Melatonin modulates the ERG circadian rhythm in crayfish

One of the most important functions modulated by melatonin is the synchronization of circadian rhythms. In crayfish (Procambarus clarkii), we have obtained evidence that the amplitude of the electrical response to light of the retinal photoreceptors the receptor potential, is modified by the action of melatonin and that the magnitude of this action depends on the circadian time of melatonin application. In contrast, the electroretinogram (ERG) circadian rhythm can be synchronized by either single or periodic melatonin application. In this work we hypothesized that, in crayfish, melatonin acts on effectors and on pacemaker of ERG circadian rhythm as a non-photic synchronizer. Melatonin could be a hormone that sends a signal of darkness to the ERG circadian system.     

Pigment dispersing hormone generates a circadian response to light in the crayfish, Procambarus clarkii

Photoreceptor cells have been identified as important structures in the organization of the circadian system responsible for the generation and expression of the electroretinogram (ERG) circadian rhythm. They are the structures where the circadian periodicity is expressed (effectors) and which transform information from external light signals to be conducted to the pacemaker in order to induce adjustments of the rhythm (synchronizers). After isolation, eyestalks perfused in a pigment dispersing hormone (PDH) solution, show significant changes in receptor potential (RP) amplitude and duration. Exogenous PDH injected into intact crayfish induces a migration of retinal shielding pigments to a light-adapted state. A single dose of PDH produces advances or delays in the circadian rhythm of response to light of visual photoreceptors. All these effects depend on the circadian phase of PDH application. Consequently, the determination of the action of exogenous PDH on photoreceptor cells proved to be very helpful in understanding some mechanisms underlying the circadian organization of crayfish.     

Erg circadian rhythm in the course of ontogeny in crayfish

• 1.1. The objective of the present work was to study the ontogeny of the ERG circadian rhythm in crayfish.
• 2.2. Long-term recordings of ERG and shielding retinal pigments position measured from the instar, the second instar, the third instar and the adult crayfish were obtained.
• 3.3. In the youngest animals (1–8 days old) an ultradian rhythm (15min-4hr periods) in the ERG amplitude was detected.
• 4.4. Older animals showed a progressive increment in the period length before they exhibited a circadian pattern. This last appeared, the first time, in 30-day-old animals and showed noticeable differences in the adult crayfish. At the same time, the crayfish began to show photomotor reflex. Later on (140-day-old crayfish) the circadian rhythm attained its final parameters.
• 5.5. The SD was used as a measure of lability in periods. The 4 hr ultradian rhythm and the 22.4 hr circadian rhythm showed the lowest SD indicating that they are the most precise period values.
• 6.6. Our results support the idea that the ERG circadian rhythm results from the coupling among high frequency (ultradian) oscillators, particularly those of 4 hr periods and that the coupling depends on the action of neurosecretions released from the sinus gland.

     

Daily variations of immunoreactive melatonin in the visual system of crayfish

In crustaceans, melatonin has been detected in the central nervous system and some other organs. The aim of this study was to analyze the melatonin content in the visual system of Procambarus clarkii, by means of radioimmunoassay, at different day-night phases. We have also studied the action of exogenous melatonin on the main properties of the electroretinogram (ERG) circadian rhythm. Experiments were conducted with 25 specimens maintained under controlled conditions of 16°C and 12 h of light alternating with 12 h of darkness. Eyes where cut in dim red light and shock frozen with liquid nitrogen and pulverized in a mortar until a homogenous powder was obtained. Melatonin was extracted with acetone, followed by centrifugation, diluted with an equal volume of equa bidest to ensure freezing at ?80°C for at least 90 min and lyophilization at the same temperature. Lyophilizates, after having been dissolved in RIA buffer, were used for determinations of melatonin. Long-term recordings of electrical responses to light (ERG) were obtained for 10 or more consecutive days. At the 5th day, a single dose of melatonin was injected and its effects on amplitude and period of the ERG circadian rhythm were measured. Melatonin concentrations differed considerably depending on the circadian time and attained a maximum during dark phase. Among the crustaceans, Procambarus clarkii represents the first case in which melatonin peaks during the night following the typical pattern known in the majority of organisms. After melatonin injection, period and amplitude of the ERG circadian rhythm were increased. This effect suggests the involvement of melatonin in the oscillators underlying the generation and expression of circadian rhythms in crayfish.     

