Excitability of Dopamine Neurons

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CNS & Neurological Disorders - Drug Targets, 2006,5, 79-97 79

1871-5273/06 $50. 00+. 00 2006 Bentham Science Publishers Ltd.

Excitability of Dopamine Neurons: Modulation and PhysiologicalConsequences

M. Marinelli*, C.N. Rudick, X-T. Hu and F.J. White

Department of Cellular & Molecular Pharmacology. Rosalind Franklin University of Medicine and Science/The

Chicago Medical School. 3333 Green Bay Road, North Chicago, IL 60064, USA

Abstract: This aim of this chapter is to review literature on the excitability and function of dopamine neurons that

originate in the midbrain and project to cortico-limbic and motor structures (A9 and A10 dopamine pathways).

Electrophysiological studies on rodent or non-human primates have shown that these dopamine neurons are silent or

spontaneously active. The spontaneously active neurons show slow regular firing, slow irregular firing or fast bursting

activity. In the first section, we will review how neuronal firing is modulated by intrinsic factors, such as impulse-

regulating somatodendritic dopamine autoreceptors, a balance between inward voltage-gated sodium and calcium currents

and outward potassiumcurrents. We will then review the major excitatory and inhibitory pathways that play important

roles in modulating dopamine cell excitability.

In the second section, we will discuss how, in addition to being modulated by intrinsic and synaptic factors, excitability of

dopamine neurons can also be modulated by life experiences. Dopamine neurons change their firing rate throughout the

developmental period, their activity can be modified by stressful life events, and the firing mode can change as a

consequence of acute or repeated exposure to psychoactive drugs. Finally, these cells change their firing pattern in

response to behaviorally relevant stimuli and learning experiences.

We will conclude by discussing how changes in the physiology of the dopamine neurons could participate in the

development or exacerbation of psychiatric conditions such as drug addiction.

Keywords: Dopamine, electrophysiology, addiction, stress, synaptic, ventral tegmental area.

1. THE MESENCEPHALIC DOPAMINE SYSTEM:BASAL ACTIVITY

This section will review the literature on excitability ofmidbrain dopamine neurons. These neurons originate in theventral tegmental area (VTA) or the substantia nigra parscompacta (SNc). They project to cortico-limbic and motor

structures. We will first discuss how the activity of theseneurons can be evaluated using electrophysiological studies.Then, we will review how dopamine neuron activity isregulated by intrinsic neuronal properties as well as byexcitatory and inhibitory inputs.

1.1. Evaluating Action Potential Activity of DopamineNeurons

1.1.a. Techniques and Considerations

Electrophysiological studies have uncovered the patternof activity of midbrain dopamine neurons. In vivo recordingshave shown how these neurons function in the context ofnormal circuitry. In vitro recordings are more reductionistic,

but they have provided valuable information to understandthe mechanisms regulating neuronal activity.

During in vivo extracellular recordings, electrical signalsare fed into a high-impedance amplifier, filtered throughdefined low and high-pass bands that significantly influence

*Address correspondence to this author at the Department of Cellular &

Molecular Pharmacology. Rosalind Franklin University of Medicine and

Science/The Chicago Medical School. 3333 Green Bay Road, NorthChicago, IL 60064, USA; Tel: 847-578-8673; Fax: 847-578-3268; E-mail:

[email protected]

the shape of the signal, and are visualized on an oscilloscopeNeurons are defined as being dopaminergic if they fulfilseveral electrophysiological requirements. Among these is atypical dopamine signature, characterized by a triphasicwaveform (+/-/+) with a duration >2.5 msec from start to end[106]. Neurons that respond to these criteria have been

defined as dopaminergic because they also respond tonumerous pharmacological or electrophysiologicamanipulations in a manner that clearly indicates theidopaminergic identity (for details, see [35, 106]). Recentlythe dopamine signature criterion for dopamine neuronidentification was revisited [262]. By performingjuxtacel lular labeling with neurobiot in combined withtyrosine hydroxylase (the rate-limiting enzyme for dopaminsynthesis) immunohistochemistry, it was reported thaneurons should only be considered dopaminergic if theiwaveform is longer than the previously-reported ones. Ishould be noted, however, that the waveform duration (andshape) is largely dependent on the filter settings applied tothe recording amplifier. In the above study by Ungless and

colleagues, the filter settings were very different from thoseused in the majority of the studies reported in the literatureBecause of this, using such a filter (300 Hz 0.8 kHz)indeed, one should use different criteria. However, using themore classical filters (50 Hz - 0.8 kHz), the long establishedcriteria remain valid. Fig. 1a shows differences in waveformproduced by different filter settings.

1.1.b. Firing modes of Dopamine Neurons

Most in vivo recordings have been performed inanesthetized rodents. Although there is minor controversy a


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80 CNS & Neurological Disorders - Drug Targets, 2006, Vol. 5, No. 1 Marinelli et al

to whether neuronal activity in anesthetized conditionsparallels that seen in freely-moving animals [86, 96, 122,148], and whether different anesthetics show similar profilesof neuronal activity [70, 181], there is general consensusthat, in vivo, dopamine neurons can be silent, orspontaneously active.

Among the spontaneously-active dopamine neurons,action potential output can show slow regular, slow irregular

or fast bursting activity, as illustrated in Fig. 1b. Bursts arecharacterized by spikes clustered in high-frequency eventsthat generally exhibit spike-frequency adaptation andaccommodation [37, 106, 107, 113]. This pattern of activityis unique to the in vivo situation and it is not observed invitro, as shown in Fig. 1b [108, 141, 219, 234], probablybecause many of the excitatory synaptic inputs responsiblefor bursting are severed.

Fig. (1). (a) In vivo extracellular recordings of a dopamine neuron in the VTA. The same neuron was recorded using different amplifier filter

settings. Solid line: 400 Hz - 0.5 kHz (as used in studies by White et al., [79]); dashed line: 50 Hz 0.8 kHz (as often used in studies by

Grace and Bunney [106]); dotted line: 300 Hz 0.5 kHz (as used in a recent study by Ungless et al. [262]). Note that, according to the filter

settings, the same neuron can exhibit waveforms of different shape and duration so that the start of the signal, the trough of the negative

peak, and the end of the signal all appear at different times. Waveforms have been aligned at the onset of the positive peak (shown by the

vertical dotted line) to simplify comparisons of waveform duration. (b) Representative traces showing different patterns of rat dopamine

neuron activity recordedin vivo (top two traces, ~300g rat) or in vitro (bottom trace, ~120g rat).In vivo , dopamine neurons exhibit bursting

activity or irregular firing; in vitro firing is regular and it lacks the irregularity or bursting activity that is typically observed in vivo. Data

obtained by M. Marinelli, for the purpose of this review.

Fig. (2).In vivo extracellular recordings of rat dopamine neurons in the VTA. (a) Firing rate histogram of dopamine neurons showing

normal-distribution of firing rates. Average rate = 4.5 0.1 Hz. A very similar pattern of activity was first reported by Grace and Bunney

[108]. (b) Distribution of the number of spikes/burst obtained during in vivo extracellular recordings. Note that columns represent the

average number of spikes/burst obtained for each cell. Each cell was recorded for a period of at least 3 min. Given that we report averages

obtained for each cell, data are not expressed as whole integers (number of spikes/burst: mean= 3.1 0.1, mode=2, median=2.7). The left

column shows the percentage of neurons exhibiting bursting consisting uniquely of 2 spikes/burst. The next columns to the right show

averages ranging from 2.5 to 8.5 spikes/burst. Note that some burst events had more than 9 spikes/burst, but these were averaged out to lower

numbers when taking into consideration the overall activity of the cell. Data expressed as independent observations (i.e. without performing

averages for each cell) can be found in previous studies that initially characterized burst firing [107].

