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The Journal of Neuroscience, June 15, 1998, 18(12):4767-4774
Aging Alters the Rhythmic Expression of Vasoactive Intestinal
Polypeptide mRNA But Not Arginine Vasopressin mRNA in the
Suprachiasmatic Nuclei of Female Rats
Kristine
Krajnak,
Michael L.
Kashon,
Katherine L.
Rosewell, and
Phyllis M.
Wise
Department of Physiology, Chandler Medical Center, University of
Kentucky, Lexington, Kentucky 40536-0084
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ABSTRACT |
Our laboratory has shown that the ability of the suprachiasmatic
nuclei (SCN) to regulate a number of rhythmic processes may be
compromised by the time females reach middle age. Therefore, we
examined the effects of aging on the rhythmic expression of two
neuropeptides synthesized in the SCN, vasoactive intestinal polypeptide
(VIP) and arginine vasopressin (AVP), using in situ hybridization. Because both VIP and AVP are outputs of the SCN, we
hypothesized that age-related changes in rhythmicity are associated with alterations in the patterns of expression of these peptides. We
found that VIP mRNA levels exhibited a 24 hr rhythm in young females,
but by the time animals were middle-aged, this rhythm was gone. The
attenuation of rhythmicity was associated with a decline in the level
of mRNA per cell and in the number of cells in the SCN producing
detectable VIP mRNA. AVP mRNA also showed a robust 24 hr rhythm in
young females. However, in contrast to VIP, the AVP rhythm was not
altered in the aging animals. The amount of mRNA per cell and the
number of cells expressing AVP mRNA also was not affected with age.
Based on these results we conclude that (1) various components of the
SCN are differentially affected by aging; and (2) age-related changes
in various rhythms may be attributable to changes in the ability of the
SCN to transmit timing information to target sites. This may explain
why the deterioration of various rhythmic processes occurs at different
rates and at different times during the aging process.
Key words:
rhythms; suprachiasmatic nuclei; aging; vasoactive
intestinal polypeptide; arginine vasopressin; females
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INTRODUCTION |
Virtually all organisms exhibit 24 hr rhythms in numerous physiological and behavioral processes. In
mammals, these rhythms are entrained to the light/dark (LD) cycle by a
circadian pacemaker located in the suprachiasmatic nuclei (SCN) (Meijer
and Rietveld, 1989 ). With age, many endogenous rhythms are blunted, and
their entrainment to the LD cycle changes (Wise, 1984; Cohen and Wise, 1988; Weiland and Wise, 1989; Monk, 1991; Copinschi and Van Cauter, 1994; Li and Satinoff, 1995; Monk et al., 1995; Van Cauter et al.,
1996; Wise et al., 1996). Evidence suggests that these disruptions may
be attributable to alterations in the circadian pacemaker. First, some
(Pittendrigh and Daan, 1974) but not all (Vaswanathan and Davis, 1995;
Duffy and Davis, 1997) studies demonstrate that the period of activity
rhythms (Ralph et al., 1990) shortens with age. Second, the ability of
aged animals to shift their activity patterns in response to photic and
nonphotic stimuli changes (Rosenberg et al., 1991; Penev et al., 1995;
Zhang et al., 1996). Third, endogenous rhythmicity in spontaneous
firing of SCN neurons in vitro is blunted (Satinoff et al.,
1993). Some of these alterations can be reversed by implanting fetal
SCN tissue into the third ventricle (Van Reeth et al., 1994;
Vaswanathan and Davis, 1995; Cai et al., 1997a,b) suggesting that these
changes in the expression of circadian rhythms result in part from
changes in SCN function.
Neurons in the SCN synthesize numerous neuropeptides (van den Pol and
Tsujimoto, 1985) that may constitute output pathways to other brain
regions. Few studies have examined the effects of aging on the rhythmic
expression of these peptides. We assessed the effects of aging on the
rhythmic gene expression of vasoactive intestinal polypeptide (VIP) and
arginine vasopressin (AVP). VIP is synthesized primarily in the ventral
SCN, whereas AVP is produced in a different population of neurons in
the dorsomedial SCN (van den Pol and Tsujimoto, 1985; Watts and
Swanson, 1987). Although these neuronal populations synapse with each
other (Kaikoku et al., 1992), little is known about how they interact.
