Amphetamine distorts stimulation-dependent dopamine overflow: effects on D2 autoreceptors, transporters, and synaptic vesicle stores

Abstract

Amphetamine (AMPH) is known to raise extracellular dopamine (DA) levels by inducing stimulation-independent DA efflux via reverse transport through the DA transporter and by inhibiting DA re-uptake. In contrast, recent studies indicate that AMPH decreases stimulation-dependent vesicular DA release. One candidate mechanism for this effect is the AMPH-mediated redistribution of DA from vesicles to the cytosol. In addition, the inhibition of stimulation-dependent release may occur because of D2 autoreceptor activation by DA that is released via reverse transport. We used the D2 receptor antagonist sulpiride and mice lacking the D2 receptor to address this issue. To evaluate carefully AMPH effects on release and uptake, we recorded stimulated DA overflow in striatal slices by using continuous amperometry and cyclic voltammetry. Recordings were fit by a random walk simulation of DA diffusion, including uptake with Michaelis-Menten kinetics, that provided estimates of DA concentration and uptake parameters. AMPH (10 microm) promoted the overflow of synaptically released DA by decreasing the apparent affinity for DA uptake (K(m) increase from 0.8 to 32 microm). The amount of DA released per pulse, however, was decreased by 82%. This release inhibition was prevented partly by superfusion with sulpiride (47% inhibition) and was reduced in D2 mutant mice (23% inhibition). When D2 autoreceptor activation was minimal, the combined effects of AMPH on DA release and uptake resulted in an enhanced overflow of exocytically released DA. Such enhancement of stimulation-dependent DA overflow may occur under conditions of low D2 receptor activity or expression, for example as a result of AMPH sensitization.

Fig. 1.

DA overflow in response to single-pulse stimulation, recorded with the same electrode at the same site by using CV and amperometry. a, The recording area in the mouse rostral, striatal slices is indicated by the stippledarea. aca, Anterior commissure, anterior;cc, corpus callosum; cp, caudate putamen.b, CV subtraction voltammograms for a calibration in 5 μm DA (top trace) and for the peak of the DA signal recording in c (bottom trace). Calibration of the electrode before and after the recording is provided for identification of the measured substance and conversion of the current into DA concentration. c, For CV, a triangular voltage wave was applied at 10 Hz. The current trace was sampled at the voltage that yielded the maximal oxidation current for DA (seeb). In this example, the time at signal peak was 180 msec, and the time at half-height (t_1/2) was 490 msec.d, For amperometry, a constant voltage of +400 mV was applied. The time at signal peak was 30 msec, and thet_1/2 was 225 msec. The thin lines in c and d are CV and amperometry simulations, respectively.

Fig. 2.

Simulated changes of signal amplitudes for increased initial DA concentration (a) and decreased apparent affinity of uptake (b). Shown are experimental changes of recorded signal peak amplitudes in response to the uptake blocker nomifensine (c).a, Normalized signal peak amplitude is plotted versus initial DA concentration for CV (white circles) and amperometry (black triangles) simulations (amplitude = 1 for 1 μm DA).V_max was 4.9 μm/sec;K_m was 0.8 μm.b, Change in simulated signal peak amplitude for increased K_m (amplitude = 1;K_m = 1 μm, with initial DA concentration of 2.9 μm andV_max of 4.9 μm/sec).c, Effects of the uptake blocker nomifensine (10 μm) on peak amplitudes of stimulated DA overflow recorded with CV (white circles; n = 5) and amperometry (black triangles; n = 5). Normalized peak amplitudes (average ± SEM) are plotted versus the time of superfusion with nomifensine. DA overflow was stimulated once per minute.

Fig. 3.

Simulation of the nomifensine effect.a, Examples of CV recordings (top) and amperometry recordings (bottom) before and after 10 min of superfusion with nomifensine. The thin lines are the corresponding simulations. b, Bar graph of the parameters (average ± SEM) forV_max, the maximal uptake rate (μm/sec); K_m, the apparent affinity (μm); and initial [DA], the initial DA concentration (μm) estimated by simulations of CV (left) and amperometry recordings (right).

Fig. 4.

Effects of AMPH on stimulated DA overflow recorded with CV and amperometry. a, CV recording of DA overflow elicited by single-pulse stimulation (1/min) during 30 min of AMPH (10 μm) superfusion. The slow rise in baseline that peaks at 19 min is attributable to DA (see subtraction voltammogram, left inset). Stimulated DA overflow decreased in amplitude and increased in t_1/2 (right inset). b, Decrease of normalized maximal signal amplitudes (average ± SEM) during 20 min of AMPH (10 μm) superfusion in amperometric recordings (black circles; n = 5) and in CV recordings (white circles; n = 10). Thelines are single exponential fits, with a time constant for CV of 3 min and a time constant for amperometry of 1.7 min.c, Increase in normalizedt_1/2 for amperometric (black circles) and CV recordings (white circles) during AMPH superfusion (up to 25 min). d, Examples of CV recordings before and after 20 min of AMPH superfusion with simulations (thin lines). The bar graphs atbottom show the estimated parameters for controls and after 15–20 min of superfusion with AMPH. e, Same as ind for amperometric recordings.

Fig. 5.

Effect of AMPH (10 μm) in the presence of the D2 receptor antagonist sulpiride (2 μm) on DA overflow amplitudes recorded with CV and amperometry.a, Normalized maximal signal amplitudes (average ± SEM) are plotted for 25 min of AMPH/sulpiride superfusion, recorded with amperometry (blackcircles;n = 6) and CV (white circles;n = 6). The horizontal line crosses at 1 for reference, and the line that follows the amperometric data is a single exponential fit (time constant, 4.3 min).b, Examples of CV recordings in sulpiride and after 25 min of AMPH/sulpiride superfusion with simulations of the data (thin lines). c, The same as inb for amperometric recordings.

Fig. 6.

Effect of AMPH (10 μm) on DA overflow amplitudes in striatal slices of D2 receptor KO mice (D2−/−) recorded with CV. a, Normalized maximal signal amplitudes (average ± SEM) are plotted for 29 min of AMPH superfusion of slices from D2 KO mice (black circles; n = 5) and slices from wild types (WT) with (white squares) and without (white circles) 2 μm sulpiride (data from Figs. 4, 5). b, Example of CV recordings from a D2 KO mouse before and after 25 min of AMPH superfusion (white circles) with simulation of the data (thin lines).