Nigrostriatal cell firing action on the dopamine transporter

Nigrostriatal cell firing action on the dopamine transporter

Manuel Rodriguez,1,4 Sergio Gonzalez,1 Ingrid Morales,1,4 Magdalena Sabate,2,4 Tomas Gonzalez-Hernandez3,4

and Jose Luis Gonzalez-Mora1

1Laboratory of Neurobiology and Experimental Neurology, Department of Physiology, Faculty of Medicine,2Department of Pharmacology and Physical Medicine and3Department of Anatomy, Faculty of Medicine, University of La Laguna, La Laguna, 38320 Tenerife, Canary Islands, Spain4Centro de Investigacion en Red sobre Enfermedades Neurodegenerativas, CIBERNED

Keywords: dopamine, dopamine transporter, firing activity, nigrostriatal cells, rat, striatum

Abstract

The influence of nigrostriatal cell firing on the dopamine transporter (DAT) activity of the rat striatum was studied in vivo withamperometric methods. Data were obtained after preventing dopamine (DA) release with a-methyl-l-tyrosine and replenishingextracellular DA with local injections. The DA cell stimulation, which under basal conditions increased extracellular DA, decreased DAafter this pre-treatment, suggesting that firing activity facilitates the DA cell uptake of DA under these circumstances (drain response).Cocaine and GBR13069 markedly decreased the drain response, suggesting that it is dependent on DAT activation. Data obtainedafter haloperidol and apomorphine administration showed that the drain response was facilitated by pre-synaptic DA receptorstimulation but that receptors are not a necessary requirement. Two components in the drain response were observed, one with ashort latency and duration that needed high-frequency stimuli, and the other with a long latency and duration that was even inducedby low-frequency stimuli. This is the first evidence showing that DAT can be activated by the firing activity in nigrostriatal cells in adirect way and without the participation of pre-synaptic DA receptors.

Introduction

Dopamine (DA) is a major neurotransmitter in the basal ganglia whereit plays a key role in different physiological functions (locomotoractivity, reward and memory), and neurological and psychiatric illness(Parkinson’s disease and schizophrenia). DA modulates the action ofglutamatergic cortical input on medium-sized spiny neurons in thestriatum (Dahlstroem & Fuxe, 1964; Ungerstedt, 1971; Fallon &Moore, 1978; Gerfen et al., 1987; Joel & Weiner, 2000), changing theactivity of the ‘direct’ (striatum–substantia nigra ⁄ internal pallidum–thalamus-cortex) and ‘indirect’ (striatum-external pallidum–subthal-amus-substantia nigra ⁄ internal pallidum–thalamus-cortex) pathways(Alexander et al., 1986; Albin et al., 1989; DeLong, 1990; Smith et al.,1998; Obeso et al., 2000b), and therefore affecting most motor andnon-motor functions of the cortex (Gerfen, 2000). Striatal DA may actboth synaptically (neurotransmission) and extrasynaptically afterdiffusion in the extracellular space (volume transmission) (Gonon,1988; Kawagoe et al., 1992; Sesack et al., 1994; Descarries et al.,1996; Schultz, 1998). Both actions are regulated by the DA transporter(DAT), a plasmalemmal protein responsible for the DA clearance fromthe extracellular space that has focused the attention of a number ofresearchers in the last 10 years (Mortensen & Amara, 2003).

Dopamine cells display a basal firing pattern (0.5–10 Hz) that, in 50–60% of cells, is transiently substituted by spike bursts of up to 35 Hz(Grace & Bunney, 1984a,b; Bunney et al., 1991; Dai & Tepper, 1998;Rodriguez & Gonzalez-Hernandez, 1999). The extrasynaptic flow ofDA across the extracellular space (and its neurotransmitter vs. volume

transmitter action) is different during basal firing and burst firing, adifference that has been attributed to changes in the probability of bothDA release (Cragg, 2003; Montague et al., 2004a,b) and DA uptake(Chergui et al., 1994; Wu et al., 2001, 2002; Cragg & Rice, 2004).Particularly efficient mechanisms for modulating the extrasynaptic DAare those involved in the regulation of the DAT activity (Chergui et al.,1994; Garris et al., 1994; Wu et al., 2001, 2002). Together with a long-term (change in hours of expression, intracellular transport, glycosyla-tion and membrane insertion of DAT protein) and a short-term (changein minutes of synaptic DAT distribution) regulation of DAT activity,there is evidence of a very fast DAT modulation (milliseconds), whichcould be particularly suitable for DAT activity adaptation to the firingpattern. Two possible mechanisms for very fast regulation have beenproposed, one involving a DATactivation by the DA stimulation of pre-synaptic D2 receptors (Dickinson et al., 1999; Hoffman et al., 1999;Benoit-Marand et al., 2001; Thompson et al., 2001;Wu et al., 2002) andthe other in which DAT is directly activated by the cell membrane after-hyperpolarization (Krueger, 1990; Sonders et al., 1997; Hoffman et al.,1999). The present work was designed to study the action of DA cellfiring onDATactivity. Thus, in-vivo amperometrymethodswere used toquantify DAT activity while the extracellular DA concentration, pre-synaptic D2 receptor stimulation and firing activity of DA cells wereindependently controlled.

