Salahpour et al. 10.1073/pnas.0707646105. |
Fig. 7. Coronal sections through the telencephalon, diencephalon, and mesencephalon of DAT-tg mice showing immunoperoxidase labeling for DAT. The distribution of labeled fibers and cellular profiles closely matches prior descriptions of DAT staining in rats (1, 2), including the following. (A) A high density of immunoreactivity in the rostral pole of the NAc, modest fibers in the superficial layers of the AC (also shown in D), and conspicuous absence of labeled axons in the PL and IL divisions of the prefrontal cortex. (B) Dense labeling in the CPu (also shown in C and F), NAc, and OT. (C and D) Moderate staining in the LS and VP. (E) Sparse immunoreactivity in the LHb and no evident labeling in the Hipp or Thal. (F) A moderate density of labeled axons in the EP and in the amygdaloid I nuclei, with fewer axons in the BLA and diffuse immunoreactivity in the CeA. (G) Fibers passing in the mfb and absence of immunoreactivity from all regions of the hypothalamus, except the ME. (H) Extensive labeling throughout the SNc and VTA. (I) Immunolabeling in perikarya and dendrites in the SNc (arrows in H and I point to the same labeled cell). 3V, third ventricle; AC, anterior cingulate cortex; ac, anterior commissure; BLA, basolateral nucleus of the amygdala; cc, corpus callosum; CeA, central nucleus of the amygdala; cp, cerebral peduncle; CPu, caudate putamen; EP, entopeduncular nucleus; f, fornix; fm, forceps minor; Hipp, hippocampus; I, intercalated nuclei of the amygdala; IL, infralimbic cortex; IP, interpeduncular nucleus; LHb, lateral habenula; LS, lateral septum; LV, lateral ventricle; ME, median eminence; mfb, medial forebrain bundle; ml, medial lemniscus; mt, mammillothalamic tract; NAc, nucleus accumbens; OT, olfactory tubercle; PL, prelimbic cortex; SNc, substantia nigra zona compacta; Thal, thalamus; VP, ventral pallidum; VTA, ventral tegmental area. (Scale bar in I represents 1,600 mm in B and C, 800 mm in A and D-H, and 100 mm in I.)
1. Ciliax BJ, Heilman C, Demchyshyn LL, Pristupa ZB, Ince E, Hersch SM, Niznik HB, Levey AI (1995) J Neurosci 15:1714-1723.
2. Freed C, Revay R, Vaughan RA, Kriek E, Grant S, Uhl GR, Kuhar MJ (1995) J Comp Neurol 359:340-349.
Fig. 8. Double immunofluorescence labeling of dopaminergic neurons from WT (A-C and G-I) and DAT-tg (D-F and J-L) mice in the midbrain (A-F) and the striatum (G-L) with anti-DAT antibody (green) and anti-TH antibody (red). There is substantial colocalization of DAT and TH immunoreactivity in the midbrain neurons of both WT (A-C) and DAT-tg (D-F) mice. The colocalization of DAT and TH is even more pronounced in the striatal projections of dopaminergic neurons (G-L).
SI Text
Binding 3[H]WIN 35428.
[3H]WIN 35,428 (83 Ci/mmol) was obtained from PerkinElmer Life Sciences. Nonradioactive WIN 35,428 was obtained from Sigma-Aldrich. Striatal tissues of wild-type and DAT-tg mice where homogenized, by using a Teflon glass homogenizer, in 2 ml of 30 mM sodium phosphate buffer (pH 7.9) containing 0.32 M sucrose (1). The homogenate was centrifuged at 1,000 rpm for 10 min at 4°C to remove tissue debris and nuclei, and the resulting supernatant was centrifuged two times at 18,000 rpm for 20 min at 4°C. The final pellet was suspended in the same buffer, and proteins were quantified by using a Bradford method (Bio-Rad Protein Assay). For saturation experiments, membrane samples (50 ml; 0.5 mg/ml final protein concentration) were incubated with increasing doses of [3H]WIN 35,428 (50 ml; range 1.25-40 nM) at room temperature for 2 h in a total volume of 150 ml of 30 mM sodium phosphate buffer (pH 7.9). Nonspecific binding was measured by using nonradioactive WIN 35,428 (40 mM) in parallel assay tubes and was subtracted from total binding to obtain specific [3H]WIN 35,428 binding. The incubation was terminated by rapid filtration over Brandel GF/C glass fiber filters that were presoaked for at least 30 min in 0.5% polyethylenimine. The filters were washed four times with ice-cold 5 mM sodium phosphate buffer (pH 7.9) and incubated overnight in 10 ml of high flash point scintillation mixture (Lefko-Fluor) before their radioactivity content was counted by a liquid scintillation counter.Histology. Light microscopic immunohistochemistry.
