Regulation of the vgf Gene in the GoldenHamster Suprachiasmatic Nucleus byLight and by the Circadian Clock

JONATHAN P. WISOR1,2 AND JOSEPH S. TAKAHASHI*1National Science Foundation Center for Biological Timing and Department of Neurobiology

and Physiology, Northwestern University, Evanston, Illinois 602082Interdepartmental Graduate Program in Neuroscience and National Multi-Site TrainingProgram for Basic Sleep Research, University of California, Los Angeles, California 90095

ABSTRACT

By using in situ hybridization in the golden hamster brain, we have found that vgfmRNAlevels are induced as a response to light stimulation in the suprachiasmatic nuclei (SCN), thesite of the mammalian circadian pacemaker. The induction exhibits delayed kinetics relativeto known light-induced immediate early genes: induction of vgfmRNA occurs over a period of3 to 9 hours after light exposure. Photic induction of vgf expression does not occur in theparaventricular nucleus (PVN) of the hypothalamus, though this nucleus expresses vgf at themRNA and protein levels. Photic induction of vgf in the SCN occurs only at circadian timeswhen light also causes a phase shift of the circadian system. The irradiance threshold of vgfinduction in the SCN closely matches that of the behavioral phase shifting response. Inaddition, basal expression of vgf in the SCN, but not in the PVN, exhibits a circadian rhythmin constant darkness. The photic induction and circadian rhythm of vgf expression areanatomically separated in the caudal and rostral portions of the SCN, respectively. Theseresults represent the first example of a delayed response to light relative to light-inducedimmediate early genes at the mRNA level in the SCN. J. Comp. Neurol. 378:229–238,1997. r 1997 Wiley-Liss, Inc.

Indexing terms: genes; immediate early; hypothalamus; in situ hybridization; protein synthesis

inhibitors; RNA

The primary circadian oscillator in mammals is local-ized in the suprachiasmatic nuclei (SCN) of the hypothala-mus (Moore, 1983; Meijer and Rietveld, 1989). Recentexperiments suggest that gene expression within the SCNmay play a central role in the regulation of circadianrhythms in mammals (Takahashi, 1993, 1995; Sassone-Corsi, 1994). Several immediate early genes are known tobe induced in the SCN by light (reviewed in Kornhauser etal., 1996), including c-fos (Rea, 1989; Aronin et al., 1990;Kornhauser et al., 1990; Rusak et al., 1990), junB (Korn-hauser et al., 1992), NGF-IA (Rusak et al., 1990), andNGF-IB (Sutin and Kilduff, 1992). One would also expectto see a class of delayed early response genes (Lanahan etal., 1992) that are regulated by immediate early genes, yetno delayed early response genes have been identified in theSCN.We now report the identification of a gene, vgf, that isinduced in the SCN with delayed kinetics relative tolight-induced immediate early genes in response to light.The vgf gene was first identified in a screen for mRNAs

induced in rat PC12 pheochromocytoma cells by nerve

growth factor (NGF; Levi et al., 1985). It was arbitrarilynamed, not as a growth factor. In fact, the function of vgf isnot known but it has been proposed to be involved inintercellular communication (Possenti et al., 1989). In PC12cells, vgf mRNA is induced as a delayed response relative toimmediate early genes and is elevated for up to 24 hours afterNGF treatment (Salton et al., 1991). The protein product ofthis gene is highly enriched in axons and presynaptic boutonsin the SCN (van den Pol et al., 1989, 1994). vgf expressionwasrecently found in the SCNat themRNAlevel (Okamura et al.,1994). Because of the enrichment of vgf in the SCN, weperformed a series of in situ hybridization experiments todetermine whether vgf is regulated by light in the SCN.

Contract grant sponsor: National Institutes of Health; Contract grantnumbers: R01MH49241, R01MH41211.*Correspondence to: Joseph S. Takahashi, Department of Neurobiology

and Physiology, Northwestern University, 2153 North Campus Dr., Evans-ton, IL 60208-3520. e-mail: j-takahashi@nwu.eduReceived 22 April 1996; Revised 6 September 1996; Accepted 20 Septem-

ber 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 378:229–238 (1997)

r 1997 WILEY-LISS, INC.

