Download Fetal tissue containing the suprachiasmatic nucleus restores

Fetal tissue containing the suprachiasmatic nucleus
restores multiple circadian rhythms in old rats
HUA LI1 AND EVELYN SATINOFF1,2
of Psychology, University of Illinois at Urbana-Champaign,
Champaign, Illinois 61820; and 2Department of Psychology and Program
in Neuroscience, University of Delaware, Newark, Deleware 19716-2577
1Department
neural transplantation; body temperature; activity; drinking;
aging; vasoactive intestinal polypeptide
AGING AFFECTS THE PERIOD, amplitude, and phase of
circadian rhythms (for review, see Ref. 25). The most
commonly reported change is diminished amplitude of,
for instance, the circadian temperature rhythm (24), of
sleep (20) and of wheel running and drinking (23).
Aging also affects cellular, metabolic, and electrophysiological properties of the suprachiasmatic nucleus
(SCN). Using 2-deoxy-D-glucose uptake as a marker,
Wise et al. (38) found a substantial decrease in metabolic activity in the SCN of aged rats. Sutin et al. (33)
found a decreased response of the immediate-early
gene c-fos in the SCN of old rats after photic stimulation. The firing rate of SCN cells in a slice preparation
is of lower amplitude in aged animals than in young
ones (28).
There is a decrease in the number of arginine vasopressin (AVP) and vasoactive intestinal polypeptide
(VIP) neurons in the SCN of old rats, as revealed by
immunocytochemical staining (7, 26). Partial SCN lesion studies show that the size of the SCN is crucial for
the expression of its pacemaker properties (27). The
decrease in SCN volume and cell number might be one
factor underlying circadian rhythm disturbances in old
rats.
The SCN in the hypothalamus is the major circadian
pacemaker in mammals. Perhaps the strongest evidence for this is that circadian rhythmicity is restored
in SCN-lesioned arrhythmic hosts after transplantation of fetal tissue containing the SCN (e.g., Ref. 18).
There has been very little behavioral work done on fetal
SCN transplants in aged rats. When fetal SCN tissue
was transplanted into old hamsters that had had their
own SCN ablated, the free-running period of these
animals lengthened to resemble that of younger hamsters (36). When grafted aged hamsters with intact
SCN were injected with triazolam, a phase-shifting
drug, activity rhythms phase-shifted more quickly than
did those of young hamsters (35). Recently, Cai et al. (3)
reported that fetal SCN grafts into middle-aged female
rats with intact SCN enabled the aged hosts to regain
their ability to show diurnal patterns of fos expression.
The present study is based on the fact that the
circadian rhythms of many aged rats become aperiodic.
In this respect they resemble the circadian rhythms of
young rats with partial SCN lesions (29). In the present
experiments we measured three circadian rhythms:
body temperature (Tb ), drinking, and activity. Our
hypothesis was that fetal transplants of SCN tissue
into intact but aperiodic aged hosts might improve
circadian rhythms just as they do in young animals
with SCN lesions.
The SCN contains several different neuropeptides
and neurotransmitters (for review, see Ref. 14). For
example, there are large populations of VIP cells in the
ventrolateral SCN (5). This area receives dense projections of neuropeptide Y (NPY)-ergic fibers from the
intergeniculate leaflet (6). There are large populations
of AVP cells in the dorsomedial SCN (34). The codistribution of dense fiber plexi characterized by VIP, AVP,
and NPY immunoreactivity in the SCN provides a
distinct neuropeptide profile for distinguishing SCN
from adjacent hypothalamic tissue and from other
brain tissues (17). A similar cytoarchitecture is preserved in fetal SCN tissue grafts (1, 17, 18). Immunocytochemical staining of these three peptides is frequently used to identify the SCN both in intact animals
and in grafted hypothalamic tissues. Therefore we used
these peptide markers in the present experiments.
0363-6119/98 $5.00 Copyright r 1998 the American Physiological Society
R1735
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Li, Hua, and Evelyn Satinoff. Fetal tissue containing
the suprachiasmatic nucleus restores multiple circadian
rhythms in old rats. Am. J. Physiol. 275 (Regulatory Integrative Comp. Physiol. 44): R1735–R1744, 1998.—The suprachiasmatic nucleus (SCN) is the major circadian pacemaker in
mammals. When fetal tissue containing the SCN is transplanted into young rats whose circadian rhythms have been
abolished by SCN lesions, the rhythms gradually reappear.
