Vaginocervical stimulation-induced release of classical neurotransmitters and nitric oxide in the nucleus of the solitary tract varies as a function of the oestrus cycle.

The effects of vaginocervical stimulation (VCS) on glutamate (GLU), aspartate (ASP), gamma-aminobutyric acid (GABA), noradrenaline (NA), arginine (ARG) and nitric oxide (NO) (citrulline) release in the nucleus of the solitary tract (nTS) were measured in anaesthetised female rats as a function of the oestrus cycle. During pro-oestrus/oestrus (P/E), but not during met-oestrus/di-oestrus (M/D), VCS significantly increased concentrations of NA, ASP, GLU, NO (citrulline) and GABA, but not ARG. Basal NA concentrations were also increased in P/E. These effects were prevented by bilateral section of either the vagus nerve or pelvic and hypogastric nerves. Vagotomy also significantly decreased basal NO concentrations in M/D and P/E while pelvic and hypogastric nerve section significantly increased GABA concentrations. Our results therefore confirm that the nTS is a relay structure for the visceral afferents sending information from the uterus into the central nervous system. The ability of VCS to trigger classical transmitter release and NO in the female is influenced by the stage of the oestrous cycle and is routed both via the vagus and pelvic/hypogastric nerves.

Release of classical transmitters and nitric oxide in the rat olfactory bulb, evoked by vaginocervical stimulation and potassium, varies with the oestrus cycle.

In vivo microdialysis was used to investigate the effects of ovariectomy and the oestrus cycle on vaginocervical stimulation-evoked classical transmitter and nitric oxide release in the olfactory bulb of anaesthetized (urethane) and conscious rats. During pro-oestrus/oestrus, vaginocervical stimulation (1 or 10 min) significantly increased concentrations of glutamate, aspartate, GABA, noradrenaline, dopamine and nitric oxide (citrulline) but failed to do so in met-oestrus/di-oestrus or following ovariectomy. Potassium chloride-evoked GABA, noradrenaline and nitric oxide release in the olfactory bulb was also significantly enhanced during pro-oestrus/oestrus. The effects of vaginocervical stimulation on olfactory bulb transmitter release during pro-oestrus/oestrus were significantly reduced by pelvic or vagus nerve section. Basal concentrations of classical transmitters and nitric oxide in the olfactory bulb did not vary across the oestrus cycle although noradrenaline and dopamine levels were reduced following ovariectomy. These results confirm our previous electrophysiological data showing that the olfactory bulb mitral cells are only excited by vaginocervical stimulation during pro-oestrus/oestrus. They also suggest that sex hormones acting primarily at the level of the olfactory bulb dramatically enhance the ability of vaginocervical stimulation to evoke release of both classical transmitters and nitric oxide in this region. Such alterations in neurochemical release in the olfactory bulb may be important for mediating plasticity changes underlying olfactory recognition of mates or offspring.

C-fos and c-jun in the paraventricular nucleus play a role in regulating peptide gene expression, oxytocin and glutamate release, and maternal behaviour.

In sheep, birth leads to the induction of maternal behaviour through brain oxytocin release. Associated with these events is an upregulation of oxytocin, opioid and corticotrophin-releasing hormone (CRH) gene expression, as well as that of the immediate early gene c-fos in the paraventricular nucleus (PVN) of the hypothalamus. We investigated the role of c-fos dimerizing with c-jun in controlling the induction of maternal behaviour, altered peptide gene expression, and oxytocin and amino acid release in this region at birth. Fluorescence-labelled antisense oligodeoxyribonucleotides (ODNs) against c-fos/c-jun were infused bilaterally in the PVN, via microdialysis probes with 100 kDa cut-off membranes, and were incorporated into 50-60% of the cells. Compared with the control (scrambled) sequences, they significantly reduced basal concentration of glutamate (to 31.7% of baseline after 10 h) and prevented birth-induced release of aspartate. In addition, antisense treatment reduced the birth-induced increase in oxytocin concentration in the PVN, but not in blood. Although all the animals were fully maternal, the antisense treatment did reduce the peak expression of two components of maternal behaviour: low-pitched bleats; and lamb sniffing. Finally, in situ hybridization histochemistry revealed that the antisense treatment significantly reduced the birth-induced upregulation of c-fos, oxytocin, CRH and preproenkephalin mRNA expression in the PVN, whilst not affecting that of arginine vasopressin. These results suggest that c-fos/c-jun transcription factors play a role in the birth-induced upregulation of oxytocin, CRH and preproenkephalin gene expression, as well as on glutamate and oxytocin release in the sheep PVN.

