Further evidence for the inhibitory action of baclofen on a prolactin-releasing factor

The mechanism of action of a specific gamma-aminobutyric acid B receptor agonist, beta-p-chlorophenyl-gamma-aminobutyric acid or baclofen, in its inhibitory action on prolactin release, was studied. Dose-response studies of the effect of baclofen on prolactin (PRL) secretion were performed in stressed male rats. Furthermore, the action of the drug was evaluated in (i) rats treated with haloperidol or alpha-methyl-p-tyrosine, (ii) stressed or suckled rats pretreated with sulpiride, and (iii) animals treated with serotonin, alone, or with alpha-methyl-p-tyrosine. Baclofen showed a clear dose-dependent inhibition of prolactin secretion in males under stress. The drug was unable to inhibit the prolactin release induced by haloperidol or alpha-methyl-p-tyrosine, although it reduced the PRL secretion induced by serotonin. It also inhibited PRL release in sulpiride-pretreated stressed or suckled rats. These results suggest that the dose-dependent effect of baclofen on PRL secretion is the consequence of an inhibition exerted on the prolactin-releasing factor component of the neuroendocrine responses evoked by stress or suckling, possibly acting at the serotonergic system.     

Development of selective tolerance to the serotonin behavioral syndrome and suppression of locomotor activity after repeated administration of either 5-MeODMT or mCPP

Repeated administration to rats of the 5-HT -selective agonist 5-methoxy-N, N-dimethyltryptamine (5-MeODMT)^1A produced tolerance to the ability of a test dose of 5-MeODMT to produce the serotonin behavioral syndrome, but not to the ability of a test dose of the 5-HT_1B -selective agonist m-chlorophenylpiperazine (mCPP) to decrease locomotor activity. Conversely, repeated administration of mCPP produced tolerance to the ability of a test dose of mCPP to decrease locomotor activity, but not to the ability of a test dose of 5-MeODMT to elicit the serotonin behavioral syndrome. The lack of cross-tolerance between these two selective agonists is consistent with the idea that the serotonin behavioral syndrome and suppression of locomotor activity are mediated by different subtypes of the 5-HT₁ receptor.     

Effects of serotonergic agents on plasma prolactin levels in pyridoxine-deficient adult male rats

Plasma concentration of prolactin was significantly reduced in pyridoxine-deficient as compared to control (pyridoxine-supplemented) adult male rats. Administration of pyridoxine to deficient rats resulted in a significant increase in plasma prolactin. The reduction in plasma prolactin in pyridoxine-deficient rats corresponded with the significantly reduced hypothalamic contents of pyridoxal phosphate and serotonin in pyridoxine-deficient rats. Plasma prolactin concentrations were also measured in response to serotonergic agents in both groups of rats. The administration of the 5HT_1A agonist (8-hydroxy 2-n-dipropylamino tetralin) resulted in a significant increase in plasma prolactin and that of the specific 5HT_1A antagonist spiroxatrine had the opposite effect. The results suggest that the hypothalamic serotonergic regulation of prolactin release is impaired in pyridoxine deficiency.     

THE PREVENTION BY INHIBITORS OF BRAIN PROTEIN SYNTHESIS OF THE HYPERACTIVITY AND HYPERPYREXIA PRODUCED IN RATS BY MONOAMINE OXIDASE INHIBITION AND THE ADMINISTRATION OF L-TRYPTOPHAN OR 5-METHOXY-N,N-DIMETHYLTRYPTAMINE

