis was not
predictive of recovery of function (Table
3). (Note: for five animals used in the
analysis CaBP-IR was not examined, and these animals are absent from
Table 2.)
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Table 3.
Transplant of half-SCN punches indicates that the
restoration of rhythmicity cannot be predicted by the SCN region
dissected for implantation
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Fifteen grafted animals were not used in the analysis of transplants
for the following reasons: the host SCN was not totally ablated
(n = 9), and the animal expressed the host rhythm; no graft was present (n = 5); and one animal had a graft
that did not express any peptide characteristic of the SCN. The latter six animals were arrhythmic.
Two examples of recovered animals are shown in Figures
6-8. The animal (B28-Q66) shown in
Figure 6 has a graft (situated slightly caudal to the lesioned SCN
site) that contains a plexus of NP and VIP cells and fibers and, at the
same level, a small cluster of CaBP cells surrounded by an area without
CaBP cells. Behavioral analysis (Fig. 8A) shows that
before lesion, this animal had a free-running period of 21.9 hr,
became arrhythmic after lesion placement, and then recovered 21 d
after transplantation with the donor period of 24.8 hr. The animal
(B33-Q67) shown in Figure 7 has a
graft situated caudal to the lesioned SCN site; the graft contains
a plexus of NP fibers with a few cells but no VIP and, slightly ventral
to the NP fibers, a large cluster of CaBP cells surrounded by an area
without CaBP cells. Behavioral analysis (Fig.
8B) shows that the
animal was rhythmic with a free-running period of 22.6 hr, was lesioned
and became arrhythmic, and then recovered ~28 d after transplantation
with the donor period of 24.0 hr.
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Figure 6.
Photomicrographs of coronal brain sections showing
the transplant containing the SCN in animal B28-Q66 (behavior shown in
Fig. 8A). A, The graft lies caudal
to the lesion site. There is a plexus of NP staining
(arrow) indicating the presence of the donor SCN.
B, In a section 50 µm from A, VIP
fibers (arrow) overlap the region of the NP fiber
plexus; the graft borders are indicated by a dashed
line. C, Section adjacent to B
showing CaBP cells at the same level as the NP and VIP fibers.
D-F, Higher magnifications of the areas
marked by arrows in A-C,
respectively. Scale bars: A-C, 200 µm;
D-F, 20 µm.
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Figure 7.
Photomicrographs of coronal brain sections showing
the transplant containing the SCN in animal B33-Q67 (behavior shown in
Fig. 8B). A, The graft (borders
are indicated by a dashed line) lies caudal to the
lesion site. There is a plexus of NP staining (arrow)
indicating the presence of the donor SCN. B, In a
section 50 µm from A, no VIP plexus was seen within
the graft. C, Section adjacent to B
showing a cluster of CaBP cells at the same level as the NP plexus.
D-F, Higher magnifications of the areas
marked by arrows in A-C,
respectively. D, Plexus of NP fibers within the graft.
E, Absence of VIP staining. F, CaBP
subnucleus within the graft, surrounded by a space devoid of CaBP cells
(asterisks). Scale bars: A-C, 200 µm;
D-F, 20 µm.
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Figure 8.
Wheel running rhythm of heterozygote tau mutants
for which the anatomical data are presented in Figures 6 and 7. The
animals received an SCN lesion (SCN-X) and
received a half-SCN transplant at the points indicated on the
left of the actogram (see Fig. 4 legend for further
details). A, The intact hamster B28-Q66 had a
free-running period of 21.9 hr (days 1-8) and became arrhythmic after
an SCN lesion (days 12-22). After transplantation, it recovered with
the donor period of 24.8 hr (days 43-71). B, The intact
hamster B33-Q67 had a period of 21.9 hr (days 1-8). After an SCN
lesion, it became arrhythmic (days 12-22). After transplantation, it
recovered with the donor period of 24.0 hr (days 61-103).
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Of the seven half-SCN grafts that did not restore rhythmicity, six
contained no detectable SCN CaBP cells but had NP and/or VIP. In one
arrhythmic animal the graft contained NP and CaBP but no VIP.