Data were obtained by pooling recordings from young adult (300-380g) nave rats across previously published [161, 162, 163, 165] or

unpublished experiments by M. Marinelli for a total of 286 neurons (a) or 214 neurons (b).


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Excitability of Dopamine Neurons CNS & Neurological Disorders - Drug Targets, 2006, Vol. 5, No. 1 81

In vivo, the firing rate (measured in spikes/sec, or Hz) isnormally-distributed across cells, (see Fig. 2a); it rangesfrom 0.5 Hz to approximately 10 Hz, with an average around4.5 Hz [106, 108]. In addition, bursting activity of dopamineneurons can exist to different degrees, from none (only asmall percentage of neurons), to moderate (most neurons) tohigh. It is important to note, however, that the degree ofbursting is on a continuum scale, and that sub-divisions into

different categories/degrees of bursting are arbitrary.Different aspects of bursting activity can be measured, suchas the amount of bursting (e.g. the proportion of spikesemitted in bursts or of the time spent bursting, the number ofburst events) or the characteristics of the bursts (e.g. numberof spikes/burst, burst duration, frequency of spikes within thebursts). Cells with lowest bursting activity generally displaynumerous single spikes (i.e. protracted periods of non-bursting activity) and a low proportion of two-spike bursts.Cells with high bursting activity exhibit a small number ofsingle spikes and numerous bursts, either two-spiked orlarger. Fig. 2b shows that most cells exhibit burst eventscharacterized by two-three spikes/burst; a smaller percentageof cells exhibit a greater number of spikes/burst.

In vitro recordings have been performed with a variety ofrecording techniques: extracellular, patch clamp (whole cellor cell-attached), or intracellular (with sharp electrodes). Theaction potential duration during patch clamp or intracellularrecordings is long (approximately 2.75 msec); in addition, inresponse to hyperpolarizing steps, dopamine neurons show aprominent hyperpolarization-activated cation current (Ih)when recorded in voltage clamp, or a voltage sag whenrecorded in current clamp. Expression of Ih in the VTA isindicative of dopamine neurons [128, 174, 187].

As mentioned above, dopamine neurons in the tissueslice (in vitro) do not spontaneously exhibit bursting activity,(see Fig. 1 b). Bursting has been observed, however in a

small percentage of neurons (18.3%) in immature animals inthe tissue slice, and so has irregular firing (28.3% ofneurons) [180]. In young adult animals, and in other studieson pre-weaning animals, instead, the pattern of output hasbeen shown to be extremely regular (pace-maker activity). Infact, in vitro, neuronal activity displays a very narrow

distribution of interspike intervals on a Gaussian curve(coefficient of variation). Situations that induce burstingactivity in the tissue slice (e.g. pharmacologicamanipulations, see below) broaden the distribution ointerspike intervals; therefore, they flatten the Gaussiancurve and increase the coefficient of variation (for examplesee [286]). This indicates that, in vitro, bursting activityconfers irregularity to spike timing. This is the opposite o

what is seen in vivo, where neurons with low or no burstingactivities exhibit a broad coefficient of variation (andtherefore irregular spiking). Instead, cells with greaterbursting activity exhibit a tighter distribution of interspikeintervals, as shown in Fig. 3. This points to the differencebetween natural burst ing in vivo and those producedartificially in vitro by pharmacological treatments in thetissue slice and suggests that, in vivo, the temporal variabilityof spike production during bursting is lower than inpharmacologically manipulated slices.

In vivo, bursting activity has been shown to increase therelease of dopamine in the terminal regions [103, 199, 243]However, given the rapid reuptake of the neurotransmitter bydopamine transporters, such phasic increases in peri-synapticdopamine are only detectable using techniques with goodtemporal resolution (e.g. fast-scan cyclic voltammetry ochronoamperometry [77, 99, 200]), unless dopaminereuptake is pharmacologically blocked [92]. When dopaminereuptake is experimentally blocked (e.g. by theadministration of nomifensine), changes in bursting do resulin changes in extracellular levels of dopamine in postsynaptic sites that are detectable with microdialysitechniques. In the absence of changes in dopamine reuptakethe accumulation of extracellular dopamine measurable withmicrodialysis techniques is the consequence of an increase inthe number of active neurons with minimal influence of theibursting pattern. In these circumstances, the increase inpopulation activity results from the activation of silentdopamine neurons after removal or reduction of inhibitoryinputs [92]. This indicates that a complex interplay betweenfiring rate, pattern of activity and number of active neuronparticipate in mainta ining phasic and tonic dopaminetransmission.

Fig. (3). Distribution of interspike intervals in rat VTA dopamine cells recorded in vivo. Figures are obtained from three different cells with

different levels of bursting activity: 80%, 45% or 15% of bursting spikes. Greater bursting activity is associated with a more narrow

distribution of interspike intervals (note the narrow distribution of interspike intervals for the cell with 80% of bursting spikes compared with

the flattened distribution for the cell firing 15% of bursting spikes). Instantaneous interspike intervals were binned in 10 msec intervals (from

0-10 msec to 2123-2140 msec) and plotted as percentage of observations (Y axis) occurring at each particular bin (in msec on the x-axis; axis

truncated at 1000 msec in the above figures). Data obtained by M. Marinelli for the purpose of this review.


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1.2. Regulation of Dopamine Neuron Activity

1.2.a. Intrinsic Modulation of Dopamine Neuron Activity

Numerous intrinsic factors regulate impulse activity ofdopamine neurons [142, 167, 192]; we will consider some ofthe most important ones, such as impulse-regulatingautoreceptors, calcium-activated potassium channels,calcium channels and Ih. Some receptors located on the

somatodendritic area of dopamine neurons require synapticinput to be activated. Therefore, although these receptors aretechnically intrinsic to the dopamine neuron, they will bediscussed in the section on synaptic modulation.

Impulse-Regulating Autoreceptors

Impulse-regulating autoreceptors play an important rolein providing an efficient auto-feedback onto neuronalactivity. These receptors are of the D2/D3 dopamine receptorfamily and are located in the somatodendritic region of VTAdopamine neurons [36, 51, 176]. They are activated bysomatodendritically-released dopamine [11, 48, 135] andreduce dopamine neuron activity by activating potassiumconductances and thereby hyperpolarizing the cell. More

precisely, they activate Gi/o proteins; the dimerdissociates from the heterotrimeric protein, binds andactivates G-protein-coupled inward-rectifying potassium(GIRK) channels [57, 127, 152, 176, 278]. Although therehave been several controversies as to whethersomatodendritically-released dopamine acts in a paracrinefashion, and diffuses over some distance prior to exerting itsaction on dopamine receptors [212], recent evidence showsthat dopamine release depends on neuronal depolarization;this exocytotic release can directly inhibit neuron excitability[12]. Nevertheless it is possible that autoreceptors do notexclusively respond to dopamine that is released from thesame neuron, because dopamine autoreceptor sensitivity hasbeen shown to occur after partial dopamine lesions [118].

In the in vivo situation, functional sensitivity of dopamineautoreceptors can be determined by examining the dose-dependent neuronal inhibition following local (intra VTA)administration of D2/D3 receptor agonists. It can also betested following systemic (intravenous) administration ofautoreceptor agonists, as, when given systemically atappropriate doses, these drugs primarily act at thesomatodendritic level [1, 98, 207]. Using these approaches,(see Fig. 4), it was shown that fast-firing dopamine neuronshave sub-sensitive impulse-regulating autoreceptors,conversely, slow-firing neurons exhibit greater sensitivity ofthese receptors [166, 276]. This effect is independent ofdifferences in neuronal inputs because slow-firing cells are

more sensitive to autoreceptor-mediated inhibition evenwhen they are driven to a faster rate by local application ofglutamate [276]. Overall, these findings suggest that thefunctional state of somatodendritic dopamine autoreceptorsdetermines the level of activity of dopamine neurons.

