Phylogeny of ants (Formicidae) based on morphology and DNA sequence data.

In order to reconstruct ants' phylogeny, we analysed DNA sequences for two nuclear genes, abdominal-A and Ultrabithorax, from 49 species of ants and two outgroups. As these genes control the development of the first segments of the abdomen in insects, which are very variable in ants (petiole, postpetiole, and gaster constriction), we hypothesized that the morphological variations between the subfamilies may be correlated with mutations of some abd-A or Ubx regions. Contrarily to our hypothesis, these sequences are highly conserved. The differences observed concern mainly third codon positions and present some saturation. Phylogenetic reconstructions were carried out using the genetic raw sequence data and by combining them with a set of morphological data (Total Evidence). Relations among subfamilies of ants remains poorly resolved with molecular data only, but adding these data to morphological characters confirms and reinforce the topology of : a Poneroid complex [Ponerinae, Cerapachyinae, Leptanillinae and army ants], a Formicoid complex [Dolichoderinae, Formicinae] and a Myrmecoid complex [Myrmicinae, Myrmeciinae, Pseudomyrmecinae, Nothomyrmeciinae]. Our molecular results allow resolution near the branch tips and three subfamilies (Dolichoderinae, Formicinae and Pseudomyrmecinae) always appear as monophyletic. The Formicinae and the Dolichoderinae have close relationships. The Camponotini appear as a strong clade inside the Formicinae. The Ponerinae are separated in two parts: the Ectatommini and all other tribes. The Cerapachyinae, Dorylinae, and Ecitoninae belong to the same clade, the Cerapachyinae being confirmed in their subfamily status. The Myrmicinae appears to be very heterogeneous, with the Attini forming a very stable and well-separated group.

Differential expression of GAD(65) and GAD(67) during the development of the rat retina.

The development of synthetic enzymes in the GABAergic system (GAD(67) and GAD(65)) of the rat retina was analyzed from birth to the 4th postnatal week by the reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. As previously observed for GABA, immunoreactive GAD(67) profiles are seen clearly in the inner retinal layers at birth. At the end of the 1st week of postnatal life, immunolabeling is detected in amacrine and/or ganglion cells and in horizontal cells. GAD(67) immunoreactivity is transiently expressed in horizontal cells and disappears during the 3rd postnatal week. GAD(65) however does not develop until the 5th postnatal day. Immunolabeling is detected in the processes layering the inner plexiform layer (IPL) before being detected in the amacrine and/or ganglion cell bodies. The appearance of transcripts for GAD coincided with the appearance of the proteins. A transient form of mRNA transcripts of the GAD(67) gene containing an extra exon (ES-exon) is also observed which disappears progressively from birth to the 4th postnatal week. This form synthesizes a truncated, enzymatically inactive protein, which could participate in the regulation of GABA synthesis from glutamate present at high levels during retinogenesis.

A molecular study of abdominal-A in the ant Myrmica rubra reveals lineage dependent evolutionary rates for a developmental gene.

We have characterized the abdominal-A locus in a Hymenopteran, the ant Myrmica rubra. The sequence of the homeotic domain of the Abdominal-A family of orthologous genes is known for a fairly large number of insects but the complete sequence of the Abdominal-A protein, is known only for a few. The two proteins of Drosophila melanogaster and Tribolium castaneum differ markedly outside the homeodomain. A comparison of the ant Abdominal-A protein sequences with those of these two insects shows that the ant and beetle sequences are very similar all along the length of the protein. The fruit fly has diverged considerably and equally from the other two insects. This divergence reflects different rates of evolution of the protein in different lineages.

Pancreatic expression of antigens for islet cell antibodies in non-obese diabetic mice.

