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

Abstract

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.

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

“…Although it has been demonstrated that CHH is synthesized in the retina [1] and XO [19], [20], neither its synthesis nor the presence of its transcripts have been observed in the various brain cells that express clock proteins. Thus, in this study, we localized the position of CHH-positive cells at two points during the circadian rhythm that were previously described as crucial for CHH transcriptional rhythm [8]. We demonstrate the presence of CHH mRNA in both the various cell clusters of the eyestalk and the brain of crayfish P .…”

“…The following reagents were used to amplify PCR products: 1 μl of 10x Taq reaction buffer (Altaenzymes), dNTP mix with each nucleotide at a final concentration of 2 mM (Altaenzymes), 2.3 mM MgCl 2 (Altaenzymes), each primer at 500 nM [8], 2 μl of cDNA that was synthesized from the brain and eyestalk, 3.5 μl of sterile water and 0.1 units of Taq DNA polymerase (Altaenzymes). After the samples were centrifuged, two drops of mineral oil were added to each tube to prevent the evaporation of the mixture.…”

“…Although the XO–SG complex is considered the main locus of neuropeptide production, CHHs have also been observed in several neural tissues, including the pericardial organs [5], [6], [7], cerebral ganglia [8], ventral nerve cord [9], and retinal tapetal cells [10], and non-neural tissues such as the foregut and hindgut [11]. CHH activity varies during the life cycle of decapod crustaceans and contributes to mechanisms underlying adaptations to stressful conditions through a dual-feedback control system [12].…”

“…In other decapods, such as Procambarus clarkii , the circadian modulation of CHH has been described in the crayfish eyestalk and retina [16]. Recently, these authors investigated the expression dynamics of CHH mRNA in the crayfish Procambarus clarkii over a day/night cycle under both entrained LD and free-running (DD) conditions [8]. Their data provided evidence of a circadian rhythm of CHH transcription.…”

“…Their data provided evidence of a circadian rhythm of CHH transcription. Intriguingly, using immunocytochemistry, they also found that CHH was expressed in the central brain in several proto- and tritocerebral cell clusters known to express canonical clock proteins, such as Timeless, Period and Clock (Bmal) [8], [17]. These observations, in combination with the finding that CHH immunoreactivity was reported in both the cytoplasm and nuclei of brain cells (similar to expression during circadian clock transcriptional repressor nuclear translocation events), suggested that there is a possible relationship between the central circadian clock and CHH.…”