Effects of dietary methionine supplementation from different sources on growth performance and meat quality of barrows and gilts.

Methionine is indispensable for growth and meat formation in pigs. However, it is still unclear that increasing dietary sulphur-containing amino acid (SAA) levels using different methionine sources affects the growth performance and meat quality of barrows and gilts. To investigate this, 144 pigs (half barrows and half gilts) were fed the control (100% SAA, CON), DL-Methionine (125% SAA, DL-Met)-supplemented, or OH-Methionine (125% SAA, OH-Met)-supplemented diets during the 11-110 kg period. The results showed that plasma methionine levels varied among treatments during the experimental phase, with increased plasma methionine levels observed following increased SAA consumption during the 25-45 kg period. In contrast, pigs fed the DL-Met diet had lower plasma methionine levels than those fed the CON diet (95-110 kg). Additionally, gilts fed the DL-Met or OH-Met diets showed decreased drip loss in longissimus lumborum muscle (LM) compared to CON-fed gilts. OH-Met-fed gilts had higher pH45min values than those fed the CON or DL-Met diets, whereas OH-Met-fed barrows had higher L45min values than those fed the CON or DL-Met diets. Moreover, increased consumption of SAA, regardless of the methionine source, tended to decrease the shear force of the LM in pigs. In conclusion, this study indicates that increasing dietary levels of SAA (+25%) appeared to improve the meat quality of gilts by decreasing drip loss and increasing meat tenderness.

Peripheral corticotropin releasing factor (CRF) and a novel CRF1 receptor agonist, stressin1-A activate CRF1 receptor expressing cholinergic and nitrergic myenteric neurons selectively in the colon of conscious rats.

Intraperitoneal (i.p.) corticotropin releasing factor (CRF) induced a CRF(1) receptor-dependent stimulation of myenteric neurons and motility in the rat proximal colon. We characterize the colonic enteric nervous system response to CRF in conscious rats. Laser capture microdissection combined with reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry in longitudinal muscle myenteric plexus whole-mount colonic preparations revealed CRF(1) receptor expression in myenteric neurons. CRF (i.p., 10 microg kg(-1)) induced Fos immunoreactivity (IR) (cells per ganglion) selectively in myenteric plexus of proximal (18.3 +/- 2.4 vs vehicle: 0.0 +/- 0.0) and distal colon (16.8 +/- 1.2 vs vehicle: 0.0 +/- 0.0), but not in that of gastric corpus, antrum, duodenum, jejunum and ileum. The selective CRF(1) agonist, stressin(1)-A (i.p., 10 microg kg(-1)) also induced Fos IR in myenteric but not in submucosal plexus of the proximal and distal colon. Fos IR induced by CRF was located in 55 +/- 1.9% and 53 +/- 5.1% of CRF(1) receptor-IR myenteric neurons and in 44 +/- 2.8% and 40 +/- 3.9% of cholinergic neurons with Dogiel type I morphology, and in 20 +/- 1.6% and 80 +/- 3.3% of nitrergic neurons in proximal and distal colon respectively. CRF and stressin(1)-A elicit defecation and diarrhoea. These data support that one mechanism through which peripherally injected CRF ligands stimulate colonic function involves a direct action on colonic cholinergic and nitrergic myenteric neurons expressing CRF(1) receptor.

Role of brainstem TRH/TRH-R1 receptors in the vagal gastric cholinergic response to various stimuli including sham-feeding.

