Light and serotonin phase-shift the circadian clock in the cricket optic lobe in vitro

Light and serotonin were found to cause phase shifts of the circadian neural activity rhythm in the optic lobe of the cricket Gryllus bimaculatus cultured in vitro. The two phase-shifting agents yielded phase-response curves different in shape. Light induced phase delay and advance in the early and late subjective night, respectively, and almost no shifts in the subjective day, whereas serotonin phase-advances the clock during the subjective day and induced delay shifts during the subjective night. The largest phase advance and delay occurred at circadian time 21 and 12, respectively, for light, and circadian time 3 and 18, respectively, for serotonin. Quipazine, a nonspecific serotonin agonist, induced phase advance and phase delay at circadian time 3 and 18, respectively, like serotonin. (±)8-OH-DPAT, a specific 5-HT_1A agonist, phase delayed by 2 h at the subjective night, but produced no significant phase shifts at the subjective day. When NAN-190, a specific 5-HT_1A antagonist, was applied together with quipazine, it completely blocked the phase delay at circadian time 18, whereas it had no effect on the advance shifts induced by quipazine. The results suggest that the phase dependency of serotonin-induced phase shifts of the clock may be partly attributable to the daily change in receptor type. Accepted: 4 July 1999     

Effects of macromolecule synthesis inhibitors on light-induced phase shift of the circadian rhythm in melatonin release from the cultured pineal organ of a teleost, ayu (Plecoglossus altivelis)

Effects of macromolecule synthesis inhibitors on the light-induced phase shift of the circadian clock in the photoreceptive pineal organ of a teleost, ayu (Plecoglosus altivelis) were investigated using melatonin release as an indicator. A single light pulse during the early- and late-subjective night delayed and advanced the phase of the circadian rhythm in melatonin release, respectively. During the late subjective-night, protein synthesis inhibitor cycloheximide (CHX) delayed the rhythm while RNA synthesis inhibitor 5,6-dichlorobenzimidazole riboside (DRB) had little effect. Light-induced phase advance was diminished by the treatment of CHX but not by DRB. During the early subjective-night, DRB, CHX, light and combination of these (DRB+light, CHX+light) all phase-delayed the rhythm. There were no additive effects of light and DRB or CHX. These results indicate that macromolecule synthesis is somehow involved in generation of circadian oscillation, and that de novo protein synthesis is required for light-induced phase shift of the circadian clock in the ayu pineal organ.     

Phase shifts of the circadian locomotor rhythm induced by pigment-dispersing factor in the cricket Gryllus bimaculatus

Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH(2), homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents.     

Pigment-dispersing factor sets the night state of the medulla bilateral neurons in the optic lobe of the cricket,Gryllus bimaculatus

Pigment-dispersing factor (PDF) is an octadeca-neuropeptide widely distributed in the insect brain and suggested to be involved in the insect circadian systems. We have examined its effects on the neuronal activity of the brain efferents in the optic stalk including medulla bilateral neurons (MBNs) in the cricket, Gryllus bimaculatus. The MBNs are visually responding interneurons connecting the bilateral medulla, which show a clear day/night change in their light responsiveness that is greater during the night. Microinjection of PDF into the optic lobe induced a significant increase in the spontaneous activity of the brain efferents and the photo-responsiveness of the MBNs during the day, while little change was induced during the night. The enhancing effects began to occur about 20 min after the injection and another 10 min was necessary to reach the maximal level. The effects of PDF were dose-dependent. When 22 nl of anti-Gryllus-PDF (1:200) IgG was injected into the medulla, the photo-responsiveness of the MBNs was suppressed in both the day and the night with greater magnitude during the night. No significant suppression was induced by injection of the same amount of IgG from normal rabbit serum. These results suggest that in the cricket optic lobe, PDF is released during the night and enhances MBNs' photo-responsiveness to set their night state.     

Functional diversities of two activity components of circadian rhythm in genetical splitting mice (CS strain)

CS mice, an inbred strain, showed two distinctive characteristics in the circadian rhythm of locomotor activity: (1) large variation in the freerunning period, and (2) spontaneous rhythm splitting under continuous darkness. In the splitting rhythm there was a positive correlation between the freerunning period of the evening component and the activity time of the morning component. The phase-shifting effect of a 15-min light pulse was examined on the two activity components of the splitting rhythm. There were significant differences in the amount of light-induced phase response between the two components. A light pulse during the late subjective night induced a phase advance shift only in the morning component, while a light pulse during the early subjective night induced a phase delay shift only in the evening component. These results indicate functional diversities of the two activity components in the circadian locomotor rhythm of CS mice, and suggest that the circadian system in CS mice consists of two mutually coupled oscillators which have different circadian periods and different responsiveness to light. The CS mouse is a useful model to explore a genetic background of oscillator coupling in the circadian system of nocturnal rodents. Accepted: 19 November 1998     

