Department of Physiology, University of Kentucky, Lexington, Kentucky 40536
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ABSTRACT |
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Quintero, Jorge E. and Douglas G. McMahon. Serotonin Modulates Glutamate Responses in Isolated Suprachiasmatic Nucleus Neurons. J. Neurophysiol. 82: 533-539, 1999. Two input pathways to the suprachiasmatic nucleus (SCN) of the hypothalamus are the glutamatergic retinohypothalamic tract and the serotonergic afferent from the midbrain raphe nucleus. To determine whether these two temporal signaling pathways can converge at the cellular level, we have investigated the effects of serotonin on glutamate-induced calcium responses of individual SCN neurons isolated in cell culture. Dispersed cultures were formed from the SCN of neonatal rats. The calcium indicator Fura-2 acetoxymethyl ester was used to assess the changes in [Ca2+]i by recording the 340-nm/380-nm excitation ratio. Application of glutamate (5 µM) to the culture caused a rapid (within 10 s) increase in the fluorescence ratio of neurons indicating a marked increase in the concentration of intracellular free calcium. However, when 5-hydroxytryptamine (5-HT; 5 µM) was coapplied with glutamate, 31% of neurons showed an overall 61% reduction in the peak of the glutamate-induced calcium increase. Application of the 5-HT7/1A receptor agonist, (±)-8-hydroxy-2-(di-n-propylamino)tetralin [(±)-8-OH-DPAT] (1 µM), also reduced the calcium elevation this time by 80% in 18% of the neurons tested. When the 5-HT7/2/1C receptor antagonist, ritanserin (800 nM), was coapplied with serotonin, it blocked modulation of the glutamate responses. Further support for the involvement of the 5-HT7 receptor was provided by the ability of the adenylate cyclase activator, forskolin (10 µM), and the cAMP analogue, 8-Br cAMP (0.5 mM), to mimic the suppressive effect of serotonin. Blocking spike-mediated cell communication with tetrodotoxin (1 µM) did not prevent the serotonergic suppression of glutamate-induced responses. These results support the hypothesis that the serotonergic modulation of photic entraining signals can occur in SCN neurons.
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INTRODUCTION |
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Endogenous daily clocks, or circadian pacemakers,
drive many daily rhythms in the physiology and behavior of eukaryotes.
In mammals, the suprachiasmatic nucleus (SCN) of the anterior
hypothalamus is the locus of a circadian pacemaker (Moore
1983; Ralph et al. 1990
; Rusak
and Zucker 1979
; Stephan and Zucker 1972
). Of
central interest in the study of circadian systems are the mechanisms by which the endogenous SCN pacemaker is synchronized to external temporal cues.
Two principal SCN input pathways are the glutamatergic
retinohypothalamic tract (RHT) and the serotonergic fibers from the midbrain raphe (Azmitia and Segal 1978; Moore et
al. 1978
; Moore and Lenn 1972
; Morin
1994
). The RHT apparently uses the neurotransmitter glutamate
to transmit photic information to the SCN (Liou et al. 1986
; reviewed by van den Pol and Dudek 1993
).
Glutamate-induced phase shifts in SCN neuronal firing rhythms and in
behavioral locomotor activity rhythms (Ding et al. 1994
;
Meijer et al. 1988
; Shirakawa and Moore
1994
) mimic the phase dependence for light-induced phase
shifts. They are mediated by activation of ionotropic glutamate receptors and liberation of nitric oxide from L-arginine,
with an increase in SCN neuronal
[Ca2+]i as a likely
intermediate step (Kornhauser et al. 1996
). Serotonergic agonists produce phase shifts in approximate antiphase to light when
agonists are applied intracerebroventricularly, into the raphe,
systemically, or in vitro (Edgar et al. 1993
;
Mintz et al. 1997
; Prosser et al. 1990
,
1993
; Shibata et al. 1992
;
Tominaga et al. 1992
).
