Effect of corticotropin releasing hormone and corticotropin releasing hormone antagonist on biosynthesis of gonadotropin relasing hormone and gonadotropin relasing hormone receptor in the hypothalamic-pituitary unit of follicular-phase ewes and contribution of kisspeptin.

This study aimed to determine the mechanisms governing Gonadotropin releasing hormone (GnRH) biosynthesis and luteinising hormone (LH) secretion in follicular-phase sheep after infusion of corticotropin releasing hormone (CRH) and/or CRH antagonist corticotropin releasing hormone nist (CRH-A) into the third cerebral ventricle. The study included two experimental approaches: first, we investigated the effect of CRH or CRH-A (α-helical CRH 9-41) on GnRH and GnRH receptor (GnRHR) biosynthesis in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VMH), stalk/median eminence (SME), and on GnRHR in the anterior pituitary (AP) using an enzyme-linked immunosorbent assay (ELISA); second, we used real-time PCR to analyse the influence of CRH and CRH-A on the levels of kisspeptin (Kiss1) mRNA in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by radioimmunoassay (RIA) and ELISA methods for measurement of LH and cortisol levels in the blood, respectively. Our results show that administration of CRH significantly decreased GnRH biosynthesis in the POA/hypothalamus. CRH also decreased GnRHR abundance in the hypothalamus and in the AP, but increased it in the POA. Furthermore, administration of CRH decreased plasma LH concentration and levels of Kiss1 mRNA in the POA and VMH/ARC as well as Kiss1r mRNA in these structures and in the SME. Significant increase in plasma cortisol concentration in the group treated with CRH was also observed. For CRH-A, all analysed effects were opposite to those induced by CRH. The study demonstrates that intracerebroventricular (i.c.v.) infusion of both CRH and CRH-A affects the GnRH/GnRHR biosynthesis and LH secretion in follicular-phase sheep conceivably via either central and peripheral mechanisms including Kiss1 neurons activity and cortisol signals. It has also been suggested that CRH and CRH-A infusion probably had effects directly at the AP.

The influence of dopaminergic system inhibition on biosynthesis of gonadotrophin-releasing hormone (GnRH) and GnRH receptor in anoestrous sheep; hierarchical role of kisspeptin and RFamide-related peptide-3 (RFRP-3).

This study aimed to explain how prolonged inhibition of central dopaminergic activity affects the cellular processes governing gonadotrophin-releasing hormone (GnRH) and LH secretion in anoestrous sheep. For this purpose, the study included two experimental approaches: first, we investigated the effect of infusion of sulpiride, a dopaminergic D2 receptor antagonist (D2R), on GnRH and GnRH receptor (GnRHR) biosynthesis in the hypothalamus and on GnRHR in the anterior pituitary using an immunoassay. This analysis was supplemented by analysis of plasma LH levels by radioimmunoassay. Second, we used real-time polymerase chain reaction to analyse the influence of sulpiride on the levels of kisspeptin (Kiss1) mRNA in the preoptic area and ventromedial hypothalamus including arcuate nucleus (VMH/ARC), and RFamide-related peptide-3 (RFRP-3) mRNA in the paraventricular nucleus (PVN) and dorsomedial hypothalamic nucleus. Sulpiride significantly increased plasma LH concentration and the levels of GnRH and GnRHR in the hypothalamic-pituitary unit. The abolition of dopaminergic activity resulted in a significant increase in transcript level of Kiss1 in VMH/ARC and a decrease of RFRP-3 in PVN. The study demonstrates that dopaminergic neurotransmission through D2R is involved in the regulatory pathways of GnRH and GnRHR biosynthesis in the hypothalamic-pituitary unit of anoestrous sheep, conceivably via mechanisms in which Kiss1 and RFRP-3 participate.

Biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in hypothalamic-pituitary unit of anoestrous and cyclic ewes.