Fig. 2 A and B: Mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and perfused transcardially with saline (0.9% NaCl) followed by 4% paraformaldehyde buffer (PFA) (pH 7.0). Brains were fixed for 4 h in 4% PFA and then immersed for 1-2 days in 30% (wt/vol) sucrose at 4°C before sectioning. The brains were frozen in OCT compound over dry ice, and tissue sections (40 mm) were prepared with a cryostat and stored in PBS solution at 4°C before histochemistry. Free-floating sections were immunostained by a 1:1,000 dilution of rat monoclonal anti-DAT antibody (Chemicon MAB369) directed against the N terminus of the DAT protein (2), followed by biotinylated anti-rat IgG as a secondary antibody. Sections were stained with VectaStain ABC peroxidase system (Vector Laboratories pk-4004). Mounted sections were scanned with a Canon 9950F scanner. For double Immunofluorescence labeling (SI Fig. 8), sections were immunostained with a 1:500 dilution of rat monoclonal anti-DAT antibody (Chemicon MAB369) and 1:500 dilution of rabbit TH antibody (Chemicon AB152), followed by labeling with secondary antibodies, a goat-anti-rat Alexa Fluo 488 (Invitrogen A11006, 1:2,000 dilution) and a goat-anti-rabbit Alexa Fluo 568 (Invitrogen A11011, 1:10,000 dilution). Mounted sections were imaged by using the Zeiss AxioVert 200M microscope with appropriate filter sets.Electron microscopic immunohistochemistry.
Mice were anesthetized with 60 mg/kg i.p. pentobarbital. Six mice (three per group) were then treated with diethyl-dithiocarbamate (DEDTC) to chelate zinc, a common source of spurious silver labeling (3). The remaining two mice did not receive DEDTC to ensure that the presence of this chemical did not alter the subcellular distribution of DAT, a supposition that was confirmed by ultrastructural examination. Mice were then perfused through the aorta with heparin saline, followed by 3.75% acrolein in 2% paraformaldehyde and then 2% paraformaldehyde as described previously (4). Brains were postfixed in 2% paraformaldehyde and then sectioned on a vibratome at 50 mm.Sections were treated with 1% sodium borohydride to reduce excess aldehydes (5), rinsed in buffer, and then placed in blocking solution to minimize nonspecific antibody binding: 3% normal goat serum, 1% BSA in 0.1 M TBS (pH 7.6). To enhance antibody penetration, the blocking solution contained Triton X-100 at 0.2% for light microscopy or 0.04% for electron microscopy. Sections were treated overnight with the rat anti-DAT antibody diluted 1:1,000 in blocking solution.
Some of the tissue sections were processed for light microscopic immunoperoxidase labeling (Fig. 2 C and D and SI Fig. 7) as described above. Most were prepared by a preembedding immunogold-silver method that affords optimal subcellular resolution (6, 7). Tissue was rinsed after primary antibody incubation and then placed in a secondary blocking solution containing 0.8% BSA and 0.1% fish gelatin in 0.01 M PBS (pH 7.4). Sections were then placed overnight in 1-nm gold-conjugated goat anti-rat IgG (Amersham Pharmacia, GE Healthcare) diluted 1:50. Sections were rinsed extensively in PBS, postfixed in 2% glutaraldehyde, and then rinsed again in PBS followed by 0.2 M sodium citrate buffer (pH 7.4). The size of bound gold particles was then enhanced by a 5- to 6-min exposure to silver solution (Amersham Pharmacia).
Sections were then prepared for electron microscopy by using standard methods (4), including postfixation in 2% osmium tetroxide, dehydration in graded ethanols and propylene oxide, and then plastic embedding using epoxy resin (EM-bed 812; Electron Microscopy Sciences). Ultrathin sections through the dorsal striatum were cut at 60 nm in serial order, collected on copper mesh grids, and counterstained with uranyl acetate and lead citrate. Sections were then examined on an FEI Morgagni transmission electron microscope and photographed by using an XP-60 digital camera (Advanced Microscopy Techniques). Micrographic images were adjusted in Adobe Photoshop for optimal brightness and contrast.
Statistical Methods for the Ultrastructural Analysis of Subcellular DAT Localization.
The primary variable of interest was the proportion of immunogold-silver particles for DAT that were expressed on the plasma membrane versus the total number of particles on the membrane and in the cytoplasm. Two models were implemented to detect a difference in this proportion between the DAT-tg and WT mice. The main analysis was based on a generalized linear mixed model. In this analysis, a logistic regression model was used in which group was treated as a fixed effect and animal was treated as a random effect. The analyses were implemented in SAS PROC GLIMMIX (version 9.0), where the degrees of freedom method was Kenward-Roger. A secondary analysis was used to confirm the robustness of the main analysis. In the second analysis, the average of the arc-sin square-root-transformed percentage (i.e., proportion on the membrane) was calculated for each individual animal. Then an independent samples t test (with 6 d.f.) was used to compare the transformed proportion difference between the DAT-tg and WT mice. The inference from this secondary analysis confirmed that of the primary generalized linear mixed model. All statistical tests were two-sided and were conducted with an alpha level = 0.05.Synaptic Plasma Membrane Fraction (SPM) of Dissected Striatum.