MATERIALS AND METHODS

Animal care and light pulse procedure

Male golden hamsters (Mesocricetus auratus, Lak:LVG[SYR]) were obtained from Charles River Laborato-ries (Kingston, NY) and maintained on LD14:10 cycles (14hours of light and 10 hours of dark each day). At 2 to 3months of age, animals were individually housed in run-ning wheel cages with ad libitum food and water asdescribed previously (Takahashi et al., 1984). They wereentrained to an LD14:10 cycle for at least 7 days and thenkept in constant darkness for a period of 7 days with theirrunning wheel activity monitored continuously. Whenhamsters are kept in constant darkness, their activitycycles are defined in terms of circadian time. The time ofonset of wheel-running activity during each cycle is de-fined as circadian time (CT) 12. All other times in the24-hour cycle are defined relative to that phase point.Thus, CT19 is 7 hours after the onset of activity, CT2 of thenext cycle is 14 hours after the onset of activity, etc. On theseventh day of continuous darkness at the circadian timesindicated in the Results section, animals were exposed to a5-minute light pulse of 503 nm monochromatic light. Alllight pulses were of saturating irradiance for the hamstercircadian system (.2.6 3 1013 photons cm22 sec21, fromNelson and Takahashi, 1991) with the exception of thoseused in the experiment in Figure 6. Dark control animalswere handled in infrared light with the aid of an infraredviewer (FJW industries) and placed in the light pulseapparatus for 5 minutes but not exposed to visible light.This type of manipulation involving infrared light expo-sure does not phase shift the golden hamster circadianclock (Takahashi et al., 1984; Nelson and Takahashi,1991). In the time between the light pulse and killing, theanimals were returned to their individual cages. For thebehavioral phase shift data in Figure 4, the animals thenremained in their cages for a total of 11 days, after whichthe steady-state phase shift was estimated (Nelson andTakahashi, 1991). For in situ hybridization experiments,animals were rapidly decapitated in infrared light (aftertime periods indicated in Results) with the aid of aninfrared viewer. Brains were dissected in red Safelight(No. 1Afilter, Kodak; Rochester, NY) until the optic nerveswere severed. The brains were quick-frozen on dry ice andstored at 280°C until sectioned. In experiments whereanimals were given anisomycin (100mg/kg, subcutaneous)or cycloheximide (65 mg/kg, subcutaneous) injections, theywere weighed in complete darkness at the time of injec-tion. All experimental procedures conform to NIH guide-lines and were approved by Northwestern University’sInstitutional Animal Care and Use Committee.

In situ hybridization

Brains were sectioned at 20 µm thickness at 220°C,thaw-mounted onto slides and subjected to in situ hybrid-ization according to the method of Suhr et al. (1989).35S-labeled vgf riboprobe was generated from the rat vgfcDNA originally isolated by Hawley et al. (1992). ThecDNA in the pBluescriptSK(1) vector was digested withPmII and EcoRV and then religated to remove approxi-mately 0.5 kb of 38-untranslated sequences. This vgfconstruct was linearized with EcoRI to produce a templateof approximately 1.5 kb for antisense riboprobe and withHindIII to produce a template of approximately 2 kb forsense riboprobe. Sense probe for vgf produced no detect-able hybridization signal in replicate experiments and

therefore will not be further discussed in the Resultssection. Sections were exposed to Kodak (Rochester, NY)autoradiographic emulsion (NTB-2) for 2 to 5 weeks at 4°Cand counterstainedwith cresyl violet for histological exami-nation.

Computer image analysis

Themethod of in situ hybridization signal quantificationwas identical to that of Kornhauser et al. (1992) withexceptions noted below. This quantification procedure waspreviously validated by comparisonwith silver grain count-ing and shown to be proportional to that measure (Korn-hauser, 1995). Briefly, darkfield microscopic projections ofbrain sections were acquired by the Image 1/AT program ofUniversal Imaging Corporation (West Chester, PA). ForSCN hybridization signal quantification, the ‘‘area bright-ness’’ of a 50 by 50 µm area of each SCN section wasmeasured to estimate the total hybridization signal in theSCN. The ‘‘area brightness’’ of a 50 by 50 µm region of theanterior hypothalamus 300 µm lateral to the SCN wasused to estimate background signal and was then sub-tracted from the SCN measurement to determine specifichybridization signal within the SCN. For the experimentsreported in Figures 2–6, the specific hybridization signalfrom ten sections containing the caudal portion of the SCN(4.4–4.6 mm rostral to the vertical zero plane of Kniggeand Joseph, 1968) was measured bilaterally and averagedto produce one data point per animal. Statistical analyseswere performed on these values. For the purpose of displayin these figures, values were normalized to the mean of alldark values in each experiment. In the experiments re-ported in Figure 8, the specific hybridization signal from20 sections encompassing the entire SCN (4.4–4.8 mmrostral to the vertical zero plane of Knigge and Joseph,1968) was measured bilaterally; the rostral ten sectionsand caudal ten sections were averaged as separate groupsto produce two data points per animal. These values werethen normalized to the mean value of the lowest circadiantime group in each experiment. These data were analyzedafter normalization andwere therefore subjected to nonpar-ametric analysis (Kruskal-Wallis H-test).For paraventricular nucleus (PVN) hybridization signal

quantification, the same brains, procedures and statisticalanalyses were used as in SCN signal quantification. PVNsignal was measured in the same sections in which caudalSCN signal was measured. In some cases, we were unableto measure the PVN hybridization signal in a section inwhich we measured SCN signal due to tissue artifact.Therefore, some PVN values for individual animals werethe average of eight or nine sections rather than of tensections.

