2.3. Ex vivo slice preparation and fast-scan cyclic voltammetry

Mice were anesthetized with isoflurane and rapidly decapitated. Brains were extracted and immersed in ice-cold, carbogen-saturated (95% O₂/5% CO₂), cutting ACSF containing the following (in mM): Sucrose (194), NaCl (30), KCl (4.5), NaHCO₃ (26), NaH₂PO₄ (1.2), dextrose (10), and MgCl₂ (1). Coronal sections (300 μm) spanning the striatum were prepared as previously described (John and Jones, 2007) and incubated for 1 h before experiments in carbogen-saturated voltammetry recording ACSF (pH 7.4) containing (in mM): NaCl (126), KCl (2.5), NaHCO₃ (25), NaH₂PO₄ (1.2), dextrose (10), HEPES (20), CaCl₂ (2.4), MgCl₂ (1.2), and L-ascorbic acid (0.4). Nr4a1-eGFP fluorescence was observed using a Stereo-Discovery V8 microscope with a GFP filter set (Zeiss) and an X-cite 120 fluorescence illuminator (Lumen Dynamics). One millisecond, monophasic electrical pulses were generated with a DS3 Constant Current Stimulator (Digitimer) and delivered through a twisted, bipolar, stainless steel stimulating electrode (Plastics One) placed 300 μm from the intended recording sites as shown in Fig. 2A. The distance between the two poles of the stimulating electrode was adjusted to 250 μm.

Dopamine release differs between striosomes and matrix in a region-dependent manner. A, Diagram of experimental configuration with an Nr4a1-eGFP slice. B, Approximate recording sites. Areas where striosome dopamine was more than or less than the corresponding matrix are represented by blue and red markers, respectively. Green markers represent no difference. C, Average dopamine transients for the striosome and matrix compartments in the dorsal striatum with representative CVs (inset). D, Corresponding color plots for dorsal striatum compartments depicting the voltammetric data with time on the X-axis, applied scan potential (E_app) on the Y-axis, and background-subtracted faradaic current on the Z-axis in pseudocolor. E, Calibrated data for individual dorsal striatum striosome-matrix pairs and summary bar graph. F, Dopamine transient decay rates (Tau) do not differ between compartments in the dorsal striatum. G, Input-Output curves examining the effect of stimulation intensity on dopamine release in the dorsal striatum. H, Average dopamine responses for the striosome and matrix compartments in the ventral striatum with representative CVs (inset). I, Corresponding color plots for ventral striatum compartments depicting the voltammetric data with time on the X-axis, applied scan potential (E_app) on the Y-axis, and background-subtracted faradaic current on the Z-axis in pseudocolor. J, Calibrated data for individual ventral striatum striosome-matrix pairs and summary bar graph. K, Dopamine transient decay rates (Tau) do not differ between compartments in the ventral striatum. L, Input-Output curves examining the effect of stimulation intensity on dopamine release in the ventral striatum. *p < 0.05, **p < 0.01. Shaded areas and error bars represent the SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Carbon fiber electrodes were made as previously described (Crowley et al., 2014) and cut to 80–120 μm. The carbon fiber electrode potential was linearly scanned as a triangle waveform from −0.4 to 1.2 and back to −0.4 V at 400 V/s. Cyclic voltammograms were collected at 10 Hz using a Chem-Clamp (Dagan Corporation) and DEMON Voltammetry and Analysis software (Yorgason et al., 2011). Dopamine release was evoked by single electrical stimulations delivered every 3 min (5 min for cocaine experiments). For experiments comparing dopamine release between compartments, the carbon fiber was placed in the matrix or striosome compartment 300 μm from the stimulating electrode. The stimulation intensity used for each experiment was selected to yield a transient peak approximately 40%–60% of the maximum transient peak (as determined from a preliminary input-output curve for each slice once stable transients were obtained but before baseline measurements) and ranged from 100 to 300 μA. Once five consecutive stable responses were collected (<10% variation in transient peak), experiments would begin. Four to five baseline measurements in the first compartment were collected and then the carbon fiber electrode was moved to the complementary compartment at the same distance from the stimulating electrode. Another four or five responses were collected in the second compartment at the same stimulation intensity as the first compartment. The order of starting compartment was counterbalanced between slices for all experiments. Dopamine transient decay rates, tau, were calculated from an exponential fit encompassing the dopamine transient peak and return to baseline with the data analysis module in DEMON. For experiments examining drug effects between compartments, data were normalized to their respective baseline/pre-drug periods. For modeling of dopamine transient kinetics (V_max and apparent K_m) in the cocaine experiments, a Michaelis-Menten-based kinetic modeling module (based on Wu et al. (2001)) in DEMON was used. Briefly, dopamine release [DAp] and dopamine affinity for the transporter (K_m; set to 0.16 μM) were held constant for the baseline determination of maximal uptake rates (V_max) for each experiment. Then following cocaine wash on, the obtained V_max for each slice was held constant and K_m was varied to fit the empirically obtained dopamine transients. The resultant K_m value reflects the affinity of dopamine for the transporter in the presence of cocaine (i.e. the apparent K_m). Extracellular dopamine concentrations were determined by post hoc calibrations against a 1 μM dopamine solution.