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The Journal of Neuroscience, August 1, 1999, 19(15):6637-6642
Pituitary Adenylate Cyclase Activating Peptide Phase Shifts
Circadian Rhythms in a Manner Similar to Light
Mary E.
Harrington1,
Sabina
Hoque1,
Adam
Hall1, 2,
Diego
Golombek3, and
Stephany
Biello4
1 Departments of Psychology and Biological Sciences,
Smith College, Northampton, Massachusetts 01063, 2 Department of Biology, Mount Holyoke College, South
Hadley, Massachusetts, 3 Departamento de
Fisiología, Facultad de Medicina, Universidad de Buenos Aires,
Paraguay 2155, (1121) Buenos Aires, Argentina, and
4 Psychology Department, University of Glasgow, Glasgow,
United Kingdom G128QB
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ABSTRACT |
The endogenous circadian pacemaker in mammals is located in the
suprachiasmatic nuclei (SCN) of the hypothalamus. Various cues can
reset circadian rhythm phase, thereby entraining the internal rhythm to
the environmental cycle, and these effects can be investigated using an
in vitro method to measure phase shifts of the SCN.
Although pituitary adenylate cyclase activating peptide (PACAP) is
localized in retinal inputs to the SCN, it has been reported to alter
clock phase only during the subjective day (Hannibal et al., 1997 ),
whereas light alters phase only in the subjective night. In this study
we show that PACAP can reset the clock in the photic pattern during the
subjective night when applied in 10 pM to 1 nM
doses. This appears to be mediated via a glutamatergic mechanism,
possibly by potentiation of NMDA currents as is seen at 10-100
pM. Given at higher doses (>10 nM), PACAP shifts in the subjective day, apparently via activation of adenylate cyclase and increased intracellular cAMP. These results indicate dose
and phase specificity of the effects of PACAP, and a new role as a
transmitter in the retinohypothalamic tract.
Key words:
cAMP; circadian; diurnal; glutamate; NMDA; PACAP; suprachiasmatic
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INTRODUCTION |
The suprachiasmatic nuclei (SCN) is
an endogenous circadian pacemaker in mammals (Klein et al., 1991).
Under constant conditions, an animal displays a free-running rhythm of
~24 hr, as determined by its internal clock. Various external cues
can reset circadian rhythm phase, thereby entraining the internal
rhythm to the environmental cycle. Light causes a phase response
pattern mimicked by various chemical and electrical treatments, such as
glutamatergic agents and optic nerve electrical stimulation (Ebling,
1996; Kornhauser et al., 1996; Meijer et al., 1996; Moore, 1997).
Another pattern of phase shifting involves advances during the
subjective day and small delays or no shifts during the subjective
night. Inputs that shift in this manner include access to a novel
running wheel, application of neuropeptide Y or serotonergic agonists,
and cAMP activators (Mrosovsky, 1995; Miller et al., 1996).
Photic information is relayed to the SCN via the retinohypothalamic
tract (Ebling, 1996; Kornhauser et al., 1996; Meijer et al., 1996;
Moore, 1997). Neurochemicals associated with the retinohypothalamic tract include glutamate acting at both NMDA and non-NMDA
receptors, and the peptides substance P and pituitary adenylate cyclase
activating peptide (PACAP) (Ebling, 1996; Hannibal et al., 1997, 1998;
Moore, 1997).
A member of the vasoactive intestinal polypeptide
(VIP)/glucagon/secretin/growth-hormone-releasing hormone superfamily,
PACAP can either activate cAMP signal cascades via stimulation of
adenylate cyclase or alter inositol phosphate levels via phospholipase
C (Masuo et al., 1993; Spengler et al., 1993; Arimura and Said, 1996).
Both forms of PACAP (PACAP-38 and PACAP-27) are widely distributed
(Masuo et al., 1993; Arimura et al., 1996). PACAP regulates hormone
release from the pituitary, pancreas, and adrenal gland, and may be
involved in spermatogenesis and neuritogenesis (Arimura et al., 1996).
Two subclasses of PACAP receptors exist in the SCN, type I and type II
(Hannibal et al., 1997). The type I receptor has high affinity for
PACAP and low affinity for VIP, whereas the type II receptor has
similar affinities for PACAP and VIP (Spengler et al., 1993; Arimura et
al., 1996).
