Regulation of serotonin levels by multiple light-entrainable endogenous rhythms
1 Department of Biological Sciences, Wellesley College, Wellesley, MA 02481,
USA
2 Universität Ulm, Neurobiologie, D-89097, Ulm, Germany
* Author for correspondence (e-mail: bbeltz{at}wellesley.edu)
Accepted 15 July 2004
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Summary |
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Key words: Homarus americanus, Crustacea, neurogenesis, 5-hydroxytryptamine, central nervous system
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Introduction |
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In the present study, we have asked whether serotonin levels in the lobster
brain are under circadian control. We are interested in this question because
serotonin regulates a variety of functions in crustaceans
(Beltz and Kravitz, 2003).
Among these actions, serotonin increases transmitter release from both
excitatory and inhibitory nerve terminals and enhances the contractility of
muscle fibers (Dudel, 1965
;
Florey and Florey, 1954
;
Glusman and Kravitz, 1982
),
modulates the sensitivity of sensory neurons
(Pasztor and Macmillan, 1990
),
increases the frequency and intensity of the heartbeat
(Battelle and Kravitz, 1978
;
Florey and Rathmayer, 1978
)
and serves as a modulator of segmental reflexes in the walking system
(Gill and Skorupski, 1996
).
Because serotonin plays so many roles, it is important to understand the
factors that influence its synthesis and availability.
Serotonin has also been implicated as a regulator of life-long neurogenesis
in the lobster brain (Benton and Beltz,
2001; Beltz et al.,
2001
), and the generation of new neurons in this system is under
circadian control, with a peak rate at dusk. This finding may be correlated
with the habits of lobsters, which are most active around dusk
(Weiss, 1970
;
Cooper and Uzmann, 1980
;
Chabot et al., 2001
). This
rhythm of neuronal proliferation is regulated by an endogenous oscillator that
is entrained by the light:dark (L:D) cycle
(Goergen et al., 2002
). We
have proposed that light may provide a dominant coordinating signal for the
many factors, including serotonin, that influence the persistent generation of
new neurons. If this hypothesis is correct, then serotonin levels should cycle
over a
24-h period, be entrainable by light and maintain a fixed phase
relationship with the rhythm of neurogenesis.
In the present study, we measured serotonin levels at various times of day in the brains of lobsters that were maintained in several different light regimes. These studies show that serotonin levels undergo circadian variations that are light entrainable and controlled by an endogenous clock. We also examined serotonin levels independently in functionally distinct brain regions (Fig. 1) and show that the serotonin rhythms observed in these areas have contrasting phases, each of which is light entrainable. This indicates that separate mechanisms control serotonin metabolism in different parts of the brain, with light as a common zeitgeber. These findings are important because they suggest that serotonin may be involved in the light-activated cascade of events that culminates in the circadian regulation of a variety of physiological functions in crustaceans, including the rate of neuronal proliferation in the brain.
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Materials and methods |
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All animals were entrained to a 12 h:12 h L:D cycle for a minimum of 2 weeks. The light intensity at the surface of the water was 611 µEinsteins m2 s1. Groups of lobsters were exposed to four different experimental protocols, all based on a 12 h:12 h L:D schedule, and brain levels of serotonin were then assessed over a 24-h period by high-performance liquid chromatography (HPLC). Group 1 was exposed to the 12 h:12 h L:D cycle. Group 2 was exposed to the same L:D cycle as Group 1 but, following the 12 h:12 h L:D exposure (entraining light regime), animals were transferred to constant darkness for 3 days without food (D:D conditions). Group 3 was exposed to an L:D regime that was phase-shifted by 4 h relative to Group 2, followed by D:D for 3 days prior to HPLC measurements. A fourth group of animals (Group 4) was subjected to the same light regime as Group 2, but in these animals the olfactory lobes (OLs), accessory lobes (ALs) and the brain remainder were separated from one another and the individual areas then assayed for serotonin content.
Changes in brain serotonin levels measured in Group 1 (L:D) did not show a
clear diurnal rhythm and were not repeatable over eight trials (see
Discussion). All HPLC data reported in this paper, therefore, are from animals
in Groups 2, 3 and 4, which were first entrained to an L:D regime and then
exposed to 3 days of D:D prior to HPLC assessments. Exposure to constant
darkness prior to HPLC measurements was aimed at preventing possible masking
effects of sudden intensity changes in light, and of feeding, on serotonin
release and also demonstrated that the rhythms observed are endogenous
(Chiu et al., 1995). The
phase-shift in the conditioning light regime for Group 3 relative to Group 2
tested whether serotonin levels are light entrainable.