Diabetes-prone NOD mice of both sexes and at different ages were compared to control mice with regard to the level of pancreatic expression of certain autoantigens: antigens for islet cell antibodies (ICA antigens) and glutamic acid decarboxylase (GAD) 67 kDa. ICA antigens were compared by immunofluorescence using serial dilutions of ICA positive human sera so that differences of fluorescence intensity were due only to differences in amounts of antigen. Pancreatic GAD67 mRNAs were compared by polymerase chain reaction followed by Southern hybridization with 32P-probes and densitometry of autoradiographic bands. GAD67 product and gamma-aminobutyric acid (GABA) were compared by immunoperoxidase staining. As compared to BALB/c, C57BL6, Swiss, or F1 mice, NOD mice displayed higher ICA antigen levels (P < 0.01) both before and after insulitis onset (at 7 days, 15 days, 1 month, 2 months). ICA antigens were scarcely detectable by the first day of life, and increased with age from 7 days to 2 months (P < 0.01; n = 10 for each strain and at each age). Both before and after insulitis onset (4 days, 7 days, 15 days, 1 month, 2 months), amounts of GAD67 mRNAs were higher (P < 0.01) in NOD mice than in BALB/c mice (n = 8 for each age in each strain). This was already noted in foetuses on Day 18 of gestation (n = 8). After birth, amounts of GAD67 mRNAs increased up to 1 month (P < 0.04) and then decreased in older mice. The staining intensity of pancreatic sections with antisera against either GAD67 or GABA was higher (P < 0.04) in islets from NOD mice than in those from control mice. Whatever the age, no significant difference was noted between female and male NOD mice with regard to ICA antigens or GAD67. The expression of ICA antigens and GAD67 was intermediate in NOD x BALB/c F1 mice when compared to parental strains. We conclude that whatever the age, NOD mice strongly express ICA antigens and GAD67. This peculiarity was detectable very early, in embryos for GAD67 but after birth for ICA antigens. The timing of antigen expression may underlie the development of diabetes. The antigen overexpression might affect early completion of self-tolerance and, during later life, might also contribute to amplification of the anti-beta cell autoimmune response due to the existence of more targets for effector mechanisms.

The rat nucleus accumbens: two levels of complexity in the distribution of glutamic acid decarboxylase (67 kDa) and preproenkephalin messenger RNA.

The distribution of messenger RNAs (mRNAs) encoding the 67 kDa isoform of glutamic acid decarboxylase (GAD67), a rate-limiting enzyme in the biosynthesis of gamma-aminobutyric acid, and preproenkephalin (PPE), the precursor of enkephalin, was analysed in the rat nucleus accumbens (NAS) taking in account its compartmentation in 'core' (NAS(core)) and 'shell' (NASshell). While GAD67 mRNA levels were more abundant (+48%) in NASshell than in NAS(core), hybridization signals for PPE mRNA were mainly found in NAScore. At a cellular level, hybridization signals for GAD67 mRNA were present in 75% and 80% of neurons NASshell and NAS(core), respectively. The level of GAD67 mRNA found higher in the NASshell corresponds in fact to a greater density of neurons in NASshell than in the NAS(core). The hybridization signals for PPE mRNA were observed in very few neurons of NASshell and in around half of the neurons in NAS(core). Besides this medio to lateral difference, GAD67 and PPE mRNAs were distributed in NAS(core) according to a rostro-caudal gradient, the content of both mRNAs being higher in the rostral part than the caudal part of this area.

GABA interneurons in the rat medial frontal cortex: characterization by quantitative in situ hybridization of the glutamic acid decarboxylase (GAD67) mRNA.

