Epigenetic-Imprinting Changes Caused by Neonatal Fasting Stress Protect From Future Fasting Stress.

Unfavourable nutritional conditions during the neonatal critical period can cause both acute metabolic disorders and severe metabolic syndromes in later life. These phenomena have been tightly related to the epigenetic modification controlling the balance between satiety and hunger in the hypothalamus. In the present study, we investigated epigenetic modification associated with both the fasting stress effects and the short-term resilience to fasting stress in the hypothalamic paraventricular nucleus (PVN) of chicks. Fasting for 24 h at 3 days of age (D) (i.e. D3) significantly increased global methylation at lysine 27 of histone 3 (H3K27) and its specific histone methyltransferase (HMT) expression level in the PVN. Because global methylation could not fully reveal the changes at specific genes, the regulation of the gene for brain-derived neurotrophic factor (Bdnf), which was recently also found to have an anorexigenic effect, was evaluated as a potential target. Chromatin immunoprecipitation assay analysis revealed that tri- (me3) and di-methylated (me2) H3K27 exhibited an instant (on D4 only) and latent increase (on both D11 and D41), respectively, at the putative promoter of Bdnf after 24 h of fasting on D3. This indicated that fasting could regulate energy-expenditure-related genes via modifying methylation at H3K27, which we suspected might be a protective mechanism for keeping the inner environment homeostatic. To test this hypothesis, a short-term repetitive fasting stress was applied to chickens, which were fasted for 24 h either on D10 only or on both D3 and D10. It was found that pre-existing fasting on D3 could induce a short-term fasting resilience, which rescued the reduction of Bdnf expression from future fasting on D10. We call this phenomenon the 'molecular memory', which was mainly conducted by HMTs and H3K27me2/me3 in the PVN. In conclusion, chicks respond to fasting with dynamic methylation at H3K27 in the PVN during the neonatal critical period. This allows the PVN to form a 'molecular memory', which keeps the individual inner environment homeostatic and resilient to future fasting over the short term.

Two receptors are involved in the central functions of kynurenic acid under an acute stress in neonatal chicks.

Intracerebroventricular (i.c.v.) injection of kynurenic acid (KYNA) had sedative and hypnotic effects during stress in neonatal chicks. However, its mechanism has not been clarified. KYNA is an endogenous antagonist of the α7 nicotinic acetylcholine (α7nACh) receptor and N-methyl-d-aspartate (NMDA) receptor. Therefore, this study clarified the mechanism of sedative and hypnotic effects of KYNA in the brain during an acute stress. In Experiment 1, to investigate the relationship between KYNA and the α7nACh receptor, KYNA was injected i.c.v. with galantamine, an agonist of the allosteric potentiating site of the α7nACh receptor. Galantamine did not attenuate the effect of KYNA, but higher levels of galantamine caused harmful effects. In Experiment 2, the role of the NMDA receptor was investigated using the NMDA receptor antagonist (+)-MK-801, d-serine which has high affinity to a co-agonist glycine site at the NMDA receptors, NMDA as the NMDA receptor agonist, and 2,3-pyridinedicarboxylic acid (QUIN), an agonist of the NMDA receptor subgroup containing the subunits NR2A and NR2B. The behavioral changes following KYNA were partially attenuated by QUIN alone. In conclusion, we suggest that KYNA functioned via the simultaneous inhibition of the α7nACh receptor and NMDA receptor subgroup containing the subunits NR2A and NR2B.

The role of GABAergic system on the inhibitory effect of ghrelin on food intake in neonatal chicks.

