Exposure of embryos to cyclically cold incubation temperatures durably affects energy metabolism and antioxidant pathways in broiler chickens.

Cyclically cold incubation temperatures have been suggested as a means to improve resistance of broiler chickens to ascites; however, the underlying mechanisms are not known. Nine hundred eggs obtained from 48 wk Ross broiler breeders were randomly assigned to 2 incubation treatments: control I eggs were incubated at 37.6°C throughout, whereas for cold I eggs the incubation temperature was reduced by 1°C for 6 h daily from 10 to 18 d of incubation. Thereafter, chickens were reared at standard temperatures or under cold exposure that was associated or not with a postnatal cold acclimation at d 5 posthatch. At hatch, hepatic catalase activity and malondialdehyde content were measured. Serum thyroid hormone and triglyceride concentrations, and muscle expression of several genes involved in the regulation of energy metabolism and oxidative stress were also measured at hatch and 5 and 25 d posthatch. Cold incubation induced modifications in antioxidant pathways with higher catalase activity, but lower expression of avian uncoupling protein 3 at hatch. However, long-term enhancement in the expression of avian uncoupling protein 3 was observed, probably caused by an increase in the expression of the transcription factor peroxisome proliferator activated receptor-γ coactivator-1α. These effects were not systematically associated with an increase in serum triiodothyronine concentrations that were observed only in chickens exposed to both cold incubation and later acclimation at 5 d with cold rearing. Our results suggest that these conditions of cyclically cold incubation resulted in the long-term in changes in antioxidant pathways and energy metabolism, which could enhance the health of chickens reared under cold conditions.

Influences of bioavailability, trophic position, and growth on methylmercury in hakes (Merluccius merluccius) from Northwestern Mediterranean and Northeastern Atlantic.

Methylmercury (MeHg) determinations in hake, its food-chain, and the surrounding waters and sediments allowed us to show that the higher length or age normalized mercury concentrations of Northwestern Mediterranean (Gulf of Lions: GoL) muscle hakes compared to its Northeastern Atlantic (Bay of Biscay: BoB) counterpart are due to both biotic and abiotic differences between their ecosystems. Bioenergetic modeling reveals that the slower growth rate of Mediterranean hake favors the MeHg bioaccumulation in the fish muscle and explains most of the difference between GOL and BoB hake populations. In addition, the waters of the Mediterranean hake habitat favor a higher MeHg exposition, due to the upper position of the thermohalocline, where MeHg is formed. Furthermore, we show that, within the Mediterranean hake population, a major increase in the biomagnification power (the slope of the relationships between logMeHg and δ(15)N), from 0.36 up to 1.12, occurs when individuals enter adulthood, resulting from the combined effects of lowering growth rate and change in feeding habits. Finally, δ(15)N normalized Hg concentrations indicate that the highest Hg concentrations are for hake from the shelf edge and the lowest are for hake from the Rhône prodelta area, suggesting a lower Hg bioavailability in inshore environments, consistent with MeHg distributions in water, sediment, and preys.

Effects of thermal manipulation during early and late embryogenesis on thermotolerance and breast muscle characteristics in broiler chickens.

Genetic selection has significantly improved the muscle development of fast-growing broiler chickens in the last 50 yr. However, improvement in muscle growth has coincided with relatively poor development of visceral systems, resulting in impaired ability to cope with high environmental temperatures. The aim of this study was to elucidate the effects of thermal manipulation (TM) during different periods of embryogenesis on chick hatchability, BW and thermoregulation upon hatching, on their ability to cope with thermal challenge at 42 d of age, and on carcass and breast meat traits. Control embryos were incubated at 37.8 degrees C. The TM embryos were incubated at 37.8 degrees C and treated for 3 h at 39.5 degrees C on the following days of embryogenesis: E8 to E10 [early (EA)], E16 to E18 [late (LA)], and both E8 to E10 and E16 to E18 (EA-LA). Body weight and body temperature (T(b)) were measured at hatching and throughout the growth period as well as during exposure of 42-d-old chickens to a thermal challenge at 35 degrees C for 6 h. The LA and EA chicks exhibited significantly lower T(b) than control chicks (37.9 vs. 38.2 degrees C) at hatching, but during the growth period, differences in T(b) between treated and control chicks decreased with age. Significant hyperthermia (over 44 degrees C) was monitored in all groups during the thermal challenge, but mortality was higher in treated than in control chickens. No effect of treatments on BW was found during the entire growth period. However, breast yield was higher in LA chickens than in controls at slaughter. The EA and EA-LA treatments slightly decreased the ultimate pH of breast meat, whereas the LA treatment had no effect. In conclusion, none of the TM conditions tested in the present study were able to improve long-term thermotolerance in chickens. Late treatment favored breast muscle growth without affecting ultimate pH and drip loss of breast meat.

