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.
These methods enable quick assessment as to whether an experimental manipulation, disease causative protein, or RNA can impact synaptic processes, and whether therapeutic compounds can restore function. These techniques provide a reliable readout of synaptic function from a group of neurons in a relatively short period of time compared to electrophysiology. This protocol can be applied to any disease of neuronal development or degeneration.
This works well for primary rodent neuronal cultures, and for neurons derived from induced pluripotent stem cells. To begin, optimize the image acquisition settings using confocal imaging acquisition software. Keep excitation power exposure time, detector gain and frame rate constant across all samples.
For time lapse imaging, select an aspect ratio of 512 by 512, and a frame rate of two images per second to minimize dye bleaching. Then select the fluorescence excitation, dichroic and emission filter combinations for GCaMP six M/GCaMP three with Fitzy and stearoyl dye with Trixi. To avoid Z drift during time lapse imaging, use the perfect focus feature of the confocal imaging acquisition software.
Next, select the time tab in the image acquisition panel. For phase one, set interval 500 milliseconds and duration three to five minutes. And for phase two, set interval 500 milliseconds and duration, five minutes.
Then to assemble the gravity perfusion apparatus for artificial cerebrospinal fluid or ACSF, load the high potassium chloride ACSF buffer into a 50 milliliter syringe at the top of the apparatus and set the flow rate to one milliliter per minute. Then, load a 35 milliliter glass dish containing neurons onto the confocal imaging stage with the end of the perfusion tubing placed at the dish edge and choose the field for imaging. 48 hours post transfection, incubate the primary cortical or motor neurons in low potassium chloride ACSF buffer for 10 minutes at 37 degrees Celsius.
Then remove the buffer by aspiration and using a pipette, load the neurons in the dark on a glass bottom Petri dish filled with ACSF containing 50 millimolar potassium chloride and 10 Micro molar styryl dye. After five minutes, remove the loading solution and bath the neurons in low potassium chloride ACSF buffer at 37 degrees Celsius for 10 minutes to eliminate the nonspecific dye loading. Then place the dish onto the imaging stage of the inverted confocal microscope and observe the cells under a 20 times air objective or 40 times oil immersion objective.
Excite the steel dye using a 546 nanometer laser and collect emission using a 570 to 620 nanometer bandpass filter. After selecting the imaging field and engaging perfect focus, take a single still image with bright field, Trixi and fluorescence marker channels to mark neuronal boundaries. Then initiate Run Now in the acquisition software and carry out the basal recording for three to five minutes to exclude variations in dye intensity.
Right at the switch to phase two, trigger the on button for the perfusion system and constantly perfuse 50 millimolar potassium chloride to the neurons to facilitate dye unloading. Carry out the recordings for five minutes then trigger the off switch for the perfusion system. Save the experiment for data analysis later using the confocal software.
48 hours post transfection with GCaMP six M, incubate the primary rodent cortical neurons with low potassium chloride ACSF for 15 minutes. Then mount the dish on the imaging platform and visualized GCaMP six M fluorescence using a Fitzy filter and a 20 times or 40 times objective. After selecting the imaging field and engaging perfect focus, take a single still image with brightfield, Fitzy, and fluorescence marker channels to mark neuronal boundaries.
Next, initiate Run Now in the acquisition software and carry out the basal recording for three to five minutes. Then perfuse ACSF containing 50 millimolar potassium chloride to the neurons as demonstrated previously and record for five minutes. When the image acquisition stops, save the experiment and proceed to data analysis using the confocal software.
For image analysis, open the time lapse images in the confocal software and align the images by clicking on Image, followed by processing, followed by align current document, then select Align to the first frame. Select regions of interest along the neurites using the ROI selection tool and also mark an ROI representing background fluorescence intensity. Next, initiate the measure function from the time measurement panel to measure raw fluorescence over time for the selected ROIs.
After measurement export the raw fluorescence intensities to the spreadsheet software. Cultured rat primary cortical neurons loaded with styryl dye are shown here. The specificity of dye loading was determined by co labeling with synaptic vesicle marker synaptophysin and the majority of sterile dye positive puncta are co positive for this marker.
The analysis of the raw intensity values over the entire imaging period reveals that synaptophysin and intensity remains constant while styryl dye intensity decreases following stimulation. For green fluorescent protein transfected control neurons, successful synaptic vesicle release resulted in the striking loss of dye fluorescence upon high potassium chloride depolarization. This representative video shows a neuron selectively unloading styryl dye in a new right region following stimulation.
In contrast, in neurons transfected with a C nine ORF 72 linked to dye peptide repeat construct to GA 50, impaired synaptic transmission is represented by retained dye fluorescence even after high potassium chloride depolarization. Representative fluorescence images of cortical neurons transfected with GCaMP six M before and after potassium chloride depolarization are shown here. Increased fluorescence values and cortical neurons transfected with GCaMP six M indicate calcium entry into neurites following potassium chloride inducted depolarization.
At the end of the baseline recording period, the neurons expressed GCaMP six M with a low fluorescence. A dramatic fluorescence increase is then observed at the start of stimulation. Make sure fluorescence baseline recordings for styryl dye and GCaMP are stable.
Always use perfect focus to avoid image drift, which would make post image data processing challenging. Further culturing of neurons is not recommended, as dishes are exposed to air during profusion. However, immuno staining or extraction of protein or RNA can assess potential causes of synaptic alterations.