Ghrelin is a gut-brain peptide that has a stimulatory effect on food intake in mammals. In contrast, this peptide decreases food intake in neonatal chicks when injected intracerebroventricularly (ICV). In mammals, neuropeptide Y (NPY) mediates the orexigenic effect of ghrelin whereas in chicks it appears that corticotrophin releasing factor (CRF) is partially involved in the inhibitory effect of ghrelin on food intake. Gamma aminobutyric acid (GABA) has a stimulatory effect on food intake in mammals and birds. In this study we investigated whether the anorectic effect of ghrelin is mediated by the GABAergic system. In Experiment 1, 3h-fasted chicks were given an ICV injection of chicken ghrelin and picrotoxin, a GABA(A) receptors antagonist. Picrotoxin decreased food intake compared to the control chicks indicating a stimulatory effect of GABA(A) receptors on food intake. However, picrotoxin did not alter the inhibitory effect of ghrelin on food intake. In Experiment 2, THIP hydrochloride, a GABA(A) receptor agonist, was used in place of picrotoxin. THIP hydrochloride appeared to partially attenuate the decrease in food intake induced by ghrelin at 30 min postinjection. In Experiment 3, the effect of ICV injection of chicken ghrelin on gene expression of glutamate decarboxylase (GAD)(1) and GAD(2), GABA synthesis enzymes in the brain stem including hypothalamus, was investigated. The ICV injection of chicken ghrelin significantly reduced GAD(2) gene expression. These findings suggest that ghrelin may decrease food intake in neonatal chicks by reducing GABA synthesis and thereby GABA release within brain feeding centers.

Changes in brain monoamine metabolism of neonatal chicks under two different acute stress conditions.

1. The purpose of the present study was to clarify brain monoamine metabolism during two different conditions of acute stress by quantifying changes in the brain of neonatal chicks exposed to either restraint with isolation, or fasting stress. 2. Under restraint with isolation-induced stress, dopaminergic metabolism was clearly stimulated. 3. During fasting stress, dopaminergic activity, serotonergic and norepinephrinergic metabolisms were stimulated. 4. It was concluded that brain monoamine metabolism of chicks is differentially affected by stressors.

L-ornithine attenuates corticotropin-releasing factor-induced stress responses acting at GABAA receptors in neonatal chicks.

I.c.v. injection of L-ornithine has been shown to have sedative and hypnotic effects on neonatal chicks exposed to acute stressful conditions. To clarify the mechanism, we conducted three experiments under strengthened stressful conditions with corticotropin-releasing factor (CRF). In Experiment 1, the effect of i.c.v. injection of CRF, L-ornithine (0.5 µmol) or CRF with L-ornithine on the stressful response of chicks was investigated. Compared with the vehicle control, CRF increased distress vocalizations and the time spent in active wakefulness. L-ornithine increased the time spent in sleeping posture, even following stimulation with CRF. In Experiment 2, dose-dependent effects of L-ornithine were investigated using i.c.v. administration with vehicle, CRF alone or CRF plus L-ornithine (0.125, 0.25 or 0.5 µmol). L-ornithine decreased the CRF-stimulated distress vocalizations in a dose-dependent manner. In Experiment 3, the chicks were injected i.c.v. with either CRF, CRF plus L-ornithine (0.5 µmol), CRF plus the γ-aminobutyric acid (GABA)A receptor antagonist picrotoxin or L-ornithine with picrotoxin. The sedative and hypnotic effects induced by L-ornithine were blocked with co-administration of picrotoxin. These results suggest that L-ornithine could attenuate CRF-stimulated stress behaviors acting at GABAA receptors.

Intracerebroventricular injection of L-proline and D-proline induces sedative and hypnotic effects by different mechanisms under an acute stressful condition in chicks.

The central effects of L-proline, D-proline and trans-4-hydroxy-L-proline were investigated by using the acute stressful model with neonatal chicks in Experiment 1. Sedative and hypnotic effects were induced by all compounds, while plasma corticosterone release under isolation stress was only attenuated by L-proline. To clarify the mechanism by which L-proline and D-proline induce sedative and hypnotic effects, the contribution of the strychnine-sensitive glycine receptor (glycine receptor) and N-methyl-D-aspartate glutamate receptor (NMDA receptor) were further investigated. In Experiments 2-3, the glycine receptor antagonist strychnine was co-injected intracerebroventricular (i.c.v.) with L-proline or D-proline. The suppression of isolation-induced stress behavior by D-proline was attenuated by strychnine. However, the suppression of stress behavior by L-proline was not attenuated. In Experiment 4, the NMDA receptor antagonist (+)-MK-801 was co-injected i.c.v. with L-proline. The suppression of stress behavior by L-proline was attenuated by (+)-MK-801. These results indicate that L-proline and D-proline differentially induce sedative and hypnotic effects through NMDA and glycine receptors, respectively.