Materials and methods

Experiments were carried out on male Sprague-Dawley rats (Panlab,Barcelona) weighing 300–350 g. Animals were housed at 22 �C, twoper cage, under normal laboratory conditions on a standard light ⁄ darkschedule (12 ⁄ 12 h with lights on from 03:00 to 15:00 h) with free

Correspondence: Dr Manuel Rodrıguez, as above.E-mail: [email protected]

Received 21 September 2006, revised 18 January 2007, accepted 5 March 2007

European Journal of Neuroscience, Vol. 25, pp. 2755–2765, 2007 doi:10.1111/j.1460-9568.2007.05510.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

access to food and water. Experiments were carried out in accordancewith the European Communities Council Directive of 24 November1986 (86 ⁄ 609 ⁄ EEC) regarding the care and use of animals forexperimental procedures and approved by the Ethical Committee ofLa Laguna University.

Experimental design

Themain aim of this workwas to study in vivo the influence of the firingpattern of nigrostriatal DA cells on DAT activity. DAT activity wasevaluated by quantifyingwith amperometry the uptake ofDA (1–5 mm)administered in the extracellular medium of the striatum with amicrocannula (fused silica capillary tube with 75 lm inner diameter)located 400 lm from the recording electrode tip (Fig. 1B). DA wasdissolved in a Ringer solution (148 mm NaCl, 2.7 mm KCl, 1.2 mm

CaCl2 and 0.8 mm MgCl2) supplemented with l-glutathione reduced(1 mm) to avoid DA oxidation. The osmolarity of the solution wasbetween 280 and 285 mOsm (auto-osmometer Osmostat OM-6020;CagaK Co. Ltd, Kyoto, Japan). Two DA fluxes were used (CMA-102microdialysis pump; CMA Microdialysis, Stockholm, Sweden), onewith a continuous DA perfusion that was regularly adjusted to hold DAlevels stable in the extracellular medium (0.1–0.3 lL ⁄ min in Fig. 1E)and the other that induced transitory increases of extracellular DA(3 lL ⁄ min for 3 s in Fig. 1D). DA needs to diffuse from the injectionpoint to the tissue surrounding the electrode and, as the electrode–cannula distance was not always the same and there are neurochemicaldifferences between neighboring striatal regions (Gerfen et al., 1987;Graybiel & Moratalla, 1989; Gerfen, 1992; Joel & Weiner, 2000), thesame DA perfusion did not always produce the same amperometricresponse. Thus, the DA dose was adjusted in each rat in order to producesimilar DA concentration increases in the tissue around the electrode tip.Different factors were taken into account in order to prevent the possiblesaturation of DAT by injected DA. The DA dose was always the lowestthat induced a suitable response. Different doses were tested at thebeginning of each experiment and the lowest DA dose necessary toinduce an amperometric response of enough amplitude to quantifyincreases and decreases of DA diffusion (and therefore to calculate boththe facilitation and inhibition of DAT activity) was selected. The DAdose per injection (�100 nL of 1–5 mm solutions) was never over0.5 nmol which, taking into account the long distance between theinjection cannula and the recording electrode (�400 lm) and the three-dimensional spherical volume cube-root function for concentrationdecrease of substances dissolved in a solution, initially seems to us a lowenough DA dose to prevent DAT activity saturation around therecording electrode. Initially, we obtained a response amplitude to DAinjection that was in the range of those normally observed after medialforebrain bundle (MFB) stimulation in rats not pre-treated with a-methyl-l-tyrosine (AMPT) (50–500 pA) (Rodriguez et al., 2006), thussuggesting that the DA concentration increase induced around therecording electrode by DA injection was similar to that obtained afterincreasing the DA cell firing (50–500 pA) (Rodriguez et al., 2006).Finally, the finding that DAT inhibitors markedly increased the DA

diffusion from the cannula to the recording electrode directly showedthat DA doses were not high enough to saturate DATactivity. However,particular care was taken to prevent possible DAT saturation when DAwas injected after cocaine or GBR13069 administration. In this case, theDA dose was adjusted to produce a current response to DA adminis-tration similar to that obtained before DAT inhibition.However, the nigrostriatal DA cell activity was modified by

