Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.     

Localization and effects of orexin on fasting motility in the rat duodenum

The orexins [orexin A (OXA) and orexin B (OXB)] are novel neuropeptides that increase food intake in rodents. The aim of this study was to determine the distribution of orexin and orexin receptors (OX1R and OX2R) in the rat duodenum and examine the effects of intravenous orexin on fasting gut motility. OXA-like immunoreactivity was found in varicose nerve fibers in myenteric and submucosal ganglia, the circular muscle, the mucosa, submucosal and myenteric neurons, and numerous endocrine cells of the mucosa. OXA neurons displayed choline acetyltransferase immunoreactivity, and a subset contained vasoactive intestinal peptide. OXA-containing endocrine cells were identified as enterochromaffin (EC) cells based on the presence of 5-hydroxytryptamine immunoreactivity. OX1R was expressed by neural elements of the gut, and EC cells expressed OX2R. OXA at 100 and 500 pmol x kg(-1) x min(-1) significantly increased the myoelectric motor complex (MMC) cycle length compared with saline. Similarly, OXB increased the MMC cycle length at 100 pmol x kg(-1) x min(-1), but there was no further effect at 500 pmol x kg(-1) x min(-1). We postulate that orexins may affect the MMC through actions on enteric neurotransmission after being released from EC cells and/or enteric neurons.     

Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.     

The regulation of hepatic protein synthesis during fasting in the rat

We have studied translational control in the model of 48 h of fasting in the rat. Our initial observations showed a paradoxical increase in ribosomal protein S6 (rpS6) phosphorylation and a decrease in eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation. These effects, which would favor an increase in protein synthesis, could be attributed to increased circulating concentrations of branched-chain amino acids in fasting. To determine what mechanisms might account for decreased hepatic translation in fasting, we examined the cap binding complex. eIF4E-bound 4E-BP1 did not increase. However, eIF4E-bound eIF4G and total cellular eIF4G were profoundly decreased in fasted liver. eIF4G mRNA levels were not lower after fasting. Based on the hypothesis that decreased eIF4G translation might account for the reduced eIF4G content, we fractionated ribosomes by sucrose density centrifugation. Immunoblotting for rpS6 showed modest polysomal disaggregation upon fasting. PCR analysis of polysome profiles revealed that a spectrum of mRNAs undergo different translational regulation in the fasted state. In particular, eIF4G was minimally affected by fasting. This indicated that reduced eIF4G abundance in fasting may be a function of its stability, whereas its recovery upon refeeding is necessarily independent of its own involvement in the cap binding complex. Western immunoblotting of polysome fractions showed that phosphorylated rpS6 was disproportionately present in translating polysomes in fed and fasted animals, consistent with a role in translational control. However, the translation of rpS8, an mRNA with a 5'-oligopyrimidine tract, did not coincide with rpS6 phosphorylation, thus dissociating rpS6 phosphorylation from the translational control of this subset of mRNAs.     

Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment

Using several antibodies against rat or human laminin and an avidin-biotin immunocytochemical protocol, laminin-like immunoreactivity was detectable in the rat nervous system in expected locations, i.e., associated with blood vessels and reactive astrocytes. However, laminin staining was also abundantly present within neuronal cell bodies in most parts of the developing and adult rat CNS. Medial septum neuronal immunoreactivity was lost after septo-hippocampal disconnection, but could be preserved or even restored by intraventricular administration of nerve growth factor. Thus, at least for medial septum neurons, this laminin-like molecule can be accumulated or produced independent of direct hippocampal (target) contact. It remains to be determined whether CNS neuronal "laminin" processes activities similar to those found for laminin in vitro.     