RESULTS

vgfmRNA is light-induced with delayedkinetics relative to immediate early

genes in the SCN

When golden hamsters are exposed to a 5-minute lightpulse during the subjective night, a phase shift of thecircadian activity rhythm occurs (Takahashi et al., 1984;Nelson and Takahashi, 1991). We examined the effect oflight pulses on the expression of the vgf gene at the mRNAlevel by in situ hybridization. When animals kept inconstant darkness for a period of 7 days were exposed to a5-minute light pulse at CT19 and killed 3 hours later, adramatic induction of vgfmRNAwas seen in comparison to

230 J.P. WISOR AND J.S. TAKAHASHI

dark control animals (Fig. 1). In order to determine thetime course of vgf induction, we performed in situ hybrid-ization on the brains of animals killed at various timepoints following a light pulse at CT19. vgf hybridizationsignal was detected with autoradiographic emulsion, andthe signal was quantified by computer imaging to producea relative measure of hybridization signal (Fig. 2). vgfinduction appeared for a period of several hours after thelight pulse, with peak levels occurring in the range of 3 to 6hours. The time course of vgf induction by a light pulsedelivered at CT14 was found to be very similar to that seenat CT19 (Fig. 2). Two-way analysis of variance indicated asignificant effect for light vs. dark at both CT14 (F 5 9.3,df 5 1,12, P , 0.01) and CT19 (F 5 25.0, df 5 1,9,P , 0.001 for 1–6 hour experiment; F 5 19.49, df 5 1,9,P , 0.005 for 6–12 hour experiment). Planned comparisonF-tests indicated that the increase in vgf hybridizationsignal in response to light was statistically significant at 3,6 and 9 hours, but not before 3 hours (Fig. 2).The time course of vgf induction is notably longer than

that of c-fos and junB mRNAs, which are maximal at 30minutes and return to baseline levels after 2 hours follow-ing a light pulse (Kornhauser et al., 1992). We performed asecond experiment to verify that the induction of vgf is notonly extended relative to induction of these genes but alsodelayed. Animals kept in constant darkness for 7 dayswere exposed to 5-minute light pulses at CT19 and killedeither 30 minutes or 3 hours later. In Figure 3, the resultsof this experiment are shown. Two-way analysis of vari-ance indicated a significant effect for light vs. dark(F 5 8.52, df 5 1,16, P , 0.025) and for time (F 5 10.76,df 5 1,16, P , 0.005). Planned comparison F-tests indi-cated no increase in vgf hybridization signal 30 minutesafter the light pulse but a statistically significant increaseat 3 hours (Fig. 3, upper panel). In this experiment, we alsoexamined the expression of vgf in the PVN, a hypothalamicnucleus that is known to express vgf at themRNA(Mahataet al., 1993a,b,c) and protein (van den Pol et al., 1989)levels. While vgf hybridization signal was detectable in thePVN, we were unable to detect an increase in hybridiza-tion signal following a light pulse (Fig. 3, bottom). Thus,the induction of vgf by a phase shifting light pulse appearsto be specific to the SCN.

Effect of protein synthesis inhibitorson vgf induction by light

The delayed time course of vgf induction suggested to usthat vgf could be acting as a delayed early response generather than an immediate early gene in the SCN. Theinduction of delayed early response genes at the mRNAlevel by extracellular stimuli requires protein synthesiswithin the cell, while immediate early gene induction doesnot (Lau andNathans, 1987). To determine the importanceof protein synthesis in vgf induction, we examined vgfinduction in the SCN by light in the presence of proteinsynthesis inhibitors. We first examined the effect of theprotein synthesis inhibitor anisomycin on vgf induction inthe SCN by light. Animals were kept in darkness for aperiod of 7 days with running wheel activity continuouslymonitored. At CT18.5 on the seventh day of darkness,hamsters were injected subcutaneously with 100mg aniso-mycin/kg body mass [a dose known to block proteinsynthesis by 92% in the rat anterior hypothalamus (Rain-bow et al., 1980) and to cause phase shifts of the circadianclock in hamsters (Takahashi and Turek, 1987)] in 0.9%saline or with 0.9% saline vehicle alone. One half-hour

after injection, animals were exposed to a 5-minute lightpulse or handled as dark controls. Three hours after thelight pulse (or handling) the animals were killed for in situ