Circadian rhythms in many rats deteriorate or disappear
with age. The rationale of the present study was that old rats
with poor circadian rhythms resemble young rats with SCN
lesions. If there is a similar mechanism underlying this
resemblance, then fetal tissue containing the SCN should
restore rhythms in old rats. Therefore, we implanted such
tissue into the third ventricle of intact aged rats with poor
circadian rhythms. Body temperature, locomotor activity,
and/or drinking were measured simultaneously within subjects. Grafts and hosts were stained immunocytochemically
for vasoactive intestinal polypeptide (VIP), arginine vasopressin (AVP), and neuropeptide Y (NPY). Of 23 SCN grafts, 14
were viable (cells observable with Nissl or peptide staining).
In 7 of the 14 aged hosts, up to three circadian rhythms were
improved or restored. VIP cells were always observable,
which was not the case for AVP cells or NPY fibers. In the
other seven hosts, no circadian rhythm was improved. Compared with the successful grafts, these unsuccessful grafts
had similar amounts of AVP and NPY staining but significantly less VIP cell and/or fiber staining. Fetal cerebellar
grafts, which do not contain any of the three peptides, did not
improve or restore any rhythms. Thus the degeneration of
circadian rhythms in aged rats may be due, at least in part, to
deterioration of the aged SCN and in particular, to a loss of
function of VIP-containing neurons.
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SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
MATERIALS AND METHODS
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Subjects and housing. Subjects were young (3–6 mo) and
old (19.5–29.6 mo) male and female Long-Evans rats bred in
the animal colonies of the University of Illinois and the
University of Delaware. Both colonies were pathogen-free,
and regular serological testing was performed to ensure that
condition. Rats with visible lesions or tumors were not used.
Food and water were available ad libitum. The rats were
maintained from birth at an ambient temperature of 23 6 1°C
on a 12:12-h light-dark cycle (LD). At the beginning of the
screening process for grafting candidates, all rats (except 3)
were maintained in constant darkness (DD). This was done
because we were mainly interested in endogenous circadian
rhythms, which can only be observed under constant conditions. The effect of SCN grafting on entrained rhythms was of
lesser interest.
Experimental design. Using periodogram analysis and gray
scales (see Data analysis), we have found over the years that
if a circadian rhythm of an old rat is poor or nonexistent, i.e.,
showing very low or no circadian power (power at 24 h), for at
least 2 wk, it does not become rhythmic again under either
LD or DD. In the present study we identified 28 old rats in
which at least one of the rhythms we measured had no power
for at least 1 mo; they became candidates for grafting.
Twenty-five of these rats were placed in DD at least 1 mo
before grafting. (One of these, OF119, was shifted from DD to
LD 2 mo postgrating.) Three other rats were housed and
grafted in LD. Tb, locomotor activity, and drinking were
continuously monitored.
Fetal tissue containing either the SCN (n 5 23) or a
comparable amount of cerebellar tissue (controls, n 5 5) was
implanted into the third ventricle of those animals. Two other
control groups received fetal SCN grafts. They were included
to examine possible interactions between the healthy host
SCN and the grafted SCN. These groups were old rats with
good rhythms (n 5 4) and 3- to 6-mo-old intact rats (n 5 6).
Measurements continued for 1–2 mo after the graft operation. The animals were then killed and their brains removed,
cut, stained, and examined microscopically. Grafts and host
SCN were stained for the presence of VIP and AVP cells and
NPY fibers and also stained with thionin.
Temperature telemeter placement. Young rats were anesthetized with ketamine-HCl (90 mg/kg) and xylazine (13 mg/kg).
Two-thirds of this dose was used for old rats. A temperature
telemeter (model VM; Mini-Mitter, Sunriver, OR) was implanted in the peritoneal cavity, and the wound was sutured.
The cylinder-shaped telemeter (diameter 1.2 cm, length 1.7
cm, weight 3 g) was calibrated at 30 and 42°C in a temperature-controlled water bath. The telemeter was accurate to
0.1°C.
Fetal tissue transplantation. Anesthetized rats were placed
in a stereotaxic device. Fetal tissue containing the SCN was
lowered to the third ventricle using the following coordinates:
anterior-posterior 21.0 mm from bregma, dorsoventral 29.0
mm from the dura, and laterally at the midline. Implants
(volume 0.5–1 mm3 ) were obtained from the brains of rat
embryos at day 17 (E17) of gestation (day 1 being the first day
after mating) to E20. Pregnant female rats were anesthetized, and an incision was made at the level of the lower
abdomen to expose the uterus. One pup at a time was taken
from the uterus. The head of the pup was removed and placed
in a sterile petri dish on ice. Under a stereoscopic zoom
microscope, the brain of the pup was dissected out. The tissue
block containing the SCN was cut out from the ventral
surface of the hypothalamus with coronal cuts just rostral
and caudal to the optic chiasm and with lateral cuts equidis-
tant from the midline. Embryonic segments from two donors
were pooled in sterile saline and injected into each host
animal using a modified 20-gauge needle. The time between
microdissection and implantation of the fetal tissue never
exceeded 1 h. Usually, 2–6 rats were grafted with pups from
one mother in one session. At the end of a grafting session, the
pregnant female was euthanized with an overdose of ketamine. The grafted rats were returned to their home cages to
recover.