Although the presence of PACAP in the retinohypothalamic tract
suggests a role in conveying photic information to the circadian pacemaker, the action of PACAP to increase cAMP suggests a role in mediating nonphotic phase shifts during the subjective day. One
study using the in vitro rat SCN preparation demonstrated that PACAP advances the phase of the firing rate rhythm during the
subjective day, an effect that can be blocked by previous treatment
with the cAMP antagonist Rp-cAMPs (Hannibal et al., 1997). Similarly,
we found that PACAP can phase advance the hamster in vitro
SCN firing rate rhythm when applied in the subjective day (Harrington
and Hoque, 1997). On the other hand, a recent report indicates that
PACAP induces increases in [Ca2+]i
independent of cAMP (Kopp et al., 1999). Here we report results of
further studies using the hamster brain slice preparation to measure
phase shifts induced by PACAP.
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MATERIALS AND METHODS |
Brain slice studies. Male golden hamsters (>1 mo of
age; Charles River Laboratories, Kingston, NY) were housed under a
14/10 light/dark cycle, and animal care was in accordance with Smith College institutional guidelines. Brain slices were prepared between Zeitgeber time (ZT) 0-10, with ZT 12 arbitrarily defined as the time
of lights off. Hamsters were administered an overdose of halothane, and
the brain was quickly dissected. A mechanical tissue chopper was used
to prepare coronal hypothalamic slices (500-µm-thick). Slices were
placed into a gas:fluid interface chamber. Tissue was maintained at the
interface between artificial CSF (ACSF) and warm, humidified
95% O2 and 5% CO2. ACSF containing
(mM): 125.2 NaCl, 3.8 KCl, 1.2 KH2PO4, 1.8 CaCl2,
1.0 MgSO4, 24.8 NaHCO3, and 10 glucose, pH 7.4, was supplemented with an antibiotic (gentamicin, 50 mg/l) and a fungicide (amphotericin, 2 mg/l) and maintained at
34.5°C. In some cases we simultaneously recorded from two SCN slices
taken from one hamster. In these cases, the two slices were given
different drug applications, so that the data in any one experimental
group always represents results from slices from different individuals.
Both PACAP-38 and Rp-cAMPS (adenosine 3',5'-cyclic monophosphothioate,
Rp-isomer) were supplied by Sigma (St. Louis, MO). AP-5
[(±)-2-amino-5-phosphonopentanoic acid] was purchased from Research
Biochemicals International (Natick, MA). All drugs were applied as a
200 nl microdrop to the SCN on the first day in vitro.
Extracellular single-unit activity in the SCN was measured using glass
micropipettes filled with ACSF advanced through the slice with a
hydraulic microdrive. The signal was amplified, filtered, and
discriminated, and firing rate was measured using a rate monitor and
computer. The spontaneous firing rate of each cell was measured for 1 min. The average firing rate of each cell was plotted versus the ZT of
recording. Recordings from individual slices were generally conducted
for 6-7 hr per day on the second day in vitro. Data were
grouped into 1 hr bins and analyzed by an ANOVA. If the ANOVA indicated significant differences (p < 0.05)
across the bins, data were summarized using a 1 hr running mean
smoother with a 15 min lag, and the time of peak firing rate calculated
as the ZT at the middle of the 1 hr bin with the highest value. The
person recording the firing rate data was blind to the drug treatments of the slice, except in one recording (Rp-cAMPS at ZT 6;
n = 1).
Patch-clamp studies. Pregnant female golden hamsters
(Charles River Laboratories) and pups were housed under a 14/10
light/dark cycle with animal care in accordance with Smith College
institutional guidelines. SCN neurons were acutely dissociated from
brain slices of 5- to 15-d-old hamster pups using the following
protocol. Between ZT 0 and 10, pups were overdosed with halothane
anesthesia and decapitated. Brains were quickly dissected in ice-cold
ACSF; 500 µm hypothalamic slices containing the SCN were prepared and
placed in ACSF bubbled with 95% O2 and 5% CO2
at room temperature for 1 hr. The SCNs were then carefully cut out with
a razor blade and digested in ACSF containing 1.5 mg/ml protease (type
XIV from bacterial Streptomyces griseus; Sigma) at 37°C
for 5 min. The tissue was then washed twice in HEPES-buffered minimum
essential medium (MEM; Sigma), and cells were dissociated by gentle
trituration in 200-300 µl of MEM with a fire-polished Pasteur
pipette. The cell suspension was deposited in drops on
poly-D-lysine-treated strips of glass coverslip, and cells
were allowed to settle for 15 min before flooding with MEM. Cells were
maintained at room temperature under a 95% O2 and 5%
CO2 atmosphere and remained viable for recording for 4-5
hr. Coverslips were positioned in the recording bath, and small SCN
neurons of 5-10 µm diameter (unipolar, bipolar, and multipolar) were
easily identified by their morphology. Cells were superfused with (in
mM): 140 NaCl, 2.5 KCl, 3 CaCl2, 10 HEPES, 3 glucose, and 0.1 µM tetrodotoxin, pH 7.4 with
NaOH. Single-channel currents were recorded from outside-out membrane
patches using standard techniques. Fire-polished, borosilicate glass
pipettes (World Precision Instruments), fabricated by a two-stage pull
(PB-7 puller; Narishige, Tokyo, Japan) and filled with a solution
containing (in mM): 120 K gluconate, 20 KCl, 5 NaCl, 2 MgCl2, 1 CaCl2, 1.1 EGTA, 1 ATP
