Questo lavoro è stato finanziato dal NINDS e American Heart Association a S.Ge. Questo approccio è stato originariamente stabilito e sviluppato dall&#39;autore nel laboratorio del Dr. Hongjun Song nel Johns Hopkins University. Vorremmo ringraziare il Dott. Hongjun canzone per il suo orientamento e sostegno.

1. Virus iniezione Alto titolo retrovirus ingegnerizzati (1 × 10 9 unità / ml) è prodotto da co-trasfezione di vettori retrovirali e VSVG in cellule HEK293T, seguita da ultracentrifugazione di supernatante virale. Per le fonti e metodi di produzione, vedere la dimostrazione eccellente Giove (3). Nota: Giovani adulti (4-6 settimane) femminile C57BL / 6 topi (Charles River) si trovano in condizioni standard. Tutte le procedure seguite la Guida Consiglio Nazionale delle Ricerche per la cura e l&#39;uso di animali da laboratorio in un protocollo approvato dalla Stony Brook University IACUC. <ol><li> 2ul disgelo dei retrovirus congelati per animale sul ghiaccio. </li><li> Anestetizzare (100 ketamina xylazina mcg + 10 mcg per grammo di peso corporeo) e montare l&#39;animale su un telaio stereotassico (Steolting). Rimuovere i capelli sulla testa e pulire la pelle con il 70% di etanolo. </li><li> Esporre il cranio e quattro fori poco profondi con un trapano odontoiatrico (punta 0,6 millimetri) alle seguenti coordinate: <ul><li> anterioposterior = -2 mm dal bregma; laterale = ± 1,6 mm; ventrale = 2,5 mm; anterioposterior = mm -3 dal bregma; laterale = ± 2,6 mm; ventrale = 3,2 mm. </li><li> anterioposterior = -2 mm dal bregma; laterale = ± 1,6 mm; ventrale = 2,5 mm; anterioposterior = mm -3 dal bregma; laterale = ± 2,6 mm; ventrale = 3,2 mm. </li></ul></li><li> Montare un Hamilton 1ml, piatto punta della siringa e iniettare 0,5 microlitri retrovirus per sito ad un tasso di 0,25 microlitri / min nel giro dentato. (Vedi le coordinate sopra per profondità ventrale.) Pausa 2 minuti dopo ogni iniezione prima di ritirarsi lentamente la siringa per evitare il riflusso del fluido. Tra le iniezioni, brevemente risciacquare la superficie esterna della punta della siringa con PBS sterile e un applicatore per eliminare tracce di sangue. </li><li> Chiudere la ferita con materiale di sutura chirurgico sterile (tipo P-1; Dimensione 6-0) e rispedire l&#39;animale verso condizioni abitative standard fino a quando le fasi di tempo desiderato dopo l&#39;iniezione. </li></ol> 2. Fetta di preparazione Per ottenere fettine acute di alta qualità da topi adulti, si usa una soluzione di taglio per la preparazione modificato slice (vedi sotto). <ol><li> Pre-chill soluzione di taglio di 0-4 ° C su ghiaccio e bolla con il 95% O 2 -5% di CO 2 per almeno 30 minuti. </li><li> Anestetizzare e transcardially profumato un animale con ghiaccio-freddo di ossigeno soluzione satura di taglio. </li><li> Rimuovere rapidamente il cervello in una petri-piatto con carta da filtro pre-bagnate con il freddo, la soluzione ossigenata di taglio. Tagliare il cervelletto e tagliare una superficie di montaggio anteriore di almeno 1 mm a il (visibile) coordina iniezione. Incollare il cervello su un palco vibrotome e montarlo nella camera di taglio pieno di freddo e ossigenato soluzione di taglio. </li><li> Preparare le fette coronali o orizzontale (300-350μm) e trasferirli con un grosso pipetta ad una camera di incubazione contenente temperatura ambiente ACSF bollito con il 95% O 2 / 5% di CO 2. </li><li> Incubare le fette, bolle continuamente, a 32 ° C per 30-60 minuti. Ritorna la camera a temperatura ambiente per tutta la durata della registrazione. </li></ol> 3. Esperimenti di elettrofisiologia <ol><li> Fette sono trasferito nella camera di registrazione che viene continuamente perfuso con l&#39;ossigeno-saturi ACSF a 32 ° C - 34 ° C. </li><li> Un elettrodo di tungsteno bipolare di stimolazione è collocato nello strato molecolare del giro dentato utilizzando un microscopio a bassa obiettivo ingrandimento (10X). </li><li> DGCS neonato nella sub-zona granulare / strato di cellule granulari sono identificati mediante l&#39;espressione di GFP o dTomato. A cellula intera registrazione patchclamp viene eseguita su neuroni neonati sotto forte ingrandimento (40X). </li><li> Le proprietà di cottura delle cellule registrate sono ottenute mediante iniezione di una serie di correnti in current-clamp mode. </li><li> Stimoli elettrici sono forniti da stimolazione l&#39;elettrodo attraverso un isolatore stimolazione controllata dal software di registrazione, e le correnti post-sinaptiche evocate nella DGCS neonati sono registrati. </li></ol> 4. Analisi dei dati Ampiezze delle risposte evocate postsinaptici sono analizzate a differenti stadi di sviluppo degli adulti nati neuroni. 5. Rappresentante Risultati Utilizzando il protocollo di cui sopra, l&#39;espressione della GFP in neuroni neonati giro dentato simile alla figura 1 è comune. Si noti che i neuroni neonati sono facilmente visibili ed entrambi i dendriti e degli assoni sono robustamente etichettati. Espressione in sottili assoni DGC e piccole spine dipende dalla qualità e titolo del virus, il promotore utilizzato e la lunghezza di espressione in vivo. Nelle nostre mani, le cellule neonato e sub-organelli cellulari sono di solito visibili in pochi giorni dopo l&#39;iniezione, e l&#39;espressione della colonna vertebrale possono essere rintracciate fin dai primi stadi dello sviluppo. Wbuco celle registrazioni da neuroni neonati in fasi di tempo diversi permettono lo studio delle uniche proprietà delle celle neonato, potenziali d&#39;azione ad esempio (Figura 2a), così come il processo di integrazione sinaptica esistente in circuiti neurali durante la maturazione (Figura 2b). <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="Figura 1" /> Figura 1 2-fotone immagine confocale dei neuroni neonati in una sezione orizzontale giro dentato di un topo adulto. Le cellule verdi sono 21dpi (giorni dopo l&#39;iniezione) GFP che esprimono DGCS neonato etichettato con pUX-GFP retrovirus. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="Figura 2" /> Figura 2 a) Azione potenziale cottura proprietà di un neonato DGC a 21 dpi. B) rappresentante evocato eccitatorio correnti post-sinaptiche (EPSCs) registrati su un DGC neonato a 21 dpi.

