Role of serotonin in estrogen-progesterone induced luteinizing hormone release in ovariectomized rats

Pharmacological agents were used to manipulate the surge of luteinizing hormone (LH) induced by progesterone in ovariectomized rats primed with estradiol benzoate. The LH surge was abolished with p-chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, and restored by 5-hydroxytryptophan, a serotonin precursor. Serotonin receptor agonists, quipazine and N-N-dimethyl-5-methoxytryptamine, were also capable of inducing an LH surge in rats pretreated with PCPA. The serotonin reuptake blocker chlorimipramine was ineffective in stimulating LH release in PCPA blocked animals. Another reuptake blocker, zimelidine was only partially effective in this regard. These two reuptake blockers, as well as amitriptyline, when injected to non-PCPA treated rats led to the reduction or inhibition of the expected LH surge. Four serotonin receptor antagonists, cyproheptadine, methysergide, cinanserin and SQ-10,631, were each able to reduce or abolish the progesterone induced surge of LH. These results suggest that some of the reuptake blockers of serotonin are also capable of inhibiting receptor binding for this neurotransmitter and strongly indicate that serotonin has a stimulatory role in the steroid induced release of LH in castrated rats.     

Alteration in hypothalamic neuropeptide Y (NPY) secretion may underlie female reproductive ageing: induction of steroid-induced luteinising hormone surge by NPY in ovariectomised aged rats

A large body of evidence suggests that a defect in the hypothalamic function may be the primary cause of reproductive ageing in female rats. We have previously shown that luteinising hormone (LH)-surge associated changes in hypothalamic neuropeptide Y (NPY) gene expression and median eminence (ME) NPY levels seen in young rats do not occur in middle-aged (MA) rats. The present study examined whether hypothalamic NPY release is altered during the steroid-induced LH surge in ovariectomised (OVX) MA rats, and whether exogenous NPY initiates steroid-induced LH surge in OVX old rats. In the first study, NPY release from the ME-arcuate nucleus, as assessed by the push-pull cannula technique, was significantly increased before and during the progesterone-induced LH surge in oestrogen (E(2))-primed ovariectomised young rats (2-3 months old). This antecedent increase in NPY release seen in young rats was not apparent in MA rats (11-13 months old) in association with a delayed and attenuated LH surge. In the second study, whereas progesterone failed to induce LH surges in E(2)-primed ovariectomised old rats (23-25 months old), intracerebroventricular NPY (0.1-0.5 microg) injections at 1100, 1200 and 13.00 h resulted in LH surge induction in E(2) + progesterone-primed ovariectomised old rats. Because increased hypothalamic NPY synthesis and release is obligatory for the preovulatory LH discharge in young rats, the present findings suggest that alteration in NPY release from the ME-arcuate nucleus contributes to the delayed and reduced LH surges in MA rats and may be involved in the subsequent loss of the LH surges in old rats.     

Microdialysis measurement of neurochemical changes in the mediobasal hypothalamus of ovariectomized ewes during oestrus

