A Na(+)/Cl(-)-dependent transporter for catecholamines, identified as a norepinephrine transporter, is expressed in the brain of the teleost fish medaka (Oryzias latipes).

We report the isolation, functional characterization, and localization of a Na(+)/Cl(-)-dependent catecholamine transporter (meNET) present in the brain of the teleost fish medaka. This carrier is very similar to the human neuronal norepinephrine transporter (NET) and the human neuronal dopamine transporter (DAT), showing 70 and 64% amino acid identity, respectively. When expressed in COS-7 cells, this transporter mediates the high-affinity uptake of dopamine (K(M) = 290 nM) and norepinephrine (K(M) = 640 nM). Its pharmacological profile reveals more similarities with NET, including a high affinity for the tricyclic antidepressants desipramine (IC(50) = 0.92 nM) and nortriptyline (IC(50) = 16 nM). In situ hybridization on the medaka brain shows that meNET mRNA is present only in a subset of tyrosine hydroxylase-positive neurons found in the noradrenergic areas of the hindbrain, such as the locus ceruleus and area postrema. None of the dopaminergic areas anterior to the isthmus contains any labeled neurons. Neither reverse transcriptase-polymerase chain reaction with degenerate primers specific for gamma-aminobutyric acid transporter/NET nor autoradiographic experiments with [(125)I]3b-(4-iodophenyl)-tropane-2b-carboxylic acid methyl ester revealed an additional catecholamine transporter in the medaka brain. Uptake experiments with medaka brain synaptosomes show an endogenous transport with a pharmacological profile identical to that of the recombinant meNET. Thus, meNET is probably the predominant--if not the only--catecholamine transporter in the medaka fish brain. In view of the highly conserved primary structures and pharmacological properties of meNET, it is tempting to speculate that a specific dopamine transport developed later in vertebrate evolution and probably accompanied the tremendous enlargement of the meso-telencephalic dopaminergic pathways in amniotes.

Distribution of the orphan nuclear receptor Nurr1 in medaka (Oryzias latipes): cues to the definition of homologous cell groups in the vertebrate brain.

The orphan nuclear receptor Nurr1 has been extensively studied in mammals and shown to contribute to the differentiation of several cell phenotypes in the nervous and endocrine systems. In this study, the gene homologous to the mammalian Nurr1 (NR4A2) was isolated in the teleost fish medaka (Oryzias latipes), and the distribution of its transcripts was analyzed within brains of embryos and adults. Nurr1 has a widespread distribution in the medaka brain. Large amounts of Nurr1 transcripts were found in the intermediate nucleus of the ventral telencephalon, preoptic magnocellular nucleus, ventral habenula, nucleus of the periventricular posterior tuberculum, and nuclei of glossopharyngeal and vagus nerves. To search for homologous cell groups between teleost fish and tetrapods brains, the co-localization of Nurr1 and tyrosine hydroxylase (TH) transcripts was analyzed. Neither Nurr1 nor TH expression was detected in the ventral midbrain, but both transcripts were present in the periventricular nucleus of the posterior tuberculum. This observation supports the hypothesis that this nucleus is homologous to dopaminergic mesencephalic nuclei of mammals. The presence of Nurr1 in the preoptic magnocellular nucleus of medaka and paraventricular hypothalamic nucleus of mammals reinforces the hypothesis of homology between these areas. TH and Nurr1 transcripts are also co-localized, among others, in the nucleus of the paraventricular organ and nucleus of the vagus nerve. This work suggests that the differentiating role of Nurr1 in the central nervous system is conserved in gnathostomes.

[Evolution and development of dopaminergic neurotransmitter systems in vertebrates]. / Evolution et développement des systèmes neuromodulateurs dopaminergiques chez les Vertébrés.

Dopamine is a widespread neurotransmitter which exerts numerous neuromodulatory actions in the vertebrate central nervous system. This pleiotropic activity relies on the organisation of dopamine-synthesizing neuronal pathways and on a multiplicity of specific membrane receptors. A comparative approach has been undertaken to gain clues on the genetic events which took place during evolution to devise the dopamine systems of modern vertebrates. The localisation and phenotype of dopamine-synthesizing neurones is determined by different gene networks in each of the dopaminergic nuclei. However, despite this amazing diversity, the overall organisation of the dopaminergic nuclei is strinkingly conserved in the main vertebrates groups. In sharp contrast, the number of dopamine receptors subtypes has been multiplied by two major steps of gene duplications during vertebrates evolution. The first one occurred in the lineage leading to agnathans, whereas the second was concomitant to the emergence of cartilaginous fish. Accordingly, three subtypes exist in D1 receptor class (D1A, D1B, D1C) in all the jawed vertebrates, with two exceptions: eutherian mammals where only two D1 subtypes are found (D1A, D1B) and archosaurs where a fourth subtype is present (D1D). Comparisons of the pharmacological and biochemical characteristics of the dopamine receptors in vertebrate groups revealed homologous features that define each of the receptor subtypes and that have been fixed after gene duplications. The comparison of the distribution of the D1 receptor transcripts in the brain of teleosts and mammals points to significant conserved or derived expression territories, revealing previously neglected aspects of dopamine physiology in vertebrates.

