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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Abstract

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.

Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Introduction

Modeling amyotrophic lateral sclerosis (ALS) in the laboratory is made uniquely challenging due to the overwhelmingly sporadic nature of over 80% of cases1, coupled with the vast number of genetic mutations known to be disease-causative2. Despite this, all cases of ALS share the unifying feature that before outright neuronal degeneration, there is dysfunctional communication between presynaptic motor neurons and postsynaptic muscle cells3,4. Clinically, as patients lose connectivity of the remaining upper and lower motor neurons, they present with features of neu....

Protocol

All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of Jefferson University.

1. Primary culture of neurons from embryonic rat cortex

NOTE: Primary cortical neurons are isolated from E17.5 rat embryos as previously described57,58. No strain bias appears to exist with the success of this culturing protocol. This method is described briefly below. The pre.......

Representative Results

Following the successful implementation of the above protocol, representative results are shown for a typical styryl dye synaptic vesicle release experiment. Cultured rat primary cortical neurons were loaded with dye using the method described in section 6. The specificity of dye loading was determined by co-labeling with synaptic vesicle marker synaptophysin. A majority of styryl dye positive puncta are co-positive for this marker (Figure 2A). To determine whether the settings used for styr.......

Discussion

Three steps common to both methods described are of crucial importance for experimental success and quantifiable outcomes. First, preparation of fresh aCSF before each round of experiments is essential, following the attached instructions. Failure to do so may prevent appropriate neuronal depolarization. A sample of untreated control neurons should constantly be tested before stimulation of any experimental groups to ensure proper cellular depolarization and provide a benchmark for positive results obtained in that imagi.......

Acknowledgements

We would like to acknowledge the present and former members of the Jefferson Weinberg ALS Center for critical feedback and suggestions for optimizing these techniques and their analyses. This work was supported by funding from the NIH (RF1-AG057882-01 and R21-NS0103118 to D.T), the NINDS (R56-NS092572 and R01-NS109150 to P.P), the Muscular Dystrophy Association (D.T.), the Robert Packard Center for ALS Research (D.T.), the Family Strong 4 ALS foundation and the Farber Family Foundation (B.K.J., K.K, and P.P).

....

Materials

NameCompanyCatalog NumberComments
20x air objectiveNikonFor imaging
40x oil immersion objectiveNikonFor imaging
B27 supplementThermo Scientific17504044Neuronal growth supplement
BD Syringes without Needle, 50 mLThermo Scientific13-689-8Part of gravity perfusion assembly
Biosafety cell culture hoodBakerSterilGARD III SG403AAsceptic cell culturing, transfection, and dye loading
b-MercaptoethanolMillipore SigmaM3148For culturing and maintenance of neuronal cultures
Bovine Serum AlbuminMillipore SigmaA9418For preparing neuronal cultures
Calcium chloride dihydrateMillipore Sigma223506Component of aCSF solutions
Cell culture CO2 incubatorThermo Scientific13-998-123For culturing and maintenance of neurons
CentrifugeEppendorf5810RFor neuronal culture preparation
Confocal microscopeNikonEclipse Ti +A1R coreFor fluorescence imaging
CoolSNAP ES2  CCD cameraPhotometricsFor image acquisition
D-GlucoseMillipore SigmaG8270Component of aCSF solutions
DNaseMillipore SigmaD5025For neuronal culture preparation
Female, timed-pregnancy Sprague Dawley ratsCharles river400SASSDFor preparing embryonic cortical and spinal motor neuron cultures
FITC Filter cubeNikon77032509For imaging Gcamp calcium transients
FM4-64 styryl dyeInvitrogenT13320For imaging synaptic vesicle release
Glass bottom petri dishes (Thickness #1.5)CellVisD35-10-1.5-NFor growth of neurons on imaging-compatible culture dish
Glass Pasteur pipetteGrainger52NK56For preparing neuronal cultures
Hank's Balanced Salt Solution (HBSS)Millipore SigmaH6648For preparing neuronal cultures
HEPESMillipore SigmaH3375Component of aCSF solutions
High KCl artifical cerebrospinal fluid (aCSF)For imaging. Please see recipes*
horse serumMillipore SigmaH1138For culturing and maintenance of neurons
Laminar flow dissection hoodNUAIRENU-301-630For preparing neuronal cultures
LamininThermo Scientific23017015For preparing neuronal cultures
Leibovitz's L-15 MediumThermo Scientific11415064For preparing neuronal cultures
Leibovitz's L-15 Medium, no phenol redThermo Scientific21083027For preparing neuronal cultures
L-Glutamine (200 mM)Thermo Scientific25030149Neuronal culture supplement
Lipofectamine 2000 Transfection ReagentThermo Scientific11668019For neuronal transfections
Low KCl artifical cerebrospinal fluid (aCSF)For imaging. Please see recipes*
Magnesium chlorideMillipore Sigma208337Component of aCSF solutions
Microsoft ExcelMicrosoftSoftware for data analysis/normalization
Nalgene  Filter Units, 0.2 µm PESThermo Scientific565-0020Filter unit for aCSF solution
Neurobasal mediumThermo Scientific21103049For culturing and maintenance of neuronal cultures
NIS-Elements Advanced ResearchNikonSoftware for image capture and analysis
Nunc 15 mL Conical tubesThermo Scientific339650For preparing neuronal culture and buffer solutions
Nunc 50 mL conical tubesThermo Scientific339652For preparing neuronal culture and buffer solutions
OptiprepMillipore SigmaD1556For preparing neuronal cultures
PapainMillipore SigmaP4762For preparing neuronal cultures
Penicillin-Streptomycin (10,000 U/mL)Thermo Scientific15140122To prevent bacterial contamination of neuronal cultures
Perfusion systemWarner InstrumentsSF-77BFor exchange of aCSF
Perfusion tubingCole-ParmerUX-30526-14Part of gravity perfusion assembly
pGP-CMV-Gcamp6m plasmidAddgene40754For imaging calcium transients
Poly-D-lysine hydrobromideMillipore SigmaP7886Coating agent for glass bottom petri dishes
Potassium chlorideMillipore SigmaP3911Component of aCSF solutions
Sodium bicarbonateMillipore SigmaS5761Component of aCSF solutions
Sodium ChlorideMillipore SigmaS9888Component of aCSF solutions
Stage Top IncubatorTokai HitFor incubation of live neurons during imaging period
TRITC Filter cubeNikon77032809For imaging FM4-64
Trypsin InhibitorMillipore SigmaT6414For preparing neuronal cultures
Trypsin-EDTA (0.25%), phenol redThermo Scientific25200056For preparing neuronal cultures
Vibration Isolation tableNew PortVIP320X2430-135520Table/stand for microscope

References

  1. Gibson, S. B., et al. The evolving genetic risk for sporadic ALS. Neurology. 89 (3), 226-233 (2017).
  2. Kim, G., Gautier, O., Tassoni-Tsuchida, E., Ma, X. R., Gitler, A. D. ALS genetics: Gains, losses, and implications for future therapies. <....

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