Inhibition by pentobarbitone and urethane of the in vitro response of the adenohypophysis to luteinising hormone-releasing hormone in male rats.

1 The effect of urethane (ethyl carbamate) and sodium pentobarbitone on the luteinising hormone-releasing hormone (LH-RH)-stimulated secretion of luteinising hormone (LH) was investigated with hemipituitaries obtained from male rats and incubated in vitro. 2 Urethane and pentobarbitone were added to the incubation medium to provide final concentrations of 2.2 mg or 4 micrograms/ml and 100 micrograms or 0.1 microgram/ml respectively. The high doses of these anaesthetics blocked the LH-RH stimulated secretion of LH. The low doses significantly reduced the amounts of LH released in response to LH-RH but did not block the response completely. 3 Both concentrations of urethane reduced the basal release of LH. 4 The inhibitory action of the anaesthetics was reversible. 5 The results indicate that the two anaesthetics most commonly used in neuroendocrine experiments have a significant inhibitory action on the release of LH from the adenohypophysis.

Relationship between duration of urethane or pentobarbitone anaesthesia in male and female rats and adenohypophyseal response to luteinising hormone-releasing hormone.

1 Luteinizing hormone releasing hormone (50 ng/100 g body weight) was injected into male and female rats which had been anaesthetized with either urethane (1.2 g/kg) or pentobarbitone (45 g/kg) for 15, 100 or 240 min and serial blood samples were taken for estimation of plasma luteinizing hormone concentrations. 2 The rats anaesthetized with urethane tended to show a greater response to the releasing hormone than those given pentobarbitone. 3 The magnitude of the responses observed in male rats and in female rats anaesthetized with pentobarbitone did not change as the period of anaesthesia prior to injection of releasing hormone was lengthened from 15 min to 4 h. By contrast, in the groups of female rats anaesthetized with urethane the magnitude of the response to luteinizing hormone releasing hormone was related to the length of pretreatment anaesthesia. Thus both dioestrous and pro-oestrous rats given releasing hormone only 15 min after the onset of urethane anaesthesia had significantly (P less than 0.001 and less than 0.05 respectively) higher concentrations of luteinizing hormone (LH) in the plasma than rats treated with the releasing hormone after 240 min of anaesthesia. 4 These effects were not due to a differential action of the anaesthetics on the mechanism for clearing LH from the plasma.

Electrochemical deposition of iron onto rat oxytocinergic neurones does not result in milk-ejection.

Exposure of the preoptic-tuberoinfundibular system to iron ions causes ovulation in the pro-oestrous rat. This electrochemical treatment has been used frequently to study the way that the hypothalamus triggers secretion of gonadotrophic hormone and most investigators have assumed that the effect is mediated by the direct excitation of those neurones exposed to the cation. The present paper reports experiments designed to confirm that iron directly excites hypothalamic neurones to the firing frequencies essential for ovulation. Because of the difficulty of measuring input-output relationships in the diffuse preoptic-tuberoinfundibular system the experiments were performed on the anatomically distinct oxytocinergic neurones of the paraventricular-hypophysial tract in lactating rats. These neurones were simulated electrically (from 10 to 50 Hz for between 1 and 300 s) and subjected to the electrochemical treatment by depositing iron ions from the tip of the stimulating electrode (up to 250 muA anodal current for between 60 and 180 s). Changes in intramammary pressure were used to indicate oxytocin release and mammary glands were calibrated for sensitivity by intravenous injection of oxytocin. Electrical stimulation in excess of about 15 Hz invariably caused release of sufficient oxytocin to cause a rise in intramammary pressure. In contrast, no changes in pressure were observed during or after the electrochemical deposition of iron. The results suggest that the response of hypothalamic neurones to electrochemical treatment is not the same as their response to electrical stimulation.

Anaesthesia with alphaxalone plus alphadolone acetate blocks the oestrogen-stimulated LH surge and impairs pulsatile LH secretion in ovariectomized female rats.

