Topographic studies of the effects of microinjections of muscimol on the hypothalamic control of feed intake in sheep

Ten sheep were used to define the anatomical basis for the feeding systems sensitive to gamma-aminobutyric acid, by using intrahypothalamic microinjections of the gamma-aminobutyric acid agonist, muscimol. In satiated sheep, 1 microL of muscimol (0.5 nmol/microL) elicited feeding when injected into paraventricular, ventromedial, and anterior hypothalamic areas. Similar injections into 39 sites tested in 6-h fasted sheep failed to decrease feed intake. The data suggest that neurons sensitive to gamma-aminobutyric acid in medial hypothalamus may be involved in the initiation of feeding.     

A role for hypothalamic malonyl-CoA in the control of food intake

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.     

Selected hormonal and neurotransmitter mechanisms regulating feed intake in sheep

Appetite control is a major issue in normal growth and in suboptimal growth performance settings. A number of hormones, in particular leptin, activate or inhibit orexigenic or anorexigenic neurotransmitters within the arcuate nucleus of the hypothalamus, where feed intake regulation is integrated. Examples of appetite regulatory neurotransmitters are the stimulatory neurotransmitters neuropeptide Y (NPY), agouti-related protein (AgRP), orexin and melanin-concentrating hormone and the inhibitory neurotransmitter, melanocyte-stimulating hormone (MSH). Examination of messenger RNA (using in situ hybridization and real-time PCR) and proteins (using immunohistochemistry) for these neurotransmitters in ruminants has indicated that physiological regulation occurs in response to fasting for several of these critical genes and proteins, especially AgRP and NPY. Moreover, intracerebroventricular injection of each of the four stimulatory neurotransmitters can increase feed intake in sheep and may also regulate either growth hormone, luteinizing hormone, cortisol or other hormones. In contrast, both leptin and MSH are inhibitory to feed intake in ruminants. Interestingly, the natural melanocortin-4 receptor (MC4R) antagonist, AgRP, as well as NPY can prevent the inhibition of feed intake after injection of endotoxin (to model disease suppression of appetite). Thus, knowledge of the mechanisms regulating feed intake in the hypothalamus may lead to mechanisms to increase feed intake in normal growing animals and prevent the wasting effects of severe disease in animals.     

Mechanisms of seizure control mediated by gamma-aminobutyric acid: role of the substantia nigra

The substantia nigra has been identified as a critical site at which gamma-aminobutyric acid (GABA) agonist drugs act to reduce susceptibility to a number of types of experimentally induced generalized seizures. Moreover, the ability of systemically administered GABA-elevating agents to protect against maximal electroshock seizures is directly correlated with an increase in GABA specifically in the nerve-terminal compartment of substantia nigra. The significance of these findings is discussed in terms of the role of specific nigral synapses for the control of seizure propagation. Evidence from lesion studies, as well as studies with opiates and substance P analogs, further supports the hypothesis that Inhibition of nigral efferents reduces susceptibility to generalized seizures. Inhibition of nigral outflow causes a decreased sensitivity to chemoconvulsants without precluding the animal's ability to exhibit any or all of the motor components of a seizure. We therefore propose that nigral outputs are capable of facilitating seizure propagation and can function as a gating mechanism for the generalization of convulsive activity.     

Effect of gamma-aminobutyric acid on excitability of tubero-infundibular neurons in rat hypothalamic slices

The effect of gamma-aminobutyric acid (GABA) on antidromically identified tubero-infundibular (TI) neurons was examined in hypothalamic slices of ovariectomized female rats. Twenty antidromically evoked spikes were obtained in the medial basal hypothalamus, including the arcuate and ventromedial nuclei, by electrical stimulation of the median eminence. Sixteen of them had a notch in the rising phase and fractionation of the initial segment (IS)- and somatodendritic (SD)-spikes was elicited by repeated stimulation at frequencies higher than 10 Hz. The application of 0.5-1.5 mM GABA to the incubation medium inhibited SD spikes in 7 of these 16 neurons. The latency, amplitude and threshold of IS spikes were not affected by GABA except for one spike whose latency fluctuated. On the remaining 9 neurons having the notch, no effect of 5-10 mM GABA was discernible. Four of 20 antidromically evoked spikes, which had a smooth rising phase and a shorter duration, were not inhibited by 5-10 mM GABA, but a fluctuation of the latency was observed in one neuron. Fifteen neurons having spontaneous unit activity were also obtained in the arcuate nucleus and its adjacent area and tested with GABA. In 10 of the 15 neurons, spontaneous unit activity disappeared following 0.1-1.5 mM GABA perfusion, while the firing rate in the remaining 5 neurons was not affected by 5-10 mM GABA. These results provide evidence for a direct inhibitory effect of GABA on TI neurons and support the involvement of GABAergic neurons in regulating neuroendocrine functions.     