Two examples of grafts that did not restore rhythmicity are shown in
Figures 9-11. The animal (B46-28T)
shown in Figure 9 has a graft (situated caudal to the lesioned SCN
site), which contains a large plexus of NP and VIP cells and fibers
and, at the same level, no CaBP cells. Behavioral analysis
(Fig. 11A) shows that the animal was rhythmic with a
free-running period of 22.6 hr, was lesioned and became arrhythmic,
and did not recover rhythmicity after transplantation. The animal
(B39-Q70) shown in Figure 10 has a
graft situated slightly caudal to the lesioned SCN site, which contains
a plexus of NP cells and fibers and a few VIP cells and fibers but no
CaBP cells. Behavioral analysis (Fig.
11B) shows that the
animal was rhythmic with a free-running period of 22.6 hr and was
lesioned and became arrhythmic, but did not recover a locomotor
activity rhythm after transplantation.
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Figure 9.
Photomicrographs of coronal brain sections of the
SCN within the grafts in animal B46-28T (behavior shown in Fig.
11A). A, The graft (borders are
indicated by a dashed line) lies caudal to the lesion
site. There is a plexus of NP staining (arrow)
indicating the presence of the donor SCN. B, In a
section 50 µm from A, a plexus of VIP fibers
(arrow) lies at the same level as the NP plexus.
C, The graft lacks CaBP cells, although many CaBP cells
can be seen in the host's hypothalamus.
D-F, Higher magnifications of the areas
marked by arrows in A-C,
respectively. D, E, Plexus of NP and VIP
cells (arrows) and fibers within the graft (open
arrows indicate nonspecific immunoreactivity within the
ventricular epithelium in E). F, No CaBP
cells can be detected within the brain parenchyma at the level of the
NP and VIP plexi (open arrows indicate nonspecific
immunoreactivity within the ventricular epithelium). Scale bars:
A-C, 200 µm;
D-F, 20 µm.
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Figure 10.
Photomicrographs of coronal brain sections of the
SCN within the grafts in animal B39-Q70 (behavior shown in Fig.
11B). A, The graft (borders are
indicated by a dashed line) lies caudal to the lesion
site. A plexus of NP staining (arrow) indicates the
location of the donor SCN. B, In a section 50 µm from
A, there are a few VIP cells and fibers
(arrow) at the same level as the NP plexus.
C, The SCN region lacks the cluster of CaBP cells,
although sparse CaBP cells can be seen in other parts of the graft.
D-F, Higher magnifications of the areas
marked by arrows in A-C,
respectively. D, E, Plexus of NP and VIP
cells (arrows) and fibers within the graft.
F, Although there is some nonspecific staining around
the edges of ventricles and blood vessels in the area where NP and
VIP are present, no CaBP cells could be detected in the SCN. Scale
bars: A, 200 µm; B, C,
100 µm; D-F, 20 µm.
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Figure 11.
Wheel running rhythm of the heterozygotes that
did not recover rhythmicity after transplantation (anatomical data are
presented in Figs. 9, 10). The animals were lesioned
(SCN-X) and received a half-SCN transplant at the
points indicated on the left of the actogram (details of
legend and analysis shown in Fig. 4). A, The intact
hamster B46-28T had a free-running period of 22.6 hr (days 1-10).
After an SCN lesion, it became arrhythmic (days 19-41).
B, Intact animal B39-Q70 had a free-running period of
22.6 hr (days 1-7). It became arrhythmic (days 10-23) after SCN
ablation.
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|
Correlation between rhythmicity and number of CaBP cells
In animals expressing circadian rhythms in which the CaBP
subregion of the SCN was present (summarized in Tables 1, 3), the
absolute power of the locomotor rhythm was significantly correlated with the number of SCN CaBP cells remaining in the partially lesioned SCN (r = 0.61; n = 23;
p = 0.002; in one animal, all the CaBP cells could not
be counted, because a section was broken) and with the number of SCN
CaBP cells in the graft (r = 0.94; n = 9; p = 0.0002; Fig.
12).
View larger version (16K):
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|
Figure 12.
There is a positive correlation between
the number of SCN CaBP profiles and the absolute power of the rhythm in
animals with a partial lesion of the SCN (A;
r = 0.61; p = 0.002) and in the
lesioned-grafted animals (B; r = 0.94; p = 0.0002).
