Adult female (225–250 grams) Long-Evans rats bilaterally ovariectomized (OVX) by the provider, were purchased from (Charles River, Wilmington, MA). OVX animals were used in order to eliminate differences over the cycle and avoid possible differences in ERβ mRNA expression and translation by E2 treatment (Shughrue and Merchenthaler, personal communication). At arrival at UCLA, all animals were housed two per cage in a partially reversed, 12/12 hr light/dark cycle (lights on at 12 midnight) and provided with food and water ad libitum. All of the experimental procedures were approved by the Chancellor’s Animal Research Committee at the University of California, Los Angeles.
rats were deeply anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with physiological saline (4°C) followed by 4% paraformaldehyde in 0.1M Sorensen’s phosphate buffer (4°C). Brains were removed, postfixed for 4–24 hrs in 4% paraformaldehyde solution and then transferred to 15% sucrose in 0.1 M phosphate buffer (pH 7.5), for cryoprotection. Coronal 30 µm sections through the ventral midbrain from four ovariectomized rats were processed simultaneously (Paxinos et al., 1985
). Every fourth section was taken through the substantia nigra in a systematic-random manner. Free-floating sections of the ventral midbrain where collected in 0.01 M phosphate buffered saline (PBS, pH 7.4). To quench endogenous peroxidase, all sections were incubated with 10% methanol containing 1% H2
for 30 min.
In situ Hybridization
rats were deeply anesthetized with sodium pentobarbital (100 mg/kg) and killed by decapitation. Brains were removed, immediately frozen in 2-methylbutane (Sigma) at a temperature of −50 to −60°C and stored at −80°C. Coronal frozen sections of the brain were serially cut at 20 µm on a cryostat, mounted on Superfrost/plus slides and stored at −70°C until processing. Slides containing fresh-frozen sections were thawed for 10 min, fixed in 4% paraformaldehyde in PBS at 4 °C for 1 hr, washed in PBS (10 mM pH 7.4) 3× for 10 min, penetrated with 0.4% Triton X-100 for 10 min and acetylated for 10 min in 100 mM triethanolamine pH 8.0 containing 0.25% (v/v) of acetic acid. The slides were rinsed in PBS (3 × for 8 min) and distilled water, dehydrated in 50% and 70% ethanol and air-dried.
Tyrosine hydroxylase (TH) is an affinity purified sheep polyclonal antibody (Chemicon International, Temecula, CA). Isulin-like growth factor-1 receptor (IGF-1R), a mouse monoclonal antibody (Chemicon Research Products, San Diego, CA) was used. For ERβ immunocytochemistry; Z8P, an affinity purified rabbit polyclonal antiserum raised against amino acid 468–485 of the C-terminal domain of the mouse ERβ (Zymed laboratories, San Franciso, CA). The Z8P antibody was wa previously characterized (Shughrue and Merchenthaler, 2001
) and ERβ-ir is present in cells that express ERβ mRNA throughout the brain of female rats including the SNpc. Another ERβ antibody used was, Ab-1, a rabbit polyclonal antiserum raised against amino acids 467–485 of the C-terminal domain of the Rat ERβ (Oncogene Research Products, San Diego, CA), a region with no homology to ERα, and recognizes ERβ of rat origin by Western blotting (Li et al., 1997
). For ERα, a rabbit polyclonal antibody raised against a peptide corresponding to amino acid 580–599 of the C-terminal domain of the mouse ERα (MC-20, Santa Cruz Biotechnology, Santa Cruz, CA). Glial fibrillary acidic protein (GFAP) immunoreactivity was detected using a rabbit polyclonal antibody raised against human GFAP (G9269; Sigma, St. Louis, MO).