inducing repetitive action potentials with electrodes located in theMFB. Electrical stimuli were provided by an S-8800 Grass model anda bipolar electrode made with two tungsten rods (100 lm diameter;A-M Systems Inc.) etched with a oxyacetylene torch flame (Bragaet al., 1977), insulated with glass (fused silica capillary, CompositeMetal Service, Hallow, UK) and placed with the tips 500 lm apart(exposed surface of the tip was 0.5 mm long). Stimuli were given in0.2–0.5 mA and 1 ms square biphasic pulses. The MFB stimulationwas performed in some experiments by simultaneously changing thestimulation rate and the duration of the stimulus train, in such a waythat all of the stimulus trains always had the same number of stimuli(isopotential stimulation; Fig. 4A). Thus, trains of 50 stimuli wereprogressively changed from 10 Hz ⁄ 5 s to 200 Hz ⁄ 0.25 s.The main problem with this method was that DA released as a

consequence of the membrane depolarization induced by MFBstimulation (action potentials reach the striatum where they activateDA release) increases the extracellular DA concentration which, addedto perfused DA, can interfere with the measurement of DAT activity.Thus, we opted for avoiding DA release during MFB stimulation bypre-treating rats with a drug (AMPT) that inhibits the tyrosinehydroxylase activity (Michael et al., 1987). AMPT (250 mg ⁄ kg i.p.)progressively eliminated the intracellular DA pool involved in theneurotransmitter release coupled to action potentials. Figure 1A showsan example of the amperometric response to MFB stimulation atdifferent times after AMPT administration. At 60 min after AMPT,MFB stimulation did not generally increase extracellular DA, althoughin some cases a very small release was observed (Fig. 1C top right).

Electrodes and electrochemical method

The DA concentration was quantified by using amperometric methods(Rodriguez et al., 2006). Briefly, the amperometric current through theelectrode was continuously measured (PowerLab system connected toa PC, ADInstruments, Castle Hill, Australia) while an oxidationpotential was held at 300 mV. In a similar way to the increase incurrent induced by the MFB stimulation (which has been proved to becaused by increases in the extracellular DA concentration) (Cherguiet al., 1994; Dugast et al., 1994; Gonon, 1997), we have recentlyreported that MFB stimulation induces a decrease in current in somestriatal regions, which is entirely caused by a DA decrease (Rodriguezet al., 2006). Electrical artefacts induced by the stimulation pulses inthe amperometric signal were withdrawn by: (i) decreasing the high-frequency components of the stimuli (square pulses were modified byshaving the pulse corners in order to obtain a stimulus waveform withthe initial and final portions of the rising and falling phases similar to

Fig. 1. Dopamine (DA) uptake activation by membrane potential. (A) An example of the effect of a-methyl-l-tyrosine (AMPT) on DA release induced by medialforebrain bundle (MFB) stimulation (values show different times after AMPT mean response ± SE). (B) Photograph of a typical microelectrode (right side) and thecannula (left side) used for the local DA perfusion in the striatum. (C) An example (10 consecutive response waves) of how DA release induced by MFBstimulation (top left) disappeared 90 min after AMPT administration (top right) and how DA perfusion increased the DA level around the electrode tip (bottom left)changing the DA-response observed before AMPT (top left vs. bottom right). (D) An example of a stimulus-response curve (stimuli of increasing duration)obtained after a DA pulse of short duration (3 lL ⁄ min for 3 s). (E) An example of drain responses obtained after a continuous DA perfusion (0.1 lL ⁄ mincontinuously adjusted to maintain stable DA levels) and of how modifications of the amperometric current induced by both DA perfusion and MFB stimulationvanished when the oxidation potential decreased from 300 to 0 mV. (F) An example of how the drain response progressively vanished when the oxidation potentialwas progressively decreased from 400 to 0 mV (values show the mean response to MFB stimulation ± SEM). (G) Direct relationship between the increase in thebackground current induced by local DA perfusion (3 lL ⁄ min for 3 s) and the amplitude of the drain response.