Abstract— In rats treated with a monoamine oxidase inhibitor, (tranylcypromine), L- tryptophan produces a stereotyped syndrome of hyperactivity and hyperpyrexia due to an increased rate of brain serotonin (5-hydroxytryptamine) synthesis. Pretreatment of rats with intraperitoneal injections of cycloheximide, acetoxycycloheximide, emetine and dehydroemetine and of mice with puromycin inhibited this syndrome. Cycloheximide also inhibited the hyperactivity caused by tranylcypromine and DL-15-hydroxtryptophan and did not affect the increased rate of brain serotonin ‘synthes’ is produced by tryptophan thus excluding a primary effect on tryptophan-5-hydroxylase. Inhibition of hyperactivity did not occur until brain protein synthesis was inhibited by greater than 65 per cent as measured by the incorporation of L-[U-¹⁴C]tyrosine into brain protein in vivo. Emetine, which has been shown to inhibit brain protein synthesis inhibited hyperactivity whereas isoemetine which did not inhibit brain protein synthesis, did not inhibit hyperactivity. Under conditions where cycloheximide inhibited hyperactivity produced by tranylcypromine and L-tryptophan, a large dose of 5-methoxy-N,N-dimethyltryptamine(5-MeODMT) still produced hyperactivity showing that the rats were still capable of the same pattern of hyperactivity. However, cycloheximide did inhibit hyperactivity due to 5-MeODMT, the degree of this inhibition being dependent upon a balance between the doses of cycloheximide and 5-MeODMT. 5-MeODMT probably acts directly within the brain to cause behavioural excitation and it seems likely that the inhibitors of brain protein synthesis interfere with the mechanism of action of brain 5HT and administered 5-MeODMT rather than upon any aspect of synthesis, storage or release of brain 5HT. It is suggested that the behaviourally excitant effects of brain 5HT and 5-MeODMT are mediated in some way by a brain protein with a short biological half-life. Such a protein may act either as a factor specifically mediating the central effects of brain 5HT or as a factor regulating the neuronal response to excitation by 5HT and 5-MeODMT.     

Effect of CNS-active drugs on TRH-induced prolactin release

The effects of several central acting drugs upon thyrotropin-releasing hormone (TRH)-induced increases in prolactin (PRL) release were compared in estrogen-primed male rats. Administration of the serotonin antagonist, p-chlorophenylalanine, or the opiate antagonist, naltrexone, did not alter TRH-induced release of PRL. Pre-treatment with either the dopamine agonist, piribedil, or the cholinergic agonist, pilocarpine, resulted in significantly reduced TRH-induced PRL release. Pilocarpine did not inhibit the TRH-induced increase in PRL release when rats were first pre-treated with the dopamine receptor blocker, haloperidol. These results indicate that the dopaminergic and cholinergic systems can modify TRH-induced release of PRL in vivo.     

Evidence that serotonin neurons stimulate secretion of prolaction releasing factor.

The purpose of the present study was to determine if serotonin was stimulatory to prolactin release by inhibition of the dopaminergic system or by stimulating release of a prolactin releasing factor (PRF). We measured the amount of prolactin secreted after administration of 30 mg/kg of 5-hydroxytryptophan (5-HTP) to male rats pretreated with fluoxetine (10 mg/kg) and compared it with the amount of prolactin released in male rats treated with αmethyl-p-tyrosine methyl ester (αMT) or various dopamine receptor blocking agents. In every experiment the serotonergic stimulus provided by 5-HTP in fluoxetine-pretreated rats released considerably more prolactin than did treatment with αMT or dopaminergic blockers. We conclude that serotonin releases prolactin not by inhibiting dopaminergic neurons but rather by stimulating the release of PRF.     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.     

The effects of opiate agonists on growth hormone and prolactin release in rats

Various opioid receptor agonists, including Met5-enkephalin amide, Leu5-enkephalin amide, [D-Ala]2-Met5-enkephalin amide, [D-Ala]2-Leu5-enkephalin amide, morphine sulfate, d-methadone hydrochloride, and l-methadone hydrochloride were administered to adult male rats by subcutaneous injection. All opioid receptor agonists except Leu5-enkephalin amide significantly stimulated growth hormone and prolactin release. Naloxone and naltrexone blocked the hormone stimulatory effects of the opioids and both naloxone and naltrexone, when administered alone, significantly reduced serum growth hormone and prolactin concentrations. The dopaminergic agonist apomorphine, but not the alpha-adrenergic agonist clonidine, blocked opiate stimulation of prolactin. Morphine sulfate caused growth hormone release in rats pretreated with alpha-methyl-p-tryosine, a catecholamine synthesis inhibitor. Cholinergic agonists, physostigmine and pilocarpine, antagonized the growth hormone and prolactin release induced by morphine sulfate. The data suggest that the opiates stimulate prolactin via an interaction with catecholaminergic neurons controlling prolactin release and stimulate growth hormone via a mechanism independent of alpha-adrenergic or general catecholaminergic influence. The mechanism through which cholinergic agonists act to inhibit opiate agonist stimulation of growth hormone is presently unknown.     

