The authors thank Yeon-Joo Jung for her experimental support during synaptosome purification and Jungjoo Park for images of synaptosomes with PS exposure. In addition, we thank all members in Chung&#39;s laboratory for helpful discussion. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2016M3C7A1905391 and NRF-2016R1C1B3006969) (W.-S. C).

<ol>
	<li>Singh, S. K., 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=Astrocytes+Assemble+Thalamocortical+Synapses+by+Bridging+NRX1alpha+and+NL1+via+Hevin.">Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1alpha and NL1 via Hevin.</a> Cell. 164 (1-2), 183-196 (2016).</li><li>Allen, N. 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. 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=Astrocytes+mediate+synapse+elimination+through+MEGF10+and+MERTK+pathways.">Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways.</a> Nature. 504 (7480), 394-400 (2013).</li><li>Schafer, D. P., 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+sculpt+postnatal+neural+circuits+in+an+activity+and+complement-dependent+manner.">Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.</a> Neuron. 74 (4), 691-705 (2012).</li><li>Ma, Z., Stork, T., Bergles, D. E., Freeman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromodulators+signal+through+astrocytes+to+alter+neural+circuit+activity+and+behaviour.">Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour.</a> Nature. 539 (7629), 428-432 (2016).</li><li>Parkhurst, C. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+engage+unique+molecular+programs+to+engulf+pruned+neuronal+debris+from+distinct+subsets+of+neurons.">Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons.</a> Genes Dev. 28 (1), 20-33 (2014).</li><li>Jones, R. S., Minogue, A. M., Connor, T. J., Lynch, 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=Amyloid-beta-induced+astrocytic+phagocytosis+is+mediated+by+CD36,+CD47+and+RAGE.">Amyloid-beta-induced astrocytic phagocytosis is mediated by CD36, CD47 and RAGE.</a> J Neuroimmune Pharmacol. 8 (1), 301-311 (2013).</li><li>Wang, Y., 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=TREM2+lipid+sensing+sustains+the+microglial+response+in+an+Alzheimer's+disease+model.">TREM2 lipid sensing sustains the microglial response in an Alzheimer's disease model.</a> Cell. 160 (6), 1061-1071 (2015).</li><li>Sekar, A., 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=Schizophrenia+risk+from+complex+variation+of+complement+component+4.">Schizophrenia risk from complex variation of complement component 4.</a> Nature. 530 (7589), 177-183 (2016).</li><li>Hong, 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=Complement+and+microglia+mediate+early+synapse+loss+in+Alzheimer+mouse+models.">Complement and microglia mediate early synapse loss in Alzheimer mouse models.</a> Science. 352 (6286), 712-716 (2016).</li><li>Chung, W. 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. H., Simonsson, M., Erlandsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+degradation+in+phagocytic+astrocytes+can+be+enhanced+by+lysosomal+acidification.">Slow degradation in phagocytic astrocytes can be enhanced by lysosomal acidification.</a> Glia. , (2015).</li><li>Xiao, Q., 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=Enhancing+astrocytic+lysosome+biogenesis+facilitates+Abeta+clearance+and+attenuates+amyloid+plaque+pathogenesis.">Enhancing astrocytic lysosome biogenesis facilitates Abeta clearance and attenuates amyloid plaque pathogenesis.</a> J Neurosci. 34 (29), 9607-9620 (2014).</li><li>Lu, T. Y., 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=Axon+degeneration+induces+glial+responses+through+Draper-TRAF4-JNK+signalling.">Axon degeneration induces glial responses through Draper-TRAF4-JNK signalling.</a> Nat Commun. 8, 14355 (2017).</li><li>Dunkley, P. R., Jarvie, P. E., Robinson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+Percoll+gradient+procedure+for+preparation+of+synaptosomes.">A rapid Percoll gradient procedure for preparation of synaptosomes.</a> Nat Protoc. 3 (11), 1718-1728 (2008).</li><li>Stigliani, 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=Glia+re-sealed+particles+freshly+prepared+from+adult+rat+brain+are+competent+for+exocytotic+release+of+glutamate.">Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate.</a> J Neurochem. 96 (3), 656-668 (2006).</li><li>Foo, L. C., 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=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71 (5), 799-811 (2011).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>Cahoy, J. D., 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=A+transcriptome+database+for+astrocytes,+neurons,+and+oligodendrocytes:+a+new+resource+for+understanding+brain+development+and+function.">A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.</a> J Neurosci. 28 (1), 264-278 (2008).</li></ol>

The authors have nothing to disclose.

In this article, we present methods for a long-term live-imaging in vitro phagocytosis assay using purified glial cells and pH indicator-conjugated synaptosomes. We show that compared with microglia, astrocytes possess different engulfment and degradation capacity during the phagocytosis of synaptosomes. In addition, our data suggest that astrocyte-secreted factors, which contain bridging molecules such as GAS6, ProteinS, and MEGE8, are essential for efficient PS-dependent phagocytosis of glial cells in the brai...

