Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Involvement of Melatonin in the Regulation of the Circadian System in Crayfish.

Melatonin (MEL) is an ancient molecule, broadly distributed in nature from unicellular to multicellular species. MEL is an indoleamine that acts on a wide variety of cellular targets regulating different physiological functions. This review is focused on the role played by this molecule in the regulation of the circadian rhythms in crayfish. In these species, information about internal and external time progression might be transmitted by the periodical release of MEL and other endocrine signals acting through the pacemaker. We describe documented and original evidence in support of this hypothesis that also suggests that the rhythmic release of MEL contributes to the reinforcement of the temporal organization of nocturnal or diurnal circadian oscillators. Finally, we discuss how MEL might coordinate functions that converge in the performance of complex behaviors, such as the agonistic responses to establish social dominance status in Procambarus clarkii and the burrowing behavior in the secondary digging crayfish P. acanthophorus.

Circadian rhythm in melatonin release as a mechanism to reinforce the temporal organization of the circadian system in crayfish.

Melatonin (MEL) is a conserved molecule with respect to its synthesis pathway and functions. In crayfish, MEL content in eyestalks (Ey) increases at night under the photoperiod, and this indoleamine synchronizes the circadian rhythm of electroretinogram amplitude, which is expressed by retinas and controlled by the cerebroid ganglion (CG). The aim of this study was to determine whether MEL content in eyestalks and CG or circulating MEL in hemolymph (He) follows a circadian rhythm under a free-running condition; in addition, it was tested whether MEL might directly influence the spontaneous electrical activity of the CG. Crayfish were maintained under constant darkness and temperature, a condition suitable for studying the intrinsic properties of circadian systems. MEL was quantified in samples obtained from He, Ey, and CG by means of an enzyme-linked immunosorbent assay, and the effect of exogenous MEL on CG spontaneous activity was evaluated by electrophysiological recording. Variation of MEL content in He, Ey, and CG followed a circadian rhythm that peaked at the same circadian time (CT). In addition, a single dose of MEL injected into the crayfish at different CTs reduced the level of spontaneous electrical activity in the CG. Results suggest that the circadian increase in MEL content directly affects the CG, reducing its spontaneous electrical activity, and that MEL might act as a periodical signal to reinforce the organization of the circadian system in crayfish.

Desensitization and recovery of crayfish photoreceptors. Dependency on circadian time, and pigment-dispersing hormone.

In this work, we studied the characteristics of recovery from desensitization of the light-elicited current of crayfish. Applying a two-flash protocol, we found that the first flash triggers a current that activates with a noticeable latency, reaches a peak value, and thereafter decays along a single exponential time course. In comparison with the first-elicited current, the current elicited by the second flash not only presents an expected smaller peak current, depending on the time between flashes, but it also displays a different latency and decay time constant. Recovery of the first flash values of these current parameters depends on the circadian time at which the experiments are conducted, and on the presence of pigment-dispersing hormone. Our data also suggest the existence of distinctive desensitized states, whose induction depends on circadian time and the presence of pigment-dispersing hormone.

Modeling some properties of circadian rhythms.

Mathematical models have been very useful in biological research. From the interaction of biology and mathematics, new problems have emerged that have generated advances in the theory, suggested further experimental work and motivated plausible conjectures. From our perspective, it is absolutely necessary to incorporate modeling tools in the study of circadian rhythms and that without a solid mathematical framework a real understanding of them will not be possible. Our interest is to study the main process underlying the synchronization in the pacemaker of a circadian system: these mechanisms should be conserved in all living beings. Indeed, from an evolutionary perspective, it seems reasonable to assume that either they have a common origin or that they emerge from similar selection circumstances. We propose a general framework to understand the emergence of synchronization as a robust characteristic of some cooperative systems of non-linear coupled oscillators. In a first approximation to the problem we vary the topology of the network and the strength of the interactions among oscillators. In order to study the emergent dynamics, we carried out some numerical computations. The results are consistent with experiments reported in the literature. Finally, we proposed a theoretical framework to study the phenomenon of synchronization in the context of circadian rhythms: the dissipative synchronization of nonautonomous dynamical systems.

