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NEUROPHARMACOLOGY
Laboratory of Neurobiology and Experimental Neurology, Department of Physiology (M.R., I.M., I.G., S.G., J.L.G.-M.) and Department of Anatomy (T.G.-H.), Faculty of Medicine, University of La Laguna, La Laguna, Tenerife, Canary Islands, Spain
Received March 16, 2006; accepted July 5, 2006.
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Abstract |
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-methyl-L-tyrosine, cocaine, 
-butyrolactone, and haloperidol) suggests that regional differences for both DA release/DA uptake and DA cell firing autoregulation are behind the striatal fountain-drain matrix. The high diversity of DA activity observed in the striatum is a new framework for analyzing experimental and clinical phenomena.
; Albin et al., 1989; DeLong, 1990; Smith et al., 1998; Obeso et al., 2000b). The striatum is massively innervated by mesostriatal dopamine (DA) cells, which, by modulating the cortical action on medium-sized spiny neurons (Dahlstroem and Fuxe, 1964; Ungerstedt, 1971; Fallon and Moore, 1978; Gerfen et al., 1987b; Joel and Weiner, 2000), influence motor and nonmotor functions of the cortex (Gerfen, 2000). At a subcellular level, DA actions are induced at both the synaptic cleft (where DA behaves as a short-lasting neurotransmitter) and the extracellular nonsynaptic space (where it behaves as a long-lasting volume transmitter) (Gonon, 1988; Kawagoe et al., 1992; Sesack et al., 1994; Descarries et al., 1996; Schultz, 1998). The role of each DA pool and the interaction between them is still being researched.
Based on the distribution of different neurochemical markers, the striatum has been segregated into two intricately related compartments, the striosoma (i.e., patch), which has a high density for substance P, dynorphin, and neurotensin markers, and the matrix which has a high density for cholinergic enzymes and calbindin (Goldman-Rakic, 1982; Groves et al., 1988; Graybiel, 1990). There is also a nonhomogeneous DA distribution across the striatum, with local heterogeneity for DA innervation (Voorn et al., 1986; Gerfen et al., 1987a,b; Graybiel et al., 1987; Langer et al., 1991; Gerfen, 1992a,b; Joel and Weiner, 2000), DA concentration (Voorn et al., 1986), tyrosine hydroxylase level (Gerfen et al., 1987b, 1992a), DA receptor (Gerfen et al., 1990), and DA transporter (DAT) (Graybiel et al., 1993; Gonzalez-Hernandez et al., 2004) density. A part of this heterogeneity has been linked to the striosoma/matrix dichotomy (Gerfen et al., 1987b; Graybiel and Moratalla, 1989; Halpain et al., 1990; Hanley and Bolam, 1997). There are electrochemical works suggesting regional diversity for striatal DA transmission (May and Wightman, 1989; Peters and Michael, 2000), but these differences have not been systematically evaluated. In the present work, the regional diversity for DA activity was studied in the striatum by quantifying extrasynaptic DA with a selective and sensitive amperometric method that made it possible to analyze the local heterogeneity of DA response to medial forebrain bundle (MFB) stimulation and its modulation by DA receptor and DAT activity.
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Materials and Methods |
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Electrodes and Electrochemical Methods. DA concentration was quantified here with two high-temporal resolution (<1 s) electrochemical methods, amperometry (Dugast et al., 1994; Gonon, 1997; Benoit-Marand et al., 2001; Puopolo et al., 2001) and fast cyclic voltammetry (FCV) (May et al., 1988; Wightman et al., 1988; Wightman and Zimmerman, 1990; Garris and Wightman, 1994; Wu et al., 2001). The recording electrode was a carbon fiber microelectrode (8-µm diameter and 100 µm long), whose tip was pulled to 
1 µm (Fu and Lorden, 1996) to minimize tissue damage (Fig. 1A). As an additional precaution, the electrode was very slowly introduced in the striatal tissue (20 µm/min) with a micromanipulator (MO-8; Narishige, Tokyo, Japan) modified in our laboratory to include a stepper motor and a digital controller for vertical displacement (1-µm resolution; Sylvac, Crissier, Switzerland). The response to MFB stimulation was tested every 50 µm (half the length of the carbon fiber electrodes) and 5 min after the last movement. Electrodes were pretreated for 20 s with a 70-Hz triangular wave (0-2.3 V versus Ag/AgCl) to optimize their in vivo electrochemical behavior. This treatment causes a very small decrease in the time resolution of electrode (compare 2.5-V-treated electrodes in Fig. 1B with that obtained in untreated electrodes and in 2.9-V-treated electrodes). The sensitivity of the electrodes was approximately 50 times higher (>1 nM) than that obtained with untreated electrodes. The reference electrode (silver wire in vitro coated with a layer of silver chloride by applying +5 V for 3 min) was placed inside a pulled glass capillary tube (filled with a 3 M potassium chloride solution saturated with AgCl; Fluka, Madrid, Spain) with a 300-µm-diameter tip.