In situ hybridization of mRNA encoding one isoform of glutamic acid decarboxylase (GAD67) was performed in the rat medial frontal cortex (MFC) to characterize GABA interneurons. Qualitatively, the labelling obtained with a [35S]cDNA probe was in register with neurons and was never associated with glial cells. No obvious differences in the density of labelled cells were observed between the different areas of the MFC examined (infralimbic, prelimbic, anterior cingulate and precentral medial) and between the various cortical layers. Grain counting was performed on single cells in the various layers of the prelimbic and the anterior cingulate area, two main areas of the MFC. According to their grain density, neurons were arbitrarily classified as low, high and very high GAD67 mRNA content. The neurons with the high GAD67 mRNA content corresponded to around 50% of the labelled cells in all the layers and in both areas. In the prelimbic area, the neuronal population with a low GAD67 mRNA content varied from 50% in layers I and II-III to 40% in layers V-VI whereas the very high GAD67 mRNA content neurons corresponded to around 5% of the labelled neurons in all layers. In the anterior cingulate area the neuronal population showing low GAD67 mRNA content varied from 35% in layers I and II-III to 20% in layers V-VI. In this area, neurons with a very high GAD67 mRNA content were more numerous than in the prelimbic area: they varied from 15% in layers I and II-III to 30% in layers V-VI. Parallel to the presence of very highly labelled cells, GAD enzymatic activity measured both in the presence and in the absence of pyridoxal 5'-phosphate was higher in the anterior cingulate area than in the prelimbic area. The heterogeneity of GAD67 mRNA content at the cellular level might underlie the existence of subpopulations of GABA interneurons in the MFC and suggests a higher GABAergic inhibitory control in the anterior cingulate area than in the prelimbic area.

Expression of GAD mRNA in GABA interneurons of the rat medial frontal cortex.

The distribution of glutamic acid decarboxylase (GAD) mRNA containing cells was studied in the rat medial frontal cortex (MFC). The neurons labelled by the 35S-labelled cDNA probe were distributed uniformly throughout all the layers and represented 16% of the total neuronal population. It was possible to distinguish two cell populations expressing high and low levels of GAD mRNA corresponding to 63% and 27% of labelled cells, respectively. Concerning the laminar distribution of these two populations of GAD mRNA containing neurons, no marked difference was observed between the various areas of the MFC.

Parallel decrease of glutamic acid decarboxylase and preproenkephalin mRNA in the rat striatum following chronic treatment with a dopaminergic D1 antagonist and D2 agonist.

The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long-term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (-30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (-38 and -57%, respectively) as well as in the ventral (-70 and -60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (-41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.

Rat brain glutamic acid decarboxylase sequence deduced from a cloned cDNA.

A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme. The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli. In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented. The predicted protein is 593 amino acids in length. The first 557 residues display a 95% identity when compared with the corresponding cat sequence. However, the deduced amino acid sequence of the carboxy-terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.

[In situ hybridization on tissue section: a new method for studying the amacrine cells of the retina and the centers of the visual system]. / L'hybridation in situ sur coupe de tissu (HIS): une nouvelle méthode d'étude des cellules amacrines de la rétine et des centres du système visuel.

In situ hybridization of nucleic acid probes, encoding the key enzymes of the synthesis of neurotransmitters, with sections of retina is a new method giving complementary informations to the data obtained by immunocytochemistry and autoradiography. It will allow a better characterization of amacrin cells, whatever might be their type, and a refined understanding of their function. It is an accurate tool to investigate the mechanisms of retinal degenerations and spontaneous or experimental microphtalmia.

Neurons containing messenger RNA encoding glutamate decarboxylase in rat hypothalamus demonstrated by in situ hybridization, with special emphasis on cell groups in medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus.

Previous deafferentation studies have suggested that most hypothalamic GABAergic innervation originates from neurons within the hypothalamus. We have investigated the distribution of GABAergic cell groups in the rat hypothalamus by means of the in situ hybridization technique, using a cDNA probe for messenger RNA encoding glutamate decarboxylase. Several major GABAergic cell groups were demonstrated, including cells of the tuberomammillary nucleus, arcuate nucleus, suprachiasmatic nucleus, medial preoptic area, anterior hypothalamic area, the dorsomedial hypothalamic nucleus, perifornical area, and lateral hypothalamic area. The most prominent glutamate decarboxylase mRNA-containing cell groups were located in the medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus, and were composed of small- to medium-sized neurons. Compared to previously well-characterized GABAergic cell groups in the tuberomammillary nucleus, reticular thalamic nucleus, and non-pyramidal cells of cerebral cortex, the cells of these GABAergic groups demonstrated only weak cDNA labelling, indicating that they contain lower levels of glutamate decarboxylase mRNA. Several types of control experiments supported the specificity of this cDNA labelling, and the GABAergic nature of these cell populations was further supported by detection of glutamate decarboxylase and GABA immunoreactivity. Abundance of GABAergic cells in many hypothalamic nuclei indicates that GABA represents quantitatively the most important transmitter of hypothalamic neurons, and may be involved in neuroendocrine and autonomic regulatory functions.