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.

Effect of pigment dispersing hormone on the electrical activity of crayfish visual photoreceptors during the 24-h cycle.

Visual photoreceptors are structures involved in the expression and synchronization of crayfish circadian rhythm of sensitivity to light (electroretinogram, ERG). Considering the relevant role of Pigment dispersing hormone (PDH) in the invertebrate circadian system organization, we study the effect of this substance on the electrical activity of crayfish visual photoreceptors during the 24-h cycle. The study demonstrates that: (1) PDH affects the electrical response to light of crayfish visual photoreceptor cells in a circadian time-dependent manner. (2) The kinetics of the light-elicited current of crayfish visual photoreceptor cells, as well as the ionic permeability underlying the electrical response to light vary over the 24-h cycle. (3) PDH modifies the kinetics and ionic permeability underlying the light-elicited current of crayfish visual photoreceptor cells in a circadian time-dependent manner.

MT2-like melatonin receptor modulates amplitude receptor potential in visual cells of crayfish during a 24-hour cycle.

Retinular photoreceptors are structures involved in the expression and synchronization of the circadian rhythm of sensitivity to light in crayfish. To determine whether melatonin possesses a differential effect upon the receptor potential (RP) amplitude of retinular photoreceptors circadian time (CT)-dependent, we conducted experiments by means of applying melatonin every 2h during a 24-hour cycle. Melatonin with 100 nM increased RP amplitude during subjective day to a greater degree than during subjective night. To determine whether MT(2) melatonin receptors regulate the melatonin-produced effect, we carried out two experiments, circadian times (CTs) 6 and 18, which included the following: (1) application of the MT(2) receptor selective agonist 8-M-PDOT and antagonist DH97, and (2) the specific binding of [(125)I]-melatonin in eyestalk membranes. The amount of 10 nM of 8-M-PDOT increased RP amplitude in a similar manner to melatonin, and 1 nM DH97 abolished the increase produced by melatonin and 8-M-PDOT. Binding of [(125)I]-melatonin was saturable and specific. Scatchard analysis revealed an affinity constant (K(d)) of 1.1 nM and a total number of binding sites (B(max)) of 6 fmol/mg protein at CT 6, and a K(d) of 1.46 nM and B(max) of 7 fmol/mg protein at CT 18. Our results indicate that melatonin increased RP amplitude of crayfish retinular photoreceptors through MT(2)-like melatonin receptors. These data support the idea that melatonin is a signal of darkness for the circadian system in crayfish retinular cells.

La desincronización interna como promotora de enfermedad y problemas de conducta / Internal desynchrony as promotor of disease and behavioral disturbance

