Chronic dexamethasone-treatment alters mineralocorticoid receptor, truncated trkB and selected glutamate receptor subunit mRNA expression in the porcine hippocampus.

Prepubertal boars (n = 4/treatment) were killed 24 h after a 5 day course of intravenous injections of dexamethasone (Dex, 1 and 5 mg kg(-1)), or saline vehicle. Gene expression was quantified in brain sections following in situ hybridisation histochemistry. The objective was to determine whether chronic glucocorticoid treatment would alter the expression of mRNAs for gluco- and mineralocorticoid receptors (GR and MR), brain-derived neurotrophic factor (BDNF), its receptor, trkB, and selected ionotropic glutamate receptor (iGluR) subunits in the hippocampus. Although Dex did not alter GR message, the higher dose reduced MR mRNA in all hippocampal subfields studied. There was no effect of Dex on the expression of BDNF, or the full-length form of its receptor but there was evidence to suggest that mRNA for the truncated form of trkB was increased. Expression of mRNA for glutamate receptor subunits was either unaffected (NR1) or decreased (GluR2 and GluR3). These findings indicate that acute and chronic glucocorticoid treatment has differential effects on hippocampal gene expression in the porcine brain.

The differential effects of intravenously administered 8-OH-DPAT on operant food intake in satiated and food-deprived pigs are mediated by central 5-HT(1A) receptors.

It has previously been shown that the intravenous administration of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), increases food intake in satiated pigs and decreases food intake in fasted pigs. The present experiments were conducted to investigate the effects of central administration of the 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-N-2-pyridinyl-cyclohexane carbox-amide maleate (WAY 100635), on the stimulant and depressant effects of 8-OH-DPAT on operant food intake in satiated and hungry pigs. In Experiment 1, 8-OH-DPAT (25 microg/kg) produced an increase in operant feeding during the first 30 min following intravenous administration to satiated pigs. The 8-OH-DPAT-induced hyperphagia was completely abolished by pretreatment with WAY 100635 (0.3 mg) administered by intracerebroventricular injection. In Experiment 2, 8-OH-DPAT (25 microg/kg) administered intravenously 15 min prior to the onset of feeding in pigs that had been fasted for 22.5 h produced a decrease in operant food intake, which was most apparent during the first 30 min of the feeding period. The hypophagic effect was completely abolished by pretreatment with WAY 100635 (0.3 mg icv) administered 30 min before the start of the feeding period. In both experiments, WAY 100635 (0.3 mg icv) did not have any significant effects on feeding. The results of the present study extend previous results in the pig and show that both the hyperphagic and the hypophagic effects of 8-OH-DPAT in satiated and fasted pigs, respectively, are mediated by central 5-HT(1A) receptors.

Down-regulation of BDNF mRNA, with no effect on trkB or glucocorticoid receptor m RNAs, in the porcine hippocampus after acute dexamethasone treatment.

Glucocorticoids bind to hippocampal mineralo-(MR) and gluco-(GR) corticoid receptors and, at high concentrations (e.g. as seen following treatment with pharmacological doses of corticosteroids or during stress), may affect hippocampal neuronal function. Such actions could involve brain-derived neurotrophic factor (BDNF), its receptor, trkB, and the excitatory neurotransmitter, glutamate. This experiment investigated the effect of a single intravenous (i.v.) injection of the synthetic glucocorticoid, dexamethasone (Dex, 5 mg kg(-1)) on gene expression for MR s, GR s, BDNF, trkB, and selected ionotropic glutamate receptor subunits (iGluRs), in the porcine hippocampus. Quantification of m RNA s in the brains of pigs (n = 4/treatment) killed 24 hours after saline or Dex administration indicated a significant Dex-induced decrease in BDNF m RNA in all hippocampal regions. However, gene expression for MR s, GR s, trkB and iGluRs was unaffected at this time-point.

Hippocampal gene expression in the pig: upregulation of corticotrophin releasing hormone mRNA following central administration of the peptide.

Corticotrophin releasing hormone (CRH) and glucocorticoids affect hypophysiotrophic regions of the brain and influence limbic system activity. Since the latter mediates emotional responses, changes in gene expression in regions such as the hippocampus may provide new information on neural stress mechanisms. In this study, mRNA for CRH and selected ionotropic glutamate receptor (iGluR) subunits (NR1, GluR2, GluR3) was quantified in the hippocampus of pigs in which stress was simulated by central administration of CRH (100 microg). Increases in hippocampal CRH mRNA were detected in the CA3 subfield 4 h later, and in the CA1, CA2 and CA3 subfields 24 h post-treatment. However, there were no associated changes in iGluR subunit mRNAs, although the ratio GluR3: GluR2 increased in the dentate gyrus after 4 h. These results, together with a recent similar finding in rats subjected to restraint, point to an involvement of hippocampal CRH in the neuronal response to stress.

