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The Journal of Neuroscience, July 1, 1998, 18(13):5045-5052
Endogenous Regulation of Serotonin Release in the Hamster
Suprachiasmatic Nucleus
Thomas E.
Dudley,
Lisa A.
DiNardo, and
J. David
Glass
Department of Biological Sciences, Kent State University, Kent,
Ohio 44242-0001
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ABSTRACT |
Serotonin (5-HT) has been strongly implicated in the regulation of
the mammalian circadian clock located in the suprachiasmatic nuclei
(SCN). However, little is known of the pattern of neuronal 5-HT release
in the SCN or of the factors involved in regulating its release. Using
in vivo microdialysis, we demonstrated the existence of
a daily rhythm in the output of 5-HT in the SCN of freely behaving
hamsters. This rhythm was characterized by a sharp increase in release
from a nadir during late midday to peak levels at the light/dark
transition. Output declined to basal levels throughout the remainder of
the night. A similar pattern also was evident under constant darkness,
with increased 5-HT output occurring at the onset of subjective night.
Locomotor activity induced by exposure to a novel running wheel had a
pronounced phase-dependent effect on 5-HT release in the SCN, with
stimulation during the light phase and suppression during the late dark
phase. Systemic application of the somatodendritic 5-HT1A
agonist BMY 7378 had a significantly greater suppressive effect on 5-HT
release in the SCN during the late dark phase compared with mid light phase, indicating that a variation in raphe autoreceptor response may
underlie the time-dependent effects of wheel running on 5-HT release.
Collectively, these results show that the daily rhythm in output of
5-HT in the SCN is generated endogenously, and that behavioral state
can strongly influence serotonergic activity in the circadian clock in
a phase-dependent manner.
Key words:
serotonin; suprachiasmatic nuclei; hamster; circadian
rhythm; microdialysis; 5-HT1A receptor
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INTRODUCTION |
The suprachiasmatic nuclei (SCN)
represent the major center for the generation and regulation of
mammalian circadian rhythms (Rusak and Zucker, 1979 ; Moore, 1983; Klein
et al., 1991). Circadian rhythms are synchronized to the daily
light/dark cycle principally by photic information relayed from the
retina to the SCN. This process is mediated by retinal input supplied
to the SCN directly by the retinohypothalamic tract (Hendrikson et al.,
1972; Moore and Lenn, 1972; Pickard, 1982; Youngstrom and Nunez, 1986;
Johnson et al., 1988) and indirectly via the geniculohypothalamic tract (Card and Moore, 1982; Johnson et al., 1989), which also is thought to
convey nonphotic entraining input to the SCN (Rusak et al., 1989;
Biello et al., 1994; Janik and Mrosovsky, 1994; Janik et al., 1995). A
third major input to the SCN is a serotonergic projection from the
raphe nuclei that terminates primarily in the retinorecipient region of
the nucleus (Azmitia and Segal, 1978; Moore et al., 1978;
Meyer-Bernstein and Morin, 1996).
Serotonin (5-HT) has been implicated in the modulation of photic
signaling in the SCN and the resetting of circadian phase. Serotonergic
agonists inhibit photically related SCN responses, including
light-induced phase shifts in free-running locomotor activity (Rea et
al., 1994; Pickard et al., 1996), light-induced Fos protein
immunoreactivity in SCN cells (Selim et al., 1993; Glass et al., 1994,
1995), electrical activity of light-responsive SCN cells (Miller and
Fuller, 1990; Ying and Rusak, 1994, 1997), and SCN field potentials
evoked by electrical stimulation of the optic nerve (Rea et al., 1994).
These agonists also have pronounced phase-shifting effects on the
circadian locomotor activity rhythm (Tominaga et al., 1992; Edgar et
al., 1993; Bobrzynska et al., 1996a; Mintz et al., 1997) and on the
circadian rhythm of neuronal activity measured from SCN slices (Prosser
et al., 1990, 1993; Medanic and Gillette, 1992; Shibata et al., 1992).
Consonant with these findings are reports that endocrine and behavioral
circadian rhythms are significantly affected by manipulating brain 5-HT levels (Honma et al., 1979; Szafarczyk et al., 1981; Levine et al.,
1986; Banky et al., 1988; Duncan et al., 1988; Smale et al., 1990;
Morin and Blanchard, 1991a,b).
In contrast to the well-characterized effects of 5-HT agonists in the
circadian system, little is known concerning the regulation of
endogenous 5-HT release in the SCN. There are reports of daily variations in indices of serotonergic activity in the SCN, including [3H]5-HT uptake (Meyer and Quay, 1976), neuronal
sensitivity to 5-HT (Mason, 1986), imipramine binding to 5-HT reuptake
sites (Wirz-Justice et al., 1983), tissue 5-HT content (Hery et al., 1982; Cagampang and Inouye, 1994), and extracellular concentrations of
the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), (Faradji et
al., 1983; Ramirez et al., 1987; Glass et al., 1992, 1993). However,
because these parameters are not accurate indicators of synaptic 5-HT
release per se, the nature of daily neuronal 5-HT output in the SCN
remains speculative. It is also unclear whether the putative daily
rhythm in 5-HT output in the SCN is driven by internal pacemakers or by
environmental influences. The aim of the present study, therefore, was
to characterize the daily profile of in vivo neuronal 5-HT
release in the SCN region of freely behaving hamsters using brain
microdialysis. We also used this methodology to evaluate the influences
of light and locomotor activity on 5-HT release in the SCN to provide
fundamental information on factors regulating serotonergic activity in
the circadian clock.
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MATERIALS AND METHODS |
Animals
Adult male Syrian hamsters (Mesocricetus auratus)
raised from breeder pairs obtained from Harlan Sprague Dawley (Madison, IL) were used for experimentation. The animals were maintained in a
temperature-controlled (22°C) vivarium under a 14/10 hr light/dark photocycle (200 lux illuminance; lights on at 7:00 A.M.) and were provided food and water ad libitum.
