This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

Tutti gli esperimenti qui descritti sono stati eseguiti in conformità alle Regno Unito Animali (Procedure Scientifiche) Act del 1986. 1. Preparazione degli strumenti, terreni di coltura, e piatti <ol><li> Preparare due forbici laboratorio in acciaio inox di dissezione e due pinze da laboratorio in acciaio inox. Autoclavare tutti gli strumenti e posto in una cappa di coltura O / N sotto luce (UV) ultravioletta per la sterilizzazione. </li><li> Fate 500 ml di mezzi essenziali (MEM) terreno minimo cultura (10% fetale Bovine Serum [FBS], 20 mM KCl, 25 NaHCO mm 3, 30 mm D-glucosio, 2 mM L-glutammina, 100 U / ml di penicillina, 100 ug / ml di streptomicina e 6 mg / ml di ampicillina) e filtro sterilizzare. Conservare a 4 ° C per mesi. </li><li> Preparare 24 piastre di coltura e contenenti 14 mm, numero 1 coprioggetto spessore. Autoclave i coprioggetti e cappotto a 100 mg / L poli-d-lisina (PDL) come descritto in precedenza 3. Luce UV sterilizzare O / N. </li></ol> 2. Preparazione di CGC Cultura Solutions <ol><li> Preparare &#39;soluzione A&#39; (100 ml) in autoclave, luce UV trattato, dH 2 O sterile contiene 250 mg di glucosio, 300 mg di albumina sierica bovina [ASC], 955 mg di tampone fosfato [PBS] (Ca 2+ e Mg + free), e 1 ml di 3,8% MgSO 4 · 7H 2 O. </li><li> Preparare &#39;soluzione B&#39; (10 ml) contenente 1 ml di 5 mg / ml di tripsina diluito in 9 ml di soluzione A. </li><li> Preparare &#39;soluzione C&#39; (10 ml) contenente 1.000 unità DNasi, 0,5 mg di inibitore della tripsina di soia, 100 ml di 3,8% MgSO4 · 7H 2 O, diluita a 10 ml con soluzione A. </li><li> Preparare &#39;soluzione D&#39; (20 ml) contenente 3,2 ml di soluzione C, diluita a 20 ml con soluzione A. </li><li> Preparare la soluzione BSA / Earle soluzione salina bilanciata (EBSS) contenente EBSS, 25 NaHCO mm 3, 3 MgSO mm 4 · 7H 2 O, e4% BSA, pH 7,4, come precedentemente descritto 3. </li><li> Preparare LME aggiungendo puro LME in 5 ml di terreno di coltura MEM per dare una concentrazione finale di 150 mM LME, riportare la soluzione a pH 7,4 utilizzando un pHmetro da banco, e filtro sterilizzare utilizzando un polietere solfone 28 mm (PES) 0,2 micron siringa filtro. </li></ol> 3. La coltura le CGC <ol><li> Raccogliere cerebella 4-7 giorni vecchi cuccioli di ratto come descritto in precedenza 3. Mettere subito in piastra Petri contenente 5 ml di soluzione A su ghiaccio. </li><li> Eliminare la soluzione in eccesso da cerebella e luogo del tessuto nel coperchio piastra di Petri. </li><li> Tagliare il tessuto finemente con una lama di rasoio ben fiammato-in almeno tre diverse direzioni. </li><li> Aggiungi il tessuto tagliato a soluzione B (lavare capsula di Petri con una soluzione per garantire il tessuto limitata è perso), e posto in bagno d&#39;acqua a 37 ° C per 5 minuti. Agitare