The ADHD-susceptibility gene lphn3.1 modulates dopaminergic neuron formation and locomotor activity during zebrafish development.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, increased impulsivity and emotion dysregulation. Linkage analysis followed by fine-mapping identified variation in the gene coding for Latrophilin 3 (LPHN3), a putative adhesion-G protein-coupled receptor, as a risk factor for ADHD. In order to validate the link between LPHN3 and ADHD, and to understand the function of LPHN3 in the etiology of the disease, we examined its ortholog lphn3.1 during zebrafish development. Loss of lphn3.1 function causes a reduction and misplacement of dopamine-positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. The behavioral phenotype can be rescued by the ADHD treatment drugs methylphenidate and atomoxetine. Together, our results implicate decreased Lphn3 activity in eliciting ADHD-like behavior, and demonstrate its correlated contribution to the development of the brain dopaminergic circuitry.

The Drosophila castor gene is involved in postembryonic brain development.

castor (cas) encodes a zink finger protein expressed in a subset of Drosophila embryonic neuroglioblasts where it controls neuronal differentiation. We show here that cas is expressed at larval and pupal stages in brain cell clusters where it participates in the elaboration of the adult structures. In particular using the MARCM system (mosaic analysis with a repressible cell marker), we show that cas is required postembryonically for correct axon pathfinding of the central complex (CX) and mushroom body (MB) neurons. The linotte (lio) gene encodes a transmembrane protein expressed at larval/pupal stage in a glial structure, the TIFR, and interacts with the no-bridge (nob) gene. We show that cas interacts genetically with lio and nob. These interactions do not involve direct transcription regulation but probably cellular communication processes.

Central brain postembryonic development in Drosophila: implication of genes expressed at the interhemispheric junction.

Postembryonic brain development of Drosophila has become recently a subject of intense investigations. In particular, the linotte (lio) mutants display strong structural defects in the mushroom bodies and the central complex. The Lio kinase is expressed in a glial structure at the interhemispheric junction of late larval and young pupal brain. With the aim of identifying new genes involved in the formation of adult central brain structures, 821 enhancer-trap Gal4 lines were generated and screened for late larval expression. We identified 167 lines showing expression at or near the interhemispheric junction of third-instar larval brain, an area from which the central complex differentiates. Adult brains from 104 of these 167 lines were analyzed through paraffin sections. This secondary screen allowed the recovery of five central brain mutants. Of 89 control lines showing various patterns of expression excluding the interhemispheric junction, only one anatomical mutant was isolated. These six mutations, which have been thoroughly characterized, affect the midline area of the adult brain with phenotypes of split central complex structures and/or fused mushroom body lobes. This work opens the way for further analysis of the molecular and cellular events involved in central brain reorganization during metamorphosis.

Cat proenkephalin-A does not contain the opioid octapeptide.

The sequence of a large cDNA fragment of proenkephalin-A from the cat adrenal medulla was obtained using reverse transcription followed by polymerase chain reaction, and cloning. This cDNA encompasses the region normally containing all the opioid peptides, except the C-terminal heptapeptide. As with other species, cat proenkephalin-A contains four conserved copies of (Met5)-enkephalin, and one of (Leu5)-enkephalin, flanked by processing sites of paired basic amino acids. However, significant differences were found in the nucleotide and deduced amino acid sequences in the region of the octapeptide. In particular, the essential tyrosyl residue is substituted by a histidyl residue, making it unlikely that the cat equivalent would have opioid activity. Furthermore, the peptide is not flanked by paired basic residues, suggesting it is not processed.

Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.

Short- and long-term alterations of gene expression in limbic structures by repeated electroconvulsive-induced seizures.

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.

Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Release of [Met]enkephalin in the central nucleus of the amygdala is increased by application of potassium in the substantia nigra.

Release of [Met]enkephalin immunoreactivity (Met-IR) in the central nucleus of the amygdala (ACE) was investigated in vivo in anesthetized rats implanted with push-pull cannulae. A stable spontaneous release of this peptide (1.3 fmol/15 min fraction) could be measured in the superfusates using a highly sensitive radioimmunoassay. The addition to the superfusion medium of cocktail of peptidase inhibitors increased three times the spontaneous release of the peptide. Superfusion with 30 mM potassium increased ten times the release of the peptide. Chemical stimulation of the substantia nigra with K+ enhanced four times the Met-IR release in the ipsilateral ACE. The dopaminergic component of the nigro-amygdaloid pathway appeared not to be directly implicated in this effect, since: d(+)amphetamine application in the ACE, which enhanced the local release of DA, remained without effect on Met-IR release and haloperidol-induced blockade of dopaminergic receptors in the ACE similarly did not affect Met-IR release.

Co-release of enkephalins and precursors with catecholamines from the perfused cat adrenal gland in situ.