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Electrode Calibration and Testing. Selectivity and sensitivity of electrochemical methods and electrodes were tested before their implantation. The active part of the carbon fiber electrode was placed 5 mm into a Teflon capillary tubing (125-µm i.d. and 5 cm in length), which was connected to the exit of a liquid switch (CMA110; CMA Microdialysis, Stockholm, Sweden), which was in turn connected to a dual syringe perfusion pump (CMA102; CMA Microdialysis). The perfusion fluid was a phosphate-buffered saline solution (148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, and 0.8 mM MgCl2), and the flow rate was 40 µl/min. Oxidizable compounds were dissolved in this solution and loaded in one of the two perfusion syringes (the other was loaded with the solution used to dissolve the test compound). The internal volume of the whole system was 40 µl.
In Vivo Preparation. Following previously reported procedures (Rodriguez and Gonzalez-Hernandez, 1999), in vivo studies were performed under chloral hydrate anesthesia (400 mg/kg i.p.) in a sound-proofed dark room (5:00 AM to 10:00 PM after turning the lights on) with the body temperature maintained between 36.5 and 37.0°C. According to previous studies (Chergui et al., 1994; Gonon, 1997), DA released during the electrical stimulation of the MFB was monitored with a carbon fiber microelectrode introduced in the striatum. The electrode was connected to an amperometry detector (BECA Instruments, Tenerife, Spain) that controlled the electrode potential (versus a reference electrode whose open tip was immersed in a saline solution in contact with the meninges through a previously made hole in the skull) by means of a potentiostat. The location of the striatal recording electrode was 0.0 to 1.2 mm anterior to bregma, 2.6 to 3.4 mm lateral to the midline, and 3.8 to 5.0 mm below the cortical surface. Activation of DA inputs to the striatum was performed by the electrical stimulation of the MFB (electrode placed in a position 4 mm anterior to lambda, 1.4 mm lateral to the midline, and 8.0 mm below the cortical surface). Electrical stimuli were provided by an S-8800 Grass model and a bipolar electrode made with two tungsten rods (100-µm diameter; A-M Systems Inc., Everett, WA) etched with a oxyacetylene torch flame (Braga et al., 1977), insulated with glass (fused silica capillary; Composite Metal Service, Hallow, UK) and placed with the tips 500 µm apart. The exposed surface of the tip was approximately 0.5 mm. Stimuli were given in 0.2 to 0.7 mA and 1-ms square biphasic pulses. The striatal DA concentration change induced by the MFB stimulation was estimated on the basis of the calibration of the carbon fiber electrodes before their implantation.
In the last experiments, the effect of DA depletion on striatal response to MFB stimulation was tested. Thus, -methyl-L-tyrosine (AMPT) was i.p. injected (250 mg/kg) during the periodic stimulation of MFB (20-30 Hz, 0.3 mA/1.0-1.5-s stimulus duration, each 20-60 s). Sixty minutes after AMPT administration, extracellular DA was replenished by injecting exogenous dopamine (5 mM) throughout a fused silica capillary tube (75-µm inner diameter) located 400 µm from the recording electrode tip. DA was dissolved 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 and whose osmolarity (auto-osmometer Osmostat OM-6020; CagaK Co. LTD, Kyoto, Japan) was between 280 and 285 mOsm. Two DA fluxes were used (CMA-102 Microdialysis pump; CMA), one which induced a fast and transitory increase of extracellular DA (3 µl/min for 3 s; Fig. 8D) and the other, which induced a slow and progressive increase of DA (continuous perfusion of 0.2 µl/min; Fig. 8E).
