Circadian Regulation of a Transcription Factor, Apc/EBP, in the Eye of Aplysia Californica

Journal of Neurochemistry, 2002, 83, 1401–1411

Circadian regulation of a transcription factor, ApC/EBP, in the eye of Aplysia californica

Samer Hattar,1 Lisa C. Lyons, Laurence Dryer2 and Arnold Eskin

Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA

Abstract Expression of ApC/EBP in the eye is also under the control of The transcription factor, ApC/EBP (Aplysia CCAAT enhancer- the circadian oscillator with circadian rhythms of ApC/EBP binding protein) is an immediate early gene that is rapidly mRNA present under constant dark conditions. Therefore, induced by serotonin and the cAMP signaling pathway. ApC/ ApC/EBP is a candidate gene for a circadian transcription EBP acts as an important link following the activation of pro- factor to mediate circadian responses activated by the cAMP tein kinase A (PKA) in the consolidation of long-term memory and cGMP second messenger signaling pathways. in Aplysia californica. In this study, we report that levels of Keywords: Aplysia californica, biological clock, C/EBP, ApC/EBP mRNA in the eye of Aplysia are modulated by cAMP, circadian rhythms, serotonin. serotonin or light. These responses of ApC/EBP to serotonin J. Neurochem. (2002) 83, 1401–1411. and light are mimicked by analogs of cAMP and cGMP.

The induction of transcriptional activators as immediate (Belvin and Yin 1997; Foulkes et al. 1997; George and response genes provides a mechanism for a cell signaling Terracol 1997; Belvin et al. 1999). pathway to affect a broad set of responses. The transcription In Aplysia, the isolated eye produces a circadian rhythm factor CCAAT enhancer-binding protein in Aplysia califor- in compound action potentials (Jacklet 1969). Treatment of nica (ApC/EBP) is an essential immediate early gene for eyes with pulses of the reversible transcription inhibitor, long-term memory consolidation (Alberini et al. 1994; DRB, phase shifts the nerve impulse rhythm demonstrating Yamamoto et al. 1999). In Aplysia, ApC/EBP is induced the importance of transcriptional regulation for the ocular via cAMP responsive element binding protein (CREB; Dash circadian rhythm (Raju et al. 1991; Khalsa et al. 1996). and Moore 1996; Bartsch et al. 1998). The capacity for ApC/ EBP to form homo- and heterodimers with different DNA binding motifs provides the ability to vastly increase the number of downstream targets affected by ApC/EBP (Bartsch et al. 2000). Recent research in mammals has shown that Received June 21, 2002; revised manuscript received September 18, homologs of ApC/EBP in the rat, C/EBPb and C/EBPd, are 2002; accepted September 23, 2002. Address correspondence and reprint requests to Arnold Eskin, also important in learning and memory highlighting evolu- Department of Biology and Biochemistry, University of Houston, 369 tionary conservation (Taubenfeld et al. 2001a; Taubenfeld Science and Research II, Houston, TX 77204–5001, USA. et al. 2001b). E-mail: [email protected] The C/EBP family of transcription factors contains a 1Present address: Department of Neuroscience, Johns Hopkins Medical bipartite basic-leucine zipper domain (b-zip domain) Institute, Howard Hughes Medical Institute, Baltimore, MD, USA. 2Present address: Avon Products R & D, Clinical Evaluations, PS & I, involved in the mediation of protein dimerization and DNA Avon Place, Suffern, NY, USA. binding. Several transcription factors belonging to the basic Abbreviations used: 5-HT, 5-hydroxytryptamine, serotonin; ApC/EBP, leucine zipper (b-zip) family have been identified either as Aplysia CCAAT enhancer binding protein; b-zip, basic leucine zipper; components of the circadian clock or as genes that are BFSW, buffered filtered artificial sea water; C/EBP, CCAAT enhancer expressed in a circadian manner. These b-zip transcription binding protein; CT, circadian time; CREB, cAMP responsive element binding protein; DBP, albumin site D-binding protein; DD, dark–dark factors include albumin site D-binding protein (DBP) in cycle; HSC, heat shock cognate gene; LD, light/dark cycle; PBS, mammals (Wuarin and Schibler 1990; Lopez-Molina et al. phosphate-buffered saline; PKA, protein kinase A; RPA, ribonuclease 1997), and Vrille (VRI), CREB and ICER in Drosophila protection assay; ZT, zeitgeber time.

