Regulation of the heparan sulfate proteoglycan, perlecan, by injury and interleukin-1alpha.

Perlecan is a specific proteoglycan that binds to amyloid precursor protein and beta-amyloid peptide, is present within amyloid deposits, and has been implicated in plaque formation. Because plaque formation is associated with local inflammation, we hypothesized that the mechanisms involved in brain inflammatory responses could influence perlecan biosynthesis. To test this hypothesis, we first studied perlecan regulation in mice after inflammation induced by a brain stab wound. Perlecan mRNA and immunoreactivity were both increased 3 days after injury. Interleukin-1alpha (IL-1alpha) is a cytokine induced after injury and plays an important role in inflammation. As such, IL-1alpha may be one of the factors participating in regulating perlecan synthesis. We thus studied perlecan regulation by IL-1alpha, in vivo. Regulation of perlecan mRNA by this cytokine was area-specific, showing up-regulation in hippocampus, whereas in striatum, perlecan mRNA was unchanged. To support this differential regulation of perlecan mRNA by IL-1alpha, basic fibroblast growth factor (bFGF), a growth factor also present in plaques, was studied in parallel. bFGF mRNA did not show any regional difference, being up-regulated in both hippocampus and striatum in vivo. In vitro, both astrocyte and microglia were immunoreactive for perlecan. Moreover, perlecan mRNA was increased in hippocampal glial cultures after IL-1alpha but not in striatal glia. These results show an increase in perlecan biosynthesis after injury and suggest a specific regulation of perlecan mRNA by IL-1alpha, which depends on brain area. Such regulation may have important implications in the understanding of regional brain variations in amyloid plaque formation.

The octadecaneuropeptide-induced response of corticotropin-releasing hormone messenger RNA levels is mediated by GABA(A) receptors and modulated by endogenous steroids.

The involvement of endogenous benzodiazepine octadecaneuropeptide in the regulation of corticotropin-releasing hormone messenger RNA expression has been studied using in situ hybridization technique. Intracerebroventricular injection of octadecaneuropeptide (4 microg/kg) induced a 26% decrease in the corticotropin-releasing hormone messenger RNA expression in the hypothalamic paraventricular nucleus. Concomitant injection of octadecaneuropeptide and i.p. injection of the GABA(A) receptor agonist muscimol (4 mg/kg) potentiated the corticotropin-releasing hormone messenger RNA decrease ( - 34%). The depressing effect of octadecaneuropeptide on corticotropin-releasing hormone gene expression was totally reversed by pretreatment of the animals with the GABA(A) receptor antagonist picrotoxin (5 mg/kg; i.p.) or by pretreatment with the benzodiazepine receptor antagonist flumazenil (4 mg/kg; i.p.). To determine the reciprocal involvement of adrenal and sexual steroids in this regulation, animals are adrenalectomized and/or castrated. Adrenalectomy reversed the effect induced by octadecaneuropeptide, which increased corticotropin-releasing hormone messenger RNA expression (+21%), while castration did not modify the negative influence of octadecaneuropeptide. When rats were adrenalectomized and castrated, the adrenalectomy influence was predominant, since octadecaneuropeptide increased significantly the hybridization signal (+18%). The involvement of neurosteroids, especially reduced metabolites of progesterone was also investigated. The concomitant injection of octadecaneuropeptide and subcutaneous injection of the 5alpha-reductase inhibitor MK-906 (14 mg/kg) to adrenalectomized and castrated rats, reduced significantly by 60% the increase of corticotropin-releasing hormone messenger RNA expression induced by octadecaneuropeptide. These results indicate that in vivo the endogenous benzodiazepine octadecaneuropeptide, via an activation of the benzodiazepine sites of the GABA(A) receptor, negatively modulates corticotropin-releasing hormone neuronal activity and that this modulation can be negatively or positively influenced by central and peripheral steroids.

Regulation of proopiomelanocortin gene expression by endogenous ligands of the GABAA receptor complex as evaluated by in situ hybridization in the rat pars intermedia.

The neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on proopiomelanocortin (POMC) neurons in the hypothalamus as well as on the melanotrope cells of the intermediate lobe (IL) of the pituitary gland. Moreover, the activation of the GABAA receptor complex by different ligands has been shown to exert a negative influence on the POMC gene expression at the hypothalamic level. In order to elucidate the in vivo regulation of the POMC mRNA levels in the intermediate lobe of the pituitary by endogenous ligands of the GABAA receptor complex, we have studied the effect of intravenous (i.v.) and intracerebroventricular (i.c.v) injections of octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI). The possible involvement of neurosteroids in the action of ODN on melanotropic cells was evaluated following inhibition of two enzymes involved in the biosynthesis of neurosteroids known as activators of G3BAA receptor complex: trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and MK-906, an inhibitor of 5 alpha-reductase. The i.v. injection of ODN produced a dose-dependent inhibition of POMC gene expression in the IL. The i.c.v. injection of ODN also depressed POMC mRNA. These effects were completely reversed by the concomitant administration of the GABAA antagonist picrotoxin. Similar results were obtained in POMC neurons in the arcuate nucleus (AN) of the hypothalamus. Trilostane administration induced an increase in POMC mRNA and also prevented the inhibitory influence of ODN. The neurosteroid pregnenolone-sulfate, a negative modulator of the GABAA receptor, also stimulated POMC gene expression. On the other hand, MK-906 produced a decrease in mRNA levels and could not reverse the effect of ODN. The results indicate that activation of the GABAA receptor complex by the endogenous benzodiazepine receptor ligand ODN can induce a negative regulation of POMC gene expression in the IL of the pituitary and neurons in the AN. The present results do not provide clear evidence that neurosteroids are involved in the action of ODN on POMC gene expression in the IL.

Effects of dehydroepiandrosterone (DHEA) on pituitary prolactin and arcuate nucleus neuron tyrosine hydroxylase mRNA levels in the rat.

It is well documented that dehydroepiandrosterone (DHEA), an adrenal androgen, is converted into potent androgens and/or estrogens in peripheral tissues. Since sex steroids are involved in the regulation of prolactin (PRL) secretion, we have studied the effect of DHEA administration on PRL mRNA levels in both adult male and female rats. Since tuberoinfundibular dopaminergic (TIDA) neurons are involved in the negative regulation of PRL, we have also evaluated the effects of DHEA on the genetic expression of tyrosine hydroxylase (TH), the limiting enzyme in catecholamine biosynthesis in TIDA neurons. Sham-operated and castrated animals of both sexes received during 2 days DHEA at the dose of 6 mg/kg/day, starting on the first day after castration. PRL and TH mRNA levels were measured by quantitative in situ hybridization. In the male rat, orchiectomy performed 3 days earlier did not modify PRL mRNA levels. DHEA administration increased the hybridization signal in both sham-operated and orchiectomized animals. In the female, ovariectomy decreased PRL mRNA levels and, as observed in the male, DHEA treatment induced an increase in the hybridization signal in both control and ovariectomized rats. In TIDA neurons, castration increased TH mRNA levels as evaluated by number of grains over labelled neurons and the number of TH-labelled cells per section in both male and female animals. In both sham-operated male rats and orchiectomized animals, DHEA decreased the hybridization signal. In the female, DHEA administration completely prevented the increase in TH mRNA levels due to ovariectomy. In sham-operated female rats, the treatment had no effect. These data clearly indicate that in both male and female rats DHEA exerts an estrogenic influence on both PRL and TH gene expression. Although these in vivo experiments do not allow to establish whether the stimulation of PRL gene expression is due to an action of the steroid on the pituitary or at the hypothalamic level or alternatively at both sites, it is likely that one of the mechanisms of action of DHEA might be related to a decrease in dopamine release following a depression of TIDA neuron activity.

Involvement of the Y2 receptor subtype in the regulation of prolactin gene expression by neuropeptide Y in the male rat.