Calcium-Activated Potassium Channels

Potassium channels come in many forms; both voltage-gated and calcium-activated small conductance potassium(SK) channels are implicated in midbrain dopamine neuronfiring. Intracellular injection of the potassium channelblocker TEA increases the firing rate of dopamine neuronsand produces burst-like activity [107]. In addition, by

combining real-time single-cell RT- PCR with slice patchclamp electrophysiology, it was shown that voltage-gated Atype potassium channels play a key role in modulating thepacemaker act ivity of dopamine neurons [157]. Thipacemaker act ivi ty is coupled with A-type potassiumchannel density and number of subunits that make up thepotassium channel, indicating that both the amount and thetype of gene expression are important in regulating

dopamine neuron firing.

Fig. (4). Dopamine neuron activity following the administration o

the D2/D3 receptor agonist quinpirole. Quinpirole produces a dose

dependent decrease in firing rate in all neurons, however, cells with

initially higher baseline firing rates require more quinpirole to be

silenced than cells with low firing rates. In this case neurons were

divided as high or low firing cells according to the median spli

(High >4.5 Hz, n=15 cells; Low


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glutamate [172, 190, 191]. Glutamate acts via three generalgroups of receptors that are expressed on dopamine neurons:AMPA, NMDA and metabotropic [46, 239, 252]. BothAMPA and NMDA receptors are ion channels that areopened by glutamate binding and allow inflow of sodiumand calcium, whereas metabotropic glutamate receptors(mGluRs) bind glutamate and modulate ion channel activityvia second messenger systems.

AMPA and NMDA receptors mediate the majority ofexcitatory input to dopamine neurons [167, 192, 270, 271].In vivo studies have shown that application of both AMPAand NMDA receptor agonists (via microiontophoresis)increases the firing rate of spontaneously-active dopamineneurons of the SNc and VTA. Both AMPA and NMDAreceptor activation increases bursting activity in the SNc,however, bursting activity in the VTA appears to beindependent of AMPA receptors [49, 50, 190, 292]. In vitrostudies in the tissue slice also show the excitatory role ofthese receptors on dopamine neurons; both application ofAMPA and NMDA evokes an inward current and increaseneuronal firing in a dose-dependent manner [177, 270, 271,287, 288]. NMDA receptor activation has been shown toincrease bursting activity only in certain conditions,particularly in the presence of hyperpolarizing currents orwhen calcium-activated potassium currents are blocked [129,130, 229]. Together, these findings underline the importantexcitatory role of AMPA and NMDA glutamate receptoractivation in dopamine neuron regulation; however, aspointed previously, it should be noted that this type ofbursting produced in the slice does not resemble thenaturally-occurring one in vivo.

Activation of mGlu receptors can have either anexcitatory or inhibitory role on dopamine neurons,depending on how the receptors are activated. Fast activationof group 1 mGlu receptors located on dopamine neurons, via

synaptically-released glutamate, produces inhibitory post-synaptic currents and mediates a slow inhibition of dopamineneurons [90]. These inhibitory currents are the consequenceof mobilization of intracellular calcium stores, which in turnactivates inhibitory calcium-dependent potassium currents.However, with prolonged activation, the inhibitory responseproduced by mGlu receptor activation desensitizes.Therefore, continuous activation of these receptors induces aslowly-developing sodium-dependent excitation, which canlead to an increase in impulse activity of dopamine cells [90,173, 178, 294]. It has also been suggested that activation ofmGlu receptors modifies dopamine neuron activityindirectly, by acting on presynaptic glutamatergic terminalsso as to modify glutamate release. As for the post-synaptic

effects, these pre-synaptic ones can also be either stimulatoryor inhibitory (according to the level of activation of thephospholipase C pathway) and produce, respective ly,enhanced or reduced NMDA-mediated excitatory currents inpost-synaptic cells [18, 101, 116]. On a similar line, at highdoses, bath application of glutamate to isolated dopamineneurons has been shown to enhance the spontaneous firing,but also to temporarily inhibit firing through two distinctcalcium-dependent mechanisms: via activation of NMDAand AMPA receptors or mGluRs [140]. Recently, it has alsobeen shown that the action of mGluRs is linked to activationof transient receptor potential channels [13].

Together, these findings indicate that bursting may beascribed, at least in part by a complex combination oglutamate receptors. Activation of these receptors can exerboth excitatory and inhibi tory roles, either direct ly, oindirectly, by modifying glutamate release or the activity oother channels.

Measuring Excitatory Synaptic Strength and Plasticity

Given the important role of excitatory glutamatergicsynapses onto dopamine neurons, recent studies haveaddressed the question as to whether these synapses canundergo synaptic plasticity. A classic way to determinesynaptic plasticity is to determine long term potentiation(LTP) of excitatory inputs using in vitro patch clamprecordings that evaluate excitatory post synaptic current(EPSCs) or potentials (EPSPs). In these experiments, achange in the strength of synaptic input is indicated by anincrease in evoked EPSC or EPSP amplitude following thepa ir ing of synapt ic st imulat ion wi th post -synaptidepolarization. Using perforated whole-cell patch clamprecordings, LTP was demonstrated in midbrain dopaminebut not GABAergic neurons [19, 193]. The increase elicited

at excitatory synapses on dopamine neurons was NMDAreceptor dependent and mGluR independent. Further studiesdemonstrate that long-term depression (LTD) is also a formof synaptic plasticity exhibited by VTA dopamine neuron[132, 133, 253]. The LTD that is elicited at excitatory inputsto dopamine neurons depends on NMDA receptor activationand requires an increase in intracellular calcium, but is Ltype calcium channel independent. Similarly, this LTD doenot rely on activation of mGluRs but can be blocked byactivation dopamine D2-class receptors, further underscoringthe role of these autoreceptors in modulating neuronaexcitability [132, 133, 253].

GABAergic Input

GABA, the major inhibitory neurotransmitter, isresponsible for most synaptically-induced inhibition odopamine neuron activity. GABAergic inputs arise mostlyfrom the striatal complex; it includes inputs from the NAccaudate nucleus, globus pallidus and ventral pallidum (foreview, see [112, 237, 238, 266, 274]). Another importanGABAergic input arises from local neurons in the midbrain[10, 115, 150].

GABA afferents from the striatal complex are oftendefined as a long-loop GABAergic pathway. Whereas thisinhibitory pathway plays an important role in certainconditions, such as after administration of psychostimulandrugs [32, 34, 79, 206], the extent to which it operates under

anesthetized basal states is controversial. Hemitransection othe long-loop pathway fails to modify basal impulse activityof both VTA and SNc dopamine cells [79, 207]. During theacute phase of kainic acid injections into the dorsal striatumthere is a transient (< 12h) decrease in the percentage oactive of SNc dopamine cells due to depolarizationinactivation, as revealed by iontophoretic administration oGABA [25]. Similar kynurinic acid lesions of the NAcproduce a transient decrease in firing rate and burst firing oVTA dopamine neurons [91].

The presence of GABA neurons within the VTA explainsmany biphasic effects produced by stimulation of brainstructures projecting to the midbrain, or by systemic


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pharmacological treatments. Thus, stimulation of afferentsonto dopamine neurons can have an initial response that isimmediately followed by an opposite effect [109, 195, 258].This is because afferents can form synapses onto bothdopamine and GABA neurons. Synapses onto dopamineneurons produce direct neuronal excitation or inhibition.Synapses onto GABA neurons will have the opposite effects;they will change the activity of GABA neurons, which in

turn will modify the activity of the dopamine neurons. Onthe same line, due to this anatomical arrangement, systemicadministration of GABAergic drugs can have aparadoxical excitatory effect on dopamine neurons [105,272].