(Na+ salt), 1 GTP (Na+ salt), and
10 HEPES, pH 7.3 with KOH, typically had resistances of 5-10 M .
Membrane patches were held at  50 to 60 mV and were exposed to
combinations of 20 µM NMDA, 100 nM glycine,
and varying PACAP concentrations by bath (volume, 200-300 µl)
exchange (flow rate, 3 ml/min). All currents were acquired using an
Axoclamp 200A interfaced to a computer running pClamp 6.1 software
(Axon Instruments, Foster City, CA). Data were filtered at 1 kHz with a
low-pass 8-pole Bessel filter, sampled at 4 kHz and analyzed using
pClamp 6.1. The duration of openings for NMDA channels at all integral
current levels was assessed throughout the 1 min exposure, a percentage
of time spent in opening was calculated, and values for exposures with
and without PACAP were compared.
In vivo experiments. Male Syrian hamsters, 40- to
60-d-old, were purchased from Charles River (Margate, Kent, UK)
and held in LD14:10. Between 11 and 12 d after arrival, animals
were anesthetized with sodium pentobarbital (~80 mg/kg, i.p.) and
fitted with a 25 gauge stainless steel guide cannula aimed at the SCN.
The cannulae were stereotaxically placed so that the tip was 2.7 mm
dorsal to the SCN and entered the brain slightly off midline at a 10° angle to avoid damaging the midsagittal sinus. Cannulae were held in
place with dental cement, and three small screws were anchored in the
skull. A pin consisting of a 10 mm length of 30 gauge stainless steel
wire was inserted into the guide cannula to prevent blockage. After
14-15 d of recovery, hamsters were placed in constant dim red light
(mean, 24 lux) provided by safelight lamps fitted with Kodak (Eastman
Kodak, Rochester, NY) filter OA 152-1491. Pilot tests indicated
that exposure to this safe light at circadian time (CT) 1, 5, 9, 13, 17, and 21 for up to 15 min had no effect on the free-running rhythm of
hamsters held in constant darkness (Biello, 1995). The onset of
wheel-running, designated as CT 12, was used as the phase reference
point when timing cannula injections. The onset was defined as 100 wheel revolutions followed by 100 wheel revolutions within the next 30 min. Phase shifts were determined by calculating a regression line for
the five onsets before the manipulation and extrapolated to the day of
the pulse. The activity onset on the pulse day as well as the following
two onsets were omitted from the regression calculation. A second
regression line was calculated from the third through seventh postpulse
onsets and projected back to the pulse day. The difference between the onsets projected from the prepulse and from the postpulse regression lines was taken as the phase shift. Microinjections were performed under a red safe light fitted with a Kodak OA 152-1491 filter. Pilot
tests indicated that exposure to this light at CT 14 for up to 15 min
had no effect on the free-running rhythm of hamsters held in constant
darkness (n = 5; S. Biello, unpublished data). PACAP or
ACSF were injected through a 1 µl Hamilton syringe connected by ~10
cm of polyethylene tubing to a 13.1 mm stainless steel injection
cannula (30 gauge). During the injections the animals were restrained
by hand. After the pin was removed from the guide cannula, the
injection cannula was inserted, the plunger was depressed over a 30 sec
period, and the injector was held in place for a further 30 sec.
Solutions were freshly prepared on the day of treatment and delivered
in a volume of 200 nl. Hamsters were allowed at least 10 d between
treatments. At the conclusion of the experiment, injections sites were
verified by an injection of 200 nl of ink. Hamsters were perfused, and
40 µm sections were cut using a cryostat. Data were only included
from hamsters if the cannula tip was within 400 µm of the SCN, the
SCN showed no apparent gliosis, ink was found to enter the SCN, and no
ink was found in the ventricular system.
