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REGULATION OF PHOTIC SIGNALING PEPTIDES IN THE MAMMALIAN SCN CIRCADIAN
CLOCK: IN VIVO MEASUREMENTS OF VIP AND GRP
JESSICA M. FRANCL AND J. DAVID GLASS
DEPARTMENT OF BIOLOGICAL SCIENCES, KENT STATE UNIVERSITY, KENT OH, USA
INTRODUCTION
RESULTS
In mammals, circadian rhythms are controlled and maintained by the biological clock,
the suprachiasmatic nuclei (SCN). The SCN receive and integrate photic and nonphotic
signals from both the outside environment and internal brain regions to generate a rhythmic
output. Interneuronal communication occurs through neuropeptide release and acts to
produce a cohesive and stable biological rhythm. Photic information (light) from the retinas
is transmitted to the SCN via the retinohypothalamic tract and utilizes glutamate and PACAP
as primary neurotransmitters. Two important photic neuropeptides intrinsic to the SCN are
vasoactive-intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP). It is thought
that release of these peptides is responsible for relaying photic information received from the
RHT within the core SCN and to pacemaker neurons of the SCN shell. The purpose of these
experiments was to characterize the daily pattern of the photic-related peptides VIP and GRP,
to determine whether this rhythm existed in constant conditions (DD), to verify their
neuronal release from SCN neurons, and to examine the effects of nonphotic (8-OH-DPAT)
and photic (NMDA) stimuli peptide release.
24 Hr Microdialysis
LD: Both peptides exhibited significant rhythmic patterns of release when analyzed in a 14L:10D light
schedule. VIP release peaked at ZT 5 (216±29%, p=0.002, Fig. 3), then dropped rapidly and remained low,
reaching a nadir at ZT 21 (81±8%). GRP release rose slowly through the dark phase to lights on, peaking at ZT
4 (207±63%, p=0.007, Fig. 4). Release then decreased to its lowest point at ZT 7 (62±13%) and rose slowly
toward lights on.
DD: VIP exhibited a significant rhythm of release under constant darkness (p=0.01, Fig. 5). VIP release was
elevated after subjective lights off (CT 12), peaking at CT 21 (150±34%). Release then declined to its lowest
point at CT 9 (45±10%). GRP release was significantly rhythmic in DD, with release rising steadily from the
start of subjective night to its highest point at CT 9 (129±13%, p=0.001, Fig. 6). Release then dropped through
the rest of the subjective night and day it nadir at CT 11 (68±6%).
METHODS
Surgery & Microdialysis
Adult male Syrian hamsters were housed under 14:10 LD schedule (unless noted) in Raturn
rodent systems (BAS) consisting of a bowl cage, position sensor, and a tether to the animal
(Fig. 1). Animals were anesthetized and were surgically implanted with a 2mm
microdialysis probe aimed at the SCN (VIP n=7, GRP n=4). After 48 hr of recovery, animals
were perfused with buffered artificial cerebral spinal fluid (ACSF, pH 7.4, BSA 0.1%) for 2448 hr at 1µl/min using a syringe pump. Samples were collected via Teflon outflow tubing
into non-stick vials using an automatic sample collector. For DD experiments, animals were
housed in constant darkness for approx. 2 wk before experimentation. Animals were fitted
with a light-proof eye cover, and surgery and microdialysis were performed as described
above (n=3/peptide). Pharmacological agents were administered to verify neuronal release of
VIP and GRP: 1 hr perfusion of Ca2+-free ACSF from ZT 2-3 (VIP n=4, GRP n=3) or
veratridine (100uM) in high K+ ACSF from ZT 13-14 for VIP (n=2) and ZT 6-7 for GRP
(n=2). Additionally, animals received a 1 hr perfusion of either 8-OH-DPAT (1.2mM) from
ZT 6-7 (n=2/peptide) or NMDA (200uM) from ZT 18-19 (GRP n=2).
Peptide Analysis
Ultra-sensitive radioimmunoassays were used to analyze samples for peptide content (VIP or
GRP, Phoenix Pharmaceuticals). Briefly, samples and known peptide standards were
incubated in a primary antibody against either VIP or GRP for 48 hr at 4°C, followed by
incubation with 125I-labeled peptide for 24 hr at 4°C. Next, samples and standards were
incubated in secondary antibody and normal rabbit serum for 90 minutes at room
temperature. Buffer was added and samples were centrifuged at 3600 rpm for 30 min at 4°C.
The supernatant was aspirated at the remaining pellets were analyzed for peptide content by
gamma counter (Packard).
Data Analysis
Data from the known assay standards were used to plot a curve, and a line was fit to the data.
This line of best fit was used to determine the peptide content of the biological samples
(Sigmaplot 11.0). Average peptide content for 24-hour LD and DD experiments was
normalized as a percentage of the daily mean for each peptide and are graphed with a
representative actogram. Differences between peak and non-peak peptide release were
analyzed using a repeated-measures ANOVA, followed by a post-hoc Dunnet’s test. For
pharmacological experiments, 2-sample baseline measurements were compared to posttreatment measurements using a repeated measures ANOVA followed by the StudentNewman-Keuls test. All significance levels were set at p<0.05. Probe position at the SCN
was verified by cresyl-violet staining of 20 µm brain sections after experimentation and is
displayed with diagrammatic probe markers (Fig. 2). Animal activity was monitored
throughout experimentation using infrared sensors and data collection software (ClockLab).
