Effects of PPT stimulation on dopamine oxidation current recorded from the striatum of wildtype (WT) and M₅ knockout (KO) mice before and after systemic administration of scopolamine (5 mg/kg, i.p.).

<p>*p<0.05 relative to wildtype control,</p>†<p>p<0.05 relative to respective within-animal control.</p

Striatal dopamine efflux following electrical stimulation of the PPT in wildtype and M₅ knockout mice.

<p>In all cases thick lines represent mean oxidation current corresponding to striatal dopamine efflux across mice, and thin lines represent ± SEM. (A) PPT-evoked striatal dopamine efflux in wildtype (black trace, n = 7) and M₅ knockout (gray trace, n = 7) mice. (B) M₅ receptor contribution to PPT-evoked striatal dopamine efflux in mice. The graph shows the difference in dopamine efflux between wildtype (n = 7) and M₅ knockout (n = 7) mice. (C) Effects of scopolamine (5 mg/kg, i.p.) pre-treatment on PPT-evoked striatal dopamine efflux in 4 of the wildtype mice shown in (A). The black trace shows PPT-evoked dopamine efflux, and the gray trace shows PPT-evoked dopamine efflux following systemic scopolamine. (D) Effects of scopolamine (5 mg/kg, i.p.) pre-treatment on PPT-evoked striatal dopamine efflux in 4 of the M₅ knockout mice shown in (A). The black trace shows PPT-evoked dopamine efflux and the gray trace shows PPT-evoked dopamine efflux following systemic scopolamine.</p

PPT stimulation sites (A) and striatal recording sites (B) used in Experiment 2.

<p>(A) Sagittal sections of the mouse brain showing tip placements of PPT stimulating electrodes in wildtype (open triangles, n = 7) and M₅ knockout (open circles, n = 7) mice. Numbers above individual sections indicate lateral distance in mm from the midline. (B) Coronal sections of the mouse brain showing tip placements of recording electrodes in wildtype (open triangles, n = 7) and M₅ knockout (open circles, n = 7) mice. Numbers above individual sections show distance in mm from bregma. Sections are adapted from the atlas of Paxinos and Franklin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027538#pone.0027538-Paxinos1" target="_blank">[38]</a>.</p

Changes in accumbal dopamine efflux produced by 50 ng intra-VTA morphine or saline in wildtype (+/+) or M₅ knockout (−/−) mice.

<p>In all cases, thick lines represent mean dopamine oxidation current across mice, and thin lines represent ±SEM. (A) Mean change in accumbal dopamine efflux in wildtype mice following 50 ng intra-VTA morphine (black line, n = 4) or 0.3 µl intra-VTA saline (gray line, n = 4). (B) Mean change in accumbal dopamine efflux in M₅ knockout mice following 50 ng intra-VTA morphine (black line, n = 4) or 0.3 µl intra-VTA saline (gray line, n = 4). (C) Effects of pre-treatment with 50 µg intra-VTA scopolamine on accumbal dopamine efflux induced by 50 ng intra-VTA morphine in wildtype mice (black line, n = 6). For comparison, data from (A) showing mean changes in accumbal dopamine efflux induced by 50 ng intra-VTA morphine without any pre-treatment in wildtype mice are reproduced (gray line, n = 4). (D) Effects of pre-treatment with naltrexone (1 mg/kg, i.p.) 5 min prior to 50 ng intra-VTA morphine in wildtype mice (black line, n = 4). For comparison, data from (A) showing mean changes in accumbal dopamine efflux induced by 50 ng intra-VTA morphine without any pre-treatment in wildtype mice are reproduced (gray line, n = 4).</p

VTA injection sites (A) and nucleus accumbens recording sites (B) used in Experiment 1.