2756 M. Rodriguez et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 2755–2765

Nigrostriatal cell firing and DAT activity 2757


those of a sigmoid waveform) and (ii) using fast biphasic stimulatingpulses [the artifact induced by each pulse was very symmetric andshort-lasting (1–2 ms), and could be withdrawn by using a movingaverage that computed each signal value as the average of the dataobtained a few milliseconds before and after it].The recording electrode was a carbon fiber microelectrode (8 lm

diameter and 50 lm long), whose tip was pulled to �1 lm (Fu &

Lorden, 1996) and slowly introduced in the striatal tissue (20 lm ⁄ min)with a micromanipulator (MO-8, Narishige with a stepper motor anddigital controller for vertical displacement) in order to minimize tissuedamage. Electrodes were pre-treated for 20 s with a 70 Hz triangularwave (0–2.3 V vs. Ag ⁄ AgCl) to optimize their in vivo electrochemicalbehavior. We have previously shown that this treatment causes a verysmall decrease in the time resolution of the electrodes and a marked

Fig. 2. Dopamine (DA) transporter and membrane potential activation of DA uptake. (A) An example of drain responses recorded during DA perfusion (whichis shown by the slow increase and the following decrease of the basal line) before (top) and after (bottom) cocaine administration (1 mm DA was injected for0.5–3 s adjusting the DA dose after cocaine administration in order to induce a similar DA increase). (B) A magnified view of superimposed drain responses tomedial forebrain bundle (MFB) stimulation before (top) and after (bottom) cocaine administration. (C–E) Effect of cocaine on slope (C), amplitude(D) and latency (E) of the drain response. (F) An example of the drain response decrease observed after GBR13069 administration. (G) Superimposed drainresponses to MFB stimulation before (top) and after (bottom) GBR13069 administration. (H–J) Effect of GBR13069 on slope (H), amplitude (I) and latency (J)of the drain response.



Fig. 3. Dopamine (DA) receptors and membrane potential activation of DA uptake. (A) Left side shows an example of drain responses recorded during DAperfusion (which is shown by the slow increase and the following decrease of the basal line) before (bottom) and after (top) haloperidol administration. Right sideshows a magnified view of superimposed drain responses to medial forebrain bundle stimulation before (bottom) and after (top) haloperidol administration. Theeffect of haloperidol on the amplitude and slope of the drain response is shown in B and C, respectively. (D) An example of the drain response observed at the endof a previous DA perfusion (left side) and during a new DA administration (right side). Note that the apomorphine administration between both DA pulses (middleposition) did not induce drain responses. (E) Superimposed drain responses during a DA perfusion before (top) and after (bottom) apomorphine administration.(F) Effect of apomorphine administration on the amplitude (top) and slope (bottom) of the drain response.



increase in their sensitivity (Rodriguez et al., 2006). In order to rule outthe possible interference of serotonin, we used a potential of 300 mVinstead of the 400 mVoxidation potential that is generally used for DAand in some experiments the amperometric response was tested atdifferent oxidation potentials. The selectivity and sensitivity ofelectrochemical methods and electrodes were tested before theirimplantation. The reference electrode (silver wire in vitro coated witha layer of silver chloride by applying +5 V for 3 min) was placed insidea pulled glass capillary tube (filled with a 3 m potassium chloridesolution saturated with AgCl; Fluka) with a 300 lm diameter tip.Electrodes were calibrated in vitro before and after their brainimplantation. Thus, the active part of the carbon-fiber electrode wasplaced 5 mm inside Teflon capillary tubing (125 lm i.d. and 5 cm inlength), which was connected to the exit of a liquid switch (CMA110;CMA Microdialysis) previously connected to a dual syringe perfusionpump (CMA102; CMA Microdialysis). The perfusion fluid was aphosphate-buffered saline solution (148 mm NaCl, 2.7 mm KCl,1.2 mm CaCl2 and 0.8 mm MgCl2) supplemented with l-glutathionereduced (1 mm) to avoid DA oxidation (40 lL ⁄ min flow rate). DAwasdissolved in this solution and loaded in one of the two perfusion syringes(the other was loaded with the solution used to dissolve the testcompound). The internal volume of the whole system was 40 lL.

Following previously reported procedures (Rodriguez & Gonzalez-Hernandez, 1999), in-vivo studies were performed under chloralhydrate anesthesia (400 mg ⁄ kg i.p.) in a sound-proofed dark room(5.00–10.00 h after turning the light on) and with the body tempera-ture maintained between 36.5 and 37.0 �C. The electrode wasconnected to an amperometry detector (Laguna, Tenerife, Spain) thatcontrolled the electrode potential (vs. a reference electrode whoseopen tip was immersed in a saline solution in contact with themeninges through a hole previously made in the skull) by means of apotentiostat. The location of the striatal recording electrode was0.0–1.2 mm anterior to bregma, 2.6–3.4 mm lateral to the midline and4.0–5.0 mm below the cortical surface.

Drugs and chemicals

Haloperidol, AMPT and reduced l-glutathione were obtained fromSigma Chemical Corp. (St Louis, MO, USA). Cocaine chlorhydratewas obtained from Avello (Barcelona, Spain) and GBR13069 wasobtained from Tocris (Bristol, UK).