Histology. The rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. After overnight
postfixation, 50-µm sections were cut on a vibratome or a
microtome. Sections through the host SCN and the graft were
saved in series. One or two series of sections were Nisslstained with thionin. The other serial sections were stained
immunocytochemically for the presence of VIP, AVP, and NPY
according to the avidin-biotin peroxidase complex method.
Sections were washed for 10 min in phosphate-buffered saline
to remove excess aldehydes and then incubated with goat
serum for 1 h to block nonspecific binding. Next, sections were
incubated overnight at 4°C in primary antisera to either AVP
(vasopressin-neurophysin II, Incstar), VIP (Incstar), or NPY
(Incstar). Sections were then incubated with biotinylated
secondary antibodies, followed by avidin-biotinylated peroxidase complex (Vectastain, Vector Laboratories). Diaminobenzidine was the chromogen substrate oxidized by horseradish
peroxidase to form an insoluble precipitate. A mixture of
glucose and glucose oxidase was used to generate the hydrogen peroxide substrate. Sections were mounted on gelatincoated slides and dried overnight. They were then dehydrated
in graded alcohols, cleared in xylene, and put on coverslips
with Permount. Control sections in which the primary antiserum was omitted completely eliminated the reaction product.
The specificity of the antibodies was assessed by preabsorbing
each antibody with a 10-µm solution of the appropriate
antigen (AVP and VIP, Sigma; NPY, Bachem), which was
sufficient in each case to block all immunoreactivity. For
thionin staining, the cut sections were mounted on slides and
air-dried overnight. Next they were hydrated through xylene,
graded alcohols, and 0.1 M acetate buffer, and then stained in
0.1% thionin (Sigma) solution for 5–10 min. Then the sections
were dehydrated, cleaned, and put on coverslips. All sections
were examined under bright- and dark-field microscopy. The
graft was considered nonviable if no cells were stained or only
gliosis was present. A viable graft could have Nissl-stained
cells but lack peptide staining.
Data collection. Tb was recorded continuously through the
implanted temperature telemeter, which emits a broad-band
radio frequency pulse at a rate proportional to its temperature. This signal was converted to a Tb value by a microcomputer and stored on disk every 10 min.
Activity was measured by monitoring cage movement. The
movement of the rat displaced a piece of transparent plastic
with an opaque dot pattern on it. This piece of plastic was
located inside a photocell. The displacement triggered electrical pulses. The number of pulses was summed, stored, and
sent to disk every 10 min. Drinking was measured using a
standard drinkometer circuit. Wires from the water bottle
and cage floor closed a circuit when the rat licked the drinking
spout. The number of licks was summed and stored every
10 min.
Data analysis. To examine the data for rhythmicity, computer-generated plots were drawn for Tb, activity, and drinking. Each 10-min data point was plotted as a black line if its
value was above the daily mean of the entire day or a blank if
its value was below the daily mean. The black line was
half-length if the value of the data point was within 0.1°C of
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SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
Table 1. Summary of experimental conditions
15 Controls
6 Young rats with SCN grafts, no changes (M, n 5 6)
4 Old with good CRs, SCN grafts, no changes (M, n 5 3; F,
n 5 1)
5 Old with poor CRs, CBL grafts, no improvements (M, n 5 3; F,
n 5 2)
23 Old, poor CRs
9 Nonviable or absent grafts; no improvements (M, n 5 6; F,
n 5 3)
14 Viable grafts
7 Improved (M, n 5 3; F, n 5 4)
7 Not improved (M, n 5 4; F, n 5 3)
CR, circadian rhythm; CBL, cerebellar; M, male; F, female; n,
sample size (total 5 38).
RESULTS
General findings. Of the 23 animals in the experimental group, 14 had viable grafts verified histologically
(Table 1). Transplants restored or improved the freerunning (n 5 6) or entrained (n 5 1) rhythms in seven
of them within 2–4 wk. Transplants of cerebellar tissue
were ineffective in improving any rhythms (n 5 4; one
graft was not viable). The improvements postgrafting
occurred in one or more rhythms. Table 2 lists those
rhythms that were restored or improved postgraft. To
be accepted as ‘‘improved,’’ the rhythms had to have
power at 24 h that was significantly greater than the
white noise level and greater than the power pregraft
(P , 0.01 in at least one rhythm that was poor or
nonexistent pregraft).