Oestrus behaviour and the luteinizing hormone (LH) surge are induced in ovariectomized ewes by oestradiol (E2) after a period of progesterone priming with a low level of E2 (Pge2) and we have previously shown that these effects are primarily mediated through their action on the mediobasal hypothalamus (MBH). The aim of the present study was to assess what neurochemical changes in the MBH are induced by these steroids that might mediate their action on oestrus behaviour and LH release. Eight ovariectomized ewes were implanted with microdialysis probes in the MBH and submitted to three artificial cycles, so that they exhibited either both oestrus behaviour and an LH surge (Pge2 + E2), and LH surge alone (E2 alone) or neither oestrus behaviour nor an LH surge (Pge2 alone). Microdialysis and blood samples were collected every 30 min from 4 h before the end of Pge2 treatment until the end of oestrus. Behavioural tests with a ram were made to assess receptivity. Dopamine (DA) levels were found to increase significantly at the termination of Pge2 treatment after both Pge2 + E2 and Pge2 treatments. When the ewes received E2 after a Pge2 + low estradiol priming (Pge2 + E2), DA levels decreased 16 h later (4 h after E2) whereas they did not change after E2 or Pge2 alone. By contrast, serotonin (5HT) levels did not change significantly during the first 24 h but then increased when ewes received E2 alone and decreased when they were treated with Pge2 + E2. γ-Aminobutyric acid (GABA) concentrations decreased significantly at the beginning of the sampling period after all treatments but this decrease lasted longer after Pge2 + E2 and was most pronounced at the beginning of receptivity. No significant long term effects of these steroid treatments were found on noradrenaline (NA), aspartate, glutamate, glycine and taurine levels. However, E2 administration was followed during the next few hours by a significant increase in glycine and to a smaller extent in glutamate and GABA. More importantly, when ewes were treated with Pge2 + E2, NA levels increased signicantly following the behavioural interactions with a ram when the ewes were sexually receptive. In contrast to this, DA levels only increased during interactions with the ram when the ewes were not receptive. 5HT levels increased after tests where the ewe was either receptive or unreceptive to the male. GABA, aspartate and glycine levels increased in the sample just preceding the test and then decreased during it. These results show that a number of neurotransmitter changes occur in the MBH during hormonal induction of oestrus. Changes mainly occured in DA, NA, 5HT and GABA concentrations during oestrus when animals were treated with Pge2 + E2 and suggest that they may be potentially involved in the control of female sexual behaviour. The changes occuring when animals received E2 alone and showed only prolactin and LH surges, but no behavioural oestrus, are much more limited. Furthermore, they do not correspond to changes after the E2 injection in the Pge2 + E2 treatment, so it is unlikely that the neurochemical changes we observed are directly related to these endocrine events.     

Ovarian-Steroid Modulation of Locus Coeruleus Activity in Female Rats: Involvement in Luteinising Hormone Regulation

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00–16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).     

The role of hypothalamic serotonin (5HT) before ovulation in immature rats treated with pregnant mare serum (PMS).

(1) PCPA methyl ester (10 mg/rat i.p.) inhibits induced ovulation in immature rats treated with pregnant mare serum (PMS). It also suppresses the preovulatory surges of LH and FSH, but not those of oestradiol or progesterone. (2) There is an increase in hypothalamic 5HT levels in the aftermoon and hypothalamic 5HIAA levels in the evening of the two days studied (days 28 and 29 of life). This occurs whether or not PMS was given on day 27. (3) The antiovulatory effects of PCPA are only seen when it is given on the afternoon or evening of the day before the pre-ovulatory gonadotrophin surge, i.e. on day 28 over the period of raised hypothalamic 5HT metabolism. (4) PCPA reduces 5HT metabolism in the hypothalamus within 2 hr of administration and its anti-ovulatory effect can be overcome by 5-hydroxytryptophan. This indicates that hypothalamic 5HT activity is essential for the gonadotrophin surge. (5) The anti-ovulatory effect of PCPA can be overcome by progesterone, LH and FSH but not oestradiol.     

Evidence for the involvement of serotonin in acute stress-induced release of luteinizing hormone in the male rat

The influence of serotonin on luteinizing hormone (LH) release caused by exposure to two acute Stressors differing in their intensity (restraint in tubes and immobilization in woodboards) was studied in adult male rats. Inhibition of serotonin synthesis with p-chlorophenylalanine (PCPA) significantly abolished LH release caused by immobilization (IMO). Administration of the serotonin antagonists mianserine and methiothepin also eliminated LH release caused by IMO without altering basal LH levels. These data represent the first evidence that a classical neurotransmitter (serotonin) is involved in LH release caused by stress in the rat.     

