Introduction

On the basis that the distance that dopamine can diffuse in extracellular space is limited to a few micrometers by high affinity uptake, we hypothesize that extracellular dopamine concentration exists as a spatially microheterogeneous distribution. We further hypothesize that changes in the kinetics of dopamine release and uptake differentially impact that distribution. We report herein on the use of voltammetry to probe the kinetically dependent features of the microheterogeneity.

Methods

Theoretical methods: We have used the following model, which couples mass transfer and saturable uptake kinetics, to consider the impact of uptake, release, and diffusion on the microheterogeneity of extracellular dopamine concentrations¹:

Numerical solutions of this equation were used to examine the distribution of dopamine concentrations near a diffusion source under various release and uptake conditions. Enhanced dopamine release and inhibition of uptake were modeled by increasing the flux at the source and increasing K_m, respectively.

Experimental methods: Electrical stimulation was applied to the medial forebrain bundle via a bipolar stimulating electrode (45 Hz, 10 s, 50 m A rms) at 30 min intervals. Pairs of carbon fiber microdisk electrodes (r = 3.5m m) were placed approximately 100 m m apart in striatum, and stimulation responses were recorded before and after administration of nomifensine (20 mg/kg, i.p.) or L-DOPA (250mg/kg, i.p. after 150 mg/kg carbidopa, i.p.). The voltammetric waveform comprised three linear segments: 0 to 1 V, 1 to -0.5 V and -0.5 to 0 V vs. Ag/AgCl. The scan rate was 300 V/s, and the applied potential was held at 0 V vs. Ag/AgCl for 200 ms between scans. The raw current was converted to concentration by postcalibration.

Results

The dimensionless concentration profiles in Figure 1A, obtained with Equation 1, show that the model predicts that enhanced release mainly affects the concentration in the immediate vicinity of the source while uptake inhibition mainly affects the concentration far from the source. In Figure 1B, the differences between the "pre-drug" and "post-drug" curves are plotted versus the "pre-drug" curve. This figure shows that the differential effects of enhanced release and inhibited uptake are predicted by the model.

During in vivo experiments, we have used the amplitude of pre-drug stimulus responses as a qualitative index of the proximity of voltammetric electrodes to sites of evoked dopamine release. Figures 1C and D show the relationship between the drug-induced increase in response amplitude and the pre-drug response amplitude. The symbols connected by tie-lines represent responses recorded simultaneously with pairs of electrodes in the same animal. In every cases the slopes of the tie-lines for L-DOPA are positive, while the slopes of the tie-lines for nomifensine are negative. This behavior is highly consistent with that predicted by Figure 1B.

Figure 1 The effects of nomifensine and L-DOPA on the responses observed at microdisk electrodes

Discussion

The correspondence between the experimental results and the theoretical predictions provides evidence that voltammetric microelectrodes afford sufficient spatial resolution to probe the differential effects of changes in the kinetics of dopamine release and uptake on the microheterogeneity of extracellular dopamine concentrations. The technical ability to resolve the behavior of extracellular dopamine concentrations at locals near to, and far from, dopamine release sites may be valuable to the investigation of the synaptic and volume modes of dopaminergic transmission. This work was supported by the NIH (Grant NS 31442).

References
1. Nicholson C (1995) Interaction Between Diffusion and Michaelis-Menten Uptake of Dopamine After Iontophoresis in the Striatum. Biophysical Journal 68: 1699-1715.