Pavlov's pioneering work established that sham-feeding induced by sight or smell of food or feeding in dogs with permanent esophagostomy stimulates gastric acid secretion through vagal pathways. Brain circuitries and transmitters involved in the central vagal regulation of gastric function have recently been unraveled. Neurons in the dorsal vagal complex including the dorsal motor nucleus of the vagus (DMN) express thyrotropin-releasing hormone (TRH) receptor and are innervated by TRH fibers originating from TRH synthesizing neurons in the raphe pallidus, raphe obscurus and the parapyramidal regions. TRH injected into the DMN or cisterna magna increases the firing of DMN neurons and gastric vagal efferent discharge, activates cholinergic neurons in gastric submucosal and myenteric plexuses, and induces a vagal-dependent, atropine-sensitive stimulation of gastric secretory (acid, pepsin) and motor functions. TRH antibody or TRH-R1 receptor oligodeoxynucleotide antisense pretreatment in the cisterna magna or DMN abolished vagal-dependent gastric secretory and motor responses to sham-feeding, 2-deoxy-D-glucose, cold exposure and chemical activation of cell bodies in medullary raphe nuclei. TRH excitatory action in the DMN is potentiated by co-released prepro-TRH-(160-169) flanking peptide, Ps4 and 5-HT, and inhibited by a number of peptides involved in the stress/immune response and inhibition of food-intake. These neuroanatomical, electrophysiological and neuropharmacological data are consistent with a physiological role of brainstem TRH in the central vagal stimulation of gastric myenteric cholinergic neurons in response to several vagal dependent stimuli including sham-feeding.

Activation of the parapyramidal region in the ventral medulla stimulates gastric acid secretion through vagal pathways in rats.

Neurons synthesizing thyrotropin-releasing hormone, substance P and serotonin in the medullary caudal raphe nuclei project to the dorsal vagal complex and play a role in the central vagal regulation of gastric function. Neurons in the parapyramidal region in the ventral medulla share similar biochemical coding and projections as those in the caudal raphe nuclei. The role of the parapyramidal region in the autonomic regulation of gastric acid secretion was investigated in urethane-anesthetized rats. Unilateral microinjection of kainate into the parapyramidal region at 10, 15 and 20 ng induced a dose-related stimulation of gastric acid secretion (net increases: 22.2+/-11.2, 40.5+/-8.5 and 89.8+/-19.4 micromol/60 min, respectively), while injection of vehicle had no effect (net change: -0.1+/-1.4 micromol/60 min). Time-course studies showed a nine-fold peak increase over basal at 30 min after parapyramidal injection of kainate (20 ng) and acid secretion returned to basal level at 70 min. Microinjections of kainate (15-20 ng) outside the parapyramidal region or into the parapyramidal region in vagotomized rats had no effect. Exposure to cold (4 degrees C) for 2 h, which is known to induce vagally mediated gastric secretory and motor responses through medullary thyrotropin-releasing hormone pathways, increased the number of Fos-positive cells in the caudal, middle and rostral parts of the parapyramidal region to 4.3+/-0.4, 9.4+/-0.9 and 18.4+/-1.6/section, respectively, compared with 0.1+/-0. 1, 0.1+/-0.0 and 0.7+/-0.6/section, respectively, in rats maintained at room temperature. Most of the Fos-labeled cells co-expressed pro-thyrotropin-releasing hormone messenger RNA signal and/or were serotonin immunoreactive. These data show that chemical activation of neurons in the parapyramidal region results in a vagal-dependent stimulation of gastric acid secretion and that acute cold exposure activates parapyramidal neurons containing pro-thyrotropin-releasing hormone and/or serotonin, suggesting a potential role of the parapyramidal region, in addition to the caudal raphe nuclei, as medullary sites involved in the vagal regulation of gastric function.

Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat.

The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the organization of limbic and motor circuits of the entopeduncular nucleus, suggesting that GABAergic transmission through the limbic pathway is regulated predominantly by glutamic acid decarboxylase-65, whereas glutamic acid decarboxylase-67 is of principal importance in the motor pathway. These data provide additional evidence that the neurons of the limbic and motor subregions of the entopeduncular nucleus are neurochemically distinct.

Localization of thyroid hormone receptor beta2 in the ventral medullary neurons that synthesize thyrotropin-releasing hormone.

