Beatriz Fuentes-Pardo

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.

Correlation between motor and electroretinographic circadian rhythms in the crayfish Procambarus bouvieri (ortmann)

Abstract 1. 1. The characteristics of both, motor and electroretinographic circadian rhythms in the crayfish Procambarus bouvieri, were examined. 2. 2. The correlation between both rhythms in intact and brainless crayfish, was obtained. 3. 3. The presence of at least two different but coupled oscillators responsible for the circadian variations in crayfish, is proposed.

Caudal photoreceptors synchronize the circadian rhythms in crayfish—I. Synchronization of ERG and locomotor circadian rhythms

Abstract 1. 1. Electroretinogram (ERG) and motor activity circadian rhythms in crayfish under constant environmental conditions were recorded. 2. 2. Changes on the circadian parameters induced by application of a single light stimulus of 2000 Ix and 10 min duration on either the eyestalk or the 6th abdominal ganglion were analyzed. 3. 3. The synchronization ability exerted by the caudal photoreceptors on the ERG and on motor circadian rhythms is discussed. 4. 4. In gangliectomized crayfish, there is a phase inversion in ERG circadian rhythm and an increment of the total amount of activity in motor circadian rhythm, but there is no evidence of synchronization when a single stimulus is applied on the place occupied by the removed 6th abdominal ganglion. 5. 5. It is proposed that extraocular photoreceptors can participate in the entrainment by light of the circadian rhythms of crayfish.

Role of the sinus gland in crayfish circadian rhythmicity—I. pseudopupil circadian rhythm

Abstract 1. 1. The objective of the present work was to determine the existence, if any, of a periodical release of sinus gland neurosecretions, the existence of a circadian rhythm in retinal pigment migration and the role of the sinus gland in the generation and maintenance of that circadian rhythm. 2. 2. Eyestalk extracts were prepared at different circadian times (CT) and injected into intact animals, changes on the pseudopupil size were measured after extract injection. It was found that the effect of the extracts on the pseudopupil depended on the CT of preparation of the extracts. 3. 3. In intact crayfish under controlled conditions, a circadian rhythm in pseudopupil size was observed. The phase of the rhythm was modified after the application of a synchronizer light stimulus. 4. 4. Crayfish that had one sinus gland removed showed loss of the pseudopupil circadian rhythm in the operated eye. After a synchronizer light stimulus, a tendency to revert to a circadian rhythm was observed. 5. 5. Crayfish with bilateral sinus gland ablation showed loss of the circadian rhythm of the pseudopupil from the recorded eye. A synchronizer light stimulus also produced a tendency towards circadian characteristics in this eye. 6. 6. Our results suggest that both retinal pigment position and sinus gland neurosecretion exhibit a circadian rhythm, and that the sinus gland is involved in the generation of the retinal pigment circadian rhythm.

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.

The phase response curve of electroretinographic circadian rhythm of crayfish

Abstract 1. 1. The photoreceptors electrical response to light (ERG) in the crayfish, shows a clear rhythm of 23.5 hr when it is obtained in complete darkness (except for the test light pulses) and controlled ambient temperature. 2. 2. If an external light (zeitgeber) of moderate intensity and duration is applied during a free running recording, an advance, a delay or no change of the rhythm will be observed, depending on the moment of application of the pulse. 3. 3. The delays and advances plotted against the circadian time of pulse application, generate the “phase response curve”. The unimodality of the curve thus obtained is interpreted as an adaptative feature of the electroretinographic circadian rhythm. 4. 4. The existence of a circuit involved in the control of sensitivity to light of the oscillators responsible for the ERG circadian rhythm, is postulated.

Role of the sinus gland in crayfish circadian rhythmicity—II. ERG circadian rhythm

Abstract 1. 1. The periodical release of sinus gland neurosecretions and the role of the sinus gland in the generation and maintenance of the circadian rhythm of the crayfish electroretinogram (ERG) were studied in isolated eyestalks and intact and surgically ablated animals. 2. 2. Eyestalk extracts were prepared at different circadian times (CT) and injected into isolated eyestalks. The ERG from the isolated eyestalks was recorded before and after extract injection. 3. 3. ERGs from both eyes of intact animals and animals with one or both sinus glands removed were recorded continuously for at least 7 days. 4. 4. The ERGs from isolated eyestalks that had been exposed to extract showed changes in the amplitude of the rhythm that were dependent on the CT of the preparation of the extracts. 5. 5. The ERG rhythms of intact animals were always in synchrony. In animals with one sinus gland removed, the ERG of the operated eye showed modifications in both amplitude and period and was desynchronized with respect to the intact eye, whose ERG rhythm was normal. After bilateral gland excision, both eyes showed amplitude and period variations of the ERG, which was also desynchronized. 6. 6. The results suggest that the sinus gland is a circadian oscillator participating in the multioscillator system that generates and maintains the ERG circadian rhythm.

ERG circadian rhythm in the course of ontogeny in crayfish

Abstract 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.

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.

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.