Myogenesis in sheep is altered by maternal feed intake during the peri-conception period

The effect of varying short-term maternal feed intake during the peri-conception period on the development of ovine fetal muscle at mid-gestation was investigated. Superovulated donor Merino ewes (n = 24) were fed a roughage/grain pelleted diet (10.1 MJME/kg dry matter) at either 1.5x maintenance (H; high) or 0.5x maintenance (L; low) from 18 days before until 6 days after ovulation. Embryos were transferred to recipient ewes (n = 60) on day 6. Singleton fetuses were collected on day 75 of gestation and placental weights, fetal body dimensions and fetal organ and muscle weights recorded. The number, type and size of muscle fibres and the dry matter, RNA, DNA and protein content in the semitendinosus muscle were determined. Maternal feed intake did not influence body dimensions, organ development or muscle weights in the fetus. However, L feed intake decreased total muscle fibre number in the fetus by approximately 20% (P = 0.06) compared to H feed intake. This resulted from a reduced secondary to primary fibre ratio (P < 0.05) and indicated that secondary fibre formation occurred at a reduced rate in L fetuses. In addition, protein:DNA ratio tended to be lower in muscles of L fetuses (P < 0.1). It is concluded that restricting feed intake over the peri-conception period reduces or delays myogenesis in fetal sheep. The potential mechanisms by which nutritional availability during this period may influence subsequent myogenic development are discussed.     

Stimulation and inhibition of food intake in sheep by centrally-administered hypothalamic releasing factors

Short-term effects of hypothalamic releasing factors on feeding behavior and digestive motility patterns were assessed in hay-fed sheep trained to eat more than half the total amount eaten over 8 h within the first 3 h after food presentation. Thyrotropin-releasing hormone (TRH) given intracerebroventricularly (ICV, 30 ng/kg) or intravenously at higher doses (IV, 3 micrograms/kg) reduced food consumption by 20 p. cent. The ICV or IV TRH-induced reduction was associated with behavioral excitation and stimulation of antroduodenal motor activity without changes in water intake. The ovine corticotropin releasing factor (oCRF 41) decreased food and water intake by 30-50% when administered ICV (60 ng/kg) but was not active when given systemically at doses up to 6 micrograms/kg. The synthetic human growth hormone releasing factor (hGRF 44) administered centrally (60 ng/kg) increased the amount of food intake and the antral motor activity without behavioral excitation. The results indicate a centrally-mediated facilitation of food intake by GRF and its inhibition by CRF which also affect water consumption. The presence of digestive motor effects suggests that extrapituitary pathways may be involved, as for TRH, in the action of both GRF and CRF.     

Characterization of the rumen microbiota and its relationship with residual feed intake in sheep