Dissection
Following the entraining light regime and 3 days in D:D, four animals from
each of Groups 24 were assayed for serotonin levels by reverse-phase
HPLC every 4 h over a 24-h period. Animals were retrieved from their
containers using night-vision goggles (Bushnell Corporation, Overland Park,
KS, USA) and placed on ice in the dark. The eyestalks were quickly removed in
dim light in order to minimize light activation of serotonergic pathways.
Brains were then dissected in cold lobster saline [462 mmol
l1 NaCl, 16 mmol l1 KCl, 34 mmol
l1 CaCl2, 17 mmol l1
MgCl2, 11 mmol l1 -D(+)-glucose
and 10 mmol l1 Hepes buffer (pH 7.4)].
HPLC methods
Following dissection, tissues were transferred to microcentrifuge tubes
containing 50 µl of 0.1 mol l1 perchloric acid. Samples
were diluted with 200 µl of mobile phase [20 mmol l1
anhydrous monobasic sodium phosphate, 1.85 mmol l1
heptanesulfonic acid sodium salt, 0.27 mmol l1 anhydrous
EDTA and 16% MeOH:4% acetonitrile (v/v) as organic modifiers (Sigma, St Louis,
MO, USA)] and homogenized manually with a pestle. The final solvent buffer was
adjusted to pH 3.25 with concentrated phosphoric acid. Homogenates were
transferred into Eppendorf tubes containing a 0.45 µm filter insert (VWR,
West Chester, PA, USA) and centrifuged for 15 min at 20 000 g
and 21°C. The clear supernatants were transferred into autosampler
microvials [Bioanalytical Systems Inc. (BAS), West Lafayette, IN, USA] and
sealed with Teflon caps. 10 µl samples were applied to a C18 reverse-phase
column (Alltech Associates Inc., Deerfield, IL, USA; 3 µm, 100x4.6
mm) via a BAS autosampler (Samplesentinel). Eluted compounds were
detected electrochemically with a BAS liquid chromatography system consisting
of a CC-5 liquid chromatography module, a PM 80 solvent delivery system and an
LC 4C amperometric detector. The detector potential was set at 625 mV and the
detection limit was in the range of 2 nA. Recovery rates were close to 100%
and no further corrections were applied. An A/D converter (A/D Instruments
Inc., Colorado Springs, CO, USA) and a strip-chart program with integrated
chromatography software (A/D Instruments Inc.; PowerChrom version 2.2.4) were
used to measure peak amplitude (Fig.
2).
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HPLC data analysis
The data for the chronograms were obtained from serially independent
measurements of serotonin levels in groups of animals killed and assayed at
each time interval. A requirement for serially independent measurements of
this nature is that the individuals in the groups are as similar to one
another, and to those in the other groups, as possible. To ensure this
equality, we used animals that had been reared under the same conditions of
temperature and nourishment and were as close in size as practically possible.
The mean body length (rostrum to telson) of all animals that were used in the
results presented here was 4.49±0.056 cm (mean ±
S.E.M.; N=72). Furthermore, in a
previous study we found that the volume of the lobster brain is linearly
correlated with body volume for the size range that we used
(Helluy et al., 1995). Given
that the individual animals, and therefore their brains, were very similar in
size, we were able to simply use the serotonin content in picomoles per brain
in the chronograms without normalizing these values to brain mass or
dimensions, which were very difficult to assess for these small tissue
volumes. When normalization of values to tissue mass was attempted, standard
errors became larger, indicating that the normalization procedure was
introducing additional variability into the results. Raw data are therefore
reported for all results.