Microdialysis-HPLC
The microdialysis-HPLC procedures developed in this laboratory
for measuring 5-HT release in the SCN of Syrian hamsters have been
described previously (Glass et al., 1995). Under sodium pentobarbital anesthesia (Nembutal, 50 mg/kg), the animals received a microdialysis probe stereotaxically aimed at the lateral margin of the midposterior aspect of the rostrocaudal axis of the SCN, which contains the largest
concentration of serotonergic fibers and terminals (Fig. 1) (Meyer-Bernstein and Morin, 1996)
(anterior-posterior, 0.0 mm from bregma; lateral, 0.3 mm from midline;
horizontal,  8.0 mm from dura, with head level). Microdialysis was
undertaken the following day. The microdialysis probes were constructed
from hemicellulose dialysis tubing with 12 kDa cutoff (230 µm OD;
Spectra-por, Fisher Scientific, Pittsburgh, PA) glued to a 26 gauge
stainless steel outer cannula containing a fused silica inner cannula
(Polymicro Technologies, Phoenix, AZ). The dialyzing tip length of the
probes was 1.0 mm. In an experiment designed to examine the extent of contamination of SCN microdialysates with 5-HT from outside the SCN,
the dialysis tips were occluded with epoxy, except for a small window
of active membrane (~200 × 300 µm) aimed medially at the SCN.
In all experiments, probes were perfused with artificial CSF (ACSF),
(in mM: 147.2 NaCl, 4.0 KCl, and 1.8 CaCl2, pH 7.2) at flow rates of 1.2 or 1.8 µl/min.
Probe position was verified histologically from 20-µm-thick frozen
sections stained with cresyl violet at the end of the experiment.
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Figure 1.
Representative coronal section through the
midposterior aspect of the SCN showing a microdialysis cannula tract
(C) at the lateral margin of the nucleus. Section
is stained with cresyl violet. 3V, Third ventricle;
OC, optic chiasm.
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Microdialysates were analyzed for 5-HT using a high-performance
liquid chromatograph with an amperometric radial flow electrochemical detector [Bioanalytical Systems (BAS), West Lafayette, IN]. The detector was set at a potential of 590 mV relative to an AgCl reference
cell. A 20 µl aliquot of microdialysate was injected directly onto a
1.0 × 100 mm reverse-phase 3 µm C-18 column (BAS). Mobile phase
consisted of 9.45 gm of monochloroacetic acid, 3.6 gm of NaOH, 0.25 gm
of Na2EDTA, and 0.2 gm of octane sulfonic acid in 1.0 l of purified distilled water, pH 3.1. Tetrahydrofuran (6 ml) was added
after filtration. Flow rate through the column was 90 µl/min, and
sensitivity (the minimal amount of 5-HT producing a signal four times
that of background) was ~500 fg (average baseline content of 5-HT in
a 20 µl sample of SCN microdialysate ranged between 5 and 10 pg). The
5-HT reuptake inhibitor citalopram (4.0 µM; Farmitalia,
Milano, Italy), was added to the ACSF perfusate in all experiments.
Authenticity of the 5-HT peak was verified by (1) coelution with
authentic standard, (2) increases after electrical stimulation of the
median raphe (Dudley and Glass, 1996) or localized perfusion with ACSF
containing citalopram or 100 mM KCl, and (3) decreases
after localized perfusion with Ca2+-free ACSF or
systemic treatment with the 5-HT1A receptor agonist 2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydro-naphthalene hydrobromide (8-OH-DPAT; Research Biochemicals, Natick, MA) or raphe
5-HT1A mixed autoreceptor agonist BMY 7378 (Research
Biochemicals).
Experimental protocol
Daily profiles of 5-HT release. Microdialysis was
undertaken in separate groups of animals maintained under light/dark
(LD) (n = 12) or constant darkness [dark/dark (DD);
n = 6]. After a 2 hr equilibration period, samples
were collected continuously at hourly intervals over 24 hr. In a
separate experiment, microdialysis also was performed over 48 hr under
LD to confirm a repeating daily cycle of 5-HT output (n = 5). For the LD groups, sample collection began at 1400 hr [zeitgeber
time (ZT) 6, with ZT 12 being the time of lights off]. For the DD
group, sample collection began between circadian times (CTs) 1-6, with
CT 12 designated as the onset of subjective night (onset of nocturnal
locomotor activity). Microdialysis under dark conditions was undertaken in the absence of visible light using infrared goggles. For animals under DD, microdialysis was initiated after at least a 2 week period of
exposure to this condition. Stereotaxic implantation of dialysis probes
in the DD group was undertaken by anesthetizing the animals in the dark
and preventing exposure of their eyes to light during surgery using a
light-proof shield. The phase of the circadian activity rhythm of
animals under DD was determined from actograms of the animals'
locomotor behavior monitored continuously using a computerized system
running Dataquest III data acquisition software (Minimitter, Sunriver,
OR). The onset of activity (CT 12) was defined as the first 6 min
interval that was preceded by at least a 6 hr period of inactivity and
was followed by a period of at least 30 min of sustained activity.
Wheel-running-induced 5-HT release. The day after probe
implantation, microdialysis was initiated with a 2 hr equilibration period followed by 1 hr of baseline collection in the home cage, collection during confinement to a 14 inch running wheel (Ancare, Bellmore, NY) for 3 hr, and transfer back to the home cage for a 1 hr
postrun collection period. The sampling interval was 20 min. This
procedure was performed in four separate groups of animals under LD,
with wheel-running commencing at ZTs 1, 4, 9, and 19 (n = 5-8 per group). Microdialysis of the ZT 19 group was conducted in
complete darkness. A magnetic switch attached to the wheel interfaced
with a Dataquest III system was used to count wheel revolutions in 20 min batches corresponding to each microdialysis sample.