Histology

After post-calibration, the electrodes were lowered to their originalcoordinates and a lesion was made by passing DC current through thecarbon fiber electrode (15 lA of cathodal DC current for 50 s). At theend of each experiment and under chloral hydrate anaesthesia, the ratswere transcardially perfused with 200 mL of 0.9% saline solutionfollowed by 400 mL of 4% paraformaldehyde in 0.1 m phosphatebuffer (pH 7.4). Brains were removed and stored in the same fixativeat 4 �C for 12–24 h and the midbrain was then cut at 50 lm with avibratome in the coronal plane and immunostained for tyrosinehydroxylase (Rodriguez & Gonzalez-Hernandez, 1999).

Results

At 1 h after administration, AMPT markedly decreased the DArelease induced by MFB stimulation (Fig. 1A and Fig. 1C top left vs.top right) until became it undetectable. Under these conditions, theelectrical MFB stimulation allowed us to modify the DA cellmembrane potential without inducing DA release in the striatum.Once this fact was verified (no DA release observed after MFBstimulation), the striatum was perfused with DA. DA perfusioninduced a transitory (after pulsatile DA administration; Fig. 1D andFig. 1C bottom left) or a more stable (after continuous DAadministration; Fig. 1E) increase of the amperometric signal. AfterDA administration, the MFB stimulus generally decreased (Fig. 1Cbottom right) the amperometric signal (instead of increasing it as aconsequence of DA release induced by the MFB stimuli beforeAMPT administration) (Fig. 1C top left). As the stimulus-responsecurve of Fig. 1D shows, the amplitude of this decrease depended onthe stimulus characteristics. Both the signal increase induced by DAperfusion and the signal decrease induced by the MFB stimulationdisappeared when the oxidation potential was decreased from 300 to0 mV (Fig. 1E), showing a direct relationship between the oxidationpotential of the electrode and the response to DA injection or to MFBstimuli (Fig. 1F). In addition, a direct relationship was observedbetween the extracellular DA level (background current in Fig. 1G)and the response amplitude to MFB stimulation (response current inFig. 1G). Taken together, these data show that the amperometricsignal decrease evoked by MFB stimulation was induced by atransitory decrease in the extracellular DA concentration (drainresponse). A more extensive characterization of the drain responsewas performed in a previous work that showed its correspondencewith a DA decrease and not with collateral non-dopaminergic-associated phenomena such as electrical artifacts, modifications inother transmitters (serotonin), changes in other extracellular sub-stances (ascorbic acid) or modifications in pH or oxygen (Rodriguezet al., 2006).The aim of the next experiment was to study the possible

involvement of DAT in the drain response. Thus, the effect of twoDAT inhibitors (cocaine and GBR13069) on the drain responseobtained during DA perfusion in rats pre-treated with AMPT (1–3 hbefore administration of the DAT inhibitor) was evaluated. The DAperfusion regime was pulsatile in the cocaine study and continuous inthe GBR13069 study. DAT inhibition decreased the drain response inboth studies. Cocaine induced a marked reduction of the drainresponse (Fig. 2A and B), decreasing its amplitude (< 20% of basalresponse; Fig. 2D) and slope (< 50% of basal response; Fig. 2C) andincreasing its maximum response latency (Fig. 2E). GBR13069induced similar effects, decreasing (50 min after administration) theamplitude (Fig. 2G and I) and slope (Fig. 2H) of the drain response,and increasing the maximum response latency (Fig. 2E). As acontinuous regime for DA perfusion was used and the temporalkinetics of GBR13069 action could be accurately followed, aninhibition of the drain response that slowly progressed after drugadministration was observed (see an example in Fig. 2F). Thus, bothcocaine and GBR13069 studies showed DAT activity as a requirementfor inducing the drain response.

Fig. 4. Dopamine (DA) receptors and membrane potential activation of DA uptake. (A) Time–frequency relationship for isopotential bursts of 50 stimuli.(B) Drain response during a stabilized DA perfusion. (C) An example of the drain response (mean ± SEM of 20 consecutive responses to an isopotential burst of35 Hz ⁄ 1.429 s). (D) Drain response to isopotential bursts (50 stimuli ⁄ burst) of decreasing duration and increasing frequency. The effect of isopotential bursts (50stimuli) of different frequencies on the amplitude of the fast (E) and slow (G) component, on the amplitude difference between the fast and slow components(J), on the latency of the slow component (F), on the latency difference between the fast and slow components (I), and on the slope of the fast component(H) is shown at the bottom of the figure. y, initial fast response; x, late slow response. MFB, medial forebrain bundle.