Feed efficiency is a highly important economic trait in sheep production and has a significant impact on the economic benefits of sheep farming. Microbial fermentation of the rumen has a vital role in the host's nutrition; the rumen microbiota might affect host feed efficiency. However, the relationship between the rumen microbiota and feed efficiency in sheep is unclear. In the present study, the microbiota of 195 Hu sheep was investigated and their residual feed intake (RFI), a commonly used measure of feed efficiency, was determined. From birth, all sheep were subjected to the same management practices. At slaughter, samples of liquid rumen contents were collected and subjected to amplicon sequencing for the 16S rDNA gene on the IonS5™XL platform. To identify the bacterial taxa differentially represented at the genus or higher taxonomy levels, we used linear discriminant analysis coupled with effect size and curve fitting. In the sheep rumen, the four most abundant phyla were Firmicutes, Bacteroidetes, Fibrobacteres, and Proteobacteria; and the dominant genera were unidentified Prevotellaceae, Fibrobacter, unidentified Lachnospiraceae, Saccharofermentans, and Succinivibrio. Pathway analysis of the 16S rDNA sequencing data from the rumen microbiota identified that carbohydrate metabolism was enriched. Using α-diversity analysis, we further identified that Observed species, ACE, Good's coverage, and Chao1 are more abundant (P < 0.01) in the low-RFI (L-RFI) group compared to the high-RFI (H-RFI) group. High-RFI sheep had a higher abundance of three bacterial taxa (Prevotellaceae, Negativicutes, and Selenomonadales), and one taxa was overrepresented in the L-RFI sheep (Succinivibrio), respectively. Furthermore, model fitting showed that Veillonellaceae, Sphaerochaeta, Negativibacillus, Saccharofermentans, and members of the Tenericutes, Kiritimatiellaeota, Deltaproteobacteria, and Campylobacterales were correlated with the sheep RFI classification and thus were indicative of a role in animal efficiency. Tax4Fun analysis revealed that metabolic pathways such as “energy metabolism,” “metabolism of cofactors and vitamins,” “poorly characterized,” and “replication recombination and repair proteins” were enriched in the rumen from H-RFI sheep, and “genetic information processing” and “lipopolysaccharide biosynthesis” were overrepresented in L-RFI sheep rumen. In addition, six Kyoto Encyclopedia of Genes and Genomes orthology pathways were identified as different between H-RFI and L-RFI groups. In conclusion, the low RFI phenotype (efficient animals) consistently (or characteristically) exhibited a more abundant and diverse microbiome in sheep.     

The role of hypothalamic input on corticotroph maturation in fetal sheep

Corticotropin-releasing hormone receptor type 1 (CRH-R1) expression and vasopressin type 1b (V1b) receptor protein decrease in late-gestation fetal sheep. Because hypothalamo-pituitary disconnection (HPD) has been demonstrated to prevent the morphological maturation of corticotrophs, we hypothesized that hypothalamic input is necessary for the maturational changes in CRH-R1 and V1b receptor levels. We measured CRH-R1 and V1b receptor expression in the anterior pituitaries of fetuses at 140 days gestational age (dGA) that underwent HPD or sham surgery at 120 dGA. CRH-R1 mRNA decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. However, CRH-R1 protein levels were elevated in HPD fetuses compared with sham and were not different from 120 dGA values. V1b protein levels decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. We conclude that hypothalamic input to the pituitary is necessary for the decrease in CRH-R1 receptor protein levels in late-gestation fetal sheep. However, hypothalamic input is not necessary for the decrease in V1b receptor expression seen in late gestation.     

Probable gamma-aminobutyric acid involvement in bisphenol A effect at the hypothalamic level in adult male rats

The aim of the present study was to investigate the effects of bisphenol A (BPA) on the neuroendocrine mechanism of control of the reproductive axis in adult male rats exposed to it during pre- and early postnatal periods. Wistar mated rats were treated with either 0.1% ethanol or BPA in their drinking water until their offspring were weaned at the age of 21 days. The estimated average dose of exposure to dams was approximately 2.5 mg/kg body weight per day of BPA. After 21 days, the pups were separated from the mother and sacrificed on 70 day of life. Gn-RH and gamma-aminobutyric acid (GABA) release from hypothalamic fragments was measured. LH, FSH, and testosterone concentrations were determined, and histological and morphometrical studies of testis were performed. Gn-RH release decreased significantly, while GABA serum levels were markedly increased by treatment. LH serum levels showed no changes, and FSH and testosterone levels decreased significantly. Histological studies showed abnormalities in the tubular organization of the germinal epithelium. The cytoarchitecture of germinal cells was apparently normal, and a reduction of the nuclear area of Leydig cells but not their number was observed. Taken all together, these results provide evidence of the effect caused by BPA on the adult male reproductive axis when exposed during pre- and postnatal period. Moreover, our findings suggest a probable GABA involvement in its effect at the hypothalamic level.     

Glutamic acid and gamma-aminobutyric acid neurotransmitters in central control of breathing.

