Male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) weighing 225–250 g were used in accordance with guidelines mandated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were housed on a 12 hr light-dark cycle either singly or in groups of four until surgeries were performed or habituation to experimental conditions was begun, after which all were singly housed. Rats used in cocaine self-administration experiments (see below) were habituated to a 12h reverse light-dark cycle. Access to food and water was provided ad libitum to all rats throughout the study. Unless stated otherwise, chemicals were purchased from Sigma Chemical Company (St. Louis, MO).
Stimulation of RMTg Fos expression
Rats that have self-administered cocaine (about 20 mg/kg/session, at 500 μg/kg per 30 μl infusion), via intravenous catheters for one or six consecutive daily two hours sessions, exhibit striking Fos expression in the RMTg, as do rats given intravenous infusions of investigator-administered cocaine (Geisler et al., 2007b
; Marinelli et al., 2008
). Furthermore, i.p. injections of methamphetamine (10 mg/kg) given 2 h prior to sacrifice also produce Fos expression in the RMTg (Colussi-Mas et al., 2007
; Zahm, unpublished). In view of these findings, sections showing Fos expression in the RMTg in the present study came from rats that received intravenous cocaine infusions (as in Geisler et al., 2007b
) or methamphetamine injections (as described above).
Several minutes after being given intraperitoneal injections of a cocktail, consisting of 45% ketamine (100mg/ml), 35% xylazine (20mg/ml) and 20% physiological saline at a dose of 0.16 ml/100g of body weight, rats were placed into a Kopf stereotaxic instrument. The skulls were exposed and small bore holes were created to allow selected brain structures to be targeted by filament-containing borosilicate glass pipettes (O.D. - 1.0 mm) pulled to tip diameters of 10–25 μm and containing the retrograde tracers Fluoro-Gold (FG; Fluorochrome, Inc., Englewood, CO; 1% in 0.1M cacodylate buffer, pH 7.4) or cholera toxin B subunit (CtB, List Biological Laboratories, Campbell, CA), or the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L; Vector, Burlingame, CA, 2.5% in 0.01 M phosphate buffer). A silver wire inserted into the pipettes contacted the solution containing the tracer, which was ejected into the brain substance using positive current pulses (7 s on, 7 s off, for 15 minutes) of 1 μA (for FG) or 4 μA (for PHA-L and CtB). After surgery the rats were kept warm until they awakened.
The results of the study are based upon the evaluation of cases in which injections of FG (3 cases), CtB (2 cases) or PHA-L (4 cases) were centered in the RMTg. Five cases in which PHA-L sites were centered in the LHb were also studied. In addition, ten, six, and eleven cases were evaluated in which FG, CtB and PHA-L injections, respectively, partially involved the RMTg but more substantially invaded adjacent structures. In addition, numerous control retrograde labeling cases were drawn from archived series of injections targeting the retrorubral field (18 cases), pedunculopontine tegmental nucleus (17 cases), laterodorsal tegmental nucleus (17 cases) and ventral tegmental area (49 cases). Structures labeled following injections of retrograde tracers into the RMTg were validated to the extent possible with available archived cases with PHA-L injections in, e.g., the prefrontal cortex, accumbens, ventral pallidum, lateral preoptic area, lateral hypothalamus, ventral tegmental area and substantia nigra compacta. Targeting of injections was done with the aid of stereotaxic coordinates which were initially acquired from the atlas of Paxinos and Watson (1998)
and refined empirically. Most abbreviations used in the text, figures and tables also are from Paxinos and Watson (1998)
Fixation of brains and immunocytochemistry
Three days after FG injections and ten days after PHA-L injections, the rats were deeply anesthetized (as above) and perfused transaortically, first with 0.01 M Sorensen's phosphate buffer (SPB; pH 7.4) containing 0.9% sodium chloride and 2.5% sucrose, followed by 0.1 M SPB (pH 7.4) containing 4% paraformaldehyde and 2.5% sucrose. The brains were removed, postfixed, infiltrated with 25% sucrose and sectioned at 50 μm. Five adjacent series of sections were collected, with each thus reflecting the structure of the entire brain from frontal pole to caudal medulla in sections spaced at intervals of 250 μm. Each series of sections was stored in a separate glass vial at −20° C in a cryoprotectant consisting of SPB containing 30% sucrose (by weight) and 30% ethylene glycol (by volume).
