Disruption of the mouse dopamine transporter gene results in spontaneous hyperlocomotion despite major adaptive changes, such as decreases in neurotransmitter and receptor levels. In homozygote mice, dopamine persists at least 100 times longer in the extracellular space, explaining the biochemical basis of the hyperdopaminergic phenotype and demonstrating the critical role of the transporter in regulating neurotransmission. The dopamine transporter is an obligatory target of cocaine and amphetamine, as these psychostimulants have no effect on locomotor activity or dopamine release and uptake in mice lacking the transporter.

The dopamine transporter (DAT) plays an important role in calibrating the duration and intensity of dopamine neurotransmission in the central nervous system. We have used a strain of mice in which the gene for the DAT has been genetically deleted to identify the DAT's homeostatic role. We find that removal of the DAT dramatically prolongs the lifetime (300 times) of extracellular dopamine. Within the time frame of neurotransmission, no other processes besides diffusion can compensate for the lack of the DAT, and the absence of the DAT produces extensive adaptive changes to control dopamine neurotransmission. Despite the absence of a clearance mechanism, dopamine extracellular levels were only 5 times greater than control animals due to a 95% reduction in content and a 75% reduction in release. Paradoxically, dopamine synthesis rates are doubled despite a decrease of 90% in the levels of tyrosine hydroxylase and degradation is markedly enhanced. Thus, the DAT not only controls the duration of extracellular dopamine signals but also plays a critical role in regulating presynaptic dopamine homeostasis. It is interesting to consider that the switch to a dopamine-deficient, but functionally hyperactive, mode of neurotransmission observed in mice lacking the DAT may represent an extreme example of neuronal plasticity resulting from long-term psychostimulant abuse.Dopamine is an important regulator of many physiological functions, including control of locomotion, cognition, affect, and neuroendocrine hormone secretion (1, 2). In the central nervous system, dopamine signaling is governed by a balance between the amount released, the duration of effects, and the responsiveness of receptors. The dopamine transporter is thought to play a central role in determining the duration of action of dopamine by rapidly taking up extracellular dopamine into presynaptic terminals after release (3). Differences in the number of uptake sites (4) in different brain regions provides dopamine with different extracellular lifetimes (5), which allows its diffusion to remote receptor sites (6). Furthermore, dopamine uptake rates are lowered by drugs of abuse such as cocaine and amphetamines, and this action is responsible for their stimulatory effects on behavior (7-9). In addition to the dynamic processes that govern dopamine neurotransmission, in the long term, dopamine is eventually inactivated by the degradative enzymes monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) (10).The physiological importance of the DAT has been mostly inferred from the behavioral and psychosocial effects of pharmacological agents that interfere with dopamine transport, such as psychostimulants and antidepressants. To examine the importance of the DAT, we created a strain of mice lacking the dopamine tranporter protein using homologous recombination (11). The most obvious phenotype of these genetically modified animals is their marked spontaneous hyperlocomotion, which is similar to animals on high doses of psychostimulants. In this work, ...

The ability to predict favorable outcomes using environmental cues is an essential part of learned behavior. Dopamine neurons in the midbrain encode such stimulus-reward relationships in a manner consistent with contemporary learning models, but it is unclear how encoding this translates into actual dopamine release in target regions. Here, we sampled dopamine levels in the rat nucleus accumbens on a rapid (100 ms) timescale using electrochemical technology during a classical conditioning procedure. Early in conditioning, transient dopamine-release events signaled a primary reward, but not predictive cues. After repeated cue-reward pairings, dopamine signals shifted in time to predictive cue onset and were no longer observed at reward delivery. In the absence of stimulus-reward conditioning, there was no shift in the dopamine signal. Consistent with proposed roles in reward prediction and incentive salience, these results indicate that rapid dopamine release provides a reward signal that is dynamically modified by associative learning.

The dopamine projection to the nucleus accumbens has been implicated in behaviors directed toward the acquisition and consumption of natural rewards. The neurochemical studies that established this link made time-averaged measurements over minutes, and so the precise temporal relationship between dopamine changes and these behaviors is not known. To resolve this, we sampled dopamine every 100 msec using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in the nucleus accumbens of rats trained to press a lever for sucrose. Cues that signal the opportunity to respond for sucrose evoked dopamine release (67 Ϯ 20 nM) with short latency (0.2 Ϯ 0.1 sec onset). When the same cues were presented to rats naive to the cue-sucrose pairing, similar dopamine signals were not observed. Thus, cue-evoked increases in dopamine in trained rats reflected a learned association between the cues and sucrose availability. Lever presses for sucrose occurred at the peak of the dopamine surges. After lever presses, and while sucrose was delivered and consumed, no further increases in dopamine were detected. Rather, dopamine returned to baseline levels. Together, the results strongly implicate subsecond dopamine signaling in the nucleus accumbens as a real-time modulator of food-seeking behavior.