Differentiation of presumed serotonergic dorsal raphe neurons in relation to behavior and wake-sleep states.

Using extracellular single unit recording, either alone or in combination with microdialysis application of drugs, we examined the characteristics of presumed serotonergic dorsal raphe neurons during wake-sleep states in the freely moving cat. Recordings were made from a total of 272 neurons in the dorsal raphe nucleus. Of these, 240 (88%) were classified as serotonergic on the basis of their typical long-duration action potential, slow discharge activity, and reduced spontaneous discharge rate during paradoxical sleep compared to during slow-wave sleep. An inhibitory response to serotonergic agonists and a slow conduction velocity were seen in all neurons of this type tested or identified by stimulation of the main ascending serotonergic pathway. These presumed serotonergic dorsal raphe neurons could be subdivided into two typical previously identified groups (types I-A and I-B) and four atypical new groups (types I-C, II-A, II-B, and II-C) according to differences in firing patterns during wake-sleep states. The typical neurons were evenly distributed in the dorsal raphe nucleus and their activity was related to the level of behavioral arousal, since they discharged regularly at a high rate during waking and at progressively slower rates during slow-wave sleep, and ceased firing either during slow-wave sleep with ponto-geniculo-occipital waves and paradoxical sleep (type I-A) or only during paradoxical sleep (type I-B). In contrast, the atypical subgroups were unevenly distributed in the dorsal raphe nucleus and exhibited firing patterns distinct from those of the typical neurons, such as sustained tonic activity during paradoxical sleep (types I-C and II-C) or showing their highest rate of tonic discharge during slow-wave sleep, with suppression of discharge during both waking and paradoxical sleep (type II-B). From these data we suggest that presumed serotonergic dorsal raphe neurons play different roles in behavioral state control and that there is functional topographic organization in the dorsal raphe nucleus.

Synaptic responsiveness of neocortical neurons to callosal volleys during paroxysmal depolarizing shifts.

Based on intracellular recordings in vivo, we investigated the responsiveness of cat neocortical neurons to callosal volleys during different phases of spontaneously occurring or electrically induced electrographic seizures, compared with control periods of slow sleep-like oscillations. Overt seizures, with spiking, triggered by pulse-trains to the callosal pathway, started with a latency of approximately 20 s after cessation of stimulation, thus contrasting with paroxysmal activity elicited by ipsilateral cortical or thalamic stimulation that is initiated immediately after electrical stimulation. During the rather long preparatory period to callosally triggered seizures, cortical neurons displayed subthreshold depolarizing runs at 4-7 Hz, associated with increased amplitudes of excitatory postsynaptic potentials. The sequential analysis of neuronal responsiveness during different components of spike-wave complexes revealed progressively increased amplitudes of callosally evoked postsynaptic excitatory responses in regular-spiking and fast-rhythmic-bursting neurons, over a period of approximately 20 ms prior to the generation of paroxysmal depolarizing shifts. These data support the concept that seizures consisting of spike-wave complexes originate within the neocortex through a progressive synaptic buildup and that their synchronization is achieved, at least partially, by cortical commissural synaptic linkages.

Spontaneous field potentials influence the activity of neocortical neurons during paroxysmal activities in vivo.

Field-potential recordings with macroelectrodes, and extra- and intracellular potentials with micropipettes were used to determine the influence of spontaneous field potentials on the activity of neocortical neurons during seizures. In vivo experiments were carried out in cats under anesthesia. Strong negative field fluctuations of up to 20 mV were associated with electroencephalogram "spikes" during spontaneously occurring paroxysmal activities. During paroxysmal events, action potentials displayed an unexpected behavior: a more hyperpolarized firing threshold and smaller amplitude than during normal activity, as determined with intracellular recordings referenced to a distant ground. Considering the transmembrane potential (the difference between extra- and intracellular potential) qualified this observation: firing threshold determined from the transmembrane potential did not decrease, and smaller action-potential amplitude was associated with depolarized firing threshold. The hyperpolarization of intracellular firing threshold was thus related to the field potentials. Similar field-potential effects on neuronal activities were observed when the paroxysmal events included very fast oscillations or ripples (80-200 Hz) that represent rapid fluctuations of field potentials (up to 5 mV in <5 ms). Neuronal firing was phase-locked to those oscillations. These results demonstrate that: (a) strong spontaneous field potentials influence neuronal behavior, and thus play an active role during paroxysmal activities; and (b) transmembrane potentials have to be used to accurately describe the behavior of neurons in conditions in which field potentials fluctuate strongly. Since neuronal activity is presumably the main generator of field potentials, and in return these potentials may increase neuronal excitability, we propose that this constitutes a positive feedback loop that is involved in the development and spread of paroxysmal activities, and that a similar feedback loop is involved in the generation of neocortical ripples. We propose a mechanism for seizure initiation involving these phenomena.

