Mouse Sebox homeobox gene expression in skin, brain, oocytes, and two-cell embryos.

Sebox is a mouse paired-like homeobox gene, previously named OG-9. Sebox genomic DNA and cDNA were cloned and sequenced. In addition, rat and human Sebox genomic DNAs were cloned and sequenced, and the predicted amino acid sequences were compared. The mouse Sebox gene was mapped to chromosome 11 near the Evi 2 locus. The mouse Sebox gene is expressed in brain, skin, ovary, and liver of mice. In the brain, the Sebox gene is expressed in the cerebral cortex and CA areas of the hippocampus, pontine nuclei, choroid plexus, and the cerebellum. Northern analysis and RNase protection assays revealed low levels of Sebox RNA in 12-day mouse embryos and higher levels in 18- and 19-day embryos. In late embryos and newborn mice, Sebox expression is localized in the epidermis. In adult mice, Sebox RNA was found in maturing oocytes and in fertilized eggs; however, the abundance of Sebox RNA is decreased in the two-cell embryo, and little or none was detected in the four-cell embryo. Hence, Sebox is a maternally expressed homeobox gene.

The mouse Nkx-1.2 homeobox gene: alternative RNA splicing at canonical and noncanonical splice sites.

A mouse homeobox gene, Nkx-1.2, (previously termed Sax-1) that is closely related to the Drosophila NK-1/S59 gene was cloned, and genomic DNA and cDNA were sequenced. Nine Nkx-1.2 cDNA clones were found that correspond to three species of Nkx-1.2 mRNA that are formed by alternative splicing at conventional 5' donor and 3' acceptor splice sites; however, seven cDNA clones were found that correspond to three species of Nkx-1.2 mRNA from testes that have novel TG/AC 5' and 3' splice sites. The consensus splice sequences are: 5' donor, CC downward arrowTGGAAG; 3' acceptor, ACTTAC downward arrow. Predicted amino acid sequences suggest that some transcripts may be translated into proteins that lack part or all of the homeodomain. At least three bands of Nkx-1.2 mRNA were found in RNA from the testes. Nkx-1.2 mRNA was shown to be present in postmeiotic germ cells of the testis and in mature spermatozoa. Nkx-1.2 mRNA also was found in regions of the adult cerebral cortex, hippocampus, diencephalon, pons/medulla, and cerebellum. Nkx-1.2 mRNA was found in embryos in highest abundance in 10-day embryos; the mRNA levels decrease during further development. Nkx-1.2 mRNA also was found in discrete zones of the embryonic mesencephalon and myelencephalon.

Homeobox gene Prx3 expression in rodent brain and extraneural tissues.

Different cDNA clones encoding a rat homeobox gene and the mouse homologue OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains). Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the "aristaless domain." Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum revealed marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also expressed the Prx3 gene. In the adult rat brain, Prx3 gene expression was restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation.

Cloning and characterization of four murine homeobox genes.

Four novel murine homeobox genes, Uncx-4.1, OG-2, OG-9, and OG-12, were cloned and partially sequenced. The amino acid sequence of the mouse Uncx-4.1 homeodomain is closely related to the sequence of the unc-4 homeodomain of Caenorhabditis elegans. However, the OG-2, OG-9, and OG-12 homeodomains are relatively diverged and are not closely related to any previously described homeodomain. Northern blot analyses revealed multiple bands of Uncx-4.1, OG-2, OG-9, and OG-12 poly(A)+ RNA in RNA from mouse embryos and adults that change during development and showed that each gene is expressed in a tissue-specific manner. OG-12 cDNAs were cloned that correspond to two alternatively spliced species of OG-12 mRNA. Three major bands of Uncx-4.1 poly(A)+ RNA were found only in RNA from adult mouse brain, but an additional band was observed in RNA from all of the other tissues tested. Major bands of OG-9 and OG-2 poly(A)+ RNA were found only in RNA from striated muscle; however, trace bands were detected in RNA from other tissues.

Heterologous sensitization of adenylate cyclase activity by serotonin in the rat cerebral cortex.