One series of sections from each case was immersed in SPB containing 0.1% Triton X-100 (SPB-t) and polyclonal antibodies raised against the relevant tracer, i.e., either anti-FG made in rabbit and used at a dilution of 1:5000, anti-CtB made in goat and used at a dilution of 1:5000, or anti-PHA-L made in goat used at a dilution of 1:10,000. The following day, after thorough rinsing in SPB-t, the sections were immersed for an hour in SPB-t containing biotinylated antibodies made in donkey against rabbit (for FG) or goat IgGs (for PHA-L and CtB) at a dilution of 1:200 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Afterward, the sections were rinsed in SPB and then immersed in SPB containing avidin-biotin-peroxidase complex (ABC - Vector Laboratories, Burlingame, CA) at a dilution of 1:200, also for an hour. After more thorough rinsing in SPB, the sections were immersed for 20–30 min in 0.05 M SPB (pH 7.4) containing 0.05% DAB, 0.04% ammonium chloride, 0.2% β-D-glucose, and 0.0004% glucose oxidase, which generates an insoluble brown reaction product, or, if the sections were destined to be reacted with a second primary antibody, in 0.025 M Tris buffer (pH 8.0) containing 0.015% 3,3'–diaminobenzidine (DAB), 0.4% nickel ammonium sulfate and 0.003% hydrogen peroxide, which generates an insoluble black reaction product.
Sections intended for additional immunohistochemical processing were then further rinsed in SPB and immersed in SPB-t containing anti-c-Fos (Fos), anti-nitric oxide synthase (Nos), anti-myu-mu opioid receptor 1A (Mor), or anti-somatostatin (Som), all made in rabbit, or anti-tyrosine hydroxylase (TH) made in mouse, all used at a dilution of 1:5000. The following morning the sections were rinsed in SPB-t and immersed for one hour in SPB-t containing a donkey antibody against mouse or rabbit IgGs, as appropriate, each used at a dilution of 1:200 (Jackson). Following further rinsing in SPB the sections were immersed for one hour in SPB containing, respectively, mouse or rabbit peroxidase-anti-peroxidase (PAP) at a dilution of 1:3000 (MP Biomedicals, Solon, OH), after which they were again rinsed thoroughly. Then the sections were immersed for 20–30 min in 0.05 M SPB (pH 7.4) containing 0.05% DAB, 0.04% ammonium chloride, 0.2% β-D-glucose, and 0.0004% glucose oxidase (brown reaction product) and, after rinsing, mounted onto gelatin coated slides, dehydrated through a series of ascending concentrations of ethanol, transferred into xylene, and coverslipped with Permount (Fisher, Pittsburgh, PA).
