Vorremmo ringraziare Ryan Maneri (Oystercatcher Productions, LLC) per le riprese la nostra procedura e Rodney McPhail (Dipartimento di Scienze Biologiche, Purdue University) per l&#39;assistenza con l&#39;editing del video dissezione. La ricerca nel laboratorio di Suter è sostenuto da finanziamenti del NIH (R01 NS049233) e il Centro Bindley Bioscience alla Purdue University di DMS

Soluzioni <ul><li> L15-ASW terreno di coltura cellulare (1l) <ul><li> 1 bustina di polvere L15 </li><li> aggiungere 800 ml di H 2 O ultrapura </li><li> NaCl 400 mM </li><li> MgSO 4 27 mM </li><li> MgCl 2 28 mM </li><li> L-Glutammina 4 mM </li><li> Gentamicina 50 mg / ml </li><li> HEPES 5 mM </li><li> Regolare a pH 7,9 </li><li> Aggiungere goccia a goccia CaCl 2 9,3 mm (arresto se precipitati) </li><li> Aggiungi H 2 O ultrapura a 1 l </li><li> Controllare osmolarità (950-1000 mg / kg) con osmometer (pressione di vapore, Wescor # 5520) </li><li> Filtro 0,22 micron con una positiva unità di filtrazione a pressione (Filtri: SVGV010RS Millipore) </li><li> Conservare a 4 ° C fino a 1 mese </li></ul></li><li> Poli-L-lisina (70-150 kD) 200 mg / ml di soluzione in magazzino sterile H 2 O ultrapura conservato a -20 ° C </li><li> 0,5 M soluzione di MgCl 2 </li></ul> 1 ° giorno: dissezione di Aplysia ganglio addominale Preparazione della soluzione enzimatica dissociazione <ol><li> Pesare 9-10 mg di dispasi enzima (Worthington, Cat: LS02111) in una provetta Eppendorf </li><li> Aggiungere 900 ml di L15-ASW terreno di coltura, e 100 ml di H 2 O sterile ultrapura </li><li> Mescolare e posto a temperatura ambiente (RT) </li></ol> Dissezione di ganglio addominale <ol><li> Riempire una siringa da 50 ml con 0,5 M MgCl 2 soluzione. </li><li> Prendere un animale da serbatoio, trattata con delicatezza per evitare la difesa dell&#39;animale risposta inchiostrazione. </li><li> Posizionare il Aplysia su un lato su una tavola di dissezione (Styrofoam bordo), parte rostrale a destra, lato caudale a sinistra. </li><li> Inserire l&#39;ago nel animale appena dietro la testa e iniettare tutta MgCl 2 soluzione nella cavità del corpo. </li><li> Strofinare delicatamente l&#39;animale per disperdere i MgCl 2 soluzione tutto cavità del corpo, attendere qualche minuto per l&#39;animale di essere completamente anestetizzata. </li><li> Attacca la testa e la coda dell&#39;animale alla scheda di dissezione con due aghi. </li><li> Usare pinze per sollevare la pelle, tagliare la pelle e lo strato muscolare con le forbici grandi verso la testa e la coda per fare una grande apertura sul lato. </li><li> Trova il ganglio addominale sotto il pennello di tipo ovaio. Usare pinze per tenere i nervi di collegamento di circa 1 cm di fronte al ganglio addominale, tagliare i nervi con le forbici dissezione di fronte alla posizione Pinza e dietro il ganglio addominale. </li><li> Ganglio posto nella soluzione dispasi e incubare per 15-16 ore a 22 ° C con una temperatura controllata bagnomaria. </li></ol> Giorno 2: Placcatura di cellule borsa Aplysia Preparazione poli-lisina rivestite coprioggetto <ol><li> In un banco di flusso, 35 millimetri preparare tre piatti di Petri come lavorare piatti per la dissezione del ganglio, riempiendole di 4 ml L15-ASW media ciascuno. Trasferire il ganglio addominale dalla soluzione dispasi a uno dei piatti di funzionare dopo 15,5 ore di digestione. </li><li> Utilizzando pinze prendere acido-pulito # 1.5 coprioggetti (22x22 mm) conservati in etanolo, rimuovere l&#39;alcol e sterilizzare coprioggetto da fiamme brevemente su becco Bunsen. Lasciateli raffreddare prima di mettere brevemente i coprioggetti in capsule di Petri. </li><li> Preparare una punta ricurva placcatura piegando una punta di giallo sopra il becco Bunsen (fiamma solo per breve tempo per evitare la fusione della plastica!). </li><li> Preparare la quantità appropriata di 20 mg / ml soluzione di poli-lisina diluendo 200 mg / ml Soluzione sterile con H 2 O ultrapura. </li><li> Coprire ogni coprioggetto con 500 ml di 20 mg / ml di poli-lisina e incubare per 20 minuti a RT. </li><li> Lavare ogni coprioggetto 3-5 volte con 500 microlitri sterile H 2 O ultrapura ciascuno. </li><li> Rimuovere l&#39;acqua dal coprioggetto; riempire ogni piatto con 4 ml di L15-ASW terreno di coltura. </li></ol> Dissociazione delle cellule borsa <ol><li> Sotto un microscopio dissezione, tagliare il ganglio addominale a metà forbici e pinze con dissezione precedentemente puliti con etanolo al 95% (Figura 1 linea 1). </li><li> Su una metà del ganglio, tagliare tra cluster borsa cellulare e le cellule hemiganglion (Figura 1 linea 2), e tagliare via le estensioni dei nervi rimasti al di là di cellule borsa (Figura 1 linea 3). Lavorare su una metà del ganglio alla volta, lasciando l&#39;altra metà nel piatto, mentre le cellule dal primo gruppo sono stati placcati. </li><li> Utilizzando due pinze (o una pinza e forbici dissezione), sacco a grappolo rilascio delle cellule mettendo da parte la guaina di tessuto connettivo che circonda il cluster di cellule borsa. Tagliare il tessuto connettivo con le forbici dissezione, se necessario. Attenzione a non stringere e toccare il cluster di cellule borsa troppo durante questo processo. </li><li> Quando la maggior parte del tessuto connettivo viene rimosso, trasferire il bgrappolo di cellule ag in un piatto nuovo a lavorare con la punta piegata giallo preparato