Well-developed grafts of various sizes were located
within the third ventricle. The grafts were always
attached to the host. Indeed, parts of the grafts often
merged with the host tissue so well that it was difficult
to determine the host-graft border (e.g., Fig. 1). The
rostral-caudal position of the grafts varied from the
level of the medial preoptic nucleus to the level of the
median eminence (Table 2).
To determine whether the size of the graft affected
functional recovery, we compared the size of the successful grafts (ranging from 0.5 3 0.25 mm to 1.75 3 0.63
mm, n 5 7, Table 2) to that of the unsuccessful grafts
(ranging from 0.6 3 0.5 mm to 1.7 3 0.75 mm, n 5 7,
Table 3) using a binomial logit link regression analysis.
The test revealed no differences in the sizes of successful and unsuccessful grafts (P , 0.14).
Six of the seven successful grafts stained for both VIP
and AVP. The seventh graft showed only VIP staining.
Peptidergic organization of the host SCN of control and
experimental animals was also examined. Although no
systematic quantification was performed, there were
Table 2. Successful transplantation: host and graft characteristics
Pre-G
Post-G
Rat
Sex
Age, mo
Lighting
T
A
D
T
A
D
Latency,
days
G Level
G Size, mm
VIP
AVP
NPY
Tumor, mm
OF122
OF119
OM64
OF117
OM44
OF112
OM82
F
F
M
F
M
F
M
19.5
23
24
24
25
27
29.6
DD
DD
DD
DD
LD
DD
DD
w
s
w
w
s
w
s
w
w
w
w
w
w
w
w
a
a
a
a
w
a
>*
s
w
>*
s
>*
s
<*
w
w
>*
>*
w
w
<*
>*
>*
>*
>*
>*
>*
10
12
14
10
15
15
30
ME
MPO/ME
SCN
SCN-RCH
ME
SCN-RCH
SCN/RCH
1.73 3 0.63
0.60 3 0.50
0.50 3 0.25
1.00 3 0.25
0.75 3 0.25
0.70 3 0.40
0.50 3 0.25
11
11
1
11
1
11
1
11
11
1
2
1
11
11
2
11
1
2
2
11
11
53534
53433
Age, age of host at time of grafting; LD, 12:12-h light-dark cycle; DD, constant darkness; Pre-G, pregraft strength of body temperature (T),
activity (A), and drinking (D) rhythms as determined by gray scales and periodograms; w, weak; s, strong; a, absent; post-G, postgraft; >*,
stronger than pregraft (P , 0.05); <*, weaker than pregraft (P , 0.05); Latency, days until improvement of rhythm was apparent; G level, level
of graft location in host brain; ME, median eminence; RCH, retrochiasmatic area; MPO, medial preoptic nucleus; SCN, suprachiasmatic
nucleus; SCN-RCH, graft extending rostrocaudally from the level of SCN to the level of RCH; SCN/RCH, graft with separate pieces at level of
the SCN and at level of RCH; G size, size of graft (length 3 width); 11, rich in peptide cells and/or fibers; 1, presence of peptide cells and/or
fibers; 2, minimal or no peptide immunoreactivity; VIP, vasoactive intestinal polypeptide; AVP, arginine vasopressin; NPY, neuropeptide Y. All
grafts were from embryonic day 18 (E18) or E17 fetal tissue.
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the daily mean Tb, or within 5% of daily mean licks and
activity measures. Consecutive days were plotted from top to
bottom. For ease of visualization, two consecutive days were
plotted on each line.
The basic tool we used to analyze the rhythmic data is the
periodogram, which describes how the variance in a time
series may be accounted for by cyclic components at different
frequencies. Periodograms reveal not only significant frequencies but also their relative strength, or power. Ten days of
continuous data were used for periodogram analysis to determine the strength of the circadian rhythm. To compare
relative power between individuals, or in the same individual
at different times, the periodograms were normalized by
equating the variance present in the data to one unit. For any
periodicity to be significant at P , 0.01, the lower limit of the
99% confidence interval of the power estimate must exceed
the white noise level. For ease of visualization, the lower limit
of the 99% confidence interval of the power estimate was
plotted instead of the actual power. To test for significant
differences between the power at the circadian range pre- and
postgrafting, we used a test procedure described by Diggle (8).
It relies on the fact that the ratio of the two power values
(spectral estimates) follows an F distribution. A graft was
defined as successful when one or more circadian rhythms
showed significantly increased power at 24 h postgraft compared with pregraft.