Direct inhibition of dopamine neurons by activation ofGABAA receptors occurs on a fast time scale. Systemic orlocal (intra VTA) application of the GABAA receptor agonistmuscimol increases dopamine neuron firing while reducingthe burst firing pattern and regularizing the firing pattern [80,83]. On the other hand, GABAA receptor antagonistsbicuculline and picrotoxin enhance both firing and burstingactivity. Studies show that the GABAA receptor agonistsmodulate dopamine neuron activity by opening ionotropicreceptors which directly allows chloride influx, therebyhyperpolarizing the cell membrane [128, 244].

In contrast to GABAA receptors, GABAB receptorsmediate inhibition of midbrain dopamine neurons via aslower time course. Local administration of the GABABreceptor agonist baclofen reduces firing and burst firing andregularizes dopamine neuron firing rhythm [82, 84].Conversely, application of GABAB receptor antagonistsincreases dopamine firing and bursting, and prevents theeffects produced by GABAB receptor activation [47, 81, 82,197]. Experiments using brain slices show that GABABreceptor agonists modulate the activity of dopamine neuronsby activating Gi/o proteins; the dimer dissociates from the

heterotrimeric protein and binds to GIRK channels, whichare differentially expressed by dopamine and non-dopamine(GABAergic) neurons within the VTA [54]. The consequentopening of these channels allows potassium outflow, whichhyperpolarizes the cell membrane thereby inhibitingneuronal activity [54, 273]; this mechanism of neuronalinhibition produced by activation of metabotropic GABABreceptor activation is similar to the one produced byactivation of dopamine D2 receptors (see above).

2. THE MESENCEPHALIC DOPAMINE SYSTEM:CHANGES DURING LIFE

This second section will review how dopamine neuronscan be modulated during the lifespan of an individual. We

will first give a brief overview of naturally-occurringchanges in dopamine neuron impulse activity. These includechanges over time, during development or naturally-occurring differences in dopamine neuron activity acrossindividuals. We will then focus on changes in neuronalactivities induced by life events, such as exposure to stressfulconditions or to addictive drugs or to behaviorally relevant(rewarding) stimuli and learning experiences.

In these sections, we refer to basal activity ofdopamine neurons to denote the general level of activity ofthese cells (as seen in the previous section, this is composed

of regular and bursting firing patterns). Instead, we refer tophasic activity of these neurons to denote rapid changes infiring pattern in response to specific stimuli.

2.1. Naturally-Occurring Differences in DopamineNeuron Activity

2.1.a. Dopamine Neurons During Development

During development, the dopamine system undergoeimportant changes. Dopaminergic innervations to theforebrain and dopamine receptors in the NAc and striatumincrease rapidly from birth to reach a peak duringadolescence, around postnatal day (PND) 40 and decreasegradually thereafter [5, 249]. Dopamine levels in the striatumincrease from birth to adolescence, although it is uncleawhether such differences are present in the NAc [3, 4, 155250]. Concerning impulse activity of midbrain dopamineneurons, developmental changes have been studied ovedifferent time periods. Firing and bursting of midbrainneurons increases progressively from birth to early (PND 2835) adolescence [205, 251], when autoreceptors in themidbrain appear to be functionally mature [268]. Activityduring mid adolescence (PND 35-42) was not assessed inthese experiments, but, recent findings from our group, (seeFig. 5), indicate that dopamine neuron activity (firing andbursting) is higher during this time than during adulthood(PND 75-90). Additional studies have also shown thadopamine neuron activity declines progressively duringadulthood [95, 154]. Taken together, we can infer thadopamine neuron activity exhibits and inverted U-shapedcurve over an individuals lifespan. Activity is low at birth, ishows a gradual increase during early adolescence, possible peak during mid adolescence, and a gradual declinethereafter to return to low levels during old age.

Fig. (5). Dopamine neuron activity in Adult (PND 75-90, n=14

cells) and Adolescent (PND 35-45, n=15 cells) rats. Adolescent rat

exhibit enhanced neuronal activity compared with Adult rats (T

test, p


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2.1.b. Inter-Individual Differences in Dopamine NeuronActivity

Dopamine neuron activities are not identical acrossindividuals. As shown in Fig. 2a, firing rates show a normaldistribution and range from low (0.5 Hz) to high (10 Hz). Asfor most biological parameters, differences are in part duesto inter-individual variance. It was recently shown thataction potential output (both firing and bursting) variesacross individuals that exhibit strong vs . low locomotoractivity when placed in a novel environment. Animals withhigh reactivity to a novel environment have high-levels ofdopamine neuron activity, whereas animals with a lowreactivity to the same environment have reduced dopamineactivity [166]. This enhanced dopamine neuronal output isparalleled by enhanced dopamine levels in the NAc [22, 119,202, 216]. Interestingly, these differences in dopamineneuronal activity across individuals are predictive ofdifferences in susceptibility to acquire cocaine self-administration behavior [166]. This suggests that the levelof activity of dopamine neurons can be a predisposingfactor that could favor addiction. This is not surprising,given the important role of these neurons in addiction-associatedbehaviors [275, 281] and will be discussed moreextensively in the next sections.

2.2. Life Events-Induced Changes in Dopamine NeuronActivity

2.2.a. Dopamine Neurons and Addictive Drugs

Effects of Addictive Drugs on Dopamine Neuron Activity

As mentioned above, the dopamine system is one of themajor players mediating the rewarding effects of addictivedrugs [85, 214, 275, 281]. Different addictive drugs have thecommon action of increasing dopamine in the striatalcomplex [23, 65, 121], however, their action on dopamine

neuron excitability varies. Acute administration ofpsychostimulant drugs such as cocaine [38, 79, 153],amphetamine [209, 269], methylphenidate [24, 88, 209] andcaffeine [241] decreases dopamine neuron activity. Thiseffect is likely due to the fact that psychostimulant drugsenhance extracellular concentrations of dopamine (either byenhancing neurotransmitter release or by preventing itsreuptake) in the somatodendritic region of dopamine neurons[23]. This increase in dopamine activates somatodendriticimpulse-regulating autoreceptors that inhibit neuronalactivity. Evidence also exists for amphetamine to have twoactions on dopamine neurons: direct via autoreceptors, andindirect via feedback from forebrain structures via differentneurotransmitters [33, 196, 198].

Although psychostimulant drugs decrease dopamineneuronal activity while the drugs are onboard, withdrawalfrom repeated administration of these drugs has oppositeeffects and produces a transient increase in the firing andbursting activity of dopamine neurons. This is seen both afterrepeated non-contingent drug exposure, via experimenter-administered injections, or voluntary drug exposure, vi aintravenous self-administration, (see Fig. 6) [163, 275].Because of its transient nature, the enhancement in dopaminecell excitability produced by withdrawal from drug exposureshould not influence the expression of addictive behaviors

Fig. (6). Dopamine neuron activity after self-administration o

saline or at different withdrawal time points from cocaine self

administration (500g/kg intravenously for 7 days, average daily

intake 15 mg/kg). Compared with animals that self-administered

saline (n=54 cells), animals that self-administered cocaine show a

transient increase in the firing rate of VTA dopamine cells at early

withdrawal times from the self-administration procedure [ANOVA

Group effect F(4,179)=9.13, p


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These differences in the duration of the neuroadaptationinduced by cocaine self-administration and withdrawal mayindicate that drug-vulnerable individuals have decreasedcapacity for recovery after exposure to drugs; given the datapresented above, it is possible that such a profile couldfacilitate the development of addiction.