Neurotransmitters are chemicals that are secreted by neurons and relay messages to target cells. The goal of in vivo electrochemistry is to provide a real-time view of neurotransmitters in the extracellular space of the brain. This may be done in brain slices or the intact brain of anesthetized animals to probe the basic functions that regulate neurotransmitter levels. In other experiments, the measurements need to be made in the brain of behaving animals so that correlations of neurotransmitter fluctuations and specific behaviors can be made. For the neurotransmitter dopamine this can be accomplished today by chemical sensing of this neurotransmitter with fast scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Dopamine is an important target because it is a central player in the brain 'reward' system, although its precise function is not understood. Proposed roles for dopamine in reward have included the mediation of hedonia (pleasure) 1 , a messenger of incentive salience (wanting) 2 , or an error signal that promotes the learning associated with goal-directed behavior 3,4 . These multiple interpretations of dopaminergic function have arisen because, until recently, a realtime view of dopamine and its actions in an awake, behaving animal was unavailable. At the same time, new electrochemical technologies are being developed to measure other neurotransmitter actions. Electrochemical approaches are well suited for this application because they allow a neurotransmitter to be measured with high time resolution, enabling its precise role in the execution of behavioral tasks to be investigated.In this review we will describe current methods to detect neurotransmitters and monitor their concentration dynamics within neural tissue. The requirements for these methods are quite stringent. They need to be sufficiently selective so that the measured responses are unequivocally due to a specific molecule. They need to be sufficiently sensitive that they can detect these substances in the physiological range. The best established methodologies are for dopamine, so the majority of the applications of the methods described herein will involve this neurotransmitter. As will be seen, the goals in measuring neurotransmitter functions are diverse. On one hand, investigators are unraveling the mechanisms that control neurotransmitter concentrations. These studies range from examining biochemical synthesis to metabolism. On the other hand, investigators are questioning how the neurotransmitter interacts with its receptors and what message it conveys. Yet a third major interest is the role of a neurotransmitter in specific behaviors. To obtain a complete view of neurotransmission and information processing, chemical sensors need to be combined with traditional neurochemical tools. We will illustrate this approach with some specific examples.

Dopamine neurotransmission has been implicated in the modulation of many cognitive processes. Both rapid (phasic) and slower (tonic) changes in its extracellular concentration contribute to its complex actions. Fast in vivo electrochemical techniques can measure extracellular dopamine on a rapid time scale but without the selectivity afforded with slower techniques that use chemical separations. Cyclic voltammetry improves chemical resolution over other electrochemical methods, and it can resolve dopamine changes in the brains of behaving rodents over short epochs (<10 s). With this method, however, selective detection of slower dopamine changes is still elusive. Here we demonstrate that principal component regression of cyclic voltammetry data enables quantification of changes in dopamine and extracellular pH. Using this method, we show that cocaine modifies dopamine release in two ways: dopamine concentration transients increase in frequency and magnitude, whereas a gradual increase in steady-state dopamine concentration occurs over 90 s.cyclic voltammetry ͉ nucleus accumbens ͉ principal component regression F ast changes in the extracellular concentration of neurotransmitter can arise from phasic neuronal firing, whereas longlasting changes are associated with tonic firing (1). Dopaminergic neurons exhibit both of these firing patterns. Phasic activity accompanies salient stimuli, whereas tonic firing regulates the steady-state extracellular concentration (2). For this reason, chemical sensors for dopamine should be able to operate on a wide range of time scales in behaving animals. Microdialysis, a commonly used in vivo chemical sampling technique, is well suited to measure the minute-to-minute changes (tonic) that occur after uptake inhibition by agents such as cocaine (3,4). In vivo voltammetry, another approach for dopamine sampling, can measure much faster events, enabling phasic dopamine changes to be measured (5).A limitation of all voltammetric techniques has been their chemical selectivity. Fast-scan cyclic voltammetry at carbonfiber microelectrodes (6) provides rapid measurements and yields a chemical signature, the cyclic voltammogram, that allows distinction among electroactive molecules that are present in the brain. The electrode is highly sensitive to dopamine relative to dihydroxyphenylacetic acid and ascorbate, two major interferants, and the voltammogram of dopamine is distinct from those for a variety of neurochemical substances, although it is the same as that for norepinephrine (7). However, measurements in behaving rats have revealed that rapid dopamine changes are usually accompanied by other rapid changes in the electrochemical signal (5,8,9). Measurements with ion-selective electrodes demonstrated that these signals arise from a change in the pH of the brain extracellular fluid (10). Therefore, an objective method is needed to resolve the detected chemical events, assign them to specific compounds, and evaluate their temporal characteristics.Voltammetric electrodes are similar to othe...

Amphetamine (AMPH) inhibits uptake and causes release of dopamine (DA) from presynaptic terminals. AMPH can act on both vesicular storage of DA and directly on the dopamine transporter (DAT). To assess the relative importance of these two processes, we have examined the releasing actions of AMPH in mice with a genetic deletion of the DAT. The sequence of actions of AMPH has been determined by following the real time changes of DA in the extracellular fluid of intact tissue with fast scan cyclic voltammetry. In striatal slices from wild-type mice, AMPH causes a gradual (approximately 30 min) increase in extracellular DA, with a concomitant disappearance of the pool of DA available for depolarization-evoked release. Conversely, in slices from mice lacking the DAT, although a similar disappearance of electrically stimulated DA release occurs, extracellular DA does not increase. Similarly, microdialysis measurements of DA after AMPH in freely moving animals show no change in mice lacking the DAT, whereas it increases 10-fold in wild-type mice. In contrast, redistribution of DA from vesicles to the cytoplasm by the use of a reserpine-like compound, Ro4-1284, does not increase extracellular DA in slices from wild-type animals; however, subsequent addition of AMPH induces rapid (<5 min) release of DA. Thus, the DAT is required for the releasing action, but not the vesicle-depleting action, of AMPH on DA neurons, and the latter represents the rate-limiting step in the effects of AMPH. Furthermore, these findings suggest that in the absence of pharmacological manipulation, such as the use of amphetamine, endogenous cytoplasmic DA normally does not reach sufficient concentrations to reverse the DAT.

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