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|
There was no significant correlation between the power of the rhythm
and the number of clusters (r = 0.06; n = 9; p = 0.87) or the size of the SCN plexus
(r = 0.33; n = 9; p = 0.38) within the graft of animals with a restored rhythm. Nor was there
a correlation between restoration of rhythmicity and number of SCN
clusters (r = 0.23; n = 16;
p = 0.37) or size of the clusters (r = 0.04; n = 16; p = 0.89) within the grafts.
| |
DISCUSSION |
Cells in the CaBP subnucleus control
locomotor rhythmicity
Previous work indicated that "... the SCN operate in an
`all-or-none' manner and suggested that even a small part of the SCN is capable of driving circadian rhythms" (van den Pol and Powley, 1979, page 323). This hypothesis has been supported in numerous studies
indicating that as long as ~25% of the SCN survive ablation, circadian rhythms are sustained (Rusak, 1977; Davis and Gorski, 1988;
Harrington et al., 1993). The present results lead us to the conclusion
that there is regional specialization of function, suggesting a
paradigm shift in our conceptualization of the SCN. Specifically, the
present lesion and transplant studies indicate that cells within the
CaBP subnucleus of the hamster SCN are necessary and sufficient for the
control of circadian locomotor rhythms. Second, other regions are not
sufficient to sustain rhythmicity of locomotor activity, even when as
much as 67% of SCN tissue remains intact after the lesion, as long as
the CaBP subregion is damaged. Finally, the strength of the rhythm is
proportional to the amount of CaBP subnucleus tissue remaining after
the lesion or present in the grafts. Two animals (one lesioned and one
grafted) in which few CaBP cells were detected were arrhythmic. The
absence of rhythmicity may be attributable to any number of factors, as has previously been reported in animals bearing whole SCN grafts (Aguilar-Roblero et al., 1994). More importantly, no animals lacking SCN CaBP cells, after lesion or in the graft, expressed locomotor rhythmicity.
These results are consistent with previous findings. We show that
analysis of which region of the SCN has been lesioned (dorsal vs
ventral or rostral vs caudal) did not suggest regional specialization. However, the use of antibodies to CaBP, NP, and VIP as markers has
permitted a detailed analysis of the locus and extent of SCN lesion
sites and has led to new conclusions. It is noteworthy that loss of
rhythms was previously reported after sparing of part of the SCN (van
den Pol and Powley, 1979). Also, small electrolytic lesions altered
both the circadian rhythm of locomotor activity and the normal temporal
pattern of gonadal regression on short days, whereas other lesions
affected one parameter but not the other (Pickard and Turek, 1985). In
their study of hamsters, detailed histological analysis of
histochemically stained sections examining retinal afferents did not
reveal regional specificity of function. However, the detailed
description of each animal bearing partial SCN lesions provided by
these authors suggests that animals that sustained locomotor rhythms
had lesions sparing the caudal aspect of the SCN, the region of CaBP
cells. Similar dissociation of various circadian rhythms has been
reported in rats (Satinoff and Prosser, 1988).
The possibility that all SCN cells are pacemakers regulating
circadian rhythmicity remains controversial
Welsh et al. (1995) cultured dispersed rat SCN cells on fixed
microelectrode arrays to record spontaneous action potentials from
individual neurons. The presence of prominent circadian rhythms in
firing rate suggested that the SCN contains a large population of
autonomous single-cell circadian oscillators (or clocks). The high
proportion of clock cells in this preparation led to the conclusion
that the capacity for generating circadian oscillations could not be
restricted to a specific peptidergic cell type. On the other hand,
Mirmiran et al. (1995) recorded spontaneous extracellular discharges of
single SCN neurons in long-term cultured organotypic SCN slices. In
this preparation, not all SCN neurons showed circadian firing rhythms.
Furthermore, neurons recorded simultaneously from the same SCN slice
were not necessarily in synchronization with each other. These authors
concluded that not all SCN neurons are pacemakers. Consistent with this
hypothesis is evidence that when the ventrolateral and dorsomedial
areas of the rat SCN are separated in vitro, the former
maintains a circadian pattern of firing, but the latter does not
(Tcheng and Gillette, 1990)
Each SCN contains 8000-10,000 cells in rats (Güdner, 1976; van
den Pol and Tsujimoto, 1985). Detailed cell counts indicate the
presence of the following peptidergic types: VIP, 2081; vasopressin, 3176; gastrin-releasing peptide, 1140; calretinin, 1370; neurotensin, 283; substance P, 168; and somatostatin, 302 (Speh et al., 1994). In
hamsters, the CaBP cells number ~250 per nucleus (LeSauter and
Silver, 1995). If all SCN cells are pacemakers, and the cells of the
CaBP region alone are necessary for the regulation of locomotor rhythmicity, the question arises of what function is served by the
remaining SCN cells.