Locomotor Activity Rhythm in the Field Mouse Mus booduga Phase-shifts to Melatonin Injections in a Dose-dependent Manner

Melatonin is known to shift the phase of the locomotor activity rhythm in the field mouse Mus booduga in accordance with a type-I phase response curve (PRC), with phase delays during the subjective day and phase advances during late subjective night and the early subjective day. At CT4 (circadian time 4; i.e. 16 hr. after activity onset) and CT22 of the circadian cycle, a single dose of melatonin (1 mg/kg) is known to evoke maximum delay and maximum advance phase-shifts, respectively. We investigated the dose-dependent responses of the circadian pacemaker of these mice to a single dose of melatonin at the times for maximum delay and maximum advance. The circadian pacemaker responsible for the locomotor activity rhythm in these mice responded to various doses of melatonin in a dose-dependent manner with the magnitude of phase shifts increasing with dose.     

Locomotor Activity Rhythm in the Field Mouse Mus booduga Phase-shifts to Melatonin Injections in a Dose-dependent Manner

Melatonin is known to shift the phase of the locomotor activity rhythm in the field mouse Mus booduga in accordance with a type-I phase response curve (PRC), with phase delays during the subjective day and phase advances during late subjective night and the early subjective day. At CT4 (circadian time 4; i.e. 16 hr. after activity onset) and CT22 of the circadian cycle, a single dose of melatonin (1 mg/kg) is known to evoke maximum delay and maximum advance phase-shifts, respectively. We investigated the dose-dependent responses of the circadian pacemaker of these mice to a single dose of melatonin at the times for maximum delay and maximum advance. The circadian pacemaker responsible for the locomotor activity rhythm in these mice responded to various doses of melatonin in a dose-dependent manner with the magnitude of phase shifts increasing with dose.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus

The photo-responsiveness of 2 groups of interneurons responding to light in the protocerebrum was investigated at 2 developmental stages, the last instar nymphs and adults, in the cricket Gryllus bimaculatus. The cricket is diurnally active during the nymphal stage but becomes nocturnal as an adult. In both adults and nymphs, light-induced responses of optic lobe light-responding interneurons that conduct light information from the optic medulla to the lobula and the cerebral lobe showed a circadian rhythm peaking during the subjective night. Amplitudes of the rhythms were not significantly different between adults and nymphs, but adults showed more stable day and night states than did nymphs. The medulla bilateral neurons that interconnect the bilateral medulla areas of the optic lobe also showed circadian rhythms in their light-induced responses in both adults and nymphs. The rhythm had a clear peak and a trough in adults, and its amplitude was significantly greater than that of nymphs. These results suggest that the 2 classes of interneurons are differentially controlled by the circadian clock. The difference might be related to their functional roles in the animal's circadian behavioral organization.     

Circadian rhythms in the mating behavior of the cockroach, Leucophaea maderae

Mating behavior of small populations of virgin males and females of the cockroach Leucophaea maderae were continuously monitored via time-lapse video recording in controlled laboratory conditions. The time of onset of copulation was found to be rhythmic in a light cycle of 12 h light alternated with 12 h of darkness, with the peak of mating behavior occurring near the light to dark transition. This rhythm persisted in constant dim red illumination and constant temperature. In constant conditions, the period of the rhythm was slightly less than 24 h, with a peak of copulation during the late subjective day. These data demonstrated that mating behavior is gated by a circadian clock. When males and females were taken from light cycles that were 12 h out of phase, a bimodal rhythm was observed with one peak in the males' late subjective day and a second peak of equal amplitude in the late subjective day of females. The results indicated that circadian systems in both males and females contribute to the circadian rhythm in copulation. Bilateral section of the optic tracts (OTX) of both males and females abolished the rhythm, but the rhythm persisted when OTX females were paired with intact males or when OTX males were paired with intact females. Furthermore, when OTX males or OTX females were paired with intact animals that were 12 h out of phase, a bimodal rhythm was still observed. These results suggested that the circadian pacemaker in the optic lobes of both male and female cockroaches participates in the control of mating, but that a pacemaker outside the optic lobes is also likely involved. Finally, it was shown that the female's olfactory response (measured by electroantennogram) to components of the male sex pheromone exhibited a circadian rhythm, but the data suggested the peripheral olfactory rhythm is not likely to be involved in the rhythm of mating behavior.     

Activity feedback to the mammalian circadian pacemaker: influence on observed measures of rhythm period length.

