Male and female mice maintained on a C57BL/6 background were used for all studies. All animals were derived from matings of DAT T356+/– parents.

Antibodies

Details of primary and secondary antibodies used in this study are summarized in Supplemental Table 1.

Generation of DAT T356M+/+ mice

Mice were generated by GenOway S.A. The point mutation was inserted into the exon 8 of the mouse Slc6a3 gene and was expressed under the control of the endogenous Slc6a3 promoter. It should be noted that the mouse orthologue of the T356M variant is T355M. The T356M terminology will be used in the manuscript to refer to this mutation to maintain consistency with the nomenclature used in previous studies referring to this variant. A neomycin-positive selection cassette flanked by loxP sites was inserted into intron 7 in a region devoid of experimentally validated regulatory regions and of predicted transcription factor binding sites conserved between mouse and human. Isolated homology fragments were used to generate the targeting vector. PCR validation was used to confirm the recombination over the 3′ homology arm. Southern blot was used to validate recombination over the 3′ and 5′ homology arms. PCR and Southern blot were used to validate recombinase-mediated excision. After linearization to enhance homologous recombination, embryonic stem (ES) cells were electroporated with the targeting vector and subjected to positive selection. The presence of the correct recombination event was validated by PCR and Southern blot. Recombined ES cell clones, derived from black-coated C57BL/6 mice, were injected into blastocysts, derived from an albino C57BL/6 mouse strain, which were then implanted in pseudo-pregnant females to produce chimeric males carrying the combined locus. Chimerism was assessed by coat color marker comparison. Male mice with more than 50% chimerism were bred with C57BL/6 mice ubiquitously expressing the Cre-recombinase to excise the loxP flanked neomycin selection cassette and to generate heterozygous mice carrying the neo-excised point mutant knockin allele. Progeny were genotyped by PCR, and the recombinase-mediated excision event was validated by Southern blot. Heterozygous animals identified by PCR screening and Southern blot were investigated by sequencing analysis to confirm the integration of the mutation.

Chronoamperometry

DA release and reuptake were measured using chronoamperometry in striatal slices. Striatal hemislices (300 μm) from 6- to 10-week-old WT and DAT T356M+/+ mice were prepared with a Vibratome (Leica VT1000S) in an ice-cold oxygenated (95% O 2 /5% CO 2 ) aCSF solution consisting of the following: 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 10 mM dextrose, 26 mM NaHCO 3 , and 0.25 mM ascorbic acid. Slices were then recovered in an oxygenated NMDG-HEPES bath (consisting of the following: 100 mM NMDG, 2.5 mM KCl, 1.2 mM NaH 2 PO 4 , 30 mM NaHCO 3 , 20 mM HEPES, 25 mM glucose, 10 mM MgSO 4 •7H 2 O, 0.5 mM CaCl 2 •2H 2 O, 5 mM L-ascorbic acid, 3 mM sodium pyruvate, 2 mM thiourea, 12 mM N-acetyl-l-cysteine adjusted to pH 7.3–7.4) for 12 minutes at 32–34°C. Slices were then moved to an oxygenated aCSF bath (as above) at 28°C for a minimum of 1 hour prior to recording. Carbon fiber electrodes were advanced angularly into the desired recording site in the striatum so that the tip of the electrode was positioned at a depth of 75–100 μm beneath the tissue surface. DA release was stimulated by a 100 ms 100–200 μA potential pulse delivered by a bipolar electrode while the carbon fiber electrode measured DA release and clearance dynamics. Data were collected with an Axopatch 200B amplifier. Decay time was calculated as time for the current to decay from 80% of peak current to 20% of peak current (t 80 –t 20 ). For these experiments, n = 17 slices for each genotype from 6 animals of each genotype.