Life on our planet is ruled by a temporary structure that governs our activities, our days and our calendars. In order to cope with a daily changing environment, organisms have developed adaptive strategies by exhibiting daily behavioral and physiological changes. Biological rhythms are properties conserved in all the levels of organization, from unicellular to prokaryotes to upper plants and mammals. A biological rhythm is defined as the recurrence of a biological phenomenon in regular intervals of time. Biological rhythms in behaviour and physiology are controled by an internal clock which synchronizes its oscillations to external time cues that have the capacity to adjust the clock's mechanism and keep it coupled to external fluctuations. The suprachiasmatic nucleus (SCN) of the hypothalamus in mammals is the master circadian clock which is mainly entrained by the light-dark cycle. The SCN transmits time signals to the brain and then to the whole body and by means of its time signals the SCN keeps a temporal order in diverse oscillations of the body and adjusted to the light-dark cycle. The correct temporal order enables an individual to adequate functioning in harmony with the external cycles. Biological rhythms have a hereditary character, thus its expression is genetically determined. All animals, plants, and probably all organism show some type of physiological rhythmic variation (metabolic rate, production of heat, flowering, etc.) that allow for the adaptation to a rhythmic environment. Biological rhythms enable individuals to anticipate and to be prepared to the demands of the prominent cyclic environmental changes, which are necessary for survival. Also, biological rhythms promote showing maximum levels of a physiological variable at the right moment when the environment requires a maximal response. In humans, an example of circadian rhythms is the sleep-wake cycle; simultaneously, a series of physiological changes are exhibited, also with circadian characteristics (close to 24 hours). Circadian oscillations are observed in the liberation of luteinizant hormone, in plasma cortisol, leptin, insulin, glucose and growth hormone just to mentions some examples. The SCN controls circadian rhythmicity via projections to the autonomic system and by controlling the hypothalamus-adenohipofisis-adrenal axis. In this way, the SCN transmits phase and period to the peripheral oscillators to maintain an internal synchrony. Modern life favors situations that oppose the time signals in the environment and promote conflicting signals to the SCN and its effectors. The consequence is that circadian oscillators uncouple from the master clock and from the external cycles leading to oscillations out of synchrony with the environment, which is known as internal desynchronization. The consequence is that physiological variables reach their peak expression at wrong moments according to environmental demands leading then to deficient responses and to disease in the long run. Also, levels of attention, learning and memory reach peak expression at wrong moments of the day leading individuals to exhibit a deficient performance at school or work. The disturbed sleep patterns promote fatigue and irritability, which difficult social interaction. Internal desynchronization results from transmeridional traveling for which people pass multiple hourly regions. This results in an abrupt change in the time schedule and a syndrome known as <>. Frequent travelers complain about difficulties to adjust their sleep-wake cycle to the new schedule, thus resulting in fatigue, increased sleepiness and reduced attention. Jet lag results from a loss of synchrony among biological rhythms and among diverse functions, which remain out of phase with the day-night cycle. This <> is the cause of general discomfort, decrement in the physical and mental performance, as well as irritability and depression. Frequently, gastrointestinal disorders are a by-product of food consumption at an unusual schedule. The state of internal desynchrony is transitory and depends on the number of time zones that were crossed; thus, adaptation to a new external cycle can take from four to seven days. Another example of internal desynchrony is observed in individuals exposed to work shifts or to nocturnal work schedules (night work). In such conditions, circadian fluctuations in behavioral, hormonal and metabolic parameters are observed but their temporary relation with the external cycles is modified. The internal synchrony is thus affected by troubled environmental signs, out of phase with the daily activities of the individual; among them are the hours of food intake, the exposure to light during resting hours, the low temperature of the night, and the forced activity when homeostatic processes indicate a need to rest. This internal desynchrony leads to gastrointestinal disorders, disturbed metabolic fluctuations, disturbed cardiovascular functions, altered menstrual cycle, sleep disorders, sleepiness, increase of work accidents, etc. Internal desynchrony is especially due to the fact that circadian fluctuations are influenced by daily external cycles, but also by homeostatic factors, and can suffer from additional disturbance by sleep deprivation. Despite years of night work experience, incapacity to adapt to night work may persist. Only a minority of shift workers achieve spontaneous adjustment of the rhythms of core body temperature, melatonin, cortisol, thyroid stimulating hormone, or prolactin secretion to shifts by nocturnal work. Therefore shift and night workers develop a propensity to smoke, drink alcoholic beverages and use stimulant products. After five years of shift or night work, health problems appear with a higher incidence than in the general population. The growing social demand of shift work makes it necessary to decide on the characteristics and forms of shifts to carry out, and up to now organizing such working schedules remaing a serious problem. The improvement of health services has increased life expectancies and thus the general population is becoming old and people survive more years. Older people ail from health and behavioral problems including a deterioration of the biological rhythms. Main alterations consist of a loss of expression of the circadian functions or a decrease of the amplitude of the rhythms, and instability of synchronization mechanisms day by day. All in all, this implies a decreased capacity of the clock to adjust to the solar day. The decreased efficacy of the aging biological clock is evident in the fragmented sleep patterns and the disturbed sleep/wake rhythms, characterized by short sleep episodes during the day and decreased sleep during the night. Some studies suggest that the disturbed circadian rhythms may be the cause of diverse diseases associated with the elderly. In conclusion, during the last 100 years we have changed our lifestyle so radically that we lack already a physiological design to adapt so quickly to modernity. We can state that our body is designed for a world that does not exist. In this article we present a review of the main alterations of the biological rhythms generated by the transmeridional trips, shift-work and aging, their behavioral and physiological consequences that lead to disease and poor mental performance. We also discuss possible strategies that need to be explored and that may help people to improve their quality of life and to prevent internal desynchrony.