delicatamente ogni paio di minuti. </li><li> Aggiungere la soluzione D (20 ml) al tubo di neutralizzare la tripsina, scossa e centrifuge a 65 xg per 5 min. </li><li> Eliminare il surnatante. </li><li> Risospendere in 4 ml di soluzione C. Triturare anche (circa 10 volte ciascuna) con tre pipette di vetro fiammati di diametro decrescente, fino a quando la sospensione è più possibile uniforme. </li><li> Lentamente e delicatamente aggiungere qualche goccia di questo omogenato in cima alla BSA / EBSS. Se inizia ad affondare attraverso la BSA / EBSS, triturare più e / o aggiungere un po &#39;di soluzione più C. L&#39;omogeneizzato dovrebbe sedersi in uno strato sopra l&#39;BSA / EBSS. Non agitare. Spin a 100 g per 5 min. </li><li> Rimuovere il surnatante e risospendere pellet (soft) in 1 ml di MEM. </li><li> Contare le cellule utilizzando un emocitometro e piastra a 800.000 cellule / vetrino in 500 ml di MEM. Inserire in termostato a 37 ° C con il 6% di CO 2. </li><li> Cambiare il medio il giorno seguente (24-36 ore dopo la preparazione). Fare una soluzione di MEM contenente 10 mM del ciclo cellulare inibitore arabinofuranosil citidina (AraC). <ol><li> Per ciascunovetrino, conservano 250 microlitri del vecchio medium in 24 pozzetti fresco, aspirare il resto, aggiungere 250 ml di normale media MEM e aspirare questo per lavare le cellule, poi aggiungete 250 ml di vecchio schienale medio alle cellule e rabboccare con 250 ml di medio-AraC contenenti. Nota: Le celle possono essere utilizzati da 6 giorni in vitro (DIV). </li></ol></li></ol> 4. selettivamente L&#39;esaurimento della microglia (eseguiti a 6 DIV) <ol><li> Aggiungere LME puro a 5 ml di un&#39;aliquota separata di MEM per dare una concentrazione finale di 150 mM LME. </li><li> Riportare la soluzione a pH 7,4 e filtro sterilizzare con un solfone polietere 28 mm (PSE) filtro da 0,2 micron siringa. </li><li> Diluire soluzione ad una concentrazione di 50 mM (2 x LME) in terreno MEM e posto in un bagno d&#39;acqua a 37 ° C per 10 min con mezzo normale MEM per colture di controllo. </li><li> Rimuovere la metà (250 ml) il terreno MEM dalle culture CGC e mantenere a 37 ° C. </li><li> Cellule Treatcon MEM terreno contenente 2 x LME (250 microlitri), cioè diluito 1: 1 dando una concentrazione di ben 25 mM, o con pre-riscaldato mezzo MEM senza LME (250 microlitri) per colture di controllo. </li><li> Incubare le cellule a 37 ° C in atmosfera umidificata con il 6% di CO 2 per 1 ora. </li><li> Lavare le cellule due volte a fresco preriscaldato medio MEM per rimuovere LME contenenti supporti, e quindi sostituire il terreno di coltura conservato e un uguale volume di fresco pre-riscaldato medio MEM per rispettivi pozzetti. </li><li> Culture Ritorna alla incubatrice (inumidito con 6% di CO 2 a 37 ° C) e lasciare riposare per 24 ore prima di ogni ulteriore trattamento. </li></ol>