Changes of the release of luteinizing hormone (LH) on the day of proestrus after lesions or stimulation of the raphe nuclei in rats

The effect of stimulation or lesion of the raphe nuclei on ovulation and on the release of luteinizing hormone (LH) on the day of proestrus was studied in unanesthetized, unrestrained rats. Electrochemical stimulation (anodic DC or 100 μA during 30 s) was applied at 12.00 h on the day of proestrus through chronically implanted stainless steel electrodes. Lesions were made by passing a cathodic current of 1 mA for 20 s through nichrome electrodes stereotaxically implanted and the rats were used 15–30 days later. Blood samples were obtained hourly from the freely behaving rats through a plastic cannula inserted into the external jugular vein. Stimulation in the medial raphe nucleus (MRn) resulted in blockade of ovulation and of the preovulatory LH release. On the contrary, no change of the normal pattern of LH surge nor in the number of ovulating rats was seen after stimulation of the dorsal raphe nucleus (DRn) or in rats stimulated in the mesecephalon outside these nuclei. Injection of p-chloropheylalamine (PCPA) into the MRn to block 5-hydroxytryptamine (5-HT) synthesis, prevented the effect of MRn stimulation, whereas injection of saline solution did not. Rats bearing lesions destroying the DRn showed decreased proestrous LH surge and blockade of ovulation whereas those with lesions of MRn ovulated normally. Rats with transverse cuts placed just behind the DRn exhibited normal LH release indicating that the effects of destroying the DRn is not due to the interruption of ascending fibers crossing the nucleus. Injections of PCPA in the DRn but not saline solution mimicked the effect of lesions. It is concluded that the serotonergic system influences the proestrus surge of LH, with the DRn playing a facilitatory role and the MRn an inhibitory role.     

Regulation of glutamic acid decarboxylase 65 and 67 gene expression by ovarian steroids: identification of two functionally distinct populations of GABA neurones in the preoptic area

GABA neurones in the preoptic area (POA) are critical for oestradiol (E2)-dependent surge release of luteinizing hormone (LH); however, it is not clear which population(s) of POA GABA neurones is involved. The goals of the present studies were: (i) to determine whether E2 regulates GABA neurones similarly in two subdivisions of the POA that play a role in LH surge release, the rostral POA region that contains the organum vasculosum of the lamina terminalis (rPOA/OVLT), and the region containing the anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN) and (ii) to determine whether GABA neurones in either or both regions exhibit temporal changes consistent with a role in the regulation of LH surge release. To accomplish these goals, we measured glutamic acid decarboxylase (GAD) 65 and 67 mRNA levels at several time points in ovariectomized (OVX), E2-treated OVX rats exhibiting LH surge release, and in E2-treated OVX rats in which LH surge release was blocked by prior administration of progesterone (P4). Our findings demonstrate that, despite their close proximity, GABA neurones in the AVPV/MPN region are regulated differently from those in the rPOA/OVLT. Only neurones in the AVPV/MPN region show temporal changes in GAD 67 mRNA expression that appear to be linked to positive-feedback effects of E2 on luteinizing hormone-releasing hormone (LHRH) and LH release. Our findings also indicate that a morning rise and an afternoon fall in GAD 67 mRNA levels marks two E2-dependent signals required for LHRH and LH surge release. Finally, our results suggest that there are distinct E2-induced signals to the rPOA/OVLT and AVPV/MPN regions and that these signals differentially regulate GAD 65 and 67 gene expression.     

Oestradiol-dependent and -independent modulation of tyrosine hydroxylase mRNA levels in subpopulations of A1 and A2 neurones with oestrogen receptor (ER)alpha and ER beta gene expression

Oestradiol (E2) induces luteinizing hormone-releasing hormone (LHRH) hypersecretion, thereby triggering LH surge release in ovariectomized (OVX) rats. Neural signals responsible for the surge are marked by a morning increase in LHRH gene expression and an afternoon increase in LHRH release. Evidence suggests that subpopulations of noradrenergic neurones may be responsible for one or both of these signals. To further investigate this issue, we examined effects of E2 on the activity of A1 and A2 noradrenergic neurones, as reflected in changes in tyrosine hydroxylase (TH) mRNA expression, on the day of LH surge release. We then used dual-label in situ hybridization to determine whether E2-induced changes occurred primarily in A1 and A2 subdivisions wherein most noradrenergic neurones expressed oestrogen receptor (ER)alpha and/or ER beta mRNA. We found that in all subdivisions, levels of TH mRNA were higher in E2- than oil-treated rats at 12.00 h. These differences resulted from a decline in TH mRNA expression in oil-treated rats, as well as a rise in levels in E2-treated rats between 10.00 h and 12.00 h. During the afternoon, TH mRNA expression in most A1 and A2 subdivisions peaked at 14.00 h when LH surge release began. However, in all but the middle and caudal A2 subdivisons, levels were similar in E2-treated and control rats at this time. This was attributable to a widespread increase in TH mRNA expression between 12.00 h and 14.00 h in OVX rats. There was no evidence that E2 induced changes in TH mRNA expression preferentially in regions wherein most neurones contained ER alpha or ER beta mRNA. Our findings suggest that E2 activation of middle and caudal A2 neurones, in conjunction with the widespread E2-independent activation of noradrenergic neurones in other subdivisions, may play a role in the induction of LH surge release.     

