Quick and simple mouse forebrain commissure anatomy
(Final)
1. Make solutions ahead of time
2. Euthanasia - use either CO2 or pentobarbital sodium; consult a veterinarian
3. Rapidly remove the brain from the skull.
4. Fix the brain in 10% formalin or 4% paraformaldehyde for at least 24 hours.
5. Trim and weigh the fixed brain to accuracy of 1mg.
6. Cut the brain in half sagittally.
7. Stain in gold chloride for 1 to 2 hours to reveal myelinated axons.
8. View the midsagittal plane and measure maximum lengths of commissures in mm.
9. Adjust for whole brain size using regression equations.
Details
1. Solutions
a. Physiological saline; 0.9% sodium chloride in distilled water; cool to 4C.
b. 10% neutral buffered formalin (easiest) - to make 1L:
900mL distilled water
4g sodium phosphate monobasic + 6.5g sodium phosphate dibasic(anhydrous)
100mL stock 40% formaldehyde solution (contains methanol as stabilizer)
OR 4% paraformaldeyde (more steps but contains no methanol) - make fresh
To make 200mL, dissolve 8g solid paraformaldehyde polymer in 150 mL distilled water heated to 70C in a large beaker with stirring; add 6 drops 1.0 M NaOH to clear the solution.
Add .44g sodium phosphate monobasic + 2.4 g anhydrous sodium phosphate dibasic
Add sufficient distilled water to make 200mL, then filter (Whatman #1 paper)
c. 0.2% buffered gold chloride stain - see Schmued (1990)
1000 mL distilled water
1.8g gold chloride [HAuCl4 . 3H2O]
0.33g NaH2PO4 . H2O + 3.6g Na2HPO4 + 9.0g NaCl
d. 2.5% sodium thiosulfate in distilled water
2. CO2 gas applied at full concentration until all breathing stops for at least 1 minute
OR Pentobarbital sodium (120 mg/Kg) injected intraperitonealy; wait until righting and then foot pinch reflexes are lost. This narcotic drug requires special permission.
OR chloral hydrate OR cervical dislocation (if experimenter is skilled at this)
3. a. Weigh the mouse to an accuracy of 0.1g; triple beam balance works well
b. Using sturdy dissecting scissors, remove the head; then rinse the incision briefly in saline and blot on paper towel. Cut the scalp up the middle from the nape of the neck to the nose. Then remove the muscle and membranous tissue from the posterior part of the skull over the cerebellum, using scissors and scraping with a scalpel blade.
c. Using small but sturdy stitch scissors, cut the skull on both sides from the foramen magnum to the olfactory bulbs and remove the flap of skull. See Figure 1. This is usually done in several steps, removing each piece of skull by lifting it away from the brain. The procedure can also be done with a pair of curved, pointed, toothless Dumont forceps. Be sure to keep the tips of the scissors or forceps away from the midline of the cerebellum and cerebral cortex.
d. Gently pry the brain from the skull, cut the trigeminal and optic nerves, and let it fall into a beaker of cold saline.
4. Remove the brain from the cold saline and immerse it in at least 20ml of fixative at room temperature. Keep this bottle sealed tightly. Agitate the bottle gently once or twice within the first hour so that the brain changes position and the solution mixes well. The bottle should have a brain code that does NOT reveal the animal's identity, so that further measures are done "blind."
5. a. Remove the brain from the fixative with a spatula and trim away parts that are often damaged during extraction, including (i) the paraflocculi of the cerebellum, (ii) the optic nerves up to the optic chiasm, (iii) the olfactory bulbs in front of the cortex. Cut the spinal cord at the base of the medulla oblongata. See Figure 2.
b. Blot the brain gently by rolling it on a non-porous plastic surface or counter top until no further drops of solution remain on the surface.
c. Place the brain into a plastic or metal weighing dish on the balance. Calculate weight to the nearest mg by subtracting the weight of the dish or using a tare function.