in precedenza. Fare attenzione a non lasciare che il gruppo di cellule sacchetto essere intrappolato dentro la punta di colore giallo o bloccati in aria / medio interfaccia. </li><li> Triturare il cluster con la punta giallo per rimuovere le cellule singolo sacchetto. Fate questo pipettando il cluster dentro e fuori la punta. Iniziare con basse forze di taglio e aumentare la forza se necessario. Utilizzare sempre la più bassa forza necessaria per rimuovere i neuroni senza uccidere le cellule troppi. </li><li> Raccogliere 3-5 cellule sane borsa e trasferirli in un piatto con un poli-lisina rivestite coprioggetto. Evitate di raccogliere le cellule morte (sono nero / trasparente al centro) e detriti del tessuto connettivo. </li><li> Ripetere il passaggio 6. e luogo circa 15-25 neuroni vivere al centro di ogni coprioggetto. Se le cellule morte e detriti vengono trasferiti anche, rimuovere il materiale indesiderato di raccoglierlo o soffiare via dalle cellule sane. Questo processo richiede circa 2-3 ore per entrambi i gruppi. </li><li> Una volta finito di tenere i piatti della cultura in panchina di flusso per almeno 2 ore a temperatura ambiente per permettere alle cellule di allegare (tenere banco flusso ventilatore spento per evitare vibrazioni indesiderate). </li><li> Porre le capsule in un incubatore a 14 ° C per una notte, o fino a quando un ulteriore uso. Coni di crescita si possono sviluppare in 4-10 ore. </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="Figura 1. Schematica del ganglio Aplysia californica addominale. Linee di colore indicano dove tagliare per ottenere gruppi di cellule borsa." /> Figura 1. Schematica del ganglio Aplysia californica addominale. Linee di colore indicano dove tagliare per ottenere gruppi di cellule borsa. Giorno 3: Impostazione da camera per immagini dal vivo di cellule borsa Aplysia Assemblaggio di immagini da camera <ol><li> Prima di utilizzare cellule borsa per esperimenti dal vivo l&#39;imaging cellulare, ispezionare le cellule utilizzando un microscopio a bassa potenza. Se le cellule non hanno sviluppato coni di crescita sana, di approvvigionamento a medio dolce e lasciarlo a temperatura ambiente per 1-2 ore. </li><li> Per la fornitura di piatti con prodotti freschi di media L15-ASW, rimuovere la metà (~ 2 ml) di media età con una linea di vuoto, e aggiungere 2 ml di nuovi L15-ASW medio per capsula di Petri. E &#39;importante non rimuovere completamente tutti i media dal piatto tutto in una volta, in modo da rimuovere le cellule dal vetrino. </li><li> Per l&#39;imaging cellulare borsa, montiamo due coprioggetto e due distanziatori di plastica in un sandwich-come camera di imaging. Il coprioggetto top nel panino viene tagliato corto per permettere un facile scambio di medie e reagenti. Durante il montaggio, coprioggetto con le cellule borsa allegata devono essere immersi in L15-ASW medio in ogni momento di impedire alle cellule di sloggiare. </li><li> Per preparare la camera, tagliati fuori ~ 2 mm dal lato destro di mm # 1 coprioggetto 22x22 con diamante penna (tagliatore di vetro) e il righello. </li><li> Usando una siringa da 1 ml, applicare grasso alto vuoto ai due lati del vetrino più brevi, e allegare distanziatori di plastica per ogni lato. Ingrassare vuoto nella parte superiore di entrambi i distanziali. </li><li> Invertire il coprioggetto e montaggio distanziatore, e posto in capsula di Petri contenente cellule borsa su un vetrino coprioggetti. Allineare e premere delicatamente verso il basso per attaccare coprioggetto dall&#39;alto verso il basso coprioggetto con la fine inversione di due Q-tips. </li><li> Utilizzando pinze, sollevare delicatamente il montaggio panino fuori della scatola di Petri, senza il supporto fuoriuscire. Allineare le due coprioggetto, se necessario, e pulire medio in eccesso con un Kimwipe. Posizionare il panino sulla parte inferiore della porta vetrino da microscopio e fissare il coperchio con cura utilizzando clip. </li><li> Pulire il centro del lato superiore del panino con Q-tips tamponò in detergente per vetri (Sparkle è usato come detergente per vetri). Rapidamente asciugare il detergente per vetri con un secondo, asciutto Q-tip per evitare linee striscia. Fare lo stesso per il coprioggetto fondo. Coprioggetto pulito, privo di linee striscia sono importanti per l&#39;imaging di alta qualità. </li></ol> Cellule borsa sul microscopio Dal Aplysia colture di cellule neuronali borsa sono a bassa densità, è meglio se il vostro setup microscopio ha funzioni di posizionamento stadio per facilitare il passaggio tra le celle. Cellule borsa può essere facilmente conservato a temperatura ambiente per diverse ore sul palco durante gli esperimenti di imaging. Cambio del mezzo periodicamente per evitare l&#39;evaporazione.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Aplysia neuroni delle cellule forniscono una borsa senza siero neuronale sistema di coltura cellulare con pochissime cellule non neuronali. Questi neuroni formano coni di crescita molto grande adatto per affrontare una serie di domande importanti nelle cellule biologiche. I neuroni delle cellule borsa può essere facilmente manipolato e ripreso a temperatura ambiente per diverse ore. Mediante microscopia a fluorescenza Speckle (FSM) si può analizzare quantitativamente i vari parametri di F-actina e polimerizzazione dei microtubuli e la din...

