Serotonin sets the day state in the neurons that control coupling between the optic lobe circadian pacemakers in the cricket Gryllus bimaculatus
Department of Physics, Biology and Informatics, Faculty of Science and Research Institute for Time Studies, Yamaguchi University, Yamaguchi 753-8512, Japan
* Author for correspondence (e-mail: tomioka{at}po.cc.yamaguchi-u.ac.jp )
Accepted 18 February 2002
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Summary |
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Key words: cricket, Gryllus bimaculatus, circadian rhythm, medulla bilateral neuron, pacemaker coupling, photo-responsiveness, serotonin
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Introduction |
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Serotonin (5-hydroxytryptamine; 5-HT) is one of the major putative
neuroactive substances in the insect optic lobe
(Nässel, 1987). Lines of
evidence from various experimental approaches suggest that serotonin plays
important physiological roles in the circadian system of insects
(Page, 1987
;
Pyza and Meinertzhagen, 1996
;
Tomioka, 1999
). In crickets,
serotonergic neurons are distributed almost the entire area of the optic
lobe's lamina and medulla neuropiles, and the serotonin content in the optic
lobe fluctuates depending on the time of day, in synchrony with circadian
changes in the sensitivity of visual interneurons
(Tomioka et al., 1993
).
Administration of exogenous serotonin shifts the phase of the cricket's optic
lobe pacemaker in vitro in a phase-dependent manner and the
phaseresponse curve caused by a serotonin pulse is quite similar in
shape to that caused by mutual coupling between bilateral clocks
(Tomioka, 1999
). Taking all
these facts together, serotonin seems to be involved in the cricket's
circadian system. Since signals required for the mutual coupling are exchanged
through a neural pathway that consists of MBNs, and since serotonin shifts the
phase of the cricket's pacemaker, in this study we tried to reveal the
physiological effects of serotonin on the MBNs, using electrophysiological
techniques. Our results show that serotonin reduces the MBNs' electrical
activity in a dose-dependent and time-dependent manner, suggesting that
serotonin not only sets the day state in the MBN but also regulates mutual
coupling between the bilaterally paired pacemakers, by modulating the MBNs'
electrical activity and responsiveness to light.
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Materials and methods |
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Electrophysiology
An adult male cricket was first anesthetized with CO2. The
walking legs, antenna and wings were removed to minimize body movement. In
some cases, the sub-esophageal ganglion was also carefully removed with a fine
pair of forceps. The head of the animal was then fixed on a specially designed
plastic platform with a 1:1 mixture of beeswax and colophonium. A small square
piece of cuticle around the compound eye was cut with a fine razor knife and
opened to expose the optic stalk, a long nerve trunk connecting the lamina
medulla complex to the lobula. The optic stalk was then cleansed from
surrounding tissues and was subdivided into fine nerve filaments using a fine
needle. The separated optic stalk filaments were finally severed vertically
near the medulla. The cut end of a nerve filament from the ventral side of the
optic stalk was sucked firmly into an electrode filled with insect Ringer's
solution (Fielden, 1960). A
silver reference electrode was placed near the tip of the suction electrode.
The cavity of the head capsule was sealed with petroleum jelly to prevent
desiccation. During the preparation, the nervous tissues were not perfused but
were bathed in a small amount of Ringer's solution. A small hole was also made
on the head capsule near the contralateral eye to allow injection into the
contralateral optic lobe (see Fig.
1). Electrical signals from the suction electrode were amplified
by an amplifier (Nihon Kohden, AVB-9) and displayed on an oscilloscope (Nihon
Kohden, VC-9). Signals were then fed into a computer (IBM, 300GL) via
an A/D converter (Cambridge Electronic Design Limited, 1401 Plus). Data were
analyzed using Spike-2 software (Version 3, Cambridge Electronic Design
Limited).
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To examine the effects of serotonin on retinal photoreceptors, simultaneous recording of the electrical activity of the optic stalk and electroretinogram (ERG) was made. ERGs were obtained using a glass electrode filled with Ringer's solution. The active electrode was positioned just beneath the surface of the cornea and a silver wire reference electrode was placed in the hemocoel of the head capsule through a small hole made with a fine needle. To elicit the ERG, light stimuli of 500 ms duration were delivered to the compound eye through a light guide. Responses were amplified by a high-gain amplifier (Tektronix, TM502A), displayed on an oscilloscope and recorded as above. The amplitude of the ERG was measured as the excursion from the baseline to its peak. In all cases, after fixing the electrode the animal was kept in the dark for at least 30 min before photic stimulation.
