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* These authors contributed equally
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.
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.
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....
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.......
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.......
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.......
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).
....Name | Company | Catalog Number | Comments |
20x air objective | Nikon | For imaging | |
40x oil immersion objective | Nikon | For imaging | |
B27 supplement | Thermo Scientific | 17504044 | Neuronal growth supplement |
BD Syringes without Needle, 50 mL | Thermo Scientific | 13-689-8 | Part of gravity perfusion assembly |
Biosafety cell culture hood | Baker | SterilGARD III SG403A | Asceptic cell culturing, transfection, and dye loading |
b-Mercaptoethanol | Millipore Sigma | M3148 | For culturing and maintenance of neuronal cultures |
Bovine Serum Albumin | Millipore Sigma | A9418 | For preparing neuronal cultures |
Calcium chloride dihydrate | Millipore Sigma | 223506 | Component of aCSF solutions |
Cell culture CO2 incubator | Thermo Scientific | 13-998-123 | For culturing and maintenance of neurons |
Centrifuge | Eppendorf | 5810R | For neuronal culture preparation |
Confocal microscope | Nikon | Eclipse Ti +A1R core | For fluorescence imaging |
CoolSNAP ES2Â CCD camera | Photometrics | For image acquisition | |
D-Glucose | Millipore Sigma | G8270 | Component of aCSF solutions |
DNase | Millipore Sigma | D5025 | For neuronal culture preparation |
Female, timed-pregnancy Sprague Dawley rats | Charles river | 400SASSD | For preparing embryonic cortical and spinal motor neuron cultures |
FITC Filter cube | Nikon | 77032509 | For imaging Gcamp calcium transients |
FM4-64 styryl dye | Invitrogen | T13320 | For imaging synaptic vesicle release |
Glass bottom petri dishes (Thickness #1.5) | CellVis | D35-10-1.5-N | For growth of neurons on imaging-compatible culture dish |
Glass Pasteur pipette | Grainger | 52NK56 | For preparing neuronal cultures |
Hank's Balanced Salt Solution (HBSS) | Millipore Sigma | H6648 | For preparing neuronal cultures |
HEPES | Millipore Sigma | H3375 | Component of aCSF solutions |
High KCl artifical cerebrospinal fluid (aCSF) | For imaging. Please see recipes* | ||
horse serum | Millipore Sigma | H1138 | For culturing and maintenance of neurons |
Laminar flow dissection hood | NUAIRE | NU-301-630 | For preparing neuronal cultures |
Laminin | Thermo Scientific | 23017015 | For preparing neuronal cultures |
Leibovitz's L-15 Medium | Thermo Scientific | 11415064 | For preparing neuronal cultures |
Leibovitz's L-15 Medium, no phenol red | Thermo Scientific | 21083027 | For preparing neuronal cultures |
L-Glutamine (200 mM) | Thermo Scientific | 25030149 | Neuronal culture supplement |
Lipofectamine 2000 Transfection Reagent | Thermo Scientific | 11668019 | For neuronal transfections |
Low KCl artifical cerebrospinal fluid (aCSF) | For imaging. Please see recipes* | ||
Magnesium chloride | Millipore Sigma | 208337 | Component of aCSF solutions |
Microsoft Excel | Microsoft | Software for data analysis/normalization | |
Nalgene Filter Units, 0.2 µm PES | Thermo Scientific | 565-0020 | Filter unit for aCSF solution |
Neurobasal medium | Thermo Scientific | 21103049 | For culturing and maintenance of neuronal cultures |
NIS-Elements Advanced Research | Nikon | Software for image capture and analysis | |
Nunc 15 mL Conical tubes | Thermo Scientific | 339650 | For preparing neuronal culture and buffer solutions |
Nunc 50 mL conical tubes | Thermo Scientific | 339652 | For preparing neuronal culture and buffer solutions |
Optiprep | Millipore Sigma | D1556 | For preparing neuronal cultures |
Papain | Millipore Sigma | P4762 | For preparing neuronal cultures |
Penicillin-Streptomycin (10,000 U/mL) | Thermo Scientific | 15140122 | To prevent bacterial contamination of neuronal cultures |
Perfusion system | Warner Instruments | SF-77B | For exchange of aCSF |
Perfusion tubing | Cole-Parmer | UX-30526-14 | Part of gravity perfusion assembly |
pGP-CMV-Gcamp6m plasmid | Addgene | 40754 | For imaging calcium transients |
Poly-D-lysine hydrobromide | Millipore Sigma | P7886 | Coating agent for glass bottom petri dishes |
Potassium chloride | Millipore Sigma | P3911 | Component of aCSF solutions |
Sodium bicarbonate | Millipore Sigma | S5761 | Component of aCSF solutions |
Sodium Chloride | Millipore Sigma | S9888 | Component of aCSF solutions |
Stage Top Incubator | Tokai Hit | For incubation of live neurons during imaging period | |
TRITC Filter cube | Nikon | 77032809 | For imaging FM4-64 |
Trypsin Inhibitor | Millipore Sigma | T6414 | For preparing neuronal cultures |
Trypsin-EDTA (0.25%), phenol red | Thermo Scientific | 25200056 | For preparing neuronal cultures |
Vibration Isolation table | New Port | VIP320X2430-135520 | Table/stand for microscope |
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