Different Critical Perinatal Periods and Hypothalamic Sites of Oestradiol Action in the Defeminisation of Luteinising Hormone Surge and Lordosis Capacity in the Rat

Female rats show a gonadotrophin‐releasing hormone (GnRH)/luteinising hormone (LH) surge in the presence of a preovulatory level of oestrogen, whereas males do not because of brain defeminisation during the developmental period by perinatal oestrogen converted from androgen. The present study aimed to identify the site(s) of oestrogen action and the critical period for defeminising the mechanism regulating the GnRH/LH surge. Animals given perinatal treatments, such as steroidal manipulations, brain local implantation of oestradiol (E₂) or administration of an NMDA antagonist, were examined for their ability to show an E₂‐induced LH surge at adulthood. Lordosis behaviour was examined to compare the mechanisms defeminising the GnRH/LH surge and sexual behaviour. A single s.c. oestradiol‐benzoate administration on either the day before birth (E21), the day of birth (D0) or day 5 (D5) postpartum completely abolished the E₂‐induced LH surge at adulthood in female rats, although the same treatment did not inhibit lordosis. Perinatal castration on E21 or D0 partially rescued the E2‐induced LH surge in genetically male rats, whereas castration from E21 to D5 totally rescued lordosis. Neonatal E₂ implantation in the anterior hypothalamus including the anteroventral periventricular nucleus (AVPV)/preoptic area (POA) abolished the E₂‐induced LH surge in female rats, whereas E₂ implantation in the mid and posterior hypothalamic regions had no inhibitory effect on the LH surge. Lordosis was not affected by neonatal E₂ implantation in any hypothalamic regions. In male rats, neonatal NMDA antagonist treatment rescued lordosis but not the LH surge. Taken together, these results suggest that an anterior hypothalamic region such as the AVPV/POA region is a perinatal site of oestrogen action where the GnRH/LH regulating system is defeminised to abolish the oestrogen‐induced surge. The mechanism for defeminisation of the GnRH/LH surge system might be different from that of sexual behaviour, in terms of the site(s) of oestrogen action and critical period, as well as the neurotransmitter system involved.     

Ovarian Steroid-Independent Diurnal Rhythm in Cyclic GMP/Nitric Oxide Efflux in the Medial Preoptic Area: Possible Role in Preovulatory and Ovarian Steroid-Induced LH Surge*

The aim of this study was to evaluate the relationship between cyclic LH hypersecretion and nitric oxide (NO) release in the medial preoptic area (MPOA), the hypothalamic site implicated in induction of LH hypersecretion. The MPOA extracellular cyclic GMP (cGMP) efflux (an index of NO release), was monitored by microdialysis. Quite unexpectedly, we observed a daily afternoon rise in the MPOA cGMP efflux in cycling female rats on proestrus and diestrus II, in ovariectomized (ovx) rats and in ovx rats treated with ovarian steroids to induce the LH surge. The daily rise in cGMP efflux occurred earlier in diestrous and in estradiol benzoate (EB)-treated ovx rats than in ovx rats. Progesterone (P) injection to estrogen-primed ovx rats further advanced the onset of the rise close to the earliest time of rise as seen on proestrus. The afternoon increase in the cGMP efflux in proestrous rats was abolished by pentobarbital treatment that blocked the LH surge. Intracerebroventricular (icv) injection of 1H-[1,2,4]oxadiazo[4,3-a]quinoxalin-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, suppressed the P-induced LH surge in EB-primed ovx rats, but not basal LH secretion in unprimed ovx rats. Analysis of brain NOS (bNOS) levels in the POA by Western blotting showed that the morning bNOS levels were higher in the POA of EB-treated rats than in unprimed ovx rats. Further, with the exception of ovx rats treated with sequential EB and P treatment, the POA bNOS levels rose significantly in the afternoon in unprimed ovx and EB-treated ovx rats. Collectively, these findings reveal a diurnal rhythm in the MPOA cGMP/NO efflux that is ovarian steroid-independent. Ovarian steroids apparently shift the timing of the afternoon rise in cGMP/NO efflux to synchronize with the activation of steroid-dependent neuronal systems responsible for the LH surge.     