Voltammetry

FSCAV was used to measure basal DA levels in the striatum. Mice between the ages of 8 and 16 weeks old were anesthetized with an i.p. injection of urethane (25% [w/v] in sterile saline) at a volume of 7 μL per 1 g body weight. A heating pad from Braintree Scientific was used to maintain mouse body temperature (37°C) throughout the duration of the experiment. Stereotaxic surgery was performed with coordinates determined with reference to bregma, according to Franklin and Paxinos (72). A pseudo Ag/AgCl reference was placed in the contralateral hemisphere. A stainless-steel stimulation electrode (diameter 0.2 mm, MS303/2-A/SPC; Plastics One) was lowered into the medial forebrain bundle. A 50 μm nafion-coated carbon fiber microelectrode (CFM), made as previously described (39), served as the working electrode and was lowered in the striatum. FSCAV was performed using instrumentation and software (WCCV 3.05) developed by Knowmad Technologies LLC. DA presence was confirmed by first collecting FSCV files. A 30-second file was collected and a stimulation applied at 5 seconds (60 Hz biphasic, 360 μA, 120 pulse stimulation, 2 ms per phase) through a linear constant current stimulus isolator (NL800A Neurolog; Digitimer Ltd.) to evoke DA release. The release was measured at the CFM in the striatum by applying a DA triangular waveform (–0.4 V to 1.4 V to -0.4 V scan rate 400 V/s). Once the presence of DA had been confirmed, FSCAV was applied for ambient DA concentration measurements. A DA FSCAV waveform was applied as previously described (39). Signals collected were processed with WCCV 3.05 software using LabVIEW 2009, which included signal deconvolution, filtering, and smoothing. The cyclic voltammogram (CV) at the third scan (following the controlled adsorption period) was extracted to integrate the DA oxidation peak between approximately 0.45 V and 0.9 V. Calibration curves were acquired prior to experiments and plotted as charge, in pC, versus DA concentration. A separate calibration set (n = 5 electrodes) was obtained using both pre- and postcalibrations to determine the factor by which the sensitivity of the electrode changes after the experiment. Each precalibration was then adjusted by this factor and used to calculate in vivo values that were specific to each electrode. Following data collection, a large voltage was applied to the electrode (~10 V for 45 s) to lesion the tissue. Mice were subsequently euthanized via cervical dislocation followed by decapitation, and the brain was removed from the skull and stored in 4% paraformaldehyde in PBS solution. Prior to sectioning, the brain was transferred into a 30% sucrose solution until it was saturated with the medium. The brain was then flash-frozen, sectioned into 20 μm slices mounted onto frosted glass slides, and the lesion identified to confirm the electrode was placed in the striatum.

Western blot

Mice were decapitated under isoflurane anesthesia at 7 to 10 weeks of age. The brain was rapidly dissected and placed in ice-cold sucrose solution consisting of the following: 210 mM sucrose, 20 mM NaCl, 2.5 mM KCl, 1.2 mM NaH 2 PO 4 , 1 mM MgCl 2 , 26 mM NaHCO 3 , and 10 mM dextrose. The striatum was then dissected on ice and snap-frozen. Dissected tissue was homogenized in 200 μL of homogenization buffer (RIPA buffer [Thermo Fisher Scientific], cOmplete protease inhibitor cocktail [MilliporeSigma], phosphatase inhibitor cocktail 3 [MilliporeSigma], 1 mM sodium orthovanadate) on ice. Protein concentrations of all samples were determined by a bicinchoninic acid (BCA) protein assay using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Samples were resolved using NuPAGE Bis-Tris precast gels (Thermo Fisher Scientific, 10% 10-well, NP0301BOX). The Precision Plus Protein Kaleidoscope Prestained Ladder was run alongside samples for protein size reference. Proteins were transferred from gels onto nitrocellulose membranes using the iBlot system (Thermo Fisher Scientific, IB23001). Western blot analysis was performed using antibodies listed in Supplemental Table 1 and visualized using Western Lightning Plus ECL. Adobe Photoshop software was used to crop full blots. Western blots were quantified using the NIH ImageJ gel analysis tool. See complete unedited blots in the supplemental material.