Serotonin also modulates the effectiveness of the light entrainment
pathway. Injection of serotonergic agents reduces light-evoked phase
shifts in the circadian rhythms of wheel running activity (Rea
et al. 1994) and in vivo in neuronal activity in the SCN (Miller and Fuller 1990
; Nishino and Koizumi
1977
), as well as decreases Fos-immunoreactivity in the SCN
(Rea et al. 1994
; Selim et al. 1993
). Use
of serotonergic agonists and antagonists has revealed that the
presynaptic 5-hydroxytryptamine1B
(5-HT1B) receptor (Pickard et al.
1996
) and the 5-HT7 receptor (Ying
and Rusak 1997
), probably located postsynaptically
(Kawahara et al. 1994
; Lovenberg et al.
1993
), contribute to the modulating actions of serotonin in the SCN.
Serotonin's action on light entrainment of SCN-driven rhythms has been
implicated as a site of neural aging in the circadian system of mammals
(Penev et al. 1997). Aging alters or disrupts the
characteristic period, phase, amplitude, and precision of circadian
rhythms in rodents and humans (Brock 1991
;
Czeisler et al. 1991
; van Coevorden et al.
1991
; Weitzman et al. 1982
), and one striking
age-related change in the rodent circadian system is the loss of
responsiveness to the 5-HT1A/7 receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)
(Penev et al. 1995
). In addition, lesioning the
serotonergic projection to the SCN in young animals mimics age-related
changes in photic responses (Morin and Blanchard 1991
;
Turek et al. 1995
). Understanding the mechanisms of
serotonin's actions in the SCN would thus contribute to elucidating
the cellular basis of aging of this highly localized brain function.
Although serotonin's effect on light-evoked responses in the SCN is
well documented, the cellular mechanisms by which serotonin modulates
photic information responses at the neuronal level are less well
characterized. We hypothesize that the serotonergic modulation of light
responses occurs through the action of serotonin on SCN neurons as well
as the presynaptic retinohypothalamic tract terminals (Pickard
et al. 1996; Pickard and Rea 1997
). We have used
calcium imaging to detect the means by which serotonin modulates responses to glutamate in dispersed cultures of neurons from the SCN.
Our results show that serotonin can act on SCN neurons to suppress
their responses to glutamate.
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METHODS |
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Cultures
Cells were cultured using modified cell culturing protocols from
Welsh et al. (1995) and Mattson et al.
(1995)
. SCN were microdissected from 400-µm hypothalamic
coronal sections of 4- to 6-day-old Sprague Dawley rats by removing a
block containing the bilateral SCN with the dorsal edge the base of the
third ventricle; the ventral margin, the optic chiasm; and a length of
~1 mm. Only one slice was taken from each animal except rarely when
it appeared that two slices contained an equal amount of SCN. Nine to
12 SCN were incubated in a solution of 0.2% trypsin dissolved in
Hank's balanced salt solution for 20 min at room temperature. The
digestion was stopped with a 1-min room temperature incubation in a
0.1% trypsin inhibitor solution. After a 30- to 60-pass trituration
with a P200 pipettor tip, cells were plated inside silicone elastomer
(Sylgard; Dow Corning, Midland, MI) rings placed on
polyethylenimine-coated 35-mm glass-bottom culture dishes (MatTek,
Ashland, MA) or glass coverslips. Cultures were maintained in 1.5 ml of
DMEM (Life Technologies, Gaithersburg, MD) with 5% horse serum, 2%
B27 (Life Technologies, Gaithersburg, MD), 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.5 mM glutamine in an incubator at 37° and
5% CO2. After 1 h, the rings were removed,
and after 3 h, 60% of the medium was replaced with fresh medium
to reduce excitotoxicity (Mattson et al. 1995
). Three
days after plating the cells, the medium in the culture was switched
from the DMEM medium to a defined medium of Neurobasal Media (Life
Technologies, Gaithersburg, MD), 2% B27, and 0.5 mM glutamine to
inhibit glial growth. Once or twice a week, 50% of the medium was
exchanged for fresh medium. For all experiments, cells were used after
they had been in culture 7-14 days. Using extracellular recordings,
Welsh et al. (1995)
showed that dispersed SCN neurons
can be in different phases of their circadian cycle even in the same
culture, so the cells in our cultures were likely also in varying
phases at the time of recording.
