Hippocampal vasopressin release evoked by N-methyl D-aspartate (NMDA) microdialysis.

Hippocampus is a brain structure containing vasopressin (AVP) fibers and specific binding sites for this peptide. There is growing evidence that AVP and its metabolites participate in glutamate-mediated plasticity of the hippocampus. The aim of the present study was to evaluate the influence of NMDA on AVP release in the rabbit hippocampus. Caudate nucleus was chosen as the reference structure. The mentioned brain structures were simultaneously microdialyzed with 0.9 % NaCl solution. AVP was determined in the outflowing fluid by radioimmunoassay. The mean basal AVP content in the fluid outflowing from the hippocampus was significantly greater than that from the caudate nucleus. The addition of K(+) into the fluid perfusing the probes implanted into the hippocampus and caudate nucleus significantly increased AVP release into the extracellular fluid of both brain structures. NMDA applied into the mentioned brain structures increased AVP release only from the hippocampus but not from the caudate nucleus. Our findings indicate a role which NMDA receptors play in AVP release into the extracellular fluid of the hippocampus.

Sex difference in rabbit eyelid conditioning.

The rabbit eyelid conditioned reflex has been used to compare associative learning in males and ovariectomized females. A new method for monitoring eyelid movements is described. Rabbits were trained on simple delay classical conditioning. Conditioned responses were recorded during 8 acquisition days and 6 days of extinction training. Analysis of variance (ANOVA) and the least significant difference (LSD) post hoc test was used to analyze the data. The results showed that males achieved significantly better learning than females during the first day of acquisition but later they slowly attained the best result, contrary to females. Moreover, extinction of the conditioned reflex was significantly faster in females than in males. It is postulated that females learn and extinguish faster than males because of a higher level of brain plasticity.

Application of infrared detection in the recording of eyelid movements in rabbits.

Classical conditioning of the eyelid reflex has been used for a long time to study associative learning in animals and humans. A new experimental procedure for rabbit's eyelid conditioning was constructed and described. A phonopneumatic stimulator generated conditioned and unconditioned stimuli and a photoelectric transducer acting in close infrared converted movements of rabbits' eyelid to electric signals. An example of acquisition and extinction training is illustrated. This method of eyelid movement monitoring is noninvasive. It may be useful for chronic studies of learning processes in rabbits when used with headpieces for microdialysis probes, electrodes or cannulas which could be implanted into brain structures.

Hippocampal vasopressin (AVP) dialysis and the conditioned eyelid reflex in rabbits.

The role of arginine vasopressin (AVP) dialyzed into the hippocampus or caudate nucleus as the reference structure in the acquisition and extinction of the conditioned eyelid reflex in rabbit was investigated. Phonopneumatic stimulator was used for the generation of conditioned and unconditioned stimuli, and for control of the recorder. Opto-electronic sensor transduced the behavioral responses. Microdialysis probes were chronically implanted into the brain structures. AVP was dialyzed into the brain structures during the extinction procedure. Restraining of the process of extinction was shown during AVP dialysis through the hippocampus and caudate nucleus but the effect in hippocampus was stronger and longer lasting than in caudate nucleus. The influence of AVP dialyzed through the hippocampus on the course of acquisition was biphasic. Some insignificant improvement of leaming was observed at the beginning of training and then compensatory, significant restraining of learning. After AVP dialysis through the caudate nucleus only the late, insignificant tendency to improve leaming was shown. The effects of AVP were dose-dependent in inversely proportional manner and long-term in nature, especially the effects in hippocampus.

Comparison of antagonistic properties of substance P analogs, spantide I, II and III, on evoked tongue jerks in rats.

OBJECTIVE: To study and evaluate the effects of perfusion through cerebral ventricles with substance P (SP) and its analogs: spantide I, II and III on evoked tongue jerks (ETJ) in male rats. METHODS: During the perfusion, stimulation of the tooth pulp caused retractive movements of the stretched tongue, the amplitudes of which were recorded. The mean amplitudes of evoked tongue jerks (ETJ) recorded during each 10 min. period of perfusion with McIlwain-Rodnight's solution and solutions containing peptides were compared. RESULTS: Perfusion of cerebral ventricles with SP caused a significant increase in the mean amplitude of evoked tongue jerks. Spantide I caused a complete respiratory arrest in all of the examined animals, so its effect on the trigemino-hypoglossal reflex could not have been tested. Spantide II, in the first two minutes, induced a transient significant decrease in ETJ amplitude, followed by an increase in ETJ in the next 8 min. SP perfused after spantide II caused a further significant increase in ETJ, as compared with control. Perfusion of cerebral ventricles with spantide III caused a significant, dose-dependent decrease in ETJ. SP perfused after spantide III caused a smaller increase in ETJ than it was observed without spantide III. CONCLUSION: Spantide III was found to be a strong antagonist of SP in trigemino-hypoglossal reflex.

