Clinical Chemistry 49:10 1763–1773 (2003) Oak Ridge Conference Detecting Subsecond Dopamine Release with Fast-Scan Cyclic Voltammetry in Vivo Donita L. Robinson,1,2,3 B. Jill Venton,1,2 Michael L.A.V. Heien,1,2 and R. Mark Wightman1,2,3* Background: Dopamine is a potent neuromodulator in chronoamperometry, differential normal-pulse voltam- the brain, influencing a variety of motivated behaviors metry, and fast-scan cyclic voltammetry. and involved in several neurologic diseases. Measure- Fast-scan cyclic voltammetry has been used exten- ments of extracellular dopamine in the brains of exper- sively to investigate the rapid events associated with imental animals have traditionally focused on a tonic neurotransmission in vivo and in vitro. It is a valuable timescale (minutes to hours). However, dopamine con- preclinical tool to evaluate drug mechanisms (1) and centrations are now known to fluctuate on a phasic animal models of disease (2) associated with dopaminer- timescale (subseconds to seconds). gic transmission. By exploring the regulation and physi- Approach: Fast-scan cyclic voltammetry provides ana- ology of neurotransmitter function with fast-scan cyclic lytical chemical measurements of phasic dopamine sig- voltammetry, novel targets for therapeutic intervention in nals in the rat brain. pathologies such as Parkinson disease, schizophrenia, and Content: Procedural aspects of the technique are dis- drug addiction may be identified. cussed, with regard to appropriate use and in compari- Relative to other available techniques, fast-scan cyclic son with other methods. Finally, examples of data voltammetry has several advantages: it measures dopa- collected using fast-scan cyclic voltammetry are summa- mine concentrations in real time, on a subsecond time- rized, including naturally occurring dopamine tran- scale; it quantifies increases and decreases in dopamine sients and signals arising from electrical stimulation of concentrations in the nanomolar to micromolar range; it dopamine neurons. uses a micrometer-dimension probe that gives fine spatial Summary: Fast-scan cyclic voltammetry offers real-time resolution with minimal tissue damage; and it provides measurements of changes in extracellular dopamine positive identification of dopamine via the cyclic voltam- concentrations in vivo. With its subsecond time resolu- mogram. As we will show in this review, these character- tion, micrometer-dimension spatial resolution, and istics make fast-scan cyclic voltammetry ideal for measur- chemical selectivity, it is the most suitable technique ing the phasic dopamine signals putatively associated currently available to measure transient concentration with burst firing of dopamine neurons in awake animals. changes of dopamine. © 2003 American Association for Clinical Chemistry Extracellular Dopamine The dynamics of the release and uptake of dopamine into Dopamine is known to be an important neurotransmitter brain extracellular space are currently under intense in- that modulates many aspects of brain circuitry. It is vestigation. It is now well known that dopamine is an therefore of interest to be able to measure dopamine in extrasynaptic messenger that functions via volume trans- awake rats to correlate neurochemistry with behavior. mission, escaping the synaptic cleft to bind to extrasyn- Over the past several decades, two general techniques aptic receptors and transporters (3–5). Furthermore, there have evolved to accomplish this: microdialysis and elec- is evidence for both tonic and phasic aspects of extracel- trochemistry. Although multiple electrochemical tech- lular dopamine (6–8), which are best measured by differ- niques exist, those used in freely moving animals are ent neurochemical techniques. tonic vs phasic extracellular dopamine Extensive data indicate that a tonic concentration of 1 2 3 Department of Chemistry, Neuroscience Center, and Center for Alco- dopamine exists in target nuclei that may play an en- hol Studies, University of North Carolina, Chapel Hill, NC 27599-3290. *Author for correspondence. Fax 919-962-2388; e-mail [email protected]. abling role in neurotransmission. In the striatum, these Received April 21, 2003; accepted July 9, 2003. tonic concentrations of dopamine are predicted to be 5–20 1763 1764 Robinson et al.: Voltammetric Measurements of Extracellular Dopamine nmol/L by microdialysis (9) and differential normal- thus, the method has slow temporal resolution and is best pulse voltammetry (10) and 50–100 nmol/L by theoretical suited for looking at concentration changes that happen estimations using fast-scan cyclic voltammetry (11) and on the minute-to-hour timescale. Because of these charac- pharmacologic studies (12). If the concentrations are teristics, microdialysis is routinely