Calcium imaging
To conduct calcium imaging, cells were loaded with Fura-2 acetoxymethyl ester (Fura-2), 4 µM by replacing the medium with an extracellular solution, pH 7.4, containing (in mM) 160 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 5 HEPES and adding the Fura-2. After 45 min at room temperature, the cells were rinsed with the above Fura-2-free solution and set aside for 20 min before imaging. Drugs were applied to the bath by a pipettor and then washed out by 10 times volume changes over a 20-min period. Imaging was paused during the wash out as indicated in the figures by breaks in the axes and traces. Increases in fluorescence ratios were never observed during the wash out (data not shown). We focused our recordings on neurons by selecting cells with a spindle-shaped morphology, delimiting ratiometric imaging to a small area of the soma to reduce extraneous signals, and viewing only cells in the selected focal plane. Cells were first identified and then imaged through a Zeiss 40× objective, with high ultraviolet (UV) light transmittance, on an Zeiss Axioskop (Carl Zeiss, Thornwood, NY) upright microscope equipped with Nomarski optics. A Sutter (Novato, CA) filter wheel was used to control the switching between 340- and 380-nm excitation filters. Emitted fluorescence passed through a 510-nm/80-nm band-pass filter to an Attofluor (Carl Zeiss, Thornwood, NY) intensified charge-coupled device (ICCD) camera for imaging. The excitation light came from a 100-W mercury lamp. All images were an average of 16 video frames. Axon Imaging Workshop (Axon Instruments, Forest City, CA) was used to control the acquisition and perform the analysis. Images were acquired every 5 s, the 340- and 380-nm images were stored, and the ratiometric values were calculated. Image analysis of [Ca2+]i was performed for ~10-30 cells per culture.
In the experiments using TTX, cells were grown on glass coverslips that were later mounted to a perfusion chamber (Warner Instruments, Hamden, CT). Drugs were introduced and washed out by rapid perfusion with the aid of a peristaltic pump (Rainin, Emeryville, CA).
Both ritanserin and forskolin were dissolved in DMSO. All of the other drugs were dissolved in extracellular solution. Glutamate, 8-Br-cAMP, forskolin, TTX, trypsin inhibitor, and trypsin were purchased from Sigma (St. Louis, MO); 5-HT, (±)-8-OH-DPAT, and ritanserin were purchased from RBI (Natick, MA); and Fura-2 was obtained from Molecular Probes (Eugene, OR).
Analysis
The change of the 340/380 fluorescence ratio was used to compare
the cells' response to drug applications because it is proportional to
the concentration of intracellular free calcium (Kao
1994). Post hoc Ca2+ calibrations
following the procedure of (Grynkiewicz et al. 1985
) indicated that the resting levels of
[Ca2+]i were between 20 and 40 nM, which are slightly lower than reported by other
investigators for rat SCN neurons (van den Pol et al. 1996
). Peak values were estimated to be between 150 and 300 nM. Cells that were included for analysis met the following criteria: an
initial response to glutamate, and for the studies using 5-HT, a
recovery of glutamate responses after the wash out of the modulator. The data were analyzed based on calculating the change in the fluorescence ratio from the baseline to the peak of the response. We
found that there was variation in the absolute ratio values among
individual cells and across experiments. Thus we analyzed the data in a
paired design in which each cells' modulated response was compared
with its own control response. Paired t-tests were used to
assess the statistical significance (P < 0.05) of the differences between treatments, and each cell was counted as an n = 1. Using this form of analysis, we also confirmed
that the 340/380 ratios could indicate dose-dependent increases in
glutamate-induced increases in
[Ca2+i] (5 µM, 0.22 ± 0.08; 10 µM, 0.30 ± 0.09, mean ± SE,
n = 7, P = 0.04, paired
t-test). Another source of variation in absolute ratios was
that early experiments in this series used a different UV collector
lens in the light source that gave lower absolute ratio values, but did
not alter the baseline or peak intracellular calcium levels or the
percentage change in modulation.
