培養中枢神経系ニューロンの樹状突起スパイン形態の解析

The overall goal of the following experiment is to assess the effects of various molecular and pharmacological manipulations on dendritic spine morphology and motility. This is achieved by transecting primary cultured cortical neurons with GFP, with or without exogenous expression of DNA plasmids to outline neuronal morphology and to manipulate endogenous protein expression and function. In the second step, cells can be treated pharmacologically and subsequently fixed into immuno stain or prepared for time-lapse imaging.

And then pharmacological treatment, which will allow for the visualization of dendritic spines and the induced changes in dendritic spine morphology and motility. Next Z Series images of fixed cells or images of live cells taken at specific time points are acquired using a confocal microscope. Detailed measurements of dendritic spine morphology can be made from 2D projections of Z series images.

The results obtained show the effect of molecular or pharmacological manipulations on dendritic spine morphology or motility based on quantitative morphological analysis of dendritic spine shape or motility. This method can help answer key questions in neuroscience such as how is synaptic plasticity regulated? How do excitatory synapsis develop?

How are synapses maintained or eliminated? How molecules, such as neurotransmitters or genes regulate synapse structure? Though this method can provide insight into the basic mechanisms that underlie the regulation of synapses, it can also be implied to investigate the contribution of disease associated genes to alter synapse structure, which is a hallmark of a number of psychiatric and neurological disorders such as autism, schizophrenia, or Alzheimer's disease.

First, prepare H-D-M-E-M by balancing the pH of DMEM with 10 millimolar sterile hippies. Warm it to 37 degrees Celsius. Next transfer 18 millimeter cover slips with cells to 600 microliters of 37 degrees Celsius prewarm, antibiotic-free media.

Incubate the cells at 37 degrees Celsius and supplement them with 5%carbon dioxide for 30 minutes. For each cover slip, add a designated amount of DNA to 50 microliters of H-D-M-E-M and allow it to stand for five minutes. Then add four microliters of lipo 2000 to another 50 microliters of H-D-M-E-M and also allow it to stand for five minutes.

Mix the two tubes thoroughly and keep it in a 37 degree Celsius humidified incubator, supplemented with 5%carbon dioxide for at least 20 minutes. Next, add the mixture dropwise to the cells. Continue to incubate the cells in the same conditions for another four hours, mix 300 microliters of feeding media prepared earlier to 400 microliters of 37 degrees Celsius.

Fresh feeding media. Then transfer the cover slips with cells to the medium containing both old and fresh feeding medium. Allow the expression of plasmids to continue for another two to three days.

Prepare a CSF with 200 micromolar D-L-A-P-V. Warm it to 37 degrees Celsius Next pre incubate. The cover slips in 900 microliters of warm A CSF for 30 to 60 minutes.

Prepare the pharmacological agent from the stock solution to a 10 x working concentration by diluting the solution with A CSF. Carefully add the agent to the cells to a final volume of 1000 microliters and a final concentration of one X agent. Fix the neurons in 800 microliters of 4%formaldehyde, 4%sucrose PBS for 10 minutes at room temperature.

Wash them twice in PBS using caution when disposing of formaldehyde waste. Then fix the neurons with 800 microliters of prefilled methanol at four degrees Celsius on an ice block for 10 minutes. Wash the cover slips twice in 800 microliters of PBS each time for 10 minutes.

Next, permeable eyes and block cells simultaneously in PBS containing 2%normal goat serum and 800 microliters of 0.1%tritton X 100 for one hour. At room temperature, add primary antibodies raised against GFP and epitope tag or endogenous proteins to PBS containing 2%normal goat serum. Then take a 15 centimeter dish, divide it into squares.

Number them and cover them with paraform. Add 80 microliters of antibody and block mixture to the paraform at one drop per square. Place the cover slip on the antibody and block mixture with the cells facing down.

Now seal the dish with param and incubate overnight at four degrees Celsius. Wash the cover slips in PB S3 times each time for 15 minutes. Dilute Alexa conjugated secondary antibodies in PBS containing 2%normal goat serum.

Incubate the secondary antibodies at room temperature for one hour and protect them from light after that. Wash the cells again in PB S3 times each time for 15 minutes. Lastly, mount the cover slips onto standard microscope slides using prolonged Antifa reagent A 63 x oil immersion objective with a numerical aperture of 1.4 is used.

Acquire a sequence of images as a Z series at predetermined increments. For example, 0.37 micrometers. Acquire fluorescent images with the appropriate laser line.

