Julio Prieto-Sagredo

effect of variation in photoperiod and light intensity on oxygen consumption, lactate concentration and behavior in crayfish procambarus clarkii and procambarus digueti

The effects of light intensity and duration on metabolic and behavioral parameters of two species of crayfish, Procambarus clarkii and Procambarus digueti, were studied. Sixty animals of each species were submitted to high irradiance conditions of two different photoperiod lengths, one normal light/dark (LD) 12:12 and one extreme LD 20:4 for 2 weeks. Hemolymph, lactate and oxygen consumption were determined throughout the experimental period. Simultaneously in 18 additional animals of each species, motor activity was individually recorded under the same control and experimental conditions. Both species showed a decrease in oxygen uptake and an increase in hemolymph lactate concentration. The statistical significance of this finding was higher for LD 20:4. This extreme condition evoked a significant decrease of motor activity in P. clarkii and a high mortality rate in P. digueti. P. digueti did not survive after the experiment, whereas P. clarkii survived and adapted to the laboratory conditions. Changes in metabolic and behavioral parameters could indicate different adaptation abilities in these species.

Crayfish Procambarus clarkii shows Circadian Variations in Different Parameters of the GSH Cycle

The present study investigated the rhythmic changes in glutathione status in midgut gland and hemolymph as well as in glutathione reductase (GR) activity in the crayfish Procambarus clarkii. In order to determine the circadian nature of these rhythms different groups of crayfish were submitted to constant‐darkness conditions for 24 or 72 h after they had spent 15 days under light–dark 12:12 cycles. The animals of the different batches were killed at 6 h intervals during a 24 h cycle. Reduced glutathione (GSH) and oxidized glutathione (GSSG) in hemolymph and midgut as well as midgut GR activity were determined in midgut gland and hemolymph by fluorometric and spectrophotometric methods. Data analysis by chronogram and single Cosinor revealed circadian rhythmicity for GSH and GSSG concentration in both tissues as well as midgut GR activity. The rhythm parameters revealed oxidative stress induced by light. The possible correlation between the glutathione rhythm and other metabolic and behavioral rhythms of crayfish as well as the importance of the glutathione circadian temporal order in the adaptation of crayfish are discussed.

Crayfish Procambarus clarkii Retina and Nervous System Exhibit Antioxidant Circadian Rhythms Coupled with Metabolic and Luminous Daily Cycles

Based on previous work in which we proposed midgut as a putative peripheral oscillator responsible for circadian reduced glutathione (GSH) crayfish status, herein we investigated the retina and optic lobe‐brain (OL‐B) circadian GSH system and its ability to deal with reactive oxygen species (ROS) produced as a consequence of metabolic rhythms and light variations. We characterized daily and antioxidant circadian variations of the different parameters of the glutathione system, including GSH, oxidized glutathione (GSSG), glutathione reductase (GR) and glutathione peroxidase (GPx), as well as metabolic and lipoperoxidative circadian oscillations in retina and OL‐B, determining internal and external GSH‐system synchrony. The results demonstrate statistically significant bi‐ and unimodal daily and circadian rhythms in all GSH‐cycle parameters, substrates and enzymes in OL‐B and retina, as well as an apparent direct effect of light on these rhythms, especially in the retina. The luminous condition appears to stimulate the GSH system to antagonize ROS and lipid peroxidation (LPO) daily and circadian rhythms occurring in both structures, oscillating with higher LPO under dark conditions. We suggest that the difference in the effect of light on GSH rhythmic mechanisms of both structures for antagonizing ROS could be due to differences in glutathione‐system coupling strength with the circadian clock.

Changes in Hemolymph Glutathione Status After Variation in Photoperiod and Light-irradiance in Crayfish Procambarus clarkii and Procambarus digueti

This work studied the effect of light‐stressors, irradiance and photoperiod length on the status of hemolymph glutathione in two species of crayfish, Procambarus clarkii and Procambarus digueti. Adult animals of each species were submitted to two experimental approaches: (1) two batches of each species were placed under low or high light irradiant conditions of light–dark (LD) 24 h cycles of two different photoperiod lengths, one normal LD 12:12 and one extreme LD 20:4 low and high irradiance for 10 weeks. Time‐dependent light changes on hemolymph glutathione concentration were determined throughout the entire experimental period; and (2) three batches of the two species were submitted to independent treatments consisting of the same LD 12:12 cycles of high and low irradiance and 20:4 high‐irradiance LD cycles. Reduced and oxidized glutathione hemolymph concentrations were determined and total glutathione was calculated. In addition midgut glutathione reductase activity in both species was determined. The two species showed different hemolymph glutathione reactivity and glutathione status for the two light parameters. Dissimilar responses of both species, as well as the rate of mortality of P. digueti represent specific differences in the metabolic responses, as well as tolerance to photo‐oxidative stress produced by light. The role of glutathione in the tolerance of crayfish to photo‐oxidative stress is discussed.