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RESULTS |
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Serotonin suppression of glutamate responses
Application of glutamate (1-10 µM) to SCN neurons (7-14 days in vitro) led to a rapid (within 10 s) increase and a peak in the [Ca2+]i followed by a more sustained elevation that gradually returned to baseline. Glutamate-induced [Ca2+]i responses were observed in 78% (134 of 172) of neurons tested. Cells that responded to glutamate had a spherical or spindle-shaped morphology while irregularly shaped, flat cells (presumed to be glia) did not respond to glutamate.
To test for serotonergic modulation of SCN neuronal glutamate responses, cells that had previously responded to glutamate were subsequently treated with 5-HT (5 µM) for 1 min before reapplying glutamate. Serotonin treatment by itself did not induce changes in the [Ca2+]i; however, 5-HT blocked or blunted the glutamate-induced increases in [Ca2+]i in 31% (41 of 134) of the glutamate-responsive SCN neurons (Fig. 1). Overall, the peak [Ca2+]i of glutamate responses in the 41 neurons was attenuated by an average of 61% in the presence of serotonin (Fig. 1B; mean of the change in the 340/380 ratio ± SD with glutamate alone: 0.72 ± 0.33; glutamate and 5-HT: 0.28 ± 0.39, P < 0.001, n = 41). Figure 1A shows a recording where serotonin completely suppressed the glutamate-induced response. This occurred in 12 of 41 cases and demonstrates that 5-HT can have a profound suppressive effect on SCN neuronal glutamate responses. The typical reduction in glutamate responses was less pronounced. This is illustrated by the other trace in Fig. 1A, which shows blunting, but not complete suppression, of the glutamate-induced calcium rise. Both recordings were from different cells in the same experiment, suggesting that the complete suppression was not a perfusion artifact. Subsequent wash out of serotonin and then reapplication of glutamate elicited responses similar in amplitude to the initial application of glutamate, demonstrating reversibility of the action of serotonin (Fig. 1A). It is interesting to note that during this recovery response to glutamate in Fig. 1A, there are secondary peaks in the calcium response. These occurred in ~17% of these responses. Their cause is unknown to us. As a control for the stability of repeated glutamate responses, we applied glutamate alone using a similar protocol. The mean change in 340/380 ratios for the first and second applications of 5 µM glutamate were not different (P = 0.86, n = 14; means = 0.19 ± 0.06 and 0.20 ± 0.11, respectively).
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5-HT7 receptor involvement
Ying and Rusak (1997) have shown that serotonin
decreased the neuronal firing rate in light-responsive neurons of the
SCN through a 5-HT7 receptor-mediated pathway.
To determine whether the same receptor pathway mediates serotonin's
effect on glutamate responses in our dispersed SCN cultures, the
5-HT7/1A agonist, (±)-8-OH-DPAT (1 µM) was
applied. (±)-8-OH-DPAT alone did not change the
[Ca2+]i. Similar to
serotonin, (±)-8-OH-DPAT reduced glutamate-induced calcium elevations
in SCN neurons (Fig. 2A).
Eighteen percent (11 of 61) of cells responded to (±)-8-OH-DPAT with a
mean reduction of 80% of the glutamate stimulation (Fig.
2C; 340/380 ratio of glutamate alone: 0.48 ± 0.13;
glutamate and (±)-8-OH-DPAT: 0.06 ± 0.13).