Evidence that Hypothalamic Neuropeptide Y Gene Expression Increases Before the Onset of the Preovulatory LH Surge*

Neuropeptide Y (NPY), a 36 amino acid residue peptide, is involved in stimulation of LHRH and LH surges on proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. Recently, we observed that NPY gene expression in the medial basal hypothalamus (MBH) was increased before the onset of the LH surge in the ovarian steroid-primed ovx rats. Since the ovarian steroidal milieu during the estrous cycle is markedly different from that prevailing after ovarian steroid injections in ovx rats, we evaluated in cycling rats the temporal relationship between MBH preproNPY mRNA levels and the preovulatory LH surge on the day of proestrus and compared that with diestrus II, concomitant with basal LH levels. PreproNPY mRNA levels in the MBH were measured by solution hybridization/RNAse protection assay, using a cRNA probe. On the day of diestrus II, preproNPY mRNA levels changed little between 1000 and 1800 h. Quite unexpectedly, preproNPY mRNA levels at 1000 h on proestrus were similar to diestrus II levels, despite additional exposure to ovarian steroids during this interval. However, from these low levels at 1000 h, the preproNPY mRNA profile displayed a biphasic rise. During the first phase, preproNPY mRNA rose significantly at 1200 h and remained elevated at 1300 and 1400 h concomitant with basal serum LH levels. Thereafter, a second rise in preproNPY mRNA began at 1500 h, peaked rapidly at 1600 h and declined significantly at 1800 h. This secondary activation of NPY gene expression occurred with a slow, two-fold increase in serum LH at 1500 h, followed by a rapid ascension to peak levels at 1800 h and was associated with an increase at 1400 h of serum progesterone levels which reached their peak at 1800 h. These results demonstrate that a dynamic, biphasic augmentation in hypothalamic NPY gene expression occurs selectively on proestrus, and that the first incremental response is observed some time before the onset of preovulatory LH hypersecretion. Because preproNPY mRNA levels at 1000 h on proestrus were similar to the low levels seen on the preceding diestrous II phase, a neural timing mechanism, and not changes in ovarian hormone levels during this phase may be responsible for the increase in NPY gene expression after 1000 h of proestrus. Because of our previous observations that progesterone can rapidly augment preproNPY mRNA in the MBH and because a rise in serum progesterone occurs hours before the onset of the LH surge, we suggest that the secondary rise in preproNPY mRNA is facilitated by this antecedent increase in serum progesterone. Cumulatively, these results are in accord with the thesis that activation of hypothalamic NPY gene expression is one of the key early neural events initiated by the neural clock that times the preovulatory LHRH and LH surges.     

Effects of p-chlorophenylalanine on hypothalamic indoleamine levels and the associated changes which occur in catecholamine dynamics and LH surges in estrogen-treated ovariectomized rats