Altered thyroid statuses are associated with autonomic disorders. Thyrotropin-releasing hormone (TRH) synthesized in medullary raphe pallidus (Rpa), raphe obscurus (Rob) and the parapyramidal regions (PPR) regulates vagal and sympathetic preganglionic motoneurons. Hypothyroidism increased TRH gene expression and c-Fos immunoreactivity (IR) in these nuclei. Whether these increases represent a direct action of thyroid hormone was studied by detecting the presence of thyroid hormone receptor beta2 (TRbeta2) in pro-TRH-synthesizing neurons in the Rpa, Rob and the PPR using immunohistochemistry with specific TRbeta2 antiserum and in situ hybridization with digoxigenin-labeled pro-TRH cRNA probe. TRbeta2 IR was widely distributed throughout the medulla and primarily localized within the cell nuclei. Particularly intense immunostaining was presented in the Rpa, Rob and the PPR neurons. The combination of immunohistochemistry with in situ hybridization revealed that all pro-TRH mRNA-positive neurons in these ventral medullary nuclei were also TRbeta2 IR positive. The numbers of TRbeta2 IR-positive neurons in each nucleus were identical in both euthyroid rats and hypothyroid rats induced by 6-n-propyl-2-thiouracil in drinking water for 4 weeks. The finding that TRbeta2 localized in pro-TRH-synthesizing neurons in the ventral medullary nuclei provides an anatomical substrate for a direct thyroid hormone action on these neurons in the regulation of TRH gene expression, which may contribute to the altered autonomic activity in different thyroid statuses.

Hyperthyroidism decreases thyrotropin-releasing hormone gene expression in the caudal raphe nuclei and the parapyramidal regions in rats.

Altered thyroid statuses are associated with autonomic disorders. Medullary thyrotropin-releasing hormone (TRH) and substance P (SP) regulate autonomic nervous activity. The influences of thyroid statuses on TRH and SP gene expressions in the caudal raphe nuclei and the parapyramidal regions were studied using quantitative in situ hybridization histochemistry. In male rats thyroidectomized (Tx) for 30 days, the serum T4 levels decreased by 64% and the medullary pro-TRH mRNA signals (silver grains per neuron) significantly increased by 32-45%. These changes were prevented by daily i.p. T4 (2 microg/100 g) injection in Tx rats. In sham operated/T4 (20 microg/100 g, daily) injected rats, T4 levels significantly increased by 88% and the silver grains decreased by 38-40%. Medullary SP mRNA signals were not significantly changed by altering thyroid status. These results support the concept that thyroid hormone regulates medullary TRH gene expression by negative feedback.

Mice overexpressing wild-type human alpha-synuclein display alterations in colonic myenteric ganglia and defecation.

BACKGROUND: Prevalent non-motor symptoms of Parkinson's disease (PD) include gastrointestinal motor impairments and advanced stage PD displays pathological aggregates of α-synuclein in colonic enteric neurons. We previously showed that 12 months old mice overexpressing human wild type (WT) α-synuclein under the Thy1 promoter (Thy1-aSyn) displayed colonic motor dysfunction. We investigated functional gut alterations at earlier ages and histological correlates. METHODS: Defecation, gastric emptying (GE), and immunostaining for α-synuclein, peripheral choline acetyltransferase (pChAT), tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) in distal colon myenteric plexuses were assessed in male Thy1-aSyn compared to littermate WT mice. KEY RESULTS: Thy1-aSyn mice aged 2.5-3 or 7-8 months old had 81% and 55% reduction in fecal pellet output, respectively, in the first 15 min of exposure to a novel environment. The reduction remained significant in the older group for 2-h, and subsequent refeeding resulted also in a 60% and 69% reduction of defecation in the first hour, respectively. Thy1-aSyn mice (8-10 months) displayed increased α-synuclein in the myenteric plexuses with abundant varicose terminals surrounding pChAT-immunoreactive (ir) neurons, and only a few, nNOS-ir neurons. There were no conspicuous changes in pChAT- and nNOS-ir neurons, or TH- and VIP-ir nerve fibers. Thy1-aSyn mice aged 4-18 months had normal GE. CONCLUSIONS & INFERENCES: The occurrence of over-production of pre-synaptic α-synuclein in colonic myenteric ganglia several months before the loss of striatal dopamine may provide an anatomical basis for interference with cholinergic neuronal activation, causing an early impairment in defecation to stimuli.

Corticotropin releasing factor in the rat colon: expression, localization and upregulation by endotoxin.

