Immunization with DISC1 protein in an animal model of ADHD influences behavior and excitatory amino acids in prefrontal cortex and striatum.

The Disrupted-in-schizophrenia 1 (DISC1) gene is involved in vulnerability to neuropsychiatric disorders. Naples high-excitability (NHE) rat model neuropsychiatric problems characterized by an unbalanced mesocortical dopamine system. Here, we assessed behavioral and neurochemical effects of immunization against multimeric rat DISC1 protein in adult NHE rats, an animal model of attention-deficit hyperactivity disorder and their Random-Bred (NRB) controls. Males of both lines received subcutaneous injections of vehicle (PB), adjuvant only (AD) or recombinant rat DISC1 protein purified from E. coli, suspended in AD (anti-DISC1) at age of 30, 45 and 60 postnatal days (pnd). At 75 pnd, the rats were exposed to a Làt maze and 2 days later to an Olton eight-arm radial maze, and horizontal (HA) and vertical activities (VA) were monitored. Non-selective (NSA) and selective spatial attention (SSA) were monitored in the Làt and in the Olton maze by duration of rearings and working memory, respectively. Post mortem neurochemistry in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum of L-Glutamate, L-Aspartate and L-Leucine was performed. All immunized rats showed a clear humoral IgM (but not IgG) immune response against the immunogen, indicating that immunological self-tolerance to DISC1 can be overcome by immunization. NHE rats exhibited a higher unspecific IgM response to adjuvant, indicating an immunological abnormality. The sole anti-DISC1 immunization-specific behavioral in the NHE rats was an increased horizontal activity in the Làt maze. Adjuvant treatment increased vertical activity in both lines, but in the NRB controls it increased rearing and decreased horizontal activity. Liquid chromatography/tandem mass spectrometry analysis of soluble or membrane-trapped neurotransmitters aspartate, glutamate and leucine revealed increased soluble aspartate levels in the ventral striatum of NRB controls after anti-DISC1 immunization. Immune activation by adjuvant independent of simultaneous DISC1 immunization led to other specific changes in NHE and control NRB rats. In DISC1-immunized NHE rats, horizontal activity in Lat maze correlated with membrane-trapped glutamate in PFc and in the NRB rats, duration of rearing in Olton maze correlated with membrane-trapped glutamate in PFc and aspartate in dorsal striatum. In addition to non-specific immune activation (by AD), the postnatal anti-DISC1 immune treatment led to behavioral changes related to mechanisms of activity and attention and had influenced amino acids and synaptic markers in striatum and neocortex in the adult NHE as well as control animals.

Prepuberal intranasal dopamine treatment in an animal model of ADHD ameliorates deficient spatial attention, working memory, amino acid transmitters and synaptic markers in prefrontal cortex, ventral and dorsal striatum.

Intranasal application of dopamine (IN-DA) has been shown to increase motor activity and to release DA in the ventral (VS) and dorsal striatum (DS) of rats. The aim of the present study was to assess the effects of IN-DA treatment on parameters of DA and excitatory amino acid (EAA) function in prepuberal rats of the Naples high-excitability (NHE) line, an animal model for attention-deficit hyperactivity disorder (ADHD) and normal random bred (NRB) controls. NHE and NRB rats were daily administered IN-DA (0.075, 0.15, 0.30 mg/kg) or vehicle for 15 days from postnatal days 28-42 and subsequently tested in the Làt maze and in the Eight-arm radial Olton maze. Soluble and membrane-trapped L-glutamate (L-Glu) and L-aspartate (L-Asp) levels as well as NMDAR1 subunit protein levels were determined after sacrifice in IN-DA- and vehicle-treated NHE and NRB rats in prefrontal cortex (PFc), DS and VS. Moreover, DA transporter (DAT) protein and tyrosine hydroxylase (TH) levels were assessed in PFc, DS, VS and mesencephalon (MES) and in ventral tegmental area (VTA) and substantia nigra, respectively. In NHE rats, IN-DA (0.30 mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15 mg/kg) increased selective spatial attention. In NHE rats, basal levels of soluble EAAs were reduced in PFc and DS relative to NRB controls, while membrane-trapped EAAs were elevated in VS. Moreover, basal NMDAR1 subunit protein levels were increased in PFc, DS and VS relative to NRB controls. In addition, DAT protein levels were elevated in PFc and VS relative to NRB controls. IN-DA led to a number of changes of EAA, NMDAR1 subunit protein, TH and DAT protein levels in PFc, DS, VS, MES and VTA, in both NHE and NRB rats with significant differences between lines. Our findings indicate that the NHE rat model of ADHD may be characterized by (1) prefrontal and striatal DAT hyperfunction, indicative of DA hyperactivty, and (2) prefrontal and striatal NMDA receptor hyperfunction indicative of net EAA hyperactivty. IN-DA had ameliorative effects on activity level, attention, and working memory, which are likely to be associated with DA action at inhibitory D2 autoreceptors, leading to a reduction in striatal DA hyperactivity and, possibly, DA action on striatal EAA levels, resulting in a decrease of striatal EAA hyperfunction (with persistence of prefrontal EAA hyperfunction). Previous studies on IN-DA treatment in rodents have indicated antidepressant, anxiolytic and anti-parkinsonian effects in relation to enhanced central DAergic activity. Our present results strengthen the prospects of potential therapeutic applications of intranasal  DA by indicating an enhancement of selective attention and working memory in a deficit model.

Putative glycine receptors in Hydra: a biochemical and behavioural study.

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.

Neuronal and glial properties coexist in a novel mouse CNS immortalized cell line.

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.

Kelletinin A, from the marine mollusc Buccinulum corneum, promotes differentiation in Hydra vulgaris.