Immunocytochemistry
Methods for processing brains as whole mounts for serotonin
immunocytochemistry were taken from Beltz and Burd
(1989). Juvenile lobsters were
maintained either on a 12 h:12 h L:D cycle for at least two weeks (Group 1
protocol) or on a 12 h:12 h L:D entraining light regime followed by 3 days in
D:D (Group 2 protocol). Animals were retrieved from their containers, placed
on ice in the dark, and the eyestalks removed in dim light. Brains were then
dissected at 3 h prior to subjective dawn (N=4) and 3 h prior to
subjective dusk (N=4) and fixed in cold 4% paraformaldehyde for
18 h.Tissues were then rinsed five times in 0.1 mol l1
phosphate buffer (PB; pH 7.4) and incubated in 0.1 mol l1 PB
with 0.3% Triton X-100 (PBTx) for 45 min, followed by incubation in rabbit
anti-serotonin antibody (1:1000; DiaSoren, Stillwater, MN, USA) for 48 h at
4°C. Following six rinses in PBTx, goat anti-rabbit Alexa 488 (1:50;
Molecular Probes, Eugene, OR, USA) was applied for 36 h, after which samples
were rinsed six more times in PB. Preparations were mounted in Gel Mount
(BiØmeda Corp., Foster City, CA, USA) and visualized using a Leica TCS
SP confocal microscope (Leica Microsystems, Germany).
In order to assess relative levels of serotonin immunoreactivity in the samples, all brains were screened during a single session and the initial laser and filter settings on the confocal microscope were maintained at the same levels throughout the entire analysis. Leica confocal quantitative software (version 2.0) was used for semi-quantitative analyses of the intensity of serotonin labeling in the OLs and ALs. These measurements were done by comparing the intensity of fluorescence (serotonin immunoreactivity) in the OLs and ALs for each time point, relative to background levels in the brain. A t-test was used for statistical analysis (SPSS Inc., Chicago, IL, USA).
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Results |
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Serotonin levels in the OLs and ALs
Experiments were also conducted to determine whether the OLs and ALs, two
midbrain regions with distinct functions and intense serotonergic innervation,
individually exhibit rhythmic changes in serotonin levels. The OLs in Group 2
animals show a clear rhythm in serotonin levels
(Fig. 5A,D), with a peak before
dusk and a sharp decline at dusk to a level that is maintained throughout the
subjective night phase and into the subjective daytime hours. The most
distinctive aspect of the OL serotonin rhythm is the peak that occurs prior to
subjective dusk, a feature that is also evident in the fluctuation of
serotonin content in the whole brain (Fig.
3). A similar pattern in serotonin levels was measured in the
brain remainder (Fig. 5B,E),
which was comprised of the protocerebral and tritocerebral areas and medial
deutocerebral regions such as soma clusters 9 and 11, the lateral antennular
neuropil and the olfactory globular tract (see
Fig. 1). The ALs, however, show
a rhythm in serotonin levels that is distinct from that of the OLs and the
brain remainder. High levels of serotonin were measured in the ALs beginning
in the hours before dusk, as in the OLs; however, high levels of serotonin
were also measured throughout the night, with a drop at subjective dawn
(Fig. 5C,F).
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If the levels of serotonin measured in each one of the OLs, ALs and the
brain remainder are all added together for each of the six time points, the
result provides a measure of the relative proportions of serotonin found in
the different brain regions over time (Fig.
6). These histograms illustrate that the serotonin content in the
OLs and ALs accounts for 70% of total brain levels. As in the whole-brain
studies (Fig. 3), the pre-dusk
peak is the most striking feature of Fig.
6. Examination of the components of this pre-dusk peak shows that
serotonin levels in the ALs rise by 18%, the OLs by greater than twofold
(123%), and the brain remainder by roughly threefold (197%) over the pre-dawn
values. These sharp increases in serotonin coincide with the period of arousal
and increased activity as lobsters undertake their nocturnal foraging and
social activities.
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Immunocytochemistry
We tested whether the brain serotonin levels measured by HPLC are also
detectable immunocytochemically. Whole mounts of brains dissected from animals
reared in the same conditions as Groups 1 and 2 and killed 3 h before
subjective dawn and dusk (the times when the serotonin trough and peak were
measured in the OLs and ALs by HPLC, respectively; see
Fig. 5) were used for these
studies. Densitometric measurements of the intensity of serotonin labeling in
the confocal images reveal detectable differences between the pre-dawn and
pre-dusk time points of both the Group 1 (L:D) and Group 2 (D:D) animals
(Table 1). The intensity of
labeling in the Group 1 brains was highly variable. Therefore, although the
means for pre-dawn and pre-dusk brains are different, the standard errors are
large and the differences are not statistically significant (see
Table 1, L:D). For the Group 2
(D:D) brains dissected at pre-dusk (15:00 h), we detected a more intense
labeling of the OLs and ALs than in the same regions of the brains of pre-dawn
(03:00 h) lobsters. Statistical analyses of the densitometric measurements for
pre-dawn vs pre-dusk ALs (P=0.050) and pre-dawn vs
pre-dusk OLs (P=0.042) indicated significant differences. Taken
together with the HPLC measurements, the immunocytochemical measurements
confirm that the levels of serotonin in the ALs and OLs of lobster brain
fluctuate rhythmically, being relatively low before dawn and high before
dusk.
