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Electrode localization in large brains: A rapid and inexpensive technique
Physiology and Behavior, Vol. 9, pp. 859-861. Brain Research Publications Inc., 1972. Printed in U.S.A.
BRIEF COMMUNICATION Electrode Localization in Large Brains: A Rapid and Inexpensive Technique I MARTIN REITE, 2 JOHN E. SWETT s AND DONALD JACKSON
Departments o f Psychiatry and Anatomy, University o f Colorado Medical Center, 4200 East Ninth Avenue Denver, Colorado 80220
(Received 12 June 1972)
REITE, M., J. E. SWETT AND D. JACKSON. Electrode localization in large brains: a rapid and inexpensive technique. PHYSIOL. BEHAV. 9(5) 859-861, 1972.-A rapid and inexpensive technique for identifying macroeleetrode positions in large brains is described. The procedure involves embedding formalin fixed brains in an agar filled lucite tube, and attaching the tube to a commercial rotary blade meat slicing machine. Uniform slices less than 1 mm thick can be obtained, and have proven adequate for gross localization of large electrodes. An optional, relatively easy, staining technique using luxol fast blue is also described. Histology technique
Electrode localization
Staining
EVERY investigator who implants electrodes in animal brain tissue must eventually examine the brain to determine the anatomical locations of the electrodes. This procedure customarily requires frozen or paraffin sections produced by highly trained personnel using elaborate histological equipment (microtomes, etc.). The purpose of this paper is to present a rapid and simple technique that we feel will provide a histological capability f.or investigators who do not have adequate funds for purchase of the large and expensive microtomes and related equipment necessary for handling large brains. We do not claim that the procedure is more reliable than a conventional freezing microtome technique, but it is vastly less expensive, and for those experiments not requiring microscopic detail, it is quite suitable for determining macroelectrode locations in brains of cats and monkeys.
Upon commencement of perfusion with 15% formalin solution, the right auricle is immediately opened to drain the venous return. Initial perfusion pressure is maintained at 150 mm/Hg and the perfusion is started within a minute of the time that the chest wall is opened. Perfusion is continued for 1 0 - 1 5 min, during which time about 1 - 2 1 of fixative is used. The brain can then be removed and stored in 15% formalin solution until it is convenient to section the brain. To insure uniform hardness of the tissue, it is advisable to store the brains for at least one week, with several changes of fresh fixative. In preparation for slicing, it is helpful to cut off the frontal or occipital poles of the brain as close to the coronal plane as possible. This allows the specimen to rest firmly on a flat surface which lies in parallel with the long axis of the electrode tracts. A lucite cylinder, 3 . 5 - 4 in. in dia. and 12 in. long (3/16 in. wall thickness), is placed upright on a 6 in. square piece of Parafilm (or aluminum foil) whose edges are folded up around the outer wall of the cylinder and secured in place with a stout rubber band. The cylinder is then placed upright in a flat bottomed stainless steel pan so that the Parafilm cap of the cylinder lies directly against the pan bottom. About 5 0 - 1 0 0 cc of a 5 . 5 - 6 % hot agar solution is poured into the cylinder and ice water is poured into the
TISSUE PREPARATION At the time of sacrifice the animals are anesthetized with sodium pentobarbital and the chest wall is opened widely to expose the heart and great vessels. While the heart is beating, the descending aorta is clamped and a glass tube 4--6 mm I. D. is inserted through a slit in the apex of the left ventricle into the ascending aorta and ligated in place.