Screening of dipeptides having central functions for excitation and sedation.

The naturally-occurring dipeptide carnosine (beta-alanyl-L-histidine) and the tripeptide glutathione (L-gammaglutamyl-L-cysteinylglycine) are found extensively in animal tissues such as brain and skeletal muscle. Central functions for excitation and sedation of them and their derivatives were screened.

L-proline is a sedative regulator of acute stress in the brain of neonatal chicks.

The purpose of the present study was to clarify the central nervous system function of amino acids during acute stress. In Experiment 1, changes in free amino acid pattern were investigated in the brain of neonatal chicks exposed to either restraint with isolation-induced or fasting stress. L-proline and L-arginine were decreased in the telencephalon and diencephalon under any stress. Since the central nervous system functions of L-arginine during the stress response has recently been reported, in Experiment 2, the effect of intracerebroventricular injection of L-proline (0.5, 1.0, 2.0 micromol) during isolation-induced stress was investigated. L-proline induced sedative and hypnotic effects in a dose-dependent manner. It is suggested that L: -proline may have an important role to attenuate the stress response in the central nervous system of chicks.

N-Methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involved in the induction of sedative effects under an acute stress in neonatal chicks.

Glutamate, an excitatory amino acid, acts at several glutamate receptor subtypes. Recently, we reported that central administration of glutathione induced hypnosis under stressful conditions in neonatal chicks. Glutathione appears to bind to the N-methyl-D-aspartate (NMDA) receptor. To clarify the involvement of each glutamate receptor subtype during stressful conditions, intracerebroventricular (i.c.v.) injection of several glutamate receptor agonists was given to chicks under social separation stress. Glutamate dose-dependently induced a hypnotic effect. NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate are characterized as ionotropic glutamate receptors (iGluRs). Although NMDA also induced a sedative effect, [corrected] the potency of NMDA for sleep-like behavior [corrected] was less than that of glutamate. AMPA tended to decrease distress vocalizations induced by acute stress and brought about a sedative effect. Kainate and (S)-3, 5-dehydroxyphenylglycine, which is a metabotropic glutamate receptor agonist, had no influence on chick behavior. Thus, it is suggested that the iGluRs, NMDA and AMPA, are important in inducing hypnosis and sedation under acute stress in chicks.

Changes in free amino acids in the brain during embryonic development in layer and broiler chickens.

Developmental changes in the levels of the excitatory amino acids L-glutamate (Glu) and L-Aspartate (Asp) and inhibitory amino acids glycine (Gly) and gamma-amino butyric acid (GABA), as well as taurine and its related amino acids L-methionine (Met), L-cysteine (Cys) and L-serine (Ser) in the brain and pectoralis muscle at various embryonic stages and hatch in broiler and layer type chickens were determined. Brain concentrations of Asp, GABA and taurine were higher than those in the muscle, but the difference in the two types was small. The concentrations of the precursors of taurine including Met, Cys and Ser were lower than that of taurine. In conclusion, the synthesis of some amino acids and their metabolites such as Asp, GABA and taurine in the chick embryo is very high in order to support brain development.

Central L-arginine reduced stress responses are mediated by L-ornithine in neonatal chicks.

Recently, we observed that central administration of L-arginine attenuated stress responses in neonatal chicks, but the contribution of nitric oxide (NO) to this response was minimal. The sedative and hypnotic effects of L-arginine may be due to L-arginine itself and/or its metabolites, excluding NO. To clarify the mechanism, the effect of intracerebroventricular (i.c.v.) injection of L-arginine metabolites on behavior under social separation stress was investigated. The i.c.v. injection of agmatine, a guanidino metabolite of L-arginine, had no effect during a 10 min behavioral test. In contrast, the i.c.v. injection of L-ornithine clearly attenuated the stress response in a dose-dependent manner, and induced sleep-like behavior. The L-ornithine concentration in the telencephalon and diencephalon increased following the i.c.v. injection of L-arginine. In addition, several free amino acids including L-alanine, glycine, L-proline and L-glutamic acid concentrations increased in the telencephalon. In conclusion, it appears that L-ornithine, produced by arginase from L-arginine in the brain, plays an important role in the sedative and hypnotic effects of L-arginine observed during a stress response. In addition, several other amino acids having a sedative effect might partly participate in the sedative and hypnotic effects of L-arginine.