Fig. 1. Photic induction of vgf mRNA in the suprachiasmaticnucleus (SCN). A: Reverse contrast image of an x-ray film autoradio-graph showing vgf hybridization signal in a coronal section (20-µm-thick) of hamster brain. The arrowhead indicates the location of thesuprachiasmatic nucleus (SCN) in the anterior hypothalamus. B, C:Darkfield photomicrographs of coronal sections hybridized for vgf andprocessed with autoradiographic emulsion. Sections were taken froman animal exposed to a 5-minute light pulse at CT19 and killed 3 hourslater (B) and a dark control animal killed at the same time (C). Thesections shown are from comparable regions of the SCN with respectto the rostral-caudal dimension. Scale bars 5 1 mm for A, 100 µm forB,C.

vgf REGULATION IN THE SUPRACHIASMATIC NUCLEUS 231

hybridization. A partial blockade of vgf induction by lightwas apparent in the presence of anisomycin (Fig. 4A).Two-way analysis of variance indicated significant effectsfor both anisomycin vs. saline (F 5 6.2, df 5 1,8, P , 0.05)and light vs. dark (F 5 69.7, df 5 1,8, P , 0.001) on vgfhybridization signal in the SCN. A planned comparisonF-test indicated that vgf induction by a light pulse issignificantly reduced in the presence of anisomycin(F 5 9.4, df 5 1,8, P , 0.025).We also examined the effect of anisomycin on the

circadian system at the behavioral level. Animals wereinjected with anisomycin (100 mg/kg, subcutaneous) orsaline at CT18.5 and exposed to light or handled as darkcontrols 30 minutes after the injection. They were thenreturned to their individual cages for a period of 11 days.The phase shifts seen in these animals are presented inFigure 4B. Phase advances were seen in response to a lightpulse at CT19, as would be expected (Takahashi et al.,1984). But when the light pulse was preceded by anisomy-cin injection, the magnitude of the phase advance wasdramatically reduced. The fact that phase delays wereseen in animals treated with anisomycin complicates thisresult. The effects of light and anisomycin may simplyhave been additive: a light-induced phase advance ofapproximately 90 minutes combined with a drug-inducedphase delay of approximately 60 minutes produces a netphase advance of 30 minutes. Nonetheless, the magnitudeof the light-induced phase shift decreases in parallel withthe reduction in vgf induction in the presence of anisomy-cin.

A second protein synthesis inhibitor, cycloheximide, isknown to block completely the phase advancing effect oflight on the hamster circadian clock (Zhang et al., 1996).We examined the effect of cycloheximide injections on vgfinduction by light. Animals were kept in darkness for aperiod of 7 days with running wheel activity continuouslymonitored. At CT18.5 on the seventh day of darkness,hamsters were injected subcutaneously with 65 mg cyclo-heximide/kg body mass (a dose known to block light-induced phase advances of the circadian clock; Zhang etal., 1996) in 0.9% saline or with 0.9% saline vehicle alone.One half-hour after injection, animals were exposed to a5-minute light pulse or handled as dark controls. Threehours after the light pulse (or handling) the animals were

Fig. 2. Time course of vgf induction in the SCN by light. vgfhybridization signal within the SCN was quantified with a computerimaging system (see Materials and Methods). Animals were light-pulsed or handled as dark controls at CT19 or CT14 and killed at thetime points indicated (x-axis). The results of two experiments, sepa-rated by a dashed line, are combined for CT19. Hybridization signalvalues represent the mean 6 S.E.M. for light groups (n 5 3) andmean 6 range for dark groups (n 5 2). Planned comparison F-testswere used to compare light and dark groups at each time point(*P , 0.05, **P , 0.01, df 5 1,12). Data for CT19 are representativeresults from two replicate sets of experiments.

Fig. 3. vgf is induced in the SCN but not in the paraventricularnucleus (PVN) as a delayed response relative to light-induced immedi-ate early genes. vgf hybridization signal within the SCN (top) andPVN (bottom) was quantified with a computer imaging system.Animals were light-pulsed or handled as dark controls at CT19 andkilled at the time points indicated (x-axis). Hybridization signal valuesrepresent the mean 1 S.E.M. (n 5 5, all groups) in the SCN (top) orPVN (bottom). Planned comparison F-tests were used to compare lightand dark groups at each time point (*P , 0.001, df 5 1,16).