t Financed by USPHS Grant No. MH 17233 and a grant from the Fluid Research Committee, University of Colorado Medical Center. 2Supported by Research Scientist Career Development Award, Type 1, No. K 1-MH-46,335-01-A 1. s Department of Anatomy 859
860
REITE, SWETT AND JACKSON
pan bottom around the outside of the cylinder to accelerate solidification of the agar. While the agar is still liquid, the flat surface of the brain is placed at the bottom of the cylinder and pressed gently against the Parafilm with the aid of tongs. After a few min, the agar will congeal, thereby holding the brain in the desired position. Then another 5 0 0 - 6 0 0 cc of hot agar is poured into the cylinder without disrupting the position or the orientation of the brain. The pan and cylinder surrounded by ice water are then transferred to a refrigerator and left there until the tissue and agar have reached a temperature of about 4 - 6 " C . A sufficient amount of agar should be used so as to immerse the brain beneath 3 - 4 in. of agar gel. When the agar becomes solidified, the lucite cylinder is attached firmly with clamps to the sliding carriage of a renovated electric meat slicing machine with a 12 in. dia. blade (Fig. 1). The cylinder, in this manner, will be converted into a guide which will hold the brain specimen in a fixed position relative to the plane of section. SECTIONING BRAIN TISSUE The edge of the lucite cylinder is positioned so that it will pass about 1 - 2 mm above the cutting edge of the blade. The tissue slice thickness can be regulated by machine adjustment. The sliding carriage is moved back and forth across the rotating blade and at the same time 3 - 4 lb of pressure is applied to the agar through the open end of the lucite cylinder. An old wine bottle with a flat bottom is ideal for this. This pressure allows the operator to obtain rather uniform tissue slices of 0.9 mm thickness. (With a new machine and blade, it is conceivable that the tissue thickness could be as small as 0.5 mm.). The cutting process is rapid and smooth providing that the blade surface and backstop, across which the agar slides, has a light coating of mineral oil or glycerine. A drop or two of mineral oil applied to the backstop after each slice is helpful. The blade must be free of nicks, and extreme caution must be used by the machine operator. We have fashioned a small nylon conveyor belt to catch the tissue slices in order that the machine operator does not have to reach behind the machine to pick up the slices and, therefore, run the risk of being badly cut.
FIG. 1. Brain slicing apparatus. General Slicing Machine Company, Model 112, rotary blade meat slicer with hollow Plexiglas cylinder held in place by two metal straps. This model is no longer available, but any commercial meat slicer with a large diameter, nonserated rotary blade should be adequate.
STAINING Although electrode tract localization can be obtained by inspecting the unstained slices, a simple myelin stain enhances contrast considerably, and is especially useful if the slices are to be photographed for permanent records. We have found the following technique, using lu×ol fast blue, to be easy and effective. The sections selected for staining are stacked in a wide mouth screw cap jar (400 cc capacity), resting on and separated by thick 2 x 2 in. gauze squares. No more than eight sections should be placed in each jar to ensure even staining. The jars are filled with a solution of 0.5 g luxol fast blue and 2.5 cc of 10% acetic acid per liter of 95% ethanol. The tissue sections are incubated in an oven at 57°C. for 2 4 - 4 8 hr. The staining solution should be replaced after 24 hr. The sections will appear as a uniform deep blue. After an initial washing in water, the stain is differentiated by soaking the tissue slices for 5 - 1 0 min in a 0.5% solution of lithium carbonate and then washing them in cold running water for about 15 min. This process is
FIG. 2. Monkey (M. nemestrina] brain section showing electrode tip (circled, at end of arrow) in basolateral amygdala. repeated until the desired staining contrast is obtained. This procedure is time consuming. It may take up to 6 - 8 hr with frequent solution changes. The entire process tends to shrink the tissue by 10-15%. When appropriate contrast is achieved, the sections can be placed in water between two large glass slides and photographed (Fig. 2). It is possible to photograph the unstained sections directly using a modification of Delgado's technique with high contrast film [2]. The staining process should be employed only when necessary. After the electrode location has been verified, the tissue slices are discarded. If stainless steel electrodes are used, it can be helpful to form a Prussian Blue spot at the electrode tip [ 1 ].
ELECTRODE LOCALIZATION
861
REFERENCES
1. Akert, K. and W. I. Welker. Problems and methods of anatomical localizations. In: Electrical Stimulation of the Brain, edited by D. Sheer. Austin, Texas: University of Texas Press, 1961, pp. 2 5 1 - 2 6 0 .
2. Delgado, J. M., R. J. Bradley, V. S. Johnston, G. Weiss, and J. D. Wallace. Implantation of multilead electrode assemblies and radio stimulation of the brain in chimpanzees. 6571st. Aeromed. Res. Lab. Tech. Rep., A R L - T R - 9 6 - 2 , HoUoman AFB, New Mexico, 1969, 19 pp.
