Elsa G. Escamilla-Chimal

The crayfish Procambarus clarkii CRY shows daily and circadian variation

SUMMARY The circadian rhythms of crayfish are entrained by blue light, through putative extra retinal photoreceptors. We investigated the presence and daily variation of CRY, a protein photosensitive to blue light spectra and ubiquitous in animals and plants, in the putative pacemakers of Procambarus clarkii, namely the eyestalk and brain, at different times of the 24 h light:dark cycles. Using different experimental light protocols and by means of qualitative/quantitative immunofluorescence anatomical and biochemical methods, we identified CRY immunoreactivity in cells located in the medulla-terminalis-hemiellipsoidal complex (MT-HB) and the anterior margin of the median protocerebrum (PR). The immunoreaction varied with the time of day and the two neural structures showed a semi-mirror image. The results of the biochemical analysis matched these variations. Western blotting demonstrated statistically significant circadian rhythms in brain CRY abundance, but no daily circadian CRY abundance oscillations in the eyestalk. These immunocytochemical and biochemical results link a specific photoreceptor molecule to circadian rhythmicity. We propose that CRY may be linked to the photoreception of the clock and to the generation of circadian rhythmicity.

putative pacemakers of crayfish show clock proteins interlocked with circadian oscillations

SUMMARY Although the molecular mechanisms that control circadian rhythms in many animals, particularly in the fly, are well known, molecular and biochemical studies addressing the location and function of the proteins and genes contributing to the cycling of the clock in crayfish Procambarus clarkii are scarce. In this study, we investigated whether three proteins that interact in the feedback loop of the molecular clock described for Drosophila are expressed in the putative circadian pacemakers of crayfish retina, eyestalk and brain and whether their expression cycles in a manner consistent with elements of the circadian clock. Here we identified PER, TIM and CLK immunoreactivity in the cytoplasm and nucleus of cells located in the retina as well as in clusters of cells and neuropils of the optic ganglia, lateral protocerebrum and brain. Brain clusters 6, 10, 9 and 11, in particular, showed Per, Tim and Clk-like immunoreactivity at the perikarya and nucleus, and these antigens colocalized at Zeitgeber time (ZT) 0 and/or ZT 12. A biochemical assay demonstrated circadian functionality of Per, Tim and Clk proteins. Both in the eyestalk and in the brain, these proteins demonstrated apparent daily and circadian rhythms. The presence and colocalization of these clock proteins in the cytoplasm and/or nucleus of several cells of retina, optic lobe and brain, depending on time, as well as their circadian oscillations, suggest interactions between positive and negative transcription factors and clock proteins similar to those forming the feedback loop of the canonical model proposed for different animals.

CIRCADIAN MODULATION OF CRUSTACEAN HYPERGLYCEMIC HORMONE IN CRAYFISH EYESTALK AND RETINA

Previous studies suggested the retina could be a putative locus of daily crustacean hyperglycemic hormone (CHH) secretion, as it possesses its own metabolic machinery and is independent of the well-known CHH eyestalk locus responsible for the circadian secretion of this peptide. However, it has been proposed that hemolymph glucose and lactate concentrations play a dual role in the regulation of CHH in crayfish. To elucidate the temporal relationship between these two different CHH production loci and to examine their relationship with glucose regulation, we investigated the expression of CHH daily and circadian rhythms in the eyestalk and retina of crayfish using biochemical methods and time series analysis. We wanted to determine whether (1) putative retina and eyestalk CHH rhythmic expressions are correlated and if the oscillations of the two metabolic products of lactate and glucose in the blood due to CHH action on the target tissue correlate, and (2) retina CHH (RCHH) and the possible retinal substrate glycogen and its product glucose are temporally correlated. We found a negative correlation between daily and circadian changes of relative CHH abundance in the retina and eyestalk. This correlation and the cross-correlation values found between eyestalk CHH and hemolymph and glucose confirm that CHH produced by the X-organ sinus gland complex is under the previously proposed dual feedback control system over the 24 h time period. However, the presence of both glycogen and glucose in the retina, the cross-correlation values found between these parameters and hemolymph lactate and glucose, as well as RCHH and hemolymph and retina metabolic markers suggest RCHH is not under the same temporal metabolic control as eyestalk CHH. Nonetheless, their expression may be linked to common rhythms-generating processes. (Author correspondence: mlfm@hp.fciencias.unam.mx; mfajul@gmail.com)

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)

The Effect of Chronic Ozone Exposure on the Activation of Endoplasmic Reticulum Stress and Apoptosis in Rat Hippocampus

The chronic exposure to low doses of ozone, like in environmental pollution, leads to a state of oxidative stress, which has been proposed to contribute to neurodegenerative disorders, including Alzheimer’s disease (AD). It induces an increase of calcium in the endoplasmic reticulum (ER), which produces ER stress. On the other hand, different studies show that, in diseases such as Alzheimer’s, there exist disturbances in protein folding where ER plays an important role. The objective of this study was to evaluate the state of chronic oxidative stress on ER stress and its relationship with apoptotic death in the hippocampus of rats exposed to low doses of ozone. We used 108 male Wistar rats randomly divided into five groups. The groups received one of the following treatments: (1) Control (air); (2) Ozone (O3) 7 days; (3) O3 15 days; (4) O3 30 days; (5) O3 60 days; and (6) O3 90 days. Two hours after each treatment, the animals were sacrificed and the hippocampus was extracted. Afterwards, the tissue was processed for western blot and immunohistochemistry using the following antibodies: ATF6, 78 kDa glucose-regulated protein (GRP78) and caspase 12. It was also subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and electronic microscopy. Our results show an increase in ATF6, GRP78 and caspase 12 as well as ER ultrastructural alterations and an increase of TUNEL positive cells after 60 and 90 days of exposure to ozone. With the obtained results, we can conclude that oxidative stress induced by chronic exposure to low doses of ozone leads to ER stress. ER stress activates ATF6 inducing the increase of GRP78 in the cytoplasm, which leads to the increase in the nuclear translocation of ATF6. Finally, the translocation creates a vicious cycle that, together with the activation of the cascade for apoptotic cell death, contributes to the maintenance of ER stress. These events potentially contribute in the neurodegeneration processes in diseases like AD.