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The ocular impulse rhythm also can be phase shifted using Eye removal either light or serotonin (5-HT) treatments. Light affects the Animals were anesthetized by injection of isotonic MgCl2 phase of the rhythm in the isolated eye through increases (MgCl2Æ6H2O, 360 mM), and eyes removed at specified times. in cGMP (Eskin et al. 1984), while 5-HT induces phase Dim red light was used for eye removal during collections in the shifts through the second messenger, cAMP (Eskin et al. dark. Eye removal took approximately 25–45 s. Eyes were immediately frozen in liquid nitrogen after their removal or they 1982; Eskin and Takahashi 1983). The involvement of the were kept in buffered filtered sea water (BFSW, instant ocean sea 5-HT/cAMP pathway in the induction of phase shifts water containing 30 mM Hepes, pH 7.65) containing streptomycin suggests that the CREB/CRE transcriptional pathway may sulfate (100 lg/mL), and penicillin G (100 units/mL). also be important in the Aplysia circadian system. Due to the potential role of b-zip transcription factors in the In situ hybridization circadian clock and the regulation of both the circadian In situ hybridization was carried out according to previously clock and ApC/EBP in the nervous system by 5-HT, the described procedures (Wilkinson et al. 1987; Levenson et al. role of ApC/EBP in the ocular circadian system of Aplysia 2000a). Eyes were fixed in 4% paraformaldehyde and 30% sucrose was investigated. In this study, we report that ApC/EBP in phosphate-buffered saline (PBS: 136 mM NaCl, 2.7 mM KCl, mRNAexpresses diurnal and circadian rhythms in the eyes 10 mM NaHPO4, 1.8 mM KH2PO4, pH 7.0) overnight at 4C. of whole animals. Moreover, damped ocular circadian Following rinses with PBS, eyes were dehydrated in a series of ethanol and then xylene solutions at room temperature. Eyes were rhythms of older animals were correlated with damped embedded in paraffin for 6 h at 60C and then transferred to fresh rhythms of ApC/EBP mRNA. Furthermore, light and 5-HT paraffin and solidified. After 10 lm sections were cut, paraffin was had phase-dependent effects on levels of ApC/EBP mRNA. removed and the sections were treated with proteinase K. Finally, the same second messengers, cGMP and cAMP, 35S-labeled antisense riboprobes were made using the Prime-It that are involved in phase shifting the ocular impulse random labeling kit (Stratagene, La Jolla, CA, USA). Hybridization rhythm also appear to be involved in regulating ApC/EBP was performed at 52C overnight. Slides were coated with the mRNA. emulsifying agent, NTB-2 (Kodak), to visualize riboprobe hybridization. After development of the emulsion, nuclei were counterstained with Hoechst (Sigma, St Louis, MO, USA). Riboprobes Materials and methods corresponding to the sense strand of ApC/EBP served as negative controls. Animal maintenance Aplysia californica (Alacrity Marine Biological Services, Redondo RNase protection assays Beach, CA, USA; Marinus, Inc., Long Beach, CA, USA; NCRR RNAextraction (four eyes per time point per experiment) was National Resource for Aplysia at the University of Miami, Miami, performed according to the guanidine hydrochloride isolation FL, USA) were maintained at 15C in Instant Ocean (Aquarium method (Stratagene). Approximately 2 lg of total eye RNAwas Systems, Mentor, OH, USA). Animals weighed between 100 and hybridized to the antisense riboprobes for ApC/EBP and heat shock 150 g and their age was estimated from shell diameter as cognate gene (HSC)at45C overnight. The ApC/EBP probe described previously (Peretz et al. 1982; Sloan et al. 1999). As it corresponds to nucleotides (nt) 808–1056 (sequence based upon was not feasible to continuously follow the rhythm of ApC/EBP Alberini et al. 1994), protecting a fragment of 248 nt. This probe with age, it was necessary to select two age groups of animals to does not distinguish between the full-length ApC/EBP mRNA study. The amplitude of the circadian ocular rhythm remains (1017 nt) and the truncated mRNA(858 nt). The ApC/EBP probe was inserted into the Pocus vector, linearized with BamHI stable in animals up to 6 months of age and then decreases 32 continuously (Sloan et al. 1999). Thus, field animals younger than (Promega, Madison, WI, USA) and synthesized using [ P]UTP approximately 7 months were classified as young animals, while with T7 RNA polymerase and the Maxiscript kit (Ambion, Austin, those animals older than 7 months were categorized as older TX, USA). The RPA II kit (Ambion) was used for all RNase animals for the purposes of age experiments. Animals received protection assays (RPAs). Protected fragments were separated from the Miami facility were of known age. Animals were kept in through electrophoresis using 5% acrylamide gels. Imaging and 400 L aerated tanks, and fed lettuce upon arrival. Animals were quantification of gels was carried out using a Fuji Phosphorimager entrained to light/dark (LD) 12 : 12 h cycles for at least 3 days or exposure to X-ray film with quantification using a computerized prior to the start of all experiments. Room lighting for entrainment image analysis system (DNAProscan Inc.). To ensure linearity of was provided by fluorescent white lights (34 watts), approximately X-ray film development, multiple exposures were carried out for 1.5 m above the surface of the water in the tank (40 cm deep). each gel. Values of ApC/EBP mRNAwere divided by the HSC For experiments under constant conditions (DD), animals were mRNAvalues for each sample to generate a normalized ApC/EBP entrained and then transferred to constant dark for at least 1 day mRNAvalue. The normalized ApC/EBP mRNAvalues were then prior to the start of the experiment. Light pulses (1200 lux; standardized to a control lane (either the peak value or a control 15 watt fluorescent white aquarium light 15 cm above the surface sample) to generate relative quantifications of the normalized ApC/ of the water, 20 cm deep) were given for 1.5 h, unless specified EBP mRNAvalues in a given experiment. Statistical analyses were otherwise, and did not affect the temperature of the seawater performed using two-tailed Student’s t-tests, ANOVAs, and the surrounding the animals. Kruskal–Wallis test for non-parametric analysis of variance.

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Treatments (p ¼ 0.900). Consequently, HSC-70 was used as an internal Experimental eyes were treated with 2 mM 8-bromo-cGMP (Sigma) loading control to normalize ApC/EBP mRNAabundance. or 8-bromo-cAMP (Sigma) dissolved in BFSW, while the contra- The diurnal rhythm observed in ApC/EBP mRNAcould lateral eyes received BFSW as a vehicle. For in vitro treatments with primarily be due to an acute effect of light on ApC/EBP at ZT 5-HT, experimental eyes were treated with various concentrations of 0. To determine if ApC/EBP also exhibited a circadian 5-HT-HCl (Sigma) while control eyes received BFSW as a vehicle. rhythm, animals were transferred to constant darkness (DD) During in vivo experiments, experimental animals were bathed in after entrainment to 12 : 12 LD cycles. After 2 days in DD, 250 lM 5-HT (Levenson et al. 2000b), while control animals were bathed in the same volume of seawater (Alberini et al. 1994). The eyes were removed from animals at 4 h intervals. ApC/EBP 5-HT concentration in the hemolymph during the 250 lM 5-HT mRNAabundance oscillated in a circadian manner (period treatment was approximately 100 nM (Levenson and Eskin, unpub- length 24–28 h) under constant conditions (Fig. 1b,c). lished data). ApC/EBP mRNAabundance peaked during the subjective night with trough levels during the subjective day.

Results mRNA oscillations were absent in isolated eyes As the circadian ocular impulse rhythm persists in isolated ApC/EBP expressed diurnal and circadian mRNA eyes, we investigated levels of ApC/EBP mRNAin isolated rhythms in vivo eyes maintained in DD for at least 1 day prior to collection. ApC/EBP mRNAwas measured in eyes obtained from intact Levels of ApC/EBP mRNAdid not oscillate when eyes were animals entrained to LD cycles. ApC/EBP mRNAexpressed a isolated and kept in vitro either in BFSW or culture media clear rhythm in animals entrained to LD (Fig. 1a). ApC/EBP (Fig. 2a). Moreover, no rhythm in ApC/EBP mRNAabun- mRNApeaked at dawn with lowest levels observed late in the dance was detected for isolated eyes maintained with the day. HSC-70 was constitutively expressed in the eyes under cerebral ganglia still attached (Fig. 2b). These results indi- both LD conditions (p ¼ 0.497) and constant conditions cate that the rhythm observed in eyes in vivo may be imposed on the eye by rhythmic factors originating outside of the eye. Alternatively, it is possible that a rhythm of ApC/EBP mRNA