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; Gonon, 1997; Staal et al., 2004), with some modifications aimed at optimizing its selectivity for DA and its time and spatial resolution. Amperometric current through the electrode was continuously measured with a time constant of 1 ms, and the detector output was digitized and recorded at 1 kHz (PowerLab system connected to a PC computer; ADInstruments, Castle Hill, Australia). It has been shown that the rapid increase in the current evoked in the striatum by MFB electrical stimulation is caused entirely by the evoked DA overflow (Chergui et al., 1994; Dugast et al., 1994; Gonon, 1997). As an additional precaution and to rule out the possible interference of serotonin, we used a potential of 200 mV instead of the 0.4-V oxidation potential that is generally used for DA (Fig. 1D). In some experiments, the amperometric response was tested at different oxidation potentials. Thus, the oxidation potential was slowly modified between 800 and -800 mV, and the response to MFB stimulation was tested every 50 mV. A three-dimensional map of data (response amplitude x poststimulus time x oxidation potential) was computed and visualized with a color plot similar to that previously used for FCV data (Peters and Michael, 2000).
The electrical artifacts induced by the stimulation pulses on the amperometric signal were initially withdrawn using two methods, one previously used in other laboratories (Chergui et al., 1994; Dugast et al., 1994; Gonon, 1997) and the other reported here. With the previously reported method, a number of stimuli (12 in Fig. 1E) are averaged, and the mean responses obtained at 0-V oxidation potential (Fig. 1F) are subtracted (electrical artifacts) from that obtained at 0.4 V (DA oxidation + electrical artifacts). Thus, the artifact can be reduced without disturbing the amplitude of the amperometric response (Fig. 1G). We also tested a new method that noticeably decreased the stimuli artifact by decreasing the high-frequency components of the stimuli (square pulses were modified by shaving the pulse corners to obtain a stimulus wave form with the initial and final portions of the rising and falling phases, similar to those of a sigmoid wave form), and fast biphasic stimulating pulses were used; the artifact induced by each pulse was very symmetric and lasted 1 to 2 ms and was withdrawn by using a moving average that computed each signal value as the average of the data obtained a few milliseconds before and after it. This new procedure was used in most studies, because it is as efficient at rejecting artifacts as the previously reported method and it does not need the averaging of a number of amperometric responses that increase the time resolution of the study. As shown in Fig. 1H, all amperometric responses to MFB stimulation (and not only the mean recording; Fig. 1G) can be used to quantify the experimental response with this method.
Fast Cyclic Voltammetry Method. FCV quantifies DA concentration by studying currents recorded when the oxidation potential is moved quickly between two boundaries (generally between 1.0 and -1.0 V). Under these conditions, most DA is oxidized in a range between 0.5 to 0.7 V. The FCV method used here was similar to that previously used by others (May et al., 1988; Wightman and Zimmerman, 1990; Garris and Wightman, 1994; Wu et al., 2002). The electrode was held at 0 V, scanned to -1 V, ramped to 1.0 V, back to -1 V, and returned to 0 V. The duration of this cycle was 15 ms, and the cycle was repeated every 100 ms. Current through the electrode was continuously digitized at 20 kHz (PowerLab system connected to a PC computer; ADInstruments).
Drug and Chemicals. Haloperidol, -butyrolactone (GBL), AMPT, L-glutathione reduced, and acetazolamide were obtained from Sigma Chemical Corp. (St. Louis, MO). Cocaine chlorohydrate was obtained from Avello (Barcelona, Spain).
Histology. To verify the location of the recording microelectrode, a lesion was made by passing direct current through the carbon fiber electrode (15 µA of cathodal direct current for 50 s). At the end of each experiment, the rats were transcardially perfused with 200 ml of 0.9% saline solution followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were removed and stored in the same fixative at 4°C for 12 to 24 h, and then the midbrain was cut at 50 µm with a vibratome in the coronal plane and stained with the formal thionine procedure.