We compared the histological data between successful and
unsuccessful grafts using the nonparametric Wilcoxon rank
sum test. To determine the effect of gender on functional
recovery rate, we used the Pearson x2 test to examine the
association of two variables expressed in a two-way contingency table. To determine the effect of age and graft size on
the functional recovery rate, a binomial logit link regression
analysis (22) was performed using SAS procedure PROC
GENMOD.
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SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
no apparent differences in the numbers of VIP and AVP
cells and NPY fibers between the aged rhythmic and
aged arrhythmic host SCN. Grafts that were successful
in restoring rhythms had significantly more VIP cell
and/or fiber staining than did the unsuccessful grafts
(P , 0.001). AVP and NPY staining was similar between successful and unsuccessful grafts (P , 0.1 and
P , 0.17, respectively).
Of the 23 old hosts with poor rhythms, 3 of 13 males
and 4 of 10 females demonstrated functional recovery
after SCN grafting. The recovery rates of the two sexes
were not significantly different at P 5 0.05 (x2 5 0.30 ,
Table 3. Unsuccessful transplantation: host and graft characteristics
Rat
Sex
Age, mo
Lighting
G Level
G Size, mm
VIP
AVP
NPY
Tumor, mm
OF118
OM113
OM93
OM106
OM91
OF116
OF113
F
M
M
M
M
F
F
23.5
24
24.3
24.3
25
25
26
DD
DD
DD
DD
LD
DD
DD
SCN-RCH
SCN
SCN-RCH
RCH-ME
SCN-RCH
SCN
SCN
1.75 3 0.63
0.87 3 0.50
1.25 3 0.63
0.75 3 0.38
1.00 3 0.50
1.70 3 0.75
0.60 3 0.50
2
2
2
2
2
1
2
1
2
2
2
2
11
2
1
1
2
11
2
11
1
43632
12 3 8 3 5
73837
For explanation of symbols, see Table 2. All grafts were from E17 fetal tissue except for that of OM113, which was E19.
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Fig. 1. Localization of neuropeptides in a functional SCN graft
(OF112). Serial sections stained for vasoactive intestinal polypeptide
(VIP; A), arginine vasopressin (VP; B), and neuropeptide Y (NPY; C).
D and E: magnified views of cell clusters in A and B, respectively. F
shows arginine vasopressin staining of a section through anterior
portion of graft at level of host suprachiasmatic nucleus (SCN). There
is an arginine vasopressin efferent fiber (large arrowhead) crossing
the graft-host border (small arrowheads). VIP cell cluster is ventral to
arginine vasopressin cell cluster (notched arrows in A and B). VIP
and arginine vasopressin cells (arrows in D and E) have spherical or
slightly elongated shapes with diameters of 5–15 µm. Fibers from
these VIP and arginine vasopressin cells ramified within clusters and
extended into other parts of the graft (arrows in A and B). NPY fibers
(A) are rich in entire graft, including the region of the arginine
vasopressin and VIP cell clusters. Calibration bar 5 200 µm (A–C) or
50 µm (D–F). G, graft; III, third ventricle; PVN, paraventricular
nucleus; RCH, retrochiasmatic area.
x21 5 3.84). The age of rats with functional recovery
ranged from 19.5 to 29.6 mo, which overlapped the age
of rats without recovery (from 23.5 to 26 mo). There was
no significant impact of age on functional recovery.
Four of five old rats with poor rhythms that had been
implanted with cerebellar grafts had large, viable,
well-vascularized grafts that did not stain for any of the
three peptides. Four of six young intact controls and
three of four old rats with good rhythms had peptidergic organization in their SCN grafts similar to the
successful grafts.
Functional SCN grafts. Grafts that improved or
restored rhythms contained neuropeptides characteristic of the SCN in normal rats. All functional grafts
contained groups of VIP-immunoreactive cells, which
sometimes formed a cluster (Fig. 1, A and D) and
sometimes were more loosely distributed (Fig. 2). These
cells gave rise to fibers that occasionally formed a
plexus surrounding the cell cluster (Fig. 1). These VIP
cells, like those seen in an intact SCN, had spherical or
slightly elongated somas, ,5–15 µm in diameter.
In six of seven grafts, sections adjacent to those
containing VIP cell clusters had AVP-positive neurons
that, like those in an intact SCN, were parvocellular
cells with diameters of 5–15 µm (Fig. 1). The AVP cells
clustered close to VIP cells and their fibers also ramified within the graft (Fig. 1, B and E). This juxtaposition of VIP and AVP cells resembles the peptidergic
organization in intact SCN.
Innervation by NPY fibers was variable. In some
grafts, the entire graft was richly innervated with NPY
fibers in some grafts, whereas in others NPY immunoreactivity was minimal or absent.
