R.M. Wightman

Regulation of transient dopamine concentration gradients in the microenvironment surrounding nerve terminals in the rat striatum.

Synaptic overflow of dopamine in the striatum has been investigated during electrical stimulation of the medial forebrain bundle in anesthetized rats. Dopamine has been detected with Nafion-coated, carbon-fiber electrodes used with fast-scan voltammetry. In accordance with previous results, dopamine synaptic overflow is a function of the stimulation frequency and the anatomical position of the carbon-fiber electrode. In some positions the concentration of dopamine is found to respond instantaneously to the stimulus when the time-delay for diffusion through the Nafion film is accounted for. In these locations the measured rates of change of dopamine are sufficiently rapid such that extracellular diffusion is not apparent. The rate of dopamine overflow can be described by a model in which each stimulus pulse causes instantaneous release, and cellular uptake decreases the concentration between stimulus pulses. Uptake is found to be described by a constant set of Michaelis-Menten kinetics at each location for concentrations of dopamine from 100 nM to 15 microM. The concentration of dopamine released per stimulus pulse is found to be greatest at low frequency (< or = 10 Hz) with stimulus trains, and with single-pulse stimulations in nomifensine-treated animals. The frequency dependence of release is not an effect of dopamine receptor activation; haloperidol (2.5 mg/kg) causes a uniform increase in release at all frequencies. The absence of diffusional effects in the measurement locations means that the constants determined with the electrode are those operant inside intact striatal tissue during stimulated overflow. These values are then extrapolated to the case where a single neuron fires alone. The extrapolation shows that while the transient concentration of dopamine may be high (200 nM) at the interface of the synapse and the extrasynaptic region, it is normally very low (< 6 nM) in the bulk of extracellular fluid.

Dopamine release and uptake are greater in female than male rat striatum as measured by fast cyclic voltammetry.

The present studies investigated sexual dimorphisms in dopamine release and uptake using fast-scan cyclic voltammetry in anesthetized rats and in brain slices. Electrical stimulation of the medial forebrain bundle of anesthetized rats at high frequency (60 Hz) elicited significantly more extracellular dopamine in the caudate nucleus of females than males. This sex difference was apparent over a range of current intensities applied to the stimulating electrode. Local electrical stimulation of brain slices in vitro verified in vivo results as more extracellular dopamine was elicited by single and 10 pulse stimulations in the caudate nucleus of females. Kinetic analysis of in vivo and in vitro dopamine overflow data indicated that dopamine release (the concentration of dopamine released per stimulus pulse) and the maximal velocity of dopamine uptake are greater in female rats, but the affinity of the transporter for dopamine was the same in males and females. None of these three parameters varied across the female estrous cycle. Linear regression analysis of dopamine release versus maximal uptake velocity data indicated a significant association of release and uptake sites in each sex and regression lines for males and females virtually overlapped. One explanation for these results is greater dopamine neuron terminal density in female caudate nucleus. These sexual dimorphisms in dopaminergic neurotransmission provide a novel, plausible mechanism to explain robust sex differences in behavioral responses of rats to psychostimulant drugs and may have implications for human neurological disorders and drug abuse.

Dopamine autoreceptor regulation of release and uptake in mouse brain slices in the absence of D3 receptors

The effects of the dopamine D(3) receptor, a putative autoreceptor, have been investigated by comparing behavioral and neurochemical properties of wild-type mice and mice with a genetic deletion of the D(3) receptor. The D(3) knock-out mice were modestly hyper-responsive to a novel environment relative to wild-type mice, and, consistent with this, quantitative in vivo microdialysis revealed elevated striatal dopamine extracellular levels. The dynamic actions of autoreceptors on electrically evoked dopamine release were examined in striatal brain slices from these animals and monitored with fast scan cyclic voltammetry at carbon-fiber microelectrodes. Quinpirole, a dopamine receptor agonist with potency at both D(2) and D(3) receptors, inhibited evoked dopamine in a dose-dependent manner with a slightly higher dose required in the knock-out animals (EC(50) of 60+/-10 nM in wild-type animals and 130+/-40 in D(3) knock-out animals; both curves had a Hill slope near 2). Dopamine synthesis inhibition with alpha-methyl-p-tyrosine caused released dopamine levels to decrease in each genotype. However, regulation of secretion by autoreceptors was still operant. Dose-response curves to quinpirole were unchanged in D(3) knock-out tissue, but secretion-regulated release exhibited a Hill slope decreased to 1 in the wild-type animals. In both genotypes, similar quinpirole-evoked increases in uptake rate were evident following synthesis inhibition. These data are consistent with the D(3) receptor having a small but significant role as a dopamine autoreceptor that partially regulates secretion, but not synthesis, in the caudate-putamen.

