Luciana Giardino

Inhibin subunits in human placenta: Localization and messenger ribonucleic acid levels during pregnancy

This study describes the difference in distribution and levels of inhibin alpha, beta A- and beta B-subunit messenger ribonucleic acids in human placenta during pregnancy. Northern blot analysis indicated that inhibin alpha messenger ribonucleic acid is present in placental extracts collected at the early stage of gestation. Hybridization to inhibin beta A messenger ribonucleic acid was detected in the first trimester but in much lower levels. However, the intensity of the hybridization signal for inhibin alpha- and beta A-subunit messenger ribonucleic acids was greater in extracts prepared from term placentas than in those from the first or second trimester of pregnancy. Low levels of inhibin beta B-subunit messenger ribonucleic acid were observed only in extracts prepared from term placenta. At both early stage and term gestation trophoblast cells showed a positive fluorescent signal with the inhibin alpha-, beta A- and beta B-subunit-specific antisera. However, whereas inhibin alpha-subunit was localized in the cytotrophoblast, inhibin beta B-subunit immunoreactivity was observed in the syncytial layer of the villi, and inhibin beta A-subunit was widely distributed. The different distribution of immunoreactive inhibin subunits was confirmed by in situ hybridization, showing the different localizations of the inhibin messenger ribonucleic acids. These results showed that (1) human placenta produces the inhibin alpha- and beta A-subunits as early as the first trimester of pregnancy, (2) messenger ribonucleic acid levels for each of the three inhibin subunits are highest at term, and (3) immunoreactive inhibin subunits are localized differently in placental villi.

Distribution of thyrotropin-releasing hormone receptor messenger RNA in the rat brain: An in situ hybridization study

Based on the recent cloning of the mouse thyrotropin-releasing hormone receptor, oligonucleotide probes complementary to the DNA sequence were constructed and used for in situ hybridization studies on the rat brain. Thyrotropin-releasing hormone receptor messenger RNA was found in many areas of the brain, mostly showing high degree of overlap with the distribution thyrotropin-releasing hormone binding sites as previously revealed in autoradiographic studies. Thus, a strong signal was observed in the accessory olfactory bulb, the perirhinal sulcus, the ventral aspects of the hippocampal formation, some amygdaloid nuclei, the diagonal band nucleus, parts of nucleus accumbens, the bed nucleus of the stria terminalis, dorsomedial, lateral and perifornical hypothalamic regions, the septohippocampal nucleus, parts of the vestibular complex, as well as many bulbar motoneurons including the facial, dorsal vagal, ambiguus and hypoglossal nuclei, the superficial layer of the spinal trigeminal nucleus, and motoneurons and dorsal horn neurons in the spinal cord. Cells within one and the same nucleus expressed varying levels of thyrotropin releasing hormone receptor messenger RNA suggesting marked differences in rate of receptor synthesis. Most of these areas receive an input by thyrotropin-releasing hormone-positive nerve endings. Taken together these results suggest that thyrotropin-releasing hormone receptors are mostly localized in the vicinity of the cell bodies which express thyrotropin-releasing hormone receptor messenger RNA and mediate the wide range of actions that have been recorded after administration of exogenous thyrotropin-releasing hormone.

Response of Hypothalamic Peptide mRNAs to Thyroidectomy

Using in situ hybridization histochemistry, we have investigated the effect of thyroid hormone on the expression of several peptide mRNAs in the hypothalamic paraventricular nucleus (PVN) of adult male rats. Hypothyroidism was induced by surgical ablation of the thyroid gland. The animals (control sham-operated, thyroidectomized, thyroidectomized+T4 replaced rats) were studied 28 and 50 days after surgery. Sections of the PVN were hybridized using synthetic oligonucleotide probes complementary to mRNA for thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), galanin (GAL), enkephalin (ENK), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and vasopressin (VP). GAL mRNA was also analyzed in the anterior paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus. At the PVN level, a feedback effect of thyroid hormone on TRH synthesis was demonstrated by the TRH mRNA increase in hypothyroidism and by its decrease in hyperthyroidism. Hypothyroidism caused a dramatic decrease in GAL mRNA in parvo- and magnocellular PVN neurons both 28 and 50 days after thyroid ablation, whereas no effect was seen in VP mRNA, the main peptide hormone coexisting with GAL. The T4 replacement prevented the GAL mRNA impairment. Hypothyroidism did not influence GAL mRNA in the anterior PVN, perifornical area or in the arcuate nucleus, whereas a decrease in GAL mRNA was observed in the dorsomedial nucleus. VIP mRNA, which is undetectable in the PVN of normal animals, was present in several PVN neurons after thyroidectomy. CRH mRNA was decreased after thyroidectomy, whereas the T4 restitution caused an upregulation. The levels of ENK or NT mRNA were not significantly affected by the thyroid status. The present results show that, in addition to TRH mRNA, other hypothalamic peptide mRNAs are affected by thyroid hormone levels.

Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in arbitrary units, is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.

Multiple neurochemical action of clozapine: A quantitative autoradiographic study of DA 2, opiate and benzodiazepine receptors in the rat brain after long-term treatment

The atypical neuroleptic clozapine has clinical and behavioral properties that differ not only from the typical compounds, but also from atypical ones. It interacts with the dopaminergic systems, but also produces effects on the serotoninergic, GABA-ergic, cholinergic systems. In spite of the amount of papers devoted to its study, the profile of the neurochemical action of this drug is still confuse. In this paper we investigated the DA 2-, opiate- and benzodiazepine-receptor modifications induced by the long term (21 days) treatment with clozapine 20 mg/kg/day in the rat brain. We found a decrease of DA 2 receptor density in the target areas of the mesolimbocortical system (ventral n. caudate-putamen, cerebral cortex except for the anterior cingulate at the most anterior level and the n. accumbens) and a decrease of opiate and benzodiazepine receptors in the cerebral cortex and in the olfactory tubercle. Opiate receptors increase in the patches of the striatum. We also compared these effects with those produced by long-term (21 days), low-dosage (0.5 mg/kg day) haloperidol.

Deficit of Galanin-Like Immunostaining in the Median Eminence of Adult Hypothyroid Rats

In this paper we describe the modification of the galanin (GAL)-like immunostaining in the hypothalamus of rats, which were made hypothyroid at 52 days after birth. On 21st day after the surgical ablation of the thyroid gland, the staining of the GAL-immunoreactive fibers in the median eminence decreased and on the 84th day disappeared almost totally. The GAL-immunoreactive distribution in other areas of the hypothalamus, e.g. the anterior hypothalamus and the dorsomedial nucleus, is only slightly affected by the absence of thyroid hormones, whereas the GAL-staining of medulla oblongata (vagal complex) is equal in both control and hypothyroid rats. In hypothyroid colchicine-treated rats, we were unable to stain GAL-immunoreactive neurons in the paraventricular nucleus (PVN). Oxytocin- and vasopressin-like material was present in the magnocellular neurons and the staining pattern in hypothyroid rats was the same as that of control animals. Our data show a marked reduction in the expression of the GAL-like immunoreactivity of the PVN and median eminence of adult hypothyroid rats. The possible role of this deficit in the pathogenesis of the GH secretion impairment that is observed in hypothyroid rats is discussed.

Muscarinic and gamma-aminobutyric acid-ergic receptor changes during vestibular compensation

SummaryInvolvement of the neurotransmitters acetyl choline and gamma-aminobutyric acid (GABA) in vestibular compensation has been suggested by electrophysiological and pharmacological experiments. In this investigation we used quantitative autoradiography to study the modification of muscarinic and benzodiazepine receptors in each nucleus of the rats vestibular nucleus complex. Tissues were examined 3, 14, 23 h and 3, 12, 37 and 90 days after unilateral surgical labyrinthectomies. The present results demonstrated a muscarinic receptor supersensitivity in the deafferented side in the suprerior vestibular nucleus 90 days after surgery. This increase was not large enough to support the cholinergic receptor supersensitivity hypothesis for vestibular compensation. The changes in the benzodiazepine receptors observed for a short time following surgery were reversed after a few days. These findings support a transient involvement of GABAergic pathways in vestibular compensation.