Nonfunctional SCN grafts. In 9 of 23 old rats with
poor rhythms, the graft could not be located or was not
viable (determined histologically). In seven old rats
that failed to resume free-running rhythms despite
viable SCN implants, the pattern of peptide immunostaining within the graft was completely lacking in two
rats and sparse in the others (Table 3). Parvocellular
VIP cells were absent in all but one graft. The graft in
that rat, OF116, contained a small number of scattered
VIP cells. However, this rat had a very large pituitary
tumor.
Pituitary tumors. In our colony, about one-third of
rats age 24 mo or older develop pituitary tumors, which
can grow quite large and at times severely depress the
ventral brain. In extreme cases, such tumors caused
SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
the animal to stop eating and drinking, resulting in
rapid weight loss and death. In the present study,
pituitary tumors were found in 8 of 32 old rats. Two old
rats with small tumors showed improvements after
SCN grafting (Table 2). From the presence of VIP cells
in the graft, we would have predicted that OF116 (Table
3) should have recovered some rhythms. However, this
rat had an extremely large pituitary tumor, ,480 mm3,
which depressed the ventral brain severely. This tumor
growth might have been responsible for the lack of
recovery in this rat.
Examples of improved rhythms in selected old rats.
All three rhythms were improved in an old female rat
(OF117) in which E17 SCN tissue was grafted when the
rat was 24 mo old (Fig. 3). Tb and activity rhythms were
poor before grafting and there was no drinking rhythm.
About 10 days postgrafting, all three rhythms became
stronger and their circadian power increased significantly over the pregraft level (P , 0.05). The graft in
this rat was richly innervated with VIP cells. There was
no evidence of AVP cells or NPY fibers.
The drinking rhythm was also improved significantly
(P , 0.05) after an E17 SCN graft at 23 mo in a female
rat, OF119 (Fig. 4). Pregraft the Tb and activity rhythms
showed significant 24-h periodicities. About 12 days
postgraft, improvement in drinking started to become
evident. The circadian power of drinking increased
significantly (P , 0.05). The period of all three freerunning rhythms lengthened slightly postgraft. When
the rat was switched to LD at 25 mo, all three rhythms
synchronized to the LD, but this looks more like
masking than entrainment (see Ref. 21). The graft of
this rat was richly innervated with VIP and NPY
(Fig. 2).
Another old rat that was kept in LD throughout the
experiment demonstrates that poor entrained rhythms
may also be improved by an SCN graft. Figure 5 shows
results in OM44, in which E18 SCN tissue was grafted
at the age of 25 mo. Pregraft, only the Tb rhythm was
robust, and it remained so for the duration of the
measurements. The activity rhythm (poor pregraft)
and the drinking rhythm (nonexistent pregraft) were
both significantly improved postgraft. Two other rats
maintained in LD showed no improvement. In one the
graft was necrotic, and in the other no peptide staining
was discernible.
There were no improvements in circadian rhythms in
old control rats bearing cerebellar grafts. No significant
changes were seen in the host rhythms after SCN
transplants in young intact controls or in old rats with
good rhythms.
DISCUSSION
Our results demonstrate that fetal SCN grafts are
able to improve or restore poor or absent circadian
rhythms in aged rats that retain their own SCN. When
all three rhythms were poor, as in OF117, all three were
significantly improved by the graft. When only two
rhythms were poor, as were activity and drinking in
OM44, they were improved or restored and the Tb
rhythm remained the same. In general, the circadian
drinking rhythm was the worst of the three rhythms
measured, and it was improved in six of seven old rats.
One old rat, OF122, was unusual in that its Tb rhythm
was the weakest pregraft. It became much stronger
after the graft, while activity and drinking rhythms
became worse. The worsening of some rhythms postoperatively was seen in old rats in all groups, including
rats with cerebellar grafts and unsuccessful SCN grafts.
We attribute this to the trauma of the anesthesia and
operation and the poorer recovery of old rats.
