Reprinted from THE JOURNAL OF COMPARATIVE NEUROLOGY

Reprinted from THE JOURNAL OF COMPARATIVE NEUROLOGY Vol. 145, No. 1, May 1972

A Stereotaxic Atlas of the Brainstem for Macaca mulatta in the Sitting Position

ORVILLE A. SMITH, KENNETH G. KASTELLA AND DAVID C. RANDALL

Regional Primate Research Center and Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195

ABSTRACT Attempts to use existing stereotaxic atlases of the lower brainstem of: monkeys for the placement of microelectrodes in the cranial nerve nuclei of awake animals have met with little success. The relationship of these nuclei to the bony structures used for orienting the head are probably altered when the position of the body is radically changed from the horizontal to the vertical (sitting) position. The atlas presented is based upon Macaca mulatta monkeys sitting in position in a primate restraint chair. This standard orientation provides a necessary frame of reference for accurate localization of lower brainstern structures.

The fundamental principle in using the stereotaxic instrument for placement of electrodes in: the deep structures of the brain is that a constant relationship exists between the bony structures of the skull (external auditory meatus, inferior orbit) and the neural structures located within the cranium. This relationship is sufficiently stable when species restrictions and weight ranges are closely observed. However, it holds only for those structures that are rigidly fixed within the cranium and cannot be altered by externally applied forces. Most stereotaxic atlases have not considered changes in the relation between bony landmarks and lower brainstem nuclei which result from alteration of body position. This discrepancy has been particularly significant in our studies involving single neuron recording from unanesthetized monkeys. These monkeys are seated in primate restraining chairs so that the lower brainstem and spinal cord is at an approximate right angle to the cerebral hemispheres. Under these conditions none of the atlases were sufficiently accurate for placing electrodes in the stem. Most atlases have been made with the monkey's body in a more horizontal position so that the stem and cord is at an angle of about 45° with respect to the hemispheres. It seemed worthwhile to prepare yet another stereotaxic atlas for the rhesus monkey to permit accurate placement of electrodes in the brainstem. of the chaired animal and to provide a standard frame of reference for the relationship between bony structures and lower brainstem nuclei.

MATERIALS AND METHODS

Seven Macaca mulatta ranging in weight from 2.85 to 7.27 kg were used for construction of the atlas. Three were used in the preparation of complete serial brain sections; the others were used to check the accuracy of the atlas based upon the sections of the first three animals.

A special primate restraining chair was constructed with a receptacle on the top plate which fit the main frame supportmember of the heavy duty Kopf stereotaxic instrument. The monkey was anesthetized with Dial‑Urethane and placed in the stereotaxic instrument. His body was allowed to hang freely, allowing a natural orientation between head and body. The height of the instrument was adjusted to maintain some body support on the seat bars. The surface of the brain was exposed and insulated stainless steel electrodes (which had previously been zeroed in the standard fashion) were introduced into the brain in order to determine anterior‑posterior and left‑right coordinates. The electrode positions varied from anterior 2.0 to posterior 13.0 and from lateral 1.0 to 4.0. The entire length of the electrode track was marked by lowering the electrode nearly to the bottom of the skull and then applying a nine volt anodal direct current while the electrode was slowly withdrawn. This left iron deposits along the extent of the track. After several such tracks were marked, a pair of bilaterally symmetrical tracks were made either far anterior or posterior for use as a leveling and'orientation guide during sectioning of the brain. The electrode carrier was then shifted to the horizontal position and the same procedure was performed at horizontal 0 (ear bar level), horizontal ‑ 5, and horizontal ‑ 10. This provided accurate localization in the horizontal (H) or dorsal‑ventral dimension.

With the animal still in this sitting position, the thorax was opened and the upper body and head were perfused through the left ventricle 'with normal saline followed by 10% formalin and 1% potassium ferricyanide. The latter reagent reacted with the iron deposits to form a vivid blue color along the electrode tracks (the Prussian Blue reaction). The brain was allowed to harden in this position for several hours before being dissected, removed and placed in 10% formalin for further fixation. At all times care was taken to maintain the original relationship between the brainstem and cerebral hemispheres. As a further aid to orientation of the brain for sectioning, an initial cut in the brain of two monkeys was made at the time of perfusion by mounting a number 11 scalpel blade in an electrode holder and slowly running the 'Carrier vertically 'and transversely in a sawing fashion.

A minimum of one week later, the brains were sectioned with a sliding microtome. The brains were mounted on a copper plate with dry ice and ethyl alcohol placed in copper receptacles attached to the sides of the plate. This kept the brains at a proper freezing temperature. Great care was taken to insure that the plane of sectioning was the same as the stereotaxic plane. Sections were cut at 50 μ.. All sections were saved and immediately mounted on slides. The slides were systematically divided into four groups for staining with Cresyl Violet, Weil, Neutral Red, or Luxol Fast Blue. Test photographs revealed that the Cresyl Violet and Luxol Fast Blue stains would give the best detail in photographs. Therefore, photographs of every second of each of these slides were taken.

All major structures were serially traced on the slides and simultaneously identified on the photographs. The detailed tracing procedure was found to be most necessary in the posterior portions of the stem where the deviations from the more usual transverse sections became pronounced. From these original photographs, 19 of the Cresyl Violet sections and eight of the Luxol Fast Blue were selected for illustration. ‑These were chosen to coincide with rapid changes in the locations of nuclear groups or fiber tracts. The intervals vary from 0.2 min to 0.6 min in the cellular stained sections and from 0.8 to 1.9 mm in the fiber stains.

DISCUSSION

The Luxol Fast Blue stain yielded a better photographic representation of fiber tracts than the Weil stain, largely because a contrasting filter could be used to emphasize the stained fibers. There was, however, some unevenness in the staining so that certain heavily myelinated tracts stained lightly or not at all. This was especially true of cranial nerves IV, V, VI and VII. The superior cerebellar peduncle (PCS) also showed uneven staining. The cellular stains were very consistent.

ACKNOWLEDGMENTS

We wish to express our gratitude to Drs. C. N. Liu, W. W. Chambers, and J. M. Sprague for reviewing the adas and to Ms. Donna Simmons and Dorothy Reese for the histological preparations. Mr. Clifford Freehe was responsible for the photography and Ms. Phyllis Wood for the preparation of the final plates.

Supported by grants RR 00166 and HE 04741 from the National Institutes of Health.