Synthesis and labeling of probes
cDNA probe containing a 593 bp coding sequence for glutamate decarboxylase 67 kD (GAD) [MASST PSPAT SSNAG ADPNT TNLRP TTYDT WCGBA HGCTR KLGLK ICGFL QRTNS LEEKS RLVSA FRERQ ASKNL LSCEN SDPGA RFRRT ETDFS NLFAQ DLLPA KNGEE QTVQF LLEVV DILLN YVRKT FDRST KVLDF HHPHQ LLEGM EGFNL ELSDH PESKE QILVD CRDTL KYGVR TGHPR FFNQL STGLD IIGLA GEWLT STANT NMFTY EIAPV FVLME QITLK KMREI IGWSN KDGDG IFSPG GAISN MYSIM AARYK YFPEV KTKGM AAVPK LVLFT SEHSH YSIKK AGAAL GFGTD NVILI KCNER GKIIP ADLEA KILDA KQKGF VPLYV NATAG TTVYG AFDPI QEIAD ICEKY NLWLH VDAAW GGGLL MSRKH RHKLS GIERA NSVTW NPHKM MGVLL QCSAI LVKEK GILOG CNQMC AGYLF QPDKQ YDVSY DTGDK AIQCG RHVDI FKFWL MWKAK GTVGF ENQIN KCLEL AEYLY AKIKN REEFE MVFNG EPEHT NVCFW YIPQS LRGVP DSPER REKLH RVAPK IKALM MESGT TMVGY QPQGD KANFF RMVIS NPAATQ SDIDF LIEEI ERLGQ DL] was generously provided to Zahm by A. J. Tobin (UCLA) and Jhou by N. Tillakaratne (UCLA). The probes were prepared from a nearly full-length-cDNA clone (3.2 Kb) isolated from a rat brain library and inserted into the bluescript (SK polylinker, Stratagene, La Jolla, CA) EcoRI site (Erlander et al., 1991
). Separate clones containing the cDNA insert in different orientations allowed sense and anti-sense probes to be transcribed. Plasmids were incorporated by heat shock into ampicillin-resistant Escherichia coli
cells, from which templates were isolated and then linearized with Sal I
. Digoxigenin-labeled or 35
S-radiolabeled probes were transcribed with T3 RNA polymerase and were hybridized and detected using, respectively, non-isotopic immunohistochemistry (Geisler et al., 2007a
) or photographic emulsion (Marcus et al., 2001
; Simmons et al., 1989
Non-isotopic hybridizations combined with immunohistochemical detection of GAD 67
These were done with free-floating vibratome sections from brains perfused as described above. Diethylpyrocarbonate (DEPC)-treated water was used for all solutions and modifications were applied to methods described by Young (1990)
. Selected series of sections from operated rats were removed from the freezer and brought to room temperature, after which they were rinsed three times for 10 minutes in SPB. The sections were next immersed in an aqueous 1% solution of sodium borohydride for 15 minutes and then rinsed in SPB every 5 minutes until bubbles were gone. Next, the sections were immersed for 15 minutes in a solution containing triethanolamine, hydrochloric acid and acetic anhydride, after which they were placed for 15 minutes at 55° C in a hybridization buffer consisting of Tris-HCl (20 nM, pH 7.4), EDTA (1 mM), NaCl (300 mM), formamide (50 %), dextran sulfate (10 %), and Denhart's soution (1×). The sections were then placed overnight at 55°C in a mixture consisting of stock probe preparation, nucleic acid mix [salmon sperm DNA (10 μg/ml), yeast total RNA (25 μg/ml), yeast tRNA (25 μg/ml)], sodium dodecyl sulfate (10 %) and sodium thiosulfate (10 %) in hybridization buffer. The dH2O, probe, and nucleic acid mix were first heated to 65°C for 5 minutes, cooled on ice for 5 minutes, and then mixed with the other reagents and brought to 55°C before the sections were added.
Following the hybridization, the sections were thrice rinsed in 4× SSC for 5 minutes at room temperature, 1X SSC three times for 5 minutes at room temperature, 0.1× SSC once for 10 minutes at 65°C and finally cooled to room temp in 0.1X SSC for one minute. The sections were then rinsed in 0.1M Tris-HCl/0.15M NaCl (pH 7.5) for 5 minutes.
Immersion of the sections in 0.1M Tris-HCl containing 0.15M NaCl, 0.3% Triton (pH 7.5) and 0.3 % normal goat serum for 30 minutes preceded overnight immersion at room temperature in the same solution to which had been added mouse anti-digoxigenin antibody-alkaline phosphatase conjugate (Dig, Roche Diagnostics Corporation, Indianapolis, IN), at a dilution of 1:1000, and rabbit anti-FG antibody, at a dilution of 1:5000 (Chemicon). This was followed by two 5 minutes rinses and reaction with reagents comprising an alkaline phosphatase substrate kit VI (Vector) for 2–5 hours in the dark. After an acceptable intensity of GAD 67 mRNA hybridization reaction product had been achieved, the sections were again rinsed in the same buffer and then immersed in SPB-t (pH 7.4) for fifteen minutes. The sections were then placed at room temperature in SPB containing biotinylated anti-rabbit at a dilution of 1:200 for 2 hours, after which they were rinsed three times for 5 minutes in SPB and then further incubated in SPB containing avidin-biotin-horseradish peroxidase conjugate (Vector) for two hours at room temperature. Further rinsing in SPB (three times at 10 minutes) preceded incubation in DAB substrate solution (as above) for 15 minutes followed by three 5 minute rinses in cold SPB. The sections were then mounted from dilute SPB onto glass slides that were then coverslipped with Permount (Fisher).