6. a. Remove the brain from the fixative and place in on a smooth surface with its dorsal side up. The surface should have a straight line on it for aligning the midline of the brain.
b. Use a fine razor blade or disposable microtome blade with a long, straight edge to cut the brain in half, starting at the longitudinal cerebral fissure and using one continuous motion from top to bottom. A blocking jig with guides perpendicular to the surface may aid in cutting. When the cut is correct, the base of the brain will be separated into two mirror-image parts. Then return the two halves to the fixative.
c. If you do not want to stain the cerebellum, cut it away from the brain stem at the peduncles and save the two halves for Nissl staining.
7. a. Rinse one half of the brain in saline at room temperature for a few seconds.
b. Place the half brain in the gold chloride solution in an incubator at 37C. Monitor the progress of the staining every 15 minutes and stop when the commissures appear clearly and have a brown or brownish purple colour. See Figure 3. The stain will work at room temperature but requires longer time. Note - the stain can be reused until exhausted.
c. Place the brain for about 5 minutes in fresh 2.5% sodium thiosulfate solution to preserve the gold chloride stain.
d. Place the stained half brain back in the fixative solution with the unstained half.
8. a. Remove the stained half and place the flat surface against a microscope slide coverslip so that it adheres firmly by surface tension.
b. Invert the slide and support it with a 3.5 cm disposable petri dish so the brain does not touch the microscope stage. Illuminate its surface with a bright light at an angle that gives a clear image through a compound microscope or stereoscope.
c. Once the best magnification is determined, be sure to record it.
d. By far the easiest method to determine length of commissures is with a microscale or graticule scale in one of the eyepieces of the microscope, which is calibrated with a microscope slide having a finely divided mm scale. For the CC and HC, maximum length is almost as good a measure to determine abnormality as cross-sectional area, because the shapes of the commissures are relatively consistent. Rotate the scale so that it is parallel to the long axis of the commissure. Place the zero mark of the graticule scale on one end of the commissure and then observe the maximum distance to the other end. For the AC, the best measure is maximum width in the horizontal dimension. See Figure 4.
Other methods of measuring commissure areas:
Camera lucida or tracing device - allows the person to see simultaneously an image of the brain and a sheet of drawing paper where the lines will be drawn. Trace the outlines of the commissures in dark ink. If you plan to measure the cross-sectional areas of the commissures, be careful to include only the commissure proper and not nearby structures. For example, the corpus callosum does not include the superior fornix, the longitudinal striae of Lancisius, or the dorsal commissure of the fornix (under the splenium). Later use a graphics tablet and computer to trace each structure and find its cross-sectional area as well as maximum length, or scan it into the computer.
Video image analysis (we use the old JAVA program from Jandel; their newer SigmaScan Pro should do the job, too). Adjust illumination to give a clear picture on the screen and then capture the image. Define an area of interest and use contrast enhancement to improve the image. Draw an outline of each commissure with the computer's mouse, looking though the microscope when in doubt about the edge where the line should be drawn, and find the area. Then draw a straight line between the two furthest points to find maximum length. Save the image as a file. If you have available a video printer, print a snapshot of the commissures and measure lengths from that print.
9. Use X = weight of trimmed and fixed brain from step 6.
Expected commissure sizes for maximum lengths
E(CC length) =
E(HC length) =
E(AC width) =
Index of abnormality = Ratio of actual measure to its value expected from brain size.
The criterion for abnormality is hopefully apparent from a frequency distribution of all animals in the study. In our lab for these measures, any ratio less than 0.6 is abnormal.
References
Conn, P. M. (Ed.) (1990) Quantitative and Qualitative Microscopy. Academic Press.
Cook, M. J. (1965) The Anatomy of the Laboratory Mouse. Academic Press.
Inoué, S. (1986) Video Microscopy. Plenum Press.
Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edition. Academic Press.
Schmued, L. C. (1990) A rapid, sensitive histochemical stain for myelin in frozen brain sections. Journal of Histochemistry and Cytochemistry, 38:717-720.
Sidman, R. L., Angevine, J. B., Jr., and Taber Pierce, E. (1971) Atlas of the Mouse Brain and Spinal Cord. Harvard University Press.
Wahlsten, D. (1983) Maternal effects on mouse brain weight. Developmental Brain Resarch, 9: 215-221.
Wahlsten, D. and Schalomon, P. M. (1994) A new hybrid mouse model for agenesis of the corpus callosum. Behavioural Brain Research, 64: 111-117.
Return to the main page.