BRIEF COMMUNICATION Electrode Localization in Large Brains: A Rapid and Inexpensive Technique I MARTIN REITE, 2 JOHN E. SWETT s AND DONALD JACKSON
Departments o f Psychiatry and Anatomy, University o f Colorado Medical Center, 4200 East Ninth Avenue Denver, Colorado 80220
(Received 12 June 1972)
REITE, M., J. E. SWETT AND D. JACKSON. Electrode localization in large brains: a rapid and inexpensive technique. PHYSIOL. BEHAV. 9(5) 859-861, 1972.-A rapid and inexpensive technique for identifying macroeleetrode positions in large brains is described. The procedure involves embedding formalin fixed brains in an agar filled lucite tube, and attaching the tube to a commercial rotary blade meat slicing machine. Uniform slices less than 1 mm thick can be obtained, and have proven adequate for gross localization of large electrodes. An optional, relatively easy, staining technique using luxol fast blue is also described. Histology technique
Electrode localization
Staining
EVERY investigator who implants electrodes in animal brain tissue must eventually examine the brain to determine the anatomical locations of the electrodes. This procedure customarily requires frozen or paraffin sections produced by highly trained personnel using elaborate histological equipment (microtomes, etc.). The purpose of this paper is to present a rapid and simple technique that we feel will provide a histological capability f.or investigators who do not have adequate funds for purchase of the large and expensive microtomes and related equipment necessary for handling large brains. We do not claim that the procedure is more reliable than a conventional freezing microtome technique, but it is vastly less expensive, and for those experiments not requiring microscopic detail, it is quite suitable for determining macroelectrode locations in brains of cats and monkeys.
Upon commencement of perfusion with 15% formalin solution, the right auricle is immediately opened to drain the venous return. Initial perfusion pressure is maintained at 150 mm/Hg and the perfusion is started within a minute of the time that the chest wall is opened. Perfusion is continued for 1 0 - 1 5 min, during which time about 1 - 2 1 of fixative is used. The brain can then be removed and stored in 15% formalin solution until it is convenient to section the brain. To insure uniform hardness of the tissue, it is advisable to store the brains for at least one week, with several changes of fresh fixative. In preparation for slicing, it is helpful to cut off the frontal or occipital poles of the brain as close to the coronal plane as possible. This allows the specimen to rest firmly on a flat surface which lies in parallel with the long axis of the electrode tracts. A lucite cylinder, 3 . 5 - 4 in. in dia. and 12 in. long (3/16 in. wall thickness), is placed upright on a 6 in. square piece of Parafilm (or aluminum foil) whose edges are folded up around the outer wall of the cylinder and secured in place with a stout rubber band. The cylinder is then placed upright in a flat bottomed stainless steel pan so that the Parafilm cap of the cylinder lies directly against the pan bottom. About 5 0 - 1 0 0 cc of a 5 . 5 - 6 % hot agar solution is poured into the cylinder and ice water is poured into the
TISSUE PREPARATION At the time of sacrifice the animals are anesthetized with sodium pentobarbital and the chest wall is opened widely to expose the heart and great vessels. While the heart is beating, the descending aorta is clamped and a glass tube 4--6 mm I. D. is inserted through a slit in the apex of the left ventricle into the ascending aorta and ligated in place.