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.

Aversive stimulus differentially triggers subsecond dopamine release in reward regions

Aversive stimuli have a powerful impact on behavior and are considered to be the opposite valence of pleasure. Recent studies have determined some populations of ventral tegmental area (VTA) dopaminergic neurons are activated by several types of aversive stimuli, whereas other distinct populations are either inhibited or unresponsive. However, it is not clear where these aversion-responsive neurons project, and whether alterations in their activity translate into dopamine release in the terminal field. Here we show unequivocally that the neurochemical and anatomical substrates responsible for the perception and processing of pleasurable stimuli within the striatum are also activated by tail pinch, a classical painful and aversive stimulus. Dopamine release is triggered in the dorsal striatum and nucleus accumbens (NAc) core by tail pinch and is time locked to the duration of the stimulus, indicating that the dorsal striatum and NAc core are neural substrates, which are involved in the perception of aversive stimuli. However, dopamine is released in the NAc shell only when tail pinch is removed, indicating that the alleviation of aversive condition could be perceived as a rewarding event.

Extracellular dopamine dynamics in rat caudate–putamen during experimenter-delivered and intracranial self-stimulation

Intracranial self-stimulation is an operant behavior whereby animals are conditioned to press a lever in order to receive an electrical stimulation of their dopamine neurons. This paradigm is thought to stimulate brain reward pathways and, as such, has been used to clarify the role of dopamine in reward. Striatal extracellular dopamine concentrations were monitored during the acquisition and maintenance of self-stimulation and compared to dopamine release generated by experimenter-delivered and yoked stimulation. Fast-scan cyclic voltammetry in conjunction with carbon-fiber microelectrodes was used to monitor evoked dopamine release in the caudate-putamen during electrical stimulation of the substantia nigra/ventral tegmental area. The sub-second temporal resolution of fast-scan cyclic voltammetry coupled with the micron spatial resolution of the microelectrodes allowed for the measurement of dopamine neurotransmission in real-time. Single experimenter-delivered stimulations, identical to those used during self-stimulation, evoked dopamine release in the caudate-putamen both before and after the self-stimulation sessions. Likewise, yoked stimulations of the substantia nigra/ventral tegmental area delivered to animals untrained to perform self-stimulation resulted in an increase in extracellular dopamine levels. During training sessions, experimenter-delivered stimulations evoked dopamine release. However, as the animals began lever-pressing, extracellular dopamine levels subsequently declined. Taken together, these results suggest that dopamine functions as an alerting device, wherein increases in extracellular dopamine are obtained by unpredicted or novel rewarding stimuli, but not by those which can be anticipated.

Heterogeneity of evoked dopamine overflow wihin the striatal and striatoamygdaloid regions

The heterogeneity of evoked dopamine overflow in vivo was examined and compared in striatal and striatoamygdaloid regions of the rat. The characteristics of appearance and disappearance rates and the maximum concentration elicited were determined from overflow curves measured by fast-scan cyclic voltammetry. Overall, the characteristics of evoked dopamine overflow were quite variable in the striatum compared to the relative uniformity of overflow in the basolateral amygdaloid nucleus. In addition, there was a significant decrease in the extracellular disappearance rate of evoked dopamine with depth in the striatum. This gradient did not alter with passage from the caudate-putamen to the nucleus accumbens and no change was observed for the appearance rate or maximum concentration. In contrast, differences in evoked dopamine overflow within the striatoamygdaloid region were sharply defined dorsoventrally and appeared to be region-specific. Dopamine terminal fields in the striatum are not clearly demarcated into the caudate-putamen and nucleus accumbens, but may exist as a continuum. The uptake of dopamine appears to be the distinguishing characteristic for the regulation of extracellular dopamine levels in the striatum and the basolateral amygdaloid nucleus.