Exposure to other drugs of abuse, such as alcohol,cannabinoids or opiates has opposite effects as those seen for

psychostimulant drugs. These drugs acutely increasedopamine cell activity [30, 102, 114, 179], when the drug isonboard, and then decrease it following withdrawal fromtheir repeated administration [8, 71, 72, 73]. The increase indopamine neuron activity produced by acute administrationof opiates is due to activation of opioid receptors; thisinhibits local GABA neurons thereby removing tonicinhibition of dopamine cells [114, 128]. In fact, animalslacking opoid receptors exhibit enhanced GABAergicinput to dopamine neurons as shown by increased frequencyof spontaneous inhibitory post-synaptic currents[168] aswell as reduced dopamine neuron activity [169].

Overall, these data indicate that dopamine neurons

respond to addictive drugs with either excitation orinhibition, according to the mechanism of action of thedrugs. The effects observed while the drug is onboard areusually the opposite of those produced during withdrawalfrom repeated drug treatment. It is possible that theseneuroadaptations represent a compensatory response suchthat repeated administration of drugs that reduce impulseactivity produces rebound increase in cell activity upondiscontinuation, whereas repeated administration of drugsthat increase impulse activity has opposite effects.Nevertheless, enhanced dopamine neuron excitabilityassociated with exposure and withdrawal to addictive drugs(either acute, or repeated) appears to be important for theinduction of addiction-associated behaviors such as

behavioral sensitization.

Effects of Addictive Drugs on Plasticity of ExcitatoryInputs onto Dopamine Neurons

The increase in dopamine cell activity observed after thediscontinuation of psychostimulant drugs mentioned above iscoupled with increased reactivity of these cells to glutamate[293]. This suggests that changes in the strength ofglutamatergic input to dopamine neurons could beresponsible for such an increase in dopamine neuron activity.Thus, excitatory synaptic inputs to dopamine cells have beenshown to undergo plasticity [19, 193], and the manner inwhich these inputs are integrated after exposure to drugs ofabuse has been the focus of numerous recent studies.

One way to evaluate the status of synaptic plasticity forexcitatory inputs onto dopamine neurons has been toquantify the ratio of AMPA to NMDA receptor-mediatedsynaptic currents (AMPA/NMDA ratio). HigherAMPA/NMDA ratios have been shown to correlate directlywith the relative degree of synaptic potentiation [217, 264].In addition, paired-pulse stimulation assays, whereby onemeasures the ratio between the amplitude of two EPSCsevoked at brief intervals, are a good method to evaluaterelease probability [75, 94, 188, 226]. Thus, the ratiobetween the amplitude of the second and first EPSC evoked

at brief intervals reflects whether there is facilitation otransmitter release (ratio >1), depression (ratio


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indicating a decrease in cue-controlled drug-seeking as well.In addition to decreasing responding to psychostimulantdrugs, other studies have also shown that activation ofGABAB receptors can inhibit certain aspects of heroin self-administration behavior [67, 289, 290]. Together, thesefindings indicate that activation of GABAB receptorsattenuates the reinforcing properties of the drugs as well asthe conditioned reinforcing properties of the drug-associated

conditioned stimuli. These effects are not attributable to ageneral decrease in motor activity or motivational drive,because similar doses of baclofen do not modify respondingfor food and do not impair locomotor activity [213, 236]. Inthese studies, the receptor agonists were administeredsystemically; therefore, it is possible that these drugs actedon brain substrates different from dopamine neurons;nevertheless, similar findings were reported after directinfusion of baclofen into the VTA [29], indicating that theeffects are, at least in part, attributable to an effect in themidbrain dopamine region.

Another way to test the effects of decreases in dopamineneuron activity on self-administration behavior is to usedrugs that modify excitatory glutamatergic transmission,which, in turn, is known to modify dopamine neuronactivity. These results, however, are not always clear,probably given the non-selective or the non-specific effectsof some of the systemically-administered receptor agonistsor antagonists (see [15, 123, 208, 220]). More selectivestudies with local infusions, however, show that directadministration of ionotropic glutamate receptor blockers intothe VTA decreases heroin self-administration [291],consistent with the idea that the level of activity of dopamineneurons could modulate self-administration behavior.

In addition to being important for self-administrationbehavior during the self-administration session, dopamineneuron activity also appears to have an important role in

drug seeking behavior. Seeking behavior is an importantaspect to study because it represents a valid model of drugrelapse in humans [137, 232]. Such studies have shown thatadministering drugs that putatively decrease dopamineneuron activity, such as autoreceptor agonists [163], or drugsthat inactivate the VTA [21, 67, 68, 171], both reduce drug-seeking behavior in animals that were trained on self-administration tasks, (see Fig. 7). This suggests that anexperimentally-induced decrease in dopamine neuronactivity could be responsible for decreasing seekingbehavior. On the other hand, treatments that are known toenhance dopamine neuron activity, such as stimulation ofafferent structures or the local infusions of glutamate ormorphine, increase seeking behavior [240, 267]. It should be

noted however, that direct stimulation of the medialforebrain bundle with repetitive 3-spike bursts, does notproduce an increase in drug seeking behavior [267]. Thissuggests a physiological increase in bursting, such as thatobtained with synaptic stimulation, i.e. several spikes/burstfollowed by long pauses as shown in Fig. 1b , might benecessary to enhance seeking behavior.

Despite the positive relationship between experimentally-induced increases in dopamine neuron activity and enhancedseeking behavior, there is no relationship between theenhanced dopamine neuron activity observed at shortwithdrawal times from psychostimulant self-administration

and drug seeking behavior. In fact, seeking behavioincreases over time, and is usually lowest at short withdrawatimes from drug intake [111, 259] when, instead, neuronaactivity is highest [163]. This dissociation could beexplained by the fact that at early withdrawal timesincreased impulse activity is often accompanied by an upregulation of dopamine transporter levels and might notranslate into a functional increase in dopaminergic

transmission [203]. In addition, we do not know the timecourse for the development of increased impulse activity indopamine cells after cocaine self-administration so it islikely that sudden changes from baseline are important totrigger seeking behavior.

Relationship Between Phasic Changes in DopamineNeuron Activity and Operant Responding for Drugs

We will further examine the relationship betweendopamine neuron activity and addiction by investigating howdopamine neurons fire in response to or anticipation of drugrewards (i.e. during self-administration tasks). As with aloperant responding, caution should be taken in interpretingthe results, because changes in firing rates prior to

performing a response could signal increased incentivewanting, expectation or anticipation, or simply motoractivation. In addition, once the drug is onboard, neuronaactivity will be influenced by the pharmacological propertieof the drug, thereby potentially masking the activity odopamine neurons during initiation of drug respondingNevertheless, careful analysis of the timing of neuronaactivity during responding to drugs that do not normallydampen neuronal firing can still give us important insightson the role of these neurons in reward-responding.

Recording dopamine neurons in freely moving smalanimals such as rodents, while they are engaged in operanresponding has only been attempted by a few investigatorsWe are aware of only two studies performed during heroinself-administration [147, 149]. These careful studies clearlyshow that neuronal activity peaks right before each selfinfusion. More precisely, for the first self-infusion, dischargrate increased before the first lever-press to obtain the drugand then remained elevated after the infusion. After thesecond infusion, phasic increases were only seen prior tolever pressing, whereas infusion delivery produced inhibitionof neuronal activity. The inhibition was followed by agradual increase in firing rate, which peaked again just priorto the next heroin infusion. The nature of the decrease inneuronal activity following the reward itself is unclear, andcould be related to behavioral (freezing behavior) opharmacological factors; however, it is clear that phasic

increases in dopamine neuron activity are associated with theactivational-motivational-drive towards the reward, but nowith the reward itself. This suggests that dopamine neuronactivity could be a driving force for goal-directedbehavior.