Little is known about CRF expression and regulation in the rat colon compared to the brain. We investigated CRF gene expression, cellular location, and regulation by endotoxin and corticosterone in the male rat colon at 6h after intraperitoneal (ip) injection. CRF mRNA level, detected by reverse transcription-polymerase chain reaction (RT-PCR) was 1.3-fold higher in the distal than proximal colon and 3.4-fold higher in the proximal colonic submucosa plus muscle layers than in mucosa. CRF immunoreactivity was located in the epithelia, lamina propria and crypts, and co-localized with tryptophan hydroxylase, a marker for enterochromaffin (EC) cells, and in enteric neurons. Lipopolysaccharide (LPS, 100 microg/kg, ip) increased defecation by 2.9-fold and upregulated CRF mRNA by 2.5-fold in the proximal and 1.1-fold in the distal colon while there was no change induced by corticosterone as monitored by quantitative PCR. LPS-induced increased CRF mRNA expression occurred in the submucosa plus muscle layers (1.5-fold) and the mucosa of proximal colon (0.9-fold). LPS increased significantly CRF immunoreactivity in the submucosal and myenteric plexuses of proximal and distal colon compared to saline groups. These results indicate that in rats, CRF is expressed in both proximal and distal colon and more prominently in enteric neurons of the submucosa plus muscle layers and subject to upregulation at the gene and protein levels by LPS through corticosteroid independent pathways. These data suggests that colonic CRF may be part of the local effector limb of the CRF(1) receptor mediated colonic alterations induced by acute stress.

Enhanced pelvic responses to stressors in female CRF-overexpressing mice.

Acute stress affects gut functions through the activation of corticotropin-releasing factor (CRF) receptors. The impact of acute stress on pelvic viscera in the context of chronic stress is not well characterized. We investigated the colonic, urinary, and locomotor responses monitored as fecal pellet output (FPO), urine voiding, and ambulatory activity, respectively, in female and male CRF-overexpressing (CRF-OE) mice, a chronic stress model, and their wild-type littermates (WTL). Female CRF-OE mice, compared with WTL, had enhanced FPO to 2-min handling (150%) and 60-min novel environment (155%) but displayed a similar response to a 60-min partial restraint stress. Female CRF-OE mice, compared with WTL, also had a significantly increased number of urine spots (7.3 +/- 1.4 vs. 1.3 +/- 0.8 spots/h) and lower locomotor activity (246.8 +/- 47.8 vs. 388.2 +/- 31.9 entries/h) to a novel environment. Male CRF-OE mice and WTL both responded to a novel environment but failed to show differences between them in colonic and locomotor responses. Male WTL, compared with female WTL, had higher FPO (113%). In female CRF-OE mice, the CRF(1)/CRF(2) receptor antagonist astressin B and the selective CRF(2) receptor agonist mouse urocortin 2 (injected peripherally) prevented the enhanced defecation without affecting urine or locomotor responses to novel environment. RT-PCR showed that CRF(1) and CRF(2) receptors are expressed in the mouse colonic tissues. The data show that chronic stress, due to continuous central CRF overdrive, renders female CRF-OE mice to have enhanced pelvic and altered behavioral responses to superimposed mild stressors and that CRF(1)-initiated colonic response is counteracted by selective activation of CRF(2) receptor.

CRF2 receptor activation prevents colorectal distension induced visceral pain and spinal ERK1/2 phosphorylation in rats.