This study was performed to explain how the molecular processes governing the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit are reflected by luteinizing hormone (LH) secretion in sheep during anoestrous period and during luteal and follicular phases of the oestrous cycle. Using an enzyme-linked immunosorbent assay (ELISA), we analyzed the levels of GnRH and GnRHR in preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk-median eminence (SME), and GnRHR in the anterior pituitary gland (AP). Radioimmunoassay has also been used to define changes in plasma LH concentrations. The study provides evidence that the levels of GnRH in the whole hypothalamus of anoestrous ewes were lower than that in sheep during the follicular phase of the oestrous cycle (POA: p < 0.001, AH: p < 0.001, VM: p < 0.01, SME: p < 0.001) and not always than in luteal phase animals (POA: p < 0.05, SME: p < 0.05). It has also been demonstrated that the GnRHR amount in the hypothalamus-anterior pituitary unit, as well as LH level, in the blood in anoestrous ewes were significantly lower than those detected in animals of both cyclic groups. Our data suggest that decrease in LH secretion during the long photoperiod in sheep may be due to low translational activity of genes encoding both GnRH and GnRHR.

Effect of short-term and prolonged stress on the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamus and GnRHR in the pituitary of ewes during various physiological states.

Using an ELISA assay, the levels of GnRH and GnRHR were analysed in the preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk/median eminence (SME); and GnRHR in the anterior pituitary gland (AP) of non-breeding and breeding sheep subjected to short-term or prolonged stress. The ELISA study was supplemented with an analysis of plasma LH concentration. Short-term footshock stimulation significantly increased GnRH levels in hypothalamus in both seasons. Prolonged stress elevated or decreased GnRH concentrations in the POA and the VM, respectively during anoestrus, and lowered GnRH amount in the POA-hypothalamus of follicular-phase sheep. An up-regulation of GnRHR levels was noted in both, anoestrous and follicular-phase animals. In the non-breeding period, a prolonged stress procedure increased GnRHR biosynthesis in the VM and decreased it in the SME and AP, while in the breeding time the quantities of GnRHR were significantly lower in the whole hypothalamus. In follicular-phase ewes the fluctuations of GnRH and GnRHR levels under short-term and prolonged stress were reflected in the changes of LH secretion, suggesting the existence of a direct relationship between GnRH and GnRH-R biosynthesis and GnRH/LH release in this period. The study showed that stress was capable of modulating the biosynthesis of GnRH and GnRHR; the pattern of changes was dependent upon the animal's physiological state and on the time course of stressor application. The obtained results indicate that the disturbances of gonadotropin secretion under stress conditions in sheep may be due to a dysfunction of GnRH and GnRHR biosynthetic pathways.

The Central Effect of ß-Endorphin and Naloxone on The Biosynthesis of GnRH and GnRH Receptor (GnRHR) in The Hypothalamic-Pituitary Unit of Follicular-Phase Ewes.

The effects of prolonged, intermittent infusion of ß-endorphin or naloxone into the third cerebral ventricle of follicular-phase ewes on the expression of genes encoding GnRH and GnRHR in the hypothalamus and GnRHR in the anterior pituitary gland (AP) were examined by an enzyme-linked immunoabsorbent assay. Activation or blockade of µ-opioid receptors significantly decreased or increased the GnRH concentration and GnRHR abundance in the hypothalamus, respectively, and affected in the same way GnRHR quantity in the AP gland. The changes in the levels of GnRH and GnRHR after treatment with ß-endorphin as well as following action of naloxone were reflected in fluctuations of plasma LH concentrations. On the basis of these results, it is suggested that ß-endorphinergic system in the hypothalamus of follicular-phase ewes affects directly or via ß-endorphin-sensitive interneurons GnRH and GnRHR biosynthesis leading to suppression in secretory activity of the hypothalamic-pituitary axis.

THE INFLUENCE OF CARBON MONOXIDE ON THE SECRETION OF MELATONIN BY PINEALOCYTES MEASURED IN VITRO.

Photoperiod is considered the most important factor entraining the circannual physiological rhythms through changing circadian patterns of melatonin (MEL) secretion from the pineal gland. The pineal gland of mammals does not respond directly to light but is controlled by light via neuronal phototransduction originating in the retina. In accordance with humoral phototransduction hypothesis, the aim of this study was to determine whether an increased concentration of CO, as a carrier of a light signal in pineal cell culture, affects the synthesis of melatonin. This study demonstrates that a commonly used carbon monoxide donor (CORM-2) markedly stimulated melatonin release from pineal cells incubated in vitro in a time-dependent manner, but the mechanism whereby CO modulates MEL release needs to be further explored.