Distribution of the mRNA encoding the four dopamine D(1) receptor subtypes in the brain of the european eel (Anguilla anguilla): comparative approach to the function of D(1) receptors in vertebrates.

Four subtypes of D(1) dopamine receptors are expressed in the brain of the European eel (Anguilla anguilla), an elopomorph teleost. To correlate this molecular multiplicity with specific localisation and functions, the distribution of the D(1) receptor transcripts was analysed by in situ hybridisation. The four D(1) receptor transcripts exhibit largely overlapping expression territories. In telencephalon, they are found in the olfactory bulb and the dorsal telencephalon (except its lateral part) but are most abundant in the subpallial areas. More caudally, the entopeduncular nucleus, preoptic nuclei, preglomerular nuclear complex, ventral thalamus, periventricular hypothalamus, optic tectum and cerebellum, all contain various amounts of D(1) receptor transcripts. Finally, D(1) receptor mRNAs are present in nuclei associated with the cranial nerves. The two D(1A) receptor subtypes are generally the most abundant and present a different distribution in several areas. The D(1B) mRNA, although present in fewer areas than D(1A) transcripts, is the most abundant in ventrolateral telencephalon and torus semicircularis. The D(1C) receptor transcript, which has not been found in mammals, is restricted to diencephalon and cerebellum. In view of the expression territories of D(1) receptor transcripts and previous data, some areas of the everted telencephalon of teleost may be homologous to regions of the tetrapod brain. In particular, D(1) expression territories of the ventral telencephalon are likely to be equivalent to striatal areas. These observations suggest an evolutionary scenario in which the D(1A) receptor subtype was highly conserved after the first gene duplication during the evolution of craniates, whereas D(1B) and D(1C), and their associated specific characteristics, appeared later, probably in the gnathostome lineage.

Developmental regulation of the serpin, protease nexin I, localization during activity-dependent polyneuronal synapse elimination in mouse skeletal muscle.

During vertebrate neuromuscular development, all muscle fibers are transiently innervated by more than one neuron. Among the numerous factors shown to potentially influence the passage from poly- to mononeuronal innervation, serine proteases and their inhibitors appear to play important roles. In this regard, protease nexin I (PNI), a potent inhibitor of the serine protease, thrombin, is highly localized to the neuromuscular junction (NMJ). In turn, thrombin is responsible for activity-dependent synapse elimination both in an in vitro model, and in vivo. In the present study, we used a monospecific anti-PNI polyclonal antibody to study the developmental kinetics of PNI expression in mouse leg skeletal muscle. By using immunoblotting, we detected PNI at embryonic day 16 (E16), as a 48-kDa band. This 48-kDa PNI band became prominent in leg muscle extracts at postnatal day 5 (P5) and remained so in extracts from adult muscle. In contrast, a higher molecular weight immunoreactive PNI band, which was sodium dodecyl sulfate- and beta-mercaptoethanol-resistant, was first detected at E16, increased at birth (P0), and then decreased at P15, i.e., after the wave of polyneuronal synapse elimination had occurred in these muscles. The results of an enzyme-linked immunosorbent assay, measuring active, complexed, and truncated PNI, correlated with Western blot data. We used immunocytochemistry to probe the localization of PNI at the NMJ and found that PNI was present in the cytoplasm of myotubes at E16, but neither then nor at birth did it colocalize with acetylcholine receptors. PNI became localized at NMJs by P5 and increased by P15, after which it remained stably concentrated there in the adult. Finally, we studied the gene expression of PNI mRNA, by using Northern blotting, and showed that PNI mRNA was present in skeletal muscle and remained stable throughout the time-course studies, suggesting that developmental regulation of muscle PNI occurs principally at the translational and/or post-translational levels. These results suggest that the localization of PNI, through a binding site or "receptor" may play an important role in differentiation and maintenance of synapse.