In order to determine the influence of neuropeptide Y (NPY) on the biosynthesis of prolactin (PRL), we have studied the effects of NPY and some NPY analogs on PRL gene expression in the male rat anterior pituitary gland. The following peptides (4 micrograms/100 g body wt): NPY, peptide YY (PYY), NPY13-36 (a Y2 receptor agonist) and [Leu31,Pro34]NPY (a Y1 receptor agonist) were injected into the left lateral ventricle of adult male rats. Control animals received only the vehicle (0.9% NaCl). All the animals were perfused with 4% paraformaldehyde 4 h after injection and processed for in situ hybridization. The intracerebroventricular injection of NPY, PYY, and NPY13-36 induced a significant increase in the hybridization signal (22-40% over control). On the other hand, the Y1 receptor agonist [Leu31,Pro34]NPY did not influence PRL mRNA levels. These data then suggest that activation of the Y2 NPY receptor subtype at the central level can positively regulate PRL gene expression.

Regulation of proopiomelanocortin gene expression by neuropeptide Y in the rat arcuate nucleus.

In the arcuate nucleus which is richly innervated by both proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons, it has been shown that NPY fibers are in synaptic contact with POMC cell bodies. In order to determine whether NPY could influence POMC neuronal activity, we have studied the effects of NPY and some NPY analogs on POMC gene expression using quantitative in situ hybridization. The following peptides NPY, [Leu31,Pro34]-NPY (a Y1 receptor agonist), and NPY13-36 (a Y2 receptor agonist) were injected into the left lateral cerebral ventricle of adult male rats 4 h before being perfused for histological procedures. The intracerebroventricular injection of NPY and NPY13-36 induced a significant decrease in the number of grains overlying the labelled neurons. On the other hand, the Y1 receptor agonist [Leu31,Pro34]-NPY did not modify POMC mRNA levels. These data then strongly suggest that NPY negatively regulates the genetic expression of POMC neurons via the Y2 NPY receptor subtype.

Effects of dehydroepiandrosterone (DHEA) on GnRH gene expression in the rat brain as studied by in situ hybridization.

Dehydroepiandrosterone (DHEA) is an adrenal androgen that is converted into potent androgens and/or estrogens in peripheral tissues. To further investigate the potential role of DHEA in reproductive functions in the rat, we have studied the effect of 2-day administration of DHEA on GnRH gene expression in brain of sham-operated and castrated animals of both sexes. In the male rat, orchiectomy induced an increase in the hybridization signal. In sham-operated animals, DHEA decreased GnRH mRNA levels induced by orchiectomy. In orchiectomized rats, DHEA also depressed the amount of mRNA levels and then reversed the increase in mRNA levels induced by orchiectomy. In female animals, as observed in the male, castration produced an increase in the hybridization signal. In both sham-operated and ovariectomized animals, DHEA administration increased mRNA levels. These data clearly indicate that DHEA administration can modify neuronal GnRH gene expression in adult rats of both sexes, the effect being inhibitory in the male and stimulating in the female. This modulation of GnRH neuronal activity, which is probably exerted following the conversion of DHEA into active sex steroids, might be at least partly responsible for modifications of the activity of the hypothalamo-pituitary-gonadal axis induced by DHEA.

Role of serotonin in the regulation of prolactin gene expression in the male rat as evaluated by in situ hybridization.

It is well documented that serotonin (5-HT) is involved in the regulation of prolactin (PRL) release as a stimulator. To evaluate the role of 5-HT on PRL gene expression, we have investigated the effects of repeated administration (during 2 days) of 5-HT, the 5-HT1 + 2 receptor antagonist methysergide, the 5-HT2 receptor antagonist ketanserin, and a combination of 5-HT and methysergide on PRL mRNA levels as measured by in situ hybridization in the adult male rat. The treatment with 5-HT induced a 14% increase in the hybridization signal, although the administration of methysergide produced a small reduction (5%) in PRL mRNA levels. On the other hand, ketanserin had no effect on the hybridization signal. The stimulatory effect of 5-HT was completely prevented by the concomitant administration of methysergide. The present results, together with previous data, indicate that 5-HT exerts a positive tonic influence not only on the release of PRL but on the biosynthesis of the hormone as evaluated by mRNA level measurements. They also strongly suggest that the role of 5-HT in the regulation of PRL secretion is mediated via activation of 5-HT1 receptors, although a dopaminergic effect of methysergide on PRL secretion cannot be totally excluded.