<p>In both (A) and (B), open circles show placements from M₅ knockout mice (n = 4) treated with 50 ng intra-VTA morphine, closed circles show placements from M₅ knockout mice treated with 0.3 µl intra-VTA saline (n = 4), open triangles show placements from wildtype mice (n = 4) treated with 50 ng intra-VTA morphine, closed triangles show placements from wildtype mice (n = 4) treated with 0.3 µl intra-VTA saline, stars show placements from wildtype mice (n = 6) pre-treated with 50 µg intra-VTA scopolamine followed by 50 ng intra-VTA morphine, and open squares show placements from wildtype mice (n = 4) pre-treated with 1 mg/kg (i.p.) naltrexone followed by 50 ng intra-VTA morphine. For clarity infusion sites are shown on both left and right side of the brain, but VTA infusions were always made on the left side of the brain. Numbers to the left of or above individual sections show distance in mm from bregma. Sections were adapted from the atlas of Paxinos and Franklin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027538#pone.0027538-Paxinos1" target="_blank">[38]</a>.</p

Diazepam Inhibits Electrically Evoked and Tonic Dopamine Release in the Nucleus Accumbens and Reverses the Effect of Amphetamine

Diazepam is a benzodiazepine receptor agonist with anxiolytic and addictive properties. Although most drugs of abuse increase the level of release of dopamine in the nucleus accumbens, here we show that diazepam not only causes the opposite effect but also prevents amphetamine from enhancing dopamine release. We used 20 min sampling <i>in vivo</i> microdialysis and subsecond fast-scan cyclic voltammetry recordings at carbon-fiber microelectrodes to show that diazepam caused a dose-dependent decrease in the level of tonic and electrically evoked dopamine release in the nucleus accumbens of urethane-anesthetized adult male Swiss mice. In fast-scan cyclic voltammetry assays, dopamine release was evoked by electrical stimulation of the ventral tegmental area. We observed that 2 and 3 mg of diazepam/kg reduced the level of electrically evoked dopamine release, and this effect was reversed by administration of the benzodiazepine receptor antagonist flumazenil in doses of 2.5 and 5 mg/kg, respectively. No significant effects on measures of dopamine re-uptake were observed. Cyclic voltammetry experiments further showed that amphetamine (5 mg/kg, intraperitoneally) caused a significant increase in the level of dopamine release and in the half-life for dopamine re-uptake. Diazepam (2 mg/kg) significantly weakened the effect of amphetamine on dopamine release without affecting dopamine re-uptake. These results suggest that the pharmacological effects of benzodiazepines have a dopaminergic component. In addition, our findings challenge the classic view that all drugs of abuse cause dopamine release in the nucleus accumbens and suggest that benzodiazepines could be useful in the treatment of addiction to other drugs that increase the level of dopamine release, such as cocaine, amphetamines, and nicotine

Monitoring In Vivo Changes in Tonic Extracellular Dopamine Level by Charge-Balancing Multiple Waveform Fast-Scan Cyclic Voltammetry

Dopamine (DA) modulates central neuronal activity through both phasic (second to second) and tonic (minutes to hours) terminal release. Conventional fast-scan cyclic voltammetry (FSCV), in combination with carbon fiber microelectrodes, has been used to measure phasic DA release in vivo by adopting a background subtraction procedure to remove background capacitive currents. However, measuring tonic changes in DA concentrations using conventional FSCV has been difficult because background capacitive currents are inherently unstable over long recording periods. To measure tonic changes in DA concentrations over several hours, we applied a novel charge-balancing multiple waveform FSCV (CBM-FSCV), combined with a dual background subtraction technique, to minimize temporal variations in background capacitive currents. Using this method, in vitro, charge variations from a reference time point were nearly zero for 48 h, whereas with conventional background subtraction, charge variations progressively increased. CBM-FSCV also demonstrated a high selectivity against 3,4-dihydroxyphenylacetic acid and ascorbic acid, two major chemical interferents in the brain, yielding a sensitivity of 85.40 ± 14.30 nA/μM and limit of detection of 5.8 ± 0.9 nM for DA while maintaining selectivity. Recorded in vivo by CBM-FSCV, pharmacological inhibition of DA reuptake (nomifensine) resulted in a 235 ± 60 nM increase in tonic extracellular DA concentrations, while inhibition of DA synthesis (α-methyl-dl-tyrosine) resulted in a 72.5 ± 4.8 nM decrease in DA concentrations over a 2 h period. This study showed that CBM-FSCV may serve as a unique voltammetric technique to monitor relatively slow changes in tonic extracellular DA concentrations in vivo over a prolonged time period