Dopamine in the nucleus accumbens (NAc) is an important neurotransmitter for reward-seeking behaviors such as intracranial selfstimulation (ICSS), although its precise role remains unclear. Here, dynamic fluctuations in extracellular dopamine were measured during ICSS in the rat NAc shell with fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Rats were trained to press a lever to deliver electrical stimulation to the substantia nigra (SNc)/ ventral tegmental area (VTA) after the random onset of a cue that predicted reward availability. Latency to respond after cue onset significantly declined across trials, indicative of learning. Dopamine release was evoked by the stimulation but also developed across trials in a time-locked fashion to the cue. Once established, the cue-evoked dopamine transients continued to grow in amplitude, although they were variable from trial to trial. The emergence of cue-evoked dopamine correlated with a decline in electrically evoked dopamine release. Extinction of ICSS resulted in a significant decline in goal-directed behavior coupled to a significant decrease in cue-evoked phasic dopamine across trials. carbon-fiber electrode ͉ cyclic voltammetry ͉ extinction ͉ nucleus accumbens shell ͉ reward I ntracranial self-stimulation (ICSS) was discovered in 1954 (1). In this paradigm, a rat depresses a lever to deliver an electric shock to electrodes implanted within the brain. Extensive mapping studies by Olds and Olds later showed that the neuroanatomical region supporting ICSS centered in the posterior MFB region of the lateral hypothalamus (2). This finding provoked considerable interest, because it identified a brain reward pathway that could be centrally activated without the need for sensory stimulation (3, 4). Although a role for several neurotransmitters has been implicated in ICSS, dopamine appears to play a primary role (5, 6), leading to the view that dopaminergic signaling is essential during goal-directed behaviors. Indeed, it was postulated that increased dopaminergic neurotransmission was necessary for the reinforcement of reward-related behavior (7).More recently, electrophysiological studies in primates have provided new insight into the role of dopaminergic neurons in reward processing (8). In response to unexpected rewards, dopamine neurons exhibit phasic firing. However, when an animal learns that a cue predicts reward, the burst of neuronal firing switches to the onset of the cue (9-12). Responses to the cue increase with repeated trials, and these paired responses of midbrain dopamine neurons follow the expectations of models of associative learning in which dopamine signaling is a reward-prediction error (12,13). Similar responses to conditioned stimuli that predict reward have also been observed for midbrain dopaminergic neurons in rats (14).A phasic increase in dopamine neuronal firing should lead to a dopamine concentration transient in terminal areas such as the nucleus accumbens (NAc). Indeed, using fast-scan cyclic voltammetry at carbon-fiber microe...

Fast-scan cyclic voltammetry with carbon-fiber microelectrodes has been successfully used to detect catecholamine release in vivo. Generally, waveforms with anodic voltage limits of 1.0 V or 1.3 V (vs. Ag/AgCl) are used for detection. The 1.0 V excursion provides good temporal resolution, but suffers from a lack of sensitivity. The 1.3 V excursion increases sensitivity, but also increases response time which can blur the detection of neurochemical events. Here, the scan rate was increased to improve the sensitivity of the 1.0 V excursion while maintaining the rapid temporal response. However, increasing scan rate increases both the desired faradaic current response and the already large charging current associated with the voltage sweep. Analog background subtraction was used to prevent the analog-to-digital converter from saturating from the high currents generated with increasing scan rate by neutralizing some of the charging current. In vitro results with the 1.0 V waveform showed approximately a four-fold increase in signal to noise ratio with maintenance of the desired faster response time by increasing scan rate up to 2400 V/s. In vivo, stable stimulated release was detected with an approximate four-fold increase in peak current. The scan rate of the 1.3 V waveform was also increased, but the signal was unstable with time in vitro and in vivo. Adapting the 1.3 V triangular wave into a sawhorse design prevented signal decay and increased the faradaic response. The use of the 1.3 V sawhorse waveform decreased the detection limit of dopamine with FSCV to 0.96 ± 0.08 nM in vitro and showed improved performance in vivo without affecting the neuronal environment. Electron microscopy showed dopamine sensitivity is in a quasi-steady state with carbon-fiber microelectrodes scanned to potentials above 1.0 V.

