Age-matched, sex-matched adult male and female mice (~25g) were used as subjects. Control mice carried one copy of cre recombinase driven by dopamine transporter regulatory elements (Zhuang et al., 2005
) and one conditional VGLUT2 allele (Hnasko et al., 2010
) [Slc6a3+/cre; Slc17a6+/lox
].Conditional VGLUT2 knockout mice were identical but carried two conditional VGLUT2 alleles [Slc6a3+/cre; Slc17a6lox/lox
].Mice were group housed in a colony maintained with a standard 12-hr light/dark cycle and given food and water ad libitum
. Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals
, as adopted by the NIH, and with approval of UCSF Institutional Animal Care and Use Committee.
Stereotaxic recombinant adeno-associated virus (AAV) injection
Methods were adapted from (Tsai et al., 2009
). Briefly, mice were anesthetized with ketamine/xylazine, placed in a stereotax (Kopf), the skull leveled, small holes drilled, and 1 µl (0.25 µl/min) of AAV5-EF1α-DIO-ChR2-mcherry (~3*1012
genomes/ml) was injected using a Hamilton syringe bilaterally into the VTA (coordinates in mm relative to bregma: −3.25 AP, −4.50 DV, & 0.5 or −0.5 ML). Mice were allowed to recover for at least 3 weeks prior to further procedures.
Mice were perfused with cold PBS followed by 4% PFA, brains were removed, post-fixed overnight, cryoprotected in 30% sucrose, frozen in superchilled isopentane, and 35-µm sections were cut on a cryostat and floated in PBS. Sections were rinsed with PBS containing 0.2% Triton-X-100, blocked 1 h w/ 4% NDS, incubated overnight at 4°C in sheep-anti-TH (Pel-Freze) and rabbit-anti-DsRed (Clontech), both at 1:2000 dilution in blocking solution. Sections were rinsed, incubated 2 hr with Cy2-conjugated anti-sheep and DyLite549-conjugated anti-rabbit secondaries (1:500, Jackson Immunoresearch), rinsed again, mounted on slides, dehydrated through alcohol/xylenes and coverslipped w/ DPX.
Images were collected using a Nikon Eclipse Ti-E motorized inverted microscope equipped with epifluorescece and a PhotometricsCoolsnap HQ2 camera or a Nikon FN1 upright C1si spectral confocal microscope. For quantification of TH/ChR2-mcherry+ neurons in the VTA, confocal images were taken of using a 60x objective. ChR2-mcherry+ neurons were identified and then scored for TH content.
Brain slice preparation
Mice were anesthetized and rapidly decapitated. Dissected brains were transferred to ice-cold ACSF containing the following (in mM): 75 sucrose, 87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 1.25 NaH2PO4, 1 ascorbic acid (saturated with 95% O2 and 5% CO2). Coronal sections of the striatum (200 µM) were cut with a vibratome (VT1200, Leica). Slices were incubated for at least 30 min in a holding chamber containing the following (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.5 CaCl2, 26 NaHCO3, 1.2 NaH2PO4, 11 glucose, 1 ascorbic acid (saturated with 95% O2 and 5% CO2). During recording, slices were superfused (2 ml/min) with this same ACSF at ~32°C but with picrotoxin (100 µṂ to block GABAA receptor-mediated synaptic currents) and without ascorbic acid.
Whole-cell voltage-clamp recordings from MSNs located in the nucleus accumbens (NAc) shell and dorsal striatum (DS) were obtained under visual control on a differential interference contrast, upright microscope with infrared illumination. Recordings were obtained using 3–6 MΩ resistance pipettes backfilled with internal solution containing the following (in mM): 120 CsCH3SO3, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 N(CH2CH3)4Cl, 2.5 Mg-ATP, 0.25 Mg-GTP, pH 7.3. Currents were measured using either an Axopatch 1D or 200A amplifier (2 kHz low-pass Bessel filter) with a DigiData 1440 interface (5 kHz digitization) and pClamp software (Molecular Devices). MSNs, identified by their morphology and hyperpolarized resting membrane potential, were voltage clamped at −70 mV. An optical fiber (200 µm core diameter, 0.2 N.A.) coupled to a diode pumped solid-state 473 nm laser was placed 200 µm from the site of recording, and was used to deliver optical stimulation at a frequency of 0.1 Hz. Stimulus intensity ranged from 1 to 30 mW with a pulse duration of 5 ms. Series resistance (5–20 MΩ) was monitored on-line with a 5 mV hyperpolarizing step (50 ms) given after each stimulus.
To quantify EPSC amplitudes, 6 sweeps were collected from each cell at each light intensity (0, 1, 2, 5, 10 mW for input/output experiment, 30 mW for maximal stimulation and pharmacology experiments). Sweeps were then averaged offline to determine EPSC amplitude at a particular intensity. Light stimulus intensities were presented in a random order and counter balanced across recordings. For AMPA receptor antagonism experiments 10 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) was used, and for DA receptor antagonism experiments 2 µM SCH23390 and 2 µM raclopride were used to respectively block D1 and D2 receptors. Experimenters were blind to the animal’s genotype during data acquisition.
Fast-scan cyclic voltammetry
Methods were adapted from previous work (Stuber et al., 2008
). T-650 carbon fiber microelectrodes (100 – 200 µm in length) were used for detection of dopamine. Electrodes were placed in the NAc shell or dorsal striatum of Slc6a3+/cre; Slc17a6+/lox
mice. The potential applied to the electrode was ramped from −0.4 V to +1.3 V to −0.4 V vs. a Ag/AgCl reference every 100 ms at a rate of 400 V/s and resulting electrochemical data was acquired using custom written software in LabVIEW. Electrochemical data was low-pass filtered at 1 kHz offline. Background subtracted cyclic voltammograms were generated immediately after optical stimulation of the slice, and were characteristic of dopamine (peak oxidation potential 600–700 mV). Current resulting from the oxidation of dopamine (monitored at the peak oxidation potential determined from cyclic voltammograms) were then converted into concentration changes using a calibration factor of 10 nA/µM DA.