Pigment dispersing hormone modulates spontaneous electrical activity of the cerebroid ganglion and synchronizes electroretinogram circadian rhythm in crayfish Procambarus clarkii

In crayfish, one very well-studied circadian rhythm is that of electroretinogram (ERG) amplitude. The cerebroid ganglion has been considered a plausible site for the circadian pacemaker of this rhythm and for the retinular photoreceptors, as the corresponding effectors. The pigment dispersing hormone (PDH) appears to synchronize ERG rhythm, but its characterization as a synchronizer cue remains incomplete. The main purposes of this work were a) to determine whether PDH acts on the cerebroid ganglion, and b) to complete its characterization as a non-photic synchronizer. Here we show that PDH increases the number of the spontaneous potentials of the cerebroid ganglion, reaching 149.92 ± 6.42% of the activity recorded in the controls, and that daily application of PDH for 15 consecutive days adjusts the ERG circadian rhythm period to 24.0 ± 0.2 h and the end of the activity period of the rhythm coincides with the injection of the hormone. In this work, we hypothesized that in crayfish, PDH transmits the “day” signal to the ERG circadian system and acts upon both the presumptive circadian pacemaker and the corresponding effectors to reinforce the synchronization of the system.     

Pigment dispersing hormone modulates spontaneous electrical activity of the cerebroid ganglion and synchronizes electroretinogram circadian rhythm in crayfish Procambarus clarkii

In crayfish, one very well-studied circadian rhythm is that of electroretinogram (ERG) amplitude. The cerebroid ganglion has been considered a plausible site for the circadian pacemaker of this rhythm and for the retinular photoreceptors, as the corresponding effectors. The pigment dispersing hormone (PDH) appears to synchronize ERG rhythm, but its characterization as a synchronizer cue remains incomplete. The main purposes of this work were a) to determine whether PDH acts on the cerebroid ganglion, and b) to complete its characterization as a non-photic synchronizer. Here we show that PDH increases the number of the spontaneous potentials of the cerebroid ganglion, reaching 149.92±6.42% of the activity recorded in the controls, and that daily application of PDH for 15 consecutive days adjusts the ERG circadian rhythm period to 24.0±0.2h and the end of the activity period of the rhythm coincides with the injection of the hormone. In this work, we hypothesized that in crayfish, PDH transmits the "day" signal to the ERG circadian system and acts upon both the presumptive circadian pacemaker and the corresponding effectors to reinforce the synchronization of the system.     

A qualitative mathematical model of the ontogeny of a circadian rhythm in crayfish

Based on experimental work on the ontogeny of the electroretinogram circadian rhythm in crayfish, we present a mathematical model simulating changes in both frequency and amplitude of the electroretinogram oscillation during several developmental stages until shortly before the adult age. Simultaneously, we propose a hypothetical oscillation in the hormonal release whose frequency is imposed on the electroretinogram oscillation. The model consists of two coupled nonlinear oscillators in which a dynamical response is obtained mainly through an Andronov-Hopf bifurcation. Through the construction of the model, a biological hypothesis about the essential elements underlying the ERG circadian rhythm and their interrelations is formulated and discussed.     

About the Existence of Circadian Activity in Cave Crayfish

Evidence of a circadian clock mechanism was found in the cave crayfish Procambarus cavernicola. Analysis of motor activity recorded in this species during 12 consecutive days in either free running (constant darkness, DD or constant light, LL) or entrainment conditions (12 h of light alternated with 12 h of darkness, 12 : 12 LD) showed a well recognized circadian rhythm. In this rhythm however, the absence of synchronization by periodical external signals was notorious. The comparison between the motor circadian rhythm in cave crayfish and epigeous crayfish Procambarus clarkii (these last studied during juvenile and adult stages), evidenced strong similitude between the motor circadian rhythm of cave crayfish and juvenile epigeous crayfish.     

