Gli autori ringraziano Jung Yeon-Joo per il suo sostegno sperimentale durante la purificazione di synaptosome e Jungjoo Park per le immagini degli synaptosomes con esposizione di PS. Inoltre, ringraziamo tutti i membri nel laboratorio di Chung per utile discussione. Questo lavoro è stato sostenuto dalla National Research Foundation di sovvenzione di Corea (NRF) finanziata dal governo coreano (MSIP) (NRF-2016M3C7A1905391 e NRF-2016R1C1B3006969) (W.-S. C).

<ol>
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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=Astrocyte+glypicans+4+and+6+promote+formation+of+excitatory+synapses+via+GluA1+AMPA+receptors.">Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors.</a> Nature. 486 (7403), 410-414 (2012).</li><li>Xu, J., Xiao, N., Xia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondin+1+accelerates+synaptogenesis+in+hippocampal+neurons+through+neuroligin+1.">Thrombospondin 1 accelerates synaptogenesis in hippocampal neurons through neuroligin 1.</a> Nat Neurosci. 13 (1), 22-24 (2010).</li><li>Chung, W. 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N., 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=Microglia+promote+learning-dependent+synapse+formation+through+brain-derived+neurotrophic+factor.">Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.</a> Cell. 155 (7), 1596-1609 (2013).</li><li>Iram, T., 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=Megf10+Is+a+Receptor+for+C1Q+That+Mediates+Clearance+of+Apoptotic+Cells+by+Astrocytes.">Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes.</a> J Neurosci. 36 (19), 5185-5192 (2016).</li><li>Tasdemir-Yilmaz, O. E., Freeman, M. 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S., 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=Novel+allele-dependent+role+for+APOE+in+controlling+the+rate+of+synapse+pruning+by+astrocytes.">Novel allele-dependent role for APOE in controlling the rate of synapse pruning by astrocytes.</a> Proc Natl Acad Sci U S A. 113 (36), 10186-10191 (2016).</li><li>Purice, M. D., Speese, S. D., Logan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+glial+clearance+of+degenerating+axons+in+aged+Drosophila+is+due+to+reduced+PI3K/Draper+activity.">Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity.</a> Nat Commun. 7, 12871 (2016).</li><li>Loov, C., Mitchell, C. 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In questo articolo, presentiamo metodi per un lungo termine test di fagocitosi live-imaging in vitro utilizzando cellule gliale purificate e sinaptosomi coniugato con indicatore di pH. Indichiamo che confrontato con microglia, astrociti possiedono diverse capacità engulfment e degradazione durante la fagocitosi dei sinaptosomi. Inoltre, i nostri dati suggeriscono che fattori Astrocita-secreto, che contengono molecole passerella come GAS6, proteine e MEGE8, sono essenziali per efficiente fagocitosi di PS-dipende...