Both VIP and AVP neurons relay circadian information to the basal
forebrain and various hypothalamic and thalamic nuclei (Watts and
Swanson, 1987). These projections regulate rhythms of
gonadotropin-releasing hormone (GnRH) release (Cheesman et al., 1977;
Osland et al., 1977; van der Beek et al., 1995; Harney et al., 1996;
Palm et al., 1997) and glucocorticoid release (Scarbrough et al., 1996; Buijs, 1997) and activity (Pickard and Turek, 1983; Sollars and Pickard, 1995; Murphy et al., 1997). Thus, age-related alterations in
VIP or AVP expression may affect the transmission of rhythmic information from the SCN to target sites. We also chose to examine the
effects of aging on these two peptides because aging differentially affects their expression in other brain regions. In the cortex, there
is a dramatic decline in VIP cell number with aging (Andreose et al.,
1994; Cha et al., 1995, 1997; Huh et al., 1997). In contrast, AVP
synthesized in the paraventricular nuclei (PVN) and supraoptic nuclei
of the hypothalamus is unaltered or mildly depressed by aging (Dobie et
al., 1991; Sladek and Olschowka, 1994), whereas AVP synthesized in the
bed nucleus of the stria terminalis is reduced (Lucassen et al., 1998).
Thus, the effects of aging on these two neuropeptides may be
region-dependent.
We assessed the effects of age on the rhythmicity of these
neuropeptides in females because cyclic reproduction, which is intimately linked to the circadian clock (Turek and Van Cauter, 1988;
Hastings, 1991), exhibits profound changes by the time animals are
middle-aged (Wise et al., 1996). In young females, the timing of the
proestrous (Legan and Karsch, 1975) and estrogen-induced (Legan et al.,
1975) surge of luteinizing hormone (LH) is tightly coupled to the LD
cycle. However, in middle-aged animals, the LH surge is delayed, and
the amplitude of the surge is attenuated (Wise, 1982; Nass et al.,
1984). We have demonstrated that the rhythmic expression of multiple
neurotransmitters and receptors (Weiland and Wise, 1986, 1989; Cohen
and Wise, 1988; Lloyd et al., 1991) is blunted by middle age,
suggesting that fundamental changes in the SCN may cause a
deterioration in the ability to maintain regular reproductive cycles.
In the present experiment, we assessed VIP and AVP mRNA levels in the
SCN of young, middle-aged, and old females. If aging involves a change
in all the essential elements (i.e., inputs, oscillators, and outputs)
of the SCN, the pattern of expression of both these peptides would be
affected. On the other hand, if only some of the components of the
clock are affected with age, we might observe differential effects on VIP compared with AVP.
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MATERIALS AND METHODS |
Animals. Young, regularly cycling (2-4 months; six
to eight animals per time point), middle-aged, irregularly cycling
(10-12 months; six animals per time point), and old, persistent
diestrous (18-20 months; six animals per time point) females were
housed in a 14/10 hr LD cycle (lights on at 0400 hr) with food and
water available ad libitum. Vaginal cytology was checked
daily for at least 2 weeks to determine the reproductive state of
animals. All animals were ovariectomized under Metofane
(methoxyflurane) inhalant anesthesia. One week later, SILASTIC capsules
containing 17- -estradiol (180 µg/ml in sesame oil; young, 20 mm
capsule; middle-aged and old, 30 mm capsule) were implanted
subcutaneously to clamp plasma estradiol at equivalent and
physiological levels in all experimental groups (Wise, 1984). This is
critical because ovarian steroids modulate the period of circadian
activity (Morin et al., 1977; Takahashi and Menaker 1980; Albers et
al., 1981) and the precise pattern of expression of VIP mRNA (Krajnak
et al., 1997). Animals were killed at the following times after
estrogen treatment: 2400 hr (day 1) and 0300, 0800, 1200, 1600, 2000, and 2300 hr (day 2).