Behavioral effects of betacarbolines in pigs:. anxiety or aversion?

Betacarbolines are often considered to be anxiogenic and may, therefore, have similar behavioral effects to those of corticotrophin releasing hormone (CRH); however, their actions have been little studied in pigs. This investigation was concerned with the effects of ethyl-beta-carboline-3-carboxylate (BCCE) and noreleagnine (NOR) on operant feeding, cortisol release, and overt behavior in swine, all of which are known to be affected by CRH in this species. Three experiments are described in which BCCE or NOR were given intravenously to prepubertal boars (n = 7). In Experiment 1, 400 microg/kg, but not 100 or 200 microg/kg, BCCE produced a rapid inhibition of ingestive activity whereas NOR (100, 200, or 400 microg/kg) was without effect. In Experiment 2, both BCCE and NOR increased plasma cortisol, but not growth hormone, concentrations. In Experiment 3, a high dose of BCCE (2 mg/kg) produced transient arousal and a sustained increase in respiration rate and plasma cortisol. These results indicate that although the responses of pigs to BCCE and CRH are similar in some respects, there are also marked behavioral differences. The possibility that BCCE has aversive rather than anxiogenic actions in this species is discussed.

Gene expression in the forebrain of dexamethasone-treated pigs: effects on stress neuropeptides in the hypothalamus and hippocampus and glutamate receptor subunits in the hippocampus.

Gene expression studies advance our understanding of the effects of stress and glucocorticoids on brain function and give a new direction to animal welfare research. In this context, the presence of messenger RNA s (m RNA s) for corticotrophin releasing hormone (CRH) and vasopressin (VP) in the porcine hypothalamus has recently been documented. This study investigated the expression of CRH, VP and ionotropic glutamate receptor (iGluR) subunit m RNA s in the brains of pigs treated with the synthetic glucocorticoid dexamethasone (Dex; 5 mg kg(-1)i.v.). In the hypothalamus, VP, but not CRH, m RNA was reduced 3 hours after Dex. In the hippocampus, expression of m RNA s for some iGluR subunits appeared to be differentially regulated 6 hours after Dex. In addition, CRH message was detected in the hippocampus and significantly upregulated in the CA1 region 3 hours after Dex. The relevance of these findings to stress neurobiology of the growing pig is discussed.

Endogenous monoamine oxidase inhibitory activity and HPA activation in the pig.

Previously we have shown that an increase in endogenous monoamine oxidase A inhibitory activity (MAO-AI), measured in human saliva, both precedes and predicts psychological stress-induced activation of the hypothalamic pituitary adrenal (HPA) axis, as determined by the cortisol response. We now report the relationship between endogenous MAO-AI and the cortisol response in the plasma of prepubertal pigs (n=5 or 6) under two experimental paradigms of HPA activation. In the first condition, pigs were physically restrained (snaring) for 15 minutes. Blood samples were taken from indwelling catheters at intervals before and after snaring (a sampling period of about 1 hour), and at the same time intervals on a separate day to provide baseline measures. Both cortisol concentration and percentage MAO-AI were determined in each plasma sample. There was a pronounced cortisol response on the snaring day (cortisol peaked 30 minutes after the start of the snaring). There was also a significant MAO-AI response to snaring which peaked 15 minutes after the start of the stress challenge. In the second experimental paradigm, bacterial endotoxin (LPS: 20microg/pig) was used to induce HPA activation and plasma cortisol and MAO-AI were determined. This time, however, the cortisol response was not preceded by any change in MAO-AI. We conclude that generation of MAO-AI, which is associated with HPA activation induced by psychological stress, is not a component of the pathways involved in immunological stimulation of the HPA axis.

Behavioral and hormonal effects of centrally injected "anxiogenic" neuropeptides in growing pigs.