The effects of kelletinin A [ribityl pentakis (p-hydroxybenzoate)] (KA), a natural compound isolated from the marine gastropod Buccinulum corneum, were studied in vivo in Hydra vulgaris during regeneration. KA caused a marked increase of regenerated tentacle numbers (ATN) and promoted transdifferentiation of epithelial cells into battery cells, and nematocyte differentiation. Morphological data were correlated to changes in acid and alkaline phosphatase levels, enzymes that have been described as regeneration markers.

The Drosophila melanogaster lipase homologs: a gene family with tissue and developmental specific expression.

We report the molecular cloning of Drosophila genes encoding putative lipase homologs, Dm lip1, lip2 and lip3, the definition of their structure and the expression patterns during development. These Drosophila lipases are related to acid lipases, with a common GHSQG motif, within a more general consensus GXSXG, identified as the active site shared by all the members of lipase superfamily. The lip1 and lip3 genes are transcribed in different tissues and developmental stages, suggesting that they have different functions. The lip1 gene, coding for a protein similar to digestive lipases, is expressed in ovaries and early embryos and, with a different sized transcript, in all the other developmental stages. The lip3 gene, whose translation product is more similar to lysosomal acid lipases, is expressed only during the larval period. The lip2 gene seems non-functional. The Drosophila putative lipases do not show similarity with the Drosophila yolk proteins that are reported to have sequence similarity with lipoprotein lipases, but share a consistent similarity with lepidopteran proteins reported as egg specific or yolk proteins, probably corresponding to lipase homologs. The results reported here are discussed in relation to the evolution and functions of lipases within the between species.

Functional organization of the promoter region of the mouse F3 axonal glycoprotein gene.

F3 is a developmentally regulated adhesive glycoprotein expressed by subpopulations of central and peripheral neurons which mediates neurite growth and fasciculation via cis- and trans-interactions with cell-surface or matrix components. We previously reported on the characterization of the F3 gene 5' flanking region in which we identified promoter and enhancer elements. Here, we report on the functional organization of the F3 gene regulatory regions. We show that the F3 promoter is built of linearly arranged positive and negative elements scattered through the 5' flanking region of the F3 gene and the 1st exon (exon 0). Neural- and cell type-specific expression of F3 appears to be governed by elements located in the most proximal promoter region which includes a neural-specific enhancer. In retardation assays, all these cis-acting elements bind nuclear proteins, three of which interact with the identified enhancer element while a single species interacts with sequences located within exon 0. Some of these proteins are also specifically expressed within the brain, indicating that they could correspond to neural-specific trans-acting factors. Elements located immediately upstream of the cell type-specific enhancer and within exon 0 are responsible for regulation of F3 expression by cAMP and retinoic acid.

Dopamine transporter gene expression in rat mesencephalic dopaminergic neurons is increased by direct interaction with target striatal cells in vitro.

By using a semi-quantitative reverse transcriptase-PCR assay (RT-PCR) we have analyzed dopamine transporter (DAT), tyrosine hydroxylase (TH) and synaptic vesicle monoamine transporter (VMAT2) gene expression in rat mesencephalic (MES) primary cultures. Consistent with previous data obtained during rat MES ontogeny, the onset of DAT transcription in vitro is delayed in embryonic day (E)13, but not in E16, MES neurons when compared to that of TH and VMAT2. In co-culture, the addition of target striatal cells (STR) to E13 MES selectively increases DAT mRNA level in DA neurons during the first 3 days in vitro; cortical cells are ineffective. On the contrary, DAT gene does not appear up-regulated in E16 MES co-cultured with target STR cells, indicating that MES DA neurons respond to STR stimulation only at defined developmental stages. Up-regulation of DAT mRNA level by STR in E13 MES seems to require direct cell interactions since target cells do not exert their effect on DAT transcription when are separated from MES cells by a porous barrier, which only allows diffusion of soluble molecules. Thus maturation of DA neurotransmission in vitro appears to follow a developmental program which can be specifically modulated by their target STR cells.

Target cells modulate dopamine transporter gene expression during brain development.

We have analysed the expression of the dopamine transporter (DAT) gene and compared it with that of tyrosine hydroxylase, neuronal GABA transporter and synaptic vesicle monoamine transporter genes during pre- and post-natal development of rat mesencephalic dopaminergic (DA) neurones. Our results show that DAT transcripts are not detectable until embryonic day (E) 15, whilst those of the other genes analysed are already present at E12. In vitro, the level of DAT gene transcription in mesencephalic E13 DA neurones is increased in coculture with target striatal cells. Thus striatal targets cells regulate, at the transcriptional level, a key step of dopaminergic neurotransmission during DA neurone development.

A membrane guanylate cyclase Drosophila homolog gene exhibits maternal and zygotic expression.

A putative membrane form of guanylate cyclase gene was identified in region 32 of the second chromosome of Drosophila melanogaster. The Drosophila protein has a single hydrophobic sequence that divides the protein into a putative extracellular region and a cytoplasmic catalytic region which contains tyrosine kinase and cyclase domains showing varying degrees of conservation with other guanylate and adenylate cyclases. The gene shows a very interrupted organization with small exons separated by small introns and a low level of expression. Transcripts have been localized in situ during oogenesis in the germarium and later in stages 10-14 of egg chamber development. The putative maternal transcript can be seen in very early embryos and reappears later as a product of zygotic gene expression.

A cDNA clone homologous to Drosophila F elements from an imaginal discs poly(A)+ library.

A cDNA clone (F-id1) homologous to Drosophila F elements has been isolated from a cDNA library prepared from poly(A)+ RNA derived from imaginal discs. The F-id1 insert is identical to the 3' end terminus of Fw, an element inserted at the white locus upon irradiation. The isolation of F-id1 provides the first evidence that F elements are expressed in the fruit fly life.