Short fasting dramatically decreases rat duodenal secretory responsiveness to orexin A but not to VIP or melatonin

Orexins are involved in the central nervous control of appetite and behavior, and in addition, they are present in endocrine cells and/or neurons in the intestine. The role of these peptides in peripheral regulation of intestinal secretion has not been investigated. We thus compared the effects of orexin A and some established secretagogues on duodenal HCO3- secretion in fed rats with effects in rats exposed to short (overnight) food deprivation. Rats were anesthetized with thiobarbiturate, a 12-mm segment of proximal duodenum with intact blood supply was cannulated in situ, and the alkaline secretion was titrated by pH stat. Secretagogues were supplied specifically to the duodenum by close intra-arterial infusion. Orexin A (60-600 pmol x kg(-1) x h(-1)) caused marked and dose-dependent stimulation of the duodenal secretion in fed animals but did not affect secretion in overnight food-deprived animals. Similarly, short fasting caused a 100-fold increase in the amount of the muscarinic agonist bethanechol (from 50 to 5,000 nmol x kg(-1) x h(-1)) required for stimulation of the secretion. In contrast, the secretory responses to VIP (50-1,000 pmol x kg(-1) x h(-1)) and melatonin (20-200 nmol x kg(-1) x h(-1)) were not affected. The appetite-regulating peptide orexin A is thus a stimulant of intestinal secretion, but the response to this peptide as well as the muscarinic agonist bethanechol is markedly dependent on previous intake of food. Overnight fasting is a standard experimental procedure in studies of gastrointestinal function and pathophysiology in humans and animals. Studies made on neuroendocrine control of intestinal secretion may require reevaluation with respect to feeding status.     

Regional distribution of ethanol in rat brain

The cerebral distribution of a low ip dose of ethanol (ETOH) was studied using a double-barrelled, membrane-tipped perfusion cannula in rats. The cannulas were perfused with physiological solution in freely-moving animals at a rate of 19 μl/min for 5 min at 5, 10, and 15 min and subsequently at 15 min intervals for the remainder of 2 hrs after 1 g/kg ETOH. Peak blood ETOH levels (in mg/ 100 ml) after the single dose were 4 times those found in the lateral ventricle, 6–7 times those found in the reticular formation, cerebral cortex, and amygdala, and 9–11 times those found in the caudate and lateral hypothalamus. Peak levels were reached earliest in the lateral ventricle and reticular formation. In a related study, homogenized (“whole”) brain ETOH levels were found to be similar to blood levels while flushed (“bloodless”) brain ETOH levels were approximately 20% lower than those found in blood and “whole” brain. It is concluded that there is a significant differential distribution of ETOH in the rat brain after a low dose of ETOH, and that this unequal brain ETOH distribution may influence the behavioral effects of the drug.     

Orexin与胃肠活动的调节

Orexin除了主要分布于中枢神经系统的下丘脑外,还广泛分布于包括外周的胃肠组织,如胃肠神经丛和内分泌细胞等部位。Orexin在促进食欲的同时,还能通过中枢和外周途径调节胃肠运动和消化液的分泌。     

Subcellular distribution of biopterin in rat brain: the biochemical basis of its stability

Rat brain biopterin, the hydroxylase cofactor, was observed to distribute equally across regional subcellular fractions, rather than to codistribute neuronally with tyrosine and tryptophan hydroxylases for which it functions. Over a 24 h period with light/dark phasing, which some groups have shown to result in cycling of biopterin levels in striate and certain other regions, only the biopterin associated with the crude nuclear fraction of the striate (not associated with neurotransmitter synthesis) demonstrated a diurnal cycle. The selectivity of this perturbation response to the striate nuclear fraction suggests that (1) multiple subcellular loci of biopterin might exist independently in rat brain neurons and (2) the pterin's availability for neurotransmitter biosynthesis is limited beyond its apparent regional concentration. The demonstration of multiple independent sources of neuronal biopterin may be relevant to understanding why regional levels have been so resistant to efforts at pharmacological manipulation (only amphetamine and CRF have changed striate biopterin levels). It also shows that changes in regional hydroxylase cofactor levels may not be related to neurotransmitter synthesis, but instead may result from another presently unknown demand for the cofactor at a disparate neuronal site.     

Cloning of rat parkin cDNA and distribution of parkin in rat brain

The rat parkin cDNA sequence was characterized after screening a rat hypothalamus cDNA library with a 32P-labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465-amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N-terminal ubiquitin and the ring-IBR (in between ring)-ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity-purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295-311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52-kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin-immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin-enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early-onset parkinsonism.