Eight adult monkeys (Macaca fascicularis) were used in this study. Seven received retrograde tracer injections, and one was used only for examination of the anatomy of the PMRF. The experimental procedures were approved by the IACUC of The Ohio State University and subject care was according to the NIH Guide for the Care and Use of Laboratory Animals.
For retrograde tracing of reticulospinal projection neurons, each monkey was initially anesthetized with ketamine hydrochloride injected intramuscularly (10mg/kg) and then maintained with 2% isoflurane anesthesia inhaled endotracheally. Under aseptic conditions, the animal was then placed in a Kopf stereotaxic apparatus and a laminectomy was performed over the cervical enlargement. The dura mater was incised longitudinally with ophthalmic surgical scissors to expose the spinal cord from the C3 – C5 cervical segments. A 5μl Hamilton syringe was mounted in a custom-built electrode carrier attached to the posterior part of the stereotaxic frame. This arrangement allowed the needle to be adjusted anteroposteriorly and mediolaterally and inserted perpendicular to the surface of the cord at the desired site.
In the first three animals, several pressure injections of 2.5 % wheat germ agglutinin conjugated to horseradish peroxidase (0.5-1.5ul per injection,WGA-HRP; Sigma Chemicals) were made into both sides of the spinal cord at multiple cervical levels in order to broadly define the origins of the reticulospinal tracts. One of these three subjects had been previously implanted with a stainless steel recording chamber and was trained to perform an instructed delay reaching task (case M121-12). This subject had 52 penetrations with recording microelectrodes as described in Davidson and Buford (2006)
. In the next four animals, 1% cholera toxin subunit B (CTB, Sigma Chemicals) was injected at several locations on one side of the cervical spinal cord in order to determine the ipsilateral and contralateral contributions of the PMRF. The number of injections per case ranged from 5 to 18. In the more medial injection sites, the tracer was deposited at depths of 2.5-mm and 1.5-mm from the surface of the spinal cord, while at the lateral sites a single injection was made 1.5-mm deep only. After each injection, the Hamilton syringe was kept still for 2 minutes to reduce the potential for backflux of tracer into the general cerebrospinal fluid compartment. After the injections, the dura was folded back in place, the opening was covered with GelFilm ™ and the laminectomy packed with Gelfoam ™, and muscles and skin were sutured in layers.
The subjects recovered from anesthesia and received analgesics (Buprenorphine 0.01 mg/kg i.m. BID, ibuprofen 7-10 mg/kg p.o.) during the survival period; and all subjects recovered independent use of all four limbs. Three to four days after the injections, each animal was sedated with ketamine hydrochloride (13 mg/kg i.m.) and then deeply anesthetized with sodium pentabarbitol given i.v. to effect (2-3 ml @ 50 mg/ml). Heparin (10 ml @ 1000 USP units/ml) was administered to prevent coagulation of the blood during perfusion. Each subject was then perfused intracardially with warm phosphate buffered saline, followed by 4% paraformaldehyde, 0.01 M sodium periodate, 0.075 M lysine in phosphate buffer followed by cold 10% and 30% sucrose in phosphate buffer. The brain was removed, immersed in 30% sucrose in phosphate buffer for 2-3 days prior sectioning at 50μm on a freezing microtome.
The normal cytoarchitecture and chemoarchitecture of monkey reticular formation was determined based on analysis of a series of sagittal sections stained for Nissl and substance P immunocytochemistry in one subject that did not receive any retrograde tracer injections. Adjacent series of sections were collected for Nissl staining using cresyl violet and immunocytochemistry using antisera against substance P (raised in rabbit, Immunostar). For the immunocytochemistry, endogenous peroxidase activity was eliminated by incubating the sections first in a hydrogen peroxide mixture. The sections were rinsed in phosphate buffered saline (PBS), blocked in normal goat serum and exposed to the primary antibody to substance P (1: 6,000 dilution) for 42 hours at 4°C. Sections were then rinsed in PBS, incubated in biotinylated secondary antibody (1:100 dilution), rinsed and incubated in avidin-biotinperoxidase complex (1:50 dilution, Vector Laboratories). The reaction product was visualized using diaminobenzidine and hydrogen peroxidase. Immunocytochemical control procedures included omission of the primary antisera, which abolished any specific labeling. Cytoarchitecture and nomenclature of the monkey brainstem largely followed the descriptions from Paxinos et al. (2000)
In the three subjects where HRP was injected, the tissue was cut in the coronal plane and reacted using tetramethylbenzidine (TMB) as the chromogen with ammonium heptamolybdate as a stabilizing agent, modified from Olucha et al (1985)
. In subjects where CTB was injected, the tissue was cut in the parasagittal plane and the CTB was identified by immunohistochemistry using a goat anti-CTB antibody (ListBiological Labs, 1:5000) and secondary biotinylated anti-goat IgG (Vector Labs, SK 4600), as described previously (Sakai et al., 2000
). The biotinylated antibody was visualized by using the avidin-biotin-immunoperoxidase method with a Vectastain ABC kit (Vector Laboratories) and Vector VIP® substrate kit (Vector Laboratories SK 4600) or diaminobenzidine as the chromagen. Adjacent sections were stained for Nissl substance using cresyl violet.
To define the extent of tracer diffusion at the injection sites, serial horizontal sections through the spinal cord injection sites were plotted onto drawings magnified to 6.5X using an Aus Jena Dokumator. The spread of the tracer including the dark dense core and light diffuse halo were plotted relative to spinal cord gray and white matter tracts.
A selected series of sections for each subject was mapped using the Neurolucida analysis system (Neurolucida 7.5 software, MBF Bioscience, Inc) connected to an Olympus BH-2 microscope equipped with a digital camera (Optronics Microfire), a X-Y-Z encoder and motorized microscope stage connected to a Dell Optiplex computer. WGA-HRP positive cells contained dense blue-black granular reaction product in the neuronal cytoplasm (Mesulam, 1982
). Cells labeled with CTB had a fine powdery purple or brown precipitate characteristic of CTB cytoplasmic label (Sakai et al., 2000
). Labeled cells were identified using the 20X and 40X microscope objectives. Using Neurolucida software, labeled cells were mapped onto section outlines including the nuclear boundaries of PnO, PnC and Gi as defined from adjacent Nissl-stained sections aligned based on section outlines and blood vessels. Somata of retrogradely labeled cells were counted within these nuclei from a selection of 4 sections using the 20X and 40X microscope objectives. Cell counts were based on analysis of an equal number of parasagittal sections from each side starting at least 0.3 mm from midline. The analyzed sections ranged from 400 to 2200 μm apart and were from comparable parasagittal levels across all cases. Giant cells were separately marked based on soma size exceeding a standard 50 μm diameter circle (adapted from Newman, 1985a
). No overestimation correction was made to the cell counts since the cell counts were used for proportional comparison between ipsilateral and contralateral labeling for each case. Photomicrographs were taken using a digital camera (Optronics Microfire) on either a microscope (Olympus BH-2) or macroscope (Olympus SZH). The images were imported into Adobe Photoshop (Version 8.0, San Jose, CA) and if necessary, cropped and adjusted for contrast or brightness. Otherwise, the images were not altered.