In vitro exposure of rat cerebrocortical slices to microM concentrations of serotonin (5HT) results in an increased response of adenylate cyclase to isoproterenol (ISO). No change in the affinity of the beta-adrenoceptor toward the agonist was found after 5HT exposure when measuring ISO displacement of [3H]CGP 12177 binding. A similar increase of adenylate cyclase response was also found when using VIP as a stimulatory agent. The dose-response curve of adenylate cyclase to the GTP analogue, GppNHp, was modified by 5HT, which promotes a significantly higher maximal response without altering the potency of GppNHp. Forskolin-stimulated adenylate cyclase activity was not affected by 5HT. Serotonergic 5HT2 receptors are involved in the sensitization of adenylate cyclase to GppNHp, since the selective 5HT2 antagonist ketanserin inhibits the effect of 5HT, whereas the 5HT2 agonist DOI mimics 5HT. The involvement of 5HT2 receptor-coupled activation of protein kinase C is also demonstrated: direct protein kinase C activators such as phorbol esters and s,n-dioctanoylglycerol behave in the same manner as 5HT, while the protein kinase C inhibitor CGP 41251 prevents 5HT from increasing adenylate cyclase responsiveness to GppNHp. Moreover, in vitro exposure of cortical slices to 5HT results in reduced inhibition of adenylate cyclase by somatostatin. Since no change was observed at the receptor level and in the direct stimulation of the catalytic subunit of the enzyme, we propose that 5HT might accomplish the sensitization of adenylate cyclase through protein kinase C by inactivating the inhibitory coupling protein Gi and facilitating the interaction of the exogenous GppNHp with the stimulatory coupling protein Gs.

Structure and evolution of four POU domain genes expressed in mouse brain.

Four mouse POU domain genomic DNA clones--Brain-1, Brain-2, Brain-4, and Scip--and Brain-2 cDNA, which are expressed in adult brain, were cloned and the coding and noncoding regions of the genes were sequenced. The amino acid sequences of the four POU domains are highly conserved; sequences in other regions of the proteins also are conserved but to a lesser extent. The absence of introns from the coding regions of the four POU domain genes and the similarity of amino acid sequences of the corresponding proteins suggest that the coding region of the ancestral class III POU domain gene lacked introns and therefore may have originated by reverse transcription of a molecule of POU domain mRNA followed by insertion of the cDNA into germ cell genomic DNA. Additional duplications of the ancestral class III POU domain gene (or mRNA) would create the Brain-1, Brain-2, Brain-4, and Scip genes.

Effect of different photoperiod exposure on [3H]imipramine binding and serotonin uptake in the rat brain.

Seasonal rhythmicity in the occurrence of acute depressive episodes and the therapeutic efficacy of light exposure suggest the possible involvement of the pineal gland or other biological oscillators in the pathophysiology of depressive illness. We have performed studies to clarify whether different light/dark (LD) cycle schedules may induce changes in the biochemical targets of antidepressants in the rat CNS. In particular, we have investigated the effect of short- (LD 8:16) or long-day (LD 14:10) photoperiods on different biochemical parameters of serotonergic neurons. A significant increase in the density of [3H]imipramine ([3H]IMI) binding and in the Vmax of 5-[3H]hydroxytryptamine (5-[3H]HT) uptake was found in the hypothalamus of LD 8:16-with respect to LD 14:10-exposed rats, whereas no difference was found in the kinetic properties of postsynaptic 5-HT receptors and in 5-HT metabolism in the hypothalami and cerebral cortices of rats exposed to the two different photoperiods. A seasonal rhythm of [3H]IMI binding sites and 5-HT uptake seems to exist only in certain brain areas, such as the hypothalamus, because no differences were found in the cerebral cortex of LD 14:10- and LD 8:16-accustomed rats. [3H]IMI binding and 5-HT uptake were significantly increased in the hypothalamus of rats accustomed to a light/dark-inverted cycle (DL 10:14) and killed 6 h after the stopping of lighting in comparison to rats exposed to normal LD 14:10 cycles and killed 6 h after the beginning of lighting. Therefore, a circadian modification of the serotonergic presynaptic sites seems to be present and related to light/dark exposure. Because the existence of endogenous compounds able to modulate [3H]IMI binding and 5-HT uptake, other than 5-HT, has been postulated in the mammalian brain, the involvement of these substances in the periodic changes observed could be suggested.

Age-related changes in rat serotonergic and adrenergic systems and in receptor responsiveness to subchronic desipramine treatment.