VIP and AVP in situ hybridization. In
situ hybridization methods were the same as previously described
(Wise et al., 1992; Krajnak et al., 1997). Briefly, brains were
removed, rapidly frozen, and stored at  70°C until sectioned. Frozen
coronal sections (12 µm) through the basal forebrain and hypothalamus
were sliced, thaw-mounted onto slides, and stored at 80°C until
processed for in situ hybridization (ISH). Slides containing
sections from the middle to midcaudal SCN (three slides or six sections
per animal) were chosen for VIP ISH, and alternate slides (two slides or four sections per animal) were chosen for AVP ISH. The riboprobe for
VIP was generated using a 500 bp human cDNA directed against exons 3-6
of the VIP-peptide histidine isoleucine gene (from Dr. R. H. Goodman, Vollum Institute). The riboprobe for AVP was generated using a
241 bp cDNA directed against exon C of the rat AVP gene (provided by
Dr. T. Sherman, Georgetown University). Both riboprobes were
transcribed using 50 µM total UTP. Because each cDNA
contains a different percentage of UTP, we used different amounts of
35S-UTP to produce comparable incorporation of the
radiolabeled nucleotide (VIP, 12.5 µM 35S-UTP
and 37.5 µM unlabeled UTP and SP6 polymerase; AVP, 37.5 µM 35S-UTP and 12.5 µM
unlabeled UTP and SP6 polymerase). Slides were thawed, fixed with 4%
paraformaldehyde, and dehydrated using a series of increasing
concentrations of ethanol. Hybridization buffer (50 µl) containing
400 ng/ml labeled VIP cRNA or 200 ng/ml AVP cRNA was applied to each
slide. In preliminary studies, saturation curves were generated and
revealed that these concentrations of cRNA produced maximal labeling
without significantly increasing background. Slides were incubated in
humid chambers at 55°C for 18 hr, washed under stringent conditions,
dehydrated with ethanol, coated with Kodak NTB2 emulsion (Eastman
Kodak, Rochester, NY; diluted 1:1 with distilled water), and stored at
4°C. Slides processed for AVP were developed 5 d after emulsion
coating, and slides processed for VIP were developed 10 d after
emulsion coating. All slides were counterstained with 0.05% toluidine
blue so that individual cell bodies could be identified.
All slides were examined for the presence of labeling in the SCN. If
the SCN from an individual animal was damaged, mRNA levels were not
quantified in those slides. Therefore, in some animals, AVP was not
quantified, and in others, VIP was not quantified. Gene expression was
quantified using the Bioquant OS/2 image analysis system. Slides from a
number of animals were examined, and a single threshold for determining
grains versus background was set. The perimeter of an individual cell
was outlined, and both the area of the cell and portion covered by
grains (i.e., above threshold) were quantified. Lighting and contrast
levels were standardized before taking measurements to assure that all
slides were assessed under the same conditions. Background was assessed
by taking measurements over unlabeled cells outside the area of
interest. Cells having a value five times higher than background were
considered labeled.
Analyses. To determine whether aging altered VIP or AVP gene
expression per cell in the SCN or the number of cells labeled for these
peptides, 3 (age) × 7 (time of day) ANOVAs were performed. Planned
comparisons using one-way ANOVAs examining the effects of time on gene
expression in each age group were performed to determine whether gene
expression was rhythmic. Post hoc comparisons were made
using Newman-Kuels tests. Differences were considered significant if
p < 0.05.
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RESULTS |
VIP gene expression
Two-way ANOVA revealed a main effect of age on VIP mRNA per cell
(Fig. 1) (F(2,101) = 5.99; p < 0.004) but no effect of time (F(6,101) = 0.81; p = 0.57) and
no interaction (F(12,101) = 1.50; p = 0.14). Further analyses showed that VIP mRNA levels
per cell were lower in middle-aged and old than in young females
(p < 0.05). VIP-expressing cells in all animals
were seen predominantly in the ventrolateral SCN. However, as animals
aged, there appeared to be a loss of VIP gene expression in the most
medially located neurons (Fig. 2). The
two-way ANOVA on the number of VIP-expressing cells in the SCN revealed
a main effect of age (F(2,101) = 4.53; p < 0.02) but no effect of time
(F(6,101) = 1.27; p = 0.28) and no interaction (F(12,101) = 0.80;
p = 0.65). Further analyses showed that the number of
cells expressing VIP mRNA was lower in the middle-aged and old females
than in the young females (Fig. 3).