3V
Pharmacology
Ca2+ Block: A 1 hr pulse of Ca2+-free ACSF delivered into the SCN via reverse microdialysis from ZT 2-3
significantly reduced VIP release (55±8%, p=0.019, Fig. 7) by ZT 3 compared to baseline levels. GRP release
was immediately reduced and decreased to its lowest point between ZT 3-4 (32±8%, p=0.002, Fig. 8)
compared to baseline levels.
Veratridine: A 1 hr pulse of veratridine (200µM) in high K+-ACSF from ZT 13-14 rapidly and significantly
increased VIP release compared to baseline levels (156±28%, p=0.011, Fig. 9) within 30 min of the start of the
drug pulse. Veratridine from ZT 6-7 increased GRP release to 702±134% of baseline levels (p=0.011, Fig. 10).
8-OH-DPAT: A 1 hr pulse of 8-OH-DPAT (1.2mM) from ZT 6-7 significantly decreased VIP release to
39±19% of baseline levels (p=0.027, Fig. 11) and VIP release remained low through the remainder of the
experiment. Administration of 8-OH-DPAT significantly decreased GRP to 21±14% of baseline levels
approximately 1 hr after the start of the drug pulse (p=0.001, Fig. 12).
NMDA: A 1 hr pulse of NMDA (200µM) from ZT 18-19 significantly increased GRP release 1 hr after
conclusion of the drug administration (203±39%, p=0.019, Fig. 13) compared to baseline levels.
Figure 9. VIP release from the SCN following 1 hr reverse dialysis
of high K+ ACSF and veratridine from ZT 13-14 (n=2). *indicates
p<0.05
Figure 10. GRP release from the SCN following 1 hr reverse
dialysis of high K+ ACSF and veratridine from ZT 6-7 (n=2).
*indicates p<0.05
Figure 11. VIP release from the SCN following 1 hr reverse dialysis Figure 12. VIP release from the SCN following 1 hr reverse dialysis
of 8-OH-DPAT from ZT 6-7 (n=2). *indicates p<0.05
of 8-OH-DPAT from ZT 6-7 (n=2). *indicates p<0.05
Figure 3. Double-plotted VIP release from the SCN in LD graphed
as a percentage of the daily mean ± S.E.M. (n=7) . *indicates
p<0.05, black bars indicate the dark phase.
Figure 4. Double-plotted release of GRP from the SCN in LD
graphed as a percentage of the daily mean±S.E.M. (n=4). *indicates
p<0.05, black bars indicate the dark phase.
Figure 13. GRP release from the SCN following 1 hr reverse dialysis
of NMDA from ZT 18-19 (n=2). *indicates p<0.05
CONCLUSIONS
•The in vivo microdialysis techniques shown here provide real-time information regarding the
endogenous release of photic neuropeptides from the biological clock.
Figure 5. Double-plotted VIP release from the SCN in DD graphed
as a percentage of the daily mean ± S.E.M. (n=3) . *indicates
p<0.05, dotted line indicates time of subjective lights off.
Figure 6. Double-plotted release of GRP from the SCN in DD
graphed as a percentage of the daily mean±S.E.M. (n=3). *indicates
p<0.05, dotted line indicates time of subjective lights off.
•The photic-related peptides of the SCN, VIP and GRP, exhibited rhythmic changes in release
when analyzed under normal LD conditions in hamsters, with peak peptide release occurring
near mid-day.
•Both peptides exhibited a significant rhythm in constant darkness.
•The neuronal origin of the peptides measured was verified pharmacologically using Ca2+inhibition and K+/veratridine stimulation.
•Administration of 8-OH-DPAT, a 5-HT1A,7 receptor agonist, in mid-day decreased release of
both VIP and GRP, indicating that serotonergic/non-photic inputs to the SCN may modulate
photic information within the core region.
OC
Figure 1.
In vivo microdialysis of a freely-behaving
experimental animal in a Raturn apparatus, allowing for
uninterrupted and automatic collection of samples over 24 hr.
•Administration of NMDA, a glutamate receptor agonist, in the middle of the dark phase
increased GRP release.
Figure 2. Photomicrograph of a coronal
section
immunostained for VIP with
representative microdialysis probe sites in the
SCN. OC, optic chiasma; 3V, 3rd ventricle.
ACKNOWLEDGEMENTS
Figure 7. VIP release from the SCN following 1 hour reverse
dialysis of Ca2+-free ACSF from ZT 2-3 (n=4). *indicates p<0.05
Figure 8. GRP release from the SCN following 1 hour reverse
dialysis of Ca2+-free ACSF from ZT 2-3 (n=3). *indicates p<0.05
NIH NH35229 to JDG
We are grateful to Gagandeep Kaur for her assistance.