Le cellule gliali, che si riferiscono alle cellule non eccitabili nel cervello, sono il tipo principali delle cellule nel sistema nervoso centrale (SNC). In precedenza, le cellule glial erano considerate come semplici cellule di supporto che principalmente giocano un ruolo passivo nel mantenimento della sopravvivenza neuronale e sinaptica basale proprietà. Tuttavia, la prova emergente ha rivelato che le cellule gliali svolgono i ruoli più attivi in vari aspetti della neurobiologia, quali il mantenimento dell'omeostasi del cervello, mediando sinapsi formazione1,2,3 e sinapsi eliminazione4,5e modulante plasticità sinaptica6,7. Astrociti, microglia ed oligodendrociti sono cellule gliali del SNC. Tra queste cellule, astrociti e microglia hanno dimostrato di svolgere ruoli fagocitici inghiottendo sinapsi4,5, di cellule apoptotiche8, detriti neurali9e proteine patogene, quali amiloide beta le piastre10,11. Nel cervello in via di sviluppo, gli astrociti eliminano sinapsi nel nucleo genicolato laterale dorsale (missilistico) attraverso fagocitosi di MERTK - e MEGF10-dipendente4. Allo stesso modo, le microglia eliminano anche rivestite con C1q sinapsi durante fasi di sviluppo attraverso il classico del complemento cascata5. Interessante, è stato suggerito che i difetti in potatura sinapsi possono essere uno degli iniziatori di parecchi disordini neurologici. Ad esempio, è stato indicato che le mutazioni nel componente del complemento 4 (C4), che aumenta la potatura sinapsi complemento-mediata da microglia, sono fortemente associate con la prevalenza della schizofrenia negli esseri umani12. Una carta recente inoltre ha dimostrato che la via classica del complemento è iperattivata nelle fasi di iniziazione dell'annuncio e induce la perdita di sinapsi iniziale in questa malattia13.
Confrontato con la fagocitosi mediata da microglia, se fagocitosi mediata Astrocita contribuiscono all'avvio e alla progressione di vari disturbi neurologici è meno chiaro. Tuttavia, un recente documento suggerisce che fattori che alterano il tasso di potatura normale sinapsi dai astrocytes possono interferire con l'omeostasi del cervello e contribuiscono alla suscettibilità e patologia DC14. Il tasso di potatura di sinapsi dai astrocytes potentemente è controllato dagli isomeri di ApoE , con un allele protettivo per annuncio (ApoE2) migliorando fortemente il tasso e un allele di rischio per l'annuncio (ApoE4) abbassando significativamente il tasso. Inoltre, topi transgenici che esprimono ApoE4 accumulato C1q sinaptica molto di più rispetto a controllo o ApoE2 topi14. Questi dati suggeriscono che alterata fagocitosi Astrocita-mediata nei primi cervello dell'annuncio può indurre l'accumulo di detriti senescenti rivestite con C1q sinapsi/sinaptica che attiva la fagocitosi microglial complemento-mediata, degenerazione sinaptica di guida . L'alterata capacità fagocitica dei astrocytes nei portatori ApoE4 può anche contribuire all'accumulo incontrollato delle placche di beta amiloide nel cervello colpite AD.
Inoltre, esso ha dimostrato che le cellule gliali nel cervello invecchiato Drosophila perdono la loro capacità fagocitica grazie alla traduzione in diminuzione di Draper, un omologo di Megf10 che gli astrociti utilizzano per phagocytosing sinapsi. Il ristabilimento dei livelli di Draper salvato la capacità fagocitica delle cellule gliali, che efficacemente ha eliminato detriti axonal danneggiati nel cervello invecchiato in simile misura come quello nel cervello giovane, che indica che invecchiamento indotto da alterazioni nella capacità fagocitica di gli astrociti possono contribuire alla rottura del cervello omeostasi15.
Basato su queste nuove scoperte, modulando la capacità fagocitica dei astrocytes può essere una strategia terapeutica attraente per prevenire e curare vari disturbi neurologici. A questo proposito, ci sono stati diversi tentativi per migliorare la capacità fagocitica dei astrocytes, ad esempio, inducendo l'acidificazione dei lisosomi con nanoparticelle acide16 e che overexpressing EB (IL2RG), che può migliorare il fattore di trascrizione lisosoma biogenesi17. Nonostante questi tentativi, non è ancora chiaro come astrociti e microglia differenze nella loro cinetica fagocitica e se dovremmo aumentare o diminuire la loro capacità fagocitica in varie malattie.
In questa carta, presentiamo un'analisi novella in vitro per la rilevazione della capacità fagocitica dei astrocytes in tempo reale. I dati mostrano diversa cinetica di engulfment e degradazione in astrociti e microglia. Astrocita-condizionato mezzo (ACM), che contiene fattori secreti dagli astrociti, è essenziale per la fagocitosi efficace di astrociti e microglia. Inoltre, Megf10, un recettore fagocitico in astrocytes e un omologo di Ced-1 e Draper, gioca ruoli critici nella fagocitosi mediata Astrocita8,18.

<table>
	<tbody>
		<tr>
			<td>Synaptosome purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE healthcare life sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Start Bradford Protein Assay Kit 2</td>
			<td>BIO-RAD</td>
			<td>5000202</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator conjugation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>LPS solution</td>
			<td>DMSO100</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo red, succinimidyl ester</td>
			<td>Molecula probes</td>
			<td>P36600</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunopanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Earle&#8217;s balanced salt solution (EBSS)</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bovogen</td>
			<td>BSA025</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyrebonuclease 1 (DNase)</td>
			<td>Worthington</td>
			<td>Is002007</td>
			<td></td>
		</tr>
		<tr>
			<td>(DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>(dPBS)</td>
			<td>Welgene</td>
			<td>LB001-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Griffonia Simplicifolia Lectin(BSL-1)</td>
			<td>Vector Labs</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG+IgM(H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgM (&#956;-chain)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-binding epidermal growth factor</td>
			<td>Sigma</td>
			<td>E4643</td>
			<td></td>
		</tr>
		<tr>
			<td>Human HepaCAM antibody</td>
			<td>R&#38;D systems</td>
			<td>MAB4108</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin beta 5 monoclonal antibody (KN52)</td>
			<td>eBioscience</td>
			<td>14-0497-82</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetly-L-cyteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 hybridoma supernatant(mouse IgM)</td>
			<td>Bansal et al.23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>Is003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluristrainer 20 &#956;m</td>
			<td>PluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-mouse CD45</td>
			<td>BD Pharmingen</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-rat CD45</td>
			<td>BD Pharmingen</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Worthington</td>
			<td>LS003086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (Tube, Thinwall, Ultra-Clear)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Collect IP-ACM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (10k)</td>
			<td>PALL</td>
			<td>MAP010C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (30k)</td>
			<td>PALL</td>
			<td>MAP030C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis live imaging assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Juli stage</td>
			<td>NanoEntek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Time Series Analyzer V3 plugins</td>
			<td>https://imagej.nih.gov/ij/plugins/time-series.html</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes

Gli astrociti sono del tipo di cellule principali nel cervello e contattare direttamente le sinapsi e vasi sanguigni. Anche se le cellule microglial sono state considerate le principali cellule del sistema immunitario e fagociti solo nel cervello, recenti studi hanno dimostrato che gli astrociti anche partecipano a vari processi fagocitici, come eliminazione sinaptica inerente allo sviluppo e liquidazione dei placche di beta amiloide nel morbo di Alzheimer (annuncio). Nonostante questi risultati, l'efficienza di engulfment Astrocita e degradazione dei loro obiettivi è chiari rispetto a quello di microglia. Questa mancanza di informazioni è per lo più a causa della mancanza di un sistema di test in cui la cinetica della fagocitosi mediata da astrociti e microglia sono facilmente paragonabili. Per raggiungere questo obiettivo, abbiamo sviluppato un test a lungo termine live-imaging in vitro fagocitosi per valutare la capacità fagocitica di purificato astrociti e microglia. In questa analisi, rilevamento in tempo reale di engulfment e degradazione è possibile tramite sinaptosomi coniugato con indicatore di pH, che emettono fluorescenza rosso brillante in organelli acidi, come ad esempio i lisosomi. La nostra analisi novella fornisce rilevamento semplice ed efficace di fagocitosi attraverso live-imaging. Inoltre, questo test di fagocitosi in vitro utilizzabile come una piattaforma di screening per identificare le sostanze chimiche e composti che possono migliorare o inibire la capacità fagocitica dei astrocytes. Come disfunzione sinaptica potatura e accumulo di proteine patogene sono stati indicati per causare disturbi mentali o malattie neurodegenerative, prodotti chimici e composti che modulano la capacità fagocitica delle cellule glial dovrebbero essere utile nel trattamento di varie disturbi neurologici.

In questo in vitro test di fagocitosi con imaging di vivere a lungo termine, abbiamo usato sinaptosomi da omogenati di cervello di topo adulto, che sono stati separati nella soluzione gradiente tra soluzione gradiente 23% e 10% pendenza soluzione per ultracentrifugazione ( Figura 3). Dopo la preparazione, sinaptosomi esposti PS nella loro membrana esterna (Figura 4), suggerendo che hanno perso la loro funzione e potrebbe...

Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Questo protocollo presenta un in vitro live-imaging test di fagocitosi per misurare la capacità fagocitica dei astrocytes. Microglia e astrociti di ratto purificati vengono utilizzati insieme a sinaptosomi coniugato con indicatore di pH. Questo metodo in grado di rilevare in tempo reale engulfment e degradazione cinetica e fornisce una piattaforma di screening adatto per identificare i fattori che influenzano la fagocitosi di astrociti.

Un romanzo In Vitro Live-imaging dosaggio del Astrocyte-mediata fagocitosi utilizzando pH indicatore-coniugato sinaptosomi

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

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Research

JoVE Journal

Neuroscience

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

12.0K Views.  Korea Advanced Institute of Science and Technology. Questo protocollo presenta un in vitro live-imaging test di fagocitosi per misurare la capacità fagocitica dei astrocytes. Microglia e astrociti di ratto purificati vengono utilizzati insieme a sinaptosomi coniugato con indicatore di pH. Questo metodo in grado di rilevare in tempo reale engulfment e degradazione cinetica e fornisce una piattaforma di screening adatto per identificare i fattori che influenzano la fagocitosi di astrociti.

Video: A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

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

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Imaging in diretta degli assoni spinali dopo rizotomia

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo Imaging in diretta di divisioni cellulari singoli in mouse neuroepitelio

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Immagini dal vivo di<em&gt; Drosophila</em&gt; larvali neuroblasti

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Immagini dal vivo del ependimale Cilia nei ventricoli laterali del cervello di topo

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Nuovo metodo di analisi per Cold nocicezione in<em&gt; Drosophila</em&gt; Le larve

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Immagini di corteccia cerebrale primario celle utilizzando un sistema 2D cultura dal vivo

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

In Vitro Fagocitosi di residui del Myelin dai macrofagi derivate da midollo osseo

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sviluppo di analisi per la quantificazione alto contenuto di formazione aggregata di Sod1 mutante della proteina in cellule viventi

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...