Iodinated-NPY binding sites: Autoradiographic study in the rat brain

Several authors have described the presence of iodinated neuropeptide-Y binding sites on membranes of the mammalian CNS. In the present study we show a mapping of iodinated-NPY binding sites in the rat brain using receptor autoradiography. The sections were incubated with 125I-Bolton-Hunter coupled NPY (0.5-03 nM), in the absence or presence of 1 microM cold NPY. Some autoradiograms are studied by means of an image analyzer (VDC 501 Tesak) equipped with the host computer PDP 11 Digital, in order to enhance the contrast of the labeling. A very high density of NPY receptors is present in the limbic regions (hippocampus, amygdaloid complex, septal nuclei), in the cortex, and in some thalamic nuclei, while in some hypothalamic regions (paraventricular nucleus and median eminence) we detected a lower amount of NPY receptors. At the mesencephalic level, the substantia nigra presents a very high density of NPY receptors.

Maternal decidua and fetal membranes contain immunoreactive neuropeptide Y

The aim of the present study was to evaluate whether the various intrauterine tissues contain immunoreactive neuropeptide Y (NPY). Previous observations showed that human placenta produces NPY and that it may play a local role. Using a polyclonal NPY antiserum and an im-munofluorescent technique, sections of maternal decidua, amnion and chorion collected from a pregnant women at parturition were studied. An intense positive staining for NPY was observed in epithelial amnion cells and in chorionic cytotrophoblast. Some of the maternal decidual cells showed a weaker signal of immunoreactive NPY. In evaluating whether NPY may coexist with other hormones in these tissues, adjacent slices of decidua, amnion and chorion were stained with activin ßB subunit antiserum. In the various tissues a relevant number of cells showed positive signals for both NPY and activin. The present findings showed that the various intrauterine tissues contain NPY and that in a large number of cells of amnion, chorion and decidua the neuropeptide is colocalized with immunoreactive activin. In view of the physiological implications of NPY in the regulation of uterine contractility and of placental hor-monogenesis, the present findings indicate a large distribution of NPY in the various intrauterine tissues.

Daily Modifications of 3h-Naloxone Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study

The endogenous opioid peptides, the opiate receptors and several related behaviours, like opioid-mediated analgesia, show daily variations in different animal species including rats. The attempt to correlate the daily rhythm of opiate receptors in the central nervous system (CNS) to opiate related rhythmic phenomena requires an experimental approach with a high anatomical resolution, as the opioid distribution is very heterogeneous. In this paper we present the study of daily variations of 3H-naloxone binding sites in the different regions of the adult male rat brain, performed by means of quantitative autoradiography. Five rats are sacrificed at each investigated time of the day (0200, 0600, 1000, 1400, 1800 and 2200). The ligant is 3H-naloxone (4 nM), the quantification is performed by means of densitometric procedures (image analyzer Tesak VDC 501, computer Digital PDP 11, 3H-microscale). The statistical analysis is performed according to the single Cosinor method and the one-way analysis of variance followed by the multiple range test of Duncan. We analysed 33 different regions of the rat CNS, and the daily variations of opiate receptors are regionally selective. A circadian rhythm is found in the anterior cingulate cortex, hippocampal cortex, periventricular, medial, ventral, reticular and posterior nuclei of the thalamus, rhomboid, gelatinosus and rheuniens nuclei, lateral hypothalamus, locus coeruleus, grey substance of the pons, reticular formation of medulla oblongata, inferior olivary complex, medial part of the nucleus of the solitary tract and nucleus of the spinal tract of the trigeminal nerve. An ultradian rhythm is found in the medial and lateral preoptic areas, in the medial hypothalamus, in the medial and in the lateral nuclei of habenula. No significant variations during 24 hr according to the Cosinor analysis are found in the dorsal and lateral cerebral cortex, striatum, globus pallidus, bed nucleus of the stria terminalis, septal nuclei, lateral nucleus of the thalamus, cochlear nuclei, nucleus of the solitary tract, lateral and caudal parts, dorsal motor nucleus of the vagal nerve, XII and IX nerve nuclei. The amplitude of the daily variations observed ranges from 10 to 40%. Our results demonstrate the high anatomical selectivity of the daily modifications of 3H-naloxone binding sites in the rat CNS. They also indicate that quantitative autoradiography is a suitable and sensitive technique for these studies.