Changes in neurotransmitter release in the main olfactory bulb following an olfactory conditioning procedure in mice.

Olfactory learning is associated with substantial neural changes at the level of the accessory and main olfactory bulb, during both pheromonal learning in mated mice and lamb odour recognition in post partum sheep. These forms of learning occur during "sensitive periods" and an important question is whether similar neural changes occur in the olfactory bulb at other times. We used a classical conditioning procedure to establish an olfactory discrimination in adult mice and then measured changes in neurotransmitter levels in the main olfactory bulb in response to the presentation of the conditioned odours. Presentation of the conditioned, but not the non-conditioned, odour resulted in significant increases in the levels of certain transmitters, including glutamate from the mitral/tufted cells, GABA from the granule and periglomerular cells and noradrenaline from the centrifugal projection from the locus coeruleus. Overall, there was a decrease in the ratio of excitatory to inhibitory neurotransmitters in the olfactory bulb in response to the conditioned, but not the non-conditioned odour. Moreover, the magnitude of the decrease in this ratio was correlated with the level of behavioural response to the conditioned odour. These findings support the hypothesis that changes in the gain of the reciprocal synapses between mitral/tufted neurons and their inhibitory interneurons are a general feature of olfactory learning.

NMDA and kainate-evoked release of nitric oxide and classical transmitters in the rat striatum: in vivo evidence that nitric oxide may play a neuroprotective role.

The effects of N-methyl-D-aspartate (NMDA), kainate, S-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and KCl on striatal nitric oxide (NO), acetylcholine (ACh), dopamine (DA), serotonin (5-HT), aspartate (ASP), glutamate (GLU) and gamma-aminobutyric acid (GABA) release were measured in anaesthetized rats in vivo by microdialysis and in vitro in organotypic slice cultures. Local NMDA (1-100 microM) infusion by retrodialysis dose-dependently increased levels of classical transmitters, NO2-, NO3-, citrulline and arginine at similar thresholds (10 microM). Similar patterns of NMDA-evoked (50 microM) release were seen in striatal cultures. NMDA-evoked changes were all calcium-dependent and blocked by NMDA (APV or MK-801) but not AMPA/kainate (DNQX) receptor antagonists, excepting DA which could be prevented by both. In vivo, kainate increased NO2-, NO3-, CIT and ARG levels at 50 and 100 microM but was less potent than NMDA. Kainate also evoked significant ACh, DA and GLU release dose-dependently starting at 1-10 microM whereas 5-HT, ASP and GABA required 50 or 100 microM doses. Kainate effects were inhibited by DNQX, but not by APV, and were calcium-dependent, AMPA failed to alter NO2-, NO3-, CIT or ARG levels at 50 or 100 microM doses but dose-dependently increased ACh and DA. Similar results were seen with kainate (50 microM) and AMPA (50 microM) in vitro. KCl evoked NO2-, NO3-, CIT and ARG release as well as that of the classical transmitters in vivo and in vitro. In vivo administration of the NO synthase inhibitor L-nitroarginine (L-NARG; 100 microM) significantly reduced NO2-, NO3- and CIT levels and prevented NMDA, kainate or KCl-evoked increases. It also potentiated ACh, ASP, GLU and GABA release and reduced that of DA in response to 50 microM NMDA whereas treatment with an NO-donor (SNAP; 10 microM) significantly reduced evoked ACh, ASP and GLU release. The NO synthase inhibitor L-NARG potentiated kainate-evoked ACh release and reduced that of DA, although less potently than NMDA, but it had no effect on KCl-evoked transmitter release. Overall, these results show that both NMDA and kainate increase striatal NO release at similar dose-thresholds as for classical transmitter release suggesting that NO is dynamically released under physiological and not just pathological conditions. Reductions of striatal NO levels also potentiates calcium-dependent transmitter release in response to NMDA and, to a lesser extent, kainate, whereas increasing them reduces it. This is consistent with a role for NO as a neuroprotective agent in this region acting to desensitize NMDA receptors.