Photic stimulation
To examine the intensity-dependence of light-evoked responses of MBNs,
1000ms light flashes of varying intensities were delivered to the compound eye
through a 3.5mm diameter plastic light guide. A slide projector (ELMO, CS II)
equipped with a 150 W lamp (Philips, KP-8) was used as a light source. Light
from the slide projector was focused on one end of the light guide and the
opposite end was placed close to the compound eye. Light pulses with various
intensities were regulated by an electric shutter controlled by a stimulator
(Nihon Kohden, SEN-3210). Neutral density filters (Shonan Kogaku Co.) ranging
from 10 to 50% were placed between the shutter and the light source to
attenuate light pulses to different intensities. The maximal light intensity
(logI=0) at the surface of the compound eye was 0.4 mW
cm-2, as measured by an optical power meter (UDT instruments, Model
371).
Microinjections
Serotonin (creatinine sulfate, Sigma), a nonspecific agonist of serotonin
receptors, quipazine dimaleate (RBI), and a nonspecific antagonist of
serotonin receptors, metergoline phenylmethyl ester (Tocris Cookson), were
used in this experiment. Serotonin and quipazine were dissolved in Ringer's
solution to the desired concentrations (0.1 mmol l-1, 1
mmoll-1 and 10 mmoll-1). Metergoline was first dissolved
in dimethyl sulphoxide (DMSO) (Sigma) and further diluted with Ringer's
solution to make working solutions of the desired concentration. Injections
were made during the day (at approximately 13:00 h) and at night
(approximately 21:00 h) into the medulla area of the optic lobe using a glass
micropipette equipped with a nanoliter injector (WPI, A203XVY) mounted on a
micromanipulator (Narishige, M-3333). The volume of injected solution was
estimated by measuring the diameter of a droplet injected into mineral oil
under a microscope. The volume for single injections was 8.47-12.76 nl
(mean±S.D.=10.02±1.5 nl). The micropipette tips were checked
before and after injections in order to ensure that the injection was
performed successfully. The same volume of Ringer's solution was injected as a
control.
Data analysis
To determine the effects of injected chemicals on neural activity, the
number of spikes during the light pulse was compared before and after the
injection. The light-evoked response was estimated by subtracting the number
of spikes occurring during the 1000 ms period just before the light pulse from
that during the 1000 ms light pulse itself. Comparisons were made using the
equation: SI=100x(B-A)/B, where SI
is the suppression index and A and B represent the total
number of spikes induced by a series of light pulses with intensities of
logI=0 to -8 before (B) and after (A) injection
with chemicals. Paired or unpaired Student's t-tests were used, where
applicable, to determine the statistical significance of differences. The
number of animals (N) used for each recording is indicated.
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Results |
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A 1000 ms light pulse given to the contralateral eye induced a burst
discharge in the brain efferents. Fig.
2 shows a representative result obtained during the day. The
light-evoked response is practically entirely from the MBNs, since it has been
shown that cutting the tract in which MBNs run resulted in a loss of
light-evoked response (Tomioka et al.,
1994). It gradually increased with an increase in light intensity,
yielding an intensityresponse curve
(Fig. 3). The smallest response
was detected at an intensity of logI=-8 and reached a maximum at
logI=-1 (Figs 2,
3). With higher intensities,
light-evoked discharges were prominent even after the light pulse
(Fig. 2). There was a clear
day/night difference in the magnitude of the light-evoked responses, which
were significantly greater during the night than the day (t-test,
P<0.01); the light intensity evoking the half-maximal response
shifts from logI=-3.7 in the day to logI=-4.7 at night
(Fig. 3).
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Effects of serotonin on spontaneous and light-induced responses
Different doses of serotonin (1.0, 10 and 100 pmol in 10 nl) were injected
into the contralateral optic lobe to reveal its effect on the brain efferents
including MBNs during both day and night. In the control experiments, the same
amount of Ringer's solution was injected into the optic lobe. No changes were
observed in either spontaneous or light-induced responses following Ringer's
solution injection (Fig. 4).