Two experiments were undertaken to assess further the action of the steroid anaesthetic alphaxalone upon LH secretion in chronically ovariectomized female rats. For the first experiment, 31 rats were given two injections of oestradiol benzoate (20 micrograms/100 g body weight), each 72 h apart, to stimulate an LH surge 6 h after the second injection. However, one group of seven rats was anaesthetized with alphaxalone throughout the 6-h period and a second group of eight was similarly anaesthetized only for the last 2 h of the 6-h period. The steroid-stimulated LH surge was blocked in both groups of rats anaesthetized with alphaxalone. The second experiment involved a comparison of pulsatile LH secretion in animals which were either unanaesthetized (n = 8) or anaesthetized with alpha xalone (n = 9). In six out of nine rats the anaesthetic did not affect the maximum or minimum plasma LH concentrations but significantly slowed the frequency at which LH pulses were measured. In the remaining three anaesthetized rats the drug blocked pulsatile LH secretion. The experiments confirm that some secretion of LH continues during alphaxalone anaesthesia but indicate also that the drug has a more deleterious action upon the oestrogen-stimulated LH surge than believed hitherto.

Neonatal testosterone potentiates stimulated prolactin secretion in adult oestrogen-primed rats.

Blood samples were collected from oestrogen-primed gonadectomized adult rats before and after electrical stimulation of the preoptic part of the hypothalamus. Six groups of rats were used for the experiments. These were (a) males castrated on the first day of life, (b) males castrated after puberty, (c) females ovariectomized after puberty and (d), (e) and (f) females given testosterone propionate at birth (1.25, 0.125 and 0.0125 mg/rat respectively). Neonatal exposure of the female rats to testosterone caused a dose-dependent increase in the amounts of prolactin released to levels significantly (P less than 0.01) higher than those observed in male animals and in untreated females. The results indicate that although neonatal testosterone inhibits oestrogen-stimulated prolactin secretion in adult rats, the neuroendocrine apparatus controlling secretion of the hormone is capable of being activated to greater effect after exposure to androgens at the time of birth.

Effects of ferrous ions on preoptic area neurones and luteinizing hormone secretion in the rat.

Ferrous ions (Fe2+) were applied by microiontophoresis of 29 neurones recordid from the forebrain of ten pro-oestrous rats. The spontaneous activity of 24 cells was reduced by Fe2+ and five units did not respond. No excitations were observed. Reproducible responses could not be obtained with ferric ions (Fe3+). Microinjection of 1 mul 200 mM-Fe2+ into the preoptic area of eight pro-oestrous rats produced a sharp increase in plasma LH levels. Similar treatment with saline was without effect. Rats anaesthetized with sodium pentobarbitone during the early afternoon of pro-oestrus ovulated overnight when injected with both Fe2+ and Fe3+. The experiments do not confirm the widely held belief that raised plasma LH levels following deposition of iron in the rostral hypothalamus result from increased firing of nerve cells.

PIP: The techniques of microiontophoresis and microinjection of iron into female rats were used to test the hyposthesis that iron causes the action potential activity of neurones in the rostral hypothalamus to increase. Ferrous ions were applied by microiontophoresis to 29 neurones recorded from the forebrain of 10 proestrous rats. The spontaneous activity of 24 cells was reduced by ferrous ions. 5 units w ere unresponsive. Excitations were absent. Ferric ions were unable to reproduce this reponse. 18 proestrous rats received a microinjection of 1 mcl 200 mM ferrous ions into the preoptic area. A sharp increase in plasma levels of luteinizing hormone was produced. Controls were injected with saline without effect. Rats anesthetized with sodium pentobarbitone during the early afternoon of proestrous ovulated overnight when injected with both ferrous and ferric ions. The hypothesis that raised plasma levels of luteinizing hormone levels following deposition of iron in the rostral hypothalamus result from increased firing of nerve cells is unconfirmed.

Electrical activity of antidromically identified tuberoinfundibular neurones during stimulated release of luteinizing hormone and prolactin in pro-oestrous rats.