Dual Color Streptravidin-Biotin-Peroxidase Immunocytochemistry
For stereologic analysis of ERβ-ir, we employed the Z8P antibody, because it predominately labeled the nucleus of TH-ir SNpc DA neurons. To quantify the number of ERβ-ir, IGF-1R-ir in TH-ir SNpc DA neurons, sections were incubated in blocking solution (5% normal donkey serum (NDS), 0.3% Triton X-100 in PBS) for 1 hr in order to block non-specific binding. Then, sections were treated with 0.2% Triton X-100 (3 ×) for 10 minutes followed by 0.1 M glycine for 30 min. Sections were incubated with anti-TH (1:6000; Chemicon, Temecula, CA) overnight at 4°C, followed by biotinylated anti-sheep IgG and avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hr at RT. TH immunoreactivity was visualized by 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma). Sections were incubated for 48–72 hr at 4 °C with Z8P (2.5 µg/ml in 1% NDS, 0.3% Triton X-100 in PBS) or anti-IGF-1R (1:600 in 1% NDS, 0.3% Triton X-100 in PBS). After incubation with primary antibody, sections were incubated with biotinylated anti-rabbit IgG or biotinylated anti-mouse IgG (Vector Laboratories) for ERβ and IGF-1R, respectively for 1h at RT. Sections were then incubated in avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hr. After series of washes, ERβ and/or IGF-1R were visualized by DAB-nickel peroxidase reaction (Vector DAB substrate kit Cat # SK-4100, Vector Laboratories). Sections were mounted on Superfrost Plus slides, dehydrated through a series of graded alcohols cleared in xylene, and coverslipped with Permount mounting medium (Fisher Scientific).
Total numbers of ERβ and/or IGF-1R that are expressed in TH immunopositive neurons of the SNpc were estimated using the optical fractionator method (West et al., 1991
) with stereoinvestigator®
software (MicroBright-Field, Colchester, VT). The precision of the serial analysis was assessed by the coefficient of error (p < 0.1) (West and Gundersen, 1990
; West et al., 1996
). Briefly, each subfield of the SNpc was manually outlined at low magnification (5× objective) and a grid of 150 × 150 µm was superimposed by the software. The SNpc was delimited rostrally by the optic tract, caudally by the level of decussation of the superior cerebellar peduncle, ventrally by the pars reticulate subdivision of substantia nigra, and medially by the third cranial nerve rootlets. A 63× oil immersion objective (1.4 NA) was used to achieve optimal optical sectioning during dissector analysis. Quantitative analysis was performed by placing an unbiased optical dissector frame (50 × 50 µm = 2,500 µm2
in the x-y-axis) over the contour outlining the SNpc. The final post-processing thickness of the sections was measured by the microcator, resulting in a 15 µm averaged mounted section thickness. A 1.5 – 2 µm guard zone was used to exclude artifacts at tissue surface. For colocalization quantitative analysis, only those TH-ir cells that clearly demonstrated nuclear ERβ were counted.