Fig. 1 ApC/EBP mRNA oscillates with diurnal and circadian rhythms in the eyes of Aplysia. (a) RNase protection assay of ApC/EBP mRNA and HSC in the eyes of intact animals under 12 : 12 LD cycles. Ani- mals were entrained for at least 3 days in 12 : 12 LD cycles prior to removal of the eyes (4–6 eyes were used per time point). For collections at ZT 0 and ZT 12, eyes were removed within 15–20 min of the change in lighting conditions. HSC was used as an internal loading control, as its mRNA levels are not rhythmic in the eyes under either LD conditions (ANOVA, p ¼ 0.497) or DD conditions (ANOVA, p ¼ 0.900) Graph represents means and standard errors based upon three independent experiments. The change in ApC/EBP mRNA was shown to be statistically significant using ANOVA (p < 0.01). Data was quanti- fied by normalizing each ApC/EBP value to its corresponding HSC value. Then, all of these values were related to one another by setting the highest ratio equal to 1.0. This quantification procedure was fol- lowed in the graphs in other figures. (b) RNase protection assay of ApC/EBP mRNA abundance in eyes under constant conditions. Ani- mals were maintained for 2 days in DD, following 12 : 12 LD entrainment, prior to removal of the eyes at 4 h intervals. (c) Graph represents means and standard errors based upon six independent experiments with n ¼ 3–6 for each time point. ANOVA analysis resulted in p < 0.05. For each time course, mRNA abundance was normalized to the peak value for each time course, which was set to 100%. It was not feasible in a given experiment on ApC/EBP mRNA to have groups of eyes collected at all time points shown in the graph. Thus, more time points are included in the graph (c) than are shown in the gel (b). Similar cases to this also exist in Figs 2 and 4. Gray boxes and black boxes shown in this figure represent subjective day and subjective night, respectively.

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Fig. 2 ApC/EBP mRNA does not oscillate in isolated eyes. (a) RNase day and subjective night, respectively. (b) RNase protection assay of protection assay of ApC/EBP mRNA in isolated eyes under constant ApC/EBP mRNA in isolated eyes attached to cerebral ganglia under conditions. Eyes were isolated from animals entrained to 12 : 12 LD constant conditions. Eyes were isolated from animals entrained to cycles and placed in DD for 1 day prior to the start of collection. The 12 : 12 LD cycles and placed in DD for 1 day prior to the start of graph represents the mean of three independent experiments. For collection. The graph is the mean of eight independent experiments. each time course, mRNA abundance was normalized to the peak For each time course, mRNA abundance was normalized to the peak abundance value. Gray boxes and black boxes represent subjective abundance value. does exist in cells of the eye, but this rhythm was masked by were masking the circadian rhythm of ApC/EBP in isolated injury induced increases in levels of ApC/EBP mRNAin eyes. Accordingly, RPAs were carried out to assess ApC/EBP many cell types within the eye. Injury due to severance of mRNAlevels at different time points following isolation of nerves has been shown to produce large, rapid increases in the eye. Isolation of eyes and severance of the optic nerve ApC/EBP expression in ganglia (Alberini et al. 1994). induced high levels of ApC/EBP mRNA that persisted for at The eye of Aplysia has many different cell types, but only least 3 h following isolation of the eye, with approximately a a limited number of cells may be involved in producing the 10-fold increase in expression (Fig. 3b). However, ApC/EBP circadian rhythm (Herman and Strumwasser 1984; Barnes mRNAlevels returned to levels approximately two-fold and Jacklet 1997). To determine if isolation of the eye and above baseline approximately 24–32 h after isolation. The severance of the optic nerve induced widespread increases in return of ApC/EBP mRNA to near basal levels 32 h ApC/EBP mRNAthroughout the isolated eye, in situ following isolation of the eye diminishes the possibility of hybridization was used to localize changes in ApC/EBP a masked mRNArhythm existing in isolated eyes, as eyes mRNAin the isolated eye. Nuclei were counterstained with were left at least 1 day following isolation before collection. Hoechst (blue). Eyes that were removed from animals Another possible explanation for the lack of rhythm in ApC/ (Fig. 3a) and immediately ﬁxed showed widespread, but EBP mRNAin the isolated eye is that rhythms in individual very low levels of ApC/EBP mRNA. Much higher levels of cells drifted out of phase with one another. This possibility ApC/EBP mRNAwere detected in eyes that had been seems unlikely because in 1–2 days in vitro, the rhythms of isolated for 3 h. The widespread expression of high levels of these cells would not have drifted very far apart. In addition, ApC/EBP mRNAseen after isolation of the eye reinforced the expression of a robust circadian ocular impulse rhythm the possibility that injury responses within the eye potentially requires that the rhythms of the cells must be in phase.

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Fig. 3 Isolation of the eye induces widespread injury. (a) In situ color (yellow) was due to autoﬂuorescence from the pigmented layer of hybridization highlighting ApC/EBP mRNA abundance and spatial the eye. Nuclei were counterstained with Hoechst (blue). All sections expression in eyes immediately and 3 h after removal from an animal. shown were processed together. (b) Injury induced increases in ApC/ Control eye was hybridized with an ApC/EBP sense probe. Only little EBP mRNA at different times after isolation. Graph represents the hybridization was seen in eyes immediately after removal from the means and standard errors based upon three independent experi- animal. Intense widespread hybridization of the antisense ApC/EBP ments. Data was analyzed by the Kruskal–Wallis test for non-para- probe (white) was seen in eyes that had been isolated for 3 h. The metric analysis of the variance, p < 0.01. sense probe produced no hybridization in the eyes. The background

Therefore, ApC/EBP mRNAdoes not appear to express a determined aging increases overall transcription and transla- circadian rhythm in the isolated eye. tion rates in Aplysia (Sloan et al. 1999).