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Additional evidence for the involvement of the 5-HT7 receptor was revealed by coapplication of the 5-HT7/2/1C receptor antagonist, ritanserin (800 nM), with serotonin. Ritanserin blocked the serotonergic suppression of glutamate-induced increases in calcium concentration (Fig. 2B) in 11 of 13 cells that had previously been shown to exhibit serotonergic modulation of glutamate responses (P < 0.001, n = 13). Peak [Ca2+]i was 90% of control when ritanserin was coapplied with 5-HT. Application of ritanserin alone did not change the calcium concentration in neurons (data not shown).
Second-messenger pathway
Because the 5-HT7 receptor is positively
coupled to adenylate cyclase (Lovenberg et al. 1993;
Tsou 1994
), the next experiments tested whether
activation of adenylate cyclase could suppress responses similar to
serotonin. Forskolin (10 µM) reduced glutamate-induced calcium
responses by 46% (Fig. 3) in 42% of
neurons tested (mean change in the 340/380 fluorescence ratio with
glutamate alone: 0.19 ± 0.09; glutamate and forskolin: 0.11 ± 0.06, in 8 of 19 cells, P < 0.01, n = 8). A similar amount of DMSO (0.1%) vehicle did not cause increases
in [Ca2+]i or prevent
glutamate responses (data not shown). Application of a
membrane-permeant analogue to cAMP, 8-Br cAMP, also mimicked the
effects of serotonin. When 8-Br cAMP (500 µM) was applied, the
glutamate-induced calcium increase was attenuated in 20 of 31 cells by
an average of 41% (P < 0.001, n = 20;
Fig. 4; mean change in the 340/380 ratio
for glutamate alone: 0.63 ± 0.31; glutamate and 8-Br cAMP:
0.37 ± 0.25). In the majority of cells, 8-Br-cAMP alone had no
effect; however, in 4 of 31 cells it produced sustained calcium
elevations (data not shown).
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Effect of TTX
Although the glutamatergic RHT and serotonergic 5-HT inputs to SCN
neurons are disrupted in dispersed cultures such as ours, SCN neurons
do make synaptic contacts among themselves in such cultures
(Welsh et al. 1995). To determine whether the effects of
serotonin in our cultured cells required spike-mediated neural communication, we applied 1 µM TTX and then tested for serotonin's action on glutamate responses. In the presence of TTX,
glutamate-induced calcium responses were increased in amplitude
(P = 0.02, n = 10; ratio for glutamate
alone: 0.09 ± 0.08; ratio for glutamate and TTX: 0.20 ± 0.15); however, serotonin continued to suppress glutamate-induced [Ca2+]i elevations in 10 of 23 neurons tested. The mean reduction in responses was 46% (peak
340/380 ratio for glutamate alone: 0.82 ± 0.30; glutamate and
5-HT and TTX: 0.44 ± 0.19). The suppression of glutamate-induced
calcium levels by serotonin in the presence of TTX was significant
compared with control responses (P < 0.001, n = 23).
When a perfusion chamber was used for rapid changes of the solutions, the results were similar. Figure 5A depicts the change in the 340/380 fluorescence ratio of two cells over time in a representative experiment. With TTX present in the bath, the mean reduction in responses for 14 of 56 neurons was 63% (mean change in the 340/380 ratio for glutamate alone ± SD: 0.44 ± 0.10; glutamate and 5-HT: 0.15 ± 0.09). The suppression of glutamate-induced calcium levels by serotonin in the presence of TTX was significant compared with control responses (P < 0.001, n = 14), and the mean suppression (63%; Fig. 5B) was not significantly different from serotonin applied in the absence of TTX (61%, P > 0.05, t-test). Application of TTX alone did not change the baseline [Ca2+]i.