Records of behavior (alertness, posture, oro-nasal responses, activity level, and vocalization pattern) were made in prepubertal pigs (n = 6) during a 60-min period following central injections of equimolar (21 nmol) doses of porcine CRH (pCRH), urocortin (UCN), octadecaneuropeptide (ODN), or saline vehicle (SAL). Blood samples were also collected at 15-min intervals before, during, and after the test, and used to determine plasma cortisol, prolactin, and growth hormone concentrations. The pigs became excited and highly active after pCRH, and to a lesser extent following UCN administration, but were subdued when given ODN or SAL. None of the peptides significantly affected prolactin or growth hormone release, but both UCN, and especially pCRH, increased cortisol concentrations. The emotional responses induced by pCRH and UCN are consistent with observations in rodents, which indicate that centrally administered CRH-like peptides have anxiogenic effects. In contrast, ODN, which inhibits benzodiazepine binding at the GABA(A) receptor and is anxiogenic in rodents, lowered plasma cortisol and had no overt behavioral effects. Hence, at the dose administered, there was no evidence to indicate that ODN acted as an anxiogen in this species.

Behaviour of pigs given corticotrophin-releasing hormone in combination with flumazenil or diazepam.

The possible involvement of an endogenous benzodiazepine (BZ) inverse agonist in the activational effects of corticotrophin-releasing hormone (CRH) on behaviour was examined in pigs given porcine CRH (75 microg) intracerebroventricularly (i.c.v.) and the BZ antagonist flumazenil (FLU; 0.09 mg/kg) intravenously (i.v.). In Experiment 1, behaviour was recorded for 75 min in pigs (n=5) given i.v. FLU or saline (SAL) followed by i.c.v. CRH or vehicle (VEH). Significant changes in arousal, posture and oro-nasal activity were induced by CRH, whereas FLU alone had no effect but appeared to reduce some responses to CRH. In Experiment 2, behaviour was observed for 60 min in pigs (n=6) given i.c.v. CRH followed by i.v. FLU, VEH or diazepam (DZ; 0.2 mg/kg). Behavioural responses to CRH, however, were unaffected by FLU, whereas certain aspects of arousal, posture and oro-nasal activity were reduced by DZ; a higher dose of DZ (0.3 mg/kg) given before CRH tended to enhance these inhibitory effects. All treatments also produced similar increases in plasma cortisol. Taken together with previous findings, the negligible effect of FLU in this study suggests that endogenous ligands for the BZ binding site on the GABA(A) receptor are of little importance in regulating the behavioural actions of CRH in swine.

Effects of the 5-HT1A receptor agonist 8-OH-DPAT on operant food intake in food-deprived pigs.

The effects of the 5-HT1A agonist 8-hydroxy-2 (di-n-propylamino)tetralin (8-OH-DPAT) were investigated on operant food intake in food-deprived pigs. In Experiment 1, 8-OH-DPAT (5-20 microg/kg) administered intravenously (i.v.) 15 min prior to the occurrence of feeding produced a dose-related decrease in operant food intake in pigs that had been fasted overnight. The effects were mainly apparent during the first 30 min after the start of the feeding period. In Experiment 2, 8-OH-DPAT (25 and 50 microg/kg, i.v.) administered 60 min prior to the occurrence of feeding in pigs that were fasted overnight also produced significant decreases in food intake. The effects were mainly apparent during the first 30-40 min after the start of the feeding period. In Experiment 3, 8-OH-DPAT (20 microg/kg, i.v.) significantly increased operant feeding in satiated pigs during the first 30 min after administration. These results show that 8-OH-DPAT has complex effects on feeding behaviour in pigs, increasing operant food intake in satiated pigs, while producing a reduction in food intake in food-deprived animals.

Intravenous lysine vasopressin lowers body temperature in normal and febrile pigs.

Vasopressin has been implicated as a centrally acting endogenous antipyretic. However, in several species, including the pig, plasma vasopressin concentrations increase during the early stages of fever. This experiment investigated the effects of intravenous lysine vasopressin on core temperature in normal and febrile swine. Lysine vasopressin (20 microg/pig) stimulated cortisol release and induced a 60-min hypothermic episode in normal animals, although a 10-fold lower dose was without effect. The peptide also delayed the pyretic response to bacterial endotoxin (20 microg intravenously). It is speculated that the hypothermic action of circulating vasopressin may involve nitric oxide.

Studies of endotoxin-dependent fever in pre-pubertal pigs following acute activation of the pituitary-adrenocortical axis: towards a new hypothesis of fever regulation.

The possibility that adrenocortical activation might alter the pyretic effects of bacterial lipopolysaccharide endotoxin in growing pigs was investigated. In a series of four experiments, animals received increasing doses of porcine adrenocorticotrophic hormone ACTH (1.5, 4.5, 13.5 IU kg-1) or CRH (7 microg kg-1), all of which markedly affected cortisol release. Unexpectedly, these treatments tended to increase body temperature during the early and middle stages of the febrile response, although they did appear to induce an earlier deferscence. These results suggest that acute stress may not modify fever induced by immunological challenge, although a different situation could obtain during chronic stress. Furthermore, a hypothesis of fever regulation is proposed which attempts to reconcile the present findings with those from previous studies in swine.