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Discussion |
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In other crustaceans such as the fiddler crab Uca pugilator and
the crayfish Procambarus clarkii, fluctuating serotonin levels have
mostly been described for eyestalks
(Fingerman and Fingerman,
1977; Fingerman et al.,
1978
; Escamilla-Chimal et al.,
2001
; Fanjul-Moles and
Prieto-Sagredo, 2003
). Castanon-Cervantes et al.
(1999
) are the only authors to
our knowledge who have assessed serotonin levels over 24 h in the brain of
another crustacean. These authors have shown that in post-embryonic P.
clarkii, brain serotonin levels show a bimodal rhythm with a major peak
at night and a minor peak during the day when held in constant light. A
trimodal rhythm becomes evident in adults, reflecting an endogenous rhythm
peaking every 8 h (Castanon-Cervantes et
al., 1999
).
The lack of a clear diurnal rhythm in serotonin levels in lobsters
maintained in 12 h:12 h L:D conditions (see Materials and methods) was
contrary to the reports cited above. We reasoned that masking, a situation
where an endogenous rhythm is obscured by transient changes in the molecule
being measured due to direct stimulation by light-activated neural pathways,
was a potential cause of our result. Alternatively, serotonin content in
different parts of the brain may cycle at different times and thus obscure a
distinct single rhythm. This is one possible interpretation of the trimodal
rhythm reported in adult crayfish by Castanon-Cervantes et al.
(1999).
Constant darkness reveals an endogenous, light-entrainable, diurnal serotonergic rhythm
To exclude the influence of masking from our study, lobsters were entrained
to a 12 h:12 h L:D cycle for at least 2 weeks (the entraining light regime)
and then introduced to D:D conditions where no direct light stimulation or
feeding occurred. At the end of the third day in D:D, serotonin levels were
measured at six time points during a 24-h period. Using this D:D protocol, an
endogenous diurnal serotonergic rhythm was revealed
(Fig. 3). The most reliable
characteristics of this whole-brain rhythm are a pre-dusk serotonin peak, with
a decrease in serotonin levels to a pre-dawn serotonin trough. Altering the
light cycle for a group of lobsters so that light-off was shifted by 4 h (to
occur at 14:00 h clock time; light-on at 02:00 h clock time; see
Fig. 3B,C) had the effect of
shifting the serotonin peak and trough accordingly, showing that the
characteristic components of the whole-brain rhythm are light entrainable
(Fig. 3C).
A visual inspection of the chronograms in
Fig. 3 showed that they are not
sinusoidal, and hence analysis with the single cosinor is inappropriate
(Nelson et al., 1979;
Reinberg and Smolensky, 1983
;
Minors and Waterhouse, 1988
;
De Prins and Waldura, 1993
),
excluding a mathematical dissection of the chronograms to reveal sub-rhythms
hidden within them. However, due to the highly modular construction of the
crustacean brain, we could physically dissect the brains into three regions:
the OLs, the ALs and the brain remainder (medial regions of the protocerebrum,
deutocerebrum and tritocerebrum), all of which contain measurable levels of
serotonin.
Serotonergic rhythms in the olfactory and accessory lobes
Regional separation of the brain into the OL, AL and brain remainder and
assaying these individually reveals that not only are the changes in serotonin
levels in these areas out of phase with one another but also that the nature
of the specific patterns of these changes is characteristically different. The
changes in the serotonin levels in the OL and the brain remainder, for
example, are abrupt. That is, the levels of serotonin in these areas of the
brain during the interval preceding the peak are clearly different from the
levels at the peak itself (paired t-test; OL, P=0.016; brain
remainder, P=0.0002). Serotonin levels then decline in an exponential
fashion (Fig. 5D,E). By
contrast, a comparison of these two measurements in the ALs shows that they
change slowly and monotonically (paired t-test; AL, P=0.242;
Fig. 5F). The combination of
the gradual rise and extended plateau of serotonin level in the ALs with the
peaks and troughs of the serotonin levels in the OLs and brain remainder
explains the relative flatness of the whole-brain chronogram and histogram
(Figs 3A,
6). If we assume that changes
in serotonin levels in the brain are indicative of changes in the releasable
pool of this transmitter, then availability of serotonin in the OL and the
brain remainder changes in a pulse-like fashion in comparison with the AL.