Demonstration of GABAergic cell bodies in the suprachiasmatic nucleus: in situ hybridization of glutamic acid decarboxylase (GAD) mRNA and immunocytochemistry of GAD and GABA.

The existence of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) was demonstrated by three specific markers; mRNA coding for glutamic acid decarboxylase (GAD) and visualized by in situ hybridization using a 35S-labelled cDNA probe, and GAD protein and GABA were identified by immunocytochemistry using specific antisera. In situ hybridization demonstrated well labelled GAD mRNA positive cells throughout SCN, and GABA and GAD immunoreactive cells showed similar distributions. These results indicate that GABA is a transmitter of a large portion of the SCN neuronal population.

Similar time course changes in striatal levels of glutamic acid decarboxylase and proenkephalin mRNA following dopaminergic deafferentation in the rat.

The time course changes in levels of mRNA encoding glutamic acid decarboxylase (GAD) and proenkephalin (PPE) was analyzed in the rat striatum following unilateral lesion of substantia nigra with 6-hydroxydopamine. The levels of both GAD and PPE mRNAs increased after the dopaminergic deafferentation, reaching concomitantly a maximal twofold increase on day 25. Thereafter, the mRNA levels declined; at 4 months, the amount of PPE mRNA remained slightly elevated whereas GAD mRNA had returned to the control value, suggesting the action of a compensatory mechanism. We also observed a rise of glial fibrillary acidic protein mRNA level which reflects a reactive astrocytosis. In contrast, alpha-tubulin mRNA level remained unchanged, indicating that no significant synaptogenesis occurs in this experimental situation. No obvious modification in mRNA levels was detected in the striatum contralateral to the lesion. These results highlight the role of the modulation of gene expression in adaptive processes to dopamine deficiency in striatal efferent pathways. Its relevance to the pathophysiology of Parkinson's disease is discussed.

A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics.

Catecholaminergic systems in discrete regions of the brain are thought to be important in affective psychoses, learning and memory, reinforcement and sleep-wake cycle regulation. Tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis. Its importance is reflected in the diversity of the mechanisms that have been described which control its activity; TH levels vary both during development and as a function of the activity of the nervous system. Recently, we deduced the complete amino-acid sequence of rat TH from a complementary DNA clone encoding a functional enzyme. Here we demonstrate that, in man, TH molecules are encoded by at least three distinct messenger RNAs. The expression of these mRNAs varies in different parts of the nervous system. The sequence differences observed are confined to the 5' termini of the messengers and involve alternative splicing events. This variation has clear functional consequences for each putative form of the enzyme and could represent a novel means of regulating catecholamine levels in normal and pathological neurons.

Molecular cloning, expression and in situ hybridization of rat brain glutamic acid decarboxylase messenger RNA.

A cDNA library was generated in the expression vector lambda GT11 from rat brain poly(A)+ RNAs and screened with a GAD antiserum. Two clones reacted positively. One of them was shown to express a GAD activity which was specifically trapped on anti-GAD immunogel and was inhibited by gamma-acetylenic-GABA. Blot hybridization analysis of RNAs from rat brain revealed a single 4 kilobases band. Preliminary in situ hybridizations showed numerous cells labelled by the GAD probe such as the Purkinje and stellate cells in the cerebellar cortex and the cells of the reticular thalamic nucleus.

Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor.

A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).

Co-linear organization of Xenopus laevis and mouse mitochondrial genomes.

Cloned fragments of Xenopus laevis mitochondrial DNA and Pleurodeles waltlii mitochondrial cDNAs have been hybridized together and with mouse mtDNA. In the three cases cross-hybridization was observed. The overall organization of the X. laevis fragments appeared to be co-linear with the mouse mtDNA, most sequences being conserved except for the D-loop and the URF6 regions. The use of mouse mtDNA has enabled us to identified several mitochondrial genes in X. laevis and P. waltlii.