This protocol describes methods for visualizing dendritic spine morphologies and calcium transients in C.elegans neurons. Our approach should facilitate genetic approaches to discover determinants of a spine morphogenesis and function. Our protocol features dendritic spines in GABAergic neurons.
Spines in other classes of C.elegans neurons can also be investigated with these methods. Our protocol describes methods for immobilizing living C.elegans. It is particularly important to prevent animal movement during image acquisition, and to pick the right laser configurations to excite and record neuronal activity.
To acquire high resolution images, add three microliters of anesthetic solution, and amount 15 to 20 young adult worms on 10%agarose pads. Then apply the coverslip to immobilize the worms, and seal the coverslip edges with a melted adhesive sealant mixture. For a super-resolution acquisition, use a laser scanning confocal microscope equipped with super-resolution microscopy.
Acquire Z-stacks using the step size recommended by the manufacturer's software. Collect a series of optical sections that span the total volume of the Dorsal D or DD ventral process. Submit the Z-stacks for image processing using the manufacturer's software, and analyze images with a score higher than seven.
For Nyquist acquisition, use a laser scanning confocal microscope, and select the optimal pixel size for the wavelength of light and numerical aperture of the objective lens. Then submit the stack for 3D-deconvolution using an automatic algorithm. For image analysis, use an appropriate image processing software to create maximum intensity projections of the Z stacks.
And manually count the protrusions on the DD dendrite. Then determine the length of the scored DD dendrite to calculate the density of spines per 10 micrometers of DD dendrite. Then classify the spines as thin or mushroom, filopodial, stubby, or branched.
Using conventional techniques such as microinjection, create transgenic worms expressing the calcium sensor GCaMP in DD neurons, and Chrimson, a red-shifted channel rhodopsin in presynaptic VA neurons. Next, under a laminar hood, prepare all trans-retinal or ATR plates by adding 300 microliters of overnight-grown OP50 bacterial culture, and 0.25 microliters of ATR to each 60 millimeter nematode growth medium nutrient agar plate. Then spread the culture with a sterile glass rod.
For control plates, add 300 microliters of OP50 bacteria and 0.25 microliters of ethanol. To allow bacterial growth, incubate the plates at room temperature for 24 hours, protected from ambient light. To set up the experiment, place five L4 stage larvae on OP50-seeded ATR or control plates, and incubate the plates in the dark at 23 degrees Celsius.
After three days, use a stereo dissecting microscope to confirm the vulva development, and pick L4 stage progeny from the ATR and control plates for imaging. Next, place two microliters of 0.05 micrometer polybeads on a microscope slide. And place approximately 10 L4 larvae in the solution.
Using a platinum wire, add a small globule of super glue to the solution. Swirl the solution gently to generate filamentous strands of glue. Then add three microliters of M9 buffer.
Then apply a cover slip and seal its edges as demonstrated previously. To record evoked calcium transients in the dendritic spines, use a spinning disk confocal microscope, and adjust the microscope stage to position the DD spines in the focal plane. Then set up time-lapse acquisition to illuminate the sample with a 488 nanometer laser line in every frame for detecting GCaMP fluorescence, and a 561 nanometer laser line at periodic intervals for Chrimson excitation.
For in vivo calcium imaging, use 2D-deconvolution and image alignment to correct minor deviations from the worm movement during acquisition. Then define the DD dendritic spine as the region of interest or ROI. Duplicate the ROI and relocate to a neighboring region inside the worm to collect the background signal.
Then using appropriate software, export the GFP intensities to Excel for each time point, and subtract background fluorescence from spine ROI fluorescence. Determine the change in fluorescence by subtracting the GFP fluorescence in the frame immediately before the 561 nanometer excitation or at zero from each time point after excitation or Delta F.Then divide by F zero to determine Delta F over F zero. And graph the normalized traces.
First, determine if the data is normally distributed using a Shapiro-Wilk test. For data that are not normally distributed, use a non-parametric ANOVA with post-hoc correction for multiple testing. Alternatively, for measurements showing normal or Gaussian distribution, perform a paired parametric ANOVA test for each measurement of GCaMP fluorescence before and after each 561 nanometer pulse.
And correct for multiple comparisons in each of the two groups. Labeling of DD dendritic spines with three independent markers, cytosolic mCherry, MYR:mRuby, and LifeAct:GFP yielded an average density of 3.4 DD dendritic spines per 10 microns of DD dendrite in wild-type young adults. The GFP:Utrophin marker was excluded from this analysis because it yielded a significantly lower spine density, potentially due to interactions of utrophin with the actin cytoskeleton that drives spine morphogenesis.
The live cell imaging approach confirmed that the thin mushroom shaped morphology of the DD spines predominates in the adult compared to alternative spine shapes like filopodial, stubby, and branched. Activation of DD dendritic spines was assessed by presynaptic cholinergic signaling in transgenic worms expressing GCaMP in DD neurons, and Chrimson in presynaptic VA neurons. Transient bursts of GCaMP signal were detected in DD spines immediately after optogenetic activation of Chrimson in presynaptic VA neurons.
A control experiment in the absence of ATR confirmed that the measure to GCaMP signal depends on the optogenetic activation of Chrimson which is strictly ATR-dependent. To visualize DD spines, it is best to image animals laying on their side such as the spines that protrude are at right angles to the light path. With this method, scientists can also use pharmacological manipulations to understand the mechanisms that drive calcium transients in DD spines.