232 J.P. WISOR AND J.S. TAKAHASHI

killed for in situ hybridization. In contrast to anisomycin,cycloheximide had no effect on vgf induction by light (Fig.4C). Two-way analysis of variance indicated a significanteffect only for light vs. dark (F 5 15.7, df 5 1,16,P , 0.005).

Photic induction of vgf dependson circadian phase

All immediate early genes that are known to be inducedin the SCN by light are only induced during the subjectivenight (Kornhauser et al., 1992; Sutin and Kilduff, 1992). Toexamine the possibility that vgf is regulated in a similarfashion, we determined the effect of light pulses at differ-ent circadian times on vgf expression. The effects on thecircadian clock of light pulses delivered at different circa-dian times are shown in Figure 5 (top). The magnitude anddirection of the phase shift are dependent on the circadiantime of the pulse. We examined the effects of light pulses atthese circadian times on vgf induction using in situhybridization. Hamsters were kept in constant darknessfor 7 days. During the seventh day of darkness at each offive circadian times (CT3, CT9, CT14, CT19, CT21), ani-mals were given a 5-minute light pulse or handled as darkcontrols. Three hours after the light pulse they were killedfor in situ hybridization.As indicated by Figure 5 (bottom),vgf induction is dependent on circadian phase; it onlyoccurs in the subjective night (at CT14, CT19 and CT21)when a phase shift is induced by light. Two-way analysis ofvariance indicated that there were effects for both light vs.dark (F 5 93.8, df 5 1,10, P , 0.001) and circadian time(F 5 20.0, df 5 4,10, P , 0.001) on vgf hybridization sig-nal in the SCN and an interaction effect (F 5 18.1,df 5 4,10, P , 0.001). Planned comparison F-tests indi-cated that vgf induction was statistically significant atCT14, 19 and 21, but not at CT3 or 9 (Fig. 5, bottom). It isnoteworthy that c-fos and junB also show this samepattern of circadian phase dependence in the SCN (Korn-hauser et al., 1992).

The photic threshold for vgf induction

The sensitivity of the golden hamster circadian systemto the irradiance of a 5-minute light pulse at CT19 wasdetermined in previous experiments in this laboratory.The solid curve in Figure 6 (taken from Nelson andTakahashi, 1991) illustrates that a light pulse must be ofirradiance greater than 1010 photons cm22 sec21 to cause areliable phase shift of the hamster circadian system. In

Fig. 4. Effects of protein synthesis inhibitors on the induction of vgfin the SCN by light. A,B:Animals were injected with anisomycin (100mg/kg, subcutaneous) or saline at CT18.5 and either light-pulsed orhandled as dark controls 30 minutes later. In A, animals were killed 3hours after the light pulse. Brain sections were subjected to in situhybridization for vgfmRNAand quantified by computer. Bars indicatemean 1 S.E.M. (n 5 3, all groups). *P , 0.025 on a planned compari-son F-test of light/anisomycin vs. light/saline (df 5 1,8). Data arerepresentative results from one of two replicate experiments. In B,animals were returned to cages for 11 days and phase shifts weredetermined based on running wheel data. Bars indicate mean 1S.E.M. (n 5 4, dark/saline and light/saline; n 5 5 dark/anisomycinand light/anisomycin). C: Animals were injected with cycloheximide(65 mg/kg, subcutaneous) or saline at circadian time (CT) 18.5 andlight-pulsed or handled as dark controls 30 minutes later. Animalswere killed 3 hours after the light pulse. Brain sections were subjectedto in situ hybridization for vgf mRNA and quantified by computer.Bars indicate mean 1 S.E.M. (n 5 5, all groups). Data are representa-tive results from one of two replicate experiments.

vgf REGULATION IN THE SUPRACHIASMATIC NUCLEUS 233

order to determine the photic threshold for vgf inductionby a light pulse, we exposed animals to light pulses ofvarying irradiance ranging from 2.5 3 108 to 2.6 3 1014photons cm22 sec21 at CT19 and killed the animals 3 hoursafter the pulse for in situ hybridization. One-way analysisof variance on the data indicated that the irradiance of alight pulse had an effect on the amount of vgf hybridizationsignal in the SCN (F 5 6.7, P , 0.005, df 5 5,11). Inanimals exposed to light pulses of 2.5 3 108 and 8 3 109photons cm22 sec21, vgf hybridization signal was very closeto dark control values. vgf induction was seen with lightpulses of irradiance greater than 1010 photons cm22 sec21,with increased irradiance causing increased hybridizationsignal (Fig. 6). We conclude from these data that the photicthreshold of induction for vgf is indistinguishable fromthat of phase shifting of the circadian system at thebehavioral level. However, while behavioral phase shiftsbegin at approximately 1010 photons cm22 sec21 and reacha maximum at approximately 1013 photons cm22 sec21, vgfinduction continued to increase at the highest irradiancewe examined (1014 photons cm22 sec21) in a mannersimilar to that seen previously with c-fos in the SCN(Kornhauser et al., 1990).