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	<li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+remarkable,+yet+not+extraordinary,+human+brain+as+a+scaled-up+primate+brain+and+its+associated+cost.">The remarkable, yet not extraordinary, human brain as a scaled-up primate brain and its associated cost.</a> PNAS. 26 (109), 10661-10668 (2012).</li><li>Evans, G. J., &amp;Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+neurotransmitter+release+by+dihydropyridine-sensitive+calcium+channels+involves+tyrosine+phosphorylation.">Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation.</a> Eur J Neuro. 11 (1), 279-292 (1999).</li><li>Kingham, P. J., Cuzner, M. L., Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+pathways+mobilized+in+microglia+and+neurones+as+a+consequence+of+chromogranin+A-induced+microglial+activation.">Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.</a> J Neurochem. 73 (2), 538-547 (1999).</li><li>Contestabile, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+granule+cells+as+a+model+to+study+mechanisms+of+neuronal+apoptosis+or+survival+in+vivo+and+in+vitro.">Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro.</a> Cerebellum. 1 (1), 41-55 (2002).</li><li>Kramer, D., Minichiello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+primary+cerebellar+granule+cells.">Cell culture of primary cerebellar granule cells.</a> Methods Mol Biol. 633, 233-239 (2010).</li><li>Facci, L., Skaper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+rat+cerebellar+granule+neurons+and+application+to+identify+neuroprotective+agents.">Culture of rat cerebellar granule neurons and application to identify neuroprotective agents.</a> Methods Mol Biol. 846, 23-37 (2012).</li><li>Thiele, D. L., Kurosaka, M., Lipsky, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+of+the+accessory+cell+necessary+for+mitogen-stimulated+T+and+B+cell+responses+in+human+peripheral+blood:+delineation+by+its+sensitivity+to+the+lysosomotropic+agent,+L-leucine+methyl+ester.">Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine methyl ester.</a> J Immunology. 131 (5), 2282-2290 (1983).</li><li>Giulian, D., Vaca, K., Corpuz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+glia+release+factors+with+opposing+actions+upon+neuronal+survival.">Brain glia release factors with opposing actions upon neuronal survival.</a> J Neurosci. 13 (1), 29-37 (1993).</li><li>Guillemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtention+and+characterization+of+primary+astrocyte+and+microglial+cultures+from+adult+monkey+brains.">Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains.</a> J Neurosci Res. 49 (5), 576-591 (1997).</li><li>Hewett, J. A., Hewett SJ, ., Winkler, S., Pfeiffer SE, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+nitric+oxide+synthase+expression+in+cultures+enriched+for+mature+oligodendrocytes+is+due+to+microglia.">Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia.</a> J Neurosci Res. 56 (2), 189-198 (1999).</li><li>Jebelli, J., Hooper, C., &amp;Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+p53+activation+is+detrimental+to+neuronal+sysnapses+during+activaton-induced+inflammation:+implications+for+neurodegeneration.">Microglial p53 activation is detrimental to neuronal sysnapses during activaton-induced inflammation: implications for neurodegeneration.</a> Neurosci Lett. 7 (583), 92-97 (2014).</li><li>Frade, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+induces+release+of+glutathione+from+cultured+rats+astrocytes+&#8211;+a+possible+neuroprotective+mechanism.">Glutamate induces release of glutathione from cultured rats astrocytes &#8211; a possible neuroprotective mechanism.</a> J Neurochem. 105 (4), 1144-1152 (2008).</li><li>Hamby, M. E., Uliasz, T. F., Hewett, S. J., Hewett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+improved+procedure+for+the+removal+of+microglia+from+confluent+monolayers+of+primary+astrocytes.">Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.</a> J Neurosci Methods. 150 (1), 128-137 (2006).</li><li>Morgan, S. C., Taylor, D. L., Pocock, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+release+activators+of+neuronal+proliferation+mediated+by+activation+of+mitogen-activated+protein+kinase,+phosphatidylinositol-3-kinase/Akt+and+delta-Notch+signalling+cascades.">Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades.</a> J Neurochem. 90 (1), 89-101 (2004).</li><li>Crocker, S. J., Frausto, R. F., Whitton, J. L., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+establish+microglia-free+astrocyte+cultures:+comparison+of+matrix+metalloproteinase+expression+profiles+in+pure+cultures+of+astrocytes+and+microglia.">A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia.</a> Glia. 56 (11), 1187-1198 (2008).</li><li>Kumamaru, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+clodronate+selectively+eliminates+microglia+from+primary+astrocyte+cultures.">Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures.</a> J Neuroinflamm. 9, 116 (2012).</li></ol>