We review recent cross-disciplinary experimental and theoretical investigations on metabolism of the amino acid neurotransmitters glutamic acid and gamma-aminobutyric acid (GABA) in the brain during hypoxia and hypercapnia and their possible role in central control of breathing. The roles of classical modifiers of central chemical drive to breathing (H+ and cholinergic mechanisms) are summarized. A brief perspective on the current widespread interest in GABA and glutamate in central control is given. The basic biochemistry of these amino acids and their roles in ammonia and bicarbonate metabolism are discussed. This review further addresses recent work on central respiratory effects of inhibitory GABA and excitatory glutamate. Current understanding of the sites and mechanisms of action of these amino acids on or near the ventral surface of the medulla is reviewed. We focus particularly on tracer kinetic investigations of glutamatergic and GABAergic mechanisms in hypoxia and hypercapnia and their possible role in the ventilatory response to hypoxia. We conclude with some speculative remarks on the critical importance of these investigations and suggest specific directions of research in central mechanisms of respiratory control.     

Studies on gamma-aminobutyric acid transport in cobalt experimental epilepsy in the rat

Abstract: Crude brain homogenates and cerebral tissue slices from rats with cobalt metal implanted in right and left cerebral cortices were used to examine high- and low-affinity GABA transport. High-affinity GABA transport was maximally reduced to 34% of controls 7 days after cobalt implantation, a time that coincides with peak seizure activity in this model. Kinetic analysis of high-affinity GABA transport, using brain homogenates, revealed a significant change in V_max 7 days after cobalt implantation. (V_max= 446.4 ± 26.2 pmol/mg prot./min, cobalt, versus 787.8 ± 67.3, control). An analysis of the low-affinity system revealed no depression of K_m , or V_max parameters. Administration of valproic acid at a concentration as high as 1 mM in vitro or a dose of 300 mg/kg in vivo had no effect on high- or low-affinity GABA transport. The results obtained from cobalt-treated rats provide additional evidence for an involvement of GABA in experimental epilepsy.     

Studies of the role of the N-methyl-D-aspartate (NMDA) receptor in the hypothalamic control of prolactin secretion.

To further examine the role of excitatory amino acids in the control of prolactin (PRL) secretion, the effects of administering a specific agonist and an antagonist of the N-methyl-D-aspartate (NMDA) receptor on plasma PRL concentrations were examined in the adult male rat. Animals of the Sprague-Dawley strain weighing 250-300 g were implanted with an indwelling cardiac catheter via the right jugular vein. Blood samples were collected through the catheter at 5 min intervals for 40 min, beginning 5 min before the iv administration of drug or the saline vehicle (V). Plasma PRL and luteinizing hormone (LH) concentrations were estimated using RIAs. Groups of animals (n = 5-7) received N-methyl-D,L-aspartate (NMA), D,L-2-amino-5-phosphonopentanoic acid (AP5), AP5 and NMA, norvaline (NOR), or V. The effects of administering the NMDA receptor antagonist alone were studied on two separate occasions. Injection of NMA (4.5 mg/rat) resulted in unambiguous PRL and LH discharges. Treatment with AP5 (9 mg/rat) 1 min prior to NMA administration completely blocked the LH releasing action of NMA, but did not significantly alter the discharge of PRL. Injection of AP5, alone, generally elicited a distinct and robust discharge of PRL, although plasma LH levels in these animals remained unchanged. NOR, an amino acid structurally related to AP5, administered at a dose (5.3 mg/animal) isomolar to that of AP5, was without effect on PRL and LH secretion, as was injection of V alone. These findings suggest that neuroexcitatory amino acids acting at the NMDA receptor may play a role in modulating the activity of neuronal systems that govern the release of both PRL releasing factor (PRF) and PRL inhibiting factor (PIF) into hypophysial portal blood.     

Effects of intrahypothalamic injections of GABA, muscimol, pentobarbital, and L-glutamic acid on feed intake of satiated sheep