ApC/EBP mRNA rhythm damped with age Light and serotonin increased levels The amplitude of the ocular circadian rhythm decreases with of ApC/EBP mRNA increasing age of the animal (Sloan et al. 1999). To further One-and-a-half hour light or 5-HT treatments are sufficient to investigate the relationship between ApC/EBP expression induce phase shifts in the ocular rhythm of Aplysia (Eskin and the circadian clock, circadian rhythms of ApC/EBP 1972; Corrent et al. 1978; Eskin et al. 1984). In order to mRNAwere compared in young versus old animals. Animals investigate whether entraining agents of the circadian oscil- of known age were obtained from the University of Miami – lator also affect the expression of ApC/EBP, animals that had NIH Aplysia Resource Facility, while the age of wild animals been entrained to LD cycles were exposed to light (1200 lux) was approximated from their shell size (Peretz et al. 1982). or 250 lM 5-HT on the second day of constant darkness. In older animals (> 7 months old) from either the University Animals were exposed to 5-HT in vivo to avoid complications of Miami – NIH Aplysia Resource Facility or field animals, arising from increases in ApC/EBP mRNAdue to injury the ocular rhythm of ApC/EBP mRNAdemonstrated around (Alberini et al. 1994). Eyes were removed and the abundance a two-fold rhythm under DD conditions in older animals of ApC/EBP mRNAwas assayed using RPAs. 1.5 h light or compared with around a five-fold rhythm in young animals 5-HT treatments greatly increased the abundance of ApC/EBP (Fig. 4). The decreased amplitude of the ApC/EBP rhythm in mRNAwhen applied both in vivo (Fig. 5) or to isolated eyes older animals was significantly different as the data in the (data not shown). mRNAlevels remained elevated at the end troughs of the rhythm of older animals was significantly of 6 h 5-HT treatments. However, the ApC/EBP mRNAlevel different from that of the young animals (p < 0.01). The was not increased at the end of 6 h light pulses. Thus, the amplitude of the circadian ocular impulse rhythm damped by effect of light on ApC/EBP mRNAwas transient. 60% in older animals (Sloan et al. 1999). The circadian oscillation of ApC/EBP mRNAdamped in older animals The second messengers used for phase shifting the compared with younger animals apparently due to an circadian clock by light and 5-HT produced increases increase in the baseline levels of ApC/EBP mRNA. Basal in ApC/EBP mRNA abundance levels of ApC/EBP mRNAwere more than doubled in older Light phase shifts the ocular rhythm through increases in compared with younger Aplysia (p < 0.01). This increase in cGMP, while 5-HT phase shifts the ocular impulse rhythm basal ApC/EBP is consistent with previous research that through increases in cAMP (Eskin et al. 1982; Eskin and

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Fig. 5 Light and serotonin increase levels of ApC/EBP mRNA in the eyes of intact Aplysia. RNase protection assay (RPA) using ApC/EBP riboprobe to measure mRNA abundance in eyes of intact animals treated with 1.5 h and 6 h light (L) or serotonin (S) at CT 18 or CT 6. For 5-HT treatments, animals were bathed in 250 lM 5-HT. Eyes were isolated from animals at the end of the treatments. Eyes from control animals were isolated at the end of the 6 h treatments. 1.5 h light: n ¼ 3 experiments for each time point; 1.5 h 5-HT: n ¼ 5 for CT 18 and n ¼ 4 for CT 6; 6 h light: n ¼ 4 for CT 18 and n ¼ 1 for CT 6; 6 h 5-HT: n ¼ 1 for CT 18 and n ¼ 3 for CT 6. Six hour light treatments were also carried out at CT 3 and CT 12 with similar results.

Fig. 4 The free-running rhythm of ApC/EBP mRNA damps with age. (a) RNase protection assay of ApC/EBP mRNA and HSC mRNA abundance in older animals under DD conditions. Animals were entrained by at least 3 days of 12 : 12 LD cycles prior to transfer to constant darkness. Eyes (four per time point) were collected at appropriate times on the second and third days in DD under dim red light. No difference was observed between animals obtained from the University of Miami – NIH Aplysia Resource Facility and animals collected in the field. (b) The graph (dashed line) shown represents the means of ApC/EBP mRNA abundance in older animals (> 7 months) based upon 5 independent experiments. In this graph, only data points with at least n ¼ 3 are shown. The change in ApC/EBP mRNA over time in older animals was significant by ANOVA analysis, p < 0.01. A summary of data from Fig. 1 of ApC/EBP mRNA abundance in young animals is plotted for comparison (solid line). The peak value for each time course was set at 1.0, so no difference between the peak ApC/EBP mRNA values for young and old animals are apparent from the graph. Trough values are significantly different (two-tailed t-test Fig. 6 Elevation of cGMP and cAMP increased levels of ApC/EBP p < 0.01). Gray boxes and black boxes represent subjective day and mRNA in isolated eyes. (a) Isolated eyes were treated with 2 mM subjective night, respectively. 8-br-cAMP (left side) or 2 mM 8-br-cGMP (right side) for 1.5 h. A representative RPA is shown using the ApC/EBP and the HSC ribo- Takahashi 1983; Eskin et al. 1984). Thus levels of ApC/ probes. (b) Graph plotting means and standard errors of treated eyes compared with untreated isolated eyes from seven independent EBP mRNAwere measured in eyes treated with analogs of experiments. Increases in ApC/EBP mRNA by cAMP and cGMP cGMP and cAMP. These experiments were performed on treatments were statistically significant (t-test, p < 0.01). Data were isolated eyes due to the inherent difficulties associated with pooled for treatments at CT 4–5:30 and CT 18–19:30. exposure of whole animals to analogs of cAMP and cGMP. Eyes were isolated before ZT12 and placed in DD for at least 6 h before the start of the treatment. Experimental eyes seems unlikely given the robustness of the increase in ApC/ were treated with 2 mM 8-bromo-cGMP or 8-bromo-cAMP EBP mRNAand that eyes in these experiments were for 1.5 h, while the contralateral eyes received BFSW as a isolated between 6 and 30 h prior to treatment to minimize vehicle. 8-Bromo-cAMP (2 mM) significantly increased the effects of injury. Moreover, light and 5-HT increase ApC/EBP mRNAby 65 ± 16% ( n ¼ 7), while 8-bromo- levels of cGMP and cAMP, respectively, in the eye (Eskin cGMP (2 mM) significantly increased levels of ApC/EBP et al. 1982, 1984). These results suggest that ApC/EBP mRNAin isolated eyes by 178 ± 37% ( n ¼ 7; Fig. 6). expression may be induced with light and 5-HT via the Although these results may reflect an enhancement of the same pathways that these entraining agents use to phase injury response in the isolated eye by cAMP or cGMP, this shift the ocular impulse rhythm.