In vivo voltammetric monitoring of catecholamine release in subterritories of the nucleus accumbens shell

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes has been used to demonstrate that sub-second changes in catecholamine concentration occur within the nucleus accumbens (NAc) shell during motivated behaviors, and these fluctuations have been attributed to rapid dopamine signaling. However, FSCV cannot distinguish between dopamine and norepinephrine, and caudal regions of the NAc shell receive noradrenergic projections. Therefore, in the present study, we examined the degree to which norepinephrine contributes to catecholamine release within rostral and caudal portion of NAc shell. Analysis of tissue content revealed that dopamine was the major catecholamine detectable in the rostral NAc shell, whereas both dopamine and norepinephrine were found in the caudal subregion. To examine releasable catecholamines, electrical stimulation was used to evoke release in anesthetized rats with either stimulation of the medial forebrain bundle, a pathway containing both dopaminergic and noradrenergic projections to the NAc, or the ventral tegmental area/substantia nigra, the origin of dopaminergic projections. The catecholamines were distinguished by their responses to different pharmacological agents. The dopamine autoreceptor blocker, raclopride, as well as the monoamine and dopamine transporter blockers, cocaine and GBR 12909, increased evoked catecholamine overflow in both the rostral and caudal NAc shell. The norepinephrine autoreceptor blocker, yohimbine, and the norepinephrine transporter blocker, desipramine, increased catecholamine overflow in the caudal NAc shell without significant alteration of evoked responses in the rostral NAc shell. Thus, the neurochemical and pharmacological results show that norepinephrine signaling is restricted to caudal portions of the NAc shell. Following raclopride and cocaine or raclopride and GBR 12909, robust catecholamine transients were observed within the rostral shell but these were far less apparent in the caudal NAc shell, and they did not occur following yohimbine and desipramine. Taken together, the data demonstrate that catecholamine signals in the rostral NAc shell detected by FSCV are due to change in dopamine transmission.

Simultaneous measurement of oxygen and dopamine: Coupling of oxygen consumption and neurotransmission

Fast-scan cyclic voltammetry was used to simultaneously measure increases in dopamine concentration and decreases in O2 concentration evoked by brief electrical stimulation (two pulses at 10 Hz) in slices of rat caudate nucleus. Dopamine concentration began increasing immediately after the first pulse and reached a maximum within 200 ms of stimulation. The O2 concentration began to decrease 300-700 ms after onset of stimulus. Responses for both dopamine and O2 were dependent on external Ca2+ and were Cd2+ and tetrodotoxin sensitive. Only the O2 response was sensitive to CN- (0.15 mM). At short times after exposure to 50 microM ouabain, electrically stimulated dopamine overflow was increased by 150% and electrically stimulated changes in O2 concentration were unaffected. Maximum dopamine concentration was increased 28% by sulpiride (2 microM), 78% by L-DOPA (60 microM), 105% by nomifensine (10 microM) and unaffected by nialamide (10 microM). Maximum decrease in O2 concentration was increased by 25% by sulpiride and unaffected by nialamide, L-DOPA, or nomifensine. The decreases in O2 concentration are indicative of increased O2 consumption and are a measure of oxidative energy production evoked by electrical stimulation. The increase in dopamine is due to the release of dopamine balanced by uptake and serves as an indication of neurotransmitter activity. The results indicate that increases in oxidative energy production following electrical stimulation are dependent on external Ca2+ entry through Cd(2+)-sensitive channels. Possible mechanisms for this coupling are discussed.

Cue-evoked dopamine release in the nucleus accumbens shell tracks reinforcer magnitude during intracranial self-stimulation

The mesolimbic dopamine system is critically involved in modulating reward-seeking behavior and is transiently activated upon presentation of reward-predictive cues. It has previously been shown, using fast-scan cyclic voltammetry in behaving rats, that cues predicting a variety of reinforcers including food/water, cocaine or intracranial self-stimulation (ICSS) elicit time-locked transient fluctuations in dopamine concentration in the nucleus accumbens (NAc) shell. These dopamine transients have been found to correlate with reward-related learning and are believed to promote reward-seeking behavior. Here, we investigated the effects of varying reinforcer magnitude (intracranial stimulation parameters) on cue-evoked dopamine release in the NAc shell in rats performing ICSS. We found that the amplitude of cue-evoked dopamine is adaptable, tracks reinforcer magnitude and is significantly correlated with ICSS seeking behavior. Specifically, the concentration of cue-associated dopamine transients increased significantly with increasing reinforcer magnitude, while, at the same time, the latency to lever press decreased with reinforcer magnitude. These data support the proposed role of NAc dopamine in the facilitation of reward-seeking and provide unique insight into factors influencing the plasticity of dopaminergic signaling during behavior.