Primate injections and surgeries
Eleven Macaca fascicularis monkeys, weighing between 3 and 9 kg, were used in these experiments (Labs of Virginia, Yemassee, SC, USA; Three Springs Laboratories, Pekaski, PA, USA, and Worldwide Primates, Tallahassee, FL, USATracers). Small amounts (40 nl) of either Lucifer Yellow conjugated to dextran amine (LY; 10% Molecular Probes, Eugene, OR, USA), Fluoro-Ruby conjugated to dextran amine (FR; 4% Molecular Probes) or Fluorescein conjugated to dextran amine (FS; 10% Molecular Probes) were injected into the central nucleus, amygdalostriatal area, interstitial nucleus of the posterior limbic of the anterior commissure (IPAC), medial nucleus, accessory basal (magnocellular and parvicellular subdivisions) and basal (magnocellular and parvicellular subdivisions) nuclei of the amygdala. To confirm retrograde studies, we placed bidirectional tracers at several mediolateral and rostrocaudal levels of the midbrain DA neurons to determine the pattern of anterogradely labeled fibers in the amygdala.
All experiments were carried out according to National Institutes of Health guidelines, and reviewed by the University of Rochester Committee on Animal Research. Animals were given i.m. injections of ketamine hydrochloride (10 mg/kg) (Hospira, Inc., Lake Forest, IL, USA), intubated, and deeply anesthetized with i.v. pentobarbital (initial dose 20 mg/kg), which was maintained as needed during surgery. We performed a craniotomy to visualize cortical surface landmarks, and electrophysiologic mapping to locate internal landmarks such as the anterior commissure, striatum and amygdala (Fudge et al., 2004
; Fudge and Tucker, 2009
). Stereotaxic coordinates for these boundaries were determined, and the locations of the nuclei were estimated. The retrograde tracer was pressure-injected over 10–15 min into individual nuclei using a 0.5 μ
l Hamilton syringe (Hamilton Company, Reno, NV, USA), and the syringe was left in place for 20 min to prevent leakage of tracer up the syringe track. Only one injection of each tracer was used per animal. After injections were placed, the bone flap was replaced and the overlying musculature and skin sutured. Prophylactic antibiotics and pain medication were given for 7–10 days post-operatively.
Ten to thirteen days after surgery, animals were deeply anesthetized and killed by perfusion through the heart with 0.9% saline containing 0.5 ml of heparin sulfate (200 ml/min for 10 min), followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer/ 30% sucrose solution (100 ml/min for 1 h). The brain was removed, placed in fixative overnight, and sunk in increasing gradients of sucrose (10%, 20%, 30%). Brains were cut on a freezing microtome (50 μm sections) and saved in cryoprotectant solution (30% ethylene glycol and 30% sucrose in 0.1 M phosphate buffer) at −20 °C (Rosene et al., 1986
). Every 24th slice was used in our studies, and adjacent sections were used for determining anatomical landmarks.
Sections were thoroughly rinsed in 0.1 M phosphate buffer (pH 7.2) with 0.3% Triton-X (PB-TX). After treatment with endogenous peroxidase inhibitor for 5 min, followed by more rinses, sections were pre-incubated in a blocking solution of 10% normal goat serum in 0.1 M PB-TX (NGS-PB-TX) for 30 min. Tissue was then placed in primary antisera to LY (1:2000, Molecular Probes, rabbit), FS (1:2000, Molecular Probes, rabbit), or FR (1:1000, Molecular Probes, rabbit) for approximately 96 h at 4 °C. After thorough rinsing with 0.1 M PB-TX, and pre-incubation with 10% NGS-PB-TX, sections were incubated in biotinylated secondary anti-rabbit antibody. Tracers were visualized using the avidinbiotin reaction (Vector ABC Standard kit, Burlingame, CA, USA). Additional compartments for each case were also processed for tracer enhanced with nickel intensification (3, 3′-diaminobenzidine tetrahydrochloride with 1% nickel ammonium sulfate and 1% cobalt chloride, catalyzed by 0.03% hydrogen peroxide for 1–2 min) and counterstained with acetylcholinesterase (AChE) (Geneser-Jensen and Blackstad, 1971
) or cresyl violet.
Adjacent or near adjacent sections through the ventral midbrain were used to demarcate the dorsal and ventral tiers of the SNpc according to CABP immunoreactivity (Lavoie and Parent, 1991
; German et al., 1992
). Sections were thoroughly rinsed, and preincubated in 10% NGS-PB-TX as described above, and then incubated for 96 h in CaBP (Chemicon, 1:10,000, mouse) antisera. Sections were then rinsed, blocked and incubated in secondary anti-mouse biotinylated antibody. Following thorough rinsing, CaBP protein was visualized using the avidin–biotin reaction described above.