Noradrenergic (NA) and serotonergic (5-HT) receptor profiles were compared in the cerebral cortex of young adult (3 months old) and aged (24 months old) male Sprague Dawley rats. Beta and alpha-1 receptors were significantly decreased in 24 month old rats, whereas alpha-2 receptors remained unchanged. 5-HT-2 Postsynaptic receptors and 5-HT high affinity uptake were reduced in aged animals; on the other hand the number of 3H-imipramine (3H-IMI) recognition sites located on serotonin nerve terminals and labelled also by 3H-paroxetine (3H-PAR), were significantly higher in the cerebral cortex of old rats. The ratio 5-hydroxyindoleacetic acid (5-HIAA)/5-HT, an index of serotonin turn-over, increased in some brain areas of senescent rats. There were no age-related changes in the responsiveness of investigated binding sites to subchronic desipramine treatment; the density of beta-noradrenergic and 5-HT-2 serotonergic receptors and that of 3H-IMI binding sites was reduced both in young adult and aged rats.

Absence of [3H]SCH 23390 specific binding sites in anterior pituitary: dissociation from effects on prolactin secretion.

We extended a previous study that had shown the selective D1 receptor antagonist SCH 23390, at relatively high doses, to stimulate prolactin (PRL) secretion in the rat and weakly inhibit [3H]spiperone binding to striatum and anterior pituitary (AP) membranes. No specific [3H]SCH 23390 binding sites, up to the micromolar range, were detected in rat AP while specific, saturable [3H]SCH 23390 binding sites (low nanomolar range) were observed in the striatum. In vivo SCH 23390 (1 mg/kg s.c.) induced higher plasma PRL levels, not reversible by the D1 agonist SKF 38393. Similarly the postsynaptic serotonin (5-HT) antagonists metergoline and cyproheptadine did not influence the SCH 23390 effect on PRL. SCH 23390 was also unable to antagonize the decrease of PRL secretion induced by the selective D2 agonist LY 171555. However this latter compound prevented SCH 23390 as well as sulpiride from increasing the PRL concentrations above the control values. These data rule out the possibility that D1 or 5-HT receptors mediate the stimulation of PRL release by SCH 23390. This effect is more likely to be due to a weak indirect interaction with AP-D2 receptors, as indicated by the non-competitive inhibition of [3H]spiperone binding to AP exerted by SCH 23390. Alternatively, non-specific mechanisms triggered by the multiple behavioral changes elicited by such high doses of SCH 23390 may be involved.

Endocrine effects of 5-methoxytryptoline, 5-hydroxytryptoline and tryptoline, putative modulators of rat serotonergic system.

The effects of tryptolines (TH beta Cs), putative endogenous compounds acting on the serotonergic function, have been studied on endocrine parameters in rats. In particular, graded doses of 5-methoxytryptoline (5-MeOT), 5-hydroxytryptoline (5-OHT) and tryptoline (Tp) were ip or iv administered to the animals and the circulating titers of prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) were assayed either at 20 min or at various times after the injection of the compounds. The data herein reported show that TH beta Cs exert endocrine effects, at least in a pharmacological condition. However, the three compounds unequally affect anterior pituitary function. In fact, while 5-MeOT, 5-OHT and Tp all enhance plasma PRL concentrations in a quick, short lasting and dose-related manner, 5-MeOT and Tp induce also decreases of serum LH levels in ovariectomized rats, and 5-MeOT only is able to diminish plasma GH titers. These findings underline an endocrine effect for TH beta Cs in pharmacological conditions and may suggest a functional role for these compounds in the control of anterior pituitary function.

Effects of repeated tiapride administration on anterior pituitary dopamine receptors and prolactin release in the rat.

The substituted benzamides tiapride and sulpiride, and the classic neuroleptic haloperidol, were studied in the rat to assess their interaction with the anterior pituitary (AP) dopamine (DA) receptors both in vitro ([3H]spiperone binding) and in vivo prolactin-PRL-release). Tiapride weakly inhibited [3H]spiperone binding in both pituitary and striatal membranes with affinity 5-7 times lower than sulpiride and 400-300 times lower than haloperidol. All three drugs were more potent in displacing [3H]spiperone from striatum than from AP. In vivo, tiapride produced weak and transient stimulation of PRL release reaching a full effect at 2 mg/kg i.p. Similar doses of sulpiride produced longer-lasting effects. Haloperidol was more potent than both benzamides. In prolonged treatments (15 or 60 days), tiapride, given twice daily at 0.5 mg/kg i.p., did not modify [3H]spiperone binding in either AP or striatum, nor did it induce significant changes of basal PRL levels. The challenge with a low threshold dose of TIA (0.2 mg/kg ip) produced similar increases of PRL release in the group either treated with TIA or saline. The data indicate that the benzamides examined have low potency for interaction with DA receptors in pituitary and striatum. In particular, tiapride displayed weaker affinity for AP-DA receptors than the other drugs and induced only slight stimulation of PRL levels. Results from repeated tiapride administration indicate that the drug, at a clinically relevant dose, is unable to modify either kinetic characteristics of DA receptors in the pituitary or plasma PRL levels.