Differences in serotonergic neurotransmission between rats displaying high or low anxiety/depression-like behaviour: effects of chronic paroxetine treatment

Disturbances in serotonergic neurotransmission have been suggested to be closely interlinked with hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) system, and are likely to be involved in the pathophysiology of anxiety disorders and major depression. We therefore investigated markers of serotonergic transmission and their modulation by chronic paroxetine in rats selectively bred for high (HAB) or low (LAB) anxiety-related behaviour, both under basal conditions and in response to emotional stress. Hippocampal serotonin 1 A (5-HT1A) receptor mRNA expression was reduced in HAB rats, whereas 5-HT concentrations in hippocampal microdialysates did not differ between HAB and LAB rats under basal conditions. In the hippocampus, overall expression of serotonin transporter binding sites was increased in HAB compared with LAB rats. Exposure to emotional stress failed to increase intrahippocampal 5-HT release in HAB rats whereas LAB rats displayed a physiological, albeit small rise. Chronic paroxetine treatment markedly increased the stress-induced rise in hippocampal 5-HT in HAB, but not LAB rats. This effect may be (at least in part) related to a greater down-regulation of hippocampal serotonin transporter binding sites by paroxetine in HABs compared with LABs, while 5-HT1A receptor expression remained unaffected in this brain area. The findings indicate reduced hippocampal serotonergic transmission in HAB rats as compared with LAB rats, which is evident both at the presynaptic (5-HT release) and the postsynaptic (5-HT1A receptor) level. Chronic paroxetine enhanced the presynaptic responsivity in HAB rats, but not LAB rats, pointing to a preferential efficacy of paroxetine in rats with enhanced anxiety/depression-related behaviour.     

Effects of naloxone on the secretion of prolactin and corticosterone induced by 5-hydroxytryptophan and a serotonergic agonist, mCPP

The effects of naloxone, an opiate “pure” receptor antagonist, on the release of prolactin and corticosterone in the rat were studied following the administration of the serotonin precursor 5-hydroxytryptophan or the serotonin receptor agonist (?) -$\text{m}$-chloropnehylpiperazine. Naloxone clearly antagonizes the release of prolactin induced by 5-hydroxytryptophan administered alone at a dosage of 50 mg/Kg/b.wt. or at dosage of 30 mg/Kg/b.wt. preceded 60 minutes before injection by the administration of the serotonin uptake blocker fluoxetine. The opiate antagonist does not modify the increase in blood level of prolactin induced by (?) ?$\text{m}$-chlorohenylpiperazine. Naloxone itself does not reduce the increase in plasma level of corticosterone induced by 5-hydroxytryptophan, 5-hydroxytryptophan +fluoxetine or (?)?$\text{m}$-chlorophenylpiperazine.The results suggest that endogenous opioids may be involved in the increase in serum level of prolactin induced by 5-hydroxytryptophan and also indicate the existence of different serotonergic neurotransmitter circuits capable of modulating the release of prolactin and corticosterone. A mutual interplay between serotonergic and opiate neurons may be involved in controlling the release of prolactin, but such an interplay does not seem to occur in the secretion of corticotrophin-releasing hormone.     

