stereotaxic-microinjection-viral-vectors-expressing-cre-recombinase

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Weill Cornell Graduate School of Biomedical Sciences

Weill Cornell Medical College 

The study of inherited retinal diseases has advanced our knowledge of the cellular and molecular mechanisms involved in sensory neural signaling. Dysfunction of two specific sensory modalities, vision and proprioception, characterizes the phenotype of the rare, autosomal-recessive disorder posterior column ataxia and retinitis pigmentosa (PCARP). Using targeted DNA capture and high-throughput sequencing, we analyzed the entire 4.2 Mb candidate sequence on chromosome 1q32 to find the gene mutated in PCARP in a single family. Employing comprehensive bioinformatic analysis and filtering, we identified a single-nucleotide coding variant in the feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), a gene encoding a heme-transporter protein. Sanger sequencing confirmed the FLVCR1 mutation in this family and identified different homozygous missense mutations located within the protein's transmembrane channel segment in two other unrelated families with PCARP. To determine whether the selective pathologic features of PCARP correlated with FLVCR1 expression, we examined wild-type mouse Flvcr1 mRNA levels in the posterior column of the spinal cord and the retina via quantitative real-time reverse-transcriptase PCR. The Flvcr1 mRNA levels were most abundant in the retina, followed by the posterior column of the spinal cord and other brain regions. These results suggest that aberrant FLVCR1 causes a selective degeneration of a subpopulation of neurons in the retina and the posterior columns of the spinal cord via dysregulation of heme or iron homeostasis. This finding broadens the molecular basis of sensory neural signaling to include common mechanisms that involve proprioception and vision.

Mutations in FLVCR1 cause posterior column ataxia and retinitis pigmentosa.

L&#39;&eacute;tude des maladies h&eacute;r&eacute;ditaires de la r&eacute;tine a fait progresser notre connaissance des m&eacute;canismes cellulaires et mol&eacute;culaires impliqu&eacute;s dans la signalisation de neurones sensoriels. Dysfonctionnement de deux modalit&eacute;s sensorielles sp&eacute;cifiques, vision et proprioception, caract&eacute;rise le ph&eacute;notype de la maladie autosomique r&eacute;cessive rare colonne post&eacute;rieure ataxie et la r&eacute;tinite pigmentaire (PCARP). &Agrave; l&#39;aide de capture cibl&eacute;e de l&#39;ADN et le s&eacute;quen&ccedil;age haut d&eacute;bit, nous avons analys&eacute; la s&eacute;quence enti&egrave;re de candidat de 4,2 Mb sur le chromosome 1q32 pour trouver le g&egrave;ne mut&eacute; dans PCARP dans une seule famille. Utilisant l&#39;analyse bioinformatique complet et filtrage, nous avons identifi&eacute; une mononucl&eacute;otidiques codage variante dans le r&eacute;cepteur cellulaire sous-groupe C virus leuc&eacute;mie f&eacute;line 1 (FLVCR1), un g&egrave;ne codant pour une prot&eacute;ine h&egrave;me-transporteur. Sanger s&eacute;quen&ccedil;age a confirm&eacute; la mutation FLVCR1 de cette famille et identifi&eacute; des mutations faux-sens homozygotes diff&eacute;rentes agrave segment transmembranaire de canal de la prot&eacute;ine dans deux autres familles sans rapport avec PCARP. Pour d&eacute;terminer si les caract&eacute;ristiques pathologiques s&eacute;lectives des PCARP en corr&eacute;lation avec l&#39;expression de FLVCR1, nous avons examin&eacute; les ARNm Flvcr1 dans la colonne post&eacute;rieure de la moelle &eacute;pini&egrave;re et la r&eacute;tine via quantitative PCR en temps r&eacute;el de la transcriptase-inverse la souris sauvage. Les taux d&#39;ARNm de Flvcr1 &eacute;taient plus abondantes dans la r&eacute;tine, suivie de la colonne post&eacute;rieure de la moelle &eacute;pini&egrave;re et d&#39;autres r&eacute;gions du cerveau. Ces r&eacute;sultats sugg&egrave;rent que FLVCR1 aberrants entra&icirc;ne une d&eacute;g&eacute;n&eacute;rescence s&eacute;lective d&#39;une sous-population de neurones dans la r&eacute;tine et les colonnes post&eacute;rieures de la moelle &eacute;pini&egrave;re par l&#39;interm&eacute;diaire de d&eacute;r&egrave;glement de l&#39;hom&eacute;ostasie h&egrave;me ou fer. Cette d&eacute;couverte &eacute;largit la base mol&eacute;culaire de la signalisation neuronale sensorielle afin d&#39;inclure des m&eacute;canismes communs qui impliquent la proprioception et la vision.