photic regulation of c-fos expression in the protocerebrum of crayfish

In various species of crayfish it has been proposed that different extra-retinal photoreceptors and pacemakers could be involved in the photo entrainment mechanisms. The identity of the neural structures responsible for this phenomenon is not known. The objective of this work was to contribute to the knowledge of the neurophysiological mechanisms and structures underlying the biological clock of crayfish by studying c-fos induction in one of the neural structures proposed as a pacemaker, the brain. Using qualitative – quantitative immunohistochemical and image methods we identified light-induced c-Fos immunoreactivity in cells located in the median protocerebrum of the supraesophagic ganglion. The immunoreaction varied with circadian time. The protocerebrum showed a statistically significant c-Fos induction, especially in neurons of the dorsal anterior region and the protocerebral bridge. A statistically significant difference between the control and the experimental conditions indicated a differential sensitivity in the early subjective night. This is the first report on c-Fos expression in crayfish and indicates that this marker may be a useful tool for studying the circadian clock in this organism.

Daily Light–Dark Cycles Influence Hypoxia‐Inducible Factor 1 and Heat Shock Protein Levels in the Pacemakers of Crayfish

It has been proposed that at moderate concentrations, reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. Many of the ROS‐mediated responses, such as those mediated by the glutathione (GSH) system, protect cells against oxidative stress and reestablish “redox homeostasis.” The response to stress is controlled by specific transcription factors, such as hypoxia‐inducible factor 1 (HIF‐1), which interacts with chaperones such as heat shock proteins (HSP) that stabilize intracellular components. We have reported that the oxidation produced by extreme light cycles was coupled to cellular fluctuations in the production of free radicals and ROS in crayfish. Herein, we investigated whether ROS resulting from light–dark cycles (20:4 LD) with a long photoperiod activated HIF‐1α in the putative pacemakers of crayfish. Our results indicate that both the light resulting from equatorial and extreme daily light cycles and the constant darkness‐induced HIF‐1α and heat shock protein 70 (HSP70) appeared to regulate each other. The interaction between these proteins and the ability of crayfish to shift from the oxidative to glycolytic pathways, thereby synchronizing to extreme illumination conditions and maintaining a rhythmic predictive relationship with the environment, suggest HIF‐1 as a key factor in these rhythmic metabolic interactions.

Crustacean hyperglycemic hormone is synthesized in the eyestalk and brain of the crayfish Procambarus clarkii

Crustacean hyperglycemic hormone (CHH) is a neuropeptide that is synthesized, stored, and released by brain and eyestalk structures in decapods. CHH participates in the regulation of several mechanisms, including increasing the level of glucose in hemolymph. Although CHH mRNA levels have been quantified and the CHH protein has been localized in various structures of the crayfish P. clarkii, CHH synthesis has only been reported in the X-organ-sinus gland (XO-SG). Therefore, the aim of this study was to use in situ hybridization to determine whether CHH mRNA is located in other structures, including the putative pacemaker, eyestalk and brain, of crayfish P. clarkii at two times of day. CHH mRNA was observed in both the eyestalk and the brain of P. clarkii, indicating that CHH is synthesized in several structures in common with other crustaceans, possibly to provide metabolic support for these regions by increasing glucose levels.

Glial fibrillary acidic protein (GFAP) shows circadian oscillations in crayfish Procambarus clarkii putative pacemakers

Although several studies of glia have examined glial fibrillary acid protein (GFAP) and its relationship to the circadian rhythms of different organisms, they have not explored the daily GFAP oscillations in the putative pacemakers of the crayfish Procambarus clarkii or in other crustaceans. In this study we investigated the daily variations in GFAP concentrations in the eyestalk and brain, which are considered to be putative pacemakers in adult P. clarkii. In both structures, the glial GFAP was quantified using the indirect enzyme-linked immunosorbent assay (ELISA), and double labeling immunofluorescence was used to detect it and its co-localization with protein Period (PER), an important component of the circadian clock, in various regions of both structures. The ELISA results were analyzed using Cosinor and one-way ANOVA with Bonferroni and Scheffé’s post hoc tests. The results of this analysis showed that the GFAP levels present circadian oscillations in both structures. Moreover, GFAP was localized in different structures of the eyestalk and brain; however, co-localization with PER occurred only in the lamina ganglionaris, specifically in the cartridges of the eyestalk and in some of the cluster 9 brain cells. These results suggest that as in other invertebrates and vertebrates, glial cells could be involved in the circadian system of P. clarkii; however, thus far we cannot know whether the glial cells are only effectors, participate in afferent pathways, or are part of the circadian clock.