HPLC

Measures of the concentration of biogenic amines were obtained by the Neurochemistry Core Facility at Vanderbilt University. The striatum was collected from animals 7 to 10 weeks old in the same manner as described for Western blots and stored at -80°C. Frozen brain tissue was homogenized using a tissue dismembrator (Misonix XL-2000, Qsonica) in 100–750 μl of 0.1M TCA, which contains 10–2 M sodium acetate, 10–4 M EDTA, and 10.5 % methanol (pH 3.8). Ten microliters of homogenate were used for protein assay. Protein concentration was determined using the BSA Protein Assay Kit (Thermo Scientific). Then samples were spun in a microcentrifuge at 10,000 g for 20 minutes. The supernatant was removed for biogenic monoamines analysis. Biogenic amine concentrations were determined utilizing an Antec Decade II (oxidation: 0.65) electrochemical detector operated at 33°C. Twenty microliter samples of the supernatant were injected using a Water 2707 autosampler onto a Phenomenex Kintex C18 HPLC column (100 × 4.60 mm, 2.6 μm). Biogenic amines were eluted with a mobile phase consisting of 89.5% 0.1M TCA, 10–2 M sodium acetate, 10–4 M EDTA, and 10.5 % methanol (pH 3.8). Solvent was delivered at 0.6 ml/min using a Waters 515 HPLC pump. Using this HPLC solvent, the following biogenic amines eluted in the following order: norepinephrine, epinephrine, DOPAC, DA, 5-HIAA, HVA, 5-HT, and 3-MT. Data acquisition was managed by Empower software (Waters Corp.). Isoproterenol (5 ng/mL) was included in the homogenization buffer for use as an internal standard to quantify the biogenic amines of interest.

Weight measurements

All mice not undergoing experimental manipulation (including behavioral testing) were weighed weekly beginning 7 days after birth (P7). Animals undergoing behavioral testing were excluded from weight measurements.

Behavioral paradigms

All behavioral experiments were performed in the Vanderbilt University Neurobehavioral Core Facility. All behavioral tests were performed at the same time of day (13:00–17:00) using mice 8 to 10 weeks of age. Mice were group housed (3–5 mice/cage) on a 12-hour light/12-hour dark cycle with food and water available ad libitum. Mice were housed with corncob bedding. Mice were transferred to testing rooms followed by a 30-minue acclimation period before the start of each test. Unless otherwise stated, each apparatus used was cleaned with a 10% ethanol solution between each animal or trial to provide a standardized testing environment.

Behavior observations. Mice were placed individually into clean cages (containing bedding only) and recorded using a video camera for 15 minutes. Videos were then scored by observers blinded to genotype for the instances of the following behaviors: digging, grooming, rearing, tail flick, jumping, and climbing.

Elevated zero maze. Anxiety behaviors were assessed using an elevated zero maze (62.5 cm outer diameter, 5 cm path width, 15 cm wall height, Stoelting). Animals were placed in center of the open portion of the maze at the start of each trial. Each trial was recorded by a video camera mounted to the ceiling, and analysis was performed using ANY-maze software (Stoelting). Data analyzed included the percentage of time in closed sections, number of entries into open and closed sections, and distance traveled in the maze.

Marble burying. Cages (189 mm × 297 mm × 128 mm) were prepared by filling clean, empty cages with 4.5 cm of Diamond Soft Bedding (Envigo Teklad 7089). Mice were placed individually into filled cages for a 5-minute acclimation period. Mice were then removed from the cages, and 15 blue marbles (15 mm diameter) cleaned with a 10% ethanol solution were gently placed in the cages. Marbles were evenly spaced in a 3 × 5 configuration in each cage. A photograph was taken of each cage prior to returning mice to the cage and again 30 minutes later when mice were removed from the cage. Photographs of cages before and after testing were compared and scored by 3 independent, blinded observers. A marble was considered buried if more than two-thirds of the marble was covered with bedding at the end of 20 minutes.

Inverted screen. Mice were individually placed on a 7.5 cm × 7.5 cm wire mesh screen that was mounted horizontally on a metal rod and elevated 40 cm above the apparatus floor. The rod was then slowly rotated 180°, ensuring all animals achieved a 4-paw grasp on the wire mesh before the apparatus was fully inverted. Time to fall was measured for each animal on 3 trials. The maximum time allowed was 180 seconds.

Horizontal pole climb. The horizontal climb tests the ability of a mouse to maintain balance on a horizontal rod (cloth tape wrapped, 1.5 cm diameter) suspended between 2 platforms. The pole is suspended 40 cm above the apparatus floor. The mouse was placed on the rod, and its movements were observed for 1 minute. Time to reach either platform or time to fall was measured for 3 trials for each mouse.