We are not aware of studies examining firing patterns ofVTA dopamine neurons during self-administration ococaine. However, recent studies monitoring changes inbrain temperature (which reflect neuronal activity [146]have shown that, similar to heroin self-administrationactivity (temperature) in the VTA increases prior to cocaineself-infusions [144, 145] and decreases about one minuteafter the reward itself. As for the heroin self-administration


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studies, the first infusion of cocaine is not followed byneuronal inhibition (decrease in temperature), but bycontinuous activation. To assess the relative behavioral andpharmacological component of thi s effect , the authorsstudied temperature changes in animals that received non-

contingent cocaine infusions at the pattern mimicking self-administration. These animals show similar patterns ofactivity as those performing operant responding, but they donot show neuronal activation preceding their first daily druginfusion. Again, these results indicate that neuronalactivation coincides with drug-seeking behavior thatprecedes the actual drug intake. Together with the abovestudies, these findings suggest that dopamine neuron activityis associated with motivational arousal, or drug seeking,rather than with rewardsper se. The concept that an increasein dopamine neuron activity could favor goal-directedbehavior is in line with our findings showing that heightenedbaseline firing and bursting activity of midbrain dopaminecells is associated with enhanced acquisition of cocaine self-

administration behavior; conversely, decreased cell activityis associated with resistance to develop this behavior [161,166].

2.2.b. Dopamine Neurons and Natural Rewards

Studies examining changes in firing patterns of dopamineneurons during presentation of rewards indicate thattemporal changes in action potential activity of dopaminecells may exert a critical function in reward-relatedbehaviors [53, 104], however whether such changes in firingrate increase learning, the impact of the reinforcer, the

associative reward-learning,

the conditioned

reinforcementthe attention towards such salient stimuli, the incentivesalience of a stimulus, or a combination of these all, is still aquestion of debate [14, 64, 218, 221]. We will discuss brieflysuch studies and will limit our analysis to work centered on

dopamine neuron excitability. We refer to excellent, morethorough reviews on the role of dopamine transmission ingeneral on different aspects of reward responding [14, 41120, 143, 224, 282].

Relationship Between Phasic Changes in DopamineNeuron Activity and Pavlovian Conditioning

Numerous studies have examined the response odopamine neurons using Pavolovian conditioning. Animalare presented with a conditioned stimulus (e.g. an image orlight), which is followed by a reward (e.g. juice). Thesestudies show that a large percentage of dopamine neuronsincrease firing rate after the initial presentation of the reward(when the reward is unexpected). During the early phases o

the Pavlovian task, when the animal is learning theassociation between the conditioned stimulus and the rewarddopamine neuron activity increases phasically during boththe reward and the conditioned (reward-predicting) stimulusOnce the task is fully learned, presentation of the rewardceases to elicit increases in neuronal activity; such increaseare transferred entirely to the conditioned rewardpredicting stimulus [221, 222] so that dopamine cell activityincreases prior to a reward but not with the reward itselfThis does not depend on a time-dependent decrease insensitivity to the reward because modifying the predictability

Fig. (7). Effects of autoreceptor-selective doses of D2/D3 receptor agonist quinpirole on drug seeking behavior tested 10 days after the endof cocaine self-administration training (500g/kg intravenously for 7 days, average daily intake 15 mg/kg). Animals were submitted to a 1-

hour extinction test (responding is measured in the absence of the reinforcer) followed by a 1-hour reinstatement test (where non-reinforced

responding is measured after administration of saline or cocaine). Quinpirole, administered at doses that are known to reduce dopamine

neuronal activity, decreased drug seeking behavior in both the extinction [ANOVA group effect F(2,37)=20.9, p


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of the reward (by presenting it earlier than expected) stillproduces a phasic increase in dopamine neuronal activity. Inaddition, omitting an expected reward depresses neuronalactivity at the exact time when the reward was predicted.This suggests that dopamine neurons are sensitive to theunpredictability of the timing and occurrence of rewards. Inother words, dopamine neurons are error predictors. Thiscoding for prediction errors resembles that employed in

conditioning theories [211] and is strikingly similar to thetemporal difference model of Pavlovian learning proposedby Sutton and Barto [247]. Simplist ically, such theoriesindicate that reinforcers that occur better than predicted willinduce learning; those that are fully predicted do notcontribute to learning, and those that are worse thanpredicted (i.e. they are omitted) produce the extinction of thelearned behavior. Accordingly, it has been suggested thatdopamine neurons could act as a teaching signal that couldfacilitate the learning of reward-related responses [222].

Although Pavlovian conditioning and the temporaldifference model compute predictive signals, such predictionis not translated into action and it does not improve orinfluence reward delivery. Several other theories havetherefore been proposed to determine the relevance ofchanges in dopamine neuron activity produced duringPavlovian conditioning in reward-related behavior. Recently,a variation of the temporal difference algorithm wasproposed; this was based on an internal model approach thatuses learning experiences to make predictions of futurerewards. This theoretical model allows for Pavlovian-induced changes in dopamine neuron activity to code for theplanning of goal directed behavior [245]. Other studies,instead, have argued that the short-latency dopamineresponse during Pavlovian conditioning is too short to beable to signal reward and it was therefore proposed thatphasic increases in neuronal activity in response toconditioned stimuli could play a different role in associativelearning: they could help orient attention and behaviortowards subsequent salient stimuli [210]. This couldfacilitate associative learning [31] and could prepare theorganism for the appropriate reaction to a significant event[210]. On a similar line, because conditioned stimulipredicting rewards increase dopamine neuron activity, theycould acquire the capacity to elicit a conditioned attentionalorienting response that facilitates goal-directed behavior[117]. This was further corroborated by more recent studies[76] showing that the superior colliculus (a primitive visualstructure in the monkey), is the primary source of short-latency visual information to dopamine neurons.

It is also possible that dopamine neuron changes during

such Pavlovian tasks are responding to the incentive value ofthe conditioned stimulus, which predicts the reward, or thatthey code for expectation of the reward. Argumentsagainst the latter hypothesis are provided by results obtainedusing a delayed alternation task. After having learned thistask, dopamine neurons increase activity when exposed tothe instruction cue (which provides special information fortask performance) and the trigger stimulus (which predictsreward), but they do not increase their activity during thelapse (delay) between the instruction and the triggerstimulus. Because of the lack of sustained activation duringthe delay period, changes in neuron activity are not likely tocode for expectation of a reward, or even preparation of

movement but, rather, for attentional and motivationaprocesses that are involved with learning and cognitivebehavior [223]. Nevertheless, in situations where rewardprobability is uncertain, dopamine neurons show an increasein activity preceding a reward that increases from the time othe conditioned stimulus predicting reward to the expectedtime of reward itself, indicating that activity can be related toreward expectation, or at least to uncertainty of expectation

[89].It should be noted that all of these theories are no

mutually exclusive. In fact, additional studies, based onexisting electrophysiological, neurochemical andneuroimaging data propose that dopamine neuron activitysignal reward prediction as well as the incentive salience of areward [170, 183]. Furthermore, recent studies fromSchultzs group, indicated that dopamine neurons showresponses related to motivational salience, because they cancode the reward value, which is a hallmark of a motivationasystem [257]. Therefore, increases in dopamine neuronactivity could represent a mechanism by which rewardincrease their incentive value or salience, thereby facilitatingtheir addictive potential.

Relationship Between Phasic Changes in DopamineNeuron Activity and Operant Responding

More information on the role of dopamine and rewardcome from operant conditioning studies. During operanconditioning, an animals response is directly paired with anoutcome, and goal-directed behavior is a consequence of theanimals intention to obtain a reward (or to seek for one)Such studies could give us information on anticipation of anoutcome, the drive towards the outcome, the value of thepredictive cues, or of the reward itself.

