Selective Enhancement of Dopamine Release in the Ventral Pallidum of Methamphetamine-Sensitized Mice

Abstract

Drugs of abuse induce sensitization, which is defined as enhanced response to additional drug following a period of withdrawal. Sensitization occurs in both humans and animal models of drug reinforcement and contributes substantially to the addictive nature of drugs of abuse, because it is thought to represent enhanced motivational wanting for drug. The ventral pallidum, a key member of the reward pathway, contributes to behaviors associated with reward, such as sensitization. Dopamine inputs to the ventral pallidum have not been directly characterized. Here we provide anatomical, neurochemical, and behavioral evidence demonstrating that dopamine terminals in the ventral pallidum contribute to reward in mice. We report subregional differences in dopamine release, measured by ex vivo fast-scan cyclic voltammetry: rostral ventral pallidum exhibits increased dopamine release and uptake compared with caudal ventral pallidum, which is correlated with tissue expression of dopaminergic proteins. We then subjected mice to a methamphetamine-sensitization protocol to investigate the contribution of dopaminergic projections to the region in reward related behavior. Methamphetamine-sensitized animals displayed a 508% and 307% increase in baseline dopamine release in the rostral and caudal ventral pallidum, respectively. Augmented dopamine release in the rostral ventral pallidum was significantly correlated with sensitized locomotor activity. Moreover, this presynaptic dopaminergic plasticity occurred only in the ventral pallidum and not in the ventral or dorsal striatum, suggesting that dopamine release in the ventral pallidum may be integrally important to drug-induced sensitization.

Keywords: Dopamine; methamphetamine; sensitization; ventral pallidum; voltammetry.

Figure 1

Dopamine neuroanatomy in the ventral pallidum. Immunohistochemistry in coronal (field view) and sagittal (magnified) slices revealed the expression of dopaminergic proteins in the ventral pallidum (dotted red lines). SV2C expression was used to delineate the ventral pallidum. TH, VMAT2, and DAT expression are shown in the RVP, CVP, dorsal striatum (DSTR), and cortex (CTX). Scale bar = 200 μm.

Figure 2

Identification of ventral pallidal slices. RVP and CVP slices (blue boxes) were chosen as shown in the diagram, using the anterior commissure for reference. The most rostral slices were not measured, given the technical difficulty of targeting ventral pallidum islands in unstained tissue. Additionally, the breadth of the ventral pallidum is shown by synaptic vesicle glycoprotein 2C (SV2C) immunohistochemistry. Dotted lines delineate the border of the ventral pallidum.

Figure 3

Optimization of stimulation parameters. Pulse number (10–60), current (500–700 μA), pulse width (2–4 ms), and collection frequency (5–10 min) were varied systematically, and resultant dopamine release was measured. Optimal stimulation parameters of 60 pulses, 60 Hz, 600 μA, and 2 ms pulse width at 10 min intervals produced the most consistent release without lesioning the slice.

Figure 4

Confirmation of recording site. Slices were carefully chosen by visualization of structures in accordance with the Allen Brain Atlas (A–D, VP shown in gray). Representative recording sites demonstrate identification of ventral pallidum boundaries (dotted red lines) using the anterior commissure (green, A, B), lateral olfactory tubercle (orange, A), and the internal capside (fuschia, B). To further confirm, several slices were electrolytically lesioned at the recording site, and the tissue was cleared via CLARITY. RVP and CVP lesions in transmitted light (E, F) and DAT immunolabeled (green, G, H) representative slices are shown. Lack of significant colocalization with DAT expression is indicative of correct electrode placement. NET and DAT inhibitor nomifensine (10 μM) augmented release in RVP (I, p = 0.003, n = 3, one-way ANOVA with Newman–Keuls multiple comparison test) and CVP (J, p = 0.003, n = 3, one-way ANOVA with Newman–Keuls multiple comparison test). Selective NET inhibition with idazoxan (10 μM) did not significantly increase release in either region (I, J).

Figure 5

Dopamine release in RVP and CVP. Stimulation of RVP produced significantly greater dopamine release compared with stimulation of CVP (A–G, 1.04 μM vs 0.38 μM, respectively; p = 0.011, RVP n = 19, CVP, n = 12, two-tailed t test). Dopamine clearance is slower in CVP than RVP (H, p = 0.002, RVP n = 19, CVP n = 12, two-tailed t test). Representative color plots (A, B) and voltammagrams (E, F) are shown. Current time traces (C, D) are cumulative. Dotted lines indicate stimulation boundaries.

Figure 6

Chronic METH treatment enhances baseline dopamine release in the ventral pallidum. Mice were sensitized to METH or saline (2 mg/kg, IP, 7 days). METH pretreated animals have enhanced baseline DA release. In RVP slices, METH pretreatment augmented baseline dopamine release by 508% (A,C, p = 0.003, n = 6, two-tailed t test). In CVP slices, METH pretreatment increased dopamine release by 307% (B,D, p = 0.017, n = 8, two-tailed t test). RVP release is correlated with motor behavior (E, r² = 0.6449, p = 0.0296, n = 7, linear regression analysis), whereas there is no correlation between baseline dopamine release and motor behavior in the CVP (F, r² = 0.001, p = 0.931, n = 8, linear regression analysis).

Figure 7

METH-sensitization preferentially augments dopamine release in the ventral pallidum. FSCV of dorsal and ventral striatal regions revealed no increase in baseline dopamine release of sensitized animals (A–D). Augmented dopamine release is restricted to the ventral pallidum (E, F). Concentration time traces are averaged for 3–8 mice; dotted lines represent standard error of the mean.