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DISCUSSION |
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We have investigated the cellular mechanisms responsible for the modulation of light-induced circadian behavioral responses by serotonin. We have shown that serotonin attenuates glutamate-induced increases in neuronal [Ca2+]i in cultured SCN neurons. The 5-HT1A/7 receptor agonist, (±)-8-OH-DPAT, mimicked the effects of serotonin, and the effects of 5-HT were antagonized by ritanserin, a 5-HT7/2/1C receptor blocker. Further evidence that the cAMP-activating 5-HT7 receptor pathway plays a role in the serotonin effect comes from the attenuation of glutamate-induced elevations of [Ca2+]i by both forskolin and 8-Br cAMP. Interestingly, in the presence of TTX, serotonin still suppressed calcium elevations.
When we compared the glutamate response between cells treated with and
without TTX, the glutamate responses consistently increased, suggesting
an alteration in the network dynamics of the culture. The most likely
candidate that was affected by blocking spike-mediated action
potentials was -aminobutyric acid (GABA) because GABA is the
predominant neurotransmitter in the SCN (Moore and Speh 1993
). SCN neurons in culture establish GABAergic synapses
(Chen and van den Pol 1997
), and the loss of
GABA-mediated inhibition between cultured SCN neurons may have resulted
in the increased glutamate responses. However, with TTX present in the
bath solution, we still observed the attenuation of glutamate
responses, thereby discounting the likelihood that GABA communication
is the primary mediator for the serotonergic effect. Overall, our TTX
data are consistent with the interpretation that, although
spike-mediated communication between SCN neurons in culture can
influence the amplitude of glutamate-induced calcium responses, it does
not mediate the effects we have observed with serotonin.
Although a smaller proportion of cells was responsive to 8-OH-DPAT, the
mean suppression was higher compared with that of serotonin. Although
8-OH-DPAT is an agonist for the 5-HT7/1A
receptor, serotonin as the endogenous ligand could act on additional
receptor types in the SCN (Roca et al. 1993). Despite a
selective difference between these compounds, both serotonin's and
8-OH-DPAT's overall effect on glutamate responses is inhibitory.
Additionally, the activation of the adenylate cyclase system also
decreases the glutamate responses. Both forskolin and 8Br-cAMP were
able to block the glutamate-induced calcium elevation with a similar
mean suppression.
Serotonergic agents have been shown to modulate light-induced circadian
responses at the level of the whole animal as well as in SCN tissue
slices (Glass et al. 1995; Miller and Fuller 1990
; Rea et al. 1994
; Selim et al.
1993
; Weber et al. 1998
). Although we must be
cautious in generalizing from in vitro results, our results show that
this modulation can occur in dispersed SCN neurons, suggesting the
possibility that convergence of the RHT and raphe input pathways might
occur on clock neurons, as well as the presynaptic terminals of the RHT
(Pickard et al. 1996
). Although our TTX experiments have
shown that spike-mediated cell communication in our low-density
cultures is not necessary for serotonin to modulate glutamate
responses, we cannot rigorously exclude the possibility that 5-HT could
be acting indirectly through alternate forms of cellular communication.
Neuronal communication through gap junctions is unlikely because
extensive screening has failed to reveal morphological or molecular
evidence for neuronal gap junctions in SCN cultures (Welsh and
Reppert 1996
). Serotonin-stimulated, nonspike-mediated release
of an unknown modulator cannot be ruled out by the current results;
however, the most straightforward interpretation of our experiments is
that a subset of SCN neurons express both glutamate and modulatory 5-HT
receptors. This is apparently the case for SCN glia. Imaging
SCN-derived astrocytes, Haak and van den Pol (1997)
,
have observed synergistic and additive interactions between glutamate
and serotonin on cellular calcium levels.
On the basis of previous findings, we propose the following working
hypothesis for how serotonin suppresses glutamate-induced calcium
influx in SCN neurons. Prosser et al. (1994) have shown that 5-HT can activate protein kinase A-dependent
K+ channels in SCN neurons. This would tend to
hyperpolarize SCN neurons, could inhibit calcium influx through the
depolarization-dependent N-methyl-D-aspartate
receptor channels and thus reduce the increase in cell calcium
stimulated by glutamate released from the RHT. In this way, 5-HT could
modulate the normal cellular events associated with light-induced phase
shifts of SCN neuronal activity. Alternately, 5-HT could modulate the
action of SCN neuronal glutamate receptors or calcium channels
directly, rather than through K+ channel modulation.
