Donita L. Robinson

Monitoring Rapid Chemical Communication in the Brain

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 behavior3,4. These multiple interpretations of dopaminergic function have arisen because, until recently, a real-time 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.

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.

Transient changes in mesolimbic dopamine and their association with 'reward'

Mesolimbic dopaminergic neurons modulate complex circuitry in the ventral forebrain involved in reward processing, although the precise function of the dopaminergic input is debated. Electrophysiological measurements have revealed that mesolimbic dopaminergic neurons can fire in either tonic or phasic modes, and that phasic firing accompanies the alerting or anticipatory phases of reward. However, the neurochemical relevance of this rapid neuronal discharge within the reward processing circuitry is not yet clear, in part because of difficulty in interpretation of extracellular dopamine measurements. Herein, the nature of the information provided by different neurochemical techniques is critically discussed. Classical methods of monitoring dopamine reveal changes in extracellular dopamine resulting from tonic neuronal activity, but do not have the temporal resolution to distinguish concentration transients. However, recent advances in dopamine sensors now enable transient dopamine concentrations resulting from phasic firing to be positively identified and followed on a physiologically relevant timescale. This has enabled demonstrations of discrete, phasic dopamine signals accompanying rewarding or alerting stimuli. Thus, enhanced dopamine release at terminals appears to be coincident with phasic electrical activity at cell bodies. These accumulating data promise to help unravel the precise role of phasic dopamine transmission in reward processing.

Sub-second changes in accumbal dopamine during sexual behavior in male rats

Transient (200–900 ms), high concentrations (200–500 nM) of dopamine, measured using fast-scan cyclic voltammetry, occurred in the nucleus accumbens core of male rats at the presentation of a receptive female. Additional dopamine signals were observed during subsequent approach behavior. Background-subtracted cyclic voltammograms of the naturally-evoked signals matched those of electrically-evoked dopamine measured at the same recording sites. Administration of nomifensine amplified natural and evoked dopamine release, and increased the frequency of detectable signals. While gradual changes in dopamine concentration during sexual behavior have been well established, these findings dramatically improve the time resolution. The observed dopamine transients, probably resulting from neuronal burst firing, represent the first direct correlation of dopamine with sexual behavior on a sub-second time scale.

A ROLE FOR PRESYNAPTIC MECHANISMS IN THE ACTIONS OF NOMIFENSINE AND HALOPERIDOL

Psychomotor stimulants and neuroleptics exert multiple effects on dopaminergic signaling and produce the dopamine (DA)-related behaviors of motor activation and catalepsy, respectively. However, a clear relationship between dopaminergic activity and behavior has been very difficult to demonstrate in the awake animal, thus challenging existing notions about the mechanism of these drugs. The present study examined whether the drug-induced behaviors are linked to a presynaptic site of action, the DA transporter (DAT) for psychomotor stimulants and the DA autoreceptor for neuroleptics. Doses of nomifensine (7 mg/kg i.p.), a DA uptake inhibitor, and haloperidol (0.5 mg/kg i.p.), a dopaminergic antagonist, were selected to examine characteristic behavioral patterns for each drug: stimulant-induced motor activation in the case of nomifensine and neuroleptic-induced catalepsy in the case of haloperidol. Presynaptic mechanisms were quantified in situ from extracellular DA dynamics evoked by electrical stimulation and recorded by voltammetry in the freely moving animal. In the first experiment, the maximal concentration of electrically evoked DA ([DA](max)) measured in the caudate-putamen was found to reflect the local, instantaneous change in presynaptic DAT or DA autoreceptor activity according to the ascribed action of the drug injected. A positive temporal association was found between [DA](max) and motor activation following nomifensine (r=0.99) and a negative correlation was found between [DA](max) and catalepsy following haloperidol (r=-0.96) in the second experiment. Taken together, the results suggest that a dopaminergic presynaptic site is a target of systemically applied psychomotor stimulants and regulates the postsynaptic action of neuroleptics during behavior. This finding was made possible by a voltammetric microprobe with millisecond temporal resolution and its use in the awake animal to assess release and uptake, two key mechanisms of dopaminergic neurotransmission. Moreover, the results indicate that presynaptic mechanisms may play a more important role in DA-behavior relationships than is currently thought.