Five wethers were surgically prepared with cranial implants to study the role of gabaminergic neural pathways on the hypothalamic control of feeding behaviour in ruminants. In the first experiment, the animals were injected (1 microL) with a physiological Tyrode (0.95%) solution, muscimol (0.5 and 1.0 nmol), GABA (0.5 and 1.0 nmol), and L-glutamic acid (0.5 and 1.0 nmol). Feed intake following injections of muscimol (1.0 nmol) and L-glutamic acid (0.5 and 1.0 nmol) was twice as large as that following the Tyrode solution, at 60-min postinjections. These results, however, were not statistically significant (p = 0.12-0.15). In the second experiment, the animals were injected (1 microL) with saline, muscimol (0.8 nmol), L-glutamic acid (0.8 nmol), and pentobarbital (0.26 mumol). Fifteen minutes after the injections, pentobarbital had induced a significant feeding response when compared with control values (p less than 0.01), whereas the effect of L-glutamic acid was not significant. However, 30 min after the injections, feed intake of sheep having received L-glutamic acid was higher than that obtained with the control injections (p less than 0.01). The response to pentobarbital was stronger than that to either muscimol or L-glutamic acid. Histological analyses of brain tissue indicated that injections were performed in the ventromedial hypothalamus of four sheep and in the dorsomedial hypothalamus of the other. The data indicate that L-glutamic acid stimulates feed intake by acting either as a precursor of GABA or by a direct stimulation of glutaminergic neural pathways involved in the control of feed intake.     

Hormonal control of metabolism of gamma-aminobutyric acid in the rat hypothalamus and hippocampus

It has been established that hydrocortisone administration increased the amount of total, free, bound and synaptosomal GABA in the hypothalamus, glutamate decarboxylase activity in the homogenate and synaptosomes and time of the mediator turnover. ACTH administration increased the GABA content and glutamate decarboxylase activity in synaptosomes. The total amino acid content and time of its turnover got higher only with single hormone administration. In the hippocamp hydrocortisone administration increased the total and free GABA contents, its turnover time, glutamate decarboxylase activity in the homogenate and decreased GABA-aminotransferase activity in the homogenate and synaptosomes. The GABA level in synaptosomes grew only with multiple hormone administration. Single administration of ACTH decreased the total GABA content, glutamate decarboxylase activity in the homogenate, while its multiple administration increased the GABA level in synaptosomes followed by a decrease of GABA-aminotransferase activity in the homogenate and synaptosomes. The GABA turnover time fell with single hormone administration and grew with the multiple one. Adrenalectomy induced no changes in the GABA content and activity of its metabolism enzymes in the hypothalamus, however the bound GABA level decreased, while the turnover time increased. In the hippocamp adrenalectomy decreased total, free and synaptosomal GABA contents, glutamate decarboxylase activity in a homogenate and turnover time. Subsequent hydrocortisone administration only partly normalized the revealed changes of the GABA metabolism in the brain structures under adrenalectomy.     

Sire and liveweight affect feed intake and methane emissions of sheep confined in respiration chambers

Daily methane production and feed intake were measured on 160 adult ewes, which were the progeny of 20 sires and 3 sire types (Merino, dual-purpose and terminal) from a genetically diverse flock. All animals were housed in individual pens and fed a 50/50 mix of chaffed lucerne and oaten hays at 20 g/kg liveweight (LW), with feed refusals measured for at least 10 days before the first of three 22-h measurements in respiration chambers (RC). Feed was withdrawn at 1600 h on the day before each RC test to encourage the ewes to eat the entire ration provided for them in the RC. After the first 1-day RC test, the sheep were returned to their pens for a day, then given a second 1-day RC test, followed by another day in their pens, then a third RC test. After all animals had been tested, they were ranked according to methane emissions adjusted for feed intake in the RC and on the previous day, enabling 10 low and 10 high methane animals to be chosen for repeat measurement. No variation between sires nor consistent effects of LW on feed eaten (%FE, expressed as per cent of feed offered) was evident in the 10 days before the first RC measurement. However, significant differences between sires (equivalent to an estimated heritability of 41%) were identified for %FE during the 2^nd and 3^rd days of RC testing (2 and 4 days after the initial RC test). The analysis of all data showed that methane emissions in the RC were related to feed intake on the day of testing and the two previous days (all P<0.0005). Before correcting for feed intake on previous days, there was some variation between sires in methane yield, equivalent to an estimated heritability of 9%. Correction for feed intake on the 2 previous days halved the residual variation, allowing other effects to be detected, including effects of LW, twins reared as singles, test batch, RC and test-day effects, but estimated sire variation fell to zero. In order to avoid potential biases, statistical models of methane emissions in the RC need to consider potential confounding factors, such as those identified as significant in this study.