In the intact SCN, the 5-HT action on SCN neurons demonstrated here
likely works in concert with presynaptic effects to modulate light
entrainment. Presynaptic 5-HT1B serotonin
receptors are located on the terminals of the RHT, and activation of
these receptors inhibits light-induced phase shifts of behavioral
rhythm and expression of c-fos in the hamster (Pickard et al.
1996) and mouse (Pickard and Rea 1997
) SCN. Our
results suggest that a subpopulation of glutamate-responsive SCN
neurons also possess postsynaptic 5-HT receptors. That the convergence
of these two temporal signaling pathways may occur in neurons
postsynaptic to RHT input is further suggested by the
5-HT7/1A pharmacology of our responses. The
5-HT7 receptor is thought to be postsynaptic and
responsible for decreasing the neuronal firing of light-stimulated SCN
neurons in intact, anesthetized animals (Ying and Rusak
1997
). In our experiments, the ability of ritanserin to block
the effects of serotonin, as well as the ability of 8-OH-DPAT,
forskolin, and 8-Br cAMP to mimic serotonergic suppression of SCN
neuronal glutamate responses, support a role for the
5-HT7 receptor.
The proportion of cells whose glutamate-induced calcium elevations were
suppressed by either serotonin or 8-OH-DPAT was lower in our culture
experiments than has been reported in some other studies. In the
current study, serotonin suppressed glutamate responses in 31% of
neurons, whereas the proportion of serotonin-responsive cells in optic
nerve-stimulated SCN neurons and as measured by light-activated cell
firing is ~75% (Nishino and Koizumi 1977; Ying
and Rusak 1994
). However, previous studies were performed in
vivo, and investigators recorded from only the ventral portion of the
SCN, which is the major site of serotonergic input. In contrast, our
cultures included a heterogeneous population of SCN neurons.
Jiang et al. (1995)
have measured responses to 5-HT in
27% of neurons in SCN slice preparations, a proportion similar to the
serotonergic suppression we have observed. Taken together, these data
suggest that postsynaptic 5-HT receptors may exist on only a
subpopulation of SCN neurons, perhaps primarily retino-recipient cells.
The serotonergic input to the SCN is also an apparent site of aging in
the circadian system. Destruction of the serotonergic system in young
animals can alter photic entrainment of circadian rhythms
(Bradbury et al. 1997; Morin and Blanchard
1991
; Penev et al. 1993
) and mimic the altered
light responses seen in aged animals (Zee et al. 1992
).
Our study provides a cellular localization for 8-OH-DPAT responses onto
SCN neurons. One hypothesis consistent with our findings and those of
Penev et al. (1997)
is that a decline in the number of
8-OH-DPAT-responsive receptors on SCN neurons leads to the decline in
pharmacological responsiveness and the alterations in photic responses
observed with advanced age in rodents.
In summary, we observed that serotonin suppressed glutamate-induced calcium elevations in dispersed SCN neurons, and the 5-HT7 receptor is an element in this response. This further elucidation of the 5-HT mechanisms of the SCN provides additional means to understand the role of 5-HT in the regulation of SCN rhythms and their aging.
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ACKNOWLEDGMENTS |
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We thank Drs. Marilyn Duncan, Phyllis Wise, and Kristine Krajnak for comments on previous versions of the manuscript.
This work was supported by National Institute on Aging Grant AG-13426 to D. G. McMahon.
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FOOTNOTES |
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Address for reprint requests: D. G. McMahon, MS508 Medical Center, Dept. of Physiology, University of Kentucky, Lexington, KY 40536.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 July 1998; accepted in final form 1 April 1999.
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REFERENCES |
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