The contrasting diurnal rhythms in serotonin levels that were measured in
the OLs, ALs and brain remainder (Fig.
5) are of particular interest because these regions are
functionally distinct. The paired ALs in crustaceans receive no primary
sensory input but rather receive projections of local interneurons in clusters
9 and 11 that carry higher-order visual, mechanosensory and olfactory
information (Sandeman et al.,
1995,Sandeman et al.,
1995
; Wachowiak et al.,
1996
; J. M. Sullivan and B. S. Beltz, unpublished results). The AL
output is carried by the axons of the cluster 10 projection neurons, which
continue to proliferate throughout the animal's life
(Harzsch et al., 1999
;
Benton and Beltz, 2002
) and
which project to the hemiellipsoid bodies located in the lateral protocerebrum
(Sullivan and Beltz, 2001
).
This connectivity pattern and the fact that multimodal inputs project to this
region suggest that the ALs are involved in higher-order integration
(Sandeman et al.,
1995
,Sandeman et al.,
1995
; Sullivan and Beltz,
2001
). The OLs, on the other hand, are innervated by olfactory
receptor neurons from the first antennae (antennules). Their output, like that
of the ALs, is carried by the axons of cluster 10 neurons that project to the
lateral protocerebrum via the olfactory globular tract
(Fig. 1). However, the
olfactory projection neurons are different from those that innervate the ALs
in that they target neuropil regions of the medulla terminalis. Therefore, the
output pathways from the OLs and ALs project to separate, largely
non-overlapping regions of the lateral protocerebrum
(Sullivan and Beltz, 2001
),
further evidence of the distinctive functions of these regions.
While the connectivity and functions of the OLs and ALs are distinct, both
regions nevertheless receive a massive serotonergic innervation from the same
neuron the ipsilateral dorsal giant neuron (DGN; Sandeman and
Sandeman, 1987,
1994
;
Benton and Beltz, 2001
). The
inputoutput relationships of the DGN in these regions are not known,
but in both regions this neuron innervates each and every glomerulus
(Benton and Beltz, 2001
). The
OLs and ALs are also innervated by relatively few, smaller serotonergic
interneurons whose cell bodies are located in clusters 9 and 11
(Beltz, 1999
). However, as the
predominant serotonergic input to both areas is from the DGN, our results
suggest that the contrasting rhythms in serotonin content measured in these
areas may reflect a differential regulation of serotonin metabolism in the OL
and AL arbors of the same neuron. Many years ago, we proposed that the DGNs,
by virtue of their massive axonal arbors projecting to functionally distinct
areas, were likely to engage in localized signaling within discrete areas of
the OLs and ALs, thereby `multitasking'
(Sandeman et al., 1993
). This
concept takes on new meaning if neuronal activity is able to regulate
serotonin levels in these areas.
The logical extension of this idea is that local activity patterns imposed
by differential inputs to the OLs and ALs may be able to influence the
synthesis, degradation, uptake and release of serotonin in these regions.
Serotonin levels in the OL arbors of the DGNs could thereby be altered by
chemosensory stimulation when lobsters become aroused and begin to forage
during the hours just prior to dusk
(Weiss, 1970;
Ennis, 1983
;
Cooper and Uzmann, 1980
;
Arechiga et al., 1993
;
Chabot et al., 2001
).
Zimmer-Faust et al. (1996
)
have shown, using behavioral tests, that the activity state does influence the
sensitivity of spiny lobsters and crabs to food odorants in the water and that
such sensitivity is much higher during periods when physical activity is high.
The implication from this work is that the change in the responsiveness is
centrally and not peripherally determined. If responsiveness of lobsters also
increases abruptly during pre-dusk/dusk arousal, then the OLs may be strongly
activated during this period. It is therefore intriguing that we consistently
see the highest serotonin levels in the OLs during this pre-dusk period.