Carbon monoxide-mediated humoral pathway for the transmission of light signal to the hypothalamus.

The gaseous messenger carbon monoxide (CO) is released from the eye into ophthalmic venous blood depending on the intensity of sunlight. Numerous neurohormones and other regulatory factors permeate from venous blood into arterial blood in the perihypophyseal vascular complex (PVC) and are transferred to the brain by the humoral pathway. This study was designed to determine whether elevated CO in ophthalmic venous blood (OphVB) affects the expression of clock genes and their transcriptional factors in the hypothalamus. Mature males of a wild boar and pig crossbreed (n=24) were used for the study. Autologous plasma with increased concentrations of CO was infused into the ophthalmic sinus (OphS) of the experimental group (n=12). The expression of clock genes (Per1, Per2, Cry1, Cry2, Rev-erb α and Rev-erb ß) and the genes of their regulators (Bmal1, Npas2, Clock, Ror ß) was estimated in two hypothalamic structures involved in the reception and transmission of light signal (the preoptic area (PA) and dorsal hypothalamus (DH)). We demonstrated that the expression of clock genes and the genes of their regulatory factors in the experimental group was altered compared with control, both in the PA and DH. The response to an increased concentration of CO differed between individual genes and the hypothalamic regions. The expression of Per1 which, according to many authors, is regulated by light, was increased in animals treated with CO both in the PA and DH, and regardless of the time of day and season. In conclusion, the current results seem to confirm the hypothesis on the function of CO in humoral transfer from the eye to structures related to the reception and transmission of light signal and the effect of CO on clock gene expression.

The inhibition of experimentally induced visceral hyperalgesia by nifedipine - a voltage-gated Ca(2+) channels blocker (VGCCs) in sheep.

Present study examined the effect of VGCC L-type blocker - nifedipine given i.c.v. (0.25, 0.5, 1 and/or 2mg in toto) on the development of nociceptive behavior, clinical symptoms, plasma catecholamin concentration and reticulo-rumen motility following 5 min lasting mechanical duodenal distension (DD) in sheep. After 24h of fasting, all animals received i.m. ketamine analgesia (20 mg kg(-1)B.W) and anesthetized with pentobarbital (20 mg kg(-1)B.W., i.v. infusion) The permanent stainless steel cannula 29 mm in length and 2mm in diameter was inserted into the lateral cerebral ventricle (controlled by cerebro-spinal efflux) 10mm above the bregma and 5mm laterally from the midline sutures using stereotaxic method. Under the same general anesthesia/analgesia a T-shaped silicon cannula (inside diameter of 21 mm), was inserted into the duodenum (12 cm from pylorus). Second identical cannule was inserted into the dorsal sac of the rumen, a previously described. After surgery each animal was kept in individual boxes for 10 days prior to experiment and was treated i.m. with benzyl procaine penicillin 30,000 I.U kg(-1)B.W.)+dihydrostreptomycine sulfate (10 g kg(-1)B.W.)+prednisolone acetate (1.2 mg kg(-1)B.W.) combination and i.m. ketamine (20 mg kg(-1)B.W.) every day by seven consecutive days. Experimental DD was conducted by insertion and then distension of rubber balloon (containing 40 ml of warm water) inserted into sheep duodenum. Duodenal distension produced a significant increase in behavioral pain manifestations, tachycardia, hyperventilation, inhibition of reticulo-ruminal contractions rate (from 87.2 to 38.0% during 15-20 min), an increase of plasma catecholamine concentration (over 6.4-fold increase of epinephrine during 2h following DD, 2-times norepinephrine and 84% increase of dopamine). Nifedipine infusion administered 10 min prior to DD decreased intensity of visceral pain manifestations such as: behavioral changes, hyperventilation, reticulo-rumen motility and efficiently prevent appearance of catecholamine release. These data demonstrated that the development and persistence of duodenal hyperalgesia depends on the activation of Ca(2+) ion flux leading to neurotransmitters release and modulation of membrane excitability. It seems that nifedipine given i.c.v. 10 min prior to DD (as a source of visceral pain), inhibited specific receptors 1 subunits of VGCCs in target tissues, prevented depolarization of cell membranes and release of neurotransmitters responsible for pain sensitivity in sheep. The observed antinociceptive action of VGCCs type L blockers suggest that these channels play a crucial role in the modulation of acute visceral hyperalgesia in sheep.