The reduction of spontaneous activity in control experiments was
-2.6±4.9% (mean ± S.E.M., N=6) and 5.5±7.4%
(N=8) for day and night, respectively. The suppression index
(SI) for the light-induced response was 1.4±2.3 (mean ±
S.E.M., N=6) and 5.2±1.3 (N=8) for day and night,
respectively.
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Microinjection of serotonin into the optic lobe clearly suppressed the spontaneous activity of the brain efferents in the optic stalk (Fig. 2). This inhibitory effect of serotonin was dose dependent during both day and night: greater suppression occurred with larger doses of serotonin. A significant reduction of spontaneous discharge rate occurred at 10 pmol both for the day and night (t-test, P<0.05, compared with that of controls) (Fig. 5A). The spontaneous activity reduced by serotonin was further reduced significantly by severing the contralateral optic stalk (t-test, P<0.01) (Fig. 5B). Removal of the contralateral optic lobe also significantly reduced the spontaneous firing rate (t-test, P<0.01) (Fig. 5B). When serotonin was injected into the brain following the removal of the optic lobe, the firing rate increased by 26% relative to the optic-lobe-removed value but this increment was not statistically significant (t-test, P>0.05, N=5) (Fig. 5B).
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Serotonin exerted a pronounced suppression not only on the spontaneous activity of MBNs but also on their light-evoked response (Fig. 2). Fig. 6A,B shows average intensityresponse curves before and after injecting 10 pmol of serotonin. Serotonin shifts the intensityresponse curve downwards. This inhibitory effect was both time and dose dependent, with greater suppression during the night and at higher dosage. A significant suppression of the light-induced response was observed at 1.0 pmol both during the day and at night (t-test, P<0.01, compared with the control). The SIs were significantly greater during the night by 25-40 % than those during the day for all dosages used (t-test, P<0.01) (Fig. 6C), indicating that the sensitivity of MBNs to serotonin during the light phase was lower than that during the dark phase. Although the photo-responsiveness of MBNs was almost immediately suppressed by injected serotonin during both day and night, it recovered gradually with time. The recovery occurred faster during the night than during the day: as shown in Fig. 7, it took only 30 min to recover by 100 % from serotonergic suppression during the night, while complete recovery was not observed even 90 min after the injection during the day. Spontaneous firing frequency returned to the initial level within 30 min after injection at either time, however.
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Effects of serotonin on the electroretinogram
To examine whether the suppression of the light-evoked response by
serotonin is attributable to reduced sensitivity of the retina, the amplitude
of the electroretinogram (ERG) was compared before and after injection of
serotonin into the optic lobe. The ERGs recorded were simple cornea negative
waves (Fig. 8A,B), which were
quite similar to those reported for Teleogryllus commodus
(Rence et al., 1988). The
waveform did not change even after cutting the optic nerve
(Fig. 8A), suggesting that the
ERG originated from the retinal cells. The amplitude of the ERG during the
night (Fig. 8D) was
approximately three times that during the day
(Fig. 8C). Little change in the
ERG waveform and amplitude was observed after the injection of serotonin
during either the day or the night (Fig.
8B-D). Similar results were obtained when serotonin was injected
directly into the retina, suggesting that the inhibitory effects of serotonin
on brain efferents do not result from changes in sensitivity of the
photoreceptors, but are probably attributable to changes in responsiveness of
visual interneurons.
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Effects of serotonin-receptor agonist and antagonist on spontaneous
activity and light-induced responses
The effects of a nonspecific agonist of serotonin receptors, quipazine,
were also examined. Quipazine injected into the optic lobe caused a
dose-dependent inhibition of both spontaneous activity and the light-induced
response of the MBNs. This effect was greater during the night
(Fig. 9A,B) and clearly dose
dependent (Fig. 9C). The effect
of 100 pmol of quipazine was approximately equivalent (t-test,
P>0.05) to that of 1.0 pmol of serotonin.
