We acknowledge the following NIH grants: R01AG047101 and R37AG56510.&#160;Members of the Driscoll and Grant labs have contributed extensively to the development and fine tuning of protocols described, with rigorous experiments and strong communication.

<ol>
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The characterization of the in vivo molecular mechanisms of aggregate and organelle elimination in the form of large exophers is in its infancy. Questions as to the designation of cargoes for expulsion, the polarized collection of these cargoes within the cell, the regulation of the decision to generate exophers, the machinery that mediates extrusions, and the interaction of exophers with the degradative machinery in a neighboring cell all remain to be addressed. Furthermore, the in vivo visualization of tubular connections that can pass biological materials that include calcium, aggregates, and mitochondria is interesting and understudied biology in its own right. Questions of why certain cells are more prone to exopher production than others also are unresolved, but can begin to be genetically dissected with the approaches outlined in this protocol.
Described in detail in this protocol are the approaches to achieving reproducible scoring of exopher production, with attention to distinguishing exophers from nearby cell somas, timing of analyses to capture peak of exopher production, and strict control of growth conditions to eliminate unintended stresses that can modulate exopher levels. Both distinction of the large early exopher, or the &#8220;starry night&#8217; dispersion in the surrounding hypodermis can be quantitated as evidence of exopher production. That being said, neurons expressing mCherry under basal conditions are most often associated with 5-25% of neurons of a specific type producing an exopher. Controlled introduction of stress conditions could be applied to increase exopher production to detection as high as 90% of neurons producing extrusions, particularly useful for genetic or pharmacological screens for modifiers.
In human neurodegenerative disease, large aggregates can transfer from diseased neurons into neighboring cells to promote pathology spread. The exopher mechanism might transpire via a conserved mechanism used for aggregate extrusion across phyla. Defining the in vivo molecules that either enhance the efficiency of this process (considered more effective proteostasis control) or block it might be harnessed to influence design of novel strategies for combating multiple neurodegenerative diseases. As such, the protocol described here could be used for classical genetic mutagenesis screens, genome-wide RNAi screens that systematically knock down genes to identify enhancers and suppressors, or for drug intervention studies that identify candidate pharmacological modifiers of this process. The approach is straightforward, although somewhat laborious. Exophers are so large they can be viewed with a high-magnification dissecting microscope. Still, C. elegans neurons are relatively small and looking at their organelles or their membranes require higher power confocal images and is a slow process. Options for higher throughput could involve high content imaging approaches in multi-well plate format.
The application of a standardized approach to exopher scoring should underlie a concerted genetic dissection of the process by which neurons can organize and eliminate cellular debris.