Recent studies have demonstrated that the serotonergic and noradrenergic systems are functionally and anatomically linked and both systems have been implicated as contributors to the regulation of the phasic release of LH. Consequently, perturbations within the serotonergic system could secondarily affect noradrenergic system activity and result in a loss of phasic LHRH secretion. In the present studies we examined the effects of p-chlorophenylalanine (PCPA) on LH surges and the associated changes which occur in hypothalamic serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations. We also evaluated the effects of this drug on norepinephrine (NE) and dopamine (DA) initial concentrations, rate constants and turnover rates in the medial preoptic area (MPN), suprachiasmatic nuclei (SCN), paraventricular nuclei (PVN) and median eminence (ME). Seven days after ovariectomy, rats received estradiol (E2) capsules (day 0) and on day 1 some animals also received PCPA (250 mg/kg b. wt., i.p.) while the remainder served as controls. LH surges occurred in control animals but not in PCPA-treated rats on days 2, 3 and 4. PCPA produced a significant decline in 5-HT and 5-HIAA concentrations in all microdissected hypothalamic regions at 09.00 and 15.00 h on day 2. In control rats, there were no significant changes in initial concentrations of NE in the MPN, PVN and ME between 09.00 and 15.00 h with the exception of the SCN where a slight decline had occurred by 15.00 h. NE rate constants and turnover rates increased during the afternoon in controls in the MPN, SCN and ME and declined in the PVN concomitant with LH surges. PCPA had variable effects in suppressing NE initial concentrations depending upon the hypothalamic area studied and the time of day. More importantly, the drug abolished the diurnal rhythm in rate constants observed in controls and consequently, neither the MPN, SCN nor ME showed any increase in NE turnover rates in the afternoon of day 2. In contrast, a significant decline in rate constants and turnover rates occurred in the PVN of both control and PCPA-treated rats during the afternoon of day 2. DA initial concentrations declined in controls between 09.00 and 15.00 h in the MPN and ME but not in the SCN or PVN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prolactin secretory surge during estrus coincides with increased dopamine activity in the hypothalamus and preoptic area and is not altered by ovariectomy on proestrus

Prolactin (PRL) secretory surges have been reported on the afternoons of both proestrus and estrous in cycling rats. As neuroendocrine regulation of estrous PRL surge is poorly understood, the present study aimed to investigate the involvement of hypothalamic dopamine and serotonin as well as of plasma ovarian steroids in this hormonal surge generation. For that, we determined the concentrations of dopamine, serotonin and their respective metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA) in the mediobasal hypothalamus (MBH) and medial preoptic area (MPOA) throughout the day of estrus and correlated them with plasma PRL levels. In a second study, we evaluated the effect of ovariectomy on the morning of proestrus on PRL surges of both proestrus and estrus. Dopamine turnover, as determined by DOPAC/dopamine ratio, increased in both the MBH and MPOA coinciding with the afternoon PRL surge on estrus. In contrast, both the concentration and turnover (5-HIAA/serotonin) of serotonin within these areas were unaltered during estrus. In addition, ovariectomy reduced plasma estradiol and progesterone levels but did not alter the PRL surges on proestrus and estrus. Considering that dopamine is the main inhibitor of PRL release and that PRL auto-regulates its secretion through a short-loop feedback mechanism, our present results suggest that PRL may suppress its own secretion during the estrus surge through the activation of the dopaminergic neurons in the MBH and MPOA. In addition, the PRL surge on estrus seems do not depend on either the activity of hypothalamic serotonin or the increased secretion of ovarian steroids on proestrus.     

Rodent data and general hypothesis: antipsychotic action exerted through 5-HT2A receptor antagonism is dependent on increased serotonergic tone