Real-Time Measurements of Phasic Changes in Extracellular Dopamine Concentration in Freely Moving Rats by Fast-Scan Cyclic Voltammetry

1. Introduction Rapid, transient changes in extracellular dopamine concentrations following salient stimuli in freely moving rats have recently been detected using fast-scan cyclic voltammetry (1,2). This type of neurotransmission had not been previously observed (for any neurotransmitter), but has been implicated by electrophysiological studies. Schultz et al. (3) reported synchronous burst firing of midbrain dopaminergic neurons following presentation of liquid reinforcers or associated cues. Such firing patterns would predictably produce transient (lasting no more than a few seconds), high concentrations (high nanomolar) of extracellular dopamine in terminal regions. This phasic dopaminergic neurotransmission has been heavily implicated in associative learning and reward processing, and therefore may prove essential in understanding the reinforcing actions of drugs of abuse. Fast-scan cyclic voltammetry is a real-time electrochemical technique that can detect dopamine by its redox properties. It is capable of monitoring monoaminergic neurotransmission in the brain on a subsecond time scale while providing chemical information on the analyte. This chapter describes how the properties of fast-scan cyclic voltammetry make it uniquely suitable for making chemical measurements of phasic dopaminergic neurotransmission in freely moving animals. To emphasize the potential of this technique, three examples of its use are highlighted. First we describe experiments testing

Disparity Between Tonic and Phasic Ethanol-Induced Dopamine Increases in the Nucleus Accumbens of Rats

BACKGROUND Dopamine concentrations in the nucleus accumbens fluctuate on phasic (subsecond) and tonic (over minutes) timescales in awake rats. Acute ethanol increases tonic concentrations of dopamine, but its effect on subsecond dopamine transients has not been fully explored. METHODS We measured tonic and phasic dopamine fluctuations in the nucleus accumbens of rats in response to ethanol (within-subject cumulative dosing, 0.125 to 2 g/kg, i.v.). RESULTS Microdialysis samples yielded significant tonic increases in dopamine concentrations at 1 to 2 g/kg ethanol in each rat, while repeated saline infusions had no effect. When monitored with fast scan cyclic voltammetry, ethanol increased the frequency of dopamine transients in 6 of 16 recording sites, in contrast to the uniform effect of ethanol as measured with microdialysis. In the remaining 10 recording sites that were unresponsive to ethanol, dopamine transients either decreased in frequency or were unaffected by cumulative ethanol infusions, patterns also observed during repeated saline infusions. The responsiveness of particular recording sites to ethanol was not correlated with either core versus shell placement of the electrodes or the basal rate of dopamine transients. Importantly, the phasic response pattern to a single dose of ethanol at a particular site was qualitatively reproduced when a second dose of ethanol was administered, suggesting that the variable between-site effects reflected specific pharmacology at that recording site. CONCLUSIONS These data demonstrate that the relatively uniform dopamine concentrations obtained with microdialysis can mask a dramatic heterogeneity of phasic dopamine release within the accumbens.