The neurotoxic challenges of aggregates and dysfunctional mitochondria have long been considered to be cell-intrinsic, but more recently it has become clear that misfolded human disease proteins originating in one neuron can also spread to neighboring cells, promoting pathology1. Likewise, mammalian mitochondria can be sent out of the cell of their original production for transcellular degradation2 or for rescue of mitochondrial populations in challenged neighboring cells3. Vesicles of various sizes have generally been observed to transfer cellular materials to neighboring cells or to fluid surroundings4. Some extruded vesicles approach the size of the average neuronal soma (average touch neuron soma ~ 6 &#181;m) and can accommodate large aggregates and organelles.
A striking example of large vesicle extrusion that can carry protein aggregates and organelles occurs in C. elegans touch receptor neurons that express a high copy number reporter construct encoding a noxious aggregation-prone, degradation-resistant mCherry5. Extrusions from the touch neurons, called exophers, are ~4 &#181;m average diameter, selectively include mCherry or other aggregates, and are delivered directly into the neighboring hypodermis, which normally surrounds the touch receptor neurons. The hypodermis attempts lysosome-based degradation, but some non-digestible contents such as mCherry aggregates can be re-extruded by the hypodermis into the fluid-filled pseudocoelom of the animal, from which the mCherry can be taken up by remote scavenger cells called coelomocytes for long term storage (Figure 1, Figure 2)5.
The large extruded exopher vesicles leave the cell surrounded by touch receptor plasma membrane and can contain aggregated human disease proteins, mitochondria, and lysosomes. The process of exopher production appears to involve sorting of potentially toxic species (for example an aggregation-prone expressed mCherry is segregated from soluble, inoffensive proteins like GFP that remains mostly in the neuronal soma). In this way, directed expulsion of the threatening entities is accomplished by the neuron5. A proteostasis challenge, such as stress induced by&#160;autophagy knockdown, MG132-mediated proteasome inhibition, or transgenic expression of human disease proteins such as Huntington&#8217;s disease-associated expanded polyglutamine Q128 or Alzheimer&#8217;s disease-implicated fragment A&#946;1-42, can increase the numbers of neurons that produce exophers5.
As exophers have only recently been documented, what is known of their biology merits description. Exophers were discovered in, and are the most well studied in, C. elegans touch receptor neurons. There are six C. elegans mechanosensory touch neurons that have cell bodies distributed around the body (Figure 3A) and are called microtubule cells because their ultrastructure features distinctive 15 protofilament microtubules. The touch receptor neurons are the anterior AVM (anterior ventral microtubule neuron), ALMR, and ALML (anterior lateral microtubule neurons right and left), the more central PVM (posterior ventral microtubule neuron), and the posterior PLMR and PLML (posterior lateral microtubule neurons right and left) in the tail. Interestingly, the six touch receptor neurons produce exophers at different rates, despite expressing the same offensive transgene (Figure 3C). Of the six mechanosensory touch receptor neurons, the ALMR neuron undergoes exophergenesis more often than the other touch neurons. Quantitation of exopher numbers from touch neurons is thus usually established by focusing upon the ALMR.
Exophergenesis is a dynamic process that typically begins with swelling of the neuronal cytoplasm (Figure 1A-B). Cellular contents, organelles, or protein aggregates are collected to one side of the neuronal soma, most commonly toward the posterior end of the ALMR neuron (away from the projecting neurite), forming a pre-exopher domain (PED) (Figure 1B). The early protrusion is observed as the PED begins to project outwards, forming a recognizable protruded bud. The late bud is defined when the widest diameter of the pre-exopher domain is approximately &#8531; larger than the diameter of the constriction of the soma-exopher neck (Figure 1C).&#160;Exophers can be ejected in nearly any direction from the soma, but most exophers exit posteriorly from the cell body and remain in approximately the same focal plane as the originating soma.