The authors declare that they have no competing financial interests.

I passi più importanti per assicurare l&#39;eliminazione selettiva di successo di microglia da CGC e / o colture miste sono: 1) il mantenimento di una cultura CGC sterile e sano; 2) filtro sterilizzante medio LME contenente e ritornando la soluzione a pH 7,4; 3) mantenere i media CGC conservati e LME contenenti supporti a 37 ° C per evitare lo shock termico; e 4) che lavorano rapidamente per ridurre il tempo di cellule sono tenuti all&#39;esterno dell&#39;incubatrice. Abbiamo ...

Il cervello umano è composto una stima di 85 miliardi di neuroni e un ulteriore 85 miliardi di cellule non neuronali tra cui glia 1. Per la maggior parte degli ultimi 100 anni i neuroscienziati si sono concentrati prevalentemente sulla popolazione delle cellule neuronali, cellule gliali credendo di essere poco più di cellule di supporto passivi che hanno fornito supporto strutturale per i neuroni - da qui l&#39;etimologia greca del &#39;glia&#39; tradotto in inglese come &#39;colla&#39;. Recentemente, tuttavia, è diventato sempre più evidente che le interazioni neuronali gliali possono essere molto più fondamentale per aspetti fondamentali della neurobiologia, neurofisiologia, e la genesi e progressione di molte malattie neurodegenerative. Cellule cerebellari granulari (CGC), il più abbondante popolazione neuronale omogenea nel cervello umano, dominano il cervelletto e costituiscono oltre il 90% dei suoi componenti cellulari. Di conseguenza, queste cellule sono stati ampiamente utilizzati in vitro come sistema modello per tegli studio dello sviluppo neuronale, funzione e patologia 2-6. Tuttavia, le culture CGC contengono ancora microglia e glia altri in proporzioni probabilmente significativi. Di conseguenza, i dati che mostrano CGC putatively risposte neuronali scalo a trattamenti con le cellule differenti possono infatti insorgere - in tutto o in parte - dalla risposta secondario indiretta della vicina glia nella cultura. Per valutare questo, abbiamo selettivamente eliminato microglia da colture neuronali CGC con l&#39;ausilio di estere metilico L-leucina (LME). LME è un agente lysomotropic originariamente utilizzato per distruggere selettivamente macrofagi 7, e da allora è stato usato per esaurire anche selettivamente microglia da neurale, astrociti, e le culture gliali miste 8,9,10. LME è interiorizzato da macrofagi e microglia, in cui esso provoca un&#39;interruzione lisosomiale e la successiva apoptosi 13,14. Macrofagi e microglia sono caratteristicamente ricchi di lisosomi, causando loro di essere particularly vulnerabile in seguito all&#39;esposizione al trattamento LME. Questo protocollo fornisce un modo potente, ma semplice e facile stabilire il contributo della microglia in esperimenti utilizzando altri sistemi di coltura gliali / neuronali CGC e.

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			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
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		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</td>
		</tr>
		<tr>
			<td>L-leucine methyl ester hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>7517-19-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7510-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>27964-99-4</td>
			<td>Coat coverslips 1 day before use&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
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		<tr>
			<td>Soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
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		<tr>
			<td>Mouse anti-ED1 antibody&#160;</td>
			<td>Abcam</td>
			<td>ab31630</td>
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selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

La capacità di questa tecnica per eliminare selettivamente microglia da CGC e / o colture miste si basa sulla successiva capacità del ricercatore di identificare accuratamente e differenziare microglia dalle celle circostanti. Ciò può essere ottenuto usando una macchina per cella specifica-microglia, come isolectin-B4, come illustrato in figura 1. Come mostrato nella Figura 2, nessun cambiamento osservabile astrociti e la densità neuronale e morfologia sono stati registrati con rif...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

L&#39;esaurimento selettiva di microglia da cerebellare granuli colture cellulari Utilizzando L-leucina estere metilico

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

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Neuroscience

8.6K Views.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

Video: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotipica Culture cerebellare: sfide apoptotiche e rilevamento

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

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Neuroscienze

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging di postnatale cerebellare granuli migrazione cellulare Utilizzando confocale macroscopia

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

Preparazione di fettine di cervello acuto utilizzando un' ottimizzata N-metilico-D-metodo di ripristino protettivo glucamine

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Valutazione della vitalità neuronale utilizzando la fluoresceina diacetato-propidium ioduro a doppia colorazione nel granulo cerebrale Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modellazione di morte neuronale e degenerazione in neuroni cerebellari del granello primario del Mouse

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Quantificare la morfologia di Microglia da microfotografie di Immunohistochemistry preparato tessuto usando ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Preparazione e Immunostaining di su colture organotipiche Myelinating fetta cerebellare

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...