Verification of neuronal 5-HT release. Pharmacological
agents were used to verify the authenticity of the 5-HT peak from
chromatographed SCN microdialysates and to verify the neuronal origin
of the 5-HT. These manipulations included localized perfusions via the
microdialysis probe with ACSF containing 100 mM KCl
(n = 4), Ca2+-free ACSF with 0.2 mM EDTA (n = 5) and systemic application of 8-OH-DPAT (5 mg/kg; n = 5). For all treatments, probes
were equilibrated for 2 hr, and 1 hr of baseline samples were
subsequently collected before treatment. A liquid switch with
negligible dead space was used to change between normal ACSF and the
high-K+ or Ca2+-free solutions.
For all treatments, sample collection interval was 20 min, and sampling
was continued for 3 hr after treatment onset at ZT 4. A further
experiment was performed using intraperitoneal injection of the mixed
5-HT1A autoreceptor agonist BMY 7378 at ZT 6 (n = 5) and ZT 19 (n = 5) and saline
controls (n = 3) to examine time-of-day differences in
5-HT autoreceptor-mediated suppression of SCN 5-HT output.
Statistics. Data from the wheel-running and pharmacological
experiments were converted to percentage of the mean of the three pretreatment baseline collections (1 hr) and were analyzed using a
one-way ANOVA. Treatment effects were determined using the
Student-Newman-Keuls test. Individual 24 or 48 hr profiles of 5-HT
release were normalized by expressing values as a percentage of the
daily mean. Differences between nighttime means and the averaged
daytime mean were determined using Dunnet's test procedure for
comparing multiple group means (Zar, 1983). For all analyses, the level
of significance was set at p < 0.05.
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RESULTS |
Neuronal release of 5-HT from the SCN
The chromatographed peak corresponding to authentic 5-HT standards
was increased 73 ± 16% over baseline by perfusion of the SCN
with 100 mM KCl (p < 0.05; Fig.
2) and was decreased 50 ± 10% by
perfusion with Ca2+-free ACSF containing EDTA
(p < 0.05). This peak was also decreased 62 ± 10% by intraperitoneal injection of 8-OH-DPAT
(p < 0.05) or 22 ± 9% by intraperitoneal
injection of BMY 7378 (daytime injection; p < 0.05;
see Fig. 10). Basal peak height also was increased ~50-fold by
localized perfusion with citalopram (see Fig. 9).
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Figure 2.
Pharmacologically induced changes in SCN
microdialysate 5-HT concentrations. Top, Middle, Effects
of localized perfusions with ACSF solutions containing high
[K+] or no Ca2+, respectively,
on 5-HT output. Solid bars denote the 2 hr duration of
the treatments. Bottom, Effect of intraperitoneal
injection of 8-OH-DPAT on 5-HT output. Arrow designates
the time of injection. The sample interval was 20 min for all
treatments; n = 4-5 for each experiment;
*p < 0.05 compared with pretreatment levels.
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Daily profiles of 5-HT release in the SCN
Release under LD
Twenty-four hour profile. The output of 5-HT measured
from animals with probes located in the midposterior aspect of the SCN exhibited a diurnal fluctuation, with the nadir (83 ± 7% of the daily mean) occurring 2-3 hr before lights off and peak output (133 ± 13% of the daily mean) beginning at the time of lights off, constituting a 50% increase from the nadir
(p < 0.05; Fig. 3). Output of 5-HT decreased to basal
levels throughout the remainder of the night. To verify that the 24 hr
profile of 5-HT assessed using microdialysis is of SCN origin, an
experiment was undertaken using microdialysis probes with the dialysis
tip occluded with epoxy, except for a small window of active membrane
(~200 × 300 µm) aimed medially at the SCN (n = 3 animals). The 24 hr release profile of 5-HT measured with these
probes was equivalent to that assessed using the conventional probes
(data not shown).
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Figure 3.
Daily profile of 5-HT release in the SCN region
in hamsters maintained under 14/10 hr light/dark cycle. Solid
bar denotes the dark phase. Each point
represents the mean ± SEM for 12 animals. *p < 0.05 versus mean light phase level.
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Forty-eight hour profile
Microdialysis performed in a separate group of animals
(n = 5) continuously over 48 hr confirmed the
repetitive nature of the daily rhythm in 5-HT output in the SCN (Fig.
4). Although the rhythm symmetry was
somewhat less regular than in the 24 hr sampling group, significant
nocturnal increases in 5-HT output over both LD cycles nevertheless
began within 2 hr of lights off. There was little diminution of
microdialysate 5-HT concentration during the second day of
sampling.
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Figure 4.
Daily profile of 5-HT release in the SCN region
assessed over two consecutive 14/10 hr light/dark cycles. Solid
bars denote the dark phase. Each point
represents the mean ± SEM for 5 animals. *p < 0.05 versus mean light-phase level.
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Release Under DD
The majority of hamsters maintained under DD for  2 weeks
exhibited a stable free-running rhythm of general locomotor activity before and during the microdialysis procedure. Similar to the profile
of 5-HT release observed under LD, a rise in 5-HT output occurred
within 1 hr after the onset of subjective night (CT 12; Fig.
5). The peak 5-HT output (165 ± 61% of the subjective daily mean) constituted an ~83% increase over
the nadir. Under both LD and DD conditions, 5-HT output averaged for
the subjective night was significantly elevated over daytime levels
(Fig. 6).
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Figure 5.
Daily profile of 5-HT release from the SCN region
in hamsters maintained under constant dark (DD) for a minimum of 2 weeks. CT 12 represents the onset of subjective night as assessed by
actograms. Each point represents the mean ± SEM for 6 animals. *p < 0.05 versus mean subjective day
level.
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Figure 6.
Averaged release of 5-HT in the SCN under LD
(n = 12) and DD (n = 6). In
both conditions, overall release of 5-HT is enhanced during subjective
night. *p < 0.01 versus respective subjective
day.