Glial cells, which refer to non-excitable cells in the brain, are the major cell type in the central nervous system (CNS). Previously, glial cells were regarded as mere supporting cells that mainly play passive roles in maintaining neuronal survival and basal synaptic properties. However, emerging evidence has revealed that glial cells play more active roles in various aspects of neurobiology, such as maintaining brain homeostasis, mediating synapse formation1,2,3 and synapse elimination4,5, and modulating synaptic plasticity6,7. Glial cells in the CNS include astrocytes, microglia, and oligodendrocytes. Among these cells, astrocytes and microglia have been shown to play phagocytic roles by engulfing synapses4,5, apoptotic cells8, neural debris9, and pathogenic proteins, such as amyloid beta plaques10,11. In the developing brain, astrocytes eliminate synapses in the dorsal lateral geniculate nucleus (dLGN) through MERTK- and MEGF10-dependent phagocytosis4. Similarly, microglia also eliminate C1q-coated synapses during developmental stages through the classical complement cascade5. Interestingly, it has been suggested that defects in synapse pruning can be one of the initiators of several neurological disorders. For example, it has been shown that mutations in complement component 4 (C4), which increases complement-mediated synapse pruning by microglia, are strongly associated with the prevalence of schizophrenia in humans12. A recent paper has also shown that the classical complement pathway is hyperactivated in the initiation stages of AD and induces early synapse loss in this disease13.
Compared with microglia-mediated phagocytosis, whether astrocyte-mediated phagocytosis contributes to the initiation and progression of various neurological disorders is less clear. However, a recent paper suggests that factors that alter the rate of normal synapse pruning by astrocytes may disrupt brain homeostasis and contribute to AD susceptibility and pathology14. The rate of synapse pruning by astrocytes is powerfully controlled by ApoE isomers, with a protective allele for AD (ApoE2) strongly enhancing the rate and a risk allele for AD (ApoE4) significantly lowering the rate. Moreover, transgenic mice expressing ApoE4 accumulated much more synaptic C1q than control or ApoE2 mice14. These data suggest that impaired astrocyte-mediated phagocytosis in the early AD brain may induce the accumulation of senescent C1q-coated synapses/synaptic debris that activates complement-mediated microglial phagocytosis, driving synaptic degeneration. The impaired phagocytic capacity of astrocytes in ApoE4 carriers may also contribute to the uncontrolled accumulation of amyloid beta plaques in AD-affected brains.
In addition, it has been shown that glial cells in the aged Drosophila brain lose their phagocytic capacity due to decreased translation of Draper, a homolog of Megf10 that astrocytes use for phagocytosing synapses. Restoring Draper levels rescued the phagocytic capacity of glial cells, which efficiently cleared damaged axonal debris in the aged brain to a similar extent as that in the young brain, indicating that aging-induced alterations in the phagocytic capacity of astrocytes may contribute to disruption of brain homeostasis15.
Based on these new findings, modulating the phagocytic capacity of astrocytes may be an attractive therapeutic strategy to prevent and treat various neurological disorders. In this regard, there have been several attempts to enhance the phagocytic capacity of astrocytes, for example, by inducing acidification of lysosomes with acidic nanoparticles16 and overexpressing transcription factor EB (TFEB), which can enhance lysosome biogenesis17. Despite these attempts, it is still unclear how astrocytes and microglial cells differ in their phagocytic kinetics and whether we should increase or decrease their phagocytic capacities in various diseases.
In this paper, we present a novel in vitro assay for detecting the phagocytic capacity of astrocytes in real-time. The data show different kinetics of engulfment and degradation in astrocytes and microglia. Astrocyte-conditioned medium (ACM), which contains secreted factors from astrocytes, is essential for effective phagocytosis of both astrocytes and microglia. Furthermore, Megf10, a phagocytic receptor in astrocytes and a homolog of Ced-1 and Draper, plays critical roles in astrocyte-mediated phagocytosis8,18.

<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

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

In this in vitro phagocytosis assay with long-term live-imaging, we used synaptosomes from adult mouse brain homogenates, which were separated in the gradient solution between 23% gradient solution and 10% gradient solution by ultracentrifugation (Figure 3). After the preparation, synaptosomes exposed PS in their outer membrane (Figure 4), suggesting that they lost their function and could be recognized by PS receptors i...

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

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

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

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

a-novel-vitro-live-imaging-assay-astrocyte-mediated-phagocytosis

Research

JoVE Journal

Neuroscience

12.0K Views.  Korea Advanced Institute of Science and Technology. This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

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.

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 ...

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.

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.

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

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.

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.

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.

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.

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.

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.

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

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

Summary

Explore More Videos

Chapters in this video

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

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

Live Imaging of Dorsal Root Axons after Rhizotomy

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Live Imaging of Drosophila Larval Neuroblasts

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

Novel Assay for Cold Nociception in Drosophila Larvae

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

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

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