Expression of mRNAs for vasopressin, oxytocin and corticotrophin releasing hormone in the hypothalamus, and of cyclooxygenases-1 and -2 in the cerebral vasculature, of endotoxin-challenged pigs.

Neuropeptide and cyclooxygenase (Cox) gene expression was examined in the brains of catheterized pigs killed 30 or 120 min after intravenous injection of a low (20 microg) dose of lipopolysaccharide endotoxin (LPS), previously demonstrated to induce fever in this species. In the paraventricular hypothalamic nucleus (PVN), corticotrophin releasing hormone (CRH) mRNA was shown to be present in the pars parvocellularis but was not upregulated 30 or 120 min after 20 microg LPS, or 90 min after 60 microg LPS; there was also no change in proopiomelanocortin (POMC) message in the anterior pituitary (AP). Similarly, expression of mRNAs for lysine vasopressin (LVP) or oxytocin (OT) did not change in the PVN after LPS (20 microg), although LVP message was increased (p<0.05) at 30 min in the hypothalamic supraoptic nucleus (SON). Expression of Cox-1 and Cox-2 genes was quantified in the organum vasculosum lamina terminalis (OVLT) and choroid plexus (CP) in an attempt to determine whether altered expression of prostaglandin (PG) synthetic enzymes in brain vasculature is involved in LPS fever. Although vascular endothelial cells in both structures expressed Cox-1 and Cox-2 mRNAs, neither increased in the OVLT following LPS. However, in the CP, Cox-1 mRNA was enhanced (p<0.05) at 30 and 120 min after LPS injection and Cox-2 showed a similar (NS) change. These results provide the first description of CRH and Cox gene expression in the porcine brain. They also suggest that LPS may influence the activity of genes controlling LVP synthesis in the hypothalamus and PG production by the brain vasculature.

Effects of intravenous nitric oxide inhibitors on endotoxin-induced fever in prepubertal pigs.

1. The ability of nitric oxide inhibitors to antagonize the febrile effects of lipopolysaccharide (LPS) endotoxin (20 microg/animal i.v.) was assessed in prepubertal pigs in which deep body temperature was measured at 10-min intervals for 180 min. 2. In experiment 1, pigs (n=5) were injected with Nomega-nitro-L-arginine-methyl ester (L-NAME) (30 mg/kg i.v.) or aminoguanidine (27 mg/kg i.v.) 30 min before LPS. There was a marked tendency for L-NAME, but not aminoguanidine, to reduce LPS pyrexia. 3. In experiment 2, pigs (n=7) were injected with 2-amino-4-methylpyridine (1 mg/kg i.v.) 30 min before LPS. This drug tended to increase, rather than reduce, core temperature. 4. In experiment 3, pigs (n=5) were injected with S-methylisothiourea (10 or 15 mg/kg i.v.) concomitantly with LPS. Both doses of the drug produced a small, nonsignificant, reduction in the febrile response. 5. The results indicate that the nitric oxide inhibitors used in this study were relatively ineffective in modifying LPS fever in conscious pigs; these findings are in marked contrast with the actions, in this species, of drugs that inhibit prostaglandin production. In addition, the most effective drug, L-NAME, was the one considered to be the least selective for the inducible form of nitric oxide.

Food for thought: a critique on the hypothesis that endogenous cholecystokinin acts as a physiological satiety factor.

This review evaluates the various lines of evidence supporting the hypothesis that cholecystokinin (CCK) released from the small intestine during feeding plays a physiological satiety. Issues considered include, the effects of systemic injection of CCK on consummatory and operant feeding, the role of the vagus nerve, the effects of CCKB receptor antagonists, and the neuroendocrine responses to exogenous CCK. A critical appraisal of this research indicates that while it is clearly demonstratable that exogenous peripheral CCK can alter food intake by acting on CCKA receptors, the mechanism involved may be more closely related to the induction if aversion and nausea, rather than satiety. With regard to peripheral endogenous CCK, the available evidence also does not seem to support a role for the hormone in satiety. In particular, it is doubtful whether plasma concentrations of CCK following a meal are sufficiently high to inhibit feeding. Moreover, CCKA receptor antagonist which do not cross the blood brain barrier fail to increase meal size, as would be expected if peripheral CCK was an effective satiety factor. In addition, the recent literature concerned with the possibility that CCK may have a direct action within the brain in the control of food intake has been reviewed. These studies show that CCK administered intracerebroventicularly, or by micoinjection into discrete brain regions, also inhibits feeding via a CCKA receptor mechanism. However, the physiological relevance of these findings have yet to be determined.