In the mouse, activity is precisely timed by the circadian clock and is normally most intense in the early subjective night. Since vigorous activity (e.g., wheel running) is thought to induce phase shifts in rodents, the temporal placement of daily exercise/activity could be a determinant of observed circadian rhythm period. The relationship between spontaneous running-wheel activity and the circadian period of free-running rhythms was studied to assess this possibility. With ad libitum access to a running wheel, mice exhibited a free-running period (tau) of 23.43 +/- 0.08 hr (mean +/- SEM). When running wheels were locked, tau increased (23.88 +/- 0.04 hr, p less than 0.03), and restoration of ad libitum wheel running again produced a shorter period (tau = 23.56 +/- 0.06 hr, p less than 0.05). A survey of free-running activity patterns in a population of 100 mice revealed a significant correlation between the observed circadian period and the time of day in which spontaneous wheel running occurred (r = 0.7314, p less than 0.0001). Significantly shorter periods were observed when running was concentrated at the beginning of the subjective night (tau = 23.23 +/- 0.04), and longer periods were observed if mice ran late in the subjective night (tau = 23.89 +/- 0.04), F (1, 99) = 34.96, p less than 0.0001. It was previously believed that the period of the circadian clock was primarily responsive to externally imposed tonic or phasic events. Systematic influences of spontaneous exercise on tau demonstrate that physiological and/or behavioral determinants of circadian timekeeping exist as well.     

Circadian periods of sensitivity for ramelteon on the onset of running-wheel activity and the peak of suprachiasmatic nucleus neuronal firing rhythms in C3H/HeN mice

Ramelteon, an MT(1)/MT(2) melatonin receptor agonist, is used for the treatment of sleep-onset insomnia and circadian sleep disorders. Ramelteon phase shifts circadian rhythms in rodents and humans when given at the end of the subjective day; however, its efficacy at other circadian times is not known. Here, the authors determined in C3H/HeN mice the maximal circadian sensitivity for ramelteon in vivo on the onset of circadian running-wheel activity rhythms, and in vitro on the peak of circadian rhythm of neuronal firing in suprachiasmatic nucleus (SCN) brain slices. The phase response curve (PRC) for ramelteon (90?μg/mouse, subcutaneous [sc]) on circadian wheel-activity rhythms shows maximal sensitivity during the late mid to end of the subjective day, between CT8 and CT12 (phase advance), and late subjective night and early subjective day, between CT20 and CT2 (phase delay), using a 3-day-pulse treatment regimen in C3H/HeN mice. The PRC for ramelteon resembles that for melatonin in C3H/HeN mice, showing the same magnitude of maximal shifts at CT10 and CT2, except that the range of sensitivity for ramelteon (CT8-CT12) during the subjective day is broader. Furthermore, in SCN brain slices in vitro, ramelteon (10 pM) administered at CT10 phase advances (5.6?±?0.29?h, n?=?3) and at CT2 phase delays (-3.2?±?0.12?h, n?=?6) the peak of circadian rhythm of neuronal firing, with the shifts being significantly larger than those induced by melatonin (10 pM) at the same circadian times (CT10: 2.7?±?0.15?h, n?=?4, p?相似文献   

Optic lobe circadian pacemaker sends its information to the contralateral optic lobe in the cricket Gryllus bimaculatus

The bilaterally paired optic lobe pacemakers of the cricket Gryllus bimaculatus are mutually coupled. In the present study we recorded the neural activity conveyed from the brain toward the optic lobe with a suction electrode to examine the coupling signals. The results demonstrated that the brain efferents to the optic lobe encode the circadian information: Both in constant light (LL) and constant darkness (DD), the neural activity of brain efferents showed a clear circadian rhythm with a nocturnal peak. Since the rhythm survived the severance of the contralateral optic nerve but disappeared when the contralateral optic lobe was removed, it is apparent that the rhythm originates from the contralateral optic lobe. The amplitude of the rhythm was greater in LL than in DD, suggesting that light affects the amplitude of the rhythm. This was confirmed by the fact that the light-induced response was under circadian control, being greater during the subjective night. These data suggest that the bilaterally paired optic lobe pacemakers exchange circadian information as well as light information. The data are also consistent with the results of previous behavioral experiment.Abbreviations DD constant darkness - LD light dark cycle - LL constant light     

Characterization of an optic lobe circadian pacemaker by in situ and in vitro recording of neural activity in the cricket,Gryllus bimaculatus

Summary The nature of the circadian rhythms of the optic lamina-medulla compound eye complex was examined in male crickets Gryllus bimaculatus by recording the multiple unit activity from the optic lobe in situ and in vitro. In most in situ preparations, the neural activity of the complex was higher during the subjective night than during the subjective day, both under constant light and dark. The same pattern was also obtained from nymphal crickets, suggesting that the properties of the pacemaker are common to both nymphs and adults. In a few cases, both diurnal and nocturnal increments in the activity were simultaneously observed, indicating there are two neuronal groups conveying different circadian information. The circadian rhythm was also demonstrated in the optic lobes in vitro, providing evidence that the optic lobe contains the circadian pacemaker that is capable of generating the rhythmicity without any neural or humoral factors from the rest of the animal.Abbreviations DD constant darkness - JST Japanese standard time - LD light to dark cycle - LL constant light     

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0- 7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.     