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Effects of light and 5-HT on ApC/EBP were phase dependent The effects of light and 5-HT on the impulse rhythm from the isolated eye are phase dependent. Light and cGMP produce phase delays at CT 6 and phase advances at CT 18, while 5-HT and cAMP produce phase advances at CT 6 and phase delays at CT 18 (Eskin et al. 1982; Eskin et al. 1984). In order to establish the role of ApC/EBP in the circadian system, the effects of light and 5-HT on ApC/EBP mRNA levels were analyzed at different phases. The phase dependent effects of light and 5-HT on ApC/EBP mRNAwere assessed in vivo to avoid the injury related induction of ApC/ EBP mRNAassociated with in vitro experiments. One-and- a-half hour treatments of either light (1200 lux) or 250 lM 5-HT were given to animals on the first day of constant darkness. Immediately following treatments, ApC/EBP mRNAabundance was measured in eyes. For each time point, the ratio of experimental/control ApC/EBP mRNAwas normalized to the ratio of experimental/control ApC/EBP mRNAabundance at CT 18–19:30, which was set to 3.0. Light and 5-HT increased ApC/EBP mRNAabundance at all time points analyzed, but the magnitude of the increase was dependent on the phase of the treatment (Fig. 7a,b). The large rhythm of light induced mRNAincreases in ApC/EBP Fig. 7 Effects of entraining agents light and 5-HT on ApC/EBP mRNA. peaked at CT 18 with minimal increases in ApC/EBP mRNA (a) Summary of RNase protection assay data measuring ApC/EBP levels seen at CT 6. Likewise, 5-HT induced higher levels of mRNA abundance after 1.5 h light treatments of animals throughout ApC/EBP mRNAat CT 18 compared with CT 6 (Fig. 7b). the circadian cycle. Eyes were collected immediately upon the end of Thus, the effects of light and 5-HT on ApC/EBP mRNA treatments. For each time point, the ratio of experimental/control ApC/ appear to be regulated (gated) by the circadian oscillator. EBP mRNA was normalized to the ratio of experimental/control ApC/ EBP mRNA abundance at CT 18–19:30, which was set to 3.0. Means and standard errors of the difference in ApC/EBP RNA abundance Discussion between experimental and control animals are plotted. Light had a significant phase dependent effect on levels of ApC/EBP mRNA as Surprisingly, ApC/EBP is the first molluscan gene shown to determined by ANOVA (p < 0.01). (b) Summary of RNase protection be expressed rhythmically. ApC/EBP mRNAoscillates in assay data measuring ApC/EBP mRNA abundance after 1.5 h 5-HT eyes from intact animals with a diurnal and circadian rhythm. treatments of animals. Eyes were collected immediately upon the end However, the phase of the diurnal and circadian rhythms of treatments. 5-HT had a significant phase dependent effect on levels differ. Under LD conditions, the levels of ApC/EBP mRNA of ApC/EBP mRNA as determined by two-tailed Mann–Whitney test peak at dawn, while in constant conditions levels are highest (p < 0.05). during the subjective night (Fig. 1). This difference in phase seems to be due to the acute effect of light on levels of ApC/ in isolated eyes (Fig. 2). As the putative clock cells in the EBP as short light pulses increased levels of ApC/EBP Aplysia eye are small in size and number compared with the mRNAthroughout the circadian cycle, while ApC/EBP photoreceptor cells and other cells found in the eye, an mRNAlevels were not affected at the end of longer duration mRNArhythm in only clock cells could be easily masked (6 h) light treatments. Thus, rhythms in ApC/EBP mRNA by widespread injury effects throughout the eye (Herman normally expressed in LD cycles appear to be a consequence and Strumwasser 1984; Barnes and Jacklet 1997). Previous of the acute effects of light on ApC/EBP at dawn and the research has noted that injury in Aplysia increases the levels effects of regulation of the circadian pacemaker throughout of ApC/EBP mRNAin ganglia (Alberini et al. 1994). the rest of the day. Light also produces acute increases in Similarly, isolation of eyes with the necessary severance of c-Fos mRNA, another b-zip transcription factor implicated in the optic nerve resulted in increased levels ApC/EBP the mammalian circadian clock (Rea 1989; Rea et al. 1993; mRNAthroughout the eye as determined by RPAsand Kornhauser et al. 1996). in situ hybridization of sectioned eyes (Fig. 3). In addition, Although rhythms in compound action potentials exist in eyes that were removed from the animal with the cerebral isolated eyes, no rhythm in ApC/EBP mRNAwas observed ganglia attached and maintained in BFSW failed to show

