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.

In vivo voltammetry monitoring of electrically evoked extracellular norepinephrine in subregions of the bed nucleus of the stria terminalis.

Norepinephrine (NE) is an easily oxidized neurotransmitter that is found throughout the brain. Considerable evidence suggests that it plays an important role in neurocircuitry related to fear and anxiety responses. In certain subregions of the bed nucleus of the stria terminalis (BNST), NE is found in large amounts. In this work we probed differences in electrically evoked release of NE and its regulation by the norepinephrine transporter (NET) and the α(2)-adrenergic autoreceptor (α(2)-AR) in two regions of the BNST of anesthetized rats. NE was monitored in the dorsomedial BNST (dmBNST) and ventral BNST (vBNST) by fast-scan cyclic voltammetry at carbon fiber microelectrodes. Pharmacological agents were introduced either by systemic application (intraperitoneal injection) or by local application (iontophoresis). The iontophoresis barrels were attached to a carbon fiber microelectrode to allow simultaneous detection of evoked NE release and quantitation of iontophoretic delivery. Desipramine (DMI), an inhibitor of NET, increased evoked release and slowed clearance of released NE in both regions independent of the mode of delivery. However, the effects of DMI were more robust in the vBNST than in the dmBNST. Similarly, the α(2)-AR autoreceptor inhibitor idazoxan (IDA) enhanced NE release in both regions but to a greater extent in the vBNST by both modes of delivery. Since both local application by iontophoresis and systemic application of IDA had similar effects on NE release, our results indicate that terminal autoreceptors play a predominant role in the inhibition of subsequent release.

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.

Electrochemical Roughening of Thin-Film Platinum Macro and Microelectrodes.

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries of the metal. Using this method, a crack free, thin-film macroelectrode surface with up to 40 times increase in active surface area was obtained. The roughening is easy to do in a standard electrochemical characterization laboratory and incudes the application of voltage pulses followed by extended application of a reductive voltage in a perchloric acid solution. The protocol includes the chemical and electrochemical preparation of both a macroscale (1.2 mm diameter) and microscale (20 µm diameter) platinum disc electrode surface, roughening the electrode surface and characterizing the effects of surface roughening on electrode active surface area. This electrochemical characterization includes cyclic voltammetry and impedance spectroscopy and is demonstrated for both the macroelectrodes and the microelectrodes. Roughening increases electrode active surface area, decreases electrode impedance, increases platinum charge injection limits to those of titanium nitride electrodes of same geometry and improves substrates for adhesion of electrochemically deposited films.

Evaluation of in vitro neuronal platforms as surrogates for in vivo whole brain systems.

Quantitatively benchmarking similarities and differences between the in vivo central nervous system and in vitro neuronal cultures can qualify discrepancies in functional responses and establish the utility of in vitro platforms. In this work, extracellular electrophysiology responses of cortical neurons in awake, freely-moving animals were compared to in vitro cultures of dissociated cortical neurons. After exposure to two well-characterized drugs, atropine and ketamine, a number of key points were observed: (1) significant differences in spontaneous firing activity for in vivo and in vitro systems, (2) similar response trends in single-unit spiking activity after exposure to atropine, and (3) greater sensitivity to the effects of ketamine in vitro. While in vitro cultures of dissociated cortical neurons may be appropriate for many types of pharmacological studies, we demonstrate that for some drugs, such as ketamine, this system may not fully capture the responses observed in vivo. Understanding the functionality associated with neuronal cultures will enhance the relevance of electrophysiology data sets and more accurately frame their conclusions. Comparing in vivo and in vitro rodent systems will provide the critical framework necessary for developing and interpreting in vitro systems using human cells that strive to more closely recapitulate human in vivo function and response.

Simultaneous electrical recording of cardiac electrophysiology and contraction on chip.

Prevailing commercialized cardiac platforms for in vitro drug development utilize planar microelectrode arrays to map action potentials, or impedance sensing to record contraction in real time, but cannot record both functions on the same chip with high spatial resolution. Here we report a novel cardiac platform that can record cardiac tissue adhesion, electrophysiology, and contractility on the same chip. The platform integrates two independent yet interpenetrating sensor arrays: a microelectrode array for field potential readouts and an interdigitated electrode array for impedance readouts. Together, these arrays provide real-time, non-invasive data acquisition of both cardiac electrophysiology and contractility under physiological conditions and under drug stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were cultured as a model system, and used to validate the platform with an excitation-contraction decoupling chemical. Preliminary data using the platform to investigate the effect of the drug norepinephrine are combined with computational efforts. This platform provides a quantitative and predictive assay system that can potentially be used for comprehensive assessment of cardiac toxicity earlier in the drug discovery process.

Medullary norepinephrine neurons modulate local oxygen concentrations in the bed nucleus of the stria terminalis.

Neurovascular coupling is understood to be the underlying mechanism of functional hyperemia, but the actions of the neurotransmitters involved are not well characterized. Here we investigate the local role of the neurotransmitter norepinephrine in the ventral bed nucleus of the stria terminalis (vBNST) of the anesthetized rat by measuring O2, which is delivered during functional hyperemia. Extracellular changes in norepinephrine and O2 were simultaneously monitored using fast-scan cyclic voltammetry. Introduction of norepinephrine by electrical stimulation of the ventral noradrenergic bundle or by iontophoretic ejection induced an initial increase in O2 levels followed by a brief dip below baseline. Supporting the role of a hyperemic response, the O2 increases were absent in a brain slice containing the vBNST. Administration of selective pharmacological agents demonstrated that both phases of this response involve ß-adrenoceptor activation, where the delayed decrease in O2 is sensitive to both α- and ß-receptor subtypes. Selective lesioning of the locus coeruleus with the neurotoxin DSP-4 confirmed that these responses are caused by the noradrenergic cells originating in the nucleus of the solitary tract and A1 cell groups. Overall, these results support that non-coerulean norepinephrine release can mediate activity-induced O2 influx in a deep brain region.

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.

Probing presynaptic regulation of extracellular dopamine with iontophoresis.

Iontophoresis allows for localized drug ejections directly into brain regions of interest driven by the application of current. Our lab has previously adapted a method to quantitatively monitor iontophoretic ejections. Here those principles have been applied in vivo to modulate electrically evoked release of dopamine in anesthetized rats. A neutral, electroactive marker molecule that is ejected purely by electroosmotic flow (EOF) was used to monitor indirectly the ejection of electroinactive dopaminergic drugs (raclopride, quinpirole, and nomifensine). Electrode placements were marked with an iontophoretically ejected dye, pontamine sky blue. We show that EOF marker molecules, acetaminophen (AP) and 2-(4-nitrophenoxy) ethanol, have no effect on electrically evoked dopamine release in the striatum or the sensitivity of electrode. Additionally, we establish that a short, 30 second ejection of raclopride, quinpirole, or nomifensine with iontophoresis is sufficient to affect autoreceptor regulation and the re-uptake of dopamine. These effects vary in lifetime, indicating that this technique can be used to study receptor kinetics.