Nomifensine amplifies subsecond dopamine signals in the ventral striatum of freely-moving rats

The present experiment examined the effect of the dopamine transporter blocker nomifensine on subsecond fluctuations in dopamine concentrations, or dopamine transients, in the nucleus accumbens and olfactory tubercle. Extracellular dopamine was measured in real time using fast‐scan cyclic voltammetry at micron‐dimension carbon fibers in freely‐moving rats. Dopamine transients occurred spontaneously throughout the ventral striatum in the absence of apparent sensory input or change in behavioral response. The frequency of dopamine transients increased at the presentation of salient stimuli to the rat (food, novel odors and unexpected noises). Administration of 7 mg/kg nomifensine amplified spontaneous dopamine transients by increasing both amplitude and duration, consistent with its known action at the dopamine transporter and emphasizing the dopaminergic origin of the signals. Moreover, nomifensine increased the frequency of detected dopamine transients, both during baseline conditions and at the presentation of stimuli, but more profoundly in the nucleus accumbens than in the olfactory tubercle. This difference was not explained by nomifensine effects on the kinetics of dopamine release and uptake, as its effects on electrically‐evoked dopamine signals were similar in both regions. These findings demonstrate the heterogeneity of dopamine transients in the ventral striatum and establish that nomifensine elevates the tone of rapid dopamine signals in the brain.

Adolescent Alcohol Exposure Persistently Impacts Adult Neurobiology and Behavior

Adolescence is a developmental period when physical and cognitive abilities are optimized, when social skills are consolidated, and when sexuality, adolescent behaviors, and frontal cortical functions mature to adult levels. Adolescents also have unique responses to alcohol compared with adults, being less sensitive to ethanol sedative–motor responses that most likely contribute to binge drinking and blackouts. Population studies find that an early age of drinking onset correlates with increased lifetime risks for the development of alcohol dependence, violence, and injuries. Brain synapses, myelination, and neural circuits mature in adolescence to adult levels in parallel with increased reflection on the consequence of actions and reduced impulsivity and thrill seeking. Alcohol binge drinking could alter human development, but variations in genetics, peer groups, family structure, early life experiences, and the emergence of psychopathology in humans confound studies. As adolescence is common to mammalian species, preclinical models of binge drinking provide insight into the direct impact of alcohol on adolescent development. This review relates human findings to basic science studies, particularly the preclinical studies of the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium. These studies focus on persistent adult changes in neurobiology and behavior following adolescent intermittent ethanol (AIE), a model of underage drinking. NADIA studies and others find that AIE results in the following: increases in adult alcohol drinking, disinhibition, and social anxiety; altered adult synapses, cognition, and sleep; reduced adult neurogenesis, cholinergic, and serotonergic neurons; and increased neuroimmune gene expression and epigenetic modifiers of gene expression. Many of these effects are specific to adolescents and not found in parallel adult studies. AIE can cause a persistence of adolescent-like synaptic physiology, behavior, and sensitivity to alcohol into adulthood. Together, these findings support the hypothesis that adolescent binge drinking leads to long-lasting changes in the adult brain that increase risks of adult psychopathology, particularly for alcohol dependence.

Fast dopamine release events in the nucleus accumbens of early adolescent rats

Subsecond fluctuations in dopamine (dopamine transients) in the nucleus accumbens are often time-locked to rewards and cues and provide an important learning signal during reward processing. As the mesolimbic dopamine system undergoes dynamic changes during adolescence in the rat, it is possible that dopamine transients encode reward and stimulus presentations differently in adolescents. However, to date no measurements of dopamine transients in awake adolescents have been made. Thus, we used fast scan cyclic voltammetry to measure dopamine transients in the nucleus accumbens core of male rats (29-30 days of age) at baseline and with the presentation of various stimuli that have been shown to trigger dopamine release in adult rats. We found that dopamine transients were detectable in adolescent rats and occurred at a baseline rate similar to adult rats (71-72 days of age). However, unlike adults, adolescent rats did not reliably exhibit dopamine transients at the unexpected presentation of visual, audible and odorous stimuli. In contrast, brief interaction with another rat increased dopamine transients in both adolescent and adult rats. While this effect habituated in adults at a second interaction, it persisted in the adolescents. These data are the first demonstration of dopamine transients in adolescent rats and reveal an important divergence from adults in the occurrence of these transients that may result in differential learning about rewards.