Circadian rhythm of vgf expressionin the rostral SCN

In examining the vgf hybridization signal in the SCN ofdark control animals, we noted a circadian rhythm of basalvgf expression in the absence of light cues. This rhythm isnot apparent in the data of Figures 2–6 because of itsanatomical specificity. vgf induction by light is most robustin the caudal portion of the nucleus and as a consequencethe data on light induction (Figs. 2–6) were taken fromthat area. But when the rostral SCN of the dark controlanimals were examined, a circadian rhythm of vgf expres-sion was seen with peak expression of vgf at CT12 (Fig. 7).That the circadian rhythm of vgf expression is confined tothe rostral SCN was verified in three separate experi-ments (Fig. 8). In these experiments, animals were killedin darkness after 7 continuous days of complete darkness.The change in vgf hybridization signal across circadiantimes is clearly not as robust as the light-induced change,but is nonetheless reliable. When the data were combinedinto 6-hour bins across the circadian cycle (CT1–6, CT7–12, CT13–18, CT18–24) and analyzed by Kruskal-WallisH-test, a significant difference was found among groups inthe rostral SCN (H 5 11.8,P , 0.01, df 5 3) but not caudalSCN (H 5 5.989, P . 0.10, df 5 3). We also measured vgfhybridization signal in the PVN of these animals, but

Fig. 5. The induction of vgf in the SCN by a light pulse isdependent on circadian phase. Top: Phase shifts occurring in thegolden hamster in response to 60-minute light pulses (taken fromTakahashi et al., 1984). The dashed line represents a phase shift valueof 0. Positive values on the curve represent phase advances andnegative values represent phase delays. Bottom: vgf induction at themRNA level is circadian phase-dependent. Animals were given a5-minute light pulse (n 5 2) or handled as dark controls (n 5 2) at oneof five circadian times and killed 3 hours later. vgf hybridization signalin the SCN was quantified by computer. Each bar represents themean 1 range for two light pulsed animals. The dashed line repre-sents the mean of all dark control values.All light-pulsed group valuesare normalized to that value. Planned comparison F-tests were used tocompare the light and dark groups at each circadian time (*P , 0.01,df 5 1,10). Data are representative results from one of two replicateexperiments.

Fig. 6. The threshold for vgf induction in the SCN by a light pulseat CT19 is indistinguishable from the threshold for phase shifting ofthe circadian clock. The solid curve is the modified Naka-Rushtonfunction for steady-state phase shifts induced by 5-minute light pulses(Nelson and Takahashi, 1991). At CT19, animals were exposed to lightpulses of the irradiance indicated on the x-axis. They were killed 3hours after the light pulse. vgf hybridization signal in the SCN wasquantified by computer. Each hybridization signal value representsthe mean 6 S.E.M. for three animals. The dashed line indicates themean hybridization signal from two dark control animals. All light-pulsed group values are normalized to that value. Planned comparisonF-tests were used to compare the lowest irradiance group (2.5 3 108photons cm22 sec21) to all higher irradiance groups (*P , 0.05,df 5 1,11). Data are representative results from one of two replicateexperiments.

234 J.P. WISOR AND J.S. TAKAHASHI

found no circadian rhythm of vgf expression (Fig. 8,bottom). When the PVN data were combined into 6-hourbins across the circadian cycle (CT1–6, CT7–12, CT13–18,CT18–24) and analyzed by Kruskal-Wallis H-test, nosignificant difference was found among groups (H 5 1.161,P . 0.10, df 5 3).

Fig. 8. Comparison of basal vgf expression in the rostral SCN,caudal SCN and PVN of hamsters kept in constant darkness. In threeseparate experiments, hamsters were kept in constant darkness for 7days and then killed in darkness at the circadian times indicated onthe x-axis. vgf hybridization signal within the rostral SCN (top),caudal SCN (middle) and PVN (bottom) was quantified by computer.Each value represents the mean 6 S.E.M. of three animals (circles,squares, from two independent experiments) or mean 6 range of twoanimals (triangles, dark controls from the experiment in Fig. 5). Thecurves are regression curves fitted to the data by SigmaplotTM.