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Wheel-running-induced 5-HT release from the SCN
Novelty-induced wheel-running during the day produced a
significant increase in 5-HT output (Figs.
7, 8). The
most robust stimulation was observed at ZT 4 (5 hr after lights on), in
which wheel-running induced an averaged maximal 52.3 ± 17%
increase in 5-HT output (p < 0.05 vs baseline).
Wheel-running during the dark phase starting at ZT 19 (7 hr after
lights off) caused a maximal 16.4 ± 4% suppression in the mean
level of 5-HT release over the running session
(p < 0.05 vs baseline; Fig. 7). This was not
attributable to less active running, as the mean number of revolutions
was twofold greater than that at ZT 4 (p < 0.01). Moreover, the lack of a stimulatory effect of running on 5-HT output at ZT 19-22 was not attributable to decreasing 5-HT baseline release, because there was no significant change in baseline output over this period. The proportionate degree of maximal increase in
wheel-running-induced 5-HT release at ZT 4 measured in the presence of
citalopram in the ACSF approximated that measured in the absence of
citalopram (Fig. 9).
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Figure 7.
Profiles of novel wheel-running-induced
alterations in 5-HT release from the SCN region during midday (ZT 4-7)
and during the late dark phase (ZT 19-22). Solid bar
denotes the 3 hr period of confinement in the wheel.
*p < 0.05 relative to prerunning baseline. Data
from all animals in each group are included irrespective of running
intensity. Inset, Bars represent total
number of revolutions over the 3 hr wheel-running period for both
groups. *p < 0.05 between groups.
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Figure 8.
Time of day difference in the effect of novel
wheel running on 5-HT release averaged over the entire 3 hr running
period from the SCN region. The vertical position and length of the
solid bars denote the mean level of 5-HT release and
period of wheel running, respectively. Data from all animals in each
group are included irrespective of running intensity. Solid
bar at the top denotes the dark phase. Groups
with different letters are different from each other;
p < 0.05.
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Figure 9.
Individual profiles of 5-HT release from the SCN
collected before, during, and after 2 hr of novelty-induced
wheel-running. Solid bar denotes the period of
confinement in the wheel. A, 5-HT measured without
reuptake blocker (citalopram) added to the ACSF. B, 5-HT
measured with citalopram added to the ACSF. Note differences in
vertical axis scales. Sample interval is 20 min.
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Effect of BMY 7378 on 5-HT release from the SCN
Hamsters that were treated with the mixed 5-HT1A
autoreceptor agonist BMY 7378 at ZT 6 exhibited a 22 ± 9%
suppression of 5-HT output (p < 0.05 vs
baseline; Fig. 10). In contrast,
hamsters receiving the same dose of this drug at ZT 19 exhibited
approximately a two times greater suppression of 5-HT output (46 ± 7%; p < 0.05 vs ZT 6).
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Figure 10.
Time of day difference in the inhibitory effect
of intraperitoneal injection of BMY 7378 on 5-HT release from the SCN
region. Arrow denotes time of injection. Sampling
interval is 20 min. *p < 0.05 versus same time
point at ZT 6; !p < 0.05 versus saline
control.
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DISCUSSION |
By direct assessment of in vivo neuronal 5-HT output,
we confirm a daily rhythm of neuronal 5-HT output in the Syrian hamster SCN. In animals entrained to a daily LD cycle, this rhythm was characterized by an abrupt increase in extracellular 5-HT from low
daytime levels to peak levels at the light/dark transition. This
increase was temporally associated with active waking behaviors, and in
most of the animals, output of 5-HT subsequently declined to basal
levels during the remainder of the night. This pattern of 5-HT output
is similar to that of 5-HIAA release reported previously in the SCN of
this species (Glass et al., 1993), the rat (Faradji et al., 1983;
Ramirez et al., 1987), and the Siberian hamster (Glass et al., 1992),
in which increases in 5-HIAA output were also associated with the
light/dark transition. Our data thus support the historical view that
the increased extracellular concentration of 5-HIAA associated with
lights off reflects increased neuronal 5-HT release. It is of note that
an enhancement of 5-HT release at the light/dark transition has also
been reported in rat cerebellum (Mendlin et al., 1996), hippocampus,
striatum, amygdala, and frontal cortex using microdialysis (Rueter and
Jacobs, 1996). A nocturnal increase in 5-HT release in the ventromedial
hypothalamus also has been observed (Martin and Marsden, 1985). Thus,
the nighttime activation of 5-HT output could be a widespread signal
throughout the brain of nocturnal mammals serving to regulate multiple
functions, including those of the circadian system.
Our demonstration that the 5-HT rhythm persists in free-running
hamsters maintained under long-term DD conditions strongly suggests
that this rhythm is generated by an endogenous mechanism and is
circadian in nature. This finding is consistent with observations of a
daily rhythm in SCN tissue content of 5-HT that persists under DD,
albeit out of phase with variations observed under LD (Cagampang and
Inouye, 1994). It should be noted that these results differ from those
of an earlier study, in which tissue 5-HT content in gross dissections
of mediobasal and anterior regions of the hypothalamus displayed
diurnal variations under LD but not under DD or constant light (Ferraro
and Steger, 1990). However, because the contribution of 5-HT from the
SCN per se cannot be assessed from such dissections, and the daily
profile of 5-HT metabolism in the SCN is different from that in
adjacent regions of the hypothalamus (Glass et al., 1992), it is not
feasible to draw conclusions regarding SCN serotonergic activity from
this approach. In the present study, the rise in SCN 5-HT output
exhibited under DD began at the onset of the subjective night (the
beginning of the active period), suggesting that the circadian activity
rhythm could be an important determinant of the daily rhythm of 5-HT
output in the SCN. Additional evidence that photic cues do not drive
the SCN 5-HT rhythm is that the rhythm persists in appropriate phase on
release of hamsters from LD into constant light (J. D. Glass,
unpublished observations).