Initial indirect evidence for a facilitatory role odopamine neuron activity on goal-directed behavior can be

extrapolated from studies examining behavior aftetreatments known to modify dopamine neuron activity. Theinfusion of GABA into the VTA, which is known todecrease dopamine cell activity (see previous sections), hasbeen shown to disrupt appetitive behavior. Interestingly, thitreatment only impairs approach to an appetitive reward(sucrose), without modifying its consumption [124]. Thisuggests that impulse activity of dopamine neurons isassociated with approach, or wanting behavior, withouhaving much relevance to the liking of the reward itself.

Studies measuring dopamine neuron activity duringoperant responding are scarce. One study measureddopamine neuron activity during operant responding for asucrose reward in rats [151]. This study showed variableresponses across neurons, which exhibited excitations oinhibitions during different phases of the reward-related tes(approach, responding and consummatory phases). Otheoperant-based studies have been performed in monkeysusing spatial choice tasks, instructed spatial tasks, and spatiaalternation delayed tasks that use Pavlovian conditioning andoperant responding [223]. It should be noted that all stimulthat predict reward in some way, even during operanconditioning, are Pavlovian conditioned. As mentionedpreviously, these studies show that dopamine neuronactivate phasically in response to the instruction cue or thetrigger stimulus, however, dopamine neuron activity is no


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sustained between the lapse of time that predicts the rewardand the reward itself, nor during the motor task that isrequired to obtain the reward. The authors thus suggestedthat changes in the activity of these neurons do not code forgoal-directed behavior or reward expectation [223]. In thesetests, although animals did perform operant responses, theycould not control when they would obtain a reward; theywere always given an instruction, after which they could

perform the correct operant response that would result inreward delivery. Although a correct operant responseprobably reflects the animals intention to obtain thereward, it would also be interesting to perform operant taskswhere animals can choose when to respond for a reward.Such tests could give us insight on how different patterns ofdrug-seeking behavior are linked with dopamine neuronactivity; alas such tests would not be free of confounds, sincethey would not be able to distinguish between incentive'wanting', expectation, or motor activation.

2.2.c. Dopamine Neurons and Stress

Defining Stress

Stress is a complex term that usually carries negativeconnotations. Under a biological point of view, it is widelyaccepted that stressful events have the common feature ofelevating blood levels of glucocorticoids, the major stresshormones (for review, see [7, 16, 56, 58, 186]). Under apsychological point of view, it is generally conceived thatstressful stimuli are something that will be avoided byanimals. However, such a vision might need some revisiting.For example, in humans, it is classically accepted that someindividuals show preference for situations that enhance stresslevels (e.g. sensation-seekers [295]). In animals, similartraits exist as well [59]. Rats with enhanced stress reactivity(glucocorticoid secretion) in response to certain situationshave been shown to spend more time in these stressful

situations (e.g. a novel environment, or the open arms of aplus maze) compared with rats with reduced stress reactivity[134]. In other words, some animals choose to spend time instressful situations that promote the release of stresshormones. This self-administration of stress is furthercorroborated by studies showing that rats will self-administercorticosterone, the major glucocorticoid hormone in the rat[63, 201].

In addition to there being individuals that seek stressfulsituations (indicating that stress is not necessarily an avoidedcondition), one must consider that there are different degreesof stress. Stressors could have different effects according totheir intensity, duration and predictability. Mild stressors,such as reduction in food availability or changes in the social

environment can increase arousal and produce a state ofactivation, whereas intense stress such as the "chronic mildstress" procedure produces learned helplessness, anddepressive-like symptoms [39, 40, 125, 279]. Indeed onecould view stress as having an inverted U-shaped curve,where low to moderate stressors may produce the excitationof a system, whereas higher levels could inhibit the samesystem. This notion is corroborated by behavioral findingsshowing such an inverted U-shaped curve in response toincreasing doses of the stress hormone corticosterone [60].

Effects of Stress on Dopamine Neuron Excitability

Information discussed above suggests that stress exhibitsan inverted U-shaped curve, where low to moderate stressorare activating whereas stronger stressors are inhibitory. Thiis particularly true for the dopamine system. Dopaminelevels in the nucleus accumbens have been shown to increasefollowing acute or repeated mild stress [9, 126, 136, 256]instead they decrease following intense, chronic ounpredictable stress [66, 159].

Although most studies focused on the effects of stress ondopamine levels, a few have analyzed the effects of stress onthe impulse activity of midbrain dopamine neurons. Thesestudies are consistent with each other as they show that stresincreases dopamine neuronal transmission. Chronic exposureto cold stress, although reducing the number of detectableneurons, also enhances the proportion of neurons showinghigh levels of bursting activity [184]; this suggests that stresscould facilitate the switch from regular-firing to burst-firingactivity. A similar effect is also observed following changesin food availability or restraint stress (such conditions areconsidered stressful because they increase glucocorticoid

levels [55, 61, 62, 233]). Thus, repeated food-restriction(Fig. 8a), or a single day of complete food deprivation, (Fig8b), enhances both bursting and firing activity of dopamineneurons in the VTA of anesthetized rats. In addition, inawake rats, a 30-min restraint stress increases firing in aldopamine neurons and bursting in 80% of the neuronsinterestingly, bursting activity is preferentially increased inthose neurons with high burst rates under resting condition[6]. In awake behaving cats, the exposure to the stress of aconditioned emotional reaction, which also produces anincrease in glucocorticoid hormones, also increasedopamine neuron activity [260]. Finally, administration oglucocorticoid hormones, in the range between low and highpeaks of the circadian cycle, increases glutamate-induced

bursting activity in VTA dopamine neurons [194].

Fig. (8). Effects of stress on dopamine neuron activity. (a

Compared with ad-libitum-fed controls (n=24 cells), animals whose

daily ration of food was decreased over 12-22 days to produce

10%-decrease in body weight (n=25 cells) show heightened firing

activity of dopamine neurons (T test, p


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In addition to modifying dopamine neuron activity, stresshas also been shown to enhance excitatory synaptic inputonto dopamine cells [217]. Thus, exposure to cold swimstress enhances the AMPA/NMDA ratio recorded fromdopamine neurons following synaptic stimulation [217]; thiseffect is probably mediated by a stress-induced increase incorticotropin-releasing factor, as administration of thishormone induces a potentiation of NMDA receptor-mediated

synaptic transmission in VTA dopamine neurons [263]. Thisincrease in excitatory synaptic transmission could be one ofthe mechanisms by which stress is able to increase dopamineneuron activity.

Effects of Brief Aversive Stimuli on Dopamine NeuronExcitability

While the above-listed forms of stressors have beenshown to increase dopamine neuron activity or excitability,brief exposure to mildly aversive situations (that might notbe stressful and might not produce an increase inglucocorticoid levels, nor in dopamine release [156, 277])either increase neuronal activity, have no effects ondopamine neuron activity or more generally produce a

transient decrease in their firing rate.In awake cats, the brief presentation of aversive stimuli

(such as brief tail pinch, immersion of paws in cold water,inaccessible food or white noise) do not seem to producemajor changes in neuronal firing [242]. In rats, the pinchingpaw while the animal is anesthetized has been shown to slowdown dopamine neuron firing in 10/12 neurons [262].Pinching of the tail in similar conditions also depressesactivity in 25% of dopamine neurons projecting to corticalregions, but increases it in 65% of this mesocorticalpopulation [160]. In monkeys a similar pinching stimulushas been shown to decrease dopamine neuron activity in50% of the entire neuronal population, but to increase it in25% of the cells or to have no effects in the remainder of theneuronal population [225]. A decrease in dopamine neuronactivity is also seen when monkeys that are trained to expecta juice reward are not offered such a reward, whichrepresents an aversive stimulus [222, 224]. Non-noxious air-puffs, however, do not produce signif icant changes indopamine cell activity [182], suggesting that aversive stimulimight require greater intensity to produce any effects or thatdopamine neurons do not respond to aversive stimuli [182].