All steps were performed at 4°C in the presence of protease inhibitors (Boehringer Mannheim). Briefly, dissected striata were homogenized in 9 volumes of 4 mM Hepes (pH 7.4)/0.32 M sucrose buffer. The nuclear fraction was removed with a 900 ´ g centrifugation to give the total membrane fraction. Membranes were washed, lysed in a hypotonic solution (water plus protease inhibitors), and layered on a 4 mM Hepes discontinuous sucrose cushions of 0.8, 1.0, and 1.2 M sucrose. Samples were centrifuged in a swinging bucket rotor at 200,000 ´ g for 2 h, and the synaptic plasma membrane fraction at the interphase of 1.0 and 1.2 M sucrose was removed, returned to 0.32 M sucrose, and pelleted by centrifugation at 200,000 ´ g for 30 min. Pellets were resuspended in RIPA buffer and used for Western blot analysis as described in Materials and Methods.Fast-Scan Cyclic Voltametry (FSCV).
Cylindrical carbon fiber microelectrodes were constructed from Thornel T-650 carbon fibers (6-mm diameter; Amoco) sealed in glass capillaries (A-M Systems). The fiber was then cut to a length of 25-50 mm relative to the glass fiber seal. The electrode was backfilled with 4 M KC2H3O2 and 150 M KCl, and electrical contact was made with a stainless steel wire. A silver/silver-chloride (Ag/AgCl) reference electrode was used. FSCV was performed with locally written LabView software and a custom-designed potentiostat (UEI). A triangle waveform (-0.4 V to 1.0 V vs. Ag/AgCl, 600 V/s) was repeated at 60 Hz. The current was low-pass-filtered at 2 KHz. Cyclic voltammograms were background-subtracted with the first 10 cyclic voltammograms in a file and were plotted in false color or as current vs. time.Animals were anesthetized with ethyl ether and decapitated, and the brain was removed. It was rapidly dissected and placed in ice-cold aCSF saturated with 95% O2/5% CO2. The cerebellum and brainstem were removed with a razor blade, and the brain was mounted on a Teflon chuck such that the optical tracts were facing upward. Coronal brain slices (300 mm) were taken with a vibrating microtome (Model NVSL, World Precision Instruments). Slices were placed in an oxygenated slice chamber at 37°C for measurement. Brain slices not being used remained in ice-cold oxygenated aCSF. Dopamine release was evoked by a single biphasic current pulse (2 ms each phase) delivered through a bipolar tungsten stimulating microelectrode (FHC). The pulse (350 mA) was computer-generated, constant-current, and optically isolated (NL800, Neurolog Medical Systems) from the carbon-fiber electrode. The stimulating electrode was placed on the surface of the brain slice, and the carbon fiber microelectrode was placed 50-100 mm away at a depth of 50-75 mm.
Data analysis.
Electrodes were pre- and/or post-calibrated in a flow cell. The electrode response to a bolus of buffer containing 2 mM dopamine was used to calibrate the voltammetric signals in concentration units. The clearance of stimulated dopamine was fit to a Michaelis-Menten model using Simplex optimization with locally written software. In the fitting, the Km was fixed to literature values (8), and the Vmax was determined. To obtain an estimate of Km in both WT and WT-Tg, the inhibition of dopamine uptake by cocaine was evaluated at different doses. Because cocaine is a competitive uptake inhibitor, it alters the apparent Km value (KM)app = KM + KM/KI [I] where Km is the Michaelis-Menten constant in the absence of drug and [I] is the concentration of cocaine. Thus, a plot of (KM)app versus cocaine concentration yields the Km value as the intercept. This KM value was compared to for the two types of mice and found to be statistically equivalent. Significance was determined by using Student's t test (99% confidence interval, P < 0.05).Conditioned Place Preference (CPP).