Summary. The locomotor stimulation induced by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) in mice was regarded as a model of at least some aspects of schizophrenia. The serotonin synthesis inhibitor dl-p-chlorophenylalanine (PCPA) was used to evaluate the involvement of endogenous serotonin in (a) the induction of MK-801-induced hyperlocomotion in NMRI mice, and (b) the inhibition of MK-801-induced hyperlocomotion by each of five monoaminergic antagonists (M100907, clozapine, olanzapine, raclopride, SCH23390). Further, brain monoaminergic biochemistry was characterised in rats and mice after various drug treatments. PCPA pretreatment did not significantly reduce MK-801-induced hyperlocomotion in any of the experiments performed; however in a meta-analysis of six experiments, the locomotion displayed by MK-801-treated animals was diminished 17% by PCPA pretreatment. The selective 5-HT2A receptor antagonist M100907 exerted a dose-dependent inhibition of MK-801-induced hyperlocomotion. This effect was abolished in mice pretreated with PCPA, but could be restored in a dose-dependent manner by restitution of endogenous 5-HT by means of 5-hydroxytryptophan (5-HTP). On the other hand, the inhibition of MK-801-induced hyperlocomotion exerted by the selective dopamine D-2 receptor antagonist raclopride or the dopamine D-1 receptor antagonist SCH23390 was unaffected by PCPA pretreatment. The antipsychotics clozapine and olanzapine displayed a split profile. Hence, the inhibitory effect on MK-801-induced hyperlocomotion exerted by low doses of these compounds was diminished after PCPA pretreatment, while inhibition exerted by higher doses was unaffected by PCPA. These results suggest that (1) MK-801-induced hyperlocomotion is accompanied by an activation of, but is not fully dependent upon, brain serotonergic systems. (2) In the hypoglutamatergic state induced by MK-801, endogenous serotonin exerts a stimulatory effect on locomotion through an action at 5-HT2A receptors, an effect that is almost completely counterbalanced by a concomitant inhibitory impact on locomotion, mediated through stimulation of serotonin receptors other than 5-HT2A receptors. M100907, by blocking 5-HT2A receptors, unveils the inhibitory effect exerted on locomotion by these other serotonin receptors. (3) Dopamine D-2 receptor antagonistic properties of antipsychotic compounds, when they come into play, override 5-HT2A receptor antagonism. Possible implications for the treatment of schizophrenia with 5-HT2A receptor antagonists are discussed. It is hypothesized that treatment response to such agents is dependent on increased serotonergic tone. Accepted February 9, 1998; received December 16, 1997     

Inhibition of Steroid-Induced Galanin mRNA Expression in GnRH Neurons by Specific NMDA-Receptor Blockade

Galanin mRNA levels in GnRH neurons increase in association with a steroid-induced LH surge in female rats. Both the steroid-induced LH surge and the concomitant increase of galanin mRNA in GnRH neurons are blocked by non-specific inhibition of central nervous system activity imposed by pentobarbital and specific central alpha-adrenergic receptor blockade. Based on these observations, we hypothesized that galanin gene expression in GnRH neurons is induced whenever GnRH neurons become activated to generate an LH surge. If this were the case, then any neurotransmitter receptor blocking agent that inhibits the LH surge by central mechanisms would likewise block the associated increase in galanin mRNA in GnRH neurons. We tested this hypothesis by examining the effects of an N-methyl-D-aspartate (NMDA) receptor antagonist on the steroid-induced LH surge and on levels of galanin mRNA in GnRH neurons. Three groups of ovariectomized rats were used: Group 1-treated with estradiol and progesterone (E/P) and sacrificed at the peak of the LH surge; Group 2-treated the same as Group 1 except that dizocilpine (MK801, an NMDA receptor antagonist) was used to block the LH surge; and Group 3-treated the same as Group 1 except they received vehicle instead of E/P. Double-and single-label in situ hybridization followed by computerized image analysis were used to measure levels of galanin mRNA and GnRH mRNA in GnRH neurons [as grains/cell (g/c)]. E/P treatment induced a 3-fold increase in LH levels and a 5-fold increase in the galanin mRNA signal content of GnRH neurons. Treatment with MK801 completely prevented the LH surge in all animals and also blocked the steroid-induced increase in galanin mRNA in GnRH neurons. As assessed by 2 independent GnRH single-labeled assays, neither GnRH message content nor the number of identifiable GnRH neurons differed among the experimental groups. We conclude that the increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a steroid-evoked LH surge, and we infer that induction of galanin gene expression in GnRH neurons is induced as a consequence of synaptic activation of GnRH neurons.     