Synchronization by light of the circadian rhythm of motor activity in the crayfish

Abstract

We have studied the pattern for resetting the circadian rhythm in the spontaneous motor activity of the crayfish. Spontaneous motor activity was recorded continously at a constant temperature and under free running conditions in complete darkness. The effect of single light pulses applied at different circadian times, on the circadian rhythm of motor activity was measured in both transient stage and steady state. The results led us to construct a phase‐transition curve and phase‐response curve which were analyzed to obtain information about the oscillators which underlie the circadian rhythm of motor activity.     

Cerebroid Ganglion is the Presumptive Pacemaker of the Circadian Rhythm of Electrical Response to Light in the Crayfish

One of the most widely studied circadian rhythms in invertebrates is that of light responsiveness whose underlying mechanisms seem to involve different groups of oscillators which act as pacemakers. Although, in crayfish, there are clear circadian rhythms in the electroretinogram (ERG) amplitude, the precise location of the pacemaker system driving this rhythm is uncertain. Some data suggest that the circadian pacemaker could be located in a group of neurosecretory cells of the supraesophageal ganglion (the cerebroid ganglion or brain) and that the sinus gland plays a determinant role in the generation and expression of this rhythm through periodic release of pigment-dispersing hormone (PDH). The aim of this work is to examine the role of the brain in the expression of the ERG circadian activity. The hypothesis we test is that the electrical activity at the brain level has a circadian behavior in the firing pattern of spontaneous multiunit activity (MUA) and in visual evoked potentials (VEPs). The results indicate that there are robust circadian rhythms in both MUA, recorded from several regions of the brain, and in the averaged VEPs recorded from the protocerebrum area. These rhythms are 180° out of phase to one another. The rhythm of VEPs showed a main peak at midnight which was in close phase relationship with the ERG amplitude rhythm.     

Effect of polarized light on the ERG circadian rhythm in crayfish

• 1.1. Crayfish subjected to constant darkness and temperature displayed an electroretinographic circadian rhythm with both non-polarized and polarized light stimuli.
• 2.2. In the ERG circadian rhythm associated with polarized light there was an observed reduction in period and increment in both amplitude and activity: rest ratio.
• 3.3. The change from non-polarized to polarized light also produced phase advances or delays in the ERG circadian rhythm depending on the circadian time when the change was introduced.
• 4.4. Separate recording of HI and HII ERG components showed that HII is always less conspicuous and more easily saturable than HI circadian rhythm.
• 5.5. These results support that: (a) the detection of polarized light contributes to extend the differences between night and day; (b) the two structures involved in the generation of HI and HII ERG components, i.e. the rhabdom and the retinular cell, operate as two independent elements of the circadian system responsible of ERG circadian rhythm.

     

On the ontogeny of the motor circadian rhythm in crayfish

In this paper we attack the problem of understanding the localization of the main structures involved in the motor circadian rhythm of crayfish by analysing its ontogeny. We present experimental results giving the properties of this rhythm in young and adult crayfish. Then we construct a mathematical model (based on a previous one for the electroretinogram circadian rhythm in the same species) simulating those properties. In the process of constructing the model we clarified and made precise various hypotheses about the biological structures involved in them and about the characteristics of the oscillators present in those structures. We also formulate some hypotheses about the general properties of circadian rhythms. Finally, we propose some experiments suggested by the mathematical model.     