The exogenous DA administered to test DAT activity could act onpre-synaptic DA receptors thus modulating the DAT response to DAcell firing. Thus, the next step was to evaluate the possibleinvolvement of DA receptors in the drain response. We tested thispossibility by evaluating the effect of a D2 receptor blocking drug(haloperidol) and a DA receptor stimulating drug (apomorphine) onthe drain response. Haloperidol induced a moderate decrease ofamplitude (Fig. 3A and B) and slope (Fig. 3C) of the drain response(response studied 2–4 h after AMPT and 1–2 h after haloperidoladministration). However, apomorphine also modulated the drainresponse. By itself, apomorphine injected intraperitoneally was notable to induce drain responses (Fig. 3D), proving that this response isnot the consequence of a DA receptor activation. Apomorphineinduced a slight increase in the drain response slope (Fig. 3F bottom)and did not affect the response amplitude (Fig. 3F top) when testedafter the local DA perfusion (Fig. 3E). Taken together, the haloperidoland apomorphine experiments suggest a modulatory role for the pre-synaptic D2 receptor on the drain response to action potentials.Finally, we decided to analyse the relevance of the stimulation

characteristics on the kinetics of the drain response. In order toobtain a stable response, two requirements were observed: (i) DAdepleted by AMPT was replenished with a continuous DA perfusionregime that maintains the DA concentration within strict limits(maximum fluctuation of the background current < 30 pA; Fig. 4B),and (ii) the drain response was tested with stimulus trains of differentfrequency (10–200 Hz) and duration (5–0.25 s) but all of which hadthe same energy (all stimulus trains had 50 stimuli; isopotentialstimulation in Fig. 4A). Under these conditions, a stable andreproducible drain response was obtained (see example in Fig. 4Cwhere the mean ± SE of 20 consecutive responses to a35 Hz ⁄ 1.429 s burst is shown).A single slow-activation drain response was observed (10 Hz ⁄ 5 s

and 25 Hz ⁄ 2 s in Fig. 4D) for low-frequency ⁄ long-duration stimuli.However, for stimulation rates over 25 Hz the drain response wassegregated into two components, an initial fast component (y inFig. 4D) with a marked slope which is only activated during thestimulus train and a late slow component (x in Fig. 4D) with a lessmarked slope which often reached its maximum response after thestimulus train switch off (40 Hz ⁄ 1.25 s)160 Hz ⁄ 0.31 s in Fig. 4D).The slow component observed in high-rate stimuli corresponds to theslow response observed for low-rate stimuli, as suggested by the factthat both showed a similar time kinetic (e.g. the same peak responselatency) and by the correspondence observed between the stimulationregime curve (shown in Fig. 4A) and a time-latency curve composedby adding both the response to slow-rate stimuli and the slowcomponent of the high-rate stimuli (Fig. 4F). Thus, both responseswere grouped and will be referred to here as the slow component ofthe drain response. The slow component increased with the stimula-tion rate up to 30 Hz (filled circles in Fig. 4G) and then progressivelydecreased (open circles in Fig. 4G). However, the fast component ofthe drain response started over 15 Hz, showing a response amplitudethat grew to 100 Hz (Fig. 4E) and a response slope that increased to200 Hz (Fig. 4H). Differences between the slow and fast componentswere also observed when the amplitude of the first was subtractedfrom the last (Fig. 4J). For stimuli under 30 Hz, the amplitude of theslow component increased more than that of the fast component(Fig. 4G vs. 4E). This changed for stimuli over 30 Hz and becomenegative (the amplitude of the fast component being higher than thatof the slow component) for stimulation rates over 60 Hz (Fig. 4J).Therefore, two components could be identified in the drain responses,a slow and a fast component that showed a different activation latency(after MFB stimulation switch on) and sensitivity to the stimulation

rate. Finally, the end of the drain response (return to the basal line) wasalways similar to and independent of the stimulus characteristics(correlation between stimulus rate and slope during the basal linerecovery with r ¼ 0.036), suggesting that it is produced by the passivediffusion of DA from the surrounding extracellular space to theelectrode surface.

Discussion

In-vivo evidence for a direct action of nigrostriatal cell firing on striatalDAT activity is reported here. Cell firing induced by repetitivestimulations of the MFB activated DA uptake (drain response), aneffect that was largely dependent on DAT activity but not on pre-synaptic DA receptors (which only behaved as response modulators).Two components were identified in the drain response, a fastcomponent with short latency and duration, and a slow componentwith long latency and persistence. This is the first in-vivo evidence fora firing rate-linked activation of DAT not induced by pre-synaptic DAreceptor stimulation.The main finding of the present study was the DA uptake activation