Effects of the 5-HT1A agonist 8-OH-DPAT on operant feeding in pigs.

The effect of intravenous (i.v.) injection of 8-OH-DPAT on operant food intake in pigs has been examined in animals with food available ad lib and in pigs feeding after 4 h deprivation of food. Operant methods were used to ascertain whether any increases in food intake were due to increased motivation to feed rather than stereotypic chewing movements. In nondeprived animals i.v. injection of 8-OH-DPAT at doses of 10, 30, and 50 micrograms/kg significantly increased food intake in the 45 min following injection. In pigs feeding after 4 h deprivation, i.v. injection of 8-OH-DPAT at doses of 30 and 50 micrograms/kg, given 5 min before feeding commenced, had no effect on food intake.

The noradrenergic innervation of the rat thymus during pregnancy and in the post partum period.

The noradrenergic innervation of the rat thymus during pregnancy and the post partum period was examined by a sucrose glyoxylic acid method for catecholamines, and by high pressure liquid chromatography. Fluorescent nerves decreased in number throughout pregnancy when there was an overall loss in thymic weight due to cortical involution. These changes are maximal by parturition. There was a dramatic increase in nerves between d 21 of pregnancy and d 1 after parturition, especially in the capsule and around blood vessels in the connective tissue septa. The neonates were removed at parturition and thymic weight was rapidly regained. The increased numbers of nerves remained throughout this post partum period. Noradrenaline levels in the thymus altered in a similar pattern throughout pregnancy and the post partum period, but did not parallel thymic weight changes. The mean noradrenaline concentration in the virgin thymus was 1063 +/- 107 pg/mg protein. Levels remained similar during early pregnancy and increased significantly at d 16. Virgin levels were regained by d 21. Values peaked after parturition but rapidly decreased over the next 3 days, and remained at or below virgin levels to d 28 except for a transient rise at d 10 post partum. Adrenaline values were consistently below detection levels. This study shows that there are variations in both nerves visualised, and in neurotransmitter (noradrenaline) content in the thymus during the course of pregnancy and the post partum period. Thus thymic function could be influenced by central events (levels of catecholamines in peripheral blood) as well as local events mediated by transmitter changes in nerves.

Differential stressor effects on the concentrations of cortisol, prolactin and catecholamines in the blood of sheep.

Adult wether sheep (n = 8) were subjected to 60 minutes of psychological (isolation) or physical (transport simulation/standing in water) stress, or a control handling procedure in the home pen. Blood samples were taken before and during these treatments and the plasma concentrations of cortisol, prolactin, noradrenaline and adrenaline determined. All the stressors significantly increased plasma cortisol concentrations (P < 0.001) whereas only transport simulation increased prolactin secretion (P < 0.005). Noradrenaline concentrations showed little change in response to the various treatments, with the exception of isolation which produced a small non-significant increase (P < 0.08). All the stress procedures stimulated adrenaline release within the first 10 minutes but the most marked effects were seen after transport simulation and isolation, both of which induced a significant increase throughout the treatment period (P < 0.01).

Effect of a novel CCKA receptor antagonist (2-NAP) on the reduction in food intake produced by CCK in pigs.

The effect of a novel CCKA receptor antagonist 2-naphthalene sulphonyl-L-aspartyl-2-(phenethyl)amide, sodium salt (2-NAP) on the reduction of food intake induced by exogenous CCK, administered centrally or peripherally, has been examined in pigs. 2-NAP is hydrophilic and should not readily cross the blood-brain barrier. Intravenous (IV) 2-NAP (20 or 40 mg/kg) injected prior to IV CCK-8S (1 microgram/kg) abolished the inhibitory effect of CCK-8S on operant food intake in hungry pigs. Intravenous injections of 2-NAP (20 and 30 mg/kg) prior to the administration of intracerebroventricular (ICV) injection of CCK-8S (1 microgram) did not affect the inhibitory action of ICV CCK-8S on food intake. ICV injection of 2-NAP (5 mg) abolished the inhibitory effect on food intake of ICV CCK-8S (1 microgram). The results indicate that 2-NAP does not cross the blood-brain barrier readily and that central and peripheral administration of CCK-8S inhibits feeding by different mechanisms. Neither ICV nor IV injection of 2-NAP altered food intake when injected alone.

Kainic acid has cell-specific effects on survival of hypothalamic mouse neurones in vitro.