Hypothalamic monoamines and their catabolites in relation to the estradiol-induced luteinizing hormone surge

Monoamines and non-conjugated catabolites (serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3,4-dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), norepinephrine (NE), and dopamine (DA] were measured in the medial basal hypothalamus (MBH) and preoptic area (POA) of ovariectomized (OVX) and OVX estradiol (E2)-treated rats using high-performance liquid chromatography with electrochemical detection. These E2 treatments were sufficient to induce an LH surge. The use of MHPG/NE ratios as estimates of NE release was validated in the rat hypothalamus by the major decreases of MHPG after injection of the alpha 2-adrenergic agonist, clonidine, and by MHPG increases after the alpha 2-antagonist, yohimbine. The ratio, MHPG/NE, decreased between morning and afternoon in the MBH but not in the POA; there were no differences between OVX and E2-treated rats. Previous studies using a variety of methods indicate that NE turnover increases during LH surges. The present data suggest that unconjugated MHPG is not a sensitive measure of NE release in the rat hypothalamus, but can detect the large changes produced by stimulating or inhibiting the alpha 2-adrenergic autoreceptor. The ratios of DOPAC/DA and 5-HIAA/5-HT in the MBH decreased consistently between morning and afternoon in OVX rats, with or without E2 treatment. This suggests that the release of DA and 5-HT decreases during the day regardless of steroidal milieu.     

Evidence suggesting that the potentiating action of neuropeptide Y on luteinizing hormone (LH)-releasing hormone-induced LH release remains unaltered in aged female rats

Several lines of evidence suggest that one of the mechanisms by which the hypothalamic neuropeptide Y plays an obligatory role in the preovulatory luteinizing hormone (LH) discharge in young rats is to potentiate the action of LH-releasing hormone (LHRH) on LH release at the level of the pituitary. This study examined whether an alteration in the potentiating action of neuropeptide Y on LHRH-induced LH release may contribute to the attenuation or absence of LH surges during female reproductive ageing. Young regularly cycling (2-3-month-old) and old constant oestrous (19-20-month-old) rats ovariectomized for 7 days were primed with oestradiol-17beta-filled Silastic capsules. Two days later, rats received s.c. progesterone at 09.00 h and then were injected i.p. with either saline or pentobarbital at 13.30 hours. Pentobarbital-treated rats received i.v. pulses of neuropeptide Y, LHRH, a combination of neuropeptide Y and LHRH, or saline, every 30 min from 14.00 to 18.00 h via a jugular cannula. Hourly blood samples were collected between 11.00 and 21.00 h. In old rats, the progesterone-induced LH surge was significantly attenuated and delayed as compared to that of young rats. Pentobarbital injection completely blocked the LH surge. Neuropeptide Y pulses alone had no significant effect on LH release. In contrast, LHRH pulses increased LH release in both age groups, although the response was significantly reduced in older rats. While combined pulses of neuropeptide Y and LHRH significantly increased LH release in both young and old rats as compared to that of LHRH alone, the potentiating action of neuropeptide Y on LHRH-induced LH release remained unchanged between the two age groups. These results, together with our recent demonstration of altered hypothalamic neuropeptide Y neuronal activity in middle-aged pro-oestrous rats, suggest that a deficit in neuropeptide Y secretion and action in the hypothalamus, rather than a decrease in pituitary responsiveness to neuropeptide Y, may partially be responsible for the absence of LH surges in old rats.     

The effects of alaproclate and p-chlorophenylalanine on cued navigation performance in rats

Summary The present study investigates the effects of a serotonin (5-HT) reuptake inhibitor, alaproclate, on water maze cued navigation performance in serotonin depleted and control rats. Treatment with p-chlorophenylalanine (PCPA), a serotonin synthesis inhibitor, (400 mg/kg/day×3 i.p.) significantly depleted cerebral levels of both 5-HT (about 80% depletion) and its major metabolite 5-HIAA (about 90% depletion) for at least 7 days. PCPA treatment also slightly decreased cerebral noradrenaline and dopamine levels (16% and 22% reductions, respectively). PCPA treatment alone had no effect on the acquisition of the cued navigation version (visible platform) of the water maze task measured as the distance to find the escape platform, but it significantly increased swimming speeds of the rats. Alaproclate (20 mg/kg i.p.) increased escape distance and slightly decreased swimming speeds of the rats. The effects of alaproclate did not differ between PCPA and sham treated (arabic gum 400 mg/kg/day × 3 i.p.) rats. The results demonstrate that alaproclate induced cued navigation behavioral deficit is maintained after a marked depletion of cerebral serotonin.