We have compared the nature of the enkephalin-like material derived from proenkephalin present in the intact cat adrenal gland with the material co-released with catecholamines from the perfused adrenal in response to splanchnic nerve stimulation and to perfusions with solutions containing acetylcholine (ACh) or high potassium chloride (KCl). In cat adrenals most of the enkephalin-like material was in the form of large enkephalin-containing peptides. Free (met)enkephalin immunoreactivity represented only 25% of the total (met)enkephalin immunoreactivity as determined by enzymatic digestion of large enkephalin-containing fragments. Electrical stimulation (15 Hz) of the splanchnic nerve or perfusion of the gland with ACh (0.1 mM) or KCl (50 mM), applied for 10 min, induced an immediate release of free (met)enkephalin immunoreactivity, (met)enkephalyl-arg-phe immunoreactivity, and of large (met)enkephalin-containing peptides. The release by all three modes of stimulation followed a pattern that paralleled the output of catecholamines. A rapid fatigue of all secretory processes developed during the stimulation periods, similar to that observed for catecholamines. During splanchnic nerve stimulation, each nanomole of catecholamine output was accompanied by the output of 0.4 pmol free (met)enkephalin immunoreactivity, of 1.1 pmol total (met)enkephalin immunoreactivity and of 0.1 pmol (met)enkephalyl-arg-phe immunoreactivity. Analysis of the perfusate by high-pressure liquid chromatography revealed that (met)enkephalin, (met)enkephalyl-arg-phe and (met)enkephalyl-arg-gly-leu were released in molar ratios of 4 to 1 to 1 which is similar to the ratio found in the precursor, proenkephalin. The ratio of total (met)enkephalin immunoreactivity to free (met)enkephalin immunoreactivity in the perfusate was the same (approximately 2.7) during two successive periods of splanchnic nerve stimulation separated by 10 min. When release was evoked by increasing the K+ concentration to 50 mM-KCl, this ratio was increased more than twofold compared with that obtained by electrical stimulation of the splanchnic nerve. Analysis of the perfusate by gel filtration showed that, during splanchnic nerve stimulation, 47% of the total (met)enkephalin immunoreactivity eluted in fractions containing fragments of low molecular weight. When KCl was used as stimulus only 12% of total (met)enkephalin immunoreactivity eluted in these fractions. The results indicate that the nature of the released peptides depends on the type of stimulus used to evoke release.(ABSTRACT TRUNCATED AT 400 WORDS)

The heptapeptide Met-enkephalin-Arg6, Phe7 is released from rat striatum in vitro by high potassium.

We present evidence that the heptapeptide Met-enkephalin-Arg6, Phe7 is released from rat striatal slices following depolarization by a high concentration of KCl. The heptapeptide-immunoreactive material released in the incubation medium, which is shown by HPLC and radioimmunoassay of serial dilutions to represent authentic heptapeptide, is detectable in the incubation media only in the presence of a cocktail of peptidase inhibitors containing thiorphan (0.1 microM), captopril (1 microM) and bestatin (20 microM).

The enkephalinergic neuron: implications of a polyenkephalin precursor.

The study of the biochemical and physiological functions of the enkephalinergic cell has greatly extended our understanding of peptidergic cells in general. In the adrenal gland, the major part of the proenkephalin-derived peptides is present as intermediates in the processing of the precursor. These peptides are contained within the adrenergic chromaffin granules, from which they are released in response to stimulation of the cell. The nature of the products released depends on the nature of the stimulus, but it appears that mature granules containing completely processed peptides are preferentially released under physiological conditions. In the brain, the presence and release of the heptapeptide that comprises the carboxyl terminus of adrenal proenkephalin suggest that similar mechanisms are operating centrally. The identity of brain and adrenal proenkephalin is further supported by the purification from brain of a large fragment of the proenkephalin molecule, synenkephalin , and the occurrence in brain of this and the other proenkephalin-derived peptides in a molar ratio close to that found in the sequence of the adrenal precursor. The processing of proenkephalin in brain appears to follow the classical models first proposed for peptide hormones (Steiner et al. 1980), which may thus be generalized to include peptide neurotransmitters/neuroregulators. In addition, the results presented in this paper indicate that enkephalins may be cotransmitters in at least two diverse systems. Enkephalins and catecholamines are colocalized in the adrenergic granules of the adrenal gland. In the brain, enkephalins and oxytocin are colocalized in the magnocellular neurons of the hypothalamo-neurohypophyseal oxytocinergic pathway. In both of these systems, the enkephalins are present in a molar concentration that is less than 1% of the concentration of the principal messenger. Such colocalization , coupled with the numerous active peptides that may arise from proenkephalin, suggests many elegant but complex schemes of neurotransmitter interactions. For example, release of enkephalins in the neurohypophysis may regulate oxytocin release through an action on autoreceptors of the oxytocinergic terminal. In the adrenal the coreleased enkephalins may act by regulating presynaptically the cholinergic output of the splanchnic nerve. However, further studies are needed to define clearly the physiological roles of such cotransmission . From the abundance of proenkephalin-derived peptides in the basal ganglia, it appears that enkephalins may represent the principal transmitter in some central neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Release of the heptapeptide Met-enkephalin-Arg6-Phe7 and of the octapeptide Met-enkephalin-Arg6-Gly7-Leu8 from rat striatum in vitro and their rapid inactivation.