La vida se rige por una estructura temporal que gobierna nuestras horas, nuestros días y nuestros calendarios. Como parte de la adaptación a los ciclos de tiempo que impone el planeta, todo organismo presenta ritmos en su actividad y fisiología. Los ritmos biológicos son una propiedad conservada en todos los niveles de organización, desde organismos unicelulares procariontes hasta plantas superiores y mamíferos. De ellos, los más sólidos son aquellos asociados a los ciclos externos por la alternancia del día y la noche y por la alternancia de las estaciones del año. Los ritmos biológicos fisiológicos y conductuales son procesos dependientes de un reloj interno capaz de ajustar sus oscilaciones a claves de tiempo externas que lo mantienen sincronizado a estas fluctuaciones externas. El núcleo supraquiasmático del hipotálamo (NSQ) es en los mamíferos el principal reloj circadiano y se sincroniza principalmente por el ciclo luz-oscuridad. El NSQ transmite señales de tiempo al cerebro y de ahí al resto del organismo, y por medio de estas señales de tiempo mantiene un orden temporal en diversas funciones del cuerpo y las mantiene ajustadas al ciclo luz-oscuridad. El correcto orden temporal interno permite un adecuado funcionamiento del individuo en armonía con el medio externo y le permite exhibir respuestas adecuadas a un ambiente cambiante y predecible. El estilo de vida del hombre moderno propicia situaciones que llevan a alteraciones de nuestros ritmos biológicos que causan una desadaptación temporal, que a su vez redunda en daños a la salud, ya que afecta tanto la fisiología como la forma en que organizamos nuestra conducta. Un ejemplo de ello son los viajes a través de múltiples regiones horarias. Estos cambios de horario bruscos provocan un síndrome conocido como jet-lag, que consiste en un conflicto transitorio entre el tiempo <> y el tiempo <>, lo cual se denomina <>. El jet-lag se define como un conjunto de síntomas causados por una alteración del patrón de sueño, y de la expresión de ritmos biológicos fuera de fase entre sí y fuera de fase con el ciclo del día y la noche. Esta es la causa del malestar general, el deterioro del desempeño mental y físico, así como de la irritabilidad y depresión. Son frecuentes también las alteraciones gastrointestinales, resultado del consumo de alimento en un horario inusual. Otro ejemplo de alteraciones en los ritmos circadianos se observa en los trabajadores con turnos rotatorios o en turnos nocturnos. En estas condiciones se produce un conflicto entre las señales temporales asociadas al ciclo diurno y que transmite el reloj con las actividades y alimentos del trabajador en turnos. De este esquema de trabajo resulta una reducción de las horas de sueño y una alteración de los ritmos circadianos, que llevan a una desincronización interna. Ésta, al igual que en el caso del jet-lag, redunda en un deterioro de las funciones mentales y de la capacidad de atención y memorización, que se asocian a irritabilidad y problemas emocionales. Además, se observan consecuencias en la salud con incremento en la incidencia de malestares gastrointestinales, enfermedades cardiovasculares, obesidad y diabetes. La mejoría en los servicios de salud ha incrementado las expectativas de vida, lo que entonces enfrenta a la humanidad a una población que logra sobrevivir muchos años de su vejez con los cambios de conducta y salud propios de su edad, entre los que se incluye un deterioro de los ritmos biológicos. En este trabajo presentamos una revisión de las principales alteraciones de los ritmos biológicos generadas por los viajes transmeridionales, la vejez y el trabajo en turnos. También discutimos la relevancia de una buena adaptación de los ritmos biológicos y las consecuencias conductuales y fisiológicas que por su alteración llevan a la enfermedad y a un desempeño mental deficiente. También sugerimos estrategias que necesitan ser exploradas y que podrían ayudar prevenir la desincronización interna para mejorar la calidad de vida.

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.

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.