Hybridizations with 35S-radiolabeled GAD-67 probe combined with immunohistochemical detection of CtB
Free-floating sections were incubated overnight at room temperature in goat anti-CtB (List Biological Laboratories) at a 1:5,000 dilution in PBS with 0.25% Triton X-100 (PBT) and 0.01% sodium azide (Sigma). After the primary incubation, the sections were rinsed three times for 1 min in PBS, incubated in biotinylated donkey anti-goat IgG (Jackson) at a dilution of 1:1000 for 1 hour, rinsed three times for 1 min in PBS, and then incubated in streptavidin-conjugated CY3 (Jackson) at a dilution of 1:1000 for 30 minutes. The sections were then rinsed in PBS and mounted on gelatin-coated glass slides. All reagents were made using water treated with diethylpyrocarbonate (Sigma) to inactivate endogenous RNAse enzymes. Control experiments in which primary antibodies were omitted, or in which tissue was obtained from rats without CtB injections, resulted in no immunoreactivity above background levels, thus indicating specificity of immunostaining.
The mounted sections were air-dried, and stored in desiccated boxes at −20°C. Prior to the hybridizations, the slides were dehydrated in ascending dilutions of ethanol, cleared in xylenes for 15 minutes, and then rehydrated in descending dilutions of ethanol. To facilitate probe penetration, the slides were placed in sodium citrate buffer (pH 6.0) and heated in a microwave oven (1100 watts) for 10 minutes at 70% power (temperature of 95–100°C). The slides were then dehydrated in ascending dilutions of ethanol and air-dried. The cRNA probe was diluted to 100 cpm/ml in a hybridization solution consisting of 50% formamide, 10 mM Tris-HCl, pH 8.0, 5.0 mg tRNA (Boehringer-Mannheim, Indianapolis, IN), 10 mM dithiothreitol, 10% dextran sulfate, 0.3 M NaCl, 1 mM EDTA at pH 8, and Denhardt's solution (Sigma). Hybridization solution and a coverslip were applied to each slide, and the sections were incubated for 12–16 hours at 57°C. The coverslips were then removed and the slides were washed with sodium chloride/sodium citrate buffer (SSC, pH 7.0). The sections were then incubated in 0.002% RNAase A (Boehringer-Mannheim) with 0.5 M NaCl, 10 mM Tris-HCl, pH 8, and 1 mM EDTA, for 30 minutes and again rinsed in decreasing concentrations of SSC containing 0.25% dithiothreitol (DTT): 2× at 50°C for 1 hour, 0.2× at 55°C for 1 hour, and 0.2× for 1 hour at 60°C. The sections were next dehydrated in graded ethanols (50, 70, 80, and 90%) containing 0.3 M ammonium acetate followed by 100% ethanol and then air-dried and placed in X-ray film cassettes with BMR-2 film (Kodak, Rochester, NY) overnight. The following day the slides were dipped in NTB2 photographic emulsion (Kodak), dried, and stored with desiccant in foil-wrapped slide boxes at 4° C for 4 days. The exposed emulsion was developed with D-19 developer (Kodak).
