Optogenetic control of striatal dopamine release in rats

Abstract

Optogenetic control over neuronal firing has become an increasingly elegant method to dissect the microcircuitry of mammalian brains. To date, examination of these manipulations on neurotransmitter release has been minimal. Here we present the first in-depth analysis of optogenetic stimulation on dopamine neurotransmission in the dorsal striatum of urethane-anesthetized rats. By combining the tight spatial and temporal resolution of both optogenetics and fast-scan cyclic voltammetry we have determined the parameters necessary to control phasic dopamine release in the dorsal striatum of rats in vivo. The kinetics of optically induced dopamine release mirror established models of electrically evoked release, indicating that potential artifacts of electrical stimulation on ion channels and the dopamine transporter are negligible. Furthermore a lack of change in extracellular pH indicates that optical stimulation does not alter blood flow. Optical control over dopamine release is highly reproducible and flexible. We are able to repeatedly evoke concentrations of dopamine release as small as a single dopamine transient (50 nM). An inverted U-shaped frequency response curve exists with maximal stimulation inducing dopamine effluxes exceeding 500 nM. Taken together, these results have obvious implications for understanding the neurobiological basis of dopaminergic-based disorders and provide the framework to effectively manipulate dopamine patterns.

Figure 1

Light stimulation of SN dopamine neurons produce transient dopamine release in the striatum detected by FSCV. (A) Representative traces of dopamine signals detected in dorsal striatum of anesthetized rats in response to light stimulation. The pulse widths, which were used to induce these 3 signals, were 10, 20 and 4 ms, respectively. (B) Representative color plots, which topographically depict the voltammetric data before, during and after light stimulation, with time on the x-axis, applied scan potential on the y-axis and background-subtracted faradaic current shown on the z-axis in pseudo-color. The color scales for the first and third panels begin with +4.0 nA and ends with -2.7 nA. The second panel scale begins with +10 nA and ends with -5 nA. (C) Background-subtracted cyclic voltammograms taken from the peak of light stimulation. Every signal has an oxidation peak at +0.65 V and reduction peak at -0.2 V vs. Ag/AgCl reference, identifying the released species as dopamine.

Figure 2

Curve fits of light evoked dopamine release in rat striatum. Data from one representative subject (black lines) were fit to a Michaelis-Menten kinetic model to determine the parameters for dopamine release and uptake. Simulation lines (red) were calculated from best-fit parameters. The magnitudes of the [DA]_p, V_max and K_m for the dopamine effluxes induced by 20- and 40-pulse stimulation (4 ms pulse width) were 45 nM, 3500 nM/s, 200 nM and 95 nM, 3500 nM/s, 200 nM, respectively. The total length of light stimulation (1 s) is indicated by the red line under the efflux curves.

Figure 3

Frequency, pulse width, and train duration dependency of light induced dopamine release. (A) The total duration of the stimulation for every frequency and pulse width combination was kept the same (1 s). (B) Data from one representative rat with 4 ms pulse width stimulation at various frequencies (20, 30 or 40 Hz), maintained for either 0.5 s (solid line) or 1 s (dashed line) train duration. An optical fiber was positioned in the SN at the following coordinates: anterior-posterior, 5.6 mm; lateral, 2.0 mm; dorsal-ventral, 7.6 mm. Voltammetric recordings were performed in the striatum at the following coordinates: anterior-posterior, 2.0 mm; lateral, 1.3 mm; dorsal-ventral, 5.0 mm. The data are presented as mean ± SEM (n =5).

Figure 4

Evaluation of laser power intensity on the efficacy of dopamine release. Laser power from 0 to 5.7 mW intensities were delivered through the fiber optic to the SN and dopamine release was voltammetrically detected in the striatum. (A) Dopamine signals recorded in 1 min intervals in the striatum of a representative subject during stimulation with different laser intensity values (0.0, 0.1, 0.6, 1.3, 2.3, 3.4, 4.5, 5.7 mW from bottom). (B) Effect of laser power on dopamine release in the striatum. The data are presented as mean ± SEM and significance versus minimal triggered dopamine release (≈ 1mW) is indicated (*P < 0.05, **P < 0.01, *** P < 0.001, n =4).

Figure 5

Optical stimulation of dopamine release in the striatum is spatially restricted to the dorsal region. Dopamine traces from one representative animal are presented. The fiber optic was fixed in the SN (anterior-posterior, 5.6 mm; lateral, 2.0 mm; dorsal-ventral, 7.6 mm) while the carbon fiber recording electrode was lowered to various depths throughout the striatum to determine level of dopamine release (anterior-posterior, 1.3 mm; lateral, 2.0 mm and dorsal-ventral varied with 1: 5.0 mm; 2: 5.5 mm; 3: 6.4 mm; 4: 6.8 mm; 5: 7.4 mm).