Possible involvement of ovarian mechanisms other than estrogen-progesterone secretion in the regulation of glutamic acid decarboxylase activity of the rat fallopian tubes.

This study was performed to clarify the physiological role of the ovary in regulating the glutamic acid decarboxylase (GAD) activity in rat Fallopian tubes. To this purpose, GAD activity of the oviduct was evaluated in the following experimental conditions: immature or adult castrated (CX) rats; immature or adult CX rats treated with graded doses of estradiol benzoate (EB) or a fixed dose of EB and progesterone; adult CX rats bearing Silastic implants able to produce steady state estradiol plasma levels in the range of diestrous values; and prepubertal rats treated with ovulatory or anovulatory doses of exogenous gonadotropins (PMS and hCG). Moreover, the possible fluctuations of both gamma-aminobutyric acid (GABA) concentrations and GAD activity in the Fallopian tubes were studied during the estrous cycle. The results show that the prepubertal rat oviduct possesses a GABA content and a GAD activity analogous to those of normal diestrous rats. The GAD activity measured with the CO2 formation method was well correlated with the formation of labeled GABA, indicating that tubes of prepubertal rats are able to form the neurotransmitter by means of specific decarboxylation of glutamate. GAD activity, but not GABA levels, was increased over control values by the administration of exogenous gonadotropins. The role of the ovary in both adult and prepubertal rats to regulate this enzymatic activity is further stressed by the results of the experiments performed in CX animals which showed that ovariectomy produced a 4- to 5-fold decrease in GAD activity independent of the age of the animals. However, implantation of Silastic estradiol-containing capsules in adult CX animals or the administration of EB for 5 days in a dose range from 0.001-6.4 micrograms/day to adult ovariectomized animals and from 0.001-0.2 microgram/day to prepubertal animals did not modify GAD activity in spite of marked peripheral estrogenization of the animals evidenced by increases in uterine weight. Moreover, no variation of the enzymatic activity was observed at puberty (assessed by the age at vaginal opening). The administration of progesterone (0.2 mg) plus EB (0.01 microgram) did not produce any significant variation in GAD activity. GABA content and GAD activity of the tubes did not change during the estrous cycle. We, therefore, believe that other ovarian, still unidentified, secretions might be involved in the regulation of GAD activity in rat Fallopian tubes.

Interaction of putative endogenous tryptolines with the hypothalamic serotonergic system and prolactin secretion in adult male rats.

The effect of 5-methoxytryptoline (5-MeOT), 5-hydroxytryptoline (5-OHT) and tryptoline (Tp), putative endogenous derivatives of the tryptamines, on plasma prolactin (PRL) concentrations has been investigated in the adult male rat. The possible involvement of the hypothalamic serotonergic system has been considered in the mediation of the hormonal effect of the tryptolines. Therefore, plasma PRL levels have been evaluated in rats receiving tryptolines after different pharmacological manipulations of central serotonergic function. Although the three compounds increased the plasma titers of PRL in a dose-dependent manner and enhanced the hypothalamic content of serotonin (5HT), they appear to affect the serotonergic system through different mechanisms. In particular, 5-OHT might act at a presynaptic level, since its hyperprolactinemic effect was antagonized both by the depletion of central 5HT content after p-chlorophenylalanine and by the degeneration of serotonergic terminals after 5,7-dihydroxytryptamine. In contrast, 5-MeOT behaved as if it had a postsynaptic site of action, being counteracted by the serotonergic postsynaptic antagonists metergoline and cyproheptadine. The unsubstituted tetrahydro-beta-carboline, Tp, is probably active at both pre- and postsynaptic sites. The enhancing effect of Tp on PRL secretion was antagonized by chronic treatment with p-chlorophenylalanine, while it was also maintained in 5,7-dihydroxytryptamine-lesioned rats. These findings suggest that tryptolines may play a functional role in PRL secretion by interacting with central serotonergic systems through different biochemical mechanisms.