The present demonstration that 5-HT output in the SCN was significantly
increased by KCl-induced depolarization and was decreased by depletion
of extracellular Ca2+ indicates that the
extracellular 5-HT measured in the SCN microdialysates is largely of
synaptic origin. The degree of response achieved with these
manipulations was similar to that reported in other brain regions using
microdialysis (Mendlin et al., 1996). Additional confirmation of the
synaptic origin of 5-HT in SCN microdialysate is that localized
perfusion with the 5-HT reuptake blocker citalopram increased
microdialysate 5-HT concentrations, and that intraperitoneal administration of the 5-HT1A receptor agonist 8-OH-DPAT or
the mixed 5-HT1A autoreceptor agonist BMY 7378 decreased
5-HT output, presumably via the activation of somatodendritic
5-HT1A autoreceptors. As final proof, we have reported
previously that the extracellular concentration of 5-HT in the SCN
measured by microdialysis is increased by electrical stimulation of the
raphe (Dudley and Glass, 1996).
An important methodological consideration in this and other related
studies is that the apparent daily rhythms in the output of 5-HT and
5-HIAA measured in the SCN (generally assessed for periods of  24 hr)
could be artifacts of the in vivo sampling procedures. It is
therefore critical to confirm the persistence of the rhythm over two or
more daily cycles. This was verified for 5-HT by our continuous
microdialysis sampling over two consecutive LD cycles. Over both
cycles, significant daily fluctuations in 5-HT output were observed,
with nocturnal increases occurring 1 hr after lights off, after
nadirs at the mid to late light phase. Levels of 5-HT during the second
day of sampling were similar to those of the first day, reflecting the
viability of presynaptic serotonergic terminals at the probe site
throughout the experiment.
Behavioral state is considered an important determinant of central
serotonergic activity (Schwartz et al., 1989; Wilkinson et al., 1991;
Linthorst et al., 1995; Mendlin et al., 1996; Rueter and Jacobs, 1996).
In particular, neuronal 5-HT release has been strongly correlated with
increased levels of arousal and locomotor activity, including alert
waking and postural adjustments, as well as feeding. Thus, in view of
the pronounced in vivo effects of pharmacological
serotonergic agonists on SCN clock phase (Tominaga et al., 1992; Edgar
et al., 1993; Bobrzynska et al., 1996a) and photic entrainment
processes (Rea et al., 1994; Glass et al., 1995; Pickard et al., 1996),
it is possible that these functions could be modulated by behaviorally
induced changes in 5-HT release. As an initial approach to this
question, we sought to determine how 5-HT output in the SCN is affected
by induced locomotor activity. Our results demonstrate that
wheel-running markedly increased 5-HT release in the SCN in a
phase-dependent manner, confirming a direct link between behavior and
serotonergic activity in the circadian clock. The running-induced
output of 5-HT during midday was correlated with running intensity,
because with one exception, the active runners had a higher rate of
5-HT output than less active runners. A similar relationship has been
reported in rat cerebellum, in which increases in 5-HT release were
correlated with the duration of alert active waking at the light/dark
transition (Mendlin et al., 1996).
The phase-related differences in the effect of wheel-running on 5-HT
output in the SCN provide a novel perspective into the relationship
between behavioral state and the regulation of serotonergic activity.
The suppression of 5-HT release induced by wheel-running during the
late dark phase, in contrast to the stimulation of 5-HT seen at midday,
points to a diurnal variation in 5-HT neuronal response to behavioral
activation. The control of raphe activity involves somatodendritic
5-HT1A autoreceptors (Garrat et al., 1988; O'Connor and
Kruk, 1992), and thus, a daily variation in 5-HT1A
autoreceptor-mediated response could underlie the biphasic time-of-day
effect of wheel-running on 5-HT release in the SCN. We tested this
possibility by examining the inhibitory effect of the mixed
5-HT1A autoreceptor agonist BMY 7378 on SCN 5-HT release at
the times of greatest difference in effect of wheel-running on 5-HT
release. The substantially greater suppressive effect of BMY 7378 on
5-HT release at ZT 19, compared with ZT 6, is evidence for a daily
variation in 5-HT1A autoreceptor-mediated response. Previous evidence for a daily variation in raphe autoreceptor responsiveness is the observation of a biphasic feeding response of
rats to intraraphe injections of the 5-HT1A agonist
8-OH-DPAT with stimulation during mid to late dark phase and inhibition soon after lights off (Currie and Coscina, 1993). There is also support
for a diurnal variation in response of central 5-HT1A postsynaptic receptors (Lu and Nagayama, 1996). It is thus plausible that 5-HT output in response to strenuous wheel-running at ZT 19-22
strongly inhibited raphe activity via enhanced 5-HT1A
autoreceptor activity during the late dark phase, thus preventing the
sustained increase in 5-HT output exhibited by wheel runners during the day.
The present demonstration that appropriately timed wheel running can
stimulate neuronal 5-HT release is further evidence that increased 5-HT
output could mediate nonphotic phase shifting. Previous evidence for
this is that behavioral activity induced by wheel running, cage
changing (Mrosovsky et al., 1989; Reebs and Mrosovsky, 1989), or
treatment with the GABAA agonist triazolam (Turek and Van
Reeth, 1988; Tominaga et al., 1992; Penev et al., 1995) phase advances
the circadian activity rhythm in a similar manner as that induced by
5-HT1A agonists, with maximal shifting at approximately CT
6-8. Depletion of 5-HT in whole brain by intraperitoneal injection of
p-chloroamphetamine (Penev et al., 1995) or in the SCN by
microinjection of 5,7-DHT (Cutrera et al., 1994) or blockade of 5-HT
action by the peripheral administration of 5-HT7/2 receptor antagonists (Sumova et al., 1996) attenuates triazolam- or
arousal-induced (latter study) phase advances. However, the recent
report that depletion of 5-HT in the SCN by intra-SCN microinjection of
5,7-DHT does not block wheel-running-induced phase advances in hamsters (Bobrzynska et al., 1996a) suggests that the SCN may not be a target
for the phase-shifting action of 5-HT. This is consistent with the
observation that 8-OH-DPAT causes phase advances when microinjected
into the raphe nuclei but not the SCN or intergeniculate leaflet (Mintz
et al., 1997). Thus, it is unclear whether activity-induced increases
in 5-HT may act directly on the SCN or may exert an indirect effect on
the SCN via action at the raphe or other sites that influence SCN
function. Further assessments will be required to establish the
identity of the target(s) of 5-HT for the induction of nonphotic phase
shifts.