BACKGROUND AND AIMS: Activation of corticotropin releasing factor 1 (CRF1) receptors is involved in stress related responses and visceral pain, while activation of CRF2 receptors dampens the endocrine and some behavioural stress responses. We hypothesised that CRF2 receptor activation may influence visceral pain induced by colorectal distension (CRD) in conscious rats, and assessed the possible sites and mechanisms of action. METHODS: Male Sprague-Dawley rats were exposed to CRDs (60 mm Hg, 10 minutes twice, with a 10 minute rest interval). Visceromotor responses (VMR) were measured by electromyography or visual observation. Spinal (L6-S1) extracellular signal regulated kinase 1/2 (ERK 1/2) activation following in vivo CRD and CRF2 receptor gene expression in the T13-S1 dorsal root ganglia (DRG) and spinal cord were determined. Inferior splanchnic afferent (ISA) activity to CRD (0.4 ml, 20 seconds) was assessed by electrophysiological recording in an in vitro ISA nerve-inferior mesenteric artery (intra-arterial)-colorectal preparation. RESULTS: In controls, VMR to the second CRD was mean 31 (SEM 4)% higher than that of the first (p<0.05). The selective CRF2 agonist, human urocortin 2 (hUcn 2, at 10 and 20 microg/kg), injected intravenous after the first distension, prevented sensitisation and reduced the second response by 8 (1)% and 30 (5)% (p<0.05) compared with the first response, respectively. RT-PCR detected CRF2 receptor gene expression in the DRG and spinal cord. CRD (60 mm Hg for 10 minutes) induced phosphorylation of ERK 1/2 in neurones of lumbosacral laminae I and IIo and the response was dampened by intravenous hUcn 2. CRD, in vitro, induced robust ISA spike activity that was dose dependently blunted by hUcn 2 (1-3 microg, intra-arterially). The CRF2 receptor antagonist, astressin2-B (200 microg/kg subcutaneously or 20 microg intra-arterially) blocked the hUcn 2 inhibitory effects in vivo and in vitro. CONCLUSIONS: Peripheral injection of hUcn 2 blunts CRD induced visceral pain, colonic afferent, and spinal L6-S1 ERK 1/2 activity through CRF2 receptor activation in rats.

Hypothyroidism induces Fos-like immunoreactivity in ventral medullary neurons that synthesize TRH.

Altered thyroid statuses are associated with autonomic disorders. Thyrotropin-releasing hormone (TRH) in medullary nuclei regulates vagal efferent activity. Induction of Fos-like immunoreactivity (IR) in medullary TRH-synthesizing neurons was investigated in 24-h fasted rats with different thyroid statuses. Hypo- and hyperthyroidism were induced by 6-N-propyl-2-thiouracil (PTU) in drinking water and a daily intraperitoneal injection of thyroxine (T(4); 10 microgram. 100 g(-1). day(-1)), respectively, for 1-4 wk. The numbers of Fos-like IR positive neurons in the raphe pallidus, raphe obscurus, and parapyramidal regions, which were low in euthyroid rats (0-2/section), increased remarkably as the hypothyroidism progressed and were negatively correlated with serum T(4) levels. At the 4th wk, Fos-like IR positive neurons were 10- to 70-fold higher compared with euthyroid controls. Simultaneous T(4) replacement (2 microgram. 100 g(-1). day(-1)) prevented the increases of Fos-like IR in PTU-treated rats. Hyperthyroidism did not change the number of Fos-like IR neurons in the raphe nuclei but reduced it in the parapyramidal regions. Double immunostaining revealed that most of the Fos-like IR induced by hypothyroidism was located in the prepro-TRH IR positive neurons. The selective and sustained induction of Fos-like IR in TRH-synthesizing neurons in ventral medullary nuclei by hypothyroidism indicates that these neurons play a role in the autonomic disorders observed in altered thyroid statuses.

Acute cold exposure induces vagally mediated Fos expression in gastric myenteric neurons in conscious rats.

Acute cold exposure-induced activation of gastric myenteric neurons in conscious rats was examined on longitudinal muscle-myenteric plexus whole mount preparations. Few Fos-immunoreactive (IR) cells (<1/ganglion) were observed in 24-h fasted rats semirestrained at room temperature. Cold exposure (4 degrees C) for 1-3 h induced a time-related increase of Fos-IR cells in corpus and antral myenteric ganglia with a maximal plateau response (17 +/- 3 and 18 +/- 3 cells/ganglion, respectively) occurring at 2 h. Gastric vagotomy partly prevented, whereas bilateral cervical vagotomy completely abolished, Fos expression in the myenteric cells induced by cold exposure (2 h). Hexamethonium (20 mg/kg) also prevented 3-h cold exposure-induced myenteric Fos expression by 76-80%, whereas atropine or bretylium had no effect. Double labeling revealed that cold (3 h)-induced Fos-IR myenteric cells were mainly neurons, including a substantial number of choline acetyltransferase-containing neurons and most NADPH-diaphorase-positive neurons. These results indicate that acute cold exposure activates cholinergic as well as nitrergic neurons in the gastric myenteric ganglia through vagal nicotinic pathways in conscious rats.