Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

The overall goal of the following experiment is to study the extent of dendritic arborization in the hippocampus of a mouse model. This is achieved through two different methods. In the first method, dendrites are traced manually in real time throughout the entire thickness of each section.

Following tracing, the entire dendritic tree can be reconstructed and analyzed. Using the fan diagram analysis of various fan diagrams derived from different mice can be used as an objective means of comparison. The second method is to visualize dendritic arborization with extended depth of field imaging.

Finally, a panoramic method is used to stitch multiple high magnification images together to yield one high resolution composite image for qualitative and quantitative assessment of dendrites in the entire region of interest And demonstrating this procedure will be GI mobi. AMI research flows from my laboratory. The main advantage of these techniques over existing metals is the ease of use and speed of acquisition and data collection.

This method can help answer key questions in the field of neurobiology, such as assessment of dendritic ization in affected brain regions, and evaluation of the effects of different therapies on dendrites. In this procedure, one day after the mouse brain has been fixed in 4%Paraform aldehyde, place it in sucrose solution for dehydration for 48 hours at four degrees Celsius. Remove the brains from sucrose and place them directly on copper blocks placed on dry ice.

Fill the block with OCT and mark the orientation of the brain using the olfactory bulb as a landmark. Cut 70 micron thick sections at negative 20 degrees Celsius using a cryostat and place them in Cryoprotectant solution and keep at negative 20 degrees Celsius until use. Incubate the sections in hydrogen peroxide in methanol for 30 minutes at room temperature, followed by warming them up in 37 degrees Celsius in TBS for half an hour.

Incubate the sections with 0.3%Triton and normal horse serum for 45 minutes at room temperature prior to staining with the DCX antibody the following day. Wash the sections three consecutive times in TBS and incubate the sections with a biotinylated horse antigo for two hours at room temperature. Wash the sections three consecutive times in TBS for 10 minutes each and incubate them with a B, C light for 1.5 hours.

At room temperature, add hydrogen peroxide to the DAB solution and incubate the sections immediately in this solution for five minutes and terminate the reaction by washing them three times with ice cold TBS followed by one. Wash in TT BS at room temperature. Dehydrate the sections using a series of ethanol solutions.

Five minutes each, clear and xylene, and then cover slip using DPX. After the sections have been completely dried, clean excess DPX on the slide surfaces with a sharp blade and place them on the scanning stage of the microscope one at a time. The visualization system is composed of a microscope equipped with a scanning stage joystick and a 12 bit color camera.

Next, start the neuro lucita program and open a new data file. Place a reference point on the screen by clicking at any place with a mouse pointer to activate all of the icons from the tool panel of the program window. Then click on the joystick free icon on the toolbar and use the joystick to locate the dentate gyrus of the hippocampus.

In the first section in the tools tab of the program window, select serial section manager. Click on the new section icon on the lower left corner of the window to open the serial section setup window. Afterward, select the serial section setup window and enter the total number of sections that contain hippocampal regions.

Then select the evaluation interval and enter the thickness of the section. Start tracing the dentate gyrus region by clicking on the free hand contour drawing icon in the toolbar. Subsequently outline the dentate granular cell layer.

Select 100 x from the magnification menu. Then add a drop of immersion oil on the section and switch the objective to 100 x. Locate the dentate granule cell bodies and dendrites under this objective.

Next, focus on a selected neuron, and click on the neuron tracing icon. On the toolbar trace the circumference of the dentate granule cell body. When finished tracing right click to select finish cell body.

Now start tracing the dendrites manually and X, Y, and Z directions, and follow each branch using the joystick and Z motor knob At a bifurcation or trifurcation node, select the respective option. From the dropdown menu, trace each of the branches that arise from these nodes at the end of each branch. Right click and select ending from the dropdown menu.

Using the arrow key icons in the software window. Randomly select another dentate granule cell and repeat the tracing procedure as just demonstrated. Save the entire tracing.

Now start neuro lucita explorer for neuron analysis. Open the first NRX data file from the first mouse of the experimental group. Append the NRX file of the second mouse of the experimental group and continue until all the NRX files from this group are appended.

Under the analysis tab, select fan in diagram to open the fan in analysis window, check mark dendrites and click display for the links and branching patterns of dendrites for this group of mice. In this procedure, connect the microscope to a digital camera. Place a section on the scanning stage connected to the microscope and switch to a 10 x objective.

Next, move the stage to the area of interest. Start a video capturing program and press the record button. As soon as the record button is pressed, use the macro focus knob to move from the top to the bottom of the section for a total of four seconds Before saving the resulting video file, now use Image J freeware to convert the a VI video files into an uncompressed format.

Start an image analysis program and open the a VI file. Go to process menu and click on extended depth of field. Select the corresponding video file in the output options.

Select generate composite best focus image in the focus analysis options. Select normalized illumination and max local contrast options. Then click create to generate a resulting image, which represents all the focused pixels throughout the Z axis.

Afterward, save the resulting image. In this step, place a section on the scanning stage connected to the microscope. Move the stage to the area of interest.

Then start the image acquisition program using a 10 x objective, acquire and save high resolution images from the region of interest, making sure to have at least 10%overlap between images. Then run image composite editor to stitch the images afterward. Go to file menu and click on new panorama.

Select the folder in which the images are stored. Next, select the images that belong to the same region and press Okay. Press the export to disc button and save the image.

This image represents an extremely high resolution picture of the area of interest that could be used for analysis. This figure shows the visualization of dendritic arborization with and without EDFI methods. The traditional method of finding the best focus plane was compared with EDFI and significantly higher values of dendritic area.

Using the EDFI method were found. The quantification of length and volume occupied by dendrites in different orders of branching is shown here. The maximum length and volume were achieved in the order one.

Here is a histogram of the dendritic length of dentate granule cells. The majority of dendritic segments of DCX stain DDRs were 13 to 26 in length. This red dotted line depicts normal distribution of the data presented Following this procedure.

Other classical methods like neuro Lucid could be used to answer additional questions regarding the volume and area occupied by dendrites. This technique that we showed you here will tremendously help researchers in the field of neurobiology to not only assess the ation of neuronal structures, but also to assess non neuronal small structures like microglia in thick brain sections. After watching this video, you should have a good understanding of how to assess the extent of neuronal projections in a relatively short period of time.