Statistics. Mathematical analyses were performed using the one-way analysis of variance followed by the least significant difference test for post hoc comparisons. Analysis was performed using the Statistica program (Statsoft, Tulsa, OK). A level of p < 0.05 was considered as critical for assigning statistical significance.
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In some striatal regions, a current decrease was observed during MFB stimulation, a response that will be referred to as type II response (type IIR, Fig. 2B). The amplitude and duration of type IIR also rose when the rate (Fig. 2B) and duration (Fig. 2D) of stimuli increased. Type IIR often began after the stimulus switch-off when a low-rate stimulation was used (10 Hz in Fig. 2E). In some recordings, type IIR began during stimulation and lasted for 1 to 2 s after stimulus switch-off (40 Hz in Fig. 2E). In this case, the stimulus rate needed to induce a within-stimulus response was often higher than that needed to induce only the poststimulus response (Fig. 2F). Type IIR was selective for the oxidation potential of DA (Fig. 2G) and was not observed for potentials lower than 50 mV. The finding of type IIR suggested that MFB stimulation decreases extracellular DA in certain striatal regions.
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The response type was different and specific in each striatal position and changed when the electrode was moved to neighboring striatal regions, with type IIR being more frequently observed in the dorsolateral edges of the striatum. Striatal loci with type IR will be referred to as fountain areas and those with type IIR as drain areas. Figure 3 shows response examples recorded across a striatal tract. As can be observed, the response was very stable within each area (the response to two consecutive stimuli are shown in each region) but can be very different when the recording electrode was moved 200 µm. The frequency for each response type clearly depends on the region of the striatum that is being recorded (e.g., it is more frequent in the dorsal regions of the striatum just under the corpus callosum than in the middle striatal zones). We did not perform a systematic screening of all striatal regions, and no information about the relative frequency for each response type is provided.
Cocaine Action. The possible involvement of dopamine transporter on the different response types was tested by administering cocaine, a transporter activity inhibitor. Cocaine increased the type IR (Fig. 4, A and C), an effect observed for a wide stimulus range (200-1200 ms in the Fig. 4B example). Cocaine induced a complex modification of type IIR. In most cases, cocaine changed the type IIR responses into a type I/IIR. This change was observed in striatal regions showing a marked type IIR preceded by a small type IR (Fig. 4D) but also in regions showing a pure type IIR (Fig. 4E). In striatal areas showing a type I/IIR, the cocaine effect (Fig. 4F) was much more marked on type IR (600% increase) than on type IIR (25% decrease).
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; Walters et al., 1973; Diana et al., 1991). GBL induced a slight increase (30%) of type IR (Fig. 5, A and B) and a marked decrease (70%) of type IIR (Fig. 5, C-E). In regions showing a type I/IIR, the initial type IR increased (to 300% of the basal response), and the posterior type IIR decreased (to 25% of the basal response) after GBL administration (Fig. 5, F and G). Haloperidol Action. The possible involvement of DA cell receptors on the different response types was tested by administering haloperidol, a DA receptor antagonist that is partially selective for D2-like receptors. Haloperidol increased the type IR (Fig. 6A). Type IIR showed a marked attenuation (for example, see Fig. 6B) after haloperidol administration to approximately 40% of its basal response (Fig. 6C). In regions showing a type I/IIR, the marked increase of the initial type IR together with the decrease of type IIR made type IIR undetectable (see 30 Hz in Fig. 6D).
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pH and Type IIR. A recent FCV study has reported pH changes that could interfere with the DA signal after MFB stimulation (Venton et al., 2003). Thus, we performed different studies to evaluate the possible involvement of pH on type IIR with amperometry and FCV methods.