Single Label Immunofluorescence
To enhance the sensitivity of the immunocytochemical reaction the tyramide signal amplification method (TSA-Direct, Perkin Elmer Life and Analytical Sciences, Boston, MA) was employed. Sections were incubated 48–72 hr at 4°C with anti-TH (1/500 in 1% NDS, 0.3% Triton X-100 in PBS); anti-IGF-1R (1:600 in 1% NDS, 0.3% Triton X-100 in PBS); Z8P (1/400 in 1% NDS, 0.3% Triton X-100 in PBS) or Ab-1 (1/200 in 1% NDS, 0.3% Triton X-100 in PBS). After incubation with primary antibodies, sections were washed in PBS (3 ×) for 10 min and incubated for 1 hr at RT with the secondary antibodies. To visualize TH-ir, rodamine (RITC)-conjugated Donkey Anti-Sheep (1:100; Jackson ImmunoResearch lab, INC; Baltimore, PA) secondary antibody was used. To visualize ERβ-ir, sections were incubated with biotinylated donkey anti-rabbit serum (1:1000; Jackson ImmunoResearch Lab, West Grove, PA). These sections were then incubated with HRP-conjugated streptavidin (1:100; TSA kit in TNB buffer) for 30 min at RT, rinsed in PBS (3 ×) for 5 min, and then incubated with fluoroscein conjugated tyramide (FITC-conjugated tyramide; 1:100 in 1 × amplification diluent; TSA kit) for 10 min at RT. To visualize IGF-1R, sections were incubated with biotinylated donkey anti-mouse serum (Vector Labs; 1:200 in PBS) for 1 hr. Sections were then washed in PBS (3 ×) for 10 min and incubated with HRP-conjugated streptavidin (1:100; TSA kit in TNB Buffer) for 30 min at RT, rinsed in PBS (3 ×) for 5 min, incubated with fluoroscein conjugated tyramide (FITC-conjugated tyramide; 1:100 in 1 × amplification diluent; TSA kit) for 10 min at RT. Tissue sections were washed in PBS (3 ×) for 10 min, mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), air-dried and coverslipped using Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
For double immunofluorescence for ERα/β or IGF-1R in TH-ir nigral DA neurons, sections were incubated for 48–72 hr at 4°C with a cocktail of antisera raised in different species containing anti-TH with either IGF-1R or Ab-1 or Z8P. Double immunofluorescences for ERβ or IGF-1R in GFAP-ir glial cells, sections were processed as mentioned above. Following amplification of either IGF-1R and/or ERβ with the TSA kit as previously described above, sections were incubated in blocking solution (5% NGS in PBS for 1 hr) and incubated for 1 hr at 4 °C with rabbit anti-GFAP (G9269; Sigma, St. Louis, MO; diluted 1:50) in 1% NGS and 0.3% Triton X-100 in PBS. GFAP-ir was detected by incubating the sections for 1 hr in Texas Red –conjugated goat anti-rabbit antibody (Molecular Probes; diluted 1:1000; 1% NGS). Colocalization for ERβ or IGF-1R in GFAP-ir cells in the SN was analyzed by the (Carl Zeiss Inc, Thornwood NY) LSM 3.2 software. Z-stacks of images were taken using a 40× oil immersion objective. An area of 1000 mm2 was delineated from each Z-stack. Quantitative data is expressed as an area of a colocalized number of pixels.
The following controls were performed: omitting the primary or secondary antibodies, dilution series and nonimmune rabbit or mouse immunoglobulin G (IgG; 50 µg/µl). The following peptides were used for preabsorbance: the epitope peptide to anti-ERα (MC20, Santa Cruz Biotechnology); and peptide to block the anti-ERβ Z8P antibody and Ab-1 antibody was preabsorbed with the 19 –amino acid sequence of ERβ (amino acids 467–485)(PA1-310B; Affinity Bioreagents). All staining was blocked by preabsorption. The pattern of staining for each antibody was the same in double or single labeled sections. For double labeling, the order of primary antibodies was reversed and homologous and heterologous absorption controls were done. Nonspecific immunoflourescent staining, cross-immunostaining, or bleeding-through were not observed.
Immunofluorescence double-labeled profiles were scanned with a Zeiss Axioskop LSM 510 laser scanning confocal microscope system equipped with an Axiocam CCD camera and a digital image analysis system (Zeiss USA Inc.). The excitation source was a krypton-argon laser (Coherent, Santa Clara, CA) with output at 488,568 and 633 nm. FITC was visualized with a 488 nm excitation and a 515–540 nm bandpass emission filter. Rhodamine was visualized with a 530 nm excitation and 560–610 nm bandpass emission filter. Z-stacks were obtained by using identical settings for laser intensity, confocal aperture, scan speed and Z-stack step size (0.5 – 1.0 µm). All final images were adjusted for contrast and background using Adobe Photoshop 5.0 (Mountain View, CA).
