A Disc1 mutation differentially affects neurites and spines in hippocampal and cortical neurons.

A balanced chromosomal translocation segregating with schizophrenia and affective disorders in a large Scottish family disrupting DISC1 implicated this gene as a susceptibility gene for major mental illness. Here we study neurons derived from a genetically engineered mouse strain with a truncating lesion disrupting the endogenous Disc1 ortholog. We provide a detailed account of the consequences of this mutation on axonal and dendritic morphogenesis as well as dendritic spine development in cultured hippocampal and cortical neurons. We show that the mutation has distinct effects on these two types of neurons, supporting a cell-type specific role of Disc1 in establishing structural connections among neurons. Moreover, using a validated antibody we provide evidence indicating that Disc1 localizes primarily to Golgi apparatus-related vesicles. Our results support the notion that in vitro cultures derived from Disc1(Tm1Kara) mice provide a valuable model for future mechanistic analysis of the cellular and biochemical effects of this mutation, and can thus serve as a platform for drug discovery efforts.

SLC25A12 expression is associated with neurite outgrowth and is upregulated in the prefrontal cortex of autistic subjects.

Autism is a neurodevelopmental disorder with a strong genetic component, probably involving several genes. Genome screens have provided evidence of linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Association between autism and single-nucleotide polymorphisms in SLC25A12 has been reported in various studies. SLC25A12 encodes the mitochondrial aspartate/glutamate carrier functionally important in neurons with high-metabolic activity. Neuropathological findings and functional abnormalities in autism have been reported for Brodmann's area (BA) 46 and the cerebellum. We found that SLC25A12 was expressed more strongly in the post-mortem brain tissues of autistic subjects than in those of controls, in the BA46 prefrontal cortex but not in cerebellar granule cells. SLC25A12 expression was not modified in brain subregions of bipolar and schizophrenic patients. SLC25A12 was expressed in developing human neuronal tissues, including neocortical regions containing excitatory neurons and neocortical progenitors and the ganglionic eminences that generate neocortical inhibitory interneurons. At mid-gestation, when gyri and sulci start to develop, SLC25A12 molecular gradients were identified in the lateral prefrontal and ventral temporal cortex. These fetal structures generate regions with abnormal activity in autism, including the dorsolateral prefrontal cortex (BA46), the pars opercularis of the inferior frontal cortex and the fusiform gyrus. SLC25A12 overexpression or silencing in mouse embryonic cortical neurons also modified dendrite length and the mobility of dendritic mitochondria. Our findings suggest that SLC25A12 overexpression may be involved in the pathophysiology of autism, modifying neuronal networks in specific subregions, such as the dorsolateral prefrontal cortex and fusiform gyrus, at both pre- and postnatal stages.

Heart transplantation changes the expression of distinct gene families.

We took advantage of the combination of a rat heart transplantation model with a modified differential display RT-PCR method to identify transcriptome changes in the right atria from transplanted compared with native hearts. Based on sequence homology search, the 37 cDNAs differentially displayed both 2 and 7 days posttransplantation were categorized into 7 unknown transcripts, 16 expressed sequence tags (ESTs), and 14 partially or completely characterized genes. The last group cDNAs, validated by relative RT-PCR, belonged to diverse gene families involved in specific metabolisms, protein synthesis, cell signaling, and transcription. Furthermore, we identified differential transcripts corresponding to denervation and fetal gene reexpression. We found coordinate downregulation of genes involved in energy metabolism and protein synthesis regulation, similar to that reported for senescent skeletal muscle. From these transcriptome changes, we propose that heart transplants and senescent muscles share common molecular mechanisms.

MASH-1/RET pathway involvement in development of brain stem control of respiratory frequency in newborn mice.