Combined CtB immunolabeling and GAD 67 hybridizations - data analysis
CtB immunolabeling, visualized under epifluorescence illumination, and silver grains representing GAD 67 hybridization, visualized under darkfield illumination, were photographed using a 25× objective (Zeiss) and a digital camera (Nikon), allowing offline analysis without excessive bleaching of fluorescence. Insofar as CtB immunohistochemistry was performed on floating sections, allowing antibodies to penetrate equally into both tissue surfaces, but in situ hybridization was carried out after the sections had been mounted, GAD 67 mRNA-hybridized neurons occupied preferentially the exposed surface of the sections. Accordingly, CtB immunolabeling was photographed twice for each region of interest, once with the focal plane at the exposed tissue surface, and a second time with the focal plane at the interface of the tissue surface and the glass slide. Co-localization of CtB with GAD 67 was recorded only for CtB neurons appearing more sharply in photomicrographs shot at the exposed surface. CtB-immunoreactive neurons were regarded as expressing GAD 67 if the density of overlying silver grains was more than three times the density observed over non-GABAergic tissue regions in the same section.
Descriptions of Antibodies
The rabbit polyclonal antibody used to detect Fos was raised against a synthetic peptide corresponding to amino acids 4–17 of human c-Fos. Formerly sold by Oncogene Science (Cambridge, MA) as anti-c-Fos [Ab-5] [4–17] rabbit pAb, it is now supplied (catalogue # PC38) by Calbiochem, a division of EMD Biosciences (San Diego CA). It was raised against the synthetic peptide SGFNADYEASSSRC corresponding to amino acids 4–17 of human c-Fos, and is reported by the vendor to recognize the ~55 kDa c-Fos and ~62 kDa v-Fos proteins and not cross-react with the ~39 kDa Jun protein.
The vendor (Roche, Indianapolis, IN) reports that after immunization with digoxigenin, sheep IgG was purified by ion-exchange chromatography, and the specific IgG was isolated by immunosorption. Fab fragments obtained by papain digestion were purified by gel filtration, conjugated to the specific label, alkaline phosphatase, and stabilized in buffer. The antibody (Catalogue # 11093274910) is reported by the vendor to show 100% reactivity with digoxigenin and digoxin, but no cross-reactivity with other steroids, such as human estrogens (e.g., estradiol) or androgens (e.g., testosterone). The antibody binds only brain sections containing bound digoxigenin labeled mRNA probes, as in in situ hybridization histochemistry.
Anti-FG and anti-CtB
This rabbit polyclonal antibody raised against FluoroGold (FG, hydroxystilbamidine) was purchased as antibody-containing serum without preservative (catalogue # AB153) from Chemicon, now Bioscience Research Reagents, a division of Millipore, located Temecula, CA. The vendor states that AB153 also reacts with aminostilbamidine in frozen, 4% PFA fixed tissues. That against CtB was raised in goat against cholera toxin B subunit and sold by List Biological Laboratories, Inc. (Campbell, CA), who report that the greatest dilution of this antibody which forms an immunopreciptin band against a 0.5 mg/ml solution of cholera toxin B is 1:16, which they report is comparable to prior lots of the preparation. In our hands, sections from brains that have not received FG or CtB injections are devoid of reaction product when processed with AB153 and anti-CtB at a range of dilutions and, in brains that have received injections of the tracers, immunostaining is observed only at the injection sites, in retrogradely labeled neurons (and anterogradely labeled axons in the case of CtB) and, occasionally, microglial cells, which, however, are easily distinguished by morphology from labeled neurons.
This rabbit polyclonal antibody (Catalogue # OR-600) was provided by Gramsch Laboratories (Schwabhausen, Germany). According to the vendor, it was raised against a synthetic peptide corresponding to amino acids LENLEAETAPLP at the COOH terminus of μ-opioid receptor subtype myu-MOR-1A. In our hands, the antibody stains rat brain sections in a manner consistent with literature descriptions of the distribution of brain Mor immunoreactivity (e.g., Schulz et al., 1998
). Preabsorption with the cognate peptide abolished immunohistochemical staining.