Retrograde Tract Tracing Experiments
OVX rats were implanted with a guide cannula aimed at the striatum (coordinates: antero-posterior, bregma +0.48 mm; lateral, ± 3.0 mm; and doso-ventral, −5.0 mm, (Swanson, 1992
). A 29 gauge cannula containing the retrograde tracer Fluorogold™ (FG; 5% w/v dissolved in 0.9% saline; Fluorochrome, Denver, CO) was used to slowly eject 1 µl of FG over 5 min (Iravani et al., 2002
). The cannula was connected to a 25 µl Hamilton syringe via a plastic tube. The cannula was allowed to remain in place an additional 5 min to allow diffusion of drug away from the cannula tip. Seventh day after FG injection, animals were perfused.
Immunocytochemical labeled sections from FG injected animals were visualized with a Axioflouroskop 2 equipped with epiflourescent illumination and Axiovision 3.1
software. Digital pictures were taken with an Axiocam CCD camera mounted on the microscope (Zeiss Inc. USA). To detect the immunolabeling of either ERβ-ir and/or IGF-1R-ir in striatal projecting SNpc DA neurons, SN sections from rats injected with FG into the striatum were processed for immunofluorescence for either ERβ and/or IGF-1R immunohistochemistry as described above. The number of FG SNpc cells immunolabeled for either ERβ and/or IGF-1R were averaged at 4 different levels corresponding to −4.8 mm, −5.1 mm, −5.4 mm and −5.7 mm with respect to bregma (Paxinos et al., 1985
) and expressed as a percentage. FG was visualized with a 333 nm excitation and wideband ultraviolet emission filter at 408 nm. FITC was visualized with a 488 nm excitation and a 515–540 nm bandpass emission filter.
In-Situ Hybridization for IGF-I Receptor
The IGF-1R probe was complementary to 15 bases of 5’ untranslated region encoding the signal peptide, which correspond to the first 53 amino acids of the alpha subunit (Werner et al., 1989
). The cDNA EcoR I/Sma I fragment (265 bp) was subcloned into a pGem-3Z transcription vector (gift from Dr. D. LeRoith; National Institutes of Health, Bethesda, MD, USA), and linearized with Eco
RI (antisense) or Bam
HI (sense) and isotopically labeled riboprobes were generated by in vitro
transcription using SP6 (antisense) and T7 (sense) polymerases in the presence of 35
S-UTP and 35
S-CTP (Amersham Biosciences Corp. Piscataway, NJ). Riboprobes were purified through NucTrap purification columns (Stratagene), diluted in hybridization buffer (100 mM Tris pH 7.5, 600 mM NaCl, 1 mM EDTA, 0.1 mg/ml sonicated salmon sperm, 0.5 mg/ml t-RNA, 1× Denhardt’s, 10% dextrane sulfate, 50% formamide) to 100,000 cpm/µl and used within 48 hr.
Tissue sections were covered with hybridization buffer (80 µl) containing antisense or the sense riboprobes, coverslipped to prevent evaporation and hybridized for 16–18 hr at 58°C in a moist chamber containing 50% v/v formamide. Following hybridization, coverslips were carefully removed, sections were washed with 2 × sodium citrate (SSC) and 1 × SSC for 15 min each at room temperature and exposed to RNAse buffer (20 µg/ml RNAse A, 1 U/ml RNAse T1, 10 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA) at 37°C for 40 min. Afterwards, slides were washed in decreasing concentrations of SSC to a final stringency of 0.1 × SSC at 62 °C for 1 hr. Finally slides were washed in double distilled water, dehydrated through graded ethanols and air dried. Slides were dipped in K5 photoemulsion (Ilford, Paramus, NJ), diluted 1:1 with double-distilled water. Emulsion coated slides were air-dried in the dark for 12 hr, packed into light-tight boxes and exposed at 4°C for 21–28 days, developed (D-19 developer) and fixed (Kodak fixer) both products from (Kodak Co, Rochester, NY). The slides were counterstained with Toluidine blue, dehydrated and coverslipped with DPX (BDH Chemical Ltd. London, UK).