daily and circadian expression of cryptochrome during the ontogeny of crayfish

Cryptochromes (CRY) are proteins with a dual role in the circadian function of different animals, participating in phototransduction and light signaling to the clock and as a transcriptional repressor that provides negative feedback in the clock feedback loop. Here we characterize functional expression of CRY as a marker of the functionality of the circadian pacemaker of crayfish (Procambarus clarkii) throughout postembryonic development. Using different experimental light protocols and by means of immunofluorescence and biochemical methods, we report that, as in the adult, in young crayfish from the first embryonic stage CRY is present in cells adjacent to the eyestalk hemiellipsoidal body and the anterior margin of the brain protocerebrum. In the brain, CRY cycles after 72 h darkness, entraining to LD cycles. Meanwhile, as in the adult eye, in juveniles CRY is driven by light, showing an arrhythmic pattern in DD and cycling under LD. These results, as well as the completely different period length found in the brain circadian oscillations of 2nd post-embryonic stage and juvenile animals, suggest important changes in the properties of the crayfish pacemaker through the development. Therefore these data support a previous idea about the functionality of the circadian system from hatching. (C) 2007 Elsevier Inc. All rights reserved

putative pacemakers of crayfish show clock proteins interlocked with circadian oscillations

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

Crustacean hyperglycemic hormone is synthesized in the eyestalk and brain of the crayfish <i>Procambarus clarkii</i>

<div><p>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 <i>P</i>. <i>clarkii</i>, CHH synthesis has only been reported in the X-organ-sinus gland (XO-SG). Therefore, the aim of this study was to use <i>in situ</i> hybridization to determine whether CHH mRNA is located in other structures, including the putative pacemaker, eyestalk and brain, of crayfish <i>P</i>. <i>clarkii</i> at two times of day. CHH mRNA was observed in both the eyestalk and the brain of <i>P</i>. <i>clarkii</i>, 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.</p></div

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. 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, GRP8 and caspase 12. It was also performed 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 Alzheimer’s Disease

Photomicrographs showing CHH mRNA expression in the brain of the crayfish <i>P</i>. <i>clarkii</i> at ZT1.

<p>Panoramic view; scale bar, 400 μm (A). Amplification of cluster 6 (c6) of the protocerebrum (B) and cluster 11 (c11) of the deutocerebrum (C); both scale bars, 100 μm. Amplification of cluster 10 (c10) of the deutocerebrum; scale bar, 200 μm (D). Amplification of cluster 15 (c15) (E) and cluster 17 (c17) (F) of the tritocerebrum; both scale bars, 100 μm. Arrows indicate the cells in clusters that express CHH mRNA in the crayfish brain.</p

Retina of crayfish <i>P</i>. <i>clarkii</i> at ZT1.

<p>Photomicrographs showing CHH mRNA signal (arrows) in tapetal cells. Scale bar, 100 μm.</p

Photomicrographs showing that CHH mRNA is expressed in the eyestalk of the crayfish <i>P</i>. <i>clarkii</i> at ZT1.

<p>Panoramic view; scale bar, 400 μm (A). Amplification of lamina ganglionaris (lg); scale bar, 200 μm (B); ganglion cell layer outer (ogl). Amplification of external medulla (em); scale bar, 200 μm (C). Amplification of terminalis medulla (tm), in which you can see the XO; scale bar, 200 μm (D); and the protocerebral tract; scale bar, 100 μm (E). The arrows indicate the CHH mRNA signal in different areas in the eyestalk of crayfish.</p