Cue-Evoked Dopamine Release Rapidly Modulates D2 Neurons in the Nucleus Accumbens During Motivated Behavior.

UNLABELLED: Dopaminergic neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) fire in response to unpredicted rewards or to cues that predict reward delivery. Although it is well established that reward-related events elicit dopamine release in the NAc, the role of rapid dopamine signaling in modulating NAc neurons that respond to these events remains unclear. Here, we examined dopamine's actions in the NAc in the rat brain during an intracranial self-stimulation task in which a cue predicted lever availability for electrical stimulation of the VTA. To distinguish actions of dopamine at select receptors on NAc neurons during the task, we used a multimodal sensor that probes three aspects of neuronal communication simultaneously: neurotransmitter release, cell firing, and identification of dopamine receptor type. Consistent with prior studies, we first show dopamine release events in the NAc both at cue presentation and after lever press (LP). Distinct populations of NAc neurons encode these behavioral events at these same locations selectively. Using our multimodal sensor, we found that dopamine-mediated responses after the cue involve exclusively a subset of D2-like receptors (D2Rs), whereas dopamine-mediated responses proximal to the LP are mediated by both D1-like receptors (D1R) and D2Rs. These results demonstrate for the first time that dopamine-mediated responses after cues that predict reward availability are specifically linked to its actions at a subset of neurons in the NAc containing D2Rs. SIGNIFICANCE STATEMENT: Successful reward procurement typically involves the completion of a goal-directed behavior in response to appropriate environmental cues. Although numerous studies link the mesolimbic dopamine system with these processes, how dopamine's effects are mediated on the receptor level within a key neural substrate, the nucleus accumbens, remains elusive. Here, we used a unique multimodal sensor that reveals three aspects of neuronal interactions: neurotransmitter release, cell firing, and dopamine-receptor type. We identified a key role of D2-like receptor (D2R)-expressing neurons in response to a reward-predicting cue, whereas both the D2R and D1R types modulate responses of neurons proximal to the goal-directed action. This work provides novel insight into the unique role of D2R-mediated neuronal activity to reward-associated cues, a fundamental aspect of motivated behaviors.

Flexible software platform for fast-scan cyclic voltammetry data acquisition and analysis.

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 that 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 transistor-transistor logic-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 subsecond and multiminute time scales. Here we describe and demonstrate the utility of HDCV for in vivo experiments.

Controlled iontophoresis coupled with fast-scan cyclic voltammetry/electrophysiology in awake, freely moving animals.

Simultaneous electrochemical and electrophysiological data were recorded to evaluate the effects of controlled local application of dopaminergic agonists and antagonists in awake rats. Measurements were made with a probe consisting of a carbon-fiber microelectrode fused to three iontophoretic barrels used to introduce the drugs of interest. The probe and the manipulator used to position it in the brain of behaving animals were optimized to improve their performance. The effect of the dopamine autoreceptor on electrically stimulated release was demonstrated. Dopamine inhibited the release of endogenous dopamine whereas raclopride, a D2 antagonist, enhanced it, with similar responses in anesthetized and awake animals. We also examined changes in the firing rate of nucleus accumbens (NAc) neurons in awake animals during and after brief (15 s) iontophoretic ejections of SCH 23390 (D1 receptor antagonist) or raclopride. Changes in response to these antagonists were seen both immediately and on a prolonged time scale. Application of raclopride increased the firing rate in 40% of medium spiny neurons (MSNs), of which half responded immediately. Decreases in firing rate were observed in 46% of MSNs after SCH 23390 application. Only 11% of MSNs responded to both antagonists and one MSN (3%) showed no response to either drug. The same prolonged response in firing rate was seen for electrically stimulated and locally applied dopamine in 75% of MSNs. These results are in agreement with previously reported distributions for dopamine receptor subtypes on MSNs and probe the effects of dopamine on these cell populations.

Opposing catecholamine changes in the bed nucleus of the stria terminalis during intracranial self-stimulation and its extinction.

BACKGROUND: While studies suggest that both dopamine and norepinephrine neurotransmission support reinforcement learning, the role of dopamine has been emphasized. As a result, little is known about norepinephrine signaling during reward learning and extinction. Both dopamine and norepinephrine projections innervate distinct regions of the bed nucleus of the stria terminalis (BNST), a structure that mediates behavioral and autonomic responses to stress and anxiety. We investigated whether norepinephrine release in the ventral BNST (vBNST) and dopamine release in the dorsolateral BNST (dlBNT) correlate with reward learning during intracranial self-stimulation (ICSS). METHODS: Using fast-scan cyclic voltammetry, norepinephrine concentration changes in the vBNST (n = 12 animals) during ICSS were compared with dopamine changes in the dlBNST (n = 7 animals) and nucleus accumbens (NAc) (n = 5 animals). Electrical stimulation was in the ventral tegmental area/substantia nigra region. RESULTS: Whereas dopamine release was evoked by presentation of a cue predicting reward availability in both dlBNST and NAc, cue-evoked norepinephrine release did not occur in the vBNST. Release of both catecholamines was evoked by the electrical stimulation. Extracellular changes in norepinephrine were also studied during extinction of ICSS and compared with results obtained for dopamine. During extinction of ICSS, norepinephrine release in the vBNST occurred at the time where the stimulation was anticipated, whereas dopamine release transiently decreased. CONCLUSIONS: The data demonstrate that norepinephrine release in the vBNST differs from dopamine release in the dlBNST and the NAc in that it signals the absence of reward rather than responding to reward predictive cues.

Sources contributing to the average extracellular concentration of dopamine in the nucleus accumbens.

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 caused by 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 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 dopamine transporter (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.

Higher sensitivity dopamine measurements with faster-scan cyclic voltammetry.

Fast-scan cyclic voltammetry (FSCV) with carbon-fiber microelectrodes has been successfully used to detect catecholamine release in vivo. Generally, waveforms with anodic voltage limits of 1.0 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 4-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 4-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.

Neural encoding of cocaine-seeking behavior is coincident with phasic dopamine release in the accumbens core and shell.

Mesolimbic dopamine neurons projecting from the ventral tegmental area 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 subregions 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 in rats 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 subregions, 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.

Dynamic changes in accumbens dopamine correlate with learning during intracranial self-stimulation.

Dopamine in the nucleus accumbens (NAc) is an important neurotransmitter for reward-seeking behaviors such as intracranial self-stimulation (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. Subsequent reinstatement of ICSS was correlated with a return to preextinction transient amplitudes in response to the cue and reestablishment of ICSS behavior. The results show the dynamic nature of chemical signaling in the NAc during ICSS and provide new insight into the role of NAc dopamine in reward-related behaviors.