<table><tbody><tr><td>#1 glass coverslips 22x22 mm</td><td>Tool</td><td>VWR international</td><td>48366067</td><td></td></tr><tr><td>#1.5 glass coverslips 22x22 mm</td><td>Tool</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>35 mm Petri dishes</td><td>Tool</td><td>Falcon BD</td><td>353001</td><td></td></tr><tr><td>Filters for medium filtration</td><td>Tool</td><td>EMD Millipore</td><td>SVGV010RS</td><td></td></tr><tr><td>High vacuum grease</td><td>Tool</td><td>Dow Corning</td><td>1597418</td><td></td></tr><tr><td>Osmometer</td><td>Tool</td><td>Wescor</td><td>5520</td><td></td></tr><tr><td>Plastic shims</td><td>Tool</td><td>Small Parts, Inc.</td><td>SHSP-200</td><td></td></tr><tr><td>Calcium chloride</td><td>Reagent</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Gentamicin</td><td>Reagent</td><td>Invitrogen</td><td>15750-060</td><td></td></tr><tr><td>HEPES</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H4030</td><td></td></tr><tr><td>L15 medium</td><td>Reagent</td><td>Invitrogen</td><td>41300-039</td><td></td></tr><tr><td>L-glutamine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Magnesium chloride</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>5958-04</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>6070-12</td><td></td></tr><tr><td>Poly-L-lysine (70-150 kD) </td><td>Reagent</td><td>Sigma-Aldrich</td><td>P6282</td><td></td></tr><tr><td>Sodium chloride</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>7581</td><td></td></tr><tr><td>Neutral Protease (Dispase)</td><td>Reagent</td><td>Worthington Biochemical</td><td>LS02111</td><td></td></tr></tbody></table>