In summary, the present findings confirm the existence of an endogenous
circadian rhythm in neuronal 5-HT release in the SCN that can occur in
the absence of photic cues. The findings that 5-HT output in the SCN
can be increased by appropriately timed activity pulses and in
association with active waking behaviors at the light/dark transition
suggest that behavioral state could be an important determinant of the
circadian pattern of 5-HT release in the circadian clock.
| |
FOOTNOTES |
Received Nov. 17, 1997; revised April 10, 1998; accepted April 13, 1998.
This work was supported by National Institutes of Health Grant NS35229
to J.D.G. We are grateful to Greg Grossman and Jon Roberts for their
technical assistance.
Correspondence should be addressed to J. David Glass, Department of
Biological Sciences, Kent State University, Kent, OH 44242-0001.
| |
REFERENCES |
-
Azmitia EC,
Segal M
(1978)
An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat.
J Comp Neurol
179:641-660[ISI][Medline].
-
Banky Z,
Molnar J,
Csernus V,
Halasz B
(1988)
Further studies on circadian hormone rhythms after local pharmacological destruction of the serotonergic innervation of the rat suprachiasmatic region before the onset of the corticosterone rhythm.
Brain Res
445:222-227[ISI][Medline].
-
Biello SM,
Janik D,
Mrosovsky N
(1994)
Neuropeptide Y and behaviorally induced phase shifts.
Neuroscience
62:273-279[ISI][Medline].
-
Bobrzynska KJ,
Godfrey MH,
Mrosovsky N
(1996a)
Serotonergic stimulation and nonphotic phase-shifting in hamsters.
Physiol Behav
59:221-230[Medline].
-
Bobrzynska KJ,
Vrang N,
Mrosovsky N
(1996b)
Persistence of nonphotic phase shifts in hamsters after serotonin depletion in the suprachiasmatic nucleus.
Brain Res
741:205-214[ISI][Medline].
-
Cagampang FRA,
Inouye S-IT
(1994)
Diurnal and circadian changes of serotonin in the suprachiasmatic nuclei: regulation by light and an endogenous pacemaker.
Brain Res
639:175-179[ISI][Medline].
-
Card JP,
Moore RY
(1982)
Ventral lateral geniculate nucleus efferents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity.
J Comp Physiol
206:390-396.
-
Currie PJ,
Coscina DV
(1993)
Diurnal variations in the feeding response to 8-OH-DPAT injected into the dorsal or median raphe.
NeuroReport
4:1105-1107[Medline].
-
Cutrera RA,
Kalsbeek A,
Pevet P
(1994)
Specific destruction of the serotonergic afferents to the suprachiasmatic nuclei prevents triazolam-induced phase advances of hamster activity rhythms.
Behav Brain Res
62:21-28[ISI][Medline].
-
Dudley T, Glass JD (1996) Endogenous 5-HT release in the
Syrian hamster SCN. Proc Soc Res Biol Rhythms abstract 48.
-
Duncan WC,
Tamarkin L,
Sokolove PG,
Wehr TA
(1988)
Chronic clorgyline treatment of Syrian hamsters: an analysis of effects of the circadian pacemaker.
J Biol Rhythms
3:305-322[Abstract/Free Full Text].
-
Edgar DM,
Miller JD,
Prosser RA,
Dean RR,
Dement WC
(1993)
Serotonin and the mammalian circadian system: II. Phase-shifting rat behavioral rhythms with serotonergic agonists.
J Biol Rhythms
8:17-31[Abstract/Free Full Text].
-
Faradji H,
Cespuglio R,
Jouvert M
(1983)
Voltametric measurements of 5-hydroxyindole compounds in the suprachiasmatic nuclei. Circadian fluctuations.
Brain Res
279:111-119[ISI][Medline].
-
Ferraro JS,
Steger RW
(1990)
Diurnal variations in brain serotonin are driven by the photic cycle and are not circadian in nature.
Brain Res
512:121-124[Medline].
-
Garrat J,
Marsden CA,
Crespi F
(1988)
8-OH-DPAT can decrease 5-HT neuronal firing and release but not metabolism.
Br J Pharmacol
95:874p.
-
Glass JD,
Randolph WW,
Ferreira SA,
Rea MA,
Hauser UE,
Blank JL,
De Vries MJ
(1992)
Diurnal variation in 5-hydroxyindoleacetic acid output in the suprachiasmatic region of the Siberian hamster assessed by in vivo microdialysis: evidence for nocturnal activation of serotonin release.
Neuroendocrinology
56:582-590[ISI][Medline].
-
Glass JD,
Hauser UE,
Blank JL,
Selim M,
Rea MA
(1993)
Differential timing of amino acid and 5-HIAA rhythms in suprachiasmatic hypothalamus.
Am J Physiol
265:R504-R511[Abstract/Free Full Text].
-
Glass JD,
Selim M,
Rea MA
(1994)
Modulation of light-induced c-fos expression in the suprachiasmatic nuclei by 5-HT receptor agonists.
Brain Res
638:235-242[ISI][Medline].