Extracellular activity was recorded from tuberoinfundibular neurones in 28 urethane-anaesthetized pro-oestrous rats. In each rat the electrical activity of one antidromically identified neurone was analysed before and during electrical stimulation in the preoptic/anterior hypothalamic areas (PO/AH). This stimulation (50 Hz, 30 s on and 30 s off for 15 min) resulted in significant increases in plasma concentrations of prolactin (from 46 +/- 11 (S.E.M.) ng/ml before stimulation to 86 +/- 17 ng/ml 20 min later; P less than 0.02) and LH (27 +/- 6 to 48 +/- 11 ng/ml; P less than 0.01). A substantial proportion (11 out of 28) of the tuberoinfundibular neurones was inhibited by PO/AH stimulation. If such cells are directly involved with the secretion of anterior pituitary hormones it is probable that they synthesize and secrete inhibitory factors. We have suggested that cells inhibited by stimulation of the PO/AH are possibly the neurones which secrete prolactin inhibitory factor. The excited cells (eight out of 28) gave complex and variable responses to hypothalamic stimulation and were unable to follow applied stimuli at a ratio of more than one extra action potential for every ten stimulus pulses. We propose that this may explain why it is necessary to stimulate the PO/AH at ten times the frequency required for ovulation to occur following stimulation in the median eminence in anaesthetized pro-oestrous rats.

Effects of neonatal exposure to monosodium glutamate on the electrical activity of neurones in the mediobasal hypothalamus, and on the plasma concentrations of thyroid-stimulating hormone and prolactin, following stimulation of the rostral hypothalamus in adult female rats.

Action potentials were recorded from 174 neurones in the mediobasal hypothalamus of ovariectomized adult female rats exposed neonatally to monosodium glutamate (MSG) and from 145 neurones in control rats. All of the animals, which were anaesthetized with urethane, had been ovariectomized for at least 3 weeks and received two injections of oestradiol benzoate (20 microgram/100 g body weight, i.m.) 72 h and immediately before the recording experiments. The response of each neurone to electrical stimulation of the median eminence and rostral hypothalamus (preoptic and anterior hypothalamus areas; PO/AH) was analysed. The most striking feature of the results obtained was the significant (P less than 0.001) loss of inhibitory responses in those neurones remaining in the adult rats after neonatal treatment with MSG. The loss of inhibitory responses applied to both stimulation sites. In each rat the response of one neurone, which was antidromically identified as projecting to the median eminence, was recorded before and during stimulation of the PO/AH at 50 Hz for 30s in every min for 15 min. Before and after this stimulation blood was collected from a jugular vein for estimation by radioimmunoassay of concentrations of prolactin and TSH. In the MSG-treated rats significantly (P less than 0.05) fewer neurones were inhibited by the 50 Hz stimulation than in control rats. In control rats the plasma concentrations of prolactin nearly quadrupled as an immediate consequence of this treatment, whereas in MSG-treated rats plasma concentrations barely doubled. However, in the MSG-treated rats plasma concentrations of prolactin continued to rise after stimulation ceased, possibly as a consequence of enhanced secretion of thyrotrophin releasing hormone.

Fasting impairs LH secretion in female rats by activating an inhibitory opioid pathway.

Two experiments were undertaken to investigate the way that fasting impairs LH secretion and to assess whether endogenous opioid mechanisms might be responsible for the impairment. In the first experiment, pulsatile LH secretion was measured in a total of 51 chronically ovariectomized female rats. Initially 29 rats were subjected to food withdrawal for 24, 48, 72 or 120 h before the experiment. When compared with data collected from eight unfasted control rats, the 120-h fast was found to reduce significantly the mean peak and trough values of the LH pulses measured. However, in a subsequent study, the inhibition of pulsatile LH secretion by a 120-h fast was prevented in a group of eight rats given the opioid antagonist naloxone hydrochloride before the start of the blood-sampling period. Naloxone was without effect on pulsatile LH secretion in eight unfasted control rats. In the second experiment, plasma LH concentrations were measured before and after unilateral electrical stimulation of the ventral noradrenergic tract (VNAT) in ovariectomized female rats pretreated with oestradiol benzoate. In 17 rats VNAT stimulation caused a significant rise in plasma LH, but after a 72-h fast this rise was significantly less than in unfasted control rats. However, pretreatment of fasted rats with naloxone (n = 9) significantly enhanced the VNAT-stimulated release of LH to the control values. Naloxone did not potentiate VNAT-stimulated LH release in unfasted animals (n = 6) or LH release in control unstimulated rats (n = 12). The experiments indicate that both pulsatile LH secretion, and LH release caused by VNAT stimulation, are impaired by an acute fast.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of luteinizing hormone in ovariectomized adult rats treated neonatally with monosodium glutamate.