induced by nigrostriatal cell stimulation after preventing DA releasewith AMPT. A direct relationship was observed between thestimulation rate or the duration of the stimulus train and the amplitudeof the drain response, showing that the firing pattern of nigrostriatalcells influences DA uptake. DAT inhibition with cocaine orGBR13069 induced a marked decrease in drain response (cocainedecreased the response to less than 20% of the basal response) that,bearing in mind that these drugs were peripherally injected and neededto compete with locally perfused DA, suggests that the drain responseis entirely caused by a transient activation of DAT. Bearing in mindprevious studies, the activation of this very fast DAT response couldbe induced by the pre-synaptic D2 receptor stimulation that followsDA release (Dickinson et al., 1999; Hoffman et al., 1999; Benoit-Marand et al., 2001; Thompson et al., 2001; Wu et al., 2002) or by thecell membrane hyperpolarization that follows action potentials(Krueger, 1990; Sonders et al., 1997; Hoffman et al., 1999).Taking into account the fast response of the D2 receptor (e.g. it

inhibits DA release in 100 ms) (Kennedy et al., 1992; Benoit-Marandet al., 2001) and its ability to activate DAT (Dickinson et al., 1999;Hoffman et al., 1999; Benoit-Marand et al., 2001; Thompson et al.,2001; Wu et al., 2002), it has been suggested that these receptorsprevent an excessive extrasynaptic diffusion of DA during burst firing(Wu et al., 2002). The present data support this possibility, showingthat the blockade of DA receptors with haloperidol or their stimulationwith apomorphine modifies the DAT activation induced by MFBstimuli. However, DA receptor-acting drugs (even in the case ofhaloperidol, which has a high affinity with the D2 receptor) (Seeman& Tallerico, 1998) only had a moderate effect on DAT activationinduced by MFB stimuli. This, and the finding of the drain responseafter preventing DA release with AMPT, showed that othermechanisms apart from DA receptors are necessary to explain theDAT activation by spike firing.The other mechanism proposed for the very fast DAT activation is

the cell membrane after-hyperpolarization (Krueger, 1990; Sonderset al., 1997; Hoffman et al., 1999). As a member of the family ofNa+ ⁄ Cl–-dependent neurotransmitter transporters (Masson et al.,1999), DAT moves DA against a concentration gradient by cotrans-porting two sodium ions and one chloride ion per DA molecule(Gu et al., 1994). These ionic movements (together with themovement of DA, which at physiological pH is positively charged)produce an inward current of two net positive charges, which has beenused in in-vitro works (voltage-clamp recordings in human dopamine



transporter expressing Xenopus oocytes) to study the voltage depend-ence of DAT activity (Sonders et al., 1997). This study, together withworks on synaptosomes (Krueger, 1990) and brain slices (Hoffmanet al., 1999), has suggested that the membrane potential directlymodulates DAT activity. However, studies in DA cell cultures showedthat neither drugs causing hyperpolarization (raclopride and baclofen)nor those blocking action potentials (tetrodotoxin) modify DATactivity (Prasad & Amara, 2001). This evidence, and the markedmodification voltage needed to activate DAT function in vitro (25%modification of DA uptake after 30 mV change in membranepotential) (Sonders et al., 1997), has suggested that the membranepotential does not modulate DAT activity (Mortensen & Amara,2003). Due to the fact that in-vitro preparations cannot accuratelyresemble the striatal microenvironment that determines DAT activity(Kawagoe et al., 1992; Nirenberg et al., 1996; Hersch et al., 1997),these inconsistencies have not been satisfactorily solved (Hoffmanet al., 1999; Mayfield & Zahniser, 2001; Gulley & Zahniser, 2003;Mortensen & Amara, 2003). The present in-vivo study (microelec-trodes sharpened to produce minimal tissue disturbance and suitablefor quantifying fast and small DA modifications in the extrasynapticmicroenvironment, and rats treated with AMPT to prevent theinterfering action of DA release on DA uptake quantification)provided additional data that, in our opinion, support the membranepotential action on DAT activity.