For example, using an argon laser line exci at 488 nanometers and a band pass filter at 505 to 530 nanometers. Adjust the detector gain and offset in order to create a sharp contrast between the fluorescent signal in the spine and the background. Keep the acquisition parameters the same for all scans within the same experiment.

Metamorph software from molecular devices is used for image analysis to examine the morphologies of dendritic spines collapse Z series images with maximum projection reconstructions and background subtract images. Next, select the neuron that has at least two dendrites of 100 micrometers in total and calibrate the required distance. Then create a binary image of the neuron by adjusting the image threshold to include all spines in a manner that the threshold corresponds exactly to the outline of the spines.

Measure spines only on the secondary and tertiary dendrites to reduce variability. Make the measurements of segments between branch points. Outline each spine manually to form a closed parameter.

Measure the spine parameters including spine length, spine, breath, cross-sectional area, and dendritic spine linear density with metamorph. Then export the dendritic spine morphometric parameters into excel for quantification and statistical analysis. Pre incubate the neurons grown on 22 by 22 millimeter cover slips in 1.5 milliliters of A CSF for 30 to 60 minutes.

In a 37 degree Celsius humidified incubator, supplemented with 5%carbon dioxide. Then transfer the cells to a 37 degree Celsius enclosed imaging stage chamber to examine spine motility, choose healthy neurons with peral morphologies expressing GFPM cherry or fluorescently tucked protein to minimize photo damage. Reduce laser power to 0.5 to 1%Then acquire images using a 63 x objective and 1.4 numerical aperture with two x averaging every 10 minutes.

Image the neurons for one hour. Then perfuse the drug or vehicle to the imaging chamber using a peristaltic pump image. The neurons for another hour.

Collect Z series images at each time point at the end of each imaging session, obtain a 10 x image of the entire neuron to ascertain the level of photo damage and omit the neurons that show signs of distress from quantification Next collapse, Z Series images of each time point as maximum projection reconstructions in metamorph, depending on the image quality and level of transfection, apply a median pass filter or background subtraction to produce clear images for analysis to evaluate spine morphing and motility color code and overlay images taken at the beginning, middle, and end of the 100 minute image sessions in metamorph, analyze at least 100 micrometers of dendrite per cell. The total spine motility fraction is defined as the total number of motility events. For example, extension retraction, head morphing or protrusive motility normalized to spine number.

This method measures the frequency of events without considering their magnitudes and is a general estimate of overall motility. A protrusive event was defined as the appearance of a new transient protrusion from a spine head or dendritic shaft. A retraction event was defined as the disappearance of an existing or transient protrusion located on a spine head or dendritic shaft.

The sum of protrusion and extension events are divided by the total number of spines in the region of dendrite that is quantified to assess changes in individual spine morphology in response to drug treatment. Measure the cross-sectional area of dendritic spines or dendritic spine linear density at the beginning of the imaging session, and then at each time point immediately before and after the perfusion treatment, normalize each time point to the one hour prior to treatment time point. Then export the dendritic spine morphometric parameters into excel for quantification and statistical analysis.

Here is an example to show that the areas of the fluorescent microspheres of 0.52 and 1.0 micrometers as 0.21 micrometer squared and 0.78 micrometer squared respectively are accurately measured even for the 0.2 micrometer microspheres. The measurements are fairly close to the actual dimensions. Shown here is a representative image of a cortical neuron transfected with GFP imaged using a confocal microscope with a 63 x objective and a 1.4 numerical aperture.

Here are the detailed high resolution images of dendritic spines. A well-characterized effect of activity dependent stimuli is an increase in dendritic spine size. Here is a representative time-lapse image of a dendritic spine of a DIV 24 cortical neuron expressing EGFP imaged for 30 minutes before and after an activity dependent stimulus.

This time-lapse experiment confirms that the increase in dendretic spine size is due to activity dependent stimuli To examine basal spine, motility images are acquired at time 0.0 50 and 100 minutes. The spine extensions, retractions, protrusive, motility, and head morphing are measured separately and combined as total motility. These figures confirm that even under basal conditions, dendritic spines of cortical neurons display some level of motility Following this procedure.

Other methods like colocalization of receptors with other synaptic proteins, localization of proteins to synapses and the electrophysiological investigations can be performed in order to answer additional questions regarding the regulation of dendritic spine morphology and the coordination with synapse function show. After watching this video, you should have a good understanding of how to perform detailed analysis of dendritic spine morphology and motility to assess the effects of various molecular and pharmacological manipulations on synapse structure.