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Figure 1.
Overall levels of VIP mRNA per cell in young,
middle-aged, and old females (mean ± SEM). The 3 (age) × 7 (time
of day) ANOVA revealed a significant effect of age
(F(2,101) = 5.99; p < 0.004) on mRNA levels per cell. VIP gene expression was significantly
lower in middle-aged and old females compared with young
(p < 0.05).
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Figure 2.
This photomicrograph shows VIP labeling in the SCN
of young (A), middle-aged
(B), and old (C) females at
1200 hr. VIP labeling was seen predominantly in the ventrolateral
portion of the SCN in all animals. However, as animals age, there
appears to be a decrease in the number of VIP-expressing cells in the
medial portion of the nucleus. 3V, Third ventricle;
OC, optic chiasm. Scale bar, 25 µm.
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Figure 3.
VIP-expressing cells per section (mean ± SEM) in young, middle-aged, and old females. The two-way ANOVA
analyzing the effects of age and time of day on the number of
VIP-expressing cells revealed a main effect of age
(F(2,101) = 4.53; p < 0.02), with the number of VIP-expressing cells per section being higher
in young than in middle-aged or old females
(p < 0.05).
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To determine whether there was a rhythm in VIP gene expression in
young, middle-aged, and old females, we performed planned comparisons
using one-way ANOVAs on VIP mRNA levels at each age. In young females,
VIP gene expression was rhythmic (F(6,43) = 3.03; p < 0.02), with mRNA levels being lower at 0300, 0800, and 2000 hr than at the other time points
(p < 0.05; Fig.
4). By the time animals reached middle
age, a rhythm in VIP gene expression was no longer detectable (middle
age, F(6,31) = 0.61; p = 0.72; old, F(6,27) = 0.93, p = 0.49).
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Figure 4.
VIP mRNA levels per cell (mean ± SEM) over
time in young ( ), middle-aged ( ), and old ( ) females. One-way
ANOVA revealed that VIP mRNA was rhythmic in young females
(F(6,43) = 3.03; p < 0.02), with VIP gene expression being higher at 2400, 1200, and 2300 hr
than at other times of day (*p < 0.05). VIP mRNA
levels did not significantly fluctuate over the day in middle-aged or
old females. The black bar under the
x-axis represents the dark phase of the cycle, and the
white bar represents the light phase.
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AVP gene expression
Two-way ANOVA revealed a main effect of time on the amount of AVP
mRNA per cell (Fig. 5)
(F(6,108) = 18.73; p < 0.001)
but no effect of age (F(2,108) = 0.64;
p = 0.53) and no interaction (F(12,108) = 0.55; p = 0.87).
Post hoc analyses examining the effect of time revealed a
prominent rhythm in AVP gene expression in all groups. AVP mRNA levels
were low between 2400 and 0300 hr and exhibited significant increases
at each time point from 0800 to 1600 hr (p < 0.05). After 1600 hr, AVP mRNA returned to baseline levels, showing a
significant decrease at both 2000 and 2300 hr (p < 0.05). Planned comparisons using one-way ANOVAs to determine whether
AVP mRNA expression was rhythmic were consistent with the two-way
ANOVA; AVP gene expression was rhythmic in all age groups (young,
F(6,54) = 10.17; p < 0.001;
middle-aged, (F(6,28) = 6.99; p < 0.001; and old, F(6,26) = 3.81;
p < 0.008), with AVP mRNA levels being highest at 1600 hr and lowest at 0300 hr (p < 0.05 in all age
groups).
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Figure 5.
AVP-expressing cells per section (mean ± SEM) in young, middle-aged, and old females. Aging did not alter the
number of AVP-expressing cells in the SCN of females
(F(2,108) = 0.928; p = 0.399).
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AVP gene expression was seen predominantly in the dorsomedial portion
of the SCN in all animals examined (Fig.
6). The two-way ANOVA on the number of
cells expressing AVP mRNA per section revealed a main effect of time on
cell number (F(6,108) = 11.19; p < 0.001) but no effect of age (F(2,108) = 0.93;
p = 0.40) and no interaction (F(12,108) = 1.43; p = 0.16).