Intracerebroventricular injection of L-arginine induces sedative and hypnotic effects under an acute stress in neonatal chicks.

L-arginine participates in many important and diverse biochemical reactions associated with the normal physiology of the organism. In the present study, we investigated the effect of central administration of L-arginine on the stress response and its mechanism in neonatal chicks. Intracerebroventricular (i.c.v.) injection of L-arginine clearly attenuated the stress response in a dose-dependent manner, and induced sleep-like behavior during 10 min. To clarify the mechanism by which L-arginine induces sedative and hypnotic effects in chicks, we investigated the effects of nitric oxide (NO) synthase (NOS) inhibitors on L-arginine-induced sedative and hypnotic effects, and as well as the effects of a NO donor. L-Arginine-induced (1.9 micromol) sedative and hypnotic effects were attenuated by i.c.v. co-injection with a non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester HCl (400 nmol). In addition, the effects of L-arginine were slightly attenuated by the inactive isomer of the NOS inhibitor N(G)-nitro-D-arginine methyl ester HCl (400 nmol). The i.c.v. injection of 3-morpholinosylnomine hydrochloride, a spontaneous NO donor, had little effect on postures. The i.c.v. injection of L-arginine had no effect on NOx concentration at various brain sites. These results suggested that the contribution of NO generation via NOS may be low in the sedative and hypnotic actions of L-arginine. Therefore, L-arginine and/or its metabolites, excluding NO, may be necessary for these actions.

Relationships between the sedative and hypnotic effects of intracerebroventricular administration of L-serine and its metabolites, pyruvate and the derivative amino acids contents in the neonatal chicks under acute stressful conditions.

Intracerebroventricular (i.c.v.) injection of L-serine was shown to have sedative and hypnotic effects on neonatal chicks under acute stressful conditions. To clarify the central mechanism of these effects of L-serine, two experiments were done. First, we focused on the glycogenic pathway in which L-serine is converted into pyruvate and finally glucose. I.c.v. administration of pyruvate (0.84 micromol) did not induce any behavioral and endocrinological changes, while L-serine and glucose triggered sedative and hypnotic effects. Secondly, the relationship between the sedation by L-serine and the metabolism into other amino acids which have sedative effects was investigated in the telencephalon and diencephalon. In both brain areas, a dose-dependent increase was seen in L-serine, although other amino acids were not changed. In the present study, it was concluded that the sedative action of L-serine was not due to the action of its metabolite pyruvate, or to the action of other amino acids.

Interaction of leptin and nitric oxide on food intake in broilers and Leghorns.

Leptin, which is produced in proportion to adiposity, has been reported to regulate feeding behaviors. Previous researchers reported that inhibition of nitric oxide (NO) synthase (NOS) decreased food intake, while L-arginine attenuated this effect. Recently, studies showed that NO plays an important role as a mediator of feeding behavior induced by a variety of neuropeptides. We investigated whether the anorectic effect of leptin is mediated by nitric oxide in broilers and Leghorns. In the first experiment, leptin was intracerebroventricularly (ICV) administered into the right lateral ventricle of broilers and food intake monitored at 15-min intervals through 180 min postinjection. L-arginine attenuated the decrease in food intake induced by leptin. In the second experiment, leptin was coinjected ICV with NG-nitro-arginine methyl ester HC1 (L-NNA), a NOS inhibitor. In the following study, we investigated whether the decreased feeding induced by leptin (10 microg/l0 microl) is mediated by nitric oxide in chickens. Three week old chickens were administered two levels of leptin (A=aCSF, B=10 microg/l0 microl) into the right lateral ventricle, and nitrate and nitrite (nitric oxide metabolites) were monitored 30-min postinjection. The results showed leptin decreased NO formation significantly compared with the control group. These results suggest that NO interacts with leptin in the central nervous system to modulate feeding behavior in the chicken.