The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) and the octapeptide Met-enkephalin-Arg6-Gly7-Leu8 (MERGL) are potent opioid peptides present in the sequence of proenkephalin, the common precursor of Met- and Leu-enkephalin (ME and LE). We demonstrate that MERF and MERGL are released concomitantly with ME and LE from rat striatal slices following a depolarisation by K+. This release is a Ca2+-dependent process. While the ratios of ME to LE, MERF and MERGL found in the tissue (ME/LE = 2.6; ME/MERF = 3.1; ME/MERGL = 4.5) are in good agreement with the ratios found in the proenkephalin molecule (ME:LE:MERF:MERGL = 4:1:1:1), the amounts of MERF and MERGL recovered from the medium are low compared to those of ME and LE, suggesting a rapid degradation of released MERF and MERGL. In fact, when incubated with striatal slices, (3H-Tyr)-MERF is rapidly degraded by four classes of peptidases: the "enkephalinase", the angiotensin-converting enzyme (ACE), aminopeptidase(s) and an endopeptidase releasing the tetrapeptide Tyr-Gly-Gly-Phe (YGGF). Whereas the activities of the three former peptidases are reduced or abolished in the presence of thiorphan (0.1 microM), captopril (1 microM) and bestatin (20 microM), the amount of YGGF formed by the endopeptidase is not reduced in these conditions but actually increased.

Co-release of enkephalins and precursors with catecholamines by the perfused cat adrenal in-situ.

In the present study we have carefully determined the nature and amount of enkephalin immunoreactive product co-related with catecholamines by the cat adrenal gland perfused in-situ. Met-enkephalin immunoreactivity (IR) and total Met-enkephalin-IR obtained after digestion of large enkephalin containing peptides were released by electrical stimulation of the splanchnic nerves, with 0.1 mM acetylcholine perfusion or 50 mM K+. We have found that in response to splanchnic nerve stimulation 47% of enkephalinergic material was on the form of small peptide (Met-enkephalin, Leu-enkephalin, heptapeptide, octapeptide). When drastic stimuli like perfusion with 50 mM or 0.1 mM acetylcholine were used, the bulk of the enkephalinergic material was present on the form of large fragments of the precursor. These results are interpreted as follows: when physiological stimuli are used, mature granules containing fully processed precursor are released; when drastic chemical stimuli are applied, unmature granules containing partially processed precursor are also released.

[An immunofluorescence study of the ontogenesis of mouse thyroid organ-specific antigens: relation to fate of foetal thyroid homografts in adult recipients (author's transl)]. / Etude par immunofluorescence de l'ontogenèse des antigènes organo-spécifiques de la thyroide de souris: relation avec le devenir d'homogreffes de thyroides embryonnaires sur des receveurs adultes.

A study of the development of organ specific thyroid antigens in mice showed that these antigens make their appearance on the 17th day of gestation. This causes an increase in the specific antigenicity of the thyroid and coincides with the critical stages at which the fate of a homograft of embryonic thyroid is seen to change: embryonic thyroid tissue from 15-16 day embryo grafted on an adult recipient is accepted but if taken from a 17 day embryo it is rejected.

[Immunofluorescent study of the distribution of adult neuro-specific antigens in the chick embryo (author's transl)]. / Etude par immunofluorescence de l'apparition d'antigènes neuro-spécifiques d'adulte chez l'embryon de poulet.

The adult neuro-specific antigens have been localized by immunofluorescence techniques in diencephalon and mesencephalon of chick embryo. This study has been made using fresh or fixed tissues from embryos 72, 48 or 36 h old. At 72 h of incubation the wall of diencephalon shows marked fluorescence; at 48 h of incubation the fluorescent cells are localized in an outer layer and an inner one. In the 48 h-old embryo the reaction is more distinct and intensive in fresh tissues than in fixed tissues. At 36 h of incubation no fluorescence has been detected either in fresh tissues or in fixed tissues.

[Quantitation of nuclear RNA synthesis in the ovary of adult Tenebrio molitor]. / Etude quantitative des synthèses d'ARN nucléaires dans l'ovaire de Tenebrio molitor adulte

We studied quantitatively the uridine 5-3H nuclear incorporation in adult ovaries of Tenebrio molitor during the oöcyte maturation. In follicular cells, the rate of RNA synthesis is constant. In the oöcytes, the precursor incorporation is maximum towards the end of previtellogenesis, and low during the yolk deposition. In nurse cells, the RNA synthesis increases during the oöcyte growth and reaches a maximum value 6.5 days after the imaginal molt.