2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1401–1411 1408 S. Hattar et al. rhythms in ApC/EBP mRNA(Fig. 2). Althoughisolated (i) Entrainment pathway The responsiveness of immediate eyes with cerebral ganglia attached showed decreased injury early genes, including b-zip transcription factors, to envi- responses compared with isolated eyes without cerebral ronmental stimuli makes them suitable candidate molecules ganglia (Hattar 2000), levels of ApC/EBP mRNA were still for components of the entrainment signaling pathways for the high in eyes attached to cerebral ganglia compared with circadian oscillator. Indeed light and serotonin induced acute eyes in vivo. Presumably, injury responses still occur due to increases in ApC/EBP mRNAin the Aplysia eye suggesting a the severance of the optic nerve fibers that continue through possible role for ApC/EBP in the entrainment of the the cerebral ganglia to the rest of the central nervous system oscillator. Furthermore, analogs of cGMP and cAMP (Olson and Jacklet 1985). As isolated eyes or eyes attached increased levels of ApC/EBP mRNAin isolated eyes to cerebral ganglia were maintained for 1 day in BFSW suggesting that light and 5-HT increase ApC/EBP expression prior to sampling for circadian rhythm experiments, suffi- via the same signaling pathways involved in entraining the cient time exists for retrograde injury signals to induce circadian oscillator in the Aplysia eye (Fig. 5). If ApC/EBP increases in ApC/EBP expression (Gunstream et al. 1995). fulfills a role in the entrainment pathway, then the threshold The overall injury response, as measured by RPAs, seemed needed for entraining agents to induce ApC/EBP expression to decrease substantially by 6 h with approximately two- should be similar to the threshold needed to induce phase fold baseline levels reached by 24 h. Thus, the effect of shifts in the isolated eye as has been determined in mammals injury on levels of ApC/EBP may not be sufficient to mask for c-fos expression (Kornhauser et al. 1990). The threshold rhythms measured several days after the nerves were for 5-HT to induce increases in the levels of ApC/EBP severed. However, the two-fold increase in ApC/EBP mRNAlies between 0.01 lM and 0.1 lM (Hattar 2000) mRNAlevels due to injury that seems to persist for at similar to the threshold for phase shifting the circadian least 32 h may disrupt the low-level circadian rhythm of oscillator which also falls between 0.01 and 0.1 lM (Corrent ApC/EBP. Therefore, the possibility that the injury induced and Eskin 1982). These results are consistent with a role for increases in ApC/EBP mRNAlevels prevent the detection ApC/EBP in the entrainment pathway. of circadian mRNArhythms in the isolated eyes cannot be ruled out. (ii) Circadian oscillator ApC/EBP expressed both diurnal Alternatively, rhythms of ApC/EBP mRNA in vivo may and circadian mRNArhythms. Similar to many recently not arise within the eye but be driven by factors outside of identified b-zip transcription factors, such as DBP and the eye. Potentially, a diurnal rhythm of 5-HT in the VRILLE (Blau and Young 1999; Yamaguchi et al. 2000), hemolymph (Levenson et al. 1999) could cause the diurnal ApC/EBP may function as both a component of the rhythm of ApC/EBP expression. However, this rhythm of entrainment pathway to the circadian oscillator and as a 5-HT fails to account for the circadian rhythm in ApC/EBP transcription factor controlled by the core circadian mRNAin the eye, as the hemolymph 5-HT levels do not vary oscillator. In support of this possibility, the amplitude of under constant conditions. Additional factors such as amino the ocular nerve impulse rhythms were damped approxi- acids, glucose or other hormones in the hemolymph (Eskin mately 60% in older Aplysia (Sloan et al. 1999), concor- 1982) could influence the circadian expression of ApC/EBP dant with the extent of the decreased amplitude of ApC/ mRNA. Additionally, it must be considered that in the EBP mRNAoscillations under both LD and DD conditions isolated eye, the cells responsible for generation of the ApC/ in older animals. The circadian oscillation of ApC/EBP EBP mRNArhythm may drift out of phase, although this mRNAdamped in older animals compared with younger possibility remains unlikely because a robust ocular impulse animals apparently due to an increase in the baseline levels rhythm is still maintained. of ApC/EBP mRNA(Fig. 4). This increase in basal ApC/ Circadian rhythms in ApC/EBP mRNAin the Aplysia EBP transcription is consistent with previous research that eye raise the possibility that the mRNArhythm of ApC/ determined aging increased transcription and translation EBP is related to that of the circadian oscillator and the rates in Aplysia (Sloan et al. 1999). It remains to be ocular impulse rhythm. These rhythms could be related in determined whether the damped rhythm in ApC/EBP several ways: (i) entrainment pathway: ApC/EBP may expression underlies the decreased amplitude of the ocular regulate the circadian oscillator which then regulates the rhythm with ApC/EBP functioning in the core oscillator or ocular impulse rhythm (ii) circadian oscillator: ApC/EBP whether the damped mRNArhythm represents a transcrip- may be regulated as a component of the core circadian tional output from a damped circadian oscillator. oscillator, and (iii) output pathways: (a) the circadian Several lines of evidence are inconsistent with ApC/EBP oscillator regulates the mRNArhythm of ApC/EBP (output as a component of the oscillator: (a) In vitro isolated eyes rhythms), which in turn regulates the ocular impulse maintain ocular impulse rhythms, while no rhythm of ApC/ rhythm, or (b) the circadian oscillator independently EBP mRNAexists in isolated eyes and (b) the phase of the regulates the rhythm of ApC/EBP mRNAand the ocular in vivo rhythm of ApC/EBP mRNAis not consistent with impulse rhythm. the light PRC of the ocular impulse rhythm (Hattar 2000).

2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1401–1411 Circadian regulation of ApC/EBP 1409

For example, based upon the mRNArhythm, light at CT 18 ApC/EBP seen in older animals could reﬂect the position of should result in little or no phase shifts as this is the peak of both these rhythms as outputs of a damped circadian the mRNArhythm, while light administered at CT 3 (trough) oscillator. Based upon current evidence, it is impossible to should induce large phase delays. However, experimentally, determine whether ApC/EBP regulates the ocular impulse light (1.5 h or 6 h) administered in vivo phase advances the output or whether these are completely independent path- rhythm at CT18 while light (6 h) has no effect on the rhythm ways, although the persistence of the ocular impulse rhythm at CT 3 (Hattar 2000). Thus, it is unlikely that ApC/EBP is in vitro in the absence of an ApC/EBP mRNArhythm the component of the core oscillator mechanism affected by suggests that these may be independent output pathways of light. However, further research is needed to determine if the circadian oscillator. ApC/EBP protein is involved in the core oscillator. Poten- tially, additional regulation of ApC/EBP protein levels and/or Model of ApC/EBP in the circadian clock DNAbinding ability would result in a delay between In the circadian clock of the Aplysia eye, ApC/EBP may increases in ApC/EBP mRNAlevels and the active tran- potentially be a component of the entrainment pathway or scription factor. Therefore, a role for ApC/EBP in the core on an output pathway or both. The model presented in oscillator cannot be completely ruled out. Fig. 8 depicts the potential role(s) of ApC/EBP in the circadian clock in the Aplysia eye. Serotonin and light, (iii) Output pathway The circadian rhythm of ApC/EBP and through cAMP and cGMP, respectively, affect ApC/EBP the circadian rhythm of ocular impulses may represent mRNAlevels (i) and phase shift (ii) the ocular rhythm of either dependent or independent outputs of the circadian Aplysia. The effects of light and serotonin on ApC/EBP oscillator. In general, the evidence cited for the role of ApC/ mRNAabundance were phase dependent, indicating that the EBP in the entrainment pathway is also consistent with circadian oscillator somehow gates or regulates the effects ApC/EBP on an output pathway. If the rhythm of ApC/EBP of light and 5-HT on ApC/EBP. The gating mechanism of mRNAlies solely on an output pathway of the circadian the circadian oscillator on the entrainment pathways oscillator, then the circadian oscillator must modulate the occurs prior to the rhythmic transcription of ApC/EBP acute effect of light on ApC/EBP mRNAlevels. In support (iii). Moreover, the circadian oscillator has to regulate ApC/ of this hypothesis, although short light pulses always EBP as evidenced by the circadian rhythm seen for ApC/ increase ApC/EBP mRNAabundance, even during subject- EBP mRNAlevels (iv). ive day, the extent of the light-induced mRNAincreases Research on long-term sensitization in Aplysia has were phase dependent (Fig. 7). elucidated much of the pathway leading to CREB The correlation that exists between the dampened ocular activation via the second messenger cAMP (reviewed in impulse rhythm and the decreased amplitude rhythm of Carew 1996). Activation of CREB leads to increased

Fig. 8 Model of possible roles of ApC/EBP in the circadian clock. As a component of the circadian clock, ApC/EBP potentially functions in the entrainment pathway to the oscillator and/or an output path from the oscillator. With the capacity to integrate signals from different signaling pathways, ApC/EBP may serve an important coordi- nation function within the circadian clock. See text for discussion. Roman numerals i–vi refer to points in the text.