The effect of time on cell number was similar to that seen on AVP mRNA
levels (Fig. 7). The number of cells
expressing AVP mRNA was low between 2400 and 0800 hr and increased to
reach peak levels by 1600 hr (p < 0.05). The number of AVP-expressing cells then began to fall and decreased significantly by 2300 hr (p < 0.05). Aging did
not alter this pattern, nor did it affect the number of cells
expressing AVP mRNA (Fig. 8).
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Figure 6.
These photomicrographs show AVP mRNA labeling in
the SCN of young (A), middle-aged
(B), and old (C) females at
1200 hr. AVP expression was seen primarily within the dorsomedial
portion of the SCN in all groups of animals. 3V, Third
ventricle; OC, optic chiasm. Scale bar, 25 µm.
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Figure 7.
AVP-expressing cells per section (mean ± SEM) over time in young (), middle-aged (), and old females
(). The two-way ANOVA examining the effects of age and time of day
on AVP cell number revealed a significant effect of time
(F(6,108) = 11.19; p < 0.001), with the number of AVP-expressing cells gradually increasing
between 0300 and 1600 hr and then declining to baseline levels by 2300 hr (different letters are significantly different from
each other; p < 0.05). The black
bar under the x-axis represents the dark phase
of the cycle, and the white bar represents the light
phase.
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Figure 8.
AVP mRNA levels per cell (mean ± SEM) over
time in young (), middle-aged (), and old females (). The 3 (age) × 7 (time of day) ANOVA revealed a main effect of time on AVP
mRNA levels (F(6,108) = 18.73;
p < 0.001), with AVP mRNA levels increasing
between 0300 and 1600 hr and then declining to baseline levels by 2300 hr (different letters are significantly different from
each other; p < 0.05). The black
bar under the x-axis represents the dark phase
of the cycle, and the white bar represents the light
phase.
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DISCUSSION |
Previous studies demonstrate that aging alters the expression of
numerous physiological and behavioral rhythms that are driven by the
SCN. This raises the possibility that age-related deterioration of the
pacemaker itself may underlie the changes in rhythmicity of these
diverse physiological endpoints. Therefore, the purpose of this study
was to examine the effects of aging on two major peptides synthesized
within the SCN, VIP and AVP, to determine whether the rhythmic
expression of their mRNAs was altered.
We focused our attention on these two neuropeptides because (1) they
are critical neuropeptides synthesized rhythmically in the SCN (Inouye
et al., 1993); this rhythmic expression dictates the ability of the SCN
to interpret environmental cues (Kiss et al., 1984; Bosler and Beaudet,
1985; Hisano et al., 1988; Ibata et al., 1989) and drive multiple
outputs (Osland et al., 1977; Sodersten et al., 1983; Sollars and
Pickard, 1995; van der Beek et al., 1995; Harney et al., 1996;
Scarbrough et al., 1996; Palm et al., 1997); and (2) these peptides
also modulate the timing of GnRH secretion (van der Beek et al., 1995;
Huhman and van der Beek, 1996; Palm et al., 1997), which is critical to
cyclic reproduction in females. Our study was performed in females
because reproductive success in this sex is intimately tied to the SCN
and begins to display overt signs of senescence by middle age (Wise et
al., 1996, 1997). Our results clearly demonstrate a selective
age-related alteration in VIP gene expression in the absence of any
change in AVP gene expression.
The rhythm of VIP mRNA was not detectable by the time animals reached
middle age. In young females, VIP mRNA showed a peak at ~1200 hr and
a smaller rise during the midevening. This finding is consistent
with our previous work, which showed that mRNA levels in females are
high during the day, and that this daytime rise is coincident with the
timing of the LH surge in young females (Krajnak et al., 1997). Other
studies also indicate that if VIP activity in young females is blocked
in the hour just before the surge with either antisense
oligonucleotides to VIP (Harney et al., 1996) or VIP antibodies (van
der Beek et al., 1995), the timing of the surge is delayed, and the
amplitude is attenuated. Thus, the loss of the daytime peak of VIP mRNA
in middle-aged animals may be responsible for the delay in the timing
and attenuation in the amplitude of the LH surge.