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The application of a nonspecific serotonin-receptor antagonist, metergoline, caused no significant alteration in MBN activity or photosensitivity at dosages of 1.0 and 10 pmol: the SI values for 1.0 and 10 pmol metergoline during the day were -0.5±4.5 (mean ± S.E.M., N=2) and -2±4.8 (N=4), respectively. However, at 100 pmol a substantial increase of both spontaneous and light-induced activity was observed during both the day and night. The spontaneous activity was increased by 56±7.3 % (N=5) during the day and by 34±6.4 % (N=4) during the night, while the SI values during the day and night were -35±6.0 (N=5) and -19±4.5 (N=6), respectively. These changes were statistically significant compared with those of controls (t-test, P<0.01). Unlike serotonin and quipazine, the effect of metergoline was greater during the day than at night (Fig. 10A,B). When injected together with serotonin, metergoline effectively antagonized the effects of serotonin on MBN photosensitivity (Fig. 10C-E).
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Discussion |
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Injection of serotonin into the optic lobe resulted in a reduction of the
spontaneous activity in the brain efferents. When serotonin was injected into
the brain after removal of the optic lobe, the spontaneous activity of the
brain efferents increased. These results suggest that reduction of spontaneous
impulse activity induced by the injection of serotonin occurred within the
optic lobe. It is also consistent with the fact that a large number of
serotonergic neurons are located in the optic lobe
(Nässel, 1987; K. T. and
S. Tamotsu, unpublished data).
Serotonin regulates the responsiveness of MBNs
The present study demonstrates that photo-responsiveness of the MBNs was
suppressed in a dose-dependent and timedependent manner by serotonin as well
as by its non-specific serotonin-receptor agonist, quipazine, when injected
into the optic lobe. The injected volume of serotonin solution in our
experiment was one-tenth of that used to induce morphological change in the
fly's optic lobe interneurons (Pyza and Meinertzhagen, 1995), but larger than
that used to examine its effects in the honeybee visual system
(Erber and Kloppenburg, 1995;
Kloppenburg and Erber, 1995
).
It has been reported that only 0.5 nl of 10-5 mol l-1
(0.005 pmol) serotonin can effectively reduce the honeybee's
direction-specific antennal responses to stripe pattern movements
(Erber and Kloppenburg, 1995
)
and that less than 1 nl of 10-5 mol l-1 (<0.01 pmol)
serotonin modulates the response properties of motion-sensitive neurons
(Kloppenburg and Erber, 1995
).
Although we did not attempt injection of such a small amount of serotonin
solution, we can estimate the minimum effective dosage by extrapolation of the
serotonin doseresponse curve for photo-responsiveness
(Fig. 6C). The estimated
minimum effective dosage at night is less than 0.01 pmol which is quite
similar to the value used for honeybees.
Since serotonin injected into the optic lobe or retina had little effect on
the amplitude of the ERG, it is most probable that the suppression of MBN
responsiveness by serotonin occurs within the optic lobe. The fact that
application of serotonin induces morphological changes in interneurons in the
fly's optic lobe (Meinertzhagen and Pyza,
1996; Pyza and Meinertzhagen,
1996
) is consistent with this hypothesis. Our finding that no
apparent changes were observed in ERG amplitude after serotonin injection is
inconsistent with the report by Chen et al.
(1999
) for blowflies
Calliphora erythrocephala, in which serotonin injected into the
retina reduced the amplitude of the sustained negative component of the ERG,
which is derived at least partly from photoreceptor responses. There might be
species specificity on the action of serotonin in the retina.
Involvement of serotonin in the visual system has been reported for a wide
variety of animals including insects. Serotonin changes the sensitivity of
retinal photoreceptors in Aplysia californica
(Jacklet, 1991) and the locust
(Cuttle et al., 1995
). The
sensitivity of visual interneurons in the optic lobe of crickets
(Tomioka et al., 1993
;
Tomioka, 1999
) and in the
brain of bees Apis mellifera
(Kloppenburg and Erber, 1995
)
is suppressed by serotonin. Serotonin also induces morphological changes in
the fly's optic lobe interneurons to mimic a day state, which may be reflected
in the change in responsiveness of the interneurons
(Meinertzhagen and Pyza, 1996
;
Chen et al., 1999
). These facts
suggest that serotonin is a common substance regulating the sensitivity of
visual system in various ways and in a wide variety of animals.