Fig. 7. Circadian rhythm of basal vgf expression within the rostralSCN of hamsters kept in constant darkness. Darkfield photomicro-graphs showing vgf hybridization signal in the rostral SCN of ham-sters kept in constant darkness for 7 days and killed in darkness atCT6 (A), CT12 (B), CT17 (C), or CT22 (D). The sections shown are fromcomparable regions of the SCN with respect to the rostral-caudaldimension. Scale bar 5 125 µm.

vgf REGULATION IN THE SUPRACHIASMATIC NUCLEUS 235

DISCUSSION

We have shown that the vgf gene is induced at themRNA level in the SCN with delayed kinetics relative toknown light-induced immediate early genes. The delayedand extended time course of induction for this geneindicates that it may be regulated by differentmechanismsthan previously studied immediate early genes. However,vgf induction shares some characteristics with immediateearly gene induction in the SCN. The induction is notblocked by injections of protein synthesis inhibitors. Thephotic induction of vgf is gated by the circadian clock: itoccurs only during the subjective night. Furthermore, theintensity-response function for vgf induction is indistin-guishable from that of c-fos. In addition to being regulatedby light, vgf expression in the SCN is regulated by thecircadian clock independently of light. The circadianrhythm of vgf expression occurs in the rostral portion ofthe SCN, while light-induced expression occurs primarilyin the caudal portion of the nucleus.We did not observe regulation of vgf by light or by the

circadian clock in the PVN. This result is not surprising,since the SCN is the site of the primary circadian clock ofthe brain and receives dense retinohypothalamic projec-tions, while the PVN serves other functions. Although wehave not made systematic observations of vgf regulationby light outside the SCN and PVN, the induction of vgfexpression by light appears to occur primarily within thecaudal SCN, where retinohypothalamic projections aremost dense in the hamster. These dense projections doextend tens of microns beyond the borders of the SCN inthe lateral and dorsal directions (Johnson et al., 1988;Card and Moore, 1991) and, in fact, we observed vgfinduction by light on or slightly outside the dorsal andlateral borders of the SCN in some cases. Thus, theanatomical location of vgf induction by light appears tooverlap with the pattern of retinohypothalamic projectionsto the SCN and its immediate vicinity. It also appears tooverlap the region in which c-fosmRNA is induced by light(Kornhauser et al., 1990). Because there are diffuse retino-hypothalamic projections terminating in areas other thanthe SCN and its immediate vicinity, it is conceivable thatvgf may be induced by light in these isolated areas.Similarly, we cannot be certain that circadian regulation ofvgf expression is confined to the rostral SCN exclusively, asother brain areas exhibit circadian rhythmicity in othervariables. But inasmuch as the PVN is representative ofvgf expressing regions outside the SCN, our results sug-gest that circadian and light regulation of vgf may beconfined to the SCN and its immediate vicinity.Other laboratories have previously shown that vgfmRNA

is regulated by external stimuli in the hypothalamus andin PC12 cells. Mahata et al. (1993a,c) found that vgfmRNAwas induced 4- to 5-fold in the hypothalamic paraventricu-lar nucleus by adrenalectomy and up to 9-fold in theparaventricular, supraoptic and retrochiasmatic nucleiand subfornical organ by salt-loading of rats. Similarly,septohippocampal lesions were found to induce vgf mRNA5- to 6-fold in the paraventricular nucleus (Mahata et al.,1993b). In PC12 cells, induction of vgf mRNA peaks 3 to 6hours after NGF treatment (Salton et al., 1991), similar tothat seen in the SCN.The effect of the protein synthesis inhibitor cyclohexi-

mide on vgf induction by NGF has been examined in PC12cells. When induction was examined 2 hours after NGF

treatment in the presence of cycloheximide, vgf inductionwas partially blocked by cycloheximide (Possenti et al.,1992). In contrast, 6 hours after NGF treatment in thepresence of cycloheximide, vgf induction was increaseddramatically by cycloheximide (Baybis and Salton, 1992).Our results with protein synthesis inhibitors are less clear,but suggest that the induction of vgf at the mRNA leveldoes not require protein synthesis. Although we did see apartial blockade of vgf induction by light in the presence ofanisomycin, cycloheximide had no detectable effect. Whilewe know that the dose of cycloheximide used in this studyblocks light induced phase advances (Zhang et al., 1996),we do not know the time course over which proteinsynthesis is blocked by the drug in the SCN. Presumably,the cycloheximide may not have begun to block proteinsynthesis until after the light pulse had caused an increasein immediate early protein synthesis in the SCN. Inaddition, the dose of anisomycin used in our experimentswas not sufficient to entirely block the effect of light on thecircadian clock; perhaps immediate early protein synthe-sis was only partially blocked by this dose. Thus, it cannotbe concluded with certainty that vgf mRNA induction bylight does not require immediate early protein synthesiswithin the SCN.Anumber of genes and proteins have been reported to be

