Figure 4.

RMTg GABA neurons modulate nucleus accumbens’ dopamine levels. (A) Adeno-associated virus (AAV) carrying hM3 DREADDs and KORD was targeted to RMTg GABAergic neurons, whereas carbon-fiber microelectrodes were implanted in the nucleus accumbens (NAc) of vGat-cre mice (n = 3) to measure dopamine transients using fast-scan cyclic voltammetry (FSCV) in freely behaving mice. (B) As a positive control, microelectrode-implanted mice received a 3 mg/kg intraperitoneal (i.p.) injection of D-amphetamine, which resulted in an increase in dopamine transients detected in the NAc. (C) To detect transients, a triangle waveform (−0.4 to +1.1 to −0.4 V vs Ag/AgCl, at 400 V/second) was applied to the carbon fiber electrode every 100 ms. During this time, 1000 samples were taken of the resulting current, and the relationship between the sample number and applied voltage is depicted in the left panel. The right panel shows the background current produced by the applied triangle waveform voltage. The voltage associated with the 300th and the 750th sample is shown at the top of the right graph. (D) Probes were placed into known concentrations of dopamine solution, and the voltage applied to calibrate the probes. Intensity plots were then generated to visualize the resulting current/voltage changes. The left colored panel represents the set of all background-subtracted cyclic voltammograms recorded over a 20-second interval. The right panel shows the individual cyclic voltammogram from the 10-second time point of this set, displaying the expected oxidation peak at +0.65 V and reduction trough at ?0.2 V. (E) Dopamine transients recorded in vivo were compared against voltammograms collected during the calibration. A principal component regression method was used to remove electrode drift and quantitatively separate dopamine transients from noise. Only voltammograms with an r² > 0.30 were considered dopamine. (F) One mechanism through which RMTg neurons could mediate morphine analgesia is by inducing dopamine release by VTA neurons. (G) Conversely, excitation of RMTg GABAergic neurons could decrease dopamine release by VTA neurons. (H) Inhibition of RMTg GABAergic neurons by SB increased dopamine transients in the NAc, (I) whereas activation by CNO significantly decreased dopamine transients (dotted blue line depicts mean value over the indicated 20-minute period, *P < 0.05, the Friedman test for one-way repeated-measures analysis of variance by ranks). RMTg, rostromedial tegmental nucleus; VTA, ventral tegmental area.