Mesolimbic dopamine neurons fire in both tonic and phasic modes resulting in detectable extracellular levels of dopamine in the nucleus accumbens (NAc). In the past, different techniques have targeted dopamine levels in the NAc to establish a basal concentration. In this study we used in vivo fast scan cyclic voltammetry (FSCV) in the NAc of awake, freely moving rats. The experiments were primarily designed to capture changes in dopamine due to phasic firing – that is, the measurement of dopamine ‘transients’. These FSCV measurements revealed for the first time that spontaneous dopamine transients constitute a major component of extracellular dopamine levels in the NAc. A series of experiments were designed to probe regulation of extracellular dopamine. Lidocaine was infused into the ventral tegmental area, the site of dopamine cell bodies, to arrest neuronal firing. While there was virtually no instantaneous change in dopamine concentration, longer sampling revealed a decrease in dopamine transients and a time-averaged decrease in the extracellular level. Dopamine transporter (DAT) inhibition using intravenous GBR12909 injections increased extracellular dopamine levels changing both frequency and size of dopamine transients in the NAc. To further unmask the mechanics governing extracellular dopamine levels we used intravenous injection of the vesicular monoamine transporter (VMAT2) inhibitor, tetrabenazine, to deplete dopamine storage and increase cytoplasmic dopamine in the nerve terminals. Tetrabenazine almost abolished phasic dopamine release but increased extracellular dopamine to ~500 nM, presumably by inducing reverse transport by DAT. Taken together, data presented here show that average extracellular dopamine in the NAc is low (20–30 nM) and largely arises from phasic dopamine transients.

Mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) are part of a complex circuit mediating cocaine-directed behaviors. However, the precise role of rapid (subsecond) dopamine release within the primary sub-regions of the NAc, the core and shell, and its relationship to NAc cell firing during this behavior remain unknown. Here, using fast-scan cyclic voltammetry (FSCV) we report rapid dopamine signaling in both the core and shell, however, significant differences were observed in the timing of dopamine release events within seconds of the cocaine reinforced response during self-administration sessions. Importantly, simultaneous voltammetric and electrophysiological recordings from the same electrode reveal that, at certain sites within both sub-regions, neurons exhibiting patterned activation were observed at locations where rapid dopamine release was present; the greater the strength of the neural signal the larger the dopamine release event. In addition, it was at those locations that electrically-evoked stimulated release was greatest. No changes in dopamine were observed where nonphasic neurons were recorded. Thus, although differences are evident in dopamine release dynamics relative to cocaine-reinforced responding within the core and shell, dopamine release is heterogeneous within each structure and varies as a function of precise neuronal targets during cocaine-seeking behavior.

Over the last several decades, fast-scan cyclic voltammetry (FSCV) has proved to be a valuable analytical tool for the real-time measurement of neurotransmitter dynamics in vitro and in vivo. Indeed, FSCV has found application in a wide variety of disciplines including electrochemistry, neurobiology and behavioral psychology. The maturation of FSCV as an in vivo technique led users to pose increasingly complex questions that require a more sophisticated experimental design. To accommodate recent and future advances in FSCV application, our lab has developed High Definition Cyclic Voltammetry (HDCV). HDCV is an electrochemical software suite, and includes data acquisition and analysis programs. The data collection program delivers greater experimental flexibility and better user feedback through live displays. It supports experiments involving multiple electrodes with customized waveforms. It is compatible with TTL-based systems that are used for monitoring animal behavior and it enables simultaneous recording of electrochemical and electrophysiological data. HDCV analysis streamlines data processing with superior filtering options, seamlessly manages behavioral events, and integrates chemometric processing. Furthermore, analysis is capable of handling single files collected over extended periods of time, allowing the user to consider biological events on both sub-second and multi-minute time scales. Here we describe and demonstrate the utility of HDCV for in vivo experiments.