Effects of transport and indomethacin on telemetered body temperature and release of cortisol and prolactin in pre-pubertal pigs.

Previous research indicates that acute physical stress (restraint) raises core temperature in growing pigs via a prostaglandin-dependent mechanism. This study investigated whether transport stress affects body temperature in pigs and whether any such changes might involve endogenous prostaglandins. Pre-pubertal boars (n = 7) were implanted with venous catheters and biotelemetry devices for the measurement of core temperature. They were transported for two hours, with and without indomethacin (IND) pre-treatment, and blood samples were taken at 15 minute intervals for the determination of plasma cortisol and prolactin concentrations. The results indicated that, contrary to predictions, body temperature tended to fall during transport and that the effect was exaggerated by IND. By contrast, cortisol concentrations increased during transport and were unaffected by IND whereas the tendency for transport to stimulate prolactin release was reversed by IND.

Comparison of the antipyretic actions of indomethacin and L-745,337, a selective cyclooxygenase-2 inhibitor, in endotoxin-treated prepubertal pigs.

1. The growing pig provides a useful, nonrodent model for studying mechanisms involved in the febrile response. 2. Indomethacin (IND) has previously been shown to prevent lipopolysaccharide (LPS) fever in prepubertal pigs. 3. This study compared the abilities of IND and L-745,337, a novel cyclooxygenase-2 (cox-2) inhibitor, to counteract the effects a low dose of LPS (20 micrograms/pig IV) on deep body temperature. Effects of IND and L-745,337 on core temperature and plasma cortisol concentrations were also examined in nonfebrile animals. 4. L-745,337 (0.3 mg/kg IV) did not alter the response to LPS, whereas both IND and L-745,337 (1.7 mg/kg) reduced the febrile effects of LPS given 60 min earlier. 5. Neither IND, nor L-745,337 (1.7 mg/kg IV) affected core temperature in nonfebrile animals whereas IND, but not L-745,337, stimulated cortisol release. 6. The results suggest that prostaglandin modulates the febrile effects of LPS in swine and that inhibition of inducible cyclooxygenase (Cox-2) suppresses fever without producing the stressful side-effects that accompany constitutive cyclooxygenase (Cox-1) inhibition, as exemplified by IND (a mixed Cox-1/Cox-2 antagonist).

Vasopressin and oxytocin gene expression in the porcine forebrain under basal conditions and following acute stress.

This study, the first using the pig, examined expression of mRNAs for vasopressin (VP), oxytocin (OT), preproenkephalin (PENK) and pro-opiomelanocortin (POMC) in the forebrain, and of POMC and prolactin in the pituitary. High basal expression of VP and OT mRNAs was present in the paraventricular (PVN) and supraoptic (SON) nuclei. In the PVN, VP was found in magnocellular regions whereas OT was also seen in the parvocellular portion; the distribution of VP and OT mRNAs in the SON was as reported in other species. The suprachiasmatic nucleus contained VP mRNA but only OT message was present in the dorsomedial SON, a structure peculiar to swine. Gene expression for PENK occurred in the caudate putamen (CPu), for POMC in the mediobasal hypothalamus (MBH) and for prolactin and POMC in the hypophysis. Following restraint, VP message increased in the magnocellular PVN, as did PENK in the CPu and POMC in the MBH.

Interrelated adrenocortical and neurohypophysial responses associated with fever in endotoxin-treated pigs.

Low intravenous doses of endotoxin [lipopolysaccharide (LPS), 0.7 microgram/kg] induce monophasic fever, increase anterior and posterior pituitary hormone release, and enhance hypothalamic c-Fos expression in pigs, all of which can be prevented by indomethacin (Ind). The present study shows that the synthetic corticosteroid dexamethasone (Dex, 5 mg/kg) has a similar action to Ind and, when given alone, lowers core temperature. In addition, the corticosteroid synthesis inhibitor metyrapone (Met, 3.3 mg/kg, every one-half hour) reduces LPS fever and amplifies the effect of LPS on vasopressin, but not on oxytocin, release. The similar actions of Dex and Ind suggest that phospholipase A2 pathways controlling prostaglandin synthesis mediate the responses of prepubertal pigs to immunological challenge with LPS. The increased vasopressin release induced when animals receiving Met are also given LPS supports findings in other nonrodent species indicating an inverse relationship between cortisol and vasopressin. The attenuation of LPS fever by Met is suggestive of an endogenous antipyretic mechanism associated with enhanced neurohypophysial vasopressin secretion.