We have studied the possible effects of monosodium glutamate (MSG) on LH secretion in ovariectomized rats. In experiment 1 MSG-treated and control rats were given oestradiol benzoate at noon and 72 h later half the rats in each group were given a second injection of oestradiol benzoate or progesterone. Blood samples were taken immediately before and 6 h after these i.m. injections. At 78 h there were no significant differences in plasma LH concentration measured in the two groups of rats given progesterone or in the two groups given a second injection of oestradiol benzoate although for both MSG-treated and control rats progesterone produced a significantly (P less than 0.01) greater LH surge than did oestradiol benzoate. In experiment 2 100 ul blood samples were collected at 5-min intervals for up to 3 h from MSG-treated and control rats. For rats showing more than one pulsatile discharge of LH, peak and trough values for plasma LH concentrations were no significantly influenced by MSG treatment. However the mean pulse height was significantly (P less than 0.001) greater in the MSG-treated group than in control rats. Pulsatile release stopped more quickly in the MSG-treated rats and their mean plasma LH concentration after 120 min of blood sampling was significantly (P less than 0.05) lower than that obtained in the control animals. Thus, although some aspects of LH secretion seem to be significantly different in MSG-treated rats, these effects may result from the greater sensitivity of the MSG-treated animals to experimental manipulation.

Central opioids: a possible role in parturition?

Pregnant rats were implanted with subcutaneous minipumps to deliver either naloxone or saline. The time-course of subsequent parturition was different between the two groups: the interval between successive births was significantly shorter for the naloxone-treated rats. This supports the hypothesis that the opioid innervation of the neurohypophysis, which is known to influence oxytocin release profoundly, has a physiological role in parturition. To test the further hypothesis that this role is particularly important in a stressful environment, pregnant rats, again implanted with minipumps, were regularly transferred, at 15-min intervals beginning with the birth of the first pup, between their normal cage and the unfamiliar environment of a glass observation chamber. No difference was noted between the time-courses of parturition for the naloxone- and saline-treated groups, although the time-courses were markedly altered from those observed in rats not subjected to an unfamiliar environment. We conclude that opioid modulation of oxytocin release may play a role in 'spacing' the delivery of successive births during normal parturition.

Stress-induced disruption of parturition in the rat may be mediated by endogenous opioids.

Plasma samples were obtained before and during parturition from conscious rats implanted chronically with a jugular cannula. Rats were either allowed to remain in their nesting cage throughout parturition, or were moved immediately following the birth of the second or third pup into an empty glass chamber. The time-course of parturition was prolonged for mother rats which were moved in mid-parturition to this unfamiliar environment. However, in rats given an i.v. injection of the opioid antagonist naloxone at the time of transfer, parturition followed a normal time-course, and plasma oxytocin levels were significantly higher than in animals injected with saline. Thus our hypothesis is that stress activates opioid pathways which delay parturition by inhibiting oxytocin release. Opioid-mediated mechanisms may similarly be responsible for some problems in human parturition.

Acute action of oestrogen on medial preoptic gamma-aminobutyric Acid neurons: correlation with oestrogen negative feedback on luteinizing hormone secretion.