Putative Pacemakers in the Eyestalk and Brain of the Crayfish Procambarus clarkii Show Circadian Oscillations in Levels of mRNA for Crustacean Hyperglycemic Hormone

Crustacean hyperglycemic hormone (CHH) synthesizing cells in the optic lobe, one of the pacemakers of the circadian system, have been shown to be present in crayfish. However, the presence of CHH in the central brain, another putative pacemaker of the multi-oscillatory circadian system, of this decapod and its circadian transcription in the optic lobe and brain have yet to be explored. Therefore, using qualitative and quantitative PCR, we isolated and cloned a CHH mRNA fragment from two putative pacemakers of the multi-oscillatory circadian system of Procambarus clarkii, the optic lobe and the central brain. This CHH transcript synchronized to daily light-dark cycles and oscillated under dark, constant conditions demonstrating statistically significant daily and circadian rhythms in both structures. Furthermore, to investigate the presence of the peptide in the central brain of this decapod, we used immunohistochemical methods. Confocal microscopy revealed the presence of CHH-IR in fibers and cells of the protocerebral and tritocerebal clusters and neuropiles, particularly in some neurons located in clusters 6, 14, 15 and 17. The presence of CHH positive neurons in structures of P. clarkii where clock proteins have been reported suggests a relationship between the circadian clockwork and CHH. This work provides new insights into the circadian regulation of CHH, a pleiotropic hormone that regulates many physiological processes such as glucose metabolism and osmoregulatory responses to stress.

Out-of-Hospital Cardiac Arrest: First Documented Experience in a Mexican Urban Setting

OBJECTIVE Out-of-hospital cardiac arrest is one of the leading causes of death in Mexico, but many survival and prognostic factors are unknown. The aim of this study was to assess out-of-hospital cardiac arrest in a Mexican city. METHODS This was a prospective, cohort study that evaluated the records of the major ambulance services in the city of Queretaro, Mexico. Means, standard deviation, and percentages for the categorical variables were obtained. Logistic regression was performed to determine the effects between interventions, times, and return of spontaneous circulation (ROSC). RESULTS For an 11-month period, 148 out-of-hospital cardiac arrest cases were recorded. The mean age of the victims was 54 +/- 22.6 years and 90 (65.3%) were males. Forty-nine cases were related to cardiac disease, 46 to other disease, 27 to trauma, 18 to terminal illnesses, and three to drowning. Twelve (8.6%) patients had a pulse upon hospital arrival, but none survived to discharge. No victims were defibrillated prior to ambulance arrival. The collapse-assessment interval was 22.5 +/- 19:1 minutes, the mean value for the ambulance response times was 13:6 +/-10:4 minutes. Basic emergency medical technicians applied chest compressions to 40 victims (27.2%), controlled the airway in 32 (21.8%), and defibrillated seven (4.8%). Chest compressions and airway control showed an OR of 8 and 12 respectively for ROSC. CONCLUSIONS The poor survival rate in this study emphasizes the need to improve efforts in provider training and public education. Authorities must promote actions to enhance prehospital emergency services capabilities, shorten response times, and provide community education to increase the chances of survival for out-of-hospital cardiac arrest victims in Mexico.

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)

PHOTOPERIODIC INDUCTION OF OVARIAN MATURATION IN CRAYFISH PROCAMBARUS CLARKII IS MEDIATED BY EXTRARETINAL PHOTORECEPTION

The current study was carried out to investigate whether thephotoperiodic induction of ovarian maturation in crayfish is based on a photosensitiverhythm related to extraretinal photoreceptors. To test this, two batches of61 juvenile crayfish Procambarus clarkiiconsisting of intact organisms and animals lacking retina and lamina were exposed to 24hlight-dark cycles of different photoperiodic schedules based on a night-breakprotocol for 3 months. Both batches of crayfish showed the greatest ovarianmaturation (size, color, degree and size of oocytes) when the light pulseinterrupted the scotophase at 21:00 and 05:00, showing a bimodal photoinduciblerhythm. Results of the current study indicate that crayfish ovarian maturationdepends on a photoinducible rhythm with two possible states that is relatedto the circadian clock of crayfish. This phenomenon is mediated by extraretinalphotoreceptors. Results are interpreted in the light of models of externalcoincidence. (Chronobiology International, 18(3),423–434, 2001)

Temperature pulses synchronise the crayfish locomotor activity rhythm

Unrestrained crayfish maintained in the dark exhibit a unimodal circadian activity that freely runs with a period of approximately 24 h rhythm that is able to entrain light–dark cycles. Although the activity rhythm has been shown to compensate for temperature, its potential for temperature synchronisation is unknown. In this work, we analysed the effects of cold-water pulses on crayfish locomotor activity. The crayfish were maintained under a 12:12 light–dark (LD) cycle and 30-min cold (5°C) water pulses were applied at three different circadian times (CTs). The crayfish were subsequently kept in darkness. Cold-water pulses applied at the beginning of the subjective night produced phase advances whereas pulses applied at the end of the subjective night produced phase delays. The magnitudes of the phase shifts were similar to those produced by light pulses but the direction was a mirror image. These findings suggest that the temperature- and light-synchronising pathways differ in crayfish.

Crayfish brain-protocerebrum and retina show serotonergic functional relationship.

The results from various studies have indicated possible functional relationships between crayfish electroretinogram (ERG) rhythmic amplitude changes and the serotonergic pathways projecting from the central brain through the optic neuropils to the eye, but to date, this functional interaction has not been proven. Here, in a set of experiments using an isolated eyestalk-brain preparation, we investigated whether there is a circadian input from the brain to retina that regulates this rhythm. We sought to determine whether the protocerebral bridge (PB) stimulation affects the ERG amplitude in accordance with the zeitgeber time (ZT) and whether 5-HT modulates the associate input. Our results showed that photic stimulation of retina produced changes in both the amplitude and the frequency of spontaneous electrical activity in the protocerebral neuropils. In addition, electrical stimulation of the medial protocerebrum, particularly the PB, produced statistically significant changes in the ERG that depended on both the time of day and the level of serotonin. This suggests that pathways between retina and PB seem to be serotonergic.