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.     

Phase and period responses to short light pulses in a wild diurnal rodent,Funambulus pennanti

Photic phase response curves (PRCs) have been extensively studied in many laboratory-bred diurnal and nocturnal rodents. However, comparatively fewer studies have addressed the effects of photic cues on wild diurnal mammals. Hence, we studied the effects of short durations of light pulses on the circadian systems of the diurnal Indian Palm squirrel, Funambulus pennanti. Adult males entrained to a light–dark cycle (12?h–12?h) were transferred to constant darkness (DD). Free-running animals were exposed to brief light pulses (250 lux) of 15?min, 3 circadian hours (CT) apart (CT 0, 3, 6, 9, 12, 15, 18 and 21). Phase shifts evoked at different phases were plotted against CT and a PRC was constructed. F. pennanti exhibited phase-dependent phase shifts at all the CTs studied, and the PRC obtained was of type 1 at the intensity of light used. Phase advances were evoked during the early subjective day and late subjective night, while phase delays occurred during the late subjective day and early subjective night, with maximum phase delay at CT 15 (?2.04?±?0.23?h), and maximum phase advance at CT 21 (1.88?±?0.31?h). No dead zone was seen at this resolution. The free-running period of the rhythm was concurrently lengthened (deceleration) during the late subjective day and early subjective night, while period shortening (acceleration) occurred during the late subjective night. The maximum deceleration was noticed at CT 15 (?0.40?±?0.09?h) and the maximum acceleration at CT 21 (0.39?±?0.07?h). A significant positive correlation exists between the phase shifts and the period changes (r?=?0.684, p?=?0.001). The shapes of both the PRC and period response curve (τRC) qualitatively resemble each other. This suggests that the palm squirrel’s circadian system is entrained both by phase and period responses to light. Thus, F. pennanti exhibits robust clock-resetting in response to light pulses.     

Circadian rhythms in the electroretinogram of the cockroach

Circadian regulation of the amplitude of the electroretinogram (ERG) of the cockroach Leucophaea maderae was investigated. Two components of the ERG exhibited circadian rhythms in amplitude. Interestingly, the peak amplitudes for the two rhythms were approximately 12 hr out of phase. The dominant corneal negative potential (the "sustained component") exhibited maximum amplitude during the subjective night. A second corneal negative potential (the "off-transient") was at a maximum during the subjective day. Intensity-response curves of the sustained component were measured at both the peak and trough of the rhythm. The results showed that the circadian rhythm in amplitude reflected a sensitivity change equivalent to 0.2-0.6 log unit of intensity. An effort was also made to identify the anatomical locus of the pacemaking oscillator for the ERG rhythm in a series of lesion experiments. Neural isolation of the optic lobe from the midbrain by bisection of the optic lobe proximal to the distal edge of the lobula had no effect on the circadian rhythm of ERG amplitude. Bisection of the optic lobe distal to the lobula abolished the ERG amplitude rhythm. These results suggest that the pacemaker is located in the optic lobe near the lobula; that its motion continues in the absence of neural connections with the rest of the nervous system; and that its regulation of ERG amplitude depends on neural pathways in the optic lobe.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

Circadian rhythm of iguana electroretinogram: the role of dopamine and melatonin

The amplitude of the b-wave of the electroretinogram (ERG) varies with a circadian rhythm in the green iguana; the amplitude is high during the day(or subjective day) and low during the night (or subjective night). Dopamine and melatonin contents in the eye are robustly rhythmic under constant conditions; dopamine levels are high during the subjective day, and melatonin levels are high during the subjective night. Dopamine and melatonin affect the amplitude of the b-wave in an antagonistic and phase-dependent manner: dopamine D2-receptor agonists injected intraocularly during the subjective night produce high-amplitude b-waves characteristic of the subjective day, whereas melatonin injected intraocularly during the subjective day reduces b-wave amplitude. Sectioning the optic nerve abolishes the circadian rhythms of b-wave amplitude and of dopamine content. The results of this study suggest that in iguana, a negative feedback loop involving dopamine and melatonin regulates the circadian rhythm of the ERG b-wave amplitude that is at least in part generated in the brain.