regulated in the SCN by light. In addition to immediateearly gene induction, changes in neuropeptide levels in theSCN are seen in response to light (Shinohara et al., 1993).But to our knowledge, vgf is the first example of a mRNAinduced with delayed kinetics relative to known immedi-ate early genes in response to light in the SCN. There isreason to believe that delayed gene induction may beimportant for phase shifting the circadian system. Theresults of experiments using the protein synthesis inhibi-tor cycloheximide indicate that protein synthesis is neces-sary over a period of several hours for light-induced phaseshifts to occur in response to light at CT19 in hamsters.Treatment with cycloheximide up to 4 hours after a lightpulse at CT19 blocks the light-induced phase shift entirely(Zhang et al., 1996). Thus, protein synthesis is necessaryfor hours after immediate early gene proteins such as Foshave been produced and their mRNAs degraded. At thislater time, mRNAs exhibiting delayed kinetics (includingvgf ) are present and presumably being translated intoactive proteins.Whether vgf induction by light is involved in the phase

shifting process remains an open question. A role for vgf inphase shifting is supported by the correlation between vgfinduction and behavioral phase shifts in two differentmeasures. Both the vgf induction and light-induced behav-ioral phase shifts occur only during the subjective night. Inaddition, vgf induction and behavioral phase shifting havethe same photic threshold. Thus, the vgf response isquantitatively correlated with light-induced behavioralphase shifts.The function of the VGF protein, however, is not known.

It is localized to axons and synaptic terminals (van den Polet al., 1989) and several lines of evidence suggest that itmay be involved in intercellular communication in someway. In PC12 cells, a VGF-immunoreactive antigen isstored in secretory vesicles and is released extracellularlyin response to depolarization, carbachol and other stimuli(Possenti et al., 1989). These observations and structuralsimilarities of VGF to secretogranins/chromogranins led

236 J.P. WISOR AND J.S. TAKAHASHI

these authors to speculate that VGF might regulate secre-tory events or have hormone-like properties itself.The functional importance of vgf in the SCN needs to be

addressed in future experiments. The role of vgf in theSCN could be studied in vgf knockout mice, although thisapproach is limited by the possibility that there could bedevelopmental abnormalities in addition to functionalabnormalities in adult animals lacking vgf. Alternatively,the role of vgf in the SCN could be studied by knockout ofvgf expression in the normal adult brain. This experimen-tal approach was used to show that induction of theimmediate early genes c-fos and junB is necessary forphotically induced phase shifts. When antisense c-fos andjunB oligonucleotides were injected into the third ventricleof rats, expression of these genes in the SCN at the proteinlevel and light-induced phase shifts of the circadian clockwere blocked (Wollnik et al., 1995). If specifically blockingvgf expression in the SCN with antisense oligonucleotideswere capable of blocking light-induced phase shifts, thefunctional importance of this gene could be ascertained.Regardless of the function of vgf, this gene may be a

useful model for studying gene regulation in the SCN andin the brain as a whole. It is likely that vgf induction occursin response to synaptic activity induced in the SCN bylight, as synaptic activity has been linked to gene induc-tion inmany neuronal systems (Morgan and Curran, 1991;Ginty et al., 1992). Similarly, the circadian rhythm of vgfexpression in the rostral SCN may be driven by theelectrical activity of SCN cells, which it closely parallels(Meijer andRietveld, 1989). The cascade of signal transduc-tion events occurring between synaptic activity and vgftranscription is not known, but one element that is likelyto be important in vgf regulation is the Ca21/cAMP re-sponse element (CRE) centered at base pair 278 of the ratvgf gene. This element has been shown to be essential forinduction of vgf by NGF in PC12 cells (Hawley et al., 1992).The CRE is capable of interacting with several transcrip-tional regulators, including jun family members and theCRE-binding protein, CREB (Sassone-Corsi et al., 1990;Ginty et al., 1992; Galien et al., 1994). Further experi-mentswill be necessary to identify which of these transcrip-tional regulators may be acting to induce vgf in the SCN.

ACKNOWLEDGMENTS

We thank Robert Hawley and David Ginty of HarvardUniversity for providing vgf cDNA clones, Alison Opperand Jim Lin for technical assistance and especially JonKornhauser for technical assistance and much helpfuladvice. Research was supported by NIH grants RO1MH49241 to J.S. Takahashi and K.E. Mayo, and RO1MH41211 to F.W. Turek and J.S. Takahashi.

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