Differences Between Aversive and Stressful Stimuli

Overall, these findings, together with those described inthe previous paragraphs, indicate that acute and shortaversive stimuli (e.g. air puffs, 15 sec paw pinch) can

produce increases, decreases or no effects in neuronalactivity and neurotransmitter release. Instead, exposure torepeated mild stress (e.g. repeated or prolonged exposure tocold, restraint, food deprivation) increases dopamine neuronactivity. It is unlikely that such increases are artifacts due torecording techniques [261] because similar stressors induce aparallel increase dopamine levels in terminal regions [164,261]. It is therefore likely that stimuli need to be presentedrepeatedly, or require long periods of time in order tobecome stressful and produce increases in dopamineneuronal firing. In addition stressors and aversivestimuli could be different in nature, and prolonged increasesin stress hormones (glucocorticoids) might be required for

stimuli to modify dopamine neuron activity. Consistent withthis idea are the findings that stress-induced increases indopamine neuron excitability are prevented by the administration of glucocorticoid receptor antagonists [217]. Inaddition, for stress to increase dopamine levels, it needs to bemoderate and presented repeatedly or for a prolonged periodof time (for review see [164]).

Possible Relevance of Stress-Induced Increases in

Dopamine Neuron Excitability

Putting aside the mechanism by which stress couldincreases dopaminergic transmission and taking into accounits relevance, it is puzzling how the effects of stress aresimilar to those produced by situations that are consideredrewarding (e.g. the unexpected presentation of a reward[224]). It is possible that stress-induced increases indopaminergic transmission could be viewed as copingmechanism that helps reduce the aversive effects of stresand thus increases the individuals ability to cope with thestressful situation. Similar to what has already been proposedfor stress hormones [164], this increase in dopamine couldreduce the aversive properties of stressors, and possibly

even make some stressors reinforcing. With repeated ointense stress, however, dopaminergic transmission coulddecrease and lead to depressive-like states [39, 40, 125, 279and offset an individuals homeostatic state.

In addition to this, the fact that dopamine cells of stressedanimals show stronger responsiveness to excitatory synapticinputs [217] could possibly result in the animals heightenedreactivity to environmental stimuli. After stressfuconditions, such a phenomenon could serve as a survivamechanism allowing animals to increase their attentiontowards behaviorally-relevant stimuli. Finally, sustainedincreases in dopamine neuron activity have been suggestedto reinforce risk-taking behavior [89]. Although veryspeculative, it is possible that stress-induced prolongedincreases in dopamine neuron activity could facilitate risktaking behavior and thereby broaden the animals ability torespond to stressful situations.

3. SUMMARY AND CONCLUSIONS

Dopamine neurons in the midbrain are spontaneouslyactive and show regular, irregular and bursting patterns oactivity. Neuronal activity is regulated by intrinsic andsynaptic factors. As for the intrinsic factors, the binding odopamine to impulse-regulating dopamine D2/D3autoreceptors located on the dopamine cell body activatesGIRK potassium currents, hyperpolarizes the cell, andthereby inhibits neuronal activity. Activation of SK

channels, produced by slight rises in intracellular calciumincreases SK potassium currents responsible for the AHPand thereby produces the neuronal inhibition that is typicaafter a train of spikes.

Differences in the strength of excitatory or inhibitorysynaptic input also produce changes in neuronal excitabilityAs for most systems, glutamatergic synapses are excitatoryand produce their effects by activating AMPA and NMDAreceptors. GABAergic synapses are largely inhibitory, andcan act on ionotropic GABAA or metabotropic GABABreceptors to produce fast or slower inhibitions of neuronaactivity respectively.


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Changes in neuronal activity of dopamine neurons areobserved throughout an individuals life. Activity is low atbirth, peaks during the adolescent period, and decreasesthereafter. Changes in the basal activity of these neurons canalso be induced by life experiences, such as exposure todrugs or stress. These are manifested as enhanced neuronalfiring, bursting, and in the strength of excitatory synapsesonto these cells. Such increases in neuronal activity are

associated with greater responding for cocaine, suggestingthat increases in the baseline activity of these neurons couldfavor addiction liability. This hypothesis is supported byobservations that reductions of dopamine cell activity arecoupled with decreases in self-administration and drug-seeking behavior.

In addition to changes in the basal activity of these cellsinduced by life events, neuronal activity can also bemodulated phasically. Phasic increases in impulse activityare observed in all forms of reinforcement-learning. Inoperant responding for drugs, neuronal activity has beenshown to increase prior each voluntary self-infusion,suggesting that phasic increases in dopamine cell activity areassociated with goal-directed behavior, or possibly themotivational drive to seek the drug. In Pavlovianconditioning for natural rewards these neurons increasebursting when presented with reward-predicting cues, orunexpected rewards, indicating that these neurons signalreward prediction errors. Such phasic increases in dopamineneuron activity during the presentation of reward-predictingcues could increase attention towards these stimuli. Overall,the phasic increases in dopamine neuron activity that precederewards could be a potential mechanism that facilitateslearning, or the incentive value of the reward-associatedcues.

Repeated or prolonged exposure to mild stressors (suchas restraint, food restriction or cold stress) has been shown to

produce increases in the basal activity of dopamine neurons.On the other hand, presentation of brief aversive stimuli(such as brief paw or tail pinches, or air puffs), producesvariable effects on dopamine neuron firing. It is possible thatstressors need to be presented repeatedly or in a prolongedmanner to produce increases in dopamine neuron activity. Itis, however, surprising how stressors could produce similareffects as those produced by rewarding stimuli. It is possiblethat such increases could serve as a coping mechanism thatcould decrease the aversive nature of the stress, increaseattention towards the environment, or possibly even increaserisk behavior. Such effects could in the short run facilitatethe individuals coping responses to the environment inconditions of stress.

In conclusion, dopamine neurons are spontaneouslyactive neurons whose activity can be modulated by diverselife events, ranging from exposure to drugs, stress, orunpredictable rewards. Ultimately if prolonged, thesechanges could lead to the development of dopamine-associated disorders, such as drug addiction. Severalhypotheses have been put forward to explain the mechanismby which addiction liability or the learning of reward-relatedtasks could be facilitated by increases in dopamine neuronactivity (either changes in the basal firing rate, or phasicchanges in response to stimuli). These include changes in

learning, the impact of the reinforcer, the associativreward-learning, the conditioned reinforcement, the attentiontowards such a salient stimulus, the incentive salience of astimulus, or a combination of these.

ACKNOWLEDGEMENTS

We thank: Frdric Ambroggi, Kristin Anstrom, KenBerridge, Lionel Dahan, Patricia DiCiano, Eugene Kiyatkin

Wolfram Schultz and Anthony West for very useful inputdiscussion, comments or the sharing of data and informationthat helped us write sections of this review. Preparation othis review was supported in part by USPHS grant DAO4093 (X-TH and FJW) and a Senior Scientist Award (DA00456) to FJW.

ABBREVIATIONS

AHP = Afterhyperpolarization

AMPA = Alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid

EGTA = Ethylene glycol-bis(2-aminoethylether)-N,N,N_N_-tetraacetic acid

EPSC = Excitatory post synaptic current

EPSP = Excitatory post synaptic potential

GIRK = G-protein-coupled inward-rectifying potassium

Ih = Hyperpolarization-activated cation current

LTP = Long term potentiation

LTD = Long-term depression

mGluRs = Metabotropic glutamate receptors

NAc = Nucleus accumbens

NMDA = N-methyl-D-aspartate

PND = Postnatal dayRT-PCR = Reverse transcriptase polymerase chain reaction

SK = Calcium-activated small conductance potassium

SNc = Substantia nigra, pars compacta

TEA = Tetraethylammonium

VTA = Ventral tegmental area

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