Respiratory abnormalities have been described in MASH-1 (mammalian achaete-scute homologous gene) and c-RET ("rearranged during transfection") mutant newborn mice. However, the neural mechanisms underlying these abnormalities have not been studied. We tested the hypothesis that the MASH-1 mutation may impair c-RET expression in brain stem neurons involved in the control of breathing. To do this, we analyzed brain stem c-RET expression and respiratory phenotype in MASH-1 +/+ wild-type, MASH-1 +/- heterozygous, and MASH-1 -/- knock-out newborn mice during the first 2 h of life. In MASH-1 -/- newborns, c-RET gene expression was absent in the noradrenergic nuclei (A2, A5, A6, A7) that contribute to modulate respiratory frequency and in scattered cells of the rostral ventrolateral medulla. The c-RET transcript levels measured by quantitative RT-PCR were lower in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ brain stems (P = 0.001 and P = 0.003, respectively). Breath durations were shorter in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ mice (P = 0.022) and were weakly correlated with c-RET transcript levels (P = 0.032). Taken together, these results provide evidence that MASH-1 is upstream of c-RET in noradrenergic brain stem neurons important for respiratory rhythm modulation.

Scorpion venom inhibits selectively Ca2+-activated K+ channels in situ.

Using patch-clamp techniques, a study was made of the component of Leiurus quinquestriatus scorpion venom which caused a blockade of one class of membrane potassium channels, the calcium activated potassium (BK) channels. This blockade was obtained on channels in their native lipidic environment and was specific for this class of channels as other types of potassium channels were not affected by this venom.

Both upstream and intragenic sequences of the human neurofilament light gene direct expression of lacZ in neurons of transgenic mouse embryos.

Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to the lacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression of lacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promote lacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression of lacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences contain cis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directs lacZ expression in embryonic neurons.

Neuronal defects in genotyped dominant megacolon (Dom) mouse embryos, a model for Hirschsprung disease.

Dominant megacolon (Dom) is one of four mutations in the mouse which can produce a phenotype similar to Hirschsprung disease in man. Here, we report that it is possible to take advantage of two microsatellite markers to genotype Dom embryos and to study enteric neuronal development in Dom embryos using whole-mount immunohistochemistry. Dom embryos present a variable defect in the ileo-caecal region, as do embryos of other murine models of Hirschsprung disease.

Environmental signals and neural crest cells.

Cell lineage analysis in both the central and peripheral nervous system of vertebrates has revealed that many neural progenitor cells are multipotent. These observations have raised the general issue of when and how such multipotent progenitors generate their various differentiated progeny. The environment of these progenitors controls the cell lineage decisions in the neural crest. This review considers the roles of the environmental signals in the context of the development of several different neural crest-derived lineages.

Potassium channels in mouse neonate dorsal root ganglion cells: a patch-clamp study.

Isolated neurons from mouse neonate dorsal root ganglia were analyzed using both whole-cell clamp and single-channel recording techniques and presented a complex repertoire of potassium (K) channels. Different types of potassium channels have been found: calcium-activated K channel presenting a large unit conductance of 260 pS in symmetrical K; voltage-dependent K channels of 130 pS without calcium-dependence; two types of inward rectifying K channels (90 and 120 pS in symmetrical K); low probability K channels; delayed rectifier channels and non-selective cationic channels.

Large unit conductance voltage chloride channels are expressed in mouse neural crest cells and embryonic dorsal root ganglion cells.

The early expression of voltage-activated chloride channels of large unitary conductance (450 pS in symmetrical 140 mM KCl) was demonstrated using patch-clamp techniques in two preparations: (i) neural crest cells isolated from 9-day-old (E9) mouse embryos and (ii) acutely isolated dorsal root ganglion cells isolated from E12 mouse embryos. Properties of these ionic channels have been analyzed using single channel recordings and the group mean of these single channels.

Cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences in transgenic mouse embryos.

Peripherin is a neuron-specific type III intermediate filament protein expressed in well-defined populations of neurons projecting towards peripheral targets. To investigate the molecular mechanisms by which a gene is expressed in a specific subset of neurons, we used a transgenic approach in order to define peripherin gene sequences that are necessary for cell-type specific expression. Transgenic mice carrying different various genomic regions of the mouse peripherin gene fused to the Escherichia coli lacZ reporter gene were generated. We used three different peripherin/lacZ constructs containing either 5.8 kb upstream sequences, or both 5.8 kb upstream and 1.1 kb intragenic sequences, or 1.1 kb intragenic sequences associated with an heterologous promoter. Analysis of lacZ gene expression in transgenic mouse embryos showed that cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences. Analysis of transgenic mouse lines expressing the construct containing both upstream and intragenic sequences showed that this transgene contains all regulatory elements essential for both spatial and temporal expression of the mouse peripherin gene during embryogenesis. Furthermore, lacZ+ positive cells isolated from these transgenic lines by fluorescence-activated cell sorting (FACS) can be stained with a peripherin antibody, demonstrating that the transgene containing both upstream and intragenic sequences is expressed in peripherin neurons. These mouse peripherin upstream and intragenic sequences can now be used to identify cis-acting regulatory elements and transcription factors involved in peripherin gene regulation.