Mutations de FLVCR1 causent une ataxie colonne post&eacute;rieure et une r&eacute;tinite pigmentaire.

Lo studio delle malattie ereditarie della retina ha avanzato la nostra conoscenza dei meccanismi cellulari e molecolari coinvolti nella segnalazione neurale sensoriale. Disfunzione delle due specifiche modalit&agrave; sensoriali, visione e propriocezione, caratterizza il fenotipo della malattia rara, autosomica recessiva colonna posteriore atassia e retinite pigmentosa (PCARP). Mediante acquisizione mirata di DNA e high-throughput sequencing, abbiamo analizzato l&#39;intera sequenza di candidato 4,2 Mb sul cromosoma 1q32 per trovare il gene mutato in PCARP in un&#39;unica famiglia. Impiegando l&#39;analisi bioinformatica completa e filtraggio, abbiamo identificato una singolo nucleotide codifica variante leucemia felina virus sottogruppo C cellular receptor 1 (FLVCR1), un gene che codifica per una proteina eme-transporter. Sanger sequenziamento ha confermato la mutazione FLVCR1 in questa famiglia e identificate diverse mutazioni missenso omozigote si trova all&#39;interno del segmento del transmembrane canale della proteina in due altre famiglie non consanguinee con PCARP. Per determinare se le caratteristiche patologiche selettive di PCARP correlata con espressione FLVCR1, abbiamo esaminato i livelli di mRNA di Flvcr1 selvaggio-tipo mouse nella colonna posteriore del midollo spinale e la retina tramite PCR quantitativa di trascrittasi inversa in tempo reale. I livelli di mRNA di Flvcr1 erano pi&ugrave; abbondanti nella retina, seguita dalla colonna posteriore del midollo spinale e altre regioni del cervello. Questi risultati suggeriscono che FLVCR1 aberrante provoca una degenerazione selettiva di una sottopopolazione di neuroni nella retina e le colonne posteriori del midollo spinale via disregolazione dell&#39;omeostasi del ferro o dell&#39;eme. Questa constatazione si allarga la base molecolare della segnalazione neurale sensoriale per includere meccanismi comuni che coinvolgono la propriocezione e la visione.

Mutazioni in FLVCR1 causano atassia della colonna posteriore e retinite pigmentosa.

Stress differentially affects hippocampal-dependent learning relevant to addiction and morphology in male and female rats. Mu opioid receptors (MORs), which are located in parvalbumin (PARV)-containing GABAergic interneurons and are trafficked in response to changes in the hormonal environment, play a critical role in promoting principal cell excitability and long-term potentiation. Here, we compared the effects of acute and chronic immobilization stress (AIS and CIS) on MOR trafficking in PARV-containing neurons in the hilus of the dentate gyrus in female and male rats using dual label immunoelectron microscopy. Following AIS, the density of MOR silver-intensified gold particles (SIGs) in the cytoplasm of PARV-labeled dendrites was significantly reduced in females (estrus stage). Conversely, AIS significantly increased the proportion of cytoplasmic MOR SIGs in PARV-labeled dendrites in male rats. CIS significantly reduced the number of PARV-labeled neurons in the dentate hilus of males but not females. However, MOR/PARV-labeled dendrites and terminals were significantly smaller in CIS females, but not males, compared with controls. Following CIS, the density of cytoplasmic MOR SIGs increased in PARV-labeled dendrites and terminals in females. Moreover, the proportion of near-plasmalemmal MOR SIGs relative to total decreased in large PARV-labeled dendrites in females. After CIS, no changes in the density or trafficking of MOR SIGs were seen in PARV-labeled dendrites or terminals in males. These data show that AIS and CIS differentially affect available MOR pools in PARV-containing interneurons in female and male rats. Furthermore, they suggest that CIS could affect principal cell excitability in a manner that maintains learning processes in females but not males. Synapse, 2013. Â© 2013 Wiley Periodicals, Inc.

