Michel Dubois-Dauphin

Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography

Sites which bind tritiated vasopressin (AVP) with high affinity were detected in the brain of male, adult rats, by light microscopic autoradiography. Their anatomical localization differed markedly from that of high affinity binding sites for tritiated oxytocin (OT) determined in the same animal. Co-labelling was minimized by using low concentrations of [3H]AVP and [3H]OT. Binding of the former occurred predominantly in several structures of the limbic system (septum, amygdala, bed nucleus of the stria terminalis, accumbens nucleus), in two hypothalamic nuclei (suprachiasmatic and dorsal tuber) and in the area of the nucleus of the solitary tract. Binding of OT was evidenced in the olfactory tubercle, the ventromedial hypothalamic nucleus, the central amygdaloid nucleus and the ventral hippocampus. The ligand specificity of the binding sites was assessed in competition experiments. Synthetic structural analogues were used, allowing to discriminate OT receptors (OH[Thr4,Gly7]OT) from V2 receptors (dDAVP and d[Tyr(Me)2]VDAVP), V1 receptors ([Phe2,Orn8]VT) and V1b receptors (desGly9d(CH2)5AVP). Our main conclusions are, firstly, that AVP and OT binding sites can be readily distinguished, and that there is virtually no overlap in their distribution in the rat brain. Second, we showed that the sites which bind AVP with high affinity in the brain are V1 receptors, different both from the renal V2 receptors and from the anterior pituitary V1b receptors. Our results support the conjecture that AVP and OT play a role in interneuronal communication in the brain.

Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study

Sites which bind oxytocin and vasopressin with high affinity were detected in the brain and upper spinal cord of 12 human subjects, using in vitro light microscopic autoradiography. Tissue sections were incubated with tritiated vasopressin, tritiated oxytocin or an iodinated oxytocin antagonist. The ligand specificity of binding was assessed with unlabelled vasopressin or oxytocin in excess, as well as in competition experiments using synthetic structural analogues. The distribution of vasopressin binding sites differed markedly from that of oxytocin binding sites in the forebrain, while there was overlap in the brainstem. Vasopressin binding sites were detected in the dorsal part of the lateral septal nucleus, in midline nuclei and adjacent intralaminar nuclei of the thalamus, in the hilus of the dentate gyrus, the dorsolateral part of the basal amygdaloid nucleus and the brainstem. The distribution of oxytocin binding sites in the brainstem has been recently reported (Loup et al., 1989). Oxytocin binding sites were also observed in the basal nucleus of Meynert, the nucleus of the vertical limb of the diagonal band of Broca, the ventral part of the lateral septal nucleus, the preoptic/anterior hypothalamic area, the posterior hypothalamic area, and variably in the globus pallidus and ventral pallidum. The presence of oxytocin and vasopressin binding sites in limbic and autonomic areas suggests a neurotransmitter or neuromodulator role for these peptides in the human central nervous system. They may also affect cholinergic transmission in the basal forebrain and consequently play a role in Alzheimers disease.

The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. Here we report that DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP‐null allele display less locomotor activity and free‐run with a shorter period than otherwise isogenic wild‐type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP−/− mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock.

Gonadal steroids regulate oxytocin receptors but not vasopressin receptors in the brain of male and female rats. An autoradiographical study.

The distribution and the amount of [3H]oxytocin binding were studied in the brain of adult rats of either sex, as well as in male and female castrates, some of which received injections of estradiol or testosterone. Intact males were treated with an aromatase inhibitor. Castration and inhibition of aromatase activity reduced, whereas estradiol and testosterone increased oxytocin binding, particularly in regions of the brain assumed to be involved in reproductive functions, such as the ventrolateral part of the hypothalamic ventromedial nucleus and the islands of Calleja and neighbouring cell groups. Binding of oxytocin to the uterus was also estrogen-dependent. In the same animals, we also studied the distribution of [3H]vasopressin binding sites present in the brain. It was similar in males and females, and was not affected by experimentally manipulating gonadal hormone levels. In immunocytochemical studies we noticed, as others had previously, that the vasopressin content of certain areas of the rat brain was affected by castration, whereas the oxytocin innervation was not. These results are discussed in relation to the possible functions of oxytocin in the brain and of the lack of correspondence between the immunocytochemical and the autoradiographic data.

Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.

Bax and Bcl‐2 Interaction in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Abstract : It has been proposed that mutations in copper/zinc‐superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl‐2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS‐linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl‐2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl‐2, Bcl‐XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl‐2 and Bcl‐XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl‐2 in transgenic mSOD1 mice, overexpression of Bcl‐2 increases the formation of Bcl‐2 : Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl‐2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl‐2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl‐2 : Bax heterodimers. Based on these findings, it may be concluded that Bcl‐2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.

Early appearance and transient expression of vasopressin receptors in the brain of rat fetus and infant. An autoradiographical and electrophysiological study

The development of vasopressin (AVP) receptors in the rat brain, spinal cord and pituitary gland was studied by in vitro light microscopic autoradiography. AVP binding sites were labeled using [3H]AVP in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the binding of [3H]AVP to oxytocin receptors was prevented by adding in the incubation medium a highly selective oxytocin agonist. Specific binding was first detected at E16 in the ventral pontine reticular formation. Many other brain areas were progressively labeled between E18 and PN5. The distribution of binding sites observed at PN5 remained unchanged until the beginning of the third postnatal week. Thereafter binding was markedly reduced or even disappeared in several areas, in particular in the facial nucleus. The adult distribution of AVP binding sites was established at the time of weaning. The properties of transient AVP binding sites in the facial nucleus were studied both by autoradiography and by electrophysiology. Non-radioactive AVP displaced [3H]AVP binding in this nucleus as efficiently as it did in the lateral septum of the adult. Single-unit extracellular recordings showed that AVP can excite facial motoneurones by interacting with receptors which are pharmacologically indistinguishable from V1 (vasopressor) type. Thus, AVP binding sites transiently expressed in the brain of fetal and infant rat probably represent functional neuronal receptors, having the same ligand selectivity and affinity than AVP binding sites present in the adult. This suggests that AVP acts not only as a neuropeptide in the adult brain but may play a significant role during maturation of the central nervous system.

Localization of oxytocin binding sites in the human brainstem and upper spinal cord: an autoradiographic study.

Two different ligands, tritiated oxytocin and a newly synthesized and monoiodinated oxytocin antagonist, were used to reveal sites which bind oxytocin in the brainstem and upper spinal cord of 12 human subjects. Tissue sections were incubated with either ligand at a concentration close to their respective dissociation constants determined in human uterus and rat brain. Specificity of binding was assessed in presence of unlabelled oxytocin in excess. Comparable results were obtained using tritiated or iodinated ligand. Labelling was most intense in the substantia nigra pars compacta, the substantiae gelatinosae of the caudal spinal trigeminal nucleus and of the dorsal horn of the upper spinal cord, as well as in the medio-dorsal region of the nucleus of the solitary tract. Binding was also detected in the rest of the nucleus of the solitary tract and in other areas, including the oral and interpolar parts of the spinal trigeminal nucleus, the hypoglossal nucleus and the area postrema. Presence of oxytocin binding sites in regions concerned with sensory, autonomic and motor processing suggests that oxytocin could act as a neurotransmitter or neuromodulator in the human central nervous system.

Rapid Generation of Stable Transgenic Embryonic Stem Cell Lines Using Modular Lentivectors

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time‐consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV‐1 element and woodchuck hepatitis virus post‐transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue‐specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.

Identification of BARD1 as Mediator between Proapoptotic Stress and p53-Dependent Apoptosis

The BRCA1-associated protein BARD1 is a putative tumor suppressor. We suggest that BARD1 is a mediator of apoptosis since (1) cell death in vivo (ischemic stroke) and in vitro is accompanied by increased levels of BARD1 protein and mRNA; (2) overexpression of BARD1 induces cell death with all features of apoptosis; and (3) BARD1-repressed cells are defective for the apoptotic response to genotoxic stress. The proapoptotic activity of BARD1 involves binding to and elevations of p53. BRCA1 is not required for but partially counteracts apoptosis induction by BARD1. A tumor-associated mutation Q564H of BARD1 is defective in apoptosis induction, thus suggesting a role of BARD1 in tumor suppression by mediating the signaling from proapoptotic stress toward induction of apoptosis.