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

Continua neurogenesi nell&#39;ippocampo del cervello dei mammiferi adulti fornisce un sistema unico modello sperimentale per lo studio dello sviluppo neuronale e di integrazione nel cervello maturo. Il protocollo presentato qui combina etichettatura retrovirali e metodi elettrofisiologici per studiare l&#39;integrazione del neonato neuroni granulari dentato nel cervello dei topi adulti. Per garantire che gli esperimenti hanno avuto successo, si raccomanda quanto segue durante le fasi critiche della procedura: Per evitare l&#39;infezione indesiderati e l&#39;infiammazione, tutti gli strumenti devono essere sterilizzati (in autoclave, qualsiasi tipo di sterilizzatore, o etanolo al 70%) prima dell&#39;intervento chirurgico. Usa PBS sterile per lavare la punta di un ago della siringa prima dell&#39;iniezione. Per l&#39;iniezione del virus, gli animali devono essere ben montato sul dispositivo stereotassico e le fonti di movimenti inutili deve essere minimizzato durante l&#39;iniezione. Assicurarsi che la testa è ben fisso e fermo, e regolare la posizione naso del mouse per livello bregma e lambda prima del calcolo delle coordinate di iniezione. Estratto del virus nella siringa rapidamente per ridurre al minimo l&#39;esposizione a temperature ambiente - i retrovirus sono particolarmente sensibili alla temperatura. Iniettare lentamente virus al tasso raccomandato per minimizzare i danni di pressione. Utilizzando la raccomandata a punta piatta siringa (vs comune punta smussata) assicurerà un uniformemente distribuita zona infezione giro dentato. Prima di preparare le fette, il taglio e la incubazione ACSF soluzione deve essere bollito con il 95% di CO O 2 -5% 2 per almeno 30 minuti per consentire l&#39;ossigeno per saturare le soluzioni. Gli animali devono essere perfusi rapidamente e il tessuto mantenuto a freddo, soluzione satura di ossigeno per una migliore qualità fetta. Per la registrazione, aumentare l&#39;intensità dello stimolo a poco a poco fino a quando c&#39;è una risposta postsinaptica. Cambiare la posizione degli elettrodi di stimolazione all&#39;interno dello strato molecolare del giro dentato, se necessario. In sintesi, l&#39;approccio raccomandato offre un modo per esplorare le proprietà distinte e possibili ruoli funzionali di neuroni adulti, nato durante la fase iniziale di integrazione in attivo, circuito maturo.