Growth performance and intestinal morphology responses in early weaned pigs to supplementation of antibiotic-free diets with an organic copper complex and spray-dried plasma protein in sanitary and nonsanitary environments.

The objective of this study was to determine the effects of addition of spray-dried plasma protein (SDPP) and Cu to nonmedicated diets on growth performance and intestinal morphology in weaned pigs reared in sanitary or nonsanitary environments. Weanling pigs (n = 192, 18 +/- 2 d of age, 6.0 +/- 0.2 kg of BW) were assigned to 8 treatments arranged factorially, including 2 dietary levels of SDPP (0 or 6% for the initial 10 d), 2 levels of added dietary Cu (0 or 200 ppm for the entire 35-d experiment), and 2 pen sanitation conditions (sanitized or nonsanitized before pig placement). The nonsanitary pen condition was created by 3 applications of swine manure slurry to all pen surfaces in 1 room and not washing or disinfecting. In an identical adjacent room, sanitary pens were washed and disinfected before weaning. There were 4 pigs per pen, and feed and water were available ad libitum. Growth performance was determined at the end of each diet formulation phase (d 10, 20, and 35 after weaning). On d 10, 1 pig per pen was euthanized, and cross sections of duodenum, jejunum, and ileum were collected for microscopic assessment of mucosal morphology. During the initial postweaning period, SDPP, and Cu supplementation improved ADG and ADFI (P < 0.001). A trend for an interaction of sanitation x dietary SDPP (P = 0.07) was observed for G:F, with a positive response to the supplement in nonsanitary pens but no response in sanitary pens. There were no interactions of SDPP and Cu for any performance variables (P > 0.30). By d 35, there were no main or interaction effects of treatment on ADG or G:F (P > 0.17). Pen sanitation condition produced morphological effects, with shorter villous length and less crypt depth observed in each intestinal segment for pigs reared in the nonsanitary pens (P < 0.05), but these effects must be considered conditional based on the potential confounding influence of separate nursery rooms. In the duodenum, reduced crypt depth with Cu supplementation (P = 0.01) and a tendency for greater villous length with SDPP supplementation (P = 0.09) were observed. In this study, SDPP and Cu supplementation improved pig growth performance during the initial 10-d postweaning. These modifications to nonmedicated diets acted independently with regard to their impacts on postweaning performance and, therefore, could have additive effects.

Interaction between nociceptin/orphanin FQ (N/OFQ) and GABA in response to feeding.

Nociceptin/orphanin FQ (N/OFQ) and gamma aminobutyric acid (GABA) agonists have been shown to increase feed intake in mammals and birds. In this study, the effect of intracerebroventricular (icv) injection of the potent NOP receptor agonists Nociceptin (1-13) NH(2), the GABA(A) receptor antagonist bicuculline, and the GABA(A) receptor agonist muscimol on feed intake in cockerels was investigated. The icv injection of N/OFQ and muscimol increases feed intake. The effect of N/OFQ on feed intake was strongly blocked by the injection of bicuculline whereas the effect of muscimol was stimulated by N/OFQ. These results suggest that N/OFQ may act at GABA(A) receptors or increases overflow of GABA in the brain of chickens to stimulate feeding.

Feeding and locomotion responses to centrally injected nociceptin/orphanin FQ in chicks.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like receptor or nociceptin receptor (NOP), has been shown to induce feeding, locomotion, anti-stress and anxiolytic effects in rodents after central nervous system injection. In this study, the effect of intracerebroventricular (icv) injection of N/OFQ on feeding and locomotion behavior was evaluated in male broiler-type chickens. The icv injection of N/OFQ caused a moderate but significant increase in feed intake similar to the classical opioid peptides in rats. It also increased feed pecking frequency and feeding time 1 h after injection. Stepping, wing flapping and preening were not affected by N/OFQ. These results suggest that N/OFQ can act within the central nervous system of chickens to increase feed intake.