Abstract The acute effects of oestrogen on the medial preoptic area (MPOA) gamma-aminobutyric acid (GABA) system were examined by delivering an intravenous bolus of 17beta-oestradiol (5 mug/100 g body wt) to conscious ovariectomized rats implanted with microdialysis probes. Fifteen-min blood samples were taken to determine the time-course of negative feedback effects of oestrogen on luteinizing hormone (LH) secretion. Two h after administration of 17beta-oestradiol, GABA release from the MPOA was significantly elevated compared with vehicle-treated controls (P<0.05). The rise in GABA levels continued until the end of the experiment, 4 h after 17beta- oestradiol, at which time it was over 50% higher than controls (P<0.01). The pulsatile pattern of LH secretion was significantly depressed 2 and 3 h after administration of 17beta-oestradiol compared with controls (P<0.05). To determine the effects of the 17beta-oestradiol treatment on pituitary responsiveness to LH-releasing hormone (LHRH), a further group of rats were given exogenous LHRH (50ng/100g body wt, intravenously) before and 3 h after vehicle or 17beta-oestradiol treatment and blood samples taken to determine the effect on LH secretion. The maximal LH response to LHRH in 17beta-oestradiol-treated rats was approximately 50% that of control-treated values. This study demonstrates the acute and potent action of 17beta-oestradiol on GABA release in the MPOA and lends support to a genomic site of action for oestrogen in modulating neural elements regulating GABA release from the MPOA. These results, showing a parallel decrease in LH secretion with increased GABA levels in the MPOA, suggest a role for GABA elements within the MPOA as a site of oestrogen negative feedback on LH secretion.

A method for recording single unit activity from the brains of foetal pigs in utero.

A technique is described for recording single unit activity from foetal miniature pigs in utero. The method involved anaesthetizing the sows with halothane and exteriorizing a sufficient length of the uterus to contain a single foetus. Both foetus and uterus were placed in a stereotaxic frame which was held independent of the mothers' support. Preliminary analysis of recordings obtained from spontaneously firing single nerve cells in the post-central cerebral cortex indicate that the patterns of spike activity in prenatal pigs are very similar to those observed in prepubertal animals.

Neonatal testosterone modifies LH secretion in the adult female rat by altering the opioid-noradrenergic interaction in the medial preoptic area.

Noradrenergic-opioid interaction in the medial preoptic area (MPOA) was examined in ovariectomised adult rats which were oestrogen-treated and had been injected neonatally with either testosterone propionate (TP) or vehicle (oil). The first experiment involved electrical stimulation of the ventral noradrenergic tract (VNAT) in anaesthetised rats. Blood samples were collected before and after the stimulation to determine plasma levels of luteinising hormone (LH). Approximately half of the animals received naloxone i.v. 15 min before the onset of stimulation. In all groups, stimulation of VNAT elicited a significant increase in plasma LH concentration. However, pretreatment with naloxone in androgenised rats, but not in oil-treated animals, almost doubled the LH increment due to stimulation. Naloxone had no effect on plasma LH concentrations in unstimulated control rats. In the second experiment hypothalamic slices containing the MPOA were preincubated with [3H]noradrenaline [( 3H]NA) and then subjected to electrical field stimulation under the conditions of (a) no drug added and (b, c) morphine superfusion without and with naloxone. The opioid agonist morphine significantly reduced the net release of [3H]NA in normal and TP-treated female rats. Addition of equimolar naloxone reversed this effect in normal females, whereas in the androgenised group it not only reversed this effect but elicited a significant increase in [3H]NA release. From these data we conclude that (1) neonatal testosterone treatment alters noradrenergic-opioid interaction regulating LH secretion in adult females and (2) the site of this change may be the presynaptic opioid input to the noradrenergic terminals in the MPOA.

Analysis of brainstem A1 and A2 noradrenergic inputs to the preoptic area using microdialysis in the rat.

Noradrenergic inputs to the preoptic area (POA) are involved in regulating a variety of homeostatic functions. However, the accurate measurement of endogenous noradrenaline (NA) release in the POA has been difficult to achieve and consequently little has been done to characterise the different noradrenergic pathways. By combining the technique of intracranial microdialysis with tissue pre-loading of [3H]NA we have developed a sensitive index of NA release in the POA [8]. Using this method we have now examined and compared the effects of electrical stimulation of the brainstem A1 and A2 cell groups on NA release in the POA. Anaesthetised proestrus rats were implanted with microdialysis probes either unilaterally or bilaterally in the POA and stimulating electrodes positioned in either the A1 or A2 regions. Electrical stimulation (10 Hz, 10s on/off for 20 min) of the A1 region resulted in repeatable, calcium-dependent increases in radioactivity outflow from the ipsilateral POA (P < 0.01). A1-evoked release was twice as large as that observed after equivalent 10 Hz electrical stimulation of the A2 region (P < 0.05). In experiments using bilateral POA microdialysis and A1 stimulation, a significant increase in release from the contralateral POA, amounting to approximately 80% of that observed in the ipsilateral POA, was observed. Experiments involving the blockade of A1-stimulated release in the ipsilateral POA by perfusion with a calcium-free medium demonstrated that increases in radioactivity measured in the contralateral POA were not originating from the ipsilateral POA.(ABSTRACT TRUNCATED AT 250 WORDS)

Oestrogen and noradrenaline modulate endogenous GABA release from slices of the rat medial preoptic area.