Implication of dopaminergic systems on GnRH and GnRHR genes expression in the hypothalamus and GnRH-R gene expression in the anterior pituitary gland of anestrous ewes.

We examined by Real-time PCR how prolonged inhibition of dopaminergic D-2 receptors (DA-2) in the hypothalamus of anestrous ewes by infusion of sulpiride into the third cerebral ventricle affected GnRH and GnRH-R gene expression in discrete parts of this structure and GnRH-R gene expression in the anterior pituitary. Blockaded DA-2 receptors significantly decreased GnRH mRNA levels in the ventromedial hypothalamus but did not evidently affect GnRH mRNA in the preoptic/ anteriorhypothalamicarea. Blockaded DA-2 receptors led to different responses in GnRH-R mRNA in various parts of the hypothalamus; increased GnRH-R mRNA levels in the preoptic/anterior hypothalamic area, and decreased GnRH-R mRNA amounts in the ventromedial hypothalamus stalk/median eminence. An infusion of sulpiride into the III-rd ventricle increased GnRH mRNA levels in the anterior pituitary gland and LH secretion. It is suggested that the increase of GnRH gene expression in the anterior pituitary gland and LH secretion in sulpiride-treated ewes are related with an increase of biosynthesis GnRH with concomitant decreased biosynthesis of GnRH-R protein in the ventromedial hypothalamus/stalk median eminence allowing to an increase of GnRH release.

The central effect of beta-endorphin and naloxone on the expression of GnRH Gene and GnRH receptor (GnRH-R) gene in the hypothalamus, and on GnRH-R gene in the anterior pituitary gland in follicular phase ewes.

The effect of prolonged intermittent infusion of beta-endorphin or naloxone into the third cerebral ventricle in ewes during the follicular phase of the estrous cycle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined by Real time-PCR. Activation of micro opioid receptors decreased GnRH mRNA levels in the hypothalamus and led to complex changes in GnRH-R mRNA: an increase of GnRH-R mRNA in the preoptic area, no change in the anterior hypothalamus and decrease in the ventromedial hypothalamus and stalk/median eminence. In beta-endorphin treated ewes the levels of GnRH-R mRNA in the anterior pituitary gland also decreased significantly. These complex changes in the levels of GnRH mRNA and GnRH-R mRNA were reflected in the decrease of LH secretion. Blockade of micro opioid receptors affected neither GnRH mRNA and GnRH-R mRNA nor LH levels secretion. These results indicate that beta-endorphin displays a suppressive effect on the expression of the GnRH gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland, but affects GnRH-R gene expression in a specific manner in the various parts of hypothalamus; altogether these events lead to the decrease in GnRH/LH secretion.

Effects of GABAB receptor modulation on gonadotropin-releasing hormone and beta-endorphin release, and on catecholaminergic activity in the ventromedial hypothalamus-infundibular nucleus region of anoestrous ewes.

To examine the role of gamma-aminobutyric acid (GABA)B receptor mediating systems in the ventromedial hypothalamus-infundibular nucleus region (VMH/NI) of anoestrous ewes in controlling gonadotropin-releasing hormone (GnRH) release, the extracellular concentrations of GnRH, beta-endorphin, norepinephrine, dopamine, 4-hydroxy-3-methoxy-glycol and 3,4-dihydroxy-phenylacetic acid were quantified during infusion of baclofen or phaclofen (agonist and antagonist of GABAB receptors, respectively) in this structure. The stimulation of GABAB receptors activates GnRH/luteinising hormone (LH) release, attenuates noradrenergic and beta-endorphinergic tone but has no evident effect on the dopaminergic system. Blockade of GABAB receptors in this structure increases the extracellular beta-endorphin concentration but has no significant influence on GnRH release or catecholaminergic activity. It is suggested that activation of GnRH/LH release in the VMH/NI of anoestrous ewes may result from a decrease of norepinephrine output and hence its inhibitory effect on GnRH secretion. Activation of GABAB receptors, as well as their blockade, did not change dopaminergic system activity, indicating that GABAB does not affect GnRH release indirectly by a GABAB receptor mechanism acting on dopaminergic neurones in the VMH/NI. Increased activity of the beta-endorphinergic system during blockade of GABAB receptors does not change GnRH release, suggesting that beta-endorphin does not play a significant role in the control of GnRH secretion in anoestrous ewes.