Cultures of mouse hypothalamus from embryonic day 15 fetuses were exposed to kainic acid (KA) to study effects on survival of selected cell populations. After 6 days in vitro, immunocytochemistry revealed destruction of dopaminergic and GABAergic neurones after KA-treatment, whereas numbers of oxytocinergic cells and neurones containing metEnkephalin were unchanged. Surviving neurones were characterized by a loss of processes or a reduction of process length. Glial cell numbers did not show any differences between groups. Amino acid concentrations decreased after KA-treatment; only glutamine and taurine levels were unaffected. We conclude that toxic effects of KA differ with respect to distinct neuronal populations of the mouse hypothalamus. Therefore, experimental data concerning neuronal destruction under in vitro conditions have to take into account the selectivity of this neurotoxic agent.

Effects of intracerebroventricular injection of dynorphin, leumorphin and alpha neo-endorphin on operant feeding in pigs.

Young pigs, which are useful experimental animals for biomedical research, were prepared with lateral intracerebroventricular (ICV) cannulae and housed individually in cages fitted with operant panels, with food and water ad lib. ICV injection of 200 micrograms of dynorphin A 1-17 or 1-13 resulted in a significant meal commencing within 2-5 min. Shorter fragments of dynorphin (1-10, 1-9, 1-8) were ineffective at inducing feeding as was dynorphin B (rimorphin). In the same situation, leumorphin and alpha neo-endorphin (200 micrograms) elicited significant feeding but beta neo-endorphin did not. Dynorphin 1-17 or 1-13, administered 5 min before feeding started, increased meal size when pigs were fed after 4-h deprivation. Naloxone ICV (0.4 mg) significantly reduced food intake in pigs feeding after 4-h deprivation and its main effect was in the second half of the meal. Naloxone also abolished the effect of ICV dynorphin. It is concluded that dynorphin and related endogenous opioids are involved in the regulation of food intake in pigs.

Effects of intracerebroventricular injection of muscimol or GABA on operant feeding in pigs.

Young pigs were prepared with lateral intracerebroventricular (ICV) cannulae. They were housed individually in cages fitted with operant panels and could obtain food and water ad lib. The GABA-A receptor agonist muscimol (25-200 nmol) ICV produced an increase in food intake in which the dose-response relation was most obvious 30-60 min after dosing. The 25-nmol dose had no effect on feeding. However, muscimol (50 nmol) caused a significant increase in feeding (p less than 0.01) during the first 30 min after injection, while the 100- and 200-nmol doses increased food intake (p less than 0.01) during the first 60 min. The effect of muscimol (100 nmol) on food intake was completely abolished by the simultaneous administration of the GABA-A receptor antagonist bicuculline (100 nmol). GABA (40-1600 nmol) ICV also produced a dose-related increase in food intake (p less than 0.01) in the 15 min after injection. Only doses of 800 nmol and above were effective. The effects of GABA (1600 nmol) were completely abolished by the simultaneous administration of bicuculline (50 nmol). Neither muscimol nor GABA influenced food intake for the 24-hr time period or water intake during any time period. The results indicate that stimulation of central GABA-A receptors induces operant feeding in the satiated pig.

Effects of the CCK receptor antagonist MK-329 on food intake in pigs.

The cholecystokinin (CCK) receptor antagonist MK-329 (previously L364,718) was administered intravenously (IV) (17.5-140 micrograms/kg) to pigs trained to make operant responses for food reinforcements after 4 hr of food deprivation. MK-329 produced a dose-related increase in food intake during the 2-hr test period, with maximum increases occurring at a dose of 70 micrograms/kg. CCK (1 micrograms/kg IV) produced a short-term reduction in feeding and this effect was completely abolished by pretreating the animals with MK-329 (70 micrograms/kg). The present results lend support to the hypothesis that endogenous CCK is involved in satiety.

Microdialysis measurement of monoamine and amino acid release from the medial preoptic region of the sheep in response to heat exposure.