The anticancer agent pb-100 concentrates in the nucleus and nucleoli of human glioblastoma cells but does not enter normal astrocytes.

Selectivity of the anticancer agent PB-100 for malignant cells, already demonstrated using cell growth and viability evaluation, is now confirmed by microscopic observations. PB-100 is easily detected inside cells by its yellow color under visible light and by its blue fluorescence; it may be measured in isolated nuclei using its characteristic UV absorbance. After short treatment of human BCNU-resistant glioblastoma cells (U 251) and normal astrocyte controls (CRL 1656), PB-100 accumulates in the malignant cell nucleus, particularly concentrating in the multiple nucleoli and rapidly inducing glioblastoma cell death, whilst, in contrast, the anticancer agent does not even enter normal cells. We had already shown that PB-100 binds to DNA of cancer cells, but not to that of normal cells. In vitro tests described in this report indicate that PB-100 binds to purine bases, but not to pyrimidines, of various ribopolymers and its binding to purine rich nucleic acid stretches is inferred.

The selective anticancer agent pb-100 inhibits interleukin-6 induced enhancement of glioblastoma cell-proliferation in-vitro.

The multifunctional cytokine interleukin-6 behaves as a growth factor for various malignancies. It is produced in significant amounts by glioblastoma cells. When exogenous IL-6 is added (pg/ml) to culture medium of human glioblastoma cells and normal (non malignant) astrocytes used as controls, it exerts a dose dependent and differential effect on these two cell lines. Enhancement of cell proliferation is twice as high for glioblastoma cells as for astrocytes. In vitro, the novel anticancer agent PB-100 (mu g/ml) dose dependently inhibits this stimulatory activity. In addition, increasing PB-100 concentrations finally induce death of the malignant cells, yet do not impede multiplication of normal astrocytes. PB-100 does not abolish IL-6 production by cells, but keeps its level down to physiological values. PB-100 should therefore find its place in therapies requiring control of IL-6 production.

The selective anticancer agents PB-100 and BG-8 are active against human melanoma cells, but do not affect non malignant fibroblasts.

When past the stage amenable to surgery, melanoma and its metastases are, as a rule, treated with chemotherapy, which is largely unsuccessful. In this report, experimental evidence is presented demonstrating that, in vitro, two selective anticancer agents, PB-100 and BG-8, dose dependently destroy human G-361 melanoma cells, but do not affect human non malignant CCD-974Sk fibroblasts used as controls. Trace metal compounds, present, often in abnormal amounts, in the cancer cell and/or its environment, are known to influence its proliferation. Assays were carried out using highly elevated amounts of ferritin, iron chloride or zinc chloride. Ferritin proved differentially mitogenic for melanoma cells and fibroblasts. Its activity was inhibited by both anticancer agents, which however tended to become less efficacious in its presence. FeCl3 was more moderately, but equally, mitogenic for malignant and normal cells, yet it impaired antiproliferative activity of PB-100 and inhibited that of BG-8. ZnCl2 exhibited a selective antiproliferative activity on the malignant melanoma cells; it did not compete with PB-100 or BG-8. Specific recognition and destruction of malignant cells by the two anticancer agents are discussed.

Serotonergic dorsal raphe neurons cease firing by disfacilitation during paradoxical sleep.

Using in vivo extracellular unit recordings combined with microdialysis infusion in the cat, we found that the cessation of discharge of presumed serotonergic dorsal raphe neurons during paradoxical sleep (PS) was completely blocked by either histamine or phenylephrine, an alpha1 adrenoceptor agonist, but not by bicuculline, a GABA receptor antagonist. In addition, application of mepyramine, a specific H1 histamine receptor antagonist, or prazosin, a specific alpha1 adrenoceptor antagonist, suppressed the spontaneous discharge of raphe neurons during both quiet waking and sleep. The present data suggest that this cessation of dorsal raphe unit activity is caused by the mechanism of disfacilitation resulting from the cessation of discharge of norepinephrine- or histamine-containing neurons during PS.

Alpha-2 adrenoceptor mediated paradoxical (REM) sleep inhibition in the cat.