The role of GabaA receptors in the neural systems of the ventromedial hypothalamus-nucleus infundibular region in the control of GnRH release in ewes during follicular phase.

To examine the role of the GABAA receptor mediating systems in the control of gonadotropin releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/NI) of the hypothalamus of ewes during the follicular phase of the estrous cycle, the extracellular concentrations of GnRH, beta-endorphin (B-END), noradrenaline (NE), dopamine (DA), and their metabolites MHPG, DOPAC and concentration of luteinizing hormone (LH) in blood plasma were quantified during local stimulation or blockade of GABAA receptors with muscimol and bicuculline, respectively. Stimulation of GABAA receptors attenuated GnRH and LH release, increased beta-endorphin outflow and dopaminergic activity but had no evident effect on noradrenergic activity. Blockade of GABAA receptors decreased beta-endorphin release but had no evident effect on the extracellular concentration of GnRH, LH levels in the blood and catecholaminergic activity. It is suggested that suppression of GnRH/LH release under muscimol treatment may result from activation of GABAA receptors on GnRH nerve terminals and through GABAA receptor mechanism activated beta-endorphinergic and dopaminergic neurons in the VEN/NI. Lack of changes in NE and MHPG concentration during stimulation or blockade of GABAA receptors suggests, that during the follicular phase of the estrous cycle the noradrenergic system in the VEN/NI is not involved in the control of GnRH/LH release by GABA.

The role of GABA(A) receptors in the neural systems of the medial preoptic area in the control of GnRH release in ewes during follicular phase.

To examine the role of gamma-aminobutyric acid (GABA)(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the medial preoptic area (MPOA) of ewes during the follicular phase of the estrous cycle, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenyl-glycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during the local infusion of muscimol and bicuculline (agonist and antagonist of GABA(A) receptors, respectively) to this structure. Stimulation of GABA(A) receptors markedly attenuated GnRH release, increased beta-endorphin release and noradrenergic system activity in the MPOA. The decrease of the luteinizing hormone (LH) concentration in blood plasma and LH pulse amplitude suggests that a GABA(A) receptor agonist in the MPOA also suppresses GnRH release from the GnRH axon terminals in the ventromedial hypothalamus/nucleus infundibularis region (VEN/NI). Blockade of GABA(A) receptors had no evident effect on GnRH/LH secretion but decreased beta-endorphin release and increased the extracellular DOPAC concentration. The suppressive influence of muscimol in the MPOA on GnRH release might be considered a net result of its direct inhibitory effect on GnRH release, indirect inhibitory influence on GnRH release through activation of the beta-endorphinergic system, and facilitation of GnRH neurons by increasing noradrenaline release. The results obtained during bicuculline perfusion on these systems' activity are not sufficiently consistent to provide a clear understanding of the lack of changes in the GnRH/LH release under blockade of GABA(A) receptors. We conclude that the MPOA in ewes during the follicular phase is an important regulatory site where stimulation of GABA(A) receptors both decreases GnRH secretion and increases beta-endorphin release.

The Involvement of GABAA receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the preoptic area in anestrous ewes.