Negative regulation of proopiomelanocortin gene expression by GABAA receptor activation in the rat arcuate nucleus.

Activation of the GABAA-benzodiazepine receptor complex has previously been shown to inhibit the release proopiomelanocortin (POMC)-related peptides from the hypothalamus and to decrease mRNA levels in POMC neurons in the arcuate nucleus. To learn more about the precise role of the GABAergic system in POMC neuron regulation, we studied the effects of the administration (2 days) of the GABAA receptor agonist muscimol and the central-type barbiturate receptor agonist pentobarbital on POMC mRNA levels measured by in situ hybridization. Treatment with pentobarbital produced a 12% decrease in the hybridization signal. Similarly, muscimol treatment decreased the signal by 20%. The concomitant administration of the two GABAA receptor agonists resulted in a decrease (28%) of mRNA levels that was significantly more marked than that induced by pentobarbital or muscimol alone. The present results, together with previous data from our laboratory, indicate that different activators of the GABAA receptor complex, including barbiturates, can negatively regulate POMC neuronal activity in the rat arcuate nucleus.

Effects of morphine and naloxone on prolactin and growth hormone gene expression in the male rat pituitary gland.

It is generally admitted that opioids can stimulate the release of both prolactin (PRL) and growth hormone (GH). In order to investigate the role of opioids in the regulation of PRL and GH gene expression in the rat pituitary, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on both PRL and GH gene expression as measured by in situ hybridization. Four-day treatment with morphine (40 mg/kg/day) produced a 12% increase in PRL mRNA levels. Conversely, naloxone (4 mg/kg/day) decreased the autoradiographic reaction by 10%. The concomitant administration of morphine and naloxone induced no significant changes in PRL gene expression. On the other hand, treatment with morphine produced a 22% decrease in GH mRNA levels, an effect which was prevented by the concomitant administration naloxone. When injected alone, naloxone did not modify the hybridization signal. These results clearly indicate that opioids are involved not only in the regulation of GH and PRL release but also in the gene expression of the two hormones. The discordance observed between the acute effects of morphine on GH release and the effect of the opioid drug on mRNA levels remains to be clarified.

Opioid regulation of proopiomelanocortin (POMC) gene expression in the rat brain as studied by in situ hybridization.

Proopiomelanocortin (POMC) is the precursor of the potent opioid peptide beta-endorphin as well as a number of other active peptides. On the basis of neuroanatomical data indicating the presence of contacts between POMC neurons in the rat arcuate nucleus, it has been proposed that POMC neurons could be autoregulated. In order to investigate the role of opiates in the regulation of POMC gene expression in the rat arcuate nucleus, we studied the effects of chronic administration of the opioid drug morphine and an opiate receptor antagonist naloxone on POMC mRNA levels as measured by in situ hybridization, 4-day treatment with naloxone (4 mg/kg/day) produced a 60% increase in the number of silver grains overlying POMC neurons. Conversely, morphine (40 mg/kg/day) also administered during 4 days decreased the hybridization signal by 30%. The concomitant administration of morphine and naloxone completely prevented the effect of morphine on POMC gene expression indicating that the inhibitory influence of morphine is likely to be mediated by opioid receptors. The data obtained clearly indicate that activation of opioid receptors decreased the biosynthetic activity of POMC neurons and that conversely opiate receptor blockade caused an increase in the activity of these neurons. They are consistent with the hypothesis of an autoregulation of the POMC neuronal system by endogenous opiate peptide(s).