Using in vivo microdialysis infusion, we have studied the adrenoceptor subtypes implicated in paradoxical sleep (PS) generation in the cat. Both epinephrine and norepinephrine inhibited PS and induced PS without atonia when applied to the caudal peri-locus coeruleus-alpha (peri-LC alpha) of the mediodorsal pontine tegmentum. This effect was mimicked by clonidine, an alpha2-selective adrenoceptor agonist, while alpha1 and beta agonists were ineffective. The norepinephrine effect was antagonized by co-application of the alpha2-selective antagonists, rauwolscine or RX 821002, but not by alpha1 or beta1 antagonists. In addition, clonidine completely blocked the PS-inducing effect of carbachol, applied to the peri-LC alpha. The present data indicate that alpha2 adrenoceptors located in the caudal peri-LC alpha play a critical role in inhibitory mechanisms of PS generation.

Increase in antidromic excitability in presumed serotonergic dorsal raphe neurons during paradoxical sleep in the cat.

Putative serotonergic dorsal raphe (DRN) neurons display a dramatic state-related change in behaviour, discharging regularly at a high rate during waking and at progressively slower rates during slow-wave sleep (SWS) and ceasing firing during paradoxical sleep (PS). Using the antidromic latency technique and extracellular recording, we have examined the change in neuronal excitability of presumed serotonergic DRN neurons during the wake-sleep cycle in freely moving cats. We found that, under normal conditions, suprathreshold stimulation of the main ascending serotonergic pathway resulted in a marked decrease in both the magnitude and variability of antidromic latency during PS, while subthreshold stimulation led to a marked increase in antidromic responsiveness during PS compared with during other behavioural states. The antidromic latency shift resulted from a change in the delay between the initial segment (IS) and soma-dendritic (SD) spikes, the antidromic latency being inversely related to the interval between the stimulus and the preceding spontaneous action potential. A marked decrease in the magnitude and variability of antidromic latency was also seen following suppression of the spontaneous discharge of DRN neurons by application of 5-HT autoreceptor agonists or muscimol, a potent GABA agonist. A marked IS-SD delay or blockage of SD spikes was, however, seen in association with the PS occurring during recovery from 5-HT autoreceptor agonist or during muscimol application. The present findings are discussed in the light of previous in vitro intracellular recording data and our recent findings of the disfacilitation mechanisms responsible for the cessation of discharge of DRN neurons during PS.

PB-100: a potent and selective inhibitor of human BCNU resistant glioblastoma cell multiplication.

Major drawbacks to present-day cancer chemotherapy are its intrinsic lack of selectivity for tumour cells, resulting in severe damage to normal rapidly dividing cells, and the widespread emergence of drug resistance. Here experimental evidence is presented demonstrating that PB-100, a beta-carboline alkaloid, selectively inhibits in vitro multiplication of human BCNU-resistant glioblastoma cells (U251), but has no effect on normal astrocyte (CRL 1656) multiplication. PB-100 activity is dose-dependent. In the presence of ferritin or CaCl2, which are highly mitogenic for glioblastoma cells, higher doses of the alkaloid are required to inhibit multiplication completely. PB-100 is one of several compounds which were selected for their specific action on cancer DNA and cells, together with lack of activity on normal DNA and cells. Both the selectivity of PB-100 and its ability to overcome drug resistance stem from its effect on cancer DNA secondary structure. This activity is described and discussed, and therapeutic applications are mentioned.

Peripheral leptin effect on food intake in young chickens is influenced by age and strain.

The acute effect of leptin on the regulation of food intake was investigated in layer and broiler chickens. In an initial study, we observed that a single intraperitoneal injection of recombinant chicken leptin (1 mg/kg BW) dramatically reduced (38%) food intake in 56-day-old layer chickens, more moderately reduced (15%) food intake in 9-day-old layer chicks, and had no significant effect in 9-day-old broiler chicks. In a subsequent study, body weight and plasma concentrations of leptin were measured weekly in layer and broiler chicks from day 1 to 35 of age and brain leptin receptor and neuropeptide Y (NPY) mRNA expression were analyzed at 1, 9, and 35 days of age. At day 1 of age, peripheral concentrations of leptin were significantly greater in layer than broiler chicks. Subsequently, despite increases in body weight and differences in growth rates between layer and broiler chicks from day 8 to day 35 of age, peripheral concentrations of leptin were constant and similar in both genotypes. Leptin receptor and NPY mRNA were expressed in brain from day 1 in chicks of both genotypes and increased significantly to day 35 of age. These observations provide evidence that the inhibitory effect of leptin on the regulation of food intake in growing chicks is an age dependent process. Furthermore, acquisition of the anorectic effect of leptin is likely to be associated with greater expression of the leptin receptor and NPY mRNAs than to changes in blood levels of leptin. Finally, this study provides evidence that chickens selected for high growth rates may be less sensitive or responsive to peripheral concentrations of leptin than chickens with low growth rates (layers), suggesting that the faster growth of broiler chicks may be related to a lessened responsiveness to anorexigenic factors.