Medial forebrain bundle stimuli induce action potentials that,moving orthodromically to the striatal terminals, activate a synapticDA release which one supposes is similar to that induced byspontaneous spikes (Gonon, 1988; Wightman & Zimmerman, 1990;Wu et al., 2001). Spontaneous spikes triggered in the initial segmentof the DA cell axon are generally followed by a long-lastinghyperpolarization (> 200 ms) that is caused by the subsequentactivation of different membrane ion channels (Kita et al., 1986;Silva et al., 1990; Shepard & Bunney, 1991; Hausser & Yung, 1994).If synaptic membrane depolarization, which is assumed to be at thebasis of synaptic DA release (this cannot be evaluated at present in in-vivo preparations), is followed in DA terminals or the distal portion ofaxons where DAT has been observed (Nirenberg et al., 1996; Herschet al., 1997) by a post-spike (Wightman & Zimmerman, 1990)hyperpolarization similar to that observed in other DA cell regions(Kita et al., 1986; Silva et al., 1990; Shepard & Bunney, 1991;Hausser & Yung, 1994; Nedergaard, 2004), then hyperpolarizationcould be at the basis of the drain response. The two components of thedrain response observed here agree with this possibility. The drainresponse was integrated by a fast component (which needed high-ratestimuli and was only active during MFB stimulation) and a slowcomponent (which had a long latency and duration, and which waseven stimulated by low-rate stimuli). In a similar way, two compo-nents have recently been found in the after-hyperpolarization of DAcells, a fast hyperpolarization (which can be activated by intense andshort-lasting depolarizing stimuli and has a response onset of �50 ms)and a slow hyperpolarization (which is even evoked by subthresholdpotentials, requires prolonged depolarizing stimuli and has a responseonset of seconds) (Nedergaard, 2004). Similarities observed for thekinetic behavior and stimulation requirement suggest that the fast andslow components of the drain response are associated with the fast andslow after-hyperpolarization, respectively.

Dopamine transporter regulation by membrane potential could havesome advantages over D2 receptor regulation. The D2 receptor actionis very fast but needs the previous release of DA (Kennedy et al.,1992; Benoit-Marand et al., 2001). Thus, this feedback mechanismonly acts after DA overloads and therefore has a limited capability toprevent DA diffusion (Wu et al., 2002). The voltage-dependent

activation of DAT proposed here is a feed-forward adaptationmechanism activated by the membrane potential (thus activatedtogether with DA release) and which will prepare DAT activity for theexpected DA increase according to the action potential trigger. Inaddition, fast and slow components could perform specific functions.The slow component of the drain response was detected for stimulusrates as low as 5 Hz, it increased its amplitude when stimulus ratesincreased to 20 Hz and decreased for rates higher than 35 Hz. Thisresponse window suggests that DAT activation during the slowcomponent could be involved in the regulation of the extracellular DAreleased under no-burst firing activity (0.5–10 Hz) (Grace & Bunney,1984a; Dai & Tepper, 1998; Rodriguez & Gonzalez-Hernandez,1999). The progressive decrease observed for stimulation rates> 35 Hz suggests a stimulus overlapping (decrease in the number ofefficient stimuli), which should be expected if, as mentioned above,the slow component is linked to the slow hyperpolarization (which hasa very soft slope during the basal line recovery). However, the fastcomponent of the drain response showed a short latency and increasedits amplitude when the stimulation rate rose to 100 Hz, whichcorresponds to the characteristics mentioned above for fast hyperpo-larization. This response could be particularly suitable when anefficient adaptation to fast bursts is required (Kiyatkin & Rebec,1998). This membrane potential-linked activation of DAT in DAterminals could be complementary to the membrane potentialdepolarization previously observed in the cell soma after DATactivation. Somatic release of DA elicits depolarizing anion-dependentinward currents that increase the firing activity of DA neurons (Ingramet al., 2002). Thus, there could be an inverse interaction between DATand membrane potential in the soma and terminals, with DAT activityincreasing membrane depolarization in the soma and post-spike after-hyperpolarization increasing DA uptake in the synaptic terminal. Thesimultaneous activation of both mechanisms by a single actionpotential could facilitate the trigger of a new spike and, at the sametime, avoids an excessive diffusion of released DA. This possibilityagrees with the well-known fact that the DA administration in thesubstantia nigra decreases the extracellular DA in the striatum(Nieoullon et al., 1977; Cheramy et al., 1981).In summary, the present data support the hypothesis that firing

patterns directly activate DAT function, an action with two compo-nents, one very fast short-lasting component that could be particularlyrelevant during burst activity, and the other that is slower and morepersistent and is probably involved in DA regulation during no-burstfiring activity. Although DAT activation by membrane potential isprobably facilitated by pre-synaptic DA receptor stimulation, this doesnot seem to be a necessary requirement. We are now studying thebiological basis for the fast and slow components of the drain responseand evaluating their functional relevance under normal (besides otherproposed regulatory mechanisms such as somatic regulation of thefiring rate; Rodriguez et al., 2003a,b) and pathological (e.g. dyskin-esias often observed in Parkinson’s disease after DA drug adminis-tration or DA cell implants; Obeso et al., 2000a; Winkler et al., 2005)conditions.

Acknowledgements

This work was supported by the Ministerio de Educacion y Ciencia de Espanaand by the Consejerıa de Educacion del Gobierno Autonomo de Canarias.

Abbreviations

AMPT, a-methyl-l-tyrosine; DA, dopamine; DAT, dopamine transporter;MFB, medial forebrain bundle.



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Nigrostriatal cell firing action on the dopamine transporter

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