The exopher can move away from the originating soma as the neck of the bud narrows into a thin filament. Exophers can remain attached to the soma via this filament (Figure 1D, arrow) and can later become detached. Cellular contents such as calcium, aggregates, and mitochondria can be transferred via this filament into the attached exopher5, although the bulk of extruded material is put into the exopher compartment by the massive budding event. Exophers are considered mature when there is no visible connecting tube or thin filament and the exopher is fully separated from the sending soma (Figure 1E).
Exophers produced by C. elegans touch neurons immediately encounter the hypodermis, the tissue that surrounds the touch neuron. Most commonly, the exopher vesicle appears to travel within the hypodermis posteriorly towards the tail, and can be fairly distant from the soma before exopher contents appear targeted for degradation (for example, the distance can be ~100 &#181;m away from the soma (Figure 1F)). The fluorescent exopher vesicle breaks up into many smaller vesicles within the hypodermis, taking on an appearance referred to as &#8220;starry night&#8221; (Figure 1G and Figure 2). In the &#8220;starry night&#8221; stage, punctate fluorescent material can be observed scattered across the hypodermal syncytium into many smaller points of fluorescence as compared to the original solitary exopher. Starry night can look punctate under low magnification and with higher magnification, can look punctate and/or networked within the hypodermis. The fluorescent signal of the starry night is typically dimmer than the exopher and the neuronally expressed fluorescence (Figure 2B-C). The dispersal of mCherry into many punctate vesicles is thought to involve phagosome maturation and fusion with the endosomal/lysosomal network of the hypodermal cell. Some exopher materials are likely degraded in the hypodermal lysosomal network, but residual species that are resistant to degradation (such as mCherry aggregates) are thrown out of the hypodermis into the pseudocoelom, a fluid compartment that can contain cellular debris. The fluorescent material is later taken in by remote scavenger cells called coelomocytes (Figure 2C), which can concentrate, store, and again attempt degradation of mCherry.
The phenomenon of aggregate extrusion and transfer appears conserved across phyla, having been reported in genetic models such as C. elegans5,6,7 and D. melanogaster8,9 as well as in multiple mammalian models. Exopher-like extrusions have been reported for mammalian cells10, an observation suggesting that conserved mechanisms might underlie aggregate and organelle expulsion. Exopher production may thus be a conserved mechanism of cellular debris management that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when imbalanced, might actively contribute to neurodegenerative disease. Identification of the molecules involved in debris discrimination and sorting, transport to a distinct subcellular locale, extrusion, formation/scission of the tubular connection linking the soma and late exopher, and recognition of the large extruded vesicle for remote degradation by a neighboring cell remain for future work. Studies in nematode and fly models will be critically important to defining mechanisms of aggregate and organelle collection and transfer, utilizing unbiased genetic approaches and powerful cell biological tools offered by these models to identify participating molecules in physiological context.
Critical first steps in deciphering mechanisms operative in exopher biology involve defining protocols for reproducible in vivo exopher quantification. The C. elegans model offers a particular advantage for such efforts since the body is transparent and exophers can be readily observed when they contain fluorescently tagged proteins or organelles. Exophers have been reported to be generated by C. elegans dopaminergic neurons PDE and CEP, ASE and ASER sensory neurons, and dye-filling amphid neurons5. Because exophers produced by touch receptor neurons are best characterized, the focus here is on the use of touch neurons for exopher analysis. However the basic approach can be applied to measure exopher production from any cell. Protocols to detect and quantitate exophers produced by C. elegans touch receptor neurons that transgenically express mCherry protein are outlined, with an emphasis on cargoes that can be monitored and temporal constraints in scoring. This article defines approaches toward in vivo exopher identification, and the quantitation of environmental and genetic conditions that modulate exopher production. Protocols emphasize critical attention to constant non-stress conditions for the determination of baseline exopher production and for comparisons across genotypes.