Effect of an anti-substance P serum on prolactin and gonadotropins in hyperprolactinemic rats

The effects of the acute injection of a rabbit anti-substance P serum (ASPS) were studied in normal rats and rats with hyperprolactinemia induced by 5-hydroxytryptophan and estradiol given as a short or chronic treatment. The anti-substance P serum decreased the release of prolactin induced by 5-hydroxytryptophan when this serotonin precursor was injected 24 h, but not 1 h, after the administration of the antiserum. ASPS reduced the hyperprolactinemia induced by short and chronic treatment with estradiol in castrated rats. This effect was observed 24 h after the injection of the antiserum. On the other hand, the injection of ASPS induced a significant decrease in LH levels in serum of intact male rats injected with 5-hydroxytryptophan 24 h after ASPS, and in castrated rats treated with short-term and chronic administration of estradiol, 24 h after the injection of the antiserum. These results suggest that substance P may have a role in the control of prolactin secretion and could play a part in the hyperprolactinemic effects of estradiol. On the other hand, substance P, under certain circumstances, may stimulate LH release.     

Effect of serotonin depletion by p-chlorophenylalanine on serum prolactin levels in estrogen-treated ovariectomized rats: insights concerning the serotoninergic, dopaminergic and opioid systems.

The stimulatory effect of serotonin on prolactin secretion is well documented, and the administration of an inhibitor of serotonin synthesis (p-chlorophenylalanine - pCPA) has the expected inhibitory action on prolactin release in most experimental situations. However, there is evidence that in certain physiological or experimental conditions, activation of the serotoninergic system can also determine inhibition of prolactin secretion. The aim of the present study was to investigate the ability of estrogen to modify the effect of pCPA on prolactin secretion and to evaluate the participation of opioid and/or dopaminergic systems in regulating pCPA-induced prolactin secretion in estradiol-treated rats. We observed that pCPA administration (200 mg/kg/day, s.c., 2 days) to ovariectomized (OVX) female rats treated with estradiol benzoate (300 microg/week for 2 weeks, or 50 microg/week for 4 weeks, s.c.) causes a significant increase in serum prolactin, whereas no effect is observed in intact rats or in OVX rats without treatment. Bromocriptine administration completely reversed prolactin values previously increased by estradiol and by pCPA [OVX rats + estradiol = 86.50 ng/ml (68.90-175.02), OVX + estradiol + pCPA = 211.30 ng/ml (142.03-311.00), OVX + estradiol + pCPA + bromocriptine = 29.35 ng/ml (23.01 - 48.74), p<0.05. Naloxone administration partially reduced estrogen-induced high prolactin concentrations, but did not affect prolactin secretion stimulation determined by pCPA. Overall, the data from this report confirm the involvement of the dopaminergic system and, to a lesser degree, of endogenous opioids in prolactin secretion stimulation determined by estradiol. Furthermore, our results suggest that the stimulatory action of pCPA on prolactin secretion in estradiol-treated OVX rats is mediated by serotonin, which may also act indirectly on dopamine neurons.     

Effect of drugs modifying central serotonergic function on the response of extensor and nonextensor rats to maximal electroshock

Influences of reduction or enhancement of serotonergic function on convulsive responses and thresholds to maximal electroshock stimulation (MES) were studied in rats classified by MES as extensors or nonextensors. In extensors, serotonin reduction decreased the tonic convulsive threshold coincident with an increased incidence of hindlimb extension (HLE). Enhancement of serotonergic function with fluoxetine, pCA, 5-HTP, fenfluramine or 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) prevented HLE. This action was antagonized by pCA, strychnine or digitoxigenin and was not related to overt behavioral disruption or an elevated tonic threshold. In nonextensors, serotonin reduction restored HLE. HLE initiated by spinal cord stimulation was not remarkably effected by any treatment. Catecholamine reduction had no effect on any part of the tonic response in either group of rats. The results suggest the activation of central serotonergic inhibitory influences during the MES tonic convulsion. The possibility of graded-intensity serotonergic attenuation of seizure spread is discussed suggesting the action of serotonergic-enhancing drugs to prevent HLE may include a mechanism similar to a naturally-occurring “mechanism” to prevent HLE in nonextensor rats.     