Some cells within the SCN may be specialized for intranuclear
communication. It has been suggested that distinct cell populations exhibit axonal arbors that are largely confined to the nucleus, because
an unusual feature of the rat SCN is the large number of local circuit
neurons in the nucleus (Moore and Card, 1985; Buijs et al., 1994). Or
intra-SCN cells may be specialized to provide afferent or efferent
information to and from pacemaker cells (Moore, 1997). Alternatively,
different cells (or cell clusters) subserve different functions. That
the SCN has separable populations of oscillators is suggested from
in vitro work. Thus, NMDA differentially shifted the
phase of VIP and vasopressin rhythms measured in the efflux, indicating
control by separate circadian oscillators (Shinohara et al., 1995).
That subsets of SCN cells regulate different rhythms is also suggested
by accumulating work characterizing specialized SCN afferents and
efferents. Thus, SCN afferent connections are topographically organized
(Moga and Moore, 1997). Also, there is a monosynaptic pathway between
the SCN and preoptic area gonadotropin-releasing hormone neurons
(Huhman and Van der Beek, 1996; van der Beek et al., 1997). The
suggestion has recently been made that the SCN has two subdivisions, a
"core" and a "shell" (Moore, 1996, 1997). Cells of the SCN
shell are small with sparse dendritic arbors, whereas the dendritic
arbors are larger and more extensive in the SCN core. Core neurons
receive retinal afferent input and project more extensively within the
SCN than do shell neurons. Moore (1996) further suggests that species
differences in the SCN tend to occur in shell rather than in core
regions. Thus, all species have VIP in the ventrolateral region and
vasopressin in the dorsomedial region. However, humans have a large
population of neurotensin neurons in the dorsolateral area; this is not
seen in other species. The CaBP region in hamster, which also contain substance P cells (Morin et al., 1992) and gastrin-releasing peptide cells (our unpublished data), fits the definition of a core area in
that it receives direct retinal input (Bryant et al., 1996; Silver et
al., 1996), although the projections of these cells are not known. The
applicability of our findings to other species cannot be ascertained at
this time. Calcium-binding proteins of the EF-hand family form a
large group of ~200 members (Heizmann and Hunziker, 1991); functional
properties similar to those of hamster CaBP cells remain to be determined.
Which properties of pacemakers are present in the
CaBP subregion?
It is generally agreed that there are three components of a
circadian system necessary for regulation of rhythmicity: an input pathway connecting the clock to the external environment, a central pacemaker, and an output pathway. As noted above, we have documented that the CaBP-IR cells receive direct retinal input (Bryant et al.,
1996; Silver et al., 1996). The present results indicate that ablation
of cells of this region results in loss of locomotor rhythmicity, and
their replacement reinstates this response. It remains to be
established whether cells of this region are pacemakers regulating all
circadian responses and/or whether their projections reach efferent
targets in the SCN and/or extra-SCN region to regulate locomotor
rhythmicity. It also remains to be determined whether pacemakers
regulating circadian rhythmicity are the CaBP cells themselves or other
as yet unidentified neurons lying within this subnucleus.
| |
FOOTNOTES |
Received Jan. 5, 1999; revised April 2, 1999; accepted April 8, 1999.
This work was supported by Air Force Office of Scientific
Research Grant F49620 and National Institutes of Health Grant
NS-37919 (to R.S.). We thank Dr. M. Ralph for the gift of the tau
mutant hamsters and Patricia Romero for technical assistance. We also thank Drs. A.-J. Silverman and L. P. Morin for their helpful
comments on an earlier version of this manuscript.
Correspondence should be addressed to Dr. Rae Silver, Psychology
Department, Mail Code 5501, Columbia University, 1190 Amsterdam Avenue,
New York, NY 10027.
| |
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TOP
ABSTRACT
INTRODUCTION