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	<tbody>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">95B Scientific CMOS camera</td>
			<td style="width:108px;">Photometrics Prime</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">1,000 &#956;L low retention tips</td>
			<td style="width:108px;">Sarstedt</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">10 mL serological pipette</td>
			<td style="width:108px;">Appleton Woods</td>
			<td style="width:128px;">CC214</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">10 &#956;L low retention tips</td>
			<td style="width:108px;">Sarstedt</td>
			<td style="width:128px;">70.1130.105</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">13% sodium hypochlorite</td>
			<td style="width:108px;">Acros Organics</td>
			<td style="width:128px;">AC219255000</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">15 mL centrifuge tubes</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">05-539-12</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">2 L erlenmeyer flasks</td>
			<td style="width:108px;">Scientific Laboratory Supplies</td>
			<td style="width:128px;">FLA4036</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">25 mL serological pipette</td>
			<td style="width:108px;">Appleton Woods</td>
			<td style="width:128px;">CC216</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">300 &#956;L low retention tips</td>
			<td style="width:108px;">Sarstedt</td>
			<td style="width:128px;">70.765.105</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">50 mL serological pipette</td>
			<td style="width:108px;">Appleton Woods</td>
			<td style="width:128px;">CC117</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">5-Fluoro-2&#39;-deoxyuridine 98%</td>
			<td style="width:108px;">Alfa Aesar</td>
			<td style="width:128px;">L16497.ME</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">9 cm sterile Petri dishes</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">11309283</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">absolute ethanol</td>
			<td style="width:108px;">Vwr</td>
			<td style="width:128px;">20821.33</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Agar</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">A1296</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">C. elegans strain wild type</td>
			<td style="width:108px;">Supplied by CGC</td>
			<td style="width:128px;">N2</td>
			<td style="width:177px;">C. elegans strain</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">calcium chloride dihydrate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">C3881</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">cholesterol</td>
			<td style="width:108px;">Acros</td>
			<td style="width:128px;">110190250</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">dibasic sodium phosphate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">S3264</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">E. coli strain OP50</td>
			<td style="width:108px;">Supplied by CGC</td>
			<td style="width:128px;">Op50</td>
			<td style="width:177px;">E coli strain</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">FBS10 Standard microscope</td>
			<td style="width:108px;">Meyer Instruments</td>
			<td style="width:128px;">KSC 410-1-100-1</td>
			<td style="width:177px;">FBS10 Standard with Plate Base, 100/100 Trinocular Head and Flip zoom</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">glass pipette 270 mm</td>
			<td style="width:108px;">Fisherbrand</td>
			<td style="width:128px;">FB50255</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Heraeus Multifuge X3R</td>
			<td style="width:108px;">Thermofisher scientific</td>
			<td style="width:128px;">75004515</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Inoculating Spreaders</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">11821741</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">LB medium capsules</td>
			<td style="width:108px;">MP biomedicals</td>
			<td style="width:128px;">3002-031</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">LDI &#8211; Laser Diode Illuminator</td>
			<td style="width:108px;">89 North</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">levamisole</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">16595-80-5</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">M4 multipette</td>
			<td style="width:108px;">Eppendorf</td>
			<td style="width:128px;">4982000012</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">magnesium sulphate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">M7506</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">monobasic potassium phosphate</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">P0662</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Multitron Standard shaking incubator</td>
			<td style="width:108px;">Infors HT</td>
			<td style="width:128px;">INFO28573</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Nalgene 1 L Centrifuge pots</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:128px;">3120-1000</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">P10 pipette</td>
			<td style="width:108px;">Eppendorf Research Plus</td>
			<td style="width:128px;">3123000020</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">P1000 pipette</td>
			<td style="width:108px;">Eppendorf Research Plus</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">P200 pipette</td>
			<td style="width:108px;">Eppendorf Research Plus</td>
			<td style="width:128px;">3123000055</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">pipeteboy 2</td>
			<td style="width:108px;">VWR</td>
			<td style="width:128px;">612-0927</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Polystyrene microbeads</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">MFCD00131491</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">RC5C plus floor mounted centrifuge</td>
			<td style="width:108px;">Sorvall</td>
			<td style="width:128px;">9900884</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Reusable ringed cytology slides</td>
			<td style="width:108px;">ThermoFisher Scientific</td>
			<td style="width:128px;">22037242</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">SK4005 zdIs5[Pmec-4GFP]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">GFP expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">sodium chloride</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">13422</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Sodium hydroxide</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:128px;">S/4880/53</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Tactrol 2 Autoclave</td>
			<td style="width:108px;">Priorclave</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Triton-X</td>
			<td style="width:108px;">Thermofisher scientific</td>
			<td style="width:128px;">28313</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">Tween 20</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:128px;">9005-64-5</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">X-Light V2 Spinning Disk Confocal Unit</td>
			<td style="width:108px;">CrestOptics</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4065 bzIs166[Pmec-4mCherry]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">mCherry expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4067 bzIs167[Pmec-4mitogfp Pmec-4mCherry4]; igIs1[Pmec-7YFP Pmec-3htt57Q128::cfp lin-15+]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">Q128 expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4509 bzIs166[Pmec-4mCherry]; bzIs168[Pmec-7LMP-1::GFP]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">mitoROGFP expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZB4528 bzIs166[Pmec-4mCherry]; zhsEx17 [Pmec-4mitoLS::ROGFP]</td>
			<td style="width:108px;">contract Driscoll lab</td>
			<td style="width:128px;"></td>
			<td style="width:177px;">autophagy marker expressed in touch neurons</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:191px;">ZEISS Axio Vert.A1</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:128px;"></td>
			<td style="width:177px;"></td>
		</tr>
	</tbody>
</table>

quantitative approaches for scoring in vivo neuronal aggregate and organelle extrusion in large exopher vesicles in c. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Watch this Scientific Journal Video about Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans at JoVE.com

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

quantitative-approaches-for-scoring-vivo-neuronal-aggregate-organelle

Research

JoVE Journal

Neuroscience

7.4K Views.  Rutgers University. This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 µm) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Video: Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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

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

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

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

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...