Daily and circadian variation in the electroretinogram of the domestic fowl: effects of melatonin

Visual and circadian function are integrally related in birds, but the precise nature of their interaction is unknown. The present study determined whether visual sensitivity measured electroretinographically (ERG) in 7-week-old cockerels varies over the time of day, whether this rhythm persists in constant darkness (DD) and whether exogenous melatonin affects this ERG rhythmicity. ERG b-wave amplitude was rhythmic in LD and persisted in DD with peak amplitude during mid- to late afternoon in LD and mid-subjective day in DD, indicating that the ERG rhythm is endogenously generated. No daily or circadian variation in a-wave amplitude was observed, and ERG component latency and durations were not rhythmic. Intramuscular injection of 10 g/kg melatonin at ZT10 in LD significantly decreased b-wave amplitude but had no effect on a-wave. Intraocular injection of 600 pg melatonin, however, had no effect on any aspect of the ERG. These data indicate that a circadian clock regulates ocular sensitivity to light and that melatonin may mediate some or all of this effect. The level at which melatonin modulates retinal sensitivity is not known, but the present data suggest a central site rather than a direct effect of the hormone in the eye.Abbreviations DD constant darkness - ERG electroretinography - EW Edinger-Westphal nuclei - IMEL iodomelatonin - IO isthmooptic nucleus - LD light-dark cycle - SCG superior cervical ganglion - SCN suprachiasmatic nuclei - vSCN visual suprachiasmatic nucleus     

SPONTANEOUS AND LIGHT-EVOKED DISCHARGE OF THE ISOLATED ABDOMINAL NERVE CORD OF CRAYFISH IN VITRO REVEALS CIRCADIAN OSCILLATIONS

We examined the well-known spontaneous discharge (SD) and lightevoked (PD) discharge of the crayfish caudal photoreceptor for the possible existence of a daily rhythm in spike frequency. To do this, we isolated the abdominal nerve cord in vitro and studied its discharge frequency in constant darkness. Single cosinor analysis revealed significant SD and PD circadian rhythms (P <. 05) with periods τ = 24.4h and 24.2h, respectively. These oscillations correspond to an endogenous circadian discharge of the caudal photoreceptor that is enhanced by light. The importance of this rhythm in the adaptive behavior of crayfish is discussed. (Chronobiology International, 18(5), 759–765, 2001)     

A qualitative model of a motor circadian rhythm

In Nature it is possible to observe diverse rhythms. Because of their adaptive characteristics, the circadian rhythms are of major importance and have been the subject of numerous experimental and theoretical studies. In this article, we give a presentation of the main results we have obtained about the motor circadian rhythm along some years of collaboration between biologists and mathematicians. We present a mathematical model simulating changes in frequency, synchronization and amplitude of the circadian oscillation during two developmental stages of the crayfish, namely, the juvenile and the adult stages. We report also some work in progress on the simulation of the phase response curve and on a simplified model of the rhythm.     

Phase Shifting the ERG Amplitude Circadian Rhythm of Juvenile Crayfish by Caudal Monochromatic Illumination

The objective of the present work was to determine the physiological mechanisms underlying the synchronization of the ERG amplitude rhythm. Chronic ERG recordings were obtained from juvenile instars of crayfish. Changes on the ERG amplitude rhythm produced when 30 min blue light illuminated the telson were determined. The PRC obtained with these data showed advances in the early subjective night and delays in the late subjective night. These phase shiftings resemble the features of curves obtained by dark pulses in other species. The relation of this curve with PRCs generated in the crayfish and other animals species are discussed.     

Phase Shifting the ERG Amplitude Circadian Rhythm of Juvenile Crayfish by Caudal Monochromatic Illumination

The objective of the present work was to determine the physiological mechanisms underlying the synchronization of the ERG amplitude rhythm. Chronic ERG recordings were obtained from juvenile instars of crayfish. Changes on the ERG amplitude rhythm produced when 30 min blue light illuminated the telson were determined. The PRC obtained with these data showed advances in the early subjective night and delays in the late subjective night. These phase shiftings resemble the features of curves obtained by dark pulses in other species. The relation of this curve with PRCs generated in the crayfish and other animals species are discussed.     