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RESULTS |
Hamster SCN cells express a reliable increase in spontaneous
firing rate for ~1 hr in the midsubjective day (ZT 6.3) in control experiments. The dose-response relationship for phase shifts in the
time of peak firing after applications of PACAP at ZT 6 is shown in
Figure 1A. Supporting
previous reports (Hannibal et al., 1997; Harrington and Hoque, 1997),
we found phase advance shifts after treatment with doses of 10 nM to 1 µM. A 1 µM dose at ZT 14 or ZT 18 did not significantly phase shift the rhythm (Fig. 1C). Thus, a 1 µM dose of PACAP appears to
induce phase advance shifts in the subjective day and no phase shifts
in the subjective night.
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Figure 1.
Dose- and phase-dependent effects of PACAP. PACAP
was applied on the first day in vitro on the phase of
the rhythm in firing rate recorded from SCN neurons on the second day.
A, Dose-response curve for PACAP applied at ZT 6, midsubjective day. B, Dose-response curve for PACAP
applied at ZT 14. Phase shifts are measured relative to control slices,
untreated, or given microdrop applications of ACSF at either ZT 6, 14, or 18, which showed peak firing at ZT 6.3 (± 0.1, n = 7; range, 6.0-6.7). Each symbol represents the
mean ± SEM phase shift of n = 3 slices.
C, Comparison of the phase-shifting responses to two
doses of PACAP (1 nM vs 1 µM) at three
phases: ZT 6, ZT 14, and ZT 18. Means ± SEMs are shown;
n = 3 in all groups.
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We reexamined the phase-shifting effects of PACAP during the subjective
night. We replicated previous results showing that a 1 µM
dose of PACAP at ZT 14 did not phase shift the rhythm (Hannibal et al.,
1997). However, lower doses of PACAP were able to induce phase delay
shifts at ZT 14, early in the subjective night (Fig. 1B). The dose-response relationship for PACAP at ZT
14 indicated maximal phase delays after applications of 1 nM PACAP. This dose of PACAP also induced a phase advance
when applied at ZT 18 on the first day in vitro, but had no
significant phase-shifting effect at ZT 6 (Fig. 1C).
Therefore, a 1 nM dose of PACAP appears to induce phase
shifts in the photic pattern.
Because previous studies indicated that PACAP can potentiate NMDA
currents (Liu and Madsen, 1997, 1998; Wu and Dun, 1997), we tested SCN
neurons for a similar effect. Outside-out patches were excised from
acutely isolated SCN neurons and held at membrane potentials of 50 to
60 mV. After exposure to 20 µM NMDA-100 nM
glycine in the recording medium, channel activity was observed in all
patches. NMDA-induced activity typically had conductance of 40-50 pS
and there were few contaminating currents. Exposures of 1 min duration
every 2 min gave consistent levels of activity, and responses were
nondesensitizing throughout the duration of the application. Inclusion
of PACAP (10 pM) in the NMDA-glycine solution consistently
resulted in an enhanced level of channel activity throughout the
exposure (Fig. 2A). The
potentiating effects of PACAP were always fully reversible, with
channel activity returning to control levels after subsequent exposure
to NMDA-glycine alone. The effects of PACAP on NMDA channel activity
were dose-dependent (Fig. 2B). At low concentrations
(1-100 pM), the inclusion of PACAP predominantly resulted
in potentiated levels of channel activity. However, at concentrations
above 1 nM, PACAP either had no effect or significantly
inhibited activity levels (Fig. 2B). Inhibitory
effects of PACAP were also reversible. By using excised membrane
patches, we demonstrate that PACAP can potentiate NMDA currents in SCN
neurons independent of cytoplasmic factors.
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Figure 2.
A, PACAP has a dual, potentiating,
and inhibitory effect on NMDA-induced channel activity in outside-out
patch recordings from SCN neurons. A patch was held at 60 mV and
exposed to 20 µM NMDA-100 nM glycine in the
absence and presence of two concentrations of PACAP. a,
Sample trace of activity in the presence of NMDA-glycine alone.
b, Trace during exposure to NMDA-glycine and 10 pM PACAP demonstrating increased activity.
c, Trace during exposure of the same patch to
NMDA-glycine and 100 nM PACAP showing the pronounced
reduction in Popen. B,
Dose-dependent effects of PACAP on NMDA channel activity. Outside-out
patches, held at 50 to 60 mV, were exposed to 20 µM
NMDA-100 nM glycine. Values for the proportion of time
spent in open configurations were assessed for channels in the absence
and presence of varying concentrations of PACAP, and potentiations and
inhibitions were calculated as percentages above and below control
activity levels. Symbols indicate means, and error bars
indicate SEMs. Each point is averaged from at least five outside-out
patch recordings.
