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    • CommentAuthorjuruble
    • CommentTimeJul 12th 2007 edited

    This protocol explains how to: measure the complexity of neurons using Sholl's Analysis.

    Protocol revised: 7/21/2006

    Protocol written by: Barbara O'Donnell

    1. Print out your images – make sure to print out the images WITH the cell body included in the picture. Some may be too large to print on one page – if this happens crop them in half and print out both sides. You’ll have to match them up and tape them back together so make sure to copy a little bit of the same middle part on both sides in order to line them up.
    2. On top of each image, center a Sholl’s transparency (transparency with a bullseye on it) around the cell body and tape it down. Make sure that ALL the dendrites are covered by the lines on the transparency. Some will be so big that they will need more than 1 transparency (you may have to cut apart transparencies and piece them together -- see Figure 1). Be careful to match up the lines so that the measurements are correct.
      figure 2
      Figure 2. Manipulating pieces of transparency to cover whole neuron (note that there are about 4-5 separate pieces taped together carefully at the top).

    3. Take a fine line Sharpie marker and make a small mark on each circle whenever a dendrite crosses over the ring. (See the Blue dots on Figure 2.) Some will be very close to each other, so come up with your own system of making multiple marks in the same area. Be careful NOT to mark the cell body when it crosses a line.

      figure 2
      Figure 2. Mark dendrite crossings.

    4. After you have made the marks, slide a piece a plain white paper under the transparency to make counting the tick marks easier (see Figure 3). I found it helpful to mark each ring with its number and use that as the starting point for counting the number of ticks.

      figure 3
      Figure 3. White paper under the transparency facilitates counting tick marks.

    5. Count the number of marks in each ring and record the data.

      There is a template for recording the results saved in
      Public > Biology > biologylom > Scholl’s Analysis Template
      (Add or delete rings as needed)

      The left (purple) side of the template is for RAW data, the right (green) side is for NORMALIZED data. You normalize data by dividing each individual result by the average of the control for that row.
    6. The information below the spreadsheet is for easy set up for graphing your results. Fill in those squares with the information from the spreadsheet. You will want to make 4 different graphs for each run of data (see the spreadsheet for more info).


    Helpful Hint

    I personally found it helpful to divide the Sholl’s Analysis into parts and do ALL of one part before moving on to the next part.

    The order I followed was this:

    1. Printing out and taping together all the images
    2. Taping down Scholl’s transparencies
    3. Making the tick marks
    4. Counting the tick marks and recording the data
    5. Averaging, normalizing, and graphing results