Chronic antidepressant treatment enhances alpha-adrenergic and serotonergic responses in the facial nucleus

In addition to their well recognized activity in blocking uptake of biogenic amines, tricyclic antidepressants have recently been shown, with chronic treatment, to alter neurotransmitter receptor sensitivity. In this study, the responsiveness of facial motoneurons to norepinephrine (NE) and serotonin (5-HT) was assessed with single unit recording and microiontophoretic techniques. Treatment of rats with daily intraperitoneal injections of several clinically effective tricyclics for 14–20 days was found to enhance responses to NE, 5-HT, and to an intravenously administered 5-HT agonist, 5-MeODMT. These changes in sensitivity were not seen in animals chronically treated with saline, chlorpromazine, or fluoxetine, and thus appear specific to antidepressants. Acute effects of tricyclics on NE and 5-HT responses were variable, dependent on the specific drug tested, and appear to have no necessary relation to the pronounced sensitization produced by chronic treatment.     

Direct action of ethanol on pituitary prolactin secretion in vitro.

The effect of ethanol on prolactin release in vitro has been studied in order to investigate the direct action of ethanol on pituitary gland of the female rats. Animals were sacrificed in diestrus 2 and pituitary glands were incubated in TC-199 medium containing dopamine, noradrenaline, serotonin, TRH or cycloheximide with or without ethanol. The total amount of prolactin after the incubation period was calculated. Alcohol significantly increased the prolactin release in all groups. Cycloheximide and dopamine decreased the prolactin synthesis, but ethanol reduced the effect of dopamine. It is concluded that part of ethanol-induced hyperprolactinaemia, is due to a direct action of the alcohol on pituitary, affecting release and/or synthesis of prolactin.     

The effect of d-fenfluramine on anterior pituitary hormone release in the rat: in vivo and in vitro studies

Administration of d-fenfluramine, a serotonin-releasing drug, to male rats induced a dose-dependent increase in both serum prolactin and corticosterone concentrations. Serum growth hormone levels increased, but not significantly, at a dose of 1.25 mg/kg i.p. and decreased significantly at higher doses. When rats were pretreated with the serotonin uptake inhibitor fluoxetine (10 mg/kg i.p.) 30 min prior to injection of d-fenfluramine (5 mg/kg i.p.), the serum prolactin response to d-fenfluramine was partially inhibited, whereas the growth hormone response was not significantly modified. Fluoxetine pretreatment increased the serum corticosterone to the same level as did d-fenfluramine. d-Fenfluramine's effect on prolactin and growth hormone release was further tested in a hypothalamic-pituitary in vitro system. The addition of d-fenfluramine (5-500 ng/mL) for 30 min to rat hypothalami resulted in an enhancement of prolactin and growth hormone-releasing activities. These were expressed as the ability of the media in which the hypothalami had been incubated to stimulate prolactin and growth hormone release by cultured pituitary cells. The data suggest that the effect of d-fenfluramine on prolactin secretion is exerted through the hypothalamus and is probably mediated, at least partially, by a serotoninergic mechanism. The mechanism of d-fenfluramine's effect on corticosterone and growth hormone release needs further evaluation.     

EVIDENCE THAT 5-METHOXY-N, N-DIMETHYL TRYPTAMINE IS A SPECIFIC SUBSTRATE FOR MAO-A IN THE RAT: IMPLICATIONS FOR THE INDOLEAMINE DEPENDENT BEHAVIOURAL SYNDROME

A simple method for the separation of 5-hydroxyindoleacetic acid (5-HIAA) and 5-methoxyindoleacetic acid (5-MeOIAA) on columns of non-ionic polystyrene (Amberlite XAD-2) is described. Administration of 5-methoxy-N, N-dimethyl-tryptamine (5-MeODMT) 2 mg/kg i. p. to rats results in a sixfold increase in brain 5-MeOIAA within 15 min. This increase is blocked by the selective inhibitor of MAO-A, clorgyline, but not by the selective inhibitor of MAO-B, deprenyl, indicating that 5-MeODMT is deaminated almost entirely by MAO-A. The apparent 5-MeOIAA concentration in the brains of L-tryptophan loaded rats is not reduced by clorgyline and deprenyl, either singly or in combination, indicating that most of this fluorescence is due to other, unidentified substances. The apparent concentration of 5-HIAA in rat brain, minus 5-MeOIAA, is unaffected by deprenyl and reduced by clorgyline. However, clorgyline and deprenyl in combination reduced 5-HIAA values below those obtained with clorgyline alone. It is concluded that very little 5-MeODMT or other 5-methoxyindoleamines are formed endogenously in rat brain, and that the stereotyped syndrome of hyperactivity and tremors produced in rats by pretreatment with MAO inhibitors and L-tryptophan is dependent on the formation of an N-substituted derivative of 5-HT which is at least partly deaminated by MAO-B to 5-HIAA.     