Role of substance P in central control of ovulation in female rats.

OBJECTIVE: To investigate the influence of substance P on gonadotropin release from the pituitary as evaluated according to the final effect, i.e. the ovulation. METHODS: Stainless steel tube was implanted into the 3rd cerebral ventricle and substance P (SP), C-terminal hexapeptide of Substance P (SP6-11), Gn-RH and adrenaline were infused on the 3rd day of the cycle (proestrus). The oviducts were then isolated on the day of estrus and the ova were counted. RESULTS: SP or SP6-11 inhibited the ovulation which was not prevented by intracerebroventricular (i.c.v.) capsaicin pretreatment. The i.c. v. administration of noradrenaline on the 7-8th day of pseudopregnancy induced the ovulation which was prevented by the injection of SP immediately before noradrenaline. CONCLUSIONS: It is suggested that noradrenergic neuros which control the ovulation by influencing the release of Gn-RH into pituitary portal vessels are affected by SP-ergic neurons.

Increase of gonadotropin-releasing hormone concentration in pituitary portal blood after substance P administration in male rats.

Substance P (SP) affects gonadotropin release from the anterior pituitary gland. In the present study we tested whether SP exerts this effect through GnRH release into pituitary portal blood in intact male rats (INT), orchidectomized rats with s.c. chronically implanted empty Silastic capsule (ORCX), testosterone capsule (ORCX + T), and 17beta-estradiol capsule (ORCX + E2). The pituitary glands were exposed by the transpharyngeal approach under urethane-chloralose anesthesia. Then, the stalk portal vessels were cut and three 30-min portal blood samples were collected. Each first sample of blood was treated as a control before 0.2 ml injection of normal saline, 5 microg, or 25 microg of SP in 0.2 ml of normal saline into the internal carotid artery. GnRH concentration in the purified portal plasma were measured by RIA. Injection of SP into the internal carotid artery caused a significant increase in GnRH concentration in pituitary portal plasma only in INT rats. The higher dose of SP markedly increased GnRH concentration in the 1st blood sample (p < 0.001) and in the 2nd blood sample GnRH concentration was lower but still significant higher than prior SP injection (p < 0.05). The lower dose of SP increased GnRH concentration later, only in the 2nd portal blood sample after intracarotid SP injection (p < 0.001). Injection of normal saline had no effect on GnRH concentration in pituitary portal blood in INT rats. In ORCX, ORCX testosterone- and estrogen-implanted rats portal plasma GnRH concentrations were not changed significantly after injection of both doses of SP. These results indicate that SP stimulates GnRH release into pituitary portal blood and the influence of SP on GnRH neurons depends on the levels of circulating gonadal steroid hormones.

Increase in vasopressin release into the hypophysial portal blood after superior cervical ganglion stimulation in rat.

The aim of the study was to investigate whether the stimulation of the superior cervical ganglion may influence vasopressin and oxytocin release into the hypophysial portal blood. In urethane-chloralose anaesthesia the pituitary gland was exposed by transpharyngeal approach in rats. The hypophysial portal vessels were transsected in the narrowing between glandular portion of the hypophysis and the infundibulum. The 15 min blood samples from the cut portal vessels were collected before and during electrical stimulation of the superior cervical ganglion. Vasopressin and oxytocin content in the plasma, were determined by radioimmunoassay. In the control samples the vasopressin content amounted to 3.2 +/- 1.03 ng/mL and oxytocin 0.75 +/- 0.3 ng/mL. Stimulation of the superior cervical ganglion evoked an increase (9.6-fold) in vasopressin concentration but not in oxytocin in the blood plasma of hypophysial portal vessels. On the basis of the results obtained, it may be presumed that the sympathetic efferents arising from the superior cervical ganglion induced only vasopressin but not in oxytocin release into the hypophysial portal blood.

Increase in cardiodepressant factor release from the posterior pituitary lobe after angiotensin II infusion into the internal carotid artery.

In our previous research the presence of a cardiodepressant factor in the medium incubating the posterior pituitary lobe 'in situ' has been demonstrated. This study presents experiments demonstrating cardiodepressant activity in medium incubating the posterior pituitary lobe 'in situ' and in dialysates of venous blood from the sella turcica region before and during angiotensin II (ANG II) infusion into the internal carotid artery in rat. Cardiodepressant activity was determined on spontaneously discharging isolated auricle of the right atrium in a two-day-old rat. It has been demonstrated that medium incubating the posterior pituitary lobe which was collected during angiotensin II infusion caused a greater decrease in auricle discharge rate than medium collected before the infusion. Angiotensin II infusion into the internal carotid artery caused a dose-dependent increase in cardiodepressant activity in dialysates of blood outflowing from the sella turcica region. The present results indicate that angiotensin II increase the release of cardiodepressant factor from the posterior pituitary lobe into blood in a dose-dependent manner.