[Genetics and respiratory control: studies in normal humans and genetically modified animals]. / Génétique et contrôle respiratoire.

INTRODUCTION: Studies into the contribution of genetic factors to respiratory control disorders are scarce, with impediments to their conduct including difficulties in characterizing these disorders, the large number of genes involved in respiratory control, and interactions between genetic and environmental factors. STATE OF THE ART: The rare congenital central hypoventilation syndrome (CCHS) has opened up the field of respiratory control genetics. Heterozygous mutations of genes involved in neural crest development were discovered recently. Studies in mutant mice have identified respiratory control disturbances related to loss of function of genes involved in neural crest development, genes encoding transcription factors, diffusible factors, and proteins contributing to neurotransmission. PERSPECTIVES: Future genetic epidemiological studies in humans and new models of mutant mice should describe genes involved in respiratory control. Better knowledge of CCHS genetics should provide guideposts for investigating the genetics of other respiratory control disorders. CONCLUSIONS: Respiratory control genetics is opening up new paths for research into respiratory physiology and pathophysiology.

[Evaluation of an information document about patients and transfusion]. / Evaluation du document d'information des patients sur la transfusion.

OBJECTIVE: To evaluate the understanding of written information contained in the information sheet for patients intended to receive an homologous transfusion and to know their opinion about this document. TYPE OF THE STUDY: A prospective cohort survey carried out by people unrelated to clinical units and transfusion services. METHODS: A document divided in two parts, the first one summarized, the second detailed, was distributed to transfused adult patients. The patients were hospitalized in the general surgery and orthopedic wards of two hospitals and in the hematology and oncology wards of two different hospitals. A questionnaire was filled out in the presence of the inquirer. RESULTS: Sixty one subjects have been enrolled, among them 53 considered the information as adequate; 53 as comforting and neutral. 53 patients considered a written information as essential and 52 estimated that both part of the information sheet (summarized and detailed) were mandatory. Conversely, a more in depth investigation revealed there was a gap between patients statements and their true understanding. CONCLUSION: The value of a written information for the patients is confirmed by the study. In addition, patients were not generally worried by this information. The partition of the document has been appreciated. It is noteworthy that a gap exist between the patient's perception of the information and their actual level of understanding.

Primate-accelerated evolutionary genes: novel routes to drug discovery in psychiatric disorders.

Novel molecular genetic approaches, at genome-scale in different species allowed characterizing genes that have undergone recent selection. The interest in this research field is not limited to the natural curiosity about our evolutionary past, but it is also to identify novel susceptibility genes for neuropsychiatic disorders by pointing specific human traits, such as behavioral and cognitive abilities. Hypotheses have been proposed to relate specific psychiatric disorders to the origin of modern humans, as evidenced by the theory of Crow about schizophrenia. In the present review, we will focus on genes that underwent positive selection in humans or displayed a human specific evolutionary pattern and which were reported as associated with psychiatric disorders. This will include the (1) DRD4 gene associated with attentiondeficit/ hyperactivity disorder, located in a locus that underwent a positive selection; the (2) GABRB2 gene, a gene associated with schizophrenia and recently reported as the target of a positive selection; (3) MARK1, a candidate gene for autism that was reported as displaying a signature of adaptative evolution in the human lineage, and (4) the ADH and ALDH2 genes which are associated with alcoholism, and for which evidence of positive selection was identified in the human lineage since the divergence between humans and chimpanzees. Identification of novel candidate genes based on recent evolution selection, coupled to genome-wide strategies designed to detect rare structural variants, could lead to a better knowledge of the molecular mechanisms of neurodevelopmental disorders and might therefore help to develop new medical chemistry.

Ret deficiency in mice impairs the development of A5 and A6 neurons and the functional maturation of the respiratory rhythm.

Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret-null mutants (Ret-/-) did not survive after birth, our experiments were performed in wild-type (wt) and Ret-/- fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret-/- fetuses, high-pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret-/- fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem 'en bloc' preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret-/- fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance.