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To test the hypothesis that PACAP phase delays the clock at ZT 14 via a
glutamatergic mechanism, possibly involving potentiation of NMDA
currents, we applied the NMDA receptor antagonists MK-801 (50 µM) or AP-5 (100 µM or 1 mM)
before PACAP. Both NMDA receptor antagonists were able to block PACAP
phase delay shifts (1 nM PACAP at ZT 14), and had no effect
when applied alone (Fig. 3B). The AMPA-kainate receptor antagonist CNQX (10 µM) had no
effect on the PACAP phase delay (1 nM PACAP at ZT 14) and
had no phase-shifting effect when applied alone. When applied in the
subjective day, MK-801 (50 µM) or AP-5 (1 mM)
had no effect on PACAP-induced phase advances (10 nM PACAP
at ZT 6). On the other hand, treatment with the cAMP antagonist
Rp-cAMPS reduced these daytime phase advances, similar to results from
the previous work using rats (Hannibal et al., 1997) (Fig.
3A). Antagonizing cAMP with Rp-cAMPS had no effect on the
PACAP-induced phase delays at ZT 14 (Fig. 3B). These findings support the conclusion that PACAP phase advances the clock in
the subjective day by increasing cAMP activation, and phase delays the
clock in the subjective night by a glutamatergic mechanism. Thus, two
different mechanisms are mediating the subjective day and subjective
night phase shifts of PACAP.
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Figure 3.
A, PACAP phase advances the clock in the
subjective day by increasing cAMP activation. B, Phase
delays the clock in the subjective night by a glutamatergic mechanism.
SCN slices were treated with either PACAP alone or PACAP applied 5 min
after application of either MK-801 (50 µM), AP-5 (100 µM or 1 mM), CNQX (10 µM), or
Rp-cAMPs (10 µM). The dose of PACAP was chosen from the
dose-response curves shown in Figure 1 (10 nM PACAP at ZT
6; 1 nM PACAP at ZT 14). Histograms indicate the mean ± SEM phase shift of n = 3 slices, except two
control groups with n = 2: AP-5 at ZT 6 and
Rp-cAMPS at ZT 14.
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Cannula microinjections of 1 nM PACAP to the area of the
SCN induced small but significant phase shifts in wheel-running
behavior. This pattern of phase shifting resembled phase shifts to
light with phase delays seen at CT 14-14.5 and advances observed at CT
18-18.5 (Fig. 4). The magnitude of these
shifts was less than those seen in the in vitro slice
preparation.
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Figure 4.
Administration of PACAP in vivo via
a cannula aimed at the SCN resets the circadian clock in a manner
similar to light. Double-plotted actograms show daily activity of
individual hamsters with the activity of each day plotted below the
previous day. Arrowheads indicate PACAP administration
on day 7. A, Sample actogram from a hamster given
PACAP at CT 14. B, Sample actogram from a hamster given
PACAP at CT 18. C, Administration of PACAP at CT 14 produced significant phase delays (n = 13; mean
phase shift, 0.51 ± 0.06 SEM) when compared with ACSF controls
(n = 13; mean phase shift, 0.07 ± 0.9 SEM)
on a paired t test (p < 0.001). PACAP microinjected at CT 18 produced small but significant
phase advances (n = 8; mean, 0.45 ± 0.39 SEM)
when compared with ACSF controls at CT 18 (n = 8;
mean, 0.05 ± 0.78 SEM) on a paired t test
(p < 0.005).
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DISCUSSION |
The doses associated with a photic effect of PACAP in this study
are in agreement with previous reports. PACAP is an exceptionally potent peptide. PACAP can increase
[Ca2+]i and stimulate insulin release
from rat pancreatic islets in doses as low as 10 14
to 1013 M (Yada et al., 1994). In
doses as low as 1012 M PACAP increases
[Ca2+]i in dissociated supraoptic
neurons and stimulates vasopressin release from supraoptic slices
(Shibuya et al., 1998). The neuroprotective effect of PACAP in cultured
rat cortical neurons shows a bell-shaped dose-response curve with
maximal effects in the picomolar range (Morio et al., 1996). Although
we have not directly measured the amount of PACAP released from hamster
retinal terminals after photic stimulation, it is likely that light
would release the lower doses (picomolar to nanomolar range) of PACAP,
because these doses altered phase in a manner similar to light.
