Michael L. Heien

Subsecond dopamine release promotes cocaine seeking

The dopamine-containing projection from the ventral tegmental area of the midbrain to the nucleus accumbens is critically involved in mediating the reinforcing properties of cocaine. Although neurons in this area respond to rewards on a subsecond timescale, neurochemical studies have only addressed the role of dopamine in drug addiction by examining changes in the tonic (minute-to-minute) levels of extracellular dopamine. To investigate the role of phasic (subsecond) dopamine signalling, we measured dopamine every 100 ms in the nucleus accumbens using electrochemical technology. Rapid changes in extracellular dopamine concentration were observed at key aspects of drug-taking behaviour in rats. Before lever presses for cocaine, there was an increase in dopamine that coincided with the initiation of drug-seeking behaviours. Notably, these behaviours could be reproduced by electrically evoking dopamine release on this timescale. After lever presses, there were further increases in dopamine concentration at the concurrent presentation of cocaine-related cues. These cues alone also elicited similar, rapid dopamine signalling, but only in animals where they had previously been paired to cocaine delivery. These findings reveal an unprecedented role for dopamine in the regulation of drug taking in real time.

Detecting Subsecond Dopamine Release with Fast-Scan Cyclic Voltammetry in Vivo

BACKGROUND Dopamine is a potent neuromodulator in the brain, influencing a variety of motivated behaviors and involved in several neurologic diseases. Measurements of extracellular dopamine in the brains of experimental animals have traditionally focused on a tonic timescale (minutes to hours). However, dopamine concentrations are now known to fluctuate on a phasic timescale (subseconds to seconds). APPROACH Fast-scan cyclic voltammetry provides analytical chemical measurements of phasic dopamine signals in the rat brain. CONTENT Procedural aspects of the technique are discussed, with regard to appropriate use and in comparison with other methods. Finally, examples of data collected using fast-scan cyclic voltammetry are summarized, including naturally occurring dopamine transients and signals arising from electrical stimulation of dopamine neurons. SUMMARY Fast-scan cyclic voltammetry offers real-time measurements of changes in extracellular dopamine concentrations in vivo. With its subsecond time resolution, micrometer-dimension spatial resolution, and chemical selectivity, it is the most suitable technique currently available to measure transient concentration changes of dopamine.

Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation

Transient surges of dopamine in the nucleus accumbens are associated with drug seeking. Using a voltammetric sensor with high temporal and spatial resolution, we demonstrate differences in the temporal profile of dopamine concentration transients caused by acute doses of nicotine, ethanol, and cocaine in the nucleus accumbens shell of freely moving rats. Despite differential release dynamics, all drug effects are uniformly inhibited by administration of rimonabant, a cannabinoid receptor (CB1) antagonist, suggesting that an increase in endocannabinoid tone facilitates the effects of commonly abused drugs on subsecond dopamine release. These time-resolved chemical measurements provide unique insight into the neurobiological effectiveness of rimonabant in treating addictive disorders.

Cannabinoids enhance subsecond dopamine release in the nucleus accumbens of awake rats

Dopaminergic neurotransmission has been highly implicated in the reinforcing properties of many substances of abuse, including marijuana. Cannabinoids activate ventral tegmental area dopaminergic neurons, the main ascending projections of the mesocorticolimbic dopamine system, and change their spiking pattern by increasing the number of impulses in a burst and elevating the frequency of bursts. Although they also increase time-averaged striatal dopamine levels for extended periods of time, little is known about the temporal structure of this change. To elucidate this, fast-scan cyclic voltammetry was used to monitor extracellular dopamine in the nucleus accumbens of freely moving rats with subsecond timescale resolution. Intravenous administration of the central cannabinoid (CB1) receptor agonist, R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl) methanone mesylate, dose-dependently produced catalepsy, decreased locomotion, and reduced the amplitude of electrically evoked dopamine release while markedly increasing the frequency of detected (nonstimulated) dopamine concentration transients. The CB1 receptor antagonist [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] reversed and prevented all agonist-induced effects but did not show effects on dopamine release when injected alone. These data demonstrate that doses of a cannabinoid agonist known to increase burst firing produce ongoing fluctuations in extracellular dopamine on a previously unrecognized temporal scale in the nucleus accumbens.

Coordinated Accumbal Dopamine Release and Neural Activity Drive Goal-Directed Behavior

Intracranial self-stimulation (ICSS) activates the neural pathways that mediate reward, including dopaminergic terminal areas such as the nucleus accumbens (NAc). However, a direct role of dopamine in ICSS-mediated reward has been questioned. Here, simultaneous voltammetric and electrophysiological recordings from the same electrode reveal that, at certain sites, the onset of anticipatory dopamine surges and changes in neuronal firing patterns during ICSS are coincident, whereas sites lacking dopamine changes also lack patterned firing. Intrashell microinfusion of a D1, but not a D2 receptor antagonist, blocks ICSS. An iontophoresis approach was implemented to explore the effect of dopamine antagonists on firing patterns without altering behavior. Similar to the microinfusion experiments, ICSS-related firing is selectively attenuated following D1 receptor blockade. This work establishes a temporal link between anticipatory rises of dopamine and firing patterns in the NAc shell during ICSS and suggests that they may play a similar role with natural rewards and during drug self-administration.

Dopamine release is heterogeneous within microenvironments of the rat nucleus accumbens

Many individual neurons within the intact brain fire in stochastic patterns that arise from interactions with the neuronal circuits that they comprise. However, the chemical communication that is evoked by these firing patterns has not been characterized because sensors suitable to monitor subsecond chemical events in micron dimensions have only recently become available. Here we employ a voltammetric sensor technology coupled with principal component regression to examine the dynamics of dopamine concentrations in the nucleus accumbens (NAc) of awake and unrestrained rats. The sensor has submillimeter dimensions and provides high temporal (0.1 s) resolution. At select locations spontaneous dopamine transient concentration changes were detected, achieving instantaneous concentrations of ∼50 nm. At other locations, transients were absent even though dopamine was available for release as shown by extracellular dopamine increases following electrical activation of dopaminergic neurons. At sites where dopamine concentration transients occur, uptake inhibition by cocaine enhances the frequency and magnitude of the rapid transients while also causing a more gradual increase in extracellular dopamine. These effects were largely absent from sites that did not support ongoing transient activity. These findings reveal an unanticipated spatial and temporal heterogeneity of dopamine transmission within the NAc that may depend upon the firing of specific subpopulations of dopamine neurons.

Spatially and Temporally Resolved Single-Cell Exocytosis Utilizing Individually Addressable Carbon Microelectrode Arrays

We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-mum-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 microm) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of high-density individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight spaces, as well as for developing multifunctional electrochemical microsensors.

Neurochemistry and electroanalytical probes.

Electroanalytical techniques have been applied to monitoring chemical events including neurotransmitter release during rodent behaviour and the release of zeptomoles of molecules from single cells. Transgenic mice models have been developed and studied to identify specific cell types in vitro. Characterization and surface modification of electroanalytical probes has enhanced the selectivity and sensitivity of measurements.

Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation

Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.

MS/MS methodology to improve subcellular mapping of cholesterol using TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60(+)/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.