Double-labeling fluorescent immunocytochemistry
Optimal dilutions for primary antibodies used in immunofluorescent studies were established in advance using single-label fluorescent labeling. In addition, control tissue from animals without tracer injections and immunohistochemical processing was examined for autofluorescence. The primary antibodies used were (1) tyrosine hydroxylase (TH; mouse, Millipore, Temecula, CA, USA) diluted to 1:5000, and antiserum to one of the following: (2) Lucifer Yellow tracer (LY; rabbit, Molecular Probes, Eugene, OR, USA) diluted to 1:3000; Fluorescein (FS; rabbit, Molecular Probes) diluted to 1:750; or Fluoro-Ruby (FR; rabbit, Molecular Probes) diluted to 1:2000. Fluorescent immunocytochemistry was run on coronal sections from the level of the mammillary bodies rostrally to the emergence of the brainstem A5 catecholamine group caudally. For each injection site, every twenty-fourth slice was pulled from storage in a −20 °C freezer and rinsed for 15 min in four successions in 0.1 M phosphate buffer with 0.3% Triton-X100 detergent (PB-TX) (pH=7.2), and then overnight in PB-TX (pH=7.2) in a 4 °C cold room. The following day, the tissue was rinsed for 5 min in a solution of: 80% 0.1 M phosphate buffer (PB) (pH=7.2), 10% methanol, and 3% hydrogen peroxide solution. The tissue was rinsed for six successions of 15 min in 0.1 M PB-TX (pH=7.2), blocked for 30 min in 10% normal goat serum (NGS) made in 0.1 M PB-TX (pH=7.2), and then incubated for four nights with two primary antibodies—TH and tracer—in a solution of 10% NGS.
After four nights, the tissue was rinsed for 15 min in six successions in 0.1 M PB-TX (pH=7.2), then blocked for 30 min in 10% NGS. The tissue then incubated at room temperature in the dark with pooled secondary antibodies for 4 h in a solution of 10% NGS (pH=7.2). Following preliminary experiments to determine that labeling from the two fluorophors was similar for each antigen, the secondary antibodies used were (1) AlexaFluor 488 nm, goat anti-mouse (against TH) at a concentration of 50 μl/10 ml of 10% NGS, plus either: (2) AlexaFluor 546 nm, goat anti-rabbit (against tracer) at a concentration of 50 microliters/10 ml of 10% normal goat serum; or Texas Red 596 nm (Genway Biotech, Inc., San Diego, CA, USA), goat anti-rabbit (against tracer, at a dilution of 1:500 in 10% NGS). After 4 h, tissue was rinsed for six successions of 15 min in 0.1 M PB (pH=7.2). Tissue was mounted on gelatin-subbed slides out of 0.1 M PB (pH=7.2), dried overnight, and coverslipped with Vectashield medium (Vector Lab, Burlingame, CA, USA). Mounted and coverslipped slides were stored in the dark at 4 °C until taken to the microscope for viewing.
Neurolucida drawings for retrograde studies
Slides containing retrograde-labeled cells were viewed and charted on an Olympus Ax70 Fluorescent Microscope equipped with cubes for viewing 488 and 546 or 596 nm wavelength antibodies. Using a video CCD camera attached to the microscope and interfaced with the computer, cells and major anatomical landmarks were drawn using the computer program, Neurolucida (MicroBrightfield Inc., Rutland, VT, USA), under 10× objective. To visualize cells in both the green and red channels, we used an exposure of 1.47 s, with a gain of 1.58. Tracer-labeled cells were charted first, and the cube was then switched to view TH-positive cells. Double labeled cells were marked based on identical position and morphology between tracer-labeled and TH-labeled cells. These drawings were transferred to Adobe Illustrator for formatting.
Identifying the dorsal and ventral tier
Adjacent or near-adjacent sections through the ventral midbrain stained with Nissl or for CABP immunoreactivity were used to identify the dorsal and ventral tier subpopulations. For those slides stained for CABPimmunoreactivity, images were projected onto a wall using a macroprojector (JENA, Germany), and the boundaries between the dorsal and ventral tiers, and the substantia nigra, pars reticulate (SNpr), were drawn by hand onto hard copies of the Neurolucida charts. The projected image was aligned with the Neurolucida drawing using major landmarks such as the cerebral peduncles, aqueduct, and blood vessels. For slides stained with Cresyl Violet, we used camera lucida techniques to project and align images onto Neurolucida paper charts. Dorsal tier and ventral tier were distinguished by soma size and dendrite orientation.
To confirm information obtained with traditional fluorescent microscopy, and better visualize the morphology of double-labeled neurons, we examined some cases using confocal microscopy. A FV1000 Olympus confocal with SIM scanner was used with Alexa Fluor 488 nm (green) and Texas Red 596 nm (red) configurations. The area of interest was first identified using a 4× objective with widened pinhole. Once double-labeled cells were seen, they were examined with oil immersion under 20× and 40× power. The objective was focused at the top and bottom of the z-plane through the cell and the area was scanned every 0.8 μm, making a stack of 14–15 slices. A Kalman setting of 6 was used for the best resolution.
We examined the distribution of labeled fibers in the amygdala using dark-field microscopy with a 10× and 20× objective. Hand-drawn charts were created using camera lucida techniques and then scanned into the computer. The distribution of labeled fibers within specific amygdala subdivisions was determined using adjacent Nissl and CABP-immunoreactive sections, which were carefully aligned with charts to match anatomic landmarks such as blood vessels (Pitkanen and Amaral, 1993