2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1401–1411 1410 S. Hattar et al. transcription of immediate early genes, including ApC/EBP Acknowledgements (Alberini et al. 1994). Interestingly, in mammals, light has The authors would like to thank Dr Jonathan Levenson for technical been shown to induce another clock gene, mPER1, through help during this research as well as Dr Greg Cahill and Dr Stuart an intact CRE element in its promoter requiring the Dryer for many helpful scientiﬁc discussions. The authors also synergistic activation of the cAMP and mitogen-activated appreciated the assistance of Ms. Carla Haramboure and Ms. Marta protein kinase pathways (Travnickova-Bendova et al. Nunez-Regueiro during this research. This work was supported by 2002). NIH grant MH 41979. Considerably less is known about the cGMP signaling pathway in Aplysia. Recent evidence suggests that the References cGMP/PKG pathway is necessary for induction of long-term excitability in sensory neurons through a pathway independ- Alberini C. M., Ghirardi M., Metz R. and Kandel E. R. (1994) C/EBP is ent of protein kinase A(PKA)(Lewin and Walters 1999). In an immediate-early gene required for the consolidation of long- term facilitation in Aplysia. Cell 76, 1099–1114. mammalian studies, the cGMP/PKG pathway has also been Barnes S. and Jacklet J. W. (1997) Ionic currents of isolated retinal shown to activate CREB phosphorylation in the hippocam- pacemaker neurons: projected daily phase differences and selective pus during late-phase long term potentiation (Lu et al. 1999). enhancement by a phase-shifting neurotransmitter. J. Neurophysiol. Potentially, increases in cGMP levels produced by light 77, 3075–3084. pulses might also lead to increased CREB phosphorylation in Bartsch D., Casadio A., Karl K. A., Serodio P. and Kandel E. R. (1998) CREB1 encodes a nuclear activator, a repressor, and a cytoplasmic Aplysia and, therefore, induction of ApC/EBP transcription. modulator that form a regulatory unit critical for long-term facili- Although the effect of cGMP on CREB could be direct tation. Cell 95, 211–223. through PKG phosphorylation of CREB, no evidence Bartsch D., Ghirardi M., Casadio A., Giustetto M., Karl K. A., Zhu H. indicates that CREB is directly phosphorylated by PKG in and Kandel E. R. (2000) Enhancement of memory-related long- Aplysia and sequence analysis reveals no consensus sites for term facilitation by ApAF, a novel transcription factor that acts downstream from both CREB1 and CREB2. Cell 103, 595–608. PKG in either the transcriptional activator form of CREB or Belvin M. P. and Yin J. C. (1997) Drosophila learning and memory: the repressor. More likely, PKG phosphorylates an interme- recent progress and new approaches. Bioessays 19, 1083–1089. diate factor that leads to subsequent CREB activation. Belvin M. P., Zhou H. and Yin J. C. (1999) The Drosophila dCREB2 Alternatively, PKG may lead to an increase in intracellular gene affects the circadian clock. Neuron 22, 777–787. Ca2+ levels causing increased adenylate cyclase activity, Blau J. and Young M. W. (1999) Cycling vrille expression is required for a functional Drosophila clock. Cell 99, 661–671. which could then induce activation of CREB and subsequent Carew T. J. (1996) Molecular enhancement of memory formation. induction of ApC/EBP expression. Neuron 16, 5–8. The results presented in this study suggest that ApC/EBP Corrent G. and Eskin A. (1982) Transmitterlike action of serotonin in may serve on an input pathway (v) to the oscillator, and yet is phase shifting a rhythm from the Aplysia eye. Am. J. Physiol. 242, regulated by the core oscillator giving this transcription R333–R338. Corrent G., McAdoo D. J. and Eskin A. (1978) Serotonin shifts the phase factor the potential to act as an output component to regulate of the circadian rhythm from the Aplysia eye. Science 202, 977–979. the transcription of target genes (vi). In addition to regulation Dash P. K. and Moore A. N. (1996) Characterization and phosphoryla- of ApC/EBP gene expression, ApC/EBP may also be subject tion of CREB-like proteins in Aplysia central nervous system. to translational or post-translational modulation. Indeed, Brain Res. Mol. Brain Res. 39, 43–51. regulation of both ApC/EBP protein stability and DNA Eskin A. (1972) Phase shifting a circadian rhythm in the eye of Aplysia by high potassium pulses. J. Comp. Physiol. [A] 80, 2573–2579. binding activity has been reported (Yamamoto et al. 1999). Eskin A. (1982) Differential effects of amino acids on the period of the Mitogen-activated protein kinase phosphorylates ApC/EBP circadian rhythm from the Aplysia eye. J. Neurobiol. 13, 231–239. protein activating its DNAbinding activity and rendering the Eskin A. and Takahashi J. S. (1983) Adenylate cyclase activation shifts protein resistant to degradation (Yamamoto et al. 1999). the phase of a circadian pacemaker. Science 220, 82–84. Consequently, for the induction and consolidation of long- Eskin A., Corrent G., Lin C. Y. and McAdoo D. J. (1982) Mechanism for shifting the phase of a circadian rhythm by serotonin: involvement term facilitation in Aplysia, both increases in ApC/EBP of cAMP. Proc. Natl Acad. Sci. USA 79, 660–664. mRNAlevels and phosphorylation of the protein are Eskin A., Takahashi J. S., Zatz M. and Block G. D. (1984) Cyclic guan- necessary (Alberini et al. 1994; Yamamoto et al. 1999). osine 3¢:5¢-monophosphate mimics the effects of light on a circadian Similarly, ApC/EBP may also serve a role as an integrating pacemaker in the eye of aplysia. J. Neurosci. 4, 2466–2471. factor for multiple signaling pathways within the circadian Foulkes N. S., Whitmore D. and Sassone-Corsi P. (1997) Rhythmic transcription: the molecular basis of circadian melatonin synthesis. clock through both transcriptional regulation of this gene as Biol. Cell 89, 487–494. well as post-translational regulation of the protein. Further George H. and Terracol R. (1997) The vrille gene of Drosophila is a studies need to be carried out to precisely deﬁne the role of maternal enhancer of decapentaplegic and encodes a new member of ApC/EBP in the Aplysia circadian system. Also, similar the bZIP family of transcription factors. Genetics 146, 1345–1363. studies to those reported here on ApC/EBP mRNAmust be Gunstream J. D., Castro G. A. and Walters E. T. (1995) Retrograde transport of plasticity signals in Aplysia sensory neurons following performed to determine if the properties of ApC/EBP protein axonal injury. J. Neurosci. 15, 439–448. are similar to those of ApC/EBP mRNA.