Leptin and insulin downregulate leptin receptor gene expression in chicken-derived leghorn male hepatoma cells.

In chickens, leptin is expressed mainly in the liver, where its receptor gene expression has also been reported, and in adipose tissue. In view of the key role played by the liver in lipogenesis in avian species, the hepatic expression of leptin may have physiological significance. In this study, we showed that leptin is constitutively expressed and secreted in a chicken-derived hepatoma cell line (LMH). Although insulin regulates leptin expression in vivo, incubation of LMH cells in the presence of 100 nM insulin for 24 or 48 h had no effect on leptin expression or its secretion in the culture medium. In addition, we developed a specific chicken leptin receptor real-time reverse transcription (RT)-PCR, and downregulation of leptin receptor gene expression by homologous and heterologous signals was demonstrated, as relative leptin receptor mRNA levels were significantly decreased after exposure of LMH cells to recombinant chicken leptin or porcine insulin. In conclusion, our results indicate that leptin is probably able to desensitize its own response in the chicken liver. Finally, the ability of insulin and leptin to regulate chicken leptin receptor gene expression suggests a direct role of leptin in the control of hepatic metabolism.

Early-age thermal conditioning reduces uncoupling protein messenger RNA expression in pectoral muscle of broiler chicks at seven days of age.

Early-age thermal conditioning (TC) by exposing young chicks to 40 C for 24 h reduces body temperature (Tb) and has been showed by others to improve long-term resistance of broilers to heat stress. Uncoupling oxidative phosphorylation in pectoral muscle mitochondria might be related to heat production. Fertile eggs were hatched under video control, and 161 pedigree chicks froml2 sires and 22 dams were immediately allocated to two groups (T, a group composed of 81 chicks exposed to TC at 5 d of age, and N, a control group of 80 nonexposed chicks). Body weights and Tb were measured at 2 and 7 d of age. Five pairs (one N and one T) of full sib chicks from families that exhibited the largest difference of Tb variation from 2 to 7 d of age between the two treatments were chosen for pectoral muscle sampling. Avian uncoupling protein (avUCP) messenger RNA expression was measured by reverse transcript-PCR coupled to southern blot in the pectoral muscle of 7-d-old broiler chicks. At 7 d of age, there were no BW differences between treatments and Tb was significantly reduced by TC (-0.13 C on average). Heritability of Tb variation between 2 and 7 d was 0.38 +/- 0.20 (SE) for T chicks and 0.35 +/- 0.17 for N chicks without a significant genetic correlation between the two environments. Expression of avUCP mRNA was significantly (85%) lower in T chicks than in N chicks. Uncoupling protein mRNA expression in pectoral muscle and Tb are quickly adjusted in broiler chicks 24 h after early thermal conditioning.

The beta-adrenergic system is involved in the regulation of the expression of avian uncoupling protein in the chicken.

Avian uncoupling protein (avUCP) is orthologous to UCP3, which is suggested to be involved in fatty acid metabolism and to limit the mitochondrial production of reactive oxygen species in mammals. In the chicken, the role and regulation of avUCP remain to be clarified. The aim of this study was to explore the control of avUCP expression by the beta-adrenergic system, known to be involved in avian thermoregulation and lipid utilization, and in UCP expression in mammals. Therefore, we measured the expression of avUCP mRNA and protein in the Pectoralis major muscle of chickens injected with the beta(2) agonist isoproterenol, and we investigated the potential pathways involved in the regulation of avUCP mRNA expression. Avian UCP mRNA expression was increased 7-fold 4h after isoproterenol injection, leading to a tendency to a 40% increase in avUCP protein 24h post-injection. This increase was preceded, 30 min after isoproterenol injection, by changes in the chicken thyroid status and in the muscular expression of PPARalpha, PPARbeta/delta, and PPARgamma coactivator-1alpha (PGC-1alpha). Moreover, the analysis of the avUCP promoter sequence suggested potential binding sites for PPARs and for thyroid hormone receptors. We also detected the activation of AMP-activated protein kinase, which has recently been reported to be involved in UCP3 regulation in mammals. This study presents for the first time evidence of beta-adrenergic control on avUCP messenger expression in chicken muscle and suggests the potential involvement of AMPK and several transcription factors in this regulation.