The rise in VIP mRNA during the middle of the dark phase (2300-2400
hr) was also attenuated in aging animals. In young males, VIP mRNA
levels and peptide concentrations in the SCN are high at night and
decline after lights on (Albers et al., 1990; Inouye et al., 1993;
Krajnak et al., 1997). Thus, it has been hypothesized that these
light-induced changes in VIP may provide environmental LD information
to the pacemaking cells in the SCN (Albers et al., 1991). However, it
is unclear whether the rise in VIP mRNA at 2300-2400 hr that we
observe in young females serves the same function. If it does, this
signal is absent by the time females reach middle age. Because VIP
neurons receive input from all major afferents to the SCN (Kiss et al.,
1984; Bosler and Beaudet, 1985; Hisano et al., 1988; Ibata et al.,
1989), this decline in mRNA could be in part responsible for the
decreased ability of both photic and nonphotic stimuli to phase shift
rhythms in aged animals (Rosenberg et al., 1991; Penev et al., 1995;
Zhang et al., 1996). Similar changes may also occur in males. Kawakami
et al. (1997) found a slight decrease in VIP mRNA at night in old males
(22-24 months) when they monitored gene expression at one time during the light phase and one time during the dark phase. The number of
VIP-immunolabeled cells within the SCN during the light phase of the
cycle may also decline in very old (33-34 months) males (Chee et al.,
1988).
Several possible mechanisms may underlie the age-related decline in the
rhythm of VIP mRNA in the SCN. First, both photic input from the retina
(Ibata et al., 1989) and serotonin (5-HT) input from the raphe (Kiss et
al., 1984; Bosler and Beaudet, 1985) regulate the rhythm of VIP gene
expression and peptide concentrations. Eliminating photic input to the
SCN of adult males by placing animals in persistent darkness (Inouye et
al., 1993) or by enucleation (Okamoto et al., 1990) results in a loss
of VIP rhythmicity and an overall increase in VIP expression. Thus, it
is possible that alterations in the retinal input to the SCN lead to
the loss of rhythmic VIP gene expression by middle age. In aged
animals, light-induced fos expression is attenuated in the SCN (Sutin
et al., 1993; Zhang et al., 1996; Cai et al., 1997), which is
concomitant with a decline in the magnitude of light-induced phase
shifts (Zhang et al., 1996). However, subjecting aged animals to
brighter light pulses partially reverses these dampened responses
(Zhang et al., 1996), suggesting that there may be age-related affects
on the ability of the retina to perceive and transmit photic
information. However, retinal degenerative mice, whose retina show
signs of aging relatively early, do not exhibit any decline in the
ability to respond to phase-shifting light pulses (Garcia-Fernandez et
al., 1995). Together, these observations suggest that the retinal
signal may be maintained with aging, but that the ability of the SCN to
receive this signal may be altered. Because retinal input appears to
synapse directly onto VIP-producing neurons (Ibata et al., 1989), and
light induces fos expression within VIP neurons (Daikoku et al., 1992;
Speh and Moore, 1996; Rominj et al., 1996), it is possible that
age-related changes in VIP alter the transmission of photic information
to other pacemaking target cells within the SCN or efferent targets in
other regions of the brain. However, the phenotype of neurons showing
reduced fos activation in aged animals needs to be determined before we
know exactly which outputs of the SCN might be altered.
VIP concentrations (Kawakami et al., 1985) and gene expression (Okamura
et al., 1995) are also modulated by 5-HT. 5-HT terminals from the raphe
synapse directly on VIP-producing neurons in the SCN (Kiss et al.,
1984), and lesioning this input results in a loss of VIP and a dramatic
decline in VIP expression within the SCN rhythmicity (Kawakami et al.,
1985, 1994; Okamura et al., 1995). Studies have shown that the rhythm
in 5-HT turnover is altered by the time females reach middle age (Cohen
and Wise, 1988) and that 5-HT projections to the SCN are diminished
(Van Luijtelaar et al., 1989). The ability of 5-HT to cause phase
shifts in activity rhythms is also reduced in aged animals (Penev et al., 1995). Thus, the age-related changes in 5-HT input to the SCN may
have dramatic effects on both the rhythmic expression of VIP and the
amount of VIP expressed.