Three-compartment commercially available place preference chambers placed into sound-attenuating cubicles were used. The time spent in each of the three compartments was recorded for 30 min. The chambers were controlled by an appropriate interface, and the data were collected by a PC (all equipment were from Med Associates). Each of three distinct compartments could be separated by a manual guillotine door, and each was illuminated with a light source of adjustable intensity. The central compartment was 7.2 cm long, 12.7 cm deep, and 12.7 cm wide, with gray walls and plastic floor. The two flanking choice compartments were 16.8 cm long, 12.7 cm deep, and 12.7 cm wide. One choice compartment was black with a stainless steel rod floor, and the other compartment was white with a stainless steel mesh floor. Each choice compartment also contained a removable, stainless steel waste pan, to which a small amount of corncob bedding was added.The experimental session began with a pretest session in which the mice's initial preference for different compartments was determined. During the pretest phase of the experiment, mice were placed into the central gray compartment and the guillotine doors were removed, so that both black and white choice compartments were accessible. Mice that spent more time in the central compartment than in any of the choice compartments were discarded. The mice were then randomized in a nonbiased fashion so that the mean group difference in time spent in the drug-associated chamber vs. the saline-associated chamber was close to zero. In the conditioning phase, mice were treated with amphetamine at the designated doses on days 1, 3, and 5 and saline on days 2, 4, and 6 of the experiment. After amphetamine or saline administration, mice were confined to the compartment designated as drug-associated or saline-associated for 30 min. In the postconditioning test, mice were handled on the test day in the same manner as on the preconditioning day and received no drug or saline administrations. With the chamber doors open, the mouse movements were automatically recorded for a total of 30 min. The test data are shown as the mean group difference between the time spent in the drug-associated chamber and the saline-associated chamber.
Evaluation of Operant Responding for Sweet Liquid Food Reward in Mice.
The procedure was adopted with modifications from ref. 9. Single housed mice were tested during the light phase of the diurnal cycle for 2-h sessions seven times a week. Experimental chambers (~23 ´ 12 ´ 19 cm) were equipped with a house light, ventilator fan, and two levers with cue lights that were located on two sides of the liquid dipper with a 17-ml cup. All equipment was obtained from Med Associates. The liquid food reward was represented by Eagle brand Borden condensed milk (Eagle Family Foods) diluted in water (396 g in 2 liters).In the first phase of operant testing, animals learned to press a lever for food reward during daily 2-h sessions. Before the first session, mice were deprived of food for 20 h, which resulted in a 15% mean reduction in body weight. Each session consisted of maximum of 100 trials, with an intertrial interval ranging from 5 s to 3 min. Each trial started with the illumination of the house light, which remained lit for the duration of the session. At the beginning of each trial, a cue lamp above the active lever was illuminated. If the lever was not pressed after 12 s, the cue lamp was extinguished and liquid food was delivered simultaneously. The goal of training was to condition the mice to associate food reward with the lever. After any response on an active lever the adjacent cue light was illuminated and the dipper with liquid food was immediately raised into the chamber. The dipper cup was released 10 s after the first head entry into the dipper. After the session, mice were given 3 g each of mouse chow in their home cage. The procedure was repeated until a minimum of 40 rewards consumed during a 2-h session. Thereafter, mouse chow was available ad libitum in the home cage. During subsequent sessions, liquid food was available under an FR1 schedule of reinforcement. The session was terminated after 100 reinforcers were delivered, or after 2 h, whichever occurred first.
The criteria for acquisition were stable daily responding (within 20% across two consecutive sessions) and a minimum of 30 responses per session. When water was substituted for liquid food in five subsequent sessions, responding extinguished rapidly. Liquid food and water were available alternately over the subsequent six sessions, resulting in greater operant responding behavior when liquid food was available than when water was available. Thereafter serial dilutions of liquid food (Stock, 1/2, 1/4, 1/8, 1/16, 1/32) were made available in a pseudorandom order over the next six sessions. Lever-pressing behavior was related to the concentration of liquid food as an inverted U-shaped function. Data are shown as mean number of lever presses during the first 15 min of the testing session.
1. Reith ME, Coffey LL (1994) Neurosci Methods 51(1):31-38.
2. Ciliax BJ, Heilman C, Demchyshyn LL, Pristupa ZB, Ince E, Hersch SM, Niznik HB, Levey AI (1995)) J Neurosci 15:1714-1723.
3. Veznedaroglu E, Milner TA (1992) Microsc Res Technol 23:100-101.
4. Sesack SR, Hawrylak VA, Matus C, Guido MA, Levey AI (1998) J Neurosci 18:2697-708.
5. Leranth C, Pickel VM (1989) Neuroanat Tract Tracing 2:129-172.
6. Chan J, Aoki C, Pickel VM (1990) J Neurosci Methods 33:113-127.
7. Hersch SM, Yi H, Heilman CJ, Edwards RH, Levey AI (1997) J Comp Neurol 388:211-227.
8. Jones SR, Gainetdinov RR, Jaber M, Giros B, Wightman RM, Caron MG (1998) Proc Natl Acad Sci USA 95: 4029-4034.
9. Caine SB, Negus SS, Mello NK (1999) Psychopharmacology (Berlin) 147, 22-24.