[Bacteriologic monitoring of 1,000 units of human packed red blood cells for Yersinia enterocolitica]. / Contrôle bactériologique de 1,000 concentrés de globules rouges humains à la recherche de Yersinia enterocolitica.

Several reports of Yersinia enterocolitica (Ye) post-transfusion septicaemia have been recently published. In order to study the prevalence of Ye contamination of packed red blood cells (PRBC) we performed a post-transfusion control on 1,000 PRBC units. We did not find any Ye contamination. This is in accordance with the rarity of the reported cases. However as they are very serious, a prevention is necessary. As classical bacteriological techniques are not feasible, the only possibility is to decrease the PRBC conservation time and to select blood donors by a careful questioning about recent GI tract disturbances.

Properties of miniature postsynaptic currents during depolarization-induced release at a cholinergic neuroneuronal synapse.

1. Miniature postsynaptic currents were analyzed at an inhibitory cholinergic neuroneuronal synapse in the buccal ganglion of Aplysia. Under double voltage-clamp, it was possible to induce postsynaptic currents by long-duration depolarizations of the presynaptic neuron and to analyze these as the linear summation of individual miniature postsynaptic currents (MPSCs). The amplitude of these miniature currents (imin) was calculated from the ratio of the variance of the noise (E2) to the mean of the postsynaptic current (Im), according to Campbell's theorem, with imin = 2E2/Im. Their decay time (tau min) was obtained from the cutoff frequencies of the power spectra obtained from the noise. 2. Neither the conductance nor the decay time of MPSCs was voltage dependent. However, imin appeared to decrease when the quantal content of the response increased. Meanwhile, tau min increased slightly with Imin. 3. Carbamylcholine was injected into the neuropile and this led to a decrease in imin and a slight increase in tau min. 4. Power spectra obtained after the application of inhibitors of acetylcholinesterase (AChE), with or without curare, suggested that acetylcholine (ACh) does not accumulate during large depolarizations. 5. The possible origin of the nonlinear relationship between the variance and the mean of the postsynaptic currents is discussed.

Mammalian neuronal differentiation: early expression of a neuronal phenotype from mouse neural crest cells in a chemically defined culture medium.

We show that mouse neural crest cells cultured in a serum-deprived chemically defined medium on appropriate culture substrata can be induced to express a neuronal phenotype. The uncommitted neural crest cells express a mesenchymal intermediate filament protein such as vimentin, but not the usual neuronal markers such as receptor sites for tetanus toxin or neurofilaments. In the chemically defined medium, receptor sites for tetanus toxin or neurofilaments can be characterized after a few hours in culture. Furthermore, these cells acquire tetrodotoxin-sensitive voltage-dependent Na+ channels and can generate action potentials. Such an in vitro system should allow us to analyze and manipulate early stages of neuronal differentiation in a mammalian embryo, at a level so far restricted to lower vertebrate embryos.

Distribution of receptors for acetylcholine and 5-hydroxytryptamine on identified leech neurones growing in culture.

The spatial distribution of receptors on identified leech neurones removed from the C.N.S. and grown in culture has been studied by applying acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) ionophoretically and by pressure. Two cells were selected: a neurone called anterior Pagoda (Ap), that shows responses to ACh, and the pressure sensory neurone (P cell), upon which 5-HT synapses form in culture. ACh receptors of Ap neurones in culture had properties similar to those of their counterparts in situ. Thus, ACh responses of Ap cells were mediated by Cl- and were blocked by curare and alpha-bungarotoxin. The cell bodies of these neurones in culture had low (10 mV/nC) and uniform sensitivity to ACh over the surface of the soma. When a sprout grew out from the Ap cell, a region of increased sensitivity appeared at its base, with a gradient of sensitivity decreasing toward the tip of the neurite. Characteristically, the base was 3-5 times more sensitive to ACh than the soma or the growth cone. Cells with multipolar processes developed a similar pattern of sensitivity for each sprout. P sensory neurones in culture showed similar distributions of sensitivity to 5-HT and ACh. Experiments made with voltage clamp suggested that the non-uniform responses to transmitter represent true differences in sensitivity. Together these findings suggest that the receptors for ACh and 5-HT have a greater density at the base of each neurite compared to that of the soma and the tip.