-
Glass JD,
Selim M,
Srkalovic G,
Rea MA
(1995)
Tryptophan loading modulates light-induced responses in the mammalian circadian system.
J Biol Rhythms
10:80-90[Abstract/Free Full Text].
-
Hendrickson AE,
Wagoner N,
Cowan WM
(1972)
An autoradiographic and electron microscope study of retinohypothalamic connections.
Z Zellforsch Mikrosk Anat
135:1-26[ISI][Medline].
-
Hery M,
Faudon M,
Dusticier G,
Hery F
(1982)
Daily variations in serotonin metabolism in the suprachiasmatic nucleus of the rat: influence of oestradiol impregnation.
J Endocrinol
94:157-166[Abstract].
-
Honma KI,
Watanabe K,
Hiroshige T
(1979)
Effects of parachlorophenylalanine and 5,6-dihydroxytryptamine on the free-running rhythms of locomotor activity and plasma corticosterone in the rat exposed to constant light.
Brain Res
169:531-544[ISI][Medline].
-
Janik D,
Mrosovsky N
(1994)
Intergeniculate lesions and behaviorally-induced shifts of circadian rhythms.
Brain Res
651:174-182[ISI][Medline].
-
Janik D,
Mikkelsen JD,
Mrosovsky N
(1995)
Cellular colocalization of fos and neuropeptide Y in the intergeniculate leaflet after nonphotic phase-shifting events.
Brain Res
698:137-145[ISI][Medline].
-
Johnson RF,
Morin LP,
Moore RY
(1988)
Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin.
Brain Res
462:301-312[ISI][Medline].
-
Johnson RF,
Moore RY,
Morin LP
(1989)
Lateral geniculate lesions alter circadian activity rhythms in the hamster.
Brain Res Bull
22:411-422[ISI][Medline].
-
Klein DC,
Moore RM,
Reppert SM
(1991)
In: Suprachiasmatic nucleus: the mind's clock. New York: Oxford UP.
-
Levine JD,
Rosenwasser AM,
Yanovski JA,
Adler NT
(1986)
Circadian activity rhythms in rats with midbrain raphe lesions.
Brain Res
384:240-249[ISI][Medline].
-
Linthorst ACE,
Flachskamm C,
Muller-Preuss P,
Holsboer F,
Reul JMHM
(1995)
Effect of bacterial endotoxin and interleukin-1
 on hippocampal serotonergic neurotransmission, behavioral activity and free corticosterone levels: an in vivo microdialysis study.
J Neurosci
15:2920-2934[Abstract]. -
Lu J-Q,
Nagayama H
(1996)
Circadian rhythm in the response of central 5-HT1A receptors to 8-OH-DPAT in rats.
Psychopharmacology
123:42-45[Medline].
-
Martin KF,
Marsden CA
(1985)
In vivo diurnal variations of 5-HT release in hypothalamic nuclei.
In: Circadian rhythms in the CNS (Redfern PH,
Campbell IC,
Davies JA,
Martin KF,
eds), pp 81-94. London: Macmillan.
-
Mason R
(1986)
Circadian variation in sensitivity of suprachiasmatic and lateral geniculate neurones to 5-hydroxytryptamine in the rat.
J Physiol (Lond)
377:1-13[Abstract/Free Full Text].
-
Medanic M,
Gillette MU
(1992)
Serotonin regulates the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day.
J Physiol (Lond)
450:629-642[Abstract/Free Full Text].
-
Mendlin A,
Martin FJ,
Rueter LE,
Jacobs BL
(1996)
Neuronal release of serotonin in the cerebellum of behaving rats: an in vivo microdialysis study.
J Neurochem
67:617-622[ISI][Medline].
-
Meyer DC,
Quay WB
(1976)
Hypothalamic and suprachiasmatic uptake of serotonin in vitro: 24 hr changes in male and proestrous female rats.
Endocrinology
98:1160-1165[Abstract].
-
Meyer-Bernstein EL,
Morin LP
(1996)
Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythm modulation.
J Neurosci
16:2097-2111[Abstract/Free Full Text].
-
Miller JD,
Fuller CA
(1990)
The response of suprachiasmatic neurons of the rat hypothalamus to photic and serotonergic stimulation.
Brain Res
515:155-162[ISI][Medline].
-
Mintz EM,
Gillespie CF,
Marvel CL,
Huhman KL,
Albers HE
(1997)
Serotonergic regulation of circadian rhythms in Syrian hamsters.
Neuroscience
79:563-569[ISI][Medline].
-
Moore RY
(1983)
Organization and function of a CNS oscillator: the suprachiasmatic nucleus.
Fed Proc
42:2783-2789[ISI][Medline].
-
Moore RY,
Lenn NJ
(1972)
A retinohypothalamic projection in the rat.
J Comp Neurol
146:1-14[ISI][Medline].
-
Moore RY,
Halaris AE,
Jones BE
(1978)
Serotonin neurons of the midbrain raphe: ascending projections.
J Comp Neurol
180:417-438[ISI][Medline].
-
Morin LP,
Blanchard J
(1991a)
Depletion of brain serotonin by 5,7-DHT modifies hamster circadian rhythm response to light.
Brain Res
566:173-185[ISI][Medline].
-
Morin LP,
Blanchard J
(1991b)
Serotonergic modulation of the hamster wheel-running rhythm: response to lighting conditions and food deprivation.
Brain Res
566:186-192[ISI][Medline].
-
Mrosovsky N,
Reebs SG,
Honrada GI,
Salmon PA
(1989)
Behavioral entrainment of circadian rhythms.
Experientia
45:696-702[ISI][Medline].
-
O'Connor JJ,
Kruk ZL
(1992)
Pharmacological characteristics of 5-hydroxytryptamine autoreceptors in rat brain slices incorporating the dorsal raphe or the suprachiasmatic nucleus.
Br J Pharmacol
106:524-532[ISI][Medline].