Time of serotonin release
The present study revealed that serotonin suppressed MBN responsiveness to
light during both the day and night, but that the amount of suppression was
greater at night with the same dosage. Recovery from the suppression also
occurred faster during the night. The greater suppression and faster recovery
during the night suggest that less endogenous serotonin is released at this
time. This possibility is supported by the finding that application of a
non-selective serotonin-receptor antagonist, metergoline, increased the MBNs
sensitivity to light during both day and night with greater effects during the
day. The increased sensitivity is consistent with the suppression of
endogenous serotonergic action by metergoline. Serotonin content in the optic
lobe fluctuates during the day/night cycle: it is high at night but
substantially reduced during the day
(Tomioka et al., 1993),
suggesting that serotonin may be related to the circadian clock system in the
cricket optic lobe. The larger content during the night could reflect a
reduced rate of serotonin release in the optic lobe
(Tomioka et al., 1993
),
although assessment of this release in vivo would be required to
confirm this assumption. Nevertheless, the data so far support the idea that
serotonin is released during the day and sets the day state in the optic lobe
interneurons.
Possible involvement of serotonin in mutual coupling between optic
lobe pacemakers
It has been shown in a wide variety of animals that serotonin shifts the
phase of the circadian clock: application of serotonin during the early
subjective night causes, for example, a phase delay in the cockroach optic
lobe circadian pacemaker (Page,
1987), whereas in Aplysia californica and the cricket
G. bimaculatus, it induces a phase delay during the subjective night,
but phase advances during the subjective day
(Koumenis and Eskin, 1992
;
Tomioka, 1999
). Since the
phaseresponse curve for serotonin is quite similar to that caused by
mutual entrainment between the two optic lobe pacemakers, serotonin is thought
to be a candidate molecule working in the coupling pathway in the cricket
G. bimaculatus (Tomioka,
1999
).
In addition to its phase-shifting effect, the present study demonstrated
that serotonin affects coupling less directly, by suppressing the sensitivity
of MBNs to light. It has been suggested that coupling signals between optic
lobe pacemakers are mediated by MBNs
(Yukizane and Tomioka, 1995;
Tomioka and Yukizane, 1997
).
Immunohistochemical labeling with anti-serotonin antibodies revealed that the
MBNs were not serotonergic themselves. Serotonin is somehow released by
serotonergic neurons under the regulation of the pacemaker and MBNs. Probably,
released serotonin in one optic lobe shifts the phase of the pacemaker on that
side in a phase-dependent manner and, at the same time, reduces the coupling
signals by suppressing the activity of MBNs, keeping, in turn, the phase of
the contralateral pacemaker stable (Fig.
11).
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The phase shifts caused by serotonin in a compound eye and optic lobe
complex isolated and kept in vitro were much larger than those
expected from the phaseresponse curve for the mutual coupling obtained
from the interaction between the experimentally dissociated pacemakers
(Tomioka et al., 1991;
Tomioka, 1993
,
1999
). Although one may
attribute this difference to the rather high concentration of serotonin used
in the phase-shifting experiment (Tomioka,
1999
), the regulation of MBN sensitivity by serotonin appears to
contribute, keeping the phase shifts in a smaller range than that in which the
phase difference between the bilateral pacemakers is normally kept.
The role of serotonin in the circadian system of many other animals has
been discussed extensively. In mammalian species, it is suggested that
serotonin is released during the night in the clock tissue, suprachiasmatic
nuclei, by the increased activity of Raphé neurons which has been
linked to arousal state, with increased Raphé neuron activity
associated with increased arousal (Prosser
et al., 1993). The activity of the serotonergic system is thought
to synchronize the pacemaker to social or behavioral changes in the
environment (Medanic and Gillet,
1992
). In Aplysia californica, the ocular circadian
pacemaker in the eye receives efferent innervation from the cerebral ganglion
of which the putative neurotransmitter is serotonin
(Takahashi et al., 1989
). The
serotonergic system is hypothesized to have two roles: carrying coupling
information from the contralateral ocular pacemaker or from oscillators
located in the central nervous system
(Lickey et al., 1983
);
mediating the effects of an extraocular light pathway on the oscillator in the
eye (Colwell, 1990
). Taking
these implications together with our present results, it is apparent that the
serotonergic system is commonly involved in the efferent regulatory pathway of
the circadian pacemaker. Our results further suggest that the serotonergic
system modulates the efferent coupling pathway to set its day state.
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Acknowledgments |
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