neuronal cell cultures from aplysia for high-resolution imaging of growth cones

Coni di crescita neuronali sono strutture altamente mobili sulla punta degli assoni in grado di rilevare segnali di orientamento per l&#39;ambiente e trasducono queste informazioni in movimento direzionale verso la cellula bersaglio appropriato. Per comprendere pienamente come le informazioni di guida viene trasmessa dalla superficie cellulare al connesse reti dinamica del citoscheletro, si ha la necessità di un sistema modello adatto per l&#39;imaging di cellule in vivo di proteine ​​dinamica ad alta risoluzione temporale e spaziale. Tipici coni di crescita vertebrati sono troppo piccoli per analizzare quantitativamente F-actina e la dinamica dei microtubuli. I neuroni dalla lepre mare Aplysia californica sono 5-10 volte più grande di neuroni vertebrati, può facilmente essere conservato a temperatura ambiente e sono cellule molto robusto per micromanipolazione e misure biofisici. I loro coni di crescita sono ben definite regioni citoplasmatiche e ben descritto sistema del citoscheletro. I corpi delle cellule neuronali possono essere microiniettati con una varietà di sonde per studiare la motilità del cono di crescita e di orientamento. Nel presente protocollo si dimostra una procedura per la dissezione del ganglio addominale, la cultura dei neuroni delle cellule borsa e la creazione di una camera di imaging per l&#39;imaging cellulare dal vivo di coni di crescita.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Aplysia californica neuroni sviluppare grandi coni di crescita nella cultura che sono eccellenti per imaging ad alta risoluzione della motilità del cono di crescita e di orientamento. Qui, vi presentiamo un protocollo per la dissezione e la placcatura dei neuroni borsa cellule Aplysia così come per la creazione di una camera per l&#39;imaging cellulare dal vivo.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.4K Views.  Purdue University. Aplysia californica neuroni sviluppare grandi coni di crescita nella cultura che sono eccellenti per imaging ad alta risoluzione della motilità del cono di crescita e di orientamento. Qui, vi presentiamo un protocollo per la dissezione e la placcatura dei neuroni borsa cellule Aplysia così come per la creazione di una camera per l'imaging cellulare dal vivo.

Video: Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-imaging di traslocazione della PKC in SF9 cellule e nei neuroni sensoriali Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Ricerca

Neuroscienze

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Alisia Preparazione ganglia per analisi elettrofisiotiche e molecolari di singoli neuroni

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Bioingegneria

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

I cambiamenti di monitoraggio della concentrazione di calcio intracellulare ed efficacia Synaptic nel Mollusco<em&gt; Aplysia</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Culture Explant neurali di<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparazione del Aplysia Senso-motorio colture cellulari neuronali

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Biologia

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolamento dei neuroni sensoriali<em&gt; Aplysia californica</em&gt; Per Bloccare Registrazioni patch di correnti glutamatergico

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Rapida genotipizzazione degli animali Seguito da Stabilire colture primarie di neuroni del cervello

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Circuiti neuronali riavvolgenti: un nuovo metodo per l&#39;estensione veloce del neurite e la connessione neuronale funzionale

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Replatazione post-differenziazione dei neuroni derivati da cellule staminali pluripotenti umane per lo screening ad alto contenuto di neuritogenesi e maturazione delle sinapsi

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

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Preparazione del Aplysia Senso-motorio colture cellulari neuronali

Isolamento dei neuroni sensoriali

Rapida genotipizzazione degli animali Seguito da Stabilire colture primarie di neuroni del cervello

Circuiti neuronali riavvolgenti: un nuovo metodo per l'estensione veloce del neurite e la connessione neuronale funzionale

Replatazione post-differenziazione dei neuroni derivati da cellule staminali pluripotenti umane per lo screening ad alto contenuto di neuritogenesi e maturazione delle sinapsi

Uso di neuroni ippocampali coltivati primari per studiare l'assemblaggio dei segmenti iniziali di Axon