Brain creatine functions to attenuate acute stress responses through GABAnergic system in chicks.

The involvement of brain creatine in the adaptation to acute stress responses was investigated in chicks. In experiment 1, brain creatine content of chicks exposed to social separation stress was significantly increased compared with control chicks. The effects of i.c.v. injection of creatine (2 mug) on vocalizations, spontaneous activity and plasma corticosterone concentration in chicks under social separation stress were investigated in experiment 2. All measurements were attenuated by the i.c.v. injection of creatine compared with the controls under separation stress. Creatine also significantly decreased the active posture, but increased the motionless eye-opened posture, compared with the control. To clarify the relationship between creatine function and GABA receptors, the i.c.v. co-injection of creatine with picrotoxin, a GABA-A receptor antagonist, or CGP54626, a GABA-B receptor antagonist, was investigated in experiments 3 and 4. The effects of creatine on vocalizations and spontaneous activity were attenuated by co-injection of picrotoxin. In this case, active postures decreased by creatine were recovered by co-injection with picrotoxin. However, these effects were not obtained with CGP54626. The results suggest that central creatine functions within the CNS to attenuate the acute stress response by acting through GABA-A receptors in chicks.

Effects of long-term dietary lipids on mature bone mineral content, collagen, crosslinks, and prostaglandin E2 production in Japanese quail.

This study investigated the effects of long-term dietary lipids on mature bone mineral content, collagen concentration, crosslink levels, bone marrow and ex vivo prostaglandin E2 (PGE2) biosynthesis, as well as the relationship of PGE2 production to these bone formation parameters. One-month-old male Japanese quail were given a basal diet containing 1 of 4 lipid sources: soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO) at 50 g/kg of the diet. At 8 mo of age, lipid treatments did not affect bone length, diameter, or weight in quail. Quail fed SBO or CF had significantly lower levels of mineral content in tibial bones compared with those given FO. Bone collagen level was significantly higher in quail consuming SBO than those given HSBO or CF. Collagen crosslink concentration was markedly increased in birds provided FO or HSBO compared with those fed SBO or CF. Prostaglandin E2 biosynthesis in bone organ culture and marrow were greatly increased in quail maintained on the SBO or CF diet compared with those given the FO or HSBO diet. Prostaglandin E2 production in the bone microenvironment was negatively correlated with tibial ash and collagen crosslinks but had a positive correlation with tibial collagen levels. These results support our previous findings that long-term exposure to diets high in SBO or CF impaired mature bone mechanical properties and histological characteristics. Further, the results suggest that long-term supplementation of SBO or CF in the diet had a significant adverse effect on mature bone metabolism, and that dietary lipids altered bone metabolism, perhaps partially by controlling the production of local regulatory factor in bone.

Differences in catecholamine metabolism and behaviour in neonatal broiler and layer chicks.

1. To clarify the difference in behavioural activities and catecholamine metabolism between layer and broiler-type chicks two experiments were conducted. 2. In experiment 1, 1-d-old male layer and broiler chicks were placed in an open-field area and their responses were investigated for 10 min. The responses of the two strains were remarkably different, with broilers being less active than layers. Vocalisations rapidly decreased in broilers whereas those of layers remained elevated during the 10 min. 3. In experiment 2, 1-d-old chicks of both strains were killed and brain catecholamine concentrations were determined in three parts of the brain: telencephalon, optic lobe and brain stem. 4. In the whole brain, dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) were significantly higher in broilers. However, the values for norepinephrine (NE), epinephrine (E) and 4-hydroxy-3-methoxyphenylacetic acid (HVA) were similar between strains. The ratios of metabolite/precursor were also calculated: HVA/DOPAC was higher in layers, while NE/DA, E/NE and DOPAC/DA were not significantly different between strains. 5. These results suggest that behavioural activities differ greatly, while there are some differences in catecholamine metabolism between the two strains.