Our data are consistent with previous graft studies
using adult rats with SCN lesions. Two such studies
demonstrated that multiple circadian rhythms can be
restored in the same individual. Boer et al. (2) found
that fetal SCN grafts restored circadian rhythms of
drinking, feeding, and wheel running in the same
SCN-lesioned rats. In an abstract, Edgar et al. (9)
reported that SCN grafts restored rhythms of sleepwake, Tb, and activity in concert in adult rats with SCN
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Fig. 2. Neuropeptide profile of a functional SCN graft (OF119)
consisting of 3 pieces. One piece (D) was at the level of medial
preoptic nucleus. The other 2 pieces attached to the top and the
bottom of the 3rd ventricle, at the level of the median eminence (ME;
A). Adjacent sections stained for Nissl (A), NPY (B), and arginine
vasopressin (C). D is a section through the anterior piece of the graft
stained for VIP. There were both a VIP cell and/or fiber cluster
(arrowhead in D) and an arginine vasopressin cell and/or fiber cluster
(arrow in C). Magnocellular arginine vasopressin fibers (arrowhead
in C) and NPY fibers (arrowhead in B) ramified extensively throughout the graft. Calibration bar 5 100 µm (C and D) or 200 µm (B) or
500 µm (A). AHP, anterior hypothalamus, posterior part; VMH,
ventromedial hypothalamic nucleus; DA, dorsal hypothalamic area.
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SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
lesions. Lehman et al. (18) reported that although
circadian locomotor rhythms were restored in SCNlesioned hamsters, reproductive responses to photoperiod and the ability to entrain to light at intensities to
which SCN-intact hamsters respond were not. Taken
together, these data indicate that the requirements for
restoring individual rhythms may be different.
Improvements in circadian rhythms were evident
from 10 to 30 days after transplantation. This time
course is comparable to the latency to recovery after
grafting in young rats and hamsters, which ranges
from several days to 2 mo (1, 18). Postgraft improvements were seen in both male (3 of 13 rats) and female
(4 of 10 rats) old hosts, at ages ranging from 19.5 to 29.6
mo. Functional grafts differed widely in size, ranging
from 0.5 3 0.25 mm to 1.75 3 0.63 mm in cross section.
There were no differences between successful and
unsuccessful grafts in these three parameters. In other
words, neither host gender, age, nor graft size were
crucial in predicting graft success.
There was no relation between graft placement in the
third ventricle and improvements in circadian rhythmicity. In two successfully transplanted animals, the grafts
were located in the third ventricle at the level of the
median eminence, distant from the host SCN. Conversely, six of seven of the unsuccessful grafts were
located at the level of the host SCN (Table 3). This
finding agrees with previous work in young SCNlesioned hamsters (37).
We noted sparse and limited neural connections
between the functional grafts and the host brain. Even
in cases where the grafts maintained physical contact
with the host SCN region, fiber connections with the
host SCN or with the SCN target areas were rarely
observed. Similar observations have been reported in
studies of grafting in SCN-lesioned animals using
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Fig. 3. Gray scales (top) and periodograms pregrafting (middle) and postgrafting (bottom) of body temperature (Tb;
T), activity (A), and drinking (D) of a successfully transplanted old female rat, OF117 in constant darkness.
Embryonic day 17 (E17) SCN tissue grafted at 24 mo (G). All 3 rhythms were poor before grafting. Periodograms
show significant but low power at 24 h for Tb and activity; there is no significant 24-h power for drinking. About 10
days postgrafting, all 3 rhythms became stronger and power at 24 h increased significantly over pregraft levels
(* P , 0.05). Dark bars at top indicate darkness. Vertical bars on left of Tb plots indicate data segment used for
computing the periodograms. Numbers on right indicate age (mo) of the host. In the periodograms the power value
plotted is the lower limit of the 99% confidence interval of the actual power. Thin horizontal line above x-axis of each
periodogram is the white noise level. Any power value higher than the white noise level is significant at P , 0.01.
SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
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Fig. 4. Same as Fig. 3 for a successfully transplanted old female rat, OF119. E17 SCN tissue grafted at 23 mo.
Pregraft there was significant 24 h periodicity in Tb and activity but none in drinking. About 12 days postgrafting,
improvement started to show in drinking. Circadian power of drinking began to improve and increased significantly
(* P , 0.05) compared with the pregrafting level. Activity did not improve. When the rat was 25 mo old the
photoperiod was switched to a 12:12-h light-dark cycle (LD). All rhythms became synchronized with LD in ,2 wk. a,
b, and c indicate data segments for the 3 periodograms.
peptidergic staining (18) or dil tract tracing (4). In
studies using xenografts and donor-specific markers,
more extensive anterior hypothalamic graft efferents
were found (16, 31). However, even with the use of such
sensitive techniques, efferents from anterior hypothalamic grafts are variable and there is no clear correlation between the pattern of innervation and recovery of
function (31).