This rabbit polyclonal antibody provided in 0.01 M phosphate buffered saline, pH 7.4, containing 15 mM sodium azide (Catalogue # N7155) was supplied by the Sigma Chemical Company (Saint Louis, MO). According to the vendor, it was raised against a synthetic peptide corresponding to amino acids 251–270 (GDNDRVFNDLWGKDNVPVILC) of nitric oxide synthase of rat brain origin conjugated to KLH (Riveros-Moreno et al., 1993
), reported by the vendor to be an immunogen sequence highly conserved in human bNos. In our hands, the antibody stains rat brain sections in a manner consistent with literature descriptions of the distribution of brain Nos immunoreactivity (e.g., Rodrigo et al., 1994
This goat polyclonal antibody raised against Phaseolus vulgaris agglutinin (E+L) was purchased (Catalogue # AS-2224) from Vector Laboratories (Burlingame, CA). According to the vendor, this antibody is produced by hyperimmunizing goats with the pure lectin. Following conventional purification steps, specific antibody was isolated by affinity chromatography on lectin-agarose columns and supplied lyophilized in buffered saline. Sections from brains that have not received PHA-L injections are devoid of reaction product when immunoprocessed with AS-2224 at optimal dilutions and, in brains that have received PHA-L injections, immunostaining is observed only at the injection sites and in anterogradely labeled axons.
This rabbit polyclonal antibody was raised against synthetic somatostatin (AGCKNFFWKTFTSC) conjugated to keyhole limpet hemocyanin with carbodiimide linker provided by ImmunoStar (Hudson, WI) as a lyophilized powder (Catalogue # 20067). The vendor reports that somatostatin immunostaining was completely abolished by preadsorption with somatostatin, somatostatin 25 and somatostatin 28, but not substance P, amylin, glucagon, insulin, neuropeptide Y or vasoactive intestinal polypeptide. In our hands, the antibody stains rat brain sections in a manner consistent with literature descriptions of the distribution of brain somatostatin immunoreactivity (e.g., Johansson et al., 1984
Anti-tyrosine hydroxylase (TH)
This mouse monoclonal antibody was raised against tyrosine hydroxylase purified from PC12 cells (Catalogue # MAB318) and purchased from the Bioscience Research Reagents (Chemicon) division of Millipore (Temecula, CA). The antibody is supplied as ascites fluid with 3% BSA and no preservative. According to the vendor, the antibody recognizes an epitope on the outside of the regulatory N-terminus of tyrosine hydroxylase. In a Western blot, the antibody recognizes a protein of approximately 59–63 kDa. It does not react with the following on Western Blots: dopamine-beta-hydroxylase, phenylalanine hydroxylase, tryptophan hydroxylase, dehydropteridine reductase, sepiapterin reductase, or phenethanolamine-N-methyl transferase. In our hands, the anti-TH antibody stains rat brain sections in a manner fully consistent with literature descriptions (e.g., Lindvall and Björklund, 1983
; Hökfelt et al., 1984
Staining for all of the antibodies described in this paper was absent when the relevant primary or secondary antibodies, ABC or PAP reagents were omitted. Immunoreactivities against Fos, Mor, Nos and Som were abolished by preadsorption with cognate amino acid sequences listed above in the sections describing the antibodies. Immunostaining with FG, CtB and PHA-L antibodies was eliminated by preadsorption, respectively, with FG, CtB and PHA-L. TH antigen was unavailable. However, specificity of anti-TH, like that of anti-NOS and anti-Som, was supported by the observed distribution of the immunoreactivity in rat brain sections, which is consistent with reports in the literature (see above).
Maps and digital images
Retrograde and anterograde labeling were plotted in representative frontal sections throughout the CNS (excluding spinal cord) with the 20× objective under brightfield optics (Nikon Optiphot) with the aid of the AccuStage digitizing system and MDPlot software (AccuStage™, Shoreview, MN). Digital micrographs were generated with the Nikon Optiphot light microscope and a QICAM Fast 1394 cooled color 12-bit digital camera (QImaging, Burnaby, BC, Candada) interfaced to Q Capture software (Q Imaging), which saved the image files in TIFF format. The images were sized and subjected to minor adjustments to brightness and contrast with the aid of Adobe Photoshop (version CS2) software. Plates were constructed with Adobe Illustrator (version CS2) software and, upon completion, exported back to TIFF format.