<ul><li> Taglio soluzione (in mm): 110 cloruro di colina, 2.5 KCl, 1.3 KH 2 PO 4, 25,0 NaHCO 3, 0,5 CaCl 2, 7 MgCl 2, 10 destrosio, 1,3 ascorbato di sodio, 0,6 piruvato di sodio, 5,0 acido kynurenic. </li><li> Artificiale liquido cerebro-spinale (ACSF, in mm): 125,0 NaCl, 25,0 NaHCO 3, 2.5 KCl, 2 CaCl 2, 1,3 KH 2 PO 4, 1.3 MgCl2, 1,3 ascorbato di sodio, 0,6 di sodio piruvato, 10 destrosio. </li><li> Soluzione interna per intero a cellule patchclamp (in mm): 120 gluconato di potassio, 15 KCl, 4 MgCl 2, 0,1 EGTA, HEPES 10,0, 4 MgATP, 0,3 Na 3 GTP, 7 fosfocreatina (pH 7.4, 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Neurogenesi si verifica nel cervello adulto dei mammiferi nella zona sub-ventricolare (SVZ) del ventricolo laterale e nella zona sub-granulare (SGZ) del giro dentato dell&#39;ippocampo tutta la vita. Rapporti precedenti hanno mostrato che la neurogenesi ippocampale adulta è associata a disturbi cerebrali diverse, quali l&#39;epilessia, la schizofrenia, depressione e ansia (1). Decifrare il processo di normale e aberrante adulti nati integrazione neurone può mettere in luce l&#39;eziologia di queste malattie e informare lo sviluppo di nuove terapie. Neurogenesi adulta SGZ specchi embrionale e post-natale sviluppo neuronale, comprese le fasi di specificazione destino, migrazione, integrazione sinaptica, e la maturazione. Tuttavia, l&#39;integrazione avviene su un prolungato periodo di 6 settimane. Iniziale di ingresso sinaptico per adulti nati in granuli SGZ dentato (DGCS) è GABAergici, seguita da ingresso glutammatergica a 14 giorni (2). I fattori specifici che regolano la formazione circuito di adulti nati neuroni nel giro dentato sono attualmente sconosciute. Il nostro laboratorio utilizza una replica con deficit vettore retrovirale basato sul virus della leucemia murina Moloney per fornire proteine ​​fluorescenti e ipotizzato geni regolatori a queste cellule proliferanti. Questa tecnica fornisce virale elevata specificità e la risoluzione per l&#39;analisi di data cellula nascita, lignaggio, morfologia e sinaptogenesi. Un tipico esperimento utilizza spesso due o tre virus che contiene un&#39;etichetta unica, transgene, promotore e gli elementi per cella singola analisi di un processo di sviluppo desiderato in vivo. Il seguente protocollo descrive un metodo per l&#39;analisi funzionale integrazione neonato utilizzando un unico neurone verde (GFP) o rosso (dTomato) retrovirus proteina fluorescente e patch-clamp elettrofisiologia.

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

Un modo per studiare l&#39;integrazione del neonato granuli dentato negli animali adulti è descritto. Questa tecnica utilizza un retrovirus progettato per etichettare i neuroni neonati, seguita da registrazioni elettrofisiologiche in vivo per determinare integrazione funzionale.

Studiare l&#39;integrazione degli adulti nati Neuroni

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

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JoVE Journal

Neuroscience

13.8K Views.  State University of New York at Stony Brook. Un modo per studiare l'integrazione del neonato granuli dentato negli animali adulti è descritto. Questa tecnica utilizza un retrovirus progettato per etichettare i neuroni neonati, seguita da registrazioni elettrofisiologiche in vivo per determinare integrazione funzionale.

Video: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Ricerca

Neuroscienze

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

Studiare le basi neurali del comportamento adattivo Locomotore in Insetti

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Selettiva trasduzione virale di neuroni olfattivi per adulti nati per cronica In vivo Optogenetic Stimolazione

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

La specifica di telencefalici neuroni glutamatergici di cellule staminali umane pluripotenti

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Metodi per studiare i meccanismi d&#39;azione di farmaci antipsicotici in<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Etichettatura differenziale di Cell-superficie e interiorizzato Proteine ​​dopo Antibody Alimentazione dal vivo colture di neuroni

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Un sistema di Microbiomechanical per lo studio della formazione del Varicosity e del recupero in centrale del neurone assoni

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Un modello in vitro per lo studio dell'aggregazione Tau utilizzando la trasduzione dei neuroni umani mediata da lentivirali

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Registrazione elettrofisiologica wireless dei neuroni da tetrodi mobili in pesci che nuotano liberamente

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...