The presence of VIP cells in the graft appeared to be
critical for functional improvements in aged rats. All
functional SCN grafts showed positive VIP staining
and had significantly more VIP cell and/or fiber staining than did the unsuccessful grafts. On the other
hand, AVP and NPY staining in grafts were not consistently correlated with functional improvements: AVP
cells were absent in one functional graft and NPY was
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SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
absent in three (Table 2). In the aged host SCN,
VIP-positive cells were frequently observed but it
seemed that the presence or absence of VIP in the host
SCN did not determine the preservation of rhythmicity
in the aged rats. Possibly, differences in characteristics
other than cell numbers, such as biochemical, electrophysiological, or connectivity properties of VIP, alone or
in combination with other peptide cells or trophic
factors, may underlie the differences in ability to sustain circadian rhythms in old age.
The importance of VIP neurons in young animals has
been noted in the transplantation literature. Sollars
and Pickard (32) found dense aggregations of VIP
neurons in SCN-lesioned hamsters in which circadian
locomotor rhythms were restored by SCN transplants.
This was true for hamster-to-hamster and mouse- or
rat-to-hamster grafts. In hamsters the presence of VIP
cells and fibers is the most consistent marker in
functional SCN grafts, whether whole tissue grafts (12)
or dispersed cell suspensions (30). Kaufman and Menaker (15) reported a high correlation between the presence of VIP cells in the SCN graft and restoration of
running-wheel rhythms in SCN-lesioned hamsters.
Our experiments were not designed to determine
whether the improved rhythms in aged rats are generated solely by the grafts or are rejuvenating the host
SCN, or some combination. Using senescent mutant
and wild-type hamsters, Hurd et al. (13) observed
various interactions between grafted and host SCN.
Because our grafts were homografts, it is not surprising
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Fig. 5. Same as Fig. 3 for a successfully transplanted old male rat, OM44. E18 SCN tissue grafted at 25 mo. This rat
was maintained in LD throughout the experiment. Pregraft there was significant circadian periodicity in Tb and
activity (* P , 0.05). About 15 days after grafting, the drinking rhythm was restored, the activity rhythm was
significantly increased, and the Tb rhythm did not change.
SCN TRANSPLANTS RESTORE CIRCADIAN RHYTHMS IN OLD RATS
to find that the improved rhythms had periods similar
to the pregrafting periods.
In summary, we have demonstrated that fetal tissue
containing the SCN can restore multiple circadian
rhythms to aged animals whose rhythms are poor or
absent. Control cerebellar grafts did not improve any
rhythms in any aged rats. One major difference between the successful grafts and unsuccessful grafts was
that the former had VIP cell and/or fiber staining
whereas the latter did not. Thus the degeneration of
circadian rhythms in aged rats may be due, at least in
part, to deterioration of the aged SCN and in particular
to a loss of function of VIP-containing neurons.
The present study is one of the few studies examining
the functional recovery of multiple circadian rhythms
simultaneously in the same individuals after implantation of fetal SCN tissue. This technique is potentially a
powerful tool for mechanistic investigations of the
circadian system and of the aging process, but much
remains to be explored. For example, many studies in
grafted adult rodents with SCN lesions show that the
presence of VIP-staining cells is an important indicator
of recovery. But does VIP play an important role in the
functional recovery following grafting in SCN-lesioned
animals or in rhythm-degenerated aged animals, or is
it simply a marker? By manipulating the material
being grafted, e.g., cultured VIP neurons, or by varying
the host brain milieu, it should be possible to determine
whether VIP is indeed a regulator of the circadian
system.
The improvement or restoration of circadian rhythms
in intact aged animals following fetal SCN grafting
strongly implies that changes in the SCN are, at least
in part, the cause of age-associated deterioration in
behavior. Our results add another dimension to the
accumulating data base showing that neural transplants can ameliorate neurological, behavioral, and
cognitive deficits associated with aging. For example,
fetal substantia nigra grafts alleviated motor deficits in
old rats (11), septal grafts improved spatial learning
ability (10), and raphe grafts improved specific electrophysiological impairments (19) in aged rats. Neural
transplantation is both a powerful tool to investigate
the aging processes and a promising therapeutic strategy in treating afflictions associated with human aging
like Parkinson’s disease and Alzheimer’s disease.
We thank Dr. JianMing Ding for teaching us immunocytochemistry and how to evaluate histological materials; Dr. Rae Silver for
teaching us fetal tissue transplantation technique; Elizabeth Peloso
and Dr. Maciek Wachulec for their generous help with the surgical
operations, data line maintenance, animal care, and data analysis;
Robert Peloso for consultation with time-series analysis; Esther
Williams for using her time-series software; and Dr. Michael Price for
writing data analysis software.
This work was supported by National Institute of Mental Health
Grant RO1-MH-41138 and an Alzheimer’s Association/Willard and
Rachael Olsen Pilot Research Grant to E. Satinoff.
Address reprint requests to E. Satinoff.
Received 5 May 1997; accepted in final form 7 August 1998.
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