Endogenous gamma-aminobutyric acid (GABA) release from the rat medial preoptic area (MPOA) was measured in an in vitro slice technique with sensitive HPLC analysis. Oestrogen is demonstrated to increase GABA activity in the ovariectomised, oestrogen-primed (OVX-EB) rat prior to the luteinising hormone (LH) surge compared with ovariectomised (OVX) animals. Noradrenaline (NA) at a concentration of 10 microM was found to significantly enhance GABA release in response to 30 mM potassium stimulation in both OVX and OVX-EB animals. A significantly greater response to NA was observed in the OVX-EB animal. No effect of NA on basal GABA release was detected. The effects of NA were blocked by the alpha-adrenergic receptor blocker phenoxybenzamine (PB). These data suggest that GABA activity is modulated both by oestrogens and noradrenergic-mediated input in the MPOA.

Effect of neonatal testosterone upon opioid receptors and the content of beta-endorphin, neuropeptide Y and neurotensin in the medial preoptic and the mediobasal hypothalamic areas of the rat brain.

The content of beta-endorphin, neuropeptide Y and neurotensin-like immunoreactivity (beta-End, NPY and NT), and the total number of opioid binding sites, were measured in the medial preoptic area (MPOA) and mediobasal hypothalamus (MBH) of ovariectomized adult rats which were oestrogen-primed. The rats had been injected neonatally with either testosterone propionate (TP) or vehicle (oil). NPY content was found to be higher in the MPOA of animals which received TP, whereas no significant difference was observed in the MBH NPY content. However, the NT concentration in the MBH of TP-treated rats was almost twice the amount detected in oil-treated rats and with this peptide no significant changes were detected in the MPOA. Finally, beta-End and the total number of opioid binding sites were reduced in both the MPOA and MBH of the rats which were exposed to TP neonatally. Since exposure to testosterone neonatally masculinises the rat hypothalamus, to the extent that female rats cannot generate oestrogen-stimulated prolactin and luteinizing hormone surges, we suggest that the neurochemical changes reported in this paper reflect some aspects of this sexual differentiation of the rat hypothalamus.

Relative effectiveness of naloxone and MR2266 in potentiating oestrogen-stimulated luteinizing hormone surges in ovariectomized female rats.

The oestrogen-stimulated surge of luteinizing hormone (LH) can be inhibited by opiates and potentiated when endogenous opiate pathways are blocked by naloxone. Experiments were undertaken to allow an assessment to be made of the opiate receptor category predominantly responsible for inhibiting LH secretion. Thus the effects of MR2266 (primarily a mu and kappa antagonist) and naloxone (primarily a mu and delta antagonist) on LH surges were compared. At a dose of 4.5 mg/kg, administered three times in the 6 h preceding the LH surge, naloxone significantly potentiated LH release whereas this dose of MR2266 was without effect. However at the lower dose of 0.5 mg/kg, administered three times also, the size of the LH surge was significantly increased by MR2266 but not by naloxone.

Electrical stimulation of the ventral noradrenergic tract (VNAT) releases hypothalamic noradrenaline and stimulates prolactin secretion in ovariectomized female rats treated with oestrogen.

Electrical stimulation of the ventral noradrenergic tract (VNAT) caused a significant fall in the hypothalamic content of noradrenaline in ovariectomized female rats which had been treated with oestrogen. No similar effect was observed in the cerebral cortex, which implies that the stimulation did not affect the dorsal noradrenergic tract. In parallel experiments with groups of similarly treated rats, VNAT stimulation was found to increase release of prolactin from the pituitary gland, and this increase was significantly enhanced in animals which had been stressed by fasting for 3 days.