Concentrations of monoamines and metabolites and amino acids were measured in microdialysis samples taken from the medial preoptic area of 5 conscious sheep before, during and after exposure to an ambient temperature of 45 degrees C. Concentrations of dopamine, noradrenaline and aspartate significantly increased, and those of the serotonin metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), significantly decreased during heat exposure and although panting was induced, body temperature did not change. Concentrations of noradrenaline and aspartate declined and 5-HIAA increased to preheat exposure levels during the 60 min after the ambient temperature was reduced but levels of dopamine and its metabolite, homovanillic acid, remained elevated. Dopamine, noradrenaline, 5-HIAA and aspartate concentrations were not significantly altered by isolation stress and did not show significant changes in the cortex following heat exposure. These experiments provide further support for the proposed roles of dopamine, noradrenaline, serotonin and aspartate in the neural control of autonomic thermoregulatory responses.

Endocrine responses and examination anxiety.

Endocrine and psychological function (measuring both affect and attitudes to study) were studied in 38 male medical students 4 weeks and 1-2 h before a major examination. Anxiety (or tension) and emotionality increased just before the examination, as did the 'denial' subscale of a 'coping' questionnaire. Serum cortisol and prolactin increased; serum testosterone and LH were unchanged. Both urinary noradrenaline and adrenaline were elevated. Increased cortisol correlated with increased prolactin across subjects; so, too, did levels of urinary noradrenaline and adrenaline, but the two sets of endocrine responses were not correlated with each other. Several of the trait scales predicted the endocrine response to the examination. The 'lie' scale of the Eysenck Personality Questionnaire correlated negatively with changes in both cortisol and prolactin, as did 'debilitating' anxiety, as defined by the Alpert-Haber scale. However, although there were no significant correlations between changes in hormone levels and those in any of the state scales, there was some relation between absolute hormone levels on the day of the examination. Measures of academic strategies or psychological responses to examinations do not predict the nature of the considerable hormonal response which occurs in this homogeneous set of high-achieving students.

Relationship between dominance hierarchy, cerebrospinal fluid levels of amine transmitter metabolites (5-hydroxyindole acetic acid and homovanillic acid) and plasma cortisol in monkeys.

The dominance hierarchy has repercussions for a monkey's sexual behaviour and endocrine state. Here we report on neural mechanisms that are sensitive to a monkey's status in the social hierarchy, and which may regulate not only its endocrine function but its sexual responsiveness to its own hormones. During the initial phase of group formation, 5-hydroxyindole acetic acid, the metabolite of serotonin, increases in the cerebrospinal fluid of monkeys which become subordinate (all groups), but decreases in monkeys which become dominant (two out of three groups) and shows no changes in intermediate-ranking animals (five out of seven). Homovanillic acid, a metabolite of dopamine, may also increase in the cerebrospinal fluid of monkeys that become subordinate (two out three groups). In the initial period of group formation these changes in transmitter metabolites do not parallel changes in cortisol. However, in the established social groups, both 5-hydroxyindole acetic acid and plasma cortisol are related to the social hierarchy, being greater in those monkeys that are subordinate, but homovanillic acid shows no consistent change. Although subordinate monkeys receive more aggression than others in their group, fluctuations in 5-hydroxyindole acetic acid do not correlate with aggressive behaviour, and are equally high on days when no aggression occurs. Dominant males, however, had higher 5-hydroxyindole acetic acid levels on days when they were involved in agonistic encounters. In the established social hierarchy therefore, elevated levels of the serotonin metabolite in cerebrospinal fluid seem reflect a "state"-dependent consequence of occupying a position of low social status.

Repeated determination of cerebrospinal fluid amine metabolites by automated direct sampling from an implanted cannula in freely moving rats.

A method is described for the automatic sampling of CSF from conscious freely moving rats and the simultaneous determination of 5-HIAA and HVA by high-pressure liquid chromatography with electrochemical detection (HPLC-EC). Timed samples (30 microliters) were taken directly from a chronically implanted cannula in the cisterna magna of adult female rats by the HPLC autoinjector. The low volume of sample permitted withdrawal of repeated samples without cross-contamination. The cannula could be used the day after implantation and remained patent for at least two weeks. Treatment with probenecid (200 mg/kg i.p.) caused an increase of both metabolites in the CSF. Pargyline (50 mg/kg i.p.) affected both the 5-HT and dopaminergic systems, reducing their metabolite levels. alpha-Methyl-p-tyrosine (200 mg/kg i.p.) acted selectively on the catecholaminergic system lowering HVA levels. The method accurately measured amine metabolite changes in the CSF due to pharmacological manipulations.