-
Penev PD,
Turek FW,
Zee PC
(1995)
A serotonin neurotoxin attenuates the phase-shifting effects of triazolam on the circadian clock in hamsters.
Brain Res
669:207-216[ISI][Medline].
-
Pickard GE
(1982)
The afferent connection of the suprachiasmatic nucleus of the golden hamster with emphasis on retinohypothalamic projection.
J Comp Neurol
211:65-83[ISI][Medline].
-
Pickard GE,
Weber ET,
Scott PA,
Riberdy AF,
Rea MA
(1996)
5HT1B receptor agonists inhibit light-induced phase shifts of behavioral circadian rhythms and expression of the immediate-early gene c-fos in the suprachiasmatic nucleus.
J Neurosci
16:8208-8220[Abstract/Free Full Text].
-
Prosser RA,
Miller JD,
Heller HC
(1990)
A serotonin agonist phase-shifts the circadian clock in the suprachiasmatic nuclei in vitro.
Brain Res
534:336-339[ISI][Medline].
-
Prosser RA,
Dean RR,
Heller HC,
Miller JD
(1993)
Serotonin and the mammalian circadian system: I. In vitro phase-shifts by serotonergic agonists and antagonists.
J Biol Rhythms
8:1-16[Abstract/Free Full Text].
-
Ramirez AD,
Ramirez VD,
Meyer DC
(1987)
The nature and magnitude of in vivo 5-hydroxyindoleacetic acid output from 5-hydroxytryptamine terminals is related to specific regions of the the suprachiasmatic nucleus.
Neuroendocrinology
46:430-438[ISI][Medline].
-
Rea MA,
Glass JD,
Colwell CS
(1994)
Serotonin modulates photic responses on the hamster suprachiasmatic nuclei.
J Neurosci
14:3635-3642[Abstract].
-
Reebs SG,
Mrosovsky N
(1989)
Effects of induced wheel-running on the circadian activity rhythm of Syrian hamsters: entrainment and phase-response curve.
J Biol Rhythms
4:39-48[Abstract/Free Full Text].
-
Rueter LE,
Jacobs BL
(1996)
Changes in forebrain serotonin at the light-dark transition: correlation with behavior.
NeuroReport
7:1107-1111[Medline].
-
Rusak B,
Zucker I
(1979)
Neural regulation of circadian rhythms.
Physiol Rev
59:449-526[Free Full Text].
-
Rusak B,
Meijer JH,
Harrington ME
(1989)
Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract.
Brain Res
493:283-291[ISI][Medline].
-
Schwartz DH,
McClane S,
Hernandez L,
Hoebel BG
(1989)
Feeding increases extracellular serotonin in the lateral hypothalamus of the rat as measured by microdialysis.
Brain Res
479:349-354[ISI][Medline].
-
Selim M,
Glass JD,
Hauser UE,
Rea MA
(1993)
Serotonergic inhibition of light-induced fos protein expression and extracellular glutamate in the suprachiasmatic nuclei.
Brain Res
621:181-188[ISI][Medline].
-
Shibata S,
Tsuneyoshi A,
Hamada T,
Tominaga K,
Watanabe S
(1992)
Phase-resetting effect of 8-OH-DPAT, a serotonin1A receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro.
Brain Res
582:353-356[ISI][Medline].
-
Smale L,
Michels KM,
Moore RY,
Morin LP
(1990)
Destruction of the hamster serotonergic system by 5,7-DHT: effects on circadian rhythm phase, entrainment and response to triazolam.
Brain Res
515:9-19[ISI][Medline].
-
Sumova A,
Maywood ES,
Selvage D,
Ebling FJP,
Hastings MH
(1996)
Serotonergic antagonists impair arousal-induced phase shifts of the circadian system of the Syrian hamster.
Brain Res
709:88-96[ISI][Medline].
-
Szafarczyk A,
Ixart G,
Alonso G,
Malaval F,
Nouglier-Soule J,
Assenmacher I
(1981)
Effects of raphe lesions on circadian ACTH, corticosterone and motor activity rhythms in free-running blinded rats.
Neurosci Lett
23:92-97.
-
Tominaga K,
Shibata S,
Ueki S,
Watanabe S
(1992)
Effects of 5-HT1A receptor agonists on the circadian rhythm of wheel-running activity in hamsters.
Eur J Pharmacol
214:79-84[ISI][Medline].
-
Turek FW,
Van Reeth O
(1988)
Altering the mammalian circadian clock with the short-acting benzodiazepine, triazolam.
Trends Neurosci
11:535-541[ISI][Medline].
-
Wilkinson LO,
Auerbach SB,
Jacobs BL
(1991)
Extracellular serotonin levels change with behavioral state but not with pyrogen-induced hyperthermia.
J Neurosci
11:2732-2741[Abstract].
-
Ying S-W,
Rusak B
(1994)
Effects of serotonergic agonists on firing rates of photically responsive cells in the hamster suprachiasmatic nucleus.
Brain Res
651:37-46[ISI][Medline].
-
Ying S-W,
Rusak B
(1997)
5-HT7 receptors mediate serotonergic effects on light-sensitive suprachiasmatic nucleus neurons.
Brain Res
755:246-254[ISI][Medline].
-
Youngstrom TG,
Nunez AA
(1986)
Comparative anatomy of the retino-hypothalamic tract in photoperiodic and nonphotoperiodic rodents.
Brain Res Bull
17:485-492[ISI][Medline].
-
Wirz-Justice A,
Krauchi K,
Morimasa T,
Willener R,
Feer H
(1983)
Circadian rhythm of (3H) imipramine binding in the rat suprachiasmatic nucleus.
Eur J Pharmacol
87:331-333[ISI][Medline].
-
Zar JH
(1983)
In: Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall.
Copyright © 1998 Society for Neuroscience 0270-6474/98/18135045-08$05.00/0
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Abstract
Introduction