Stress differentially alters mu opioid receptor density and trafficking in parvalbumin-containing interneurons in the female and male rat hippocampus.

Le stress affecte diff&eacute;remment hippocampe-d&eacute;pendantes d&#39;apprentissage pertinentes &agrave; la toxicomanie et de la morphologie chez les rats m&acirc;les et femelles. R&eacute;cepteurs opio&iuml;des MU (GPR), qui sont trouvent dans la parvalbumine (PARV)-contenant GABAergique interneurones et sont victimes de trafic en r&eacute;ponse aux changements dans l&#39;environnement hormonal, jouent un r&ocirc;le essentiel dans la promotion d&#39;excitabilit&eacute; cellulaire principal et potentialisation &agrave; long terme. Ici, nous avons compar&eacute; les effets du stress aigu et chronique immobilisation (AIS et CEI) sur la traite MOR PARV contenant des neurones dans le hile du gyrus dent&eacute; en femelle et rats m&acirc;les &agrave; l&#39;aide de double &eacute;tiquette immunomicroscopie &eacute;lectronique. Suite AIS, la densit&eacute; de MOR intensifi&eacute; argent particules d&#39;or (SIGs) dans le cytoplasme des dendrites PARV marqu&eacute;e a &eacute;t&eacute; significativement r&eacute;duite chez les femelles (phase d&#39;oestrus). &Agrave; l&#39;inverse, AIS a consid&eacute;rablement augment&eacute; la proportion de cytoplasmique MOR SIGs en dendrites PARV marqu&eacute;s chez les m&acirc;les. CIS a consid&eacute;rablement r&eacute;duit le nombre de neurones PARV marqu&eacute;s dans le gyrus hile des m&acirc;les, mais pas les femelles. Toutefois, les terminaux et les dendrites MOR/PARV-&eacute;tiquet&eacute; &eacute;tait significativement plus petits dans les femelles CIS, mais pas les hommes, compar&eacute;s aux t&eacute;moins. Suite de CIS, la densit&eacute; des SIGs cytoplasmique de MOR a augment&eacute; en dendrites PARV-&eacute;tiquet&eacute;s et terminaux chez les femelles. En outre, la proportion de pr&egrave;s de-plasmiques MOR SIGs relative au total a diminu&eacute; en grandes dendrites PARV marqu&eacute; chez les femelles. Apr&egrave;s CIS, aucun changement dans la densit&eacute; ou traite des SIGs MOR n&#39;a observ&eacute; dendrites PARV marqu&eacute;e ni bornes chez les m&acirc;les. Ces donn&eacute;es montrent que SIA et CIS diff&eacute;rentiellement affectent les piscines MOR disponibles dans PARV contenant des interneurones chez les rats m&acirc;les et femelles. En outre, ils sugg&egrave;rent que le CIS peut nuire excitabilit&eacute; cellulaire principal d&#39;une mani&egrave;re qui maintient le processus d&#39;apprentissage dans les femelles, mais pas les hommes. Synapse, 2013. &copy; 2013 Wiley p&eacute;riodiques, Inc.

Stress alt&egrave;re diff&eacute;remment la densit&eacute; des r&eacute;cepteurs opio&iuml;des mu et le trafic en parvalbumine contenant des interneurones dans l&#39;hippocampe de rats m&acirc;les et femelles.

Kathryn C. Schierberl

Graduate Program in Neuroscience

Weill Cornell Graduate School of Biomedical Sciences

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Kathryn C. Schierberl

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Graduate Program in Neuroscience

Weill Cornell Graduate School of Biomedical Sciences

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Graduate Program in Neuroscience