By contrast, serotonin levels in the ALs rise throughout subjective day to
a peak at dusk and are sustained at a high level throughout subjective night,
rather than dropping precipitously as in the OLs. In this context, it is
interesting that activity patterns in the ALs will be sensitive not only to
chemosensory activation but also to visual and mechanosensory stimulation. It
is possible that the sustained high levels of serotonin in these regions
during subjective night reflect a heightened sensitivity, and corresponding
increased activity, in the variety of sensory systems involved in nocturnal
behaviors. The decrease in serotonin levels during the early subjective
morning hours coincides with the onset of a low activity period in lobsters
(Ennis, 1983;
Chabot et al., 2001
). The fact
that serotonin has been repeatedly associated with learning and memory
mechanisms (Harvey, 2003
;
Meneses, 2003
;
Orsetti et al., 2003
;
Wolff et al., 2003
) may also
be relevant to the rhythms we measure in brain regions. Certainly, the fact
that serotonin is differentially regulated in specific brain regions may
reflect the potential importance of time-of-day performance of those
areas.
Multiple functional, entrainable circadian rhythms have also been found in
the mammalian brain. Nuclei in the olfactory bulb and the ventral hypothalamus
of rats are rhythmic, with peak expression of Per at night, while
other brain areas are only weakly rhythmic or arrhythmic
(Abe et al., 2002). It is
believed that cells within the mitral cell layer of the olfactory bulb are
competent circadian pacemakers, regulating their own gene expression and
membrane excitability (Granados-Fuentes et
al., 2004
). Such results indicate that multiple pacemaking tissues
exist and that these function semiautonomously from each other. These data, in
combination with the fact that circadian modulation of olfaction has been
reported in mammals (Amir et al.,
1999
; Funk and Amir,
2000
), suggest that the presence of an independent pacemaker in
the olfactory bulb may be related to the need for local regulation of
olfactory processing. However, how such rhythms in transcriptional or
electrical activity in the bulb relate to olfaction is not known. Therefore,
in contrast to the traditional view of a single pacemaker driving multiple
rhythms, the presence of independent pacemakers in functionally distinct brain
regions whose activities are coordinated appears to be the standard in many
tissues.
Serotonin as a regulator of neurogenesis
In both vertebrate and invertebrate species, serotonin is a potent
regulator of neurogenesis (Brezun and Daszuta,
1999,
2000
;
Benton and Beltz, 2001
;
Beltz et al., 2001
;
Jacobs, 2002
;
Radley and Jacobs, 2002
;
Malberg and Duman, 2003
). In
the brain of the American lobster, reduced serotonin levels result in a
decrease in neurogenesis among the deutocerebral local and projection neurons
(clusters 9 and 10; Fig. 1)
(Benton and Beltz, 2001
;
Beltz et al., 2001
), while
elevated serotonin levels result in an increased rate of neurogenesis (J. L.
Benton, E. M. Goergen and B. S. Beltz, unpublished results). It is also known
that a bundle of fine serotonergic fibers from the DGN terminate blindly in
the region where new projection neurons are born in cluster 10
(Beltz et al., 2001
). Serotonin
is therefore thought to be important in regulating the cell cycle period of
progenitor cells that produce neurons in the lobster. Hence, it is of
particular interest in the context of our current study of circadian
regulation of serotonin levels that the rate of neurogenesis in the lobster
follows a diurnal rhythm with the lowest rate of neurogenesis at dawn and a
peak rate at dusk; this rhythm is due to a light-entrainable, endogenous
circadian oscillation (Goergen et al.,
2002
).
Data presented here demonstrate that serotonin levels in the brain also follow an endogenous circadian rhythm that is light entrainable. Electrochemical (Fig. 3) and immunocytochemical (Table 1) analyses show that serotonin levels are at their highest in whole lobster brains prior to dusk and at their lowest in the pre-dawn period. Therefore, the peak and trough in serotonin levels in the lobster brain precede the peak and trough in the rate of neurogenesis among the projection neurons.
There are two possible interpretations of the fact that light entrains brain serotonin levels and the timing of neurogenesis. Either the day/night cycle influences these two processes via parallel regulatory mechanisms, or serotonin may be an element in the direct pathway by which light regulates neurogenesis in the lobster brain. However, as we now know that serotonin levels are regulated independently in different brain regions, it is not possible at this stage to relate endogenous fluctuations in brain serotonin levels to the rate of neurogenesis. We cannot directly measure local serotonin levels in the proliferation zone of cluster 10, because this region is very small and serotonin levels would be below the limits of detection. Nevertheless, we can test whether serotonin is directly involved in circadian regulation of neurogenesis using the knowledge that the DGN innervates the region of proliferation in cluster 10. Thus, we can manipulate levels of activity unilaterally in the DGNs and ask whether the rate of neurogenesis is altered in response to stimulation of this neuron.
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Acknowledgments |
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