Inhibition of tongue reflex in rats by tooth pulp stimulation during cerebral ventricle perfusion with (6-11) substance P analogs.

The effect of 12 C-terminal hexapeptide analogs of substance P perfused into the cerebral ventricles on the trigemino-hypoglossal reflex caused by incisor pulp stimulation in rats was investigated. A substitution of the L-isomer by D at position 6, 7, or 8 of the substance P analogs used in this study caused the most pronounced inhibition of the trigemino-hypoglossal reflex. A correlation between the degree of inhibition of the reflex and concentration of the peptide used was observed.

The increase in the cardiodepressant activity and vasopressin concentration in the sella turcica venous blood during vagal afferents stimulation or after angiotensin II infusion.

It has previously been demonstrated that the cardiodepressant activity is present in the bovine hypothalamic extract and in the fluid incubating the posterior pituitary lobe "in situ". The present study was an attempt to reveal if the cardiodepressant factor and vasopressin were simultaneously released from the pituitary into blood. The samples of venous blood flowing from the sella turcica and, for comparison, from the posterior paw were collected in anaesthetized rats. Blood from the sella turcica was collected with a fine cannula inserted into the internal maxillary vein. The concentration of vasopressin in blood plasma was determined by radioimmunoassay and cardiodepressant activity--using a biological test on a spontaneously discharged pacemaker tissue of the right auricle of the right heart atrium. Stimulation of the central ends of the cut vagus nerves or intra-arterial infusion of angiotensin II simultaneously caused an increase in the cardiodepressant activity and vasopressin concentration in the sella turcica venous blood. The cardiodepressant activity and vasopressin concentration was also enhanced to some degree in blood outflowing from the posterior paw. Present results indicate that both vasopressin and the cardiodepressant factor are released into blood from the posterior pituitary lobe.

Increase in oxytocin and vasopressin concentration in the blood outflowing from sella turcica region after superior cervical ganglion stimulation in rat.

The aim of the study was to investigate whether the stimulation of the superior cervical ganglion influences the oxytocin and vasopressin release into the blood in condition of the of the sella turcica integrity. The experiments were performed on male rats under urethane-chloralose anaesthesia. Four 0.7 ml samples of the blood from the sella turcica region flowing through a tube inserted in the maxillary interna vein were collected in the 30, 35, 60 and 90 min of the experiments. The animals were divided into three groups: 1) control, 2) after the exposition of superior cervical ganglion. 3) after the collection of the 1-st sample of the blood the superior cervical ganglion was electrically stimulated for 30 min with trains of pulses. Vasopressin (AVP) and oxytocin (OXY) were determined in the blood plasma by radioimmunoassay. Stimulation of the superior cervical ganglion evoked an significant increase of AVP and OXY release into the blood. The increase of AVP release occurred after 30 min longer latency than the increase of OXY release.

Vasopressin content in the cerebrospinal fluid and fluid perfusing cerebral ventricles in rats after the afferent vagus nerve fibres stimulation.

Experiments were carried out on male rats in urethane anaesthesia. Cerebroventricular system was perfused with McIlwain-Rodniht's solution from lateral ventricles to cerebellomedullary cistern. Both vagus nerves were cut and the central ends of the nerves were electrically stimulated during the collection of the third 30-min portion of perfusing fluid. Vasopressin (AVP) was determined by radioimmunoassay in samples of the cerebrospinal fluid (CSF) (the first portion) and in five successive samples of the perfusing fluid. AVP concentration in the CSF was several times greater than in the fluid perfusing cerebral ventricles. Alternate electrical stimulation of both vagus nerves did not change considerably the release of AVP into the fluid perfusing the cerebral ventricles in rat, although a certain upward tendency could be observed. It seems that only AVP raised in circulating blood and not in CSF, after vagus nerves stimulation may act on the central nervous structures.

Vasopressin release into the 3rd cerebral ventricle and into the blood after sciatic and trigeminal nerve stimulation.