This study examined role of GABA A receptors in the control of GnRH, beta-endorphin release and catecholaminergic system activity in the preoptic area and LH secretion in anestrous ewes. Stimulation of GABA A receptors in the medial preoptic area (MPOA) by muscimol attenuated GnRH release and dopaminergic system activity and increased extracellular noradrenaline (NE) and MHPG concentration. Muscimol has no evident effect on the extracellular concentration of beta-endorphin-like immunoreactivity (B-END-LI) in the MPOA. The decrease of LH pulse frequency and concentration of this hormone in blood plasma suggests that GABA A receptor agonist applied in the MPOA suppresses GnRH release from the GnRH axon terminals in the ventromedial hypothalamus-nucleus infundibularis region (VEN/NI) into the hypophyseal vascular system. Blockade of GABA A receptors with bicuculline did not change GnRH release, catecholaminergic activity, B-END-LI concentration in the MPOA, and LH release. The presented data indicate that activation of GABA A receptors in the MPOA decreases extracellular concentration of GnRH in this structure and LH level in the blood plasma thus suggesting that GABA may act in the MPOA to inhibit GnRH release in the VEN/NI. These results suggest that suppression of GnRH/LH release during muscimol treatment may result from activation of GABA A receptors on the GnRH perikarya and/or through GABA A receptor mechanism on the dopaminergic and noradrenergic system in the MPOA. Lack of changes in B-END-LI concentration during stimulation or blocking GABA A receptors suggests, that beta-endorphinergic system in the MPOA does not participate in the GABA A receptors mechanism modulating GnRH release.

The role of GABA(A) receptors in the neural systems of the ventromedial-infundibular region of the hypothalamus in the control of gonadotropin release during the luteal phase in ewes.

To examine the role of GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/NI) in ewes during luteal phase, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), and their metabolites: MHPG and DOPAC were quantified by local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline, respectively. Stimulation of GABA(A) receptors in the VEN/NI did not affect GnRH, beta-endorphin release or catecholaminergic system activity. Blockade of GABA(A) receptors decreased beta-endorphinergic and dopaminergic activity, and lowered the extracellular concentration of MHPG. It did not affect GnRH release or luteinizing hormone (LH) secretion. It is suggested that progesterone-induced GABAergic activity during the luteal phase may desensitize GABA(A) receptors to muscimol. Lack of changes in GnRH/LH secretion with concomitant depressed beta-endorphinergic activity corroborated the conclusion that beta-endorphin does not inhibit GnRH release from the VEN/NI during the luteal phase. The physiological significance of changes in the catecholaminergic system activity under GABA(A) receptor blockade in the control of GnRH secretion awaits to be established.

The involvement of GABA(A) receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the ventromedial-infundibular region of hypothalamus in anestrous ewes.

To examine the role of the GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/IN) of anestrous ewes, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline respectively. In most animals stimulation of GABA(A) receptors significantly attenuates GnRH release with concomitant increase of beta-endorphin and DA release, and MHPG and DOPAC levels. Blockade of the GABA(A) receptors generally did not affect GnRH and NE release but inhibited in most animals beta-endorphin release and decreased dopaminergic activity. These results suggest, that GABA may suppress GnRH release directly by GABA(A) receptor mechanism on the axon terminal of GnRH neurons or indirectly by GABA(A) receptor processes activating beta-endorphin-ergic and dopaminergic neurons in the VEN/NI. On the basis of these results in could not be distinguish between these two events. The decrease in extracellular beta-endorphin and dopamine concentration without evident changes in the GnRH level during GABA(A) receptor blockade may suggest that other neuronal systems are involved in this effect.

Changes in extracellular LHRH and beta-endorphin-like immunoreactivity in the nucleus infundibularis-median eminence of anestrous ewes under stress condition.

To clarify the effect of beta-endorphin released under stress condition on LHRH secretion, the concentration of LHRH and beta-endorphin-like-immunoreactivity (beta-END-LI) were analysed in perfusates from the nucleus infundibularis-median eminence of anestrous ewes subjected to footshocks stimulation. The dynamics of LHRH and beta-END-LI release during time-course of footshocks stimuli was altered. A brief facilitatory influence of stress on LHRH and beta-END-LI release was observed at the beginning of the stimulation on the first and third day; however the peaks of beta-END-LI were delayed about half an hour in relation to LHRH. Than on the first day of the stimulation LHRH returned to the control value but on the third day it declined below the control levels. Prolonged stress had stimulatory effect on beta-END-LI secretion during first and third day. The presented results indicate that: 1) short stress activates LHRH and beta-END-LI release, 2) prolonged stress has stimulatory effect on beta-END-LI release but leads to suppression of LHRH release. It is suggested that stress-induced beta-endorphin release may be one of the factors responsible for suppression of LHRH activity.

