JESSICA M. FRANCL AND J. DAVID GLASS

1. 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 RESULTS 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%). 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. INTRODUCTION 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. 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 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 24-48 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 post-treatment measurements using a repeated measures ANOVA followed by the Student-Newman-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). 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 11. VIP release from the SCN following 1 hr reverse dialysis of 8-OH-DPAT from ZT 6-7 (n=2). *indicates p<0.05 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 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. • 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. • Administration of NMDA, a glutamate receptor agonist, in the middle of the dark phase increased GRP release. 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. 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. 3V 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. OC Figure 2. Photomicrograph of a coronal section immunostained for VIP with representative microdialysis probe sites in the SCN. OC, optic chiasma; 3V, 3rd ventricle. 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. ACKNOWLEDGEMENTS NIH NH35229 to JDG We are grateful to Gagandeep Kaur for her assistance. 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 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