Open-field locomotion. Spontaneous locomotor activity in an open field was measured using 27 × 27 × 20.5 chambers (Med Associates) placed within sound-attenuating boxes (64 × 45 × 42 cm) that were light and air controlled. Locomotion was detected by infrared beam disruption by the body of the mouse. Sixteen evenly spaced photocells located 1 cm above the chamber floor measured horizontal movement, while 16 photocells located 4 cm above the chamber floor captured vertical movements (jumping and rearing). Animals were placed in the activity chambers, and their activity was recorded for 60 minutes. Total distance traveled was analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (GraphPad Prism 8.0.2). Data from this test were also used to measure anxiety-like behaviors. Thigmotaxis, the tendency of the mice to remain close to the walls of the open field, can be used as an index of anxiety. Percentage of time mice spent in the periphery of the open field (~52% of area) versus the center was determined.

Rotarod. Rotarod was used to assess the balance and motor coordination of the mice on a rotating rod as well as the propensity for acquisition of repetitive motor routines (73). The rotarod apparatus consists of a rubber-covered cylindrical rod (3 cm in diameter) suspended 30 cm above the apparatus floor and separated into 5 equal compartments by plastic dividers with a radius of 15 cm. The starting rotation rate was 4 rpm and increased steadily to 40 rpm over a period of 4 minutes. Mice were placed on the rod, and the timer was started. The time to fall or to complete 2 passive rotations was measured for each animal with a maximum time of 5 minutes. Animals completed 3 trials each day for 3 days. The time to fall or rotate for each day was calculated as an average over the time to fall or rotate for the 3 trials completed in a given day.

Tube test. The tube test was used to assess social dominance (a test for normal social behavior). One DAT T356M+/+ knockin mouse and one WT mouse were placed head first at opposite ends of a clear plastic tube (2.5 cm inner diameter, 13 cm length) and released simultaneously. Each trial ended when 1 mouse completely retreated from the tube or after 3 minutes. The mouse remaining in the tube was designated the winner and the retreating mouse was designated the loser. If neither animal retreated from the tube after 3 minutes, the trial was designated a draw. Each pair of animals participated in 2 trials, starting on alternating ends of the tube on each trial. A χ2 1-sample analysis was used to determine whether the scores of the mutant mice were significantly different from an outcome expected by chance (50:50 win-lose).

Three-chamber test. The 3-chamber test was used to assess social preference (a test for normal social behavior in mice). The apparatus consists of a 60 × 42 × 22 cm box divided into 3 equal-sized compartments. Removable doors initially confined the test mouse to the center chamber, where the mouse habituated for 5 minutes. After 5 minutes, the doors were removed and the mouse was allowed to freely explore the apparatus. A stimulus mouse was then introduced in an inverted wire cup in one side chamber, while a clean, empty, inverted pencil cup was introduced in the opposite chamber. The stimulus mouse was always an age- and sex-matched WT mouse, either known to the subject mouse (familiar social stimulus) or unknown to the subject mouse (novel social stimulus). The subject mouse was then allowed to explore all 3 chambers freely for 10 minutes. A research assistant blinded to genotype then coded videos for time spent in each chamber and time spent interacting with the stimulus mouse or the empty inverted wire cup.

ACT-01 reversal of hyperactivity. Mice were allowed to habituate to the test room for 15 minutes prior to treatment. Mice naive to the open-field apparatus were treated with either 50 mg/kg of ACT-01 or with vehicle via i.p. injection 30 minutes prior to testing. The same protocol for measuring spontaneous locomotor activity was then followed as is described under the Open-field locomotion section. ACT-01 was provided in-house.

GBR12909 reversal of hyperactivity. Mice were allowed to habituate to the test room for 15 minutes prior to treatment. Mice naive to the open-field apparatus were treated with either 20 mg/kg of GBR12909 or with vehicle via i.p. injection 30 minutes prior to testing. The same protocol for measuring spontaneous locomotor activity was then followed as is described under the Open-field locomotion section.

Statistics

All statistical analyses were performed using GraphPad Prism software (version 8.0.2). Statistical methods are indicated in the figure legends and Results. Data are presented as mean ± SEM. Differences are considered statistically significant at P < 0.05. Unpaired 2-tailed Student’s t test was used for 2-group comparisons, unless stated otherwise. Either 1- or 2-way ANOVA with Šidák’s test was used for multiple comparisons.

Study approval

All behavioral, biochemical, and chronoamperometry experiments were performed under a protocol approved by the Vanderbilt University Animal Care and Use Committee. For FSCAV, a protocol approved by the Institutional Animal Care and Use Committees of the University of South Carolina was followed.