Catecholaminergic activity in the medial preoptic area and nucleus infundibularis-median eminence of anestrous ewes in normal physiological state and under stress condition.

The stressful events induce in organism both excitatory and inhibitory mechanisms for rapid physiological adjustment to environmental stressors. Pull-push technique was used to determine extracellular concentrations of NE, DA, and their metabolites MHPG and DOPAC in the medial preoptic area (MPOA) and nucleus infundibularis-median eminence (NI/ ME) of anestrous ewes in normal physiological state and under stress condition. In non-stressed ewes the level of NE in the MPOA was substantially lower than in the NI/ME, whereas DA was found only in the NI/ME. No regional differences in the concentrations of MHPG, DOPAC in these structures were found. On the first day, footshock stimulation activated noradrenergic system in the MPOA and both noradrenergic and dopaminergic system in the NI/ME during the first 1.5 h followed by gradual desensitisation of these systems. On the third day during whole period of stimulation the concentration of all these neurochemical compounds were significantly lower than in controls. It indicates that prolonged intermittent stress elicits a neurochemical processes in the MPOA and NI/ME whose effects lead to the suppression of catecholamines release and their metabolism. The basal concentration and stress induced changes of NE, DA, MHPG, DOPAC in the fluid of the III-rd brain ventricle (V-III) reflect a dynamic relationship between extracellular levels of catecholamines and their metabolites in the hypothalamus and cerebral fluid. The consequences of these long-term response of catecholaminergic system in the MPOA and NI/ME may be essential for changes in LHRH release from the hypothalamus which we observed in ewes subjected to prolonged stressful experience (Tomaszewska et al., 1999).

The immune-neuro-endocrine interactions.

This article reviews data concerning the interactions between immune, endocrine and neural systems in physiological, pathophysiological and stress conditions in animals and humans. Numerous studies have provided evidence that these systems interact with each other in maintaining homeostasis. This interaction may be classified as follows: immune, endocrine and neural cell products coexist in lymphoid, endocrine and neural tissue. Endocrine and neural mediators modulate immune system activity. Immune, endocrine and neural cells express receptors for cytokines, hormones, neuropeptides and transmitters.

Extracellular monoamines and their metabolites in the mediobasal hypothalamus--median eminence of anestrous and estrous ewes during CRF treatment.

In order to clarify the effect of exogenous corticotropin-releasing factor (CRF) on catecholaminergic and serotoninergic system activity in the mediobasal hypothalamus-median eminence (MBH-ME) of ewes the changes in extracellular levels of noradrenaline (NA) and serotonin (5HT), and main metabolites of monoamines, 4-hydroxy-3-methoxyphenylglycol (MHPG), 3,4-dihydroxy-phenylacetic acid (DOPAC), homovanilic acid (HVA), and 5-hydroxy-indolo-3-acetic acid (5-HIAA) were quantified in the perfusates collected from MBH-ME. NA, 5-HT and monoamine metabolites in the perfusates were analyzed using high performance liquid chromatography with electrochemical detection. CRF induced a rise in extracellular concentration of NA and 5-HT only in the estrous ewes prior to a preovulatory LH surge. CRF treatment caused a heterogenous effect on extra-cellular concentrations of 5-HT in ewes during the preovulatory LH surge. Except for DOPAC and HVA in some estrous ewes during the preovulatory LH surge, CRF caused an increase in monoamine metabolites levels in the MBH-ME in anestrous and estrous animals. These results indicate that CRF facilities NA release in the MBH-ME during the presurge LH period in ewes, and that CRF increases metabolic activities of the monoaminergic systems in this structure in the anestrous and estrous ewes, except dopaminergic system in the ewes during the preovulatory LH surge. It is suggested that: 1) the responses of monoaminergic systems activity in the MBH-ME to CRF in large degree is dependent upon physiological state of ewes and 2) in some endocrinological phases CRF may affect LHRH and other hypothalamic hormone secretion indirectly by altering monoaminergic system activity in the MBH-ME.