The aim of the experiments was an assessment of vasopressinergic (AVP-ergic) neurons response to somatic afferent impulsation in the spinal and in the cranial nerves. Experiments were carried out on male rats in urethane anaesthesia with the perfusion cannula introduced into the 3rd cerebral ventricle and with the catheter in the internal maxillary vein, in the vicinity of the cavernous sinus. Four samples of perfusing fluid, each of about 1.5 ml/30 min., and four samples of blood, 0.8 ml each, were collected. Vasopressin (AVP) was determined by radioimmunoassay in samples of perfusing fluid from the 3rd cerebral ventricle and in blood plasma. The central end of the left sciatic, supraorbital or infraorbital nerves was electrically stimulated with an intensity which increased the respiratory rate by 10-15% during collection of the IIIrd samples. The AVP concentration in the fluid perfusing the 3rd ventricle did not change during stimulation of the nerves. On the contrary, sciatic and supraorbital nerve stimulation caused a significant increase of AVP concentration in the blood. It can be concluded that sciatic and supraorbital nerve afferentation induced AVP release from the posterior pituitary lobe and did not affect AVP-ergic neurons projecting to the ventricular ependyma and responsible for AVP release into the cerebrospinal fluid.

Vasopressin release from posterior pituitary lobe incubated in situ after preganglionic stimulation of the rat superior cervical ganglion.

In urethane-chloralose anaesthesia the pituitary gland was exposed by transpharyngeal approach in rats. The anterior lobe was removed and the posterior lobe was incubated in situ, that is in conditions of anatomical integrity of the hypothalamus with the posterior pituitary lobe. The 15-min samples of the medium incubating the posterior pituitary lobe in situ were collected. Vasopressin (AVP) content in the incubation medium was determined by radioimmunoassay. The stimulation of preganglionic fibers of the superior cervical ganglion (SCG) with alternate short (5 s) bursts of electric pulses with short (5 s) breaks did not change AVP release. However, stimulation of preganglionic fibres with alternate long (30 s) bursts of electric pulses with long (30 s) breaks evoked an increase in AVP release after some latency. Probably, at the hypothalamic or posterior pituitary level temporal summation should occur affecting vasopressinergic neurons or their endings and evoking AVP release.

Modulatory role of substance P on gonadotropin and prolactin secretion in the rabbit.

Substance P (SP) is present in large quantities in the brainstem and hypophysiotropic areas of the brain, but its roles in gonadotropin and prolactin secretion are controversial. The aim of this study was to measure luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL) release from the pituitary after either intracerebroventricular (ICV) injection or infusion of SP or its C- and N-terminal fragments in intact (INT) and ovariectomized (OVX) conscious rabbits. A single injection of SP into the 3rd cerebral ventricle (3CVT) in INT and OVX rabbits augmented plasma LH concentrations, especially when SP was applied during the initial phase of an LH peak. Injection of SP during the declining phase of LH release was not effective. Injection of SP into the 3CVT was followed by increased plasma PRL concentrations in OVX but not in INT rabbits. Both SP 1-11 and SP 1-7 failed to alter LH, FSH, and PRL secretion when the peptides were slowly infused into the 3CVT, although ICV infusion of SP 6-11 did cause a delayed increase in LH release. The results support a stimulatory role of SP on LH and prolactin release. The results further indicate that although the stimulatory effect of SP on LH is ovarian steroid-independent, in the absence of ovarian steroids, SP is stimulatory only during the rising phase of an LH pulse. A dual role of SP-ergic transmission in modulating LH secretion is discussed.

Occurrence of ovulation after intracerebroventricular infusion of substance P in 6-OHDA pretreated female rats.

Substance P (SP) infused into the third cerebral ventricle blocks spontaneous ovulation in female rats, probably through catecholaminergic neurons. The studies presented in this paper were undertaken to investigate whether SP exerts its suppressing effect on ovulation in 6-hydroxydopamine (6-OHDA) pretreated female rats. After 8-12 days following 6-OHDA pretreatment female rats were infused, on the day of proestrus, with vehicle and all animals were found to ovulate or with a solution containing 5 nmol of SP and 89% of female rats were then found to ovulate. In the group pretreated with vehicle and subsequently infused with SP, ovulation was found to occur only in 25% of animals. The obtained results indicate that spontaneous ovulation in 6-OHDA-pretreated female rats cannot be blocked by i.c.v. administration of SP, and it may be concluded that SP exerts its suppressing effect through the monoaminergic neurons.

Increase of cardiodepressant activity in medium incubating the posterior pituitary lobe in situ during vagal nerve stimulation in rat.

Previous studies have indicated that there is a cardiodepressant factor in the medium incubating the posterior pituitary lobe in situ. The cardiodepressant activity of the medium incubating the posterior pituitary lobe before and during stimulation of the vagus nerves was tested on isolated auricles of the right heart atrium of a two-day-old rat. It was found that the medium incubating the posterior pituitary lobe collected before stimulation decreased the contraction rate of the auricle by 34%, while that collected during the intermittent stimulation of the central ends of the cut vagus nerves caused a decrease of the auricle contractions frequency by 52%. The addition of cholinergic, serotoninergic, histaminergic receptor blockers or prostaglandin synthetase into Ringer-Lock's solution bathing the auricle has no effect on the changes of the contraction rate caused by the incubation medium.