RETINAL CIRCADIAN RHYTHMS IN HUMANS*

Circadian rhythms in the retina may reflect intrinsic rhythms in the eye. Previous reports on circadian variability in electrophysiological human retinal measures have been scanty, and the results have been somewhat inconsistent. We studied the circadian variation of the electrooculography (EOG), electroretinography (ERG), and visual threshold (VTH) in subjects undergoing a 36h testing period. We used an ultrashort sleep-wake cycle to balance effects of sleep and light-dark across circadian cycles. Twelve healthy volunteers (10 males, 2 females; mean age 26.3 years, standard deviation [SD] 8.0 years, range 19–40 years) participated in the study. The retinal functions and oral temperature were measured every 90 min. The EOG was measured in the light, whereas the ERG and the VTH were measured in the dark. Sleep was inferred from activity detected by an Actillume monitor. The EOG peak-to-peak responses followed a circadian rhythm, with the peak occurring late in the morning (acrophase 12:22). The ERG b-wave implicit time peaked in the early morning (acrophase 06:46). No statistically significant circadian rhythms could be demonstrated in the ERG a-wave implicit time or peak-to-peak amplitude. The VTH rhythm peaked in the early morning (acrophases 07:59 for blue and 07:32 for red stimuli). All retinal rhythms showed less-consistent acrophases than the temperature and sleep rhythms. This study demonstrated several different circadian rhythms in retinal electrophysiological and psychophysical measures of healthy subjects. As the retinal rhythms had much poorer signal-to-noise ratios than the temperature rhythm, these measures cannot be recommended as circadian markers. (Chronobiology International, 18(6), 957–971, 2001)     

Circadian rhythm of ERG in Iguana iguana: role of the pineal

In green iguanas, the pineal controls the circadian rhythm of body temperature but not the rhythm of locomotor activity. As part of a program to investigate the characteristics of this multioscillator circadian system, the authors studied the circadian rhythms of the electroretinographic response (ERG) and asked whether the pineal gland is necessary for the expression of this rhythm. ERGs from a total of 24 anesthetized juvenile iguanas were recorded under four different conditions: (a) complete darkness (DD), (b) dim light-dark cycles (dLD), (c) constant dim light (dLL), and (d) pinealectomized in DD. Results demonstrate that the b-wave component of the ERG shows a very clear circadian rhythm in DD and that this rhythm persists in dLL and entrains to dLD cycles. The ERG response is maximally sensitive during the subjective day. Pinealectomy does not abolish the circadian rhythm in ERG, demonstrating that the oscillator responsible for the ERG rhythm is located elsewhere.     

Transient Fluctuation of Serum Melatonin Rhythm is Suppressed Centrally by Vitamin B

Vitamin B₁₂ has been reported to improve sleep-wake rhythm disorders. Although the mechanism is still unclear, a change in the sensitivity of the circadian clock system to photic input is thought to be a possible mechanism of the effect. In this study, the effect of the vitamin B₁₂ on the circadian aspect of the electroretinogram (ERG) and serum melatonin level was analyzed in rats. Vitamin B₁₂, α-(5,6-dimethylbenzimidazolyl)-co-methyl-cobamide was daily administrated subcutaneously for 8 weeks to adult male Wister rats in the experimental group, and saline was given to the control group. The ERGs were recorded under dark adaptation during the night and day, and under light adaptation (0.1 lux) during the night. Blood was drawn before and after ERG recording. The amplitudes of the a-wave, fc-wave, and trough-to-peak of both waves and latencies of ERG were analyzed following various exposures to stimuli of light intensity. These parameters in the group treated with vitamin B₁₂ showed similar characteristics to the control group, and no significant difference was observed between the two groups. The melatonin levels of both groups before the measurement of ERG were similar under each measurement condition. The elevated serum melatonin concentration in the control group under dark adaptation at night was suppressed after the series of 10-msec light stimuli used for measurement of ERG. However, this suppressing effect of light pulses on melatonin level was significantly inhibited in the group treated with vitamin B₁₂. Under light adaptation during the night and under dark adaptation during the day, melatonin levels after the measurement of ERG were not different between the groups. From these results, it is suggested that vitamin B₁₂ is effective in suppressing melatonin rhythm disturbances introduced by transient light stimulation, and it affects the site more central than the retinal level. (Chronobiology International, 14(6), 549–560, 1997)