The most surprising finding of this study is that the AVP rhythm
remains completely intact in the same animals in which the rhythmic
expression of VIP was absent. The rhythm in gene expression, the number
of cells expressing AVP, and the amount of AVP mRNA per cell were
unaltered, even in the oldest group of females. These results are
consistent with data collected from aged males showing that the number
of AVP-immunopositive neurons in the SCN was not altered in males
16-18 months of age (Roozendaal et al., 1987; Lucassen et al., 1995).
These findings are remarkable in that most entrained rhythms that have
been examined show age-related alterations in rhythmic expression (Wise
et al., 1987, 1997; Satinoff et al., 1993; Sutin et al., 1993; Zhang et
al., 1996; Cai et al., 1997).
AVP projections from the SCN serve as an output, carrying timing
information to a number of target areas, including regions involved in
generation of the LH surge (Watson and Langub, 1996) and generation of
the rhythm in corticosteroid release (Buijs, 1997). Our findings
showing that neither the rhythmic expression of AVP nor the amount of
AVP expressed per cell changes during aging helps explain the
maintenance of the daily rhythm in corticosterone despite the absence
of a rhythm in corticotropin-releasing hormone (CRH) mRNA in the PVN of
aged animals (Cai and Wise, 1996). Buijs (1997) has reported a
multisynaptic pathway between SCN AVP neurons and the adrenal that
bypasses PVN CRH. In addition, our data lead us to conclude that
changes in the timing or amplitude of the LH surge seen in middle-aged
animals are unlikely attributable to changes in the AVP signal.
However, it is possible that changes in the ability of target sites to
recognize the AVP signal occur in aging females.
The rhythm in AVP synthesis and release is endogenous and driven by the
SCN (Gillette and Reppert, 1987; Inouye et al., 1993). Thus, these
rhythms are maintained in constant conditions. However, under LD
conditions, the AVP rhythm is also tightly coupled to the LD cycle
(Burbach et al., 1988; Inouye et al., 1993). Our findings show that
this rhythm remains tightly coupled to the LD cycle throughout aging.
However, other entrained rhythms regulated by the SCN and synchronized
to the LD cycle, such as glucose utilization (Wise et al., 1987), Fos
induction by the LD cycle (Sutin et al., 1993; Zhang et al., 1996; Cai
et al., 1997), and activity rhythms (Satinoff et al., 1993), lose their
tight coupling to the LD cycle during aging. In fact, when temperature,
activity, and drinking rhythms are monitored in the same elderly
animal, a deterioration in the entrainment and amplitude of one rhythm
is not necessarily correlated with a deterioration in the other rhythms
(Satinoff et al., 1993). Together these studies support the hypothesis
that age-related changes in the expression of rhythmic processes are the result of the uncoupling of various oscillators within the SCN and
not necessarily attributable to complete deterioration of the ability
of the SCN to generate rhythms.
In summary, our results show that the rhythmic expression of VIP in the
SCN is undetectable by the time females reach middle age, but AVP
expression in these same animals is unaltered. Based on these findings,
we suggest that the differential effects of age on these two outputs
may explain why certain rhythms are altered with age, whereas others
remain intact. Future studies should concentrate on changes in the
inputs to the SCN to determine how age-related alterations in these
inputs may affect the ability of the SCN to entrain rhythms to the
environment. Other studies, concentrating on how individual oscillators
are coupled and how this coupling is altered by aging, will also help
us determine why some rhythms are more resistant to the effect of aging
than others.
| |
FOOTNOTES |
Received Jan. 8, 1998; revised March 24, 1998; accepted March 31, 1998.
This work was supported by National Institutes of Health Grants AGO2224
to P.M.W., AGO5755 to K.K., and AGO5762 to M.L.K. We thank Susan Steman
and Dr. Jacob P. Harney for technical assistance. We also thank Dr.
R. H. Goodman (Vollum Institute) and Dr. T. Sherman (Georgetown
University) for supplying us with cDNAs to VIP and AVP.
Correspondence should be addressed to Dr. Kristine Krajnak, Department
of Physiology, MS508 Chandler Medical Center, University of Kentucky,
Lexington, KY 40536-0084.
| |
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Abstract
Introduction