2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1401–1411 Circadian regulation of ApC/EBP 1411

Hattar S. (2000) Role of the transcription factor ApC/EBP in the ocular Peretz B., Ringham G. and Wilson R. (1982) Age-diminished motor rhythm of Aplysia. Biology and Biochemistry, p. 172. University of neuronal function of central neuron L7 in Aplysia. J. Neurobiol. Houston, Houston. 13, 141–151. Herman K. G. and Strumwasser F. (1984) Regional specializations in the Raju U., Koumenis C., Nunez-Regueiro M. and Eskin A. (1991) eye of Aplysia, a neuronal circadian oscillator. J. Comp Neurol. Alteration of the phase and period of a circadian oscillator by a 230, 593–613. reversible transcription inhibitor. Science 253, 673–675. Jacklet J. W. (1969) Circadian rhythm of optic nerve impulses Rea M. A. (1989) Light increases Fos-related protein immunoreactivity recorded in darkness from isolated eye of Aplysia. Science 164, in the rat suprachiasmatic nuclei. Brain Res. Bull. 23, 577–581. 562–563. Rea M. A., Michel A. M. and Lutton L. M. (1993) Is Fos expression Khalsa S. B., Whitmore D., Bogart B. and Block G. D. (1996) Evidence necessary and sufficient to mediate light-induced phase advances for a central role of transcription in the timing mechanism of a of the suprachiasmatic circadian oscillator? J. Biol. Rhythms 8, circadian clock. Am. J. Physiol. 271, C1646–C1651. S59–S64. Kornhauser J. M., Nelson D. E., Mayo K. E. and Takahashi J. S. (1990) Sloan M. A., Levenson J., Tran Q., Kerbeshian M., Block G. D. and Photic and circadian regulation of c-fos gene expression in the Eskin A. (1999) Aging affects the ocular circadian pacemaker of hamster suprachiasmatic nucleus. Neuron 5, 127–134. Aplysia californica. J. Biol. Rhythms 14, 151–159. Kornhauser J. M., Mayo K. E. and Takahashi J. S. (1996) Light, Taubenfeld S. M., Milekic M. H., Monti B. and Alberini C. M. (2001a) immediate-early genes, and circadian rhythms. Behav. Genet. 26, The consolidation of new but not reactivated memory requires 221–240. hippocampal C/EBPbeta. Nat.. Neurosci. 4, 813–818. Levenson J., Byrne J. H. and Eskin A. (1999) Levels of serotonin in the Taubenfeld S. M., Wiig K. A., Monti B., Dolan B., Pollonini G. and hemolymph of Aplysia are modulated by light/dark cycles and Alberini C. M. (2001b) Fornix-dependent induction of hippocam- sensitization training. J. Neurosci. 19, 8094–8103. pal CCAAT enhancer-binding protein [beta] and [delta] co-local- Levenson J., Sherry D. M., Dryer L., Chin J., Byrne J. H. and Eskin A. izes with phosphorylated cAMP response element-binding protein (2000a) Localization of glutamate and glutamate transporters in the and accompanies long-term memory consolidation. J. Neurosci. sensory neurons of Aplysia. J. Comp. Neurol. 423, 121–131. 21, 84–91. Levenson J., Endo S., Kategaya L. S., Fernandez R. I., Brabham D. G., Travnickova-Bendova A., Cermakian N., Reppert S. M. and Sassone- Chin J., Byrne J. H. and Eskin A. (2000b) Long-term regulation of Corsi P. (2002) Bimodal regulation of mPeriod promoters by neuronal high-affinity glutamate and glutamine uptake in Aplysia. CREB-dependent signaling and CLOCK/BMAL activity. PNAS Proc. Natl Acad. Sci. USA 97, 12858–12863. 99, 7728–7733. Lewin M. R. and Walters E. T. (1999) Cyclic GMP pathway is critical Wilkinson D. G., Bailes J. A., Champion J. E. and McMahon A. P. for inducing long-term sensitization of nociceptive sensory neu- (1987) Amolecular analysis of mouse development from 8 to 10 rons. Nat. Neurosci. 2, 18–23. days post coitum detects changes only in embryonic globin Lopez-Molina L., Conquet F., Dubois-Dauphin M. and Schibler U. expression. Development 99, 493–500. (1997) The DBP gene is expressed according to a circadian rhythm Wuarin J. and Schibler U. (1990) Expression of the liver-enriched in the suprachiasmatic nucleus and influences circadian behavior. transcriptional activator protein DBP follows a stringent circadian EMBO J. 16, 6762–6771. rhythm. Cell 63, 1257–1266. Lu W. Y., Xiong Z. G., Lei S., Orser B. A., Dudek E., Browning M. D. Yamamoto N., Hegde A. N., Chain D. G. and Schwartz J. H. (1999) and MacDonald J. F. (1999) G-protein-coupled receptors act Activation and degradation of the transcription factor C/EBP dur- via protein kinase C and Src to regulate NMDAreceptors. Nat. ing long-term facilitation in Aplysia. J. Neurochem. 73, 2415– Neurosci. 2, 331–338. 2423. Olson L. M. and Jacklet J. W. (1985) The circadian pacemaker in the Yamaguchi S., Mitsui S., Yan L., Yagita K., Miyake S. and Okamura H. Aplysia eye sends axons throughout the central nervous system. (2000) Role of DBP in the circadian oscillatory mechanism. Mol. J. Neurosci. 5, 3214–3227. Cell Biol. 20, 4773–4781.

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