Although PACAP in the low nanomolar range (0.5-2 nM)
potentiates glycine-mediated NMDA currents in chick cortical neurons,
10-1000 nM concentrations of the peptide have an
inhibitory effect on this current (Liu and Madsen, 1997, 1998; Wu and
Dun, 1997; current study). For chick cortical neurons, the magnitude of
the potentiating effect of PACAP on NMDA currents depends strongly on
the amount of glycine present, suggesting that PACAP may interact with
the glycine site to produce enhancement of NMDA currents (Liu and
Madsen, 1997, 1998; Wu and Dun, 1997). Thus, the potentiation of PACAP
could be regulated by the amount of coincident glycine release.
Alternatively, although the potentiating effects of PACAP on NMDA
currents have been recorded in excised outside-out patches (Liu and
Madsen, 1997; current study), membrane-delimited modulation of NMDA
currents by other mechanisms, e.g., G-proteins (Yu et al., 1997) or
phosopholipase activation (Spengler et al., 1993) cannot be ruled out.
Of particular interest is work indicating that PACAP potentiates
glutamate-evoked release of arachidonic acid from mouse cortical
neurons via a mechanism that does not appear to involve Gs
proteins, or the cAMP-PKA pathway or the phospholipase C-PKC pathway
(Stella and Magistretti, 1996).
PACAP induced a similar pattern of phase shifts in vivo and
in vitro in this study, whereas magnitude of the shifts
varied between the two preparations. This variation could be caused by a multitude of factors, for example, the lack of feedback inhibition in
the isolated SCN.
PACAP content in the rat SCN and pineal gland exhibits a significant
diurnal rhythm in animals housed under light/dark cycles which
disappears after housing in constant darkness, suggesting an exogenous
origin of the rhythm (Fukuhara et al., 1997, 1998). These day-night
variations in the SCN and in the pineal gland support a putative role
of this peptide in entrainment to the daily photoperiod.
The mechanism through which PACAP is able to induce photic-like phase
shifts may be related to potentiation of the effects of glutamate in
the SCN. Indeed, PACAP has been shown to potentiate glutamate-induced
Fos expression in cortical neurons (Martin et al., 1995). Both light
and PACAP can induce phosphorylation of the transcription factor cAMP
response element-binding protein (CREB) in the rat SCN (Kornhauser et
al., 1996; Kopp et al., 1997), although the phase specificity of this
effect of PACAP differs from that of light (von Gall et al., 1998).
Phosphorylated CREB can induce immediate early gene transcription, an
event associated with photic phase shifts of the circadian clock
(Kornhauser et al., 1996). PACAP has also been shown to stimulate the
expression of BDNF mRNA in mouse cortical neurons, an effect blocked by
NMDA receptor antagonists (Pellegri et al., 1998).
These results allow insight into the actions of this peptide associated
with the retinohypothalamic tract, indicating that phase shifts in the
photic pattern are observed at nanomolar doses. Several more general
insights are possible. First, the action of the peptide on the
circadian clock differs with dose, in that lower doses (circa 1 nM) phase shift in the photic pattern, apparently via
potentiation of NMDA currents, whereas higher doses (circa 1 µM) phase shift in a different pattern, presumably via
activation of cAMP. Second, the dose-response relationship for the
photic effects of PACAP has a roughly bell-shaped curve (Figs.
1B, 2C), suggesting that studies testing
only relatively high doses of PACAP may miss this response completely
(Piggins et al., 1998). Neuropeptides may vary in the amount released
so as to have different cellular effects. It appears that thorough
dose-response studies conducted at several circadian phases are
necessary to understand the action of a drug on a neural system
(Miller, 1993).
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FOOTNOTES |
Received April 16, 1999; accepted May 13, 1999.
This work was supported by National Institutes of Health grants to
M.E.H., Consejo Nacional de Investigaciones Científicas y
Técnicas, Agencia Nacional de Promoción Cientifica y
Técnica, Universidad de Buenos Aires and Fundación
Antorchas (Argentina) to D.A.G., and Royal Society grant to S.M.B. We
thank Liz Cate, Jenny Siegel, and Katie Schak for technical assistance
and Victoria Flood and Donna Ewell for animal care.
Correspondence should be addressed to Mary E. Harrington, Department of
Psychology, Smith College, Northampton, MA 01063.
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ABSTRACT
INTRODUCTION