used to measure tonic indeed 20 nmol/L or above, those dopamine receptors in changes in dopamine. [Although measurements of tonic high-affinity states would be chronically occupied, which dopamine theoretically include phasic signals integrated appears to be the case for D2-like receptors (13, 14). across the sample, it has been demonstrated that micro- Additional evidence for extracellular dopamine “tone” dialysis is insensitive to dopamine transients (24, 25).] comes from microdialysis studies in 6-hydroxydopamine- lesioned rats. Basal extracellular dopamine concentrations Constant-potential amperometry. Constant-potential amper- are maintained until dopaminergic cell loss exceeds 80%, ometry is a simple electrochemical technique in which a after which both parkinsonian symptoms emerge and potential sufficient to oxidize or reduce the molecule of extracellular dopamine concentrations drop (15, 16). interest is applied to the electrode. The electrode responds Tonic extracellular dopamine would be best measured by extremely quickly to changes in analyte concentration, neurochemical techniques that offer both high sensitivity and high sampling rates can be used; therefore, constant- and a long timescale, such as microdialysis. potential amperometry offers the best temporal resolution In contrast, extracellular dopamine is known to reach among the available techniques. However, it suffers from high concentrations for brief periods (17, 18), which we poor selectivity: any molecule that can be oxidized or describe as phasic activity. These transients are likely to reduced at the potential of the electrode is detected, so arise from concerted burst firing of dopamine neurons there is no way to differentiate between molecules. Be- (19) that often occur on presentation of salient sensory cause of its lack of chemical selectivity, constant-potential input (6, 20). Fast-scan cyclic voltammetry and amperom- amperometry should never be used in the freely moving etry have been used to measure dopamine release after an preparation, where higher chemical selectivity is needed electrical stimulation of dopamine neurons that mimics to provide confidence in the identity of the analyte. tonic and phasic firing (11, 21). The amount of dopamine Nevertheless, it is appropriate for measurement of elec- released is frequency-dependent; as the stimulation ap- trically evoked dopamine release in anesthetized animals proaches frequencies achieved by burst firing, the dopa- or brain slices, and its fast sampling rate makes it ideal to mine transporter is saturated and high concentrations are study the kinetics of phasic dopamine signals. achieved. Similar naturally occurring dopamine tran- sients have been measured in the rat, sometimes reaching Fast-scan cyclic voltammetry. Fast-scan cyclic voltammetry Ͼ ␮ concentrations 1 mol/L (18, 22). These higher concen- is a more complex electrochemical technique that pro- trations are sufficient to activate the low-affinity D1- and vides good chemical selectivity while retaining subsecond D2-like receptors (23). Because the events themselves are temporal resolution. Each measurement consists of a Ͻ so brief ( 10 s), phasic extracellular dopamine would be cyclic voltammogram that serves as a chemical identifier best measured by neurochemical techniques that have to provide chemical selectivity. Thus, possible interfer- subsecond timescales. ents, such as ascorbic acid, dihydroxyphenylacetic acid (DOPAC),4 and pH shifts, can be easily distinguished neurochemical methods to measure from dopamine via the cyclic voltammogram. Because extracellular dopamine fast-scan cyclic voltammograms can be repeated every 100 High sensitivity, chemical selectivity, and fast temporal ms, changes in dopamine concentration can be monitored resolution are all desirable characteristics when measur- on the subsecond time scale. These characteristics make ing neurotransmitters in vivo. In practice, it is difficult to fast-scan cyclic voltammetry well suited for detecting achieve all of these with one method. In this section, we phasic dopamine changes in the freely moving animal. will briefly discuss the advantages and disadvantages of Additional electrochemical techniques include high- the methods used for dopamine detection in vivo, with speed chronoamperometry and differential normal-pulse reference to their appropriate applications. voltammetry. Both techniques provide more chemical selectivity than constant-potential amperometry but less Microdialysis. Microdialysis is the most widely used than fast-scan cyclic voltammetry. High-speed chrono- method to sample the chemical environment of the brain. amperometry provides measurements on the second time A dialysis probe that is permeable to small molecules is scale (usually