Elevation of serum prolactin acutely after administration of p-chloroamphetamine in rats

p-Chloroamphetamine hydrochloride (0.5-10 mg/kg, i.p.) caused a rapid (within 30 minutes), dose-related increase in serum prolactin concentration in male rats. The effect was antagonized by pretreatment with p-chlorophenylalanine, an inhibitor of serotonin synethesis, or by metergoline, a serotonin receptor antagonist. The acute elevation of serum prolactin may have been mediated by the release of serotonin by p-chloroamphetamine.     

Involvement of Opioid Receptor Subtypes in Both Stimulatory and Inhibitory Effects of the Opioid Peptides on Prolactin Secretion During Pregnancy

1. We have previously demonstrated the existence of a dual neuromodulatory regulation of prolactin secretion by the opioid system. In the present work, we evaluated the opioid receptor subtypes involved in both the stimulatory and the inhibitory regulation of prolactin secretion in pregnant rats. 2. Specific opioid agonists and antagonists were administered intracerebro ventricular (i.c.v.) to rats on day 3 and on day 19 pregnancy in rats of pretreated with mifepristone. Blood samples were obtained after decapitation at 12.00 and 18.00 h. Serum prolactin levels were measured by RIA. 3. The mu-selective agonist DAMGO and beta-endorphin caused a significant increase in serum prolactin secretion on day 3 of pregnancy, during the diurnal surge and intersurge period. Pretreatment with naloxone prevented the increase on prolactin levels induced by DAMGO. The administration of U-50,488, a kappa-selective agonist or DPDPE, a delta-selective agonist, did not modify serum prolactin concentration while the mu1-antagonist naloxonazine reduced significantly serum prolactin levels. On day 19 of pregnancy, the release of prolactin induced by mifepristone was significantly increase by naloxonazine, while the kappa-antagonist nor-binaltorfimine induced only a small but significant increase. No effect was observed after administration of the delta-antagonist naltrindole. 4. We conclude that the mu-opioid receptor seems to be more specifically involved in both the stimulatory and inhibitory regulation by the opioid system on prolactin secretion during pregnancy. The increase on serum prolactin levels on day 3 after administration of DAMGO and beta-endorphin may suggest the participation of other regulatory mechanisms as the dopaminergic and serotoninergic systems. On day 19, only the endogenous ligands delta did not participate in the regulation of prolactin secretion, while the participation of the kappa-opioid receptor was significantly less effective than the endogenous ligand mu. Our results provide evidences of an important role of the opioid system through specific receptors on the regulation of prolactin secretion during early and late pregnancy.     

Effect of quipazine on rat plasma prolactin levels

Quipazine (2-(1-piperazinyl) quinoline maleate), a serotonin agonist which also has other effects on serotonin metabolism, in doses from 2.5 – 20 mg/kg, i.p., was found to markedly increase plasma prolactin levels in male rats. This increase was blocked by the serotonin antagonists methysergide and brom-lysergic acid diethylamide and potentiated by para-chlorophenylalanine, an inhibitor of serotonin synthesis. These findings suggest that the increase in plasma prolactin levels is due to the serotonin agonist properties of quipazine. Apomorphine and 2-Br-α-ergocryptine pretreatment blocked the effect on plasma prolactin of quipazine, while apomorphine given 15 min after quipazine brought about a rapid decline in the elevated plasma prolactin levels produced by quipazine.