t Financed by USPHS Grant No. MH 17233 and a grant from the Fluid Research Committee, University of Colorado Medical Center. 2Supported by Research Scientist Career Development Award, Type 1, No. K 1-MH-46,335-01-A 1. s Department of Anatomy 859
860
REITE, SWETT AND JACKSON
pan bottom around the outside of the cylinder to accelerate solidification of the agar. While the agar is still liquid, the flat surface of the brain is placed at the bottom of the cylinder and pressed gently against the Parafilm with the aid of tongs. After a few min, the agar will congeal, thereby holding the brain in the desired position. Then another 5 0 0 - 6 0 0 cc of hot agar is poured into the cylinder without disrupting the position or the orientation of the brain. The pan and cylinder surrounded by ice water are then transferred to a refrigerator and left there until the tissue and agar have reached a temperature of about 4 - 6 " C . A sufficient amount of agar should be used so as to immerse the brain beneath 3 - 4 in. of agar gel. When the agar becomes solidified, the lucite cylinder is attached firmly with clamps to the sliding carriage of a renovated electric meat slicing machine with a 12 in. dia. blade (Fig. 1). The cylinder, in this manner, will be converted into a guide which will hold the brain specimen in a fixed position relative to the plane of section. SECTIONING BRAIN TISSUE The edge of the lucite cylinder is positioned so that it will pass about 1 - 2 mm above the cutting edge of the blade. The tissue slice thickness can be regulated by machine adjustment. The sliding carriage is moved back and forth across the rotating blade and at the same time 3 - 4 lb of pressure is applied to the agar through the open end of the lucite cylinder. An old wine bottle with a flat bottom is ideal for this. This pressure allows the operator to obtain rather uniform tissue slices of 0.9 mm thickness. (With a new machine and blade, it is conceivable that the tissue thickness could be as small as 0.5 mm.). The cutting process is rapid and smooth providing that the blade surface and backstop, across which the agar slides, has a light coating of mineral oil or glycerine. A drop or two of mineral oil applied to the backstop after each slice is helpful. The blade must be free of nicks, and extreme caution must be used by the machine operator. We have fashioned a small nylon conveyor belt to catch the tissue slices in order that the machine operator does not have to reach behind the machine to pick up the slices and, therefore, run the risk of being badly cut.
FIG. 1. Brain slicing apparatus. General Slicing Machine Company, Model 112, rotary blade meat slicer with hollow Plexiglas cylinder held in place by two metal straps. This model is no longer available, but any commercial meat slicer with a large diameter, nonserated rotary blade should be adequate.
STAINING Although electrode tract localization can be obtained by inspecting the unstained slices, a simple myelin stain enhances contrast considerably, and is especially useful if the slices are to be photographed for permanent records. We have found the following technique, using lu×ol fast blue, to be easy and effective. The sections selected for staining are stacked in a wide mouth screw cap jar (400 cc capacity), resting on and separated by thick 2 x 2 in. gauze squares. No more than eight sections should be placed in each jar to ensure even staining. The jars are filled with a solution of 0.5 g luxol fast blue and 2.5 cc of 10% acetic acid per liter of 95% ethanol. The tissue sections are incubated in an oven at 57°C. for 2 4 - 4 8 hr. The staining solution should be replaced after 24 hr. The sections will appear as a uniform deep blue. After an initial washing in water, the stain is differentiated by soaking the tissue slices for 5 - 1 0 min in a 0.5% solution of lithium carbonate and then washing them in cold running water for about 15 min. This process is
FIG. 2. Monkey (M. nemestrina] brain section showing electrode tip (circled, at end of arrow) in basolateral amygdala. repeated until the desired staining contrast is obtained. This procedure is time consuming. It may take up to 6 - 8 hr with frequent solution changes. The entire process tends to shrink the tissue by 10-15%. When appropriate contrast is achieved, the sections can be placed in water between two large glass slides and photographed (Fig. 2). It is possible to photograph the unstained sections directly using a modification of Delgado's technique with high contrast film [2]. The staining process should be employed only when necessary. After the electrode location has been verified, the tissue slices are discarded. If stainless steel electrodes are used, it can be helpful to form a Prussian Blue spot at the electrode tip [ 1 ].
ELECTRODE LOCALIZATION
861
REFERENCES
1. Akert, K. and W. I. Welker. Problems and methods of anatomical localizations. In: Electrical Stimulation of the Brain, edited by D. Sheer. Austin, Texas: University of Texas Press, 1961, pp. 2 5 1 - 2 6 0 .
2. Delgado, J. M., R. J. Bradley, V. S. Johnston, G. Weiss, and J. D. Wallace. Implantation of multilead electrode assemblies and radio stimulation of the brain in chimpanzees. 6571st. Aeromed. Res. Lab. Tech. Rep., A R L - T R - 9 6 - 2 , HoUoman AFB, New Mexico, 1969, 19 pp.