Initially, we tested in vitro the electrode sensitivity for DA and pH with amperometry methods. In this technique, electrodes showed a more marked sensitivity for DA concentration than for pH modification (Fig. 1C). In addition, no DA-pH overlapping was observed at the oxidation potential typically used for DA quantification with amperometry methods. Thus, for positive oxidation potentials, the DA wave increased with the oxidation potential (Fig. 7, D and G), whereas no response was observed for pH pulses (Fig. 7, E and H). When negative oxidation potentials were used, a small response to DA was observed for potentials lower than -300 mV (Fig. 7, D and G), whereas pH pulses induced a more marked effect, especially at -500 mV (Fig. 7, E and H). These data show that when an oxidation potential of +200 mV is used (i.e., in vivo studies), the pH change does not affect the amperometric signal (Fig. 7F), at least when pH oscillates within the boundaries previously observed in the striatum (<0.1) after the MFB stimulation (Venton et al., 2003).
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7.6) increased at oxidation potentials around -400 mV and decreased it at +600 mV (Fig. 7J). Therefore, the positive peak induced by DA at +600 mV could be reduced by a negative peak induced by a simultaneous increase of pH (Fig. 7K). In agreement with a previous study, present data suggest that care should be taken not to misinterpret pH shifts accompanying terminal activity as DA changes when FCV is used (Venton et al., 2003). In this respect, the amperometric evaluation of DA at +200 mV did not have the same pH interference problem as FCV.
Finally, acetazolamide (a carbonic anhydrase inhibitor that selectively decreases the pH response to MFB stimulation without modifying the DA response) (Venton et al., 2003) was used for in vivo testing the possible influence of pH on type IIR with amperometric methods. This amperometric study showed that after drug administration, no type IIR modifications were observed (Fig. 7A) either in the response amplitude (Fig. 7B) or in the response latency (time taken by the oxidation current to reach its minimal point) (Fig. 7C). Taken together, present data show that pH modifications cannot explain type IIR.
Tyrosine Hydroxylase Inhibition and Dopamine Action. This experiment was aimed at obtaining direct evidence of DA involvement on type IIR. Thus, the effect of -methyl-p-tyrosine (an inhibitor of the rate-limiting enzyme tyrosine hydroxylase) (Michael et al., 1987; Watanabe et al., 2005) on type IIR was evaluated before and 50 to 120 min after AMPT administration, and then (120-200 min after AMPT) DA was directly perfused in the striatum with phasic (3 µl/min x 3 s) or tonic (0.2 µl/min x 1 min) perfusion rates. AMPT dramatically reduced type IIR in three of the six rats studied, decreasing both the response amplitude (Fig. 8C, left) and the response duration (Fig. 8C, right). In the other three rats, a complete disappearance of type IIR was observed after AMPT (Fig. 8B). In rats with complete type IIR disappearance, DA perfusion increased the amperometric current, inducing a fast and transitory increase (phasic perfusion; Fig. 8D) or a slow and progressive increase (tonic perfusion; Fig. 8E). A recovery of the type IIR that disappeared after AMPT was observed after both the phasic DA administration (Fig. 8D) and the tonic DA administration (Fig. 8E). In the second case, the type IIR recovery was progressive and followed the slow increase of the amperometric current induced by DA perfusion.
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Discussion |
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Type IR. Type IR showed the typical DA response to MFB stimulation reported in previous voltammetry (Wightman et al., 1988; Garris and Wightman, 1994; Garris et al., 1994; Wu et al., 2001) and amperometry (Chergui et al., 1994; Dugast et al., 1994) studies. The extracellular DA concentration is generally the result of two opposing mechanisms: DA release and DA uptake. It has been shown that the increase of the electrochemical signal observed after MFB stimulation is induced by an excess of DA release that, after saturating DAT, diffuses until the recording electrode (DA overflow) (Wightman and Zimmerman, 1990; Gonon, 1997; Wu et al., 2001). From this perspective, the rapid decrease of the electrochemical signal observed after the MFB stimulation switch-off (which corresponds to a fast DA concentration decrease) is induced by an efficient activity of DAT (although DA diffusion may play a minor role); the type IR increase induced by cocaine is secondary to a DA uptake inhibition (thus facilitating DA overflow) (Greco and Garris, 2003); and the type IR increase induced by haloperidol is secondary to a blockade of the receptor-mediated presynaptic self-inhibition (thus facilitating DA release and inhibiting DA uptake) (Benoit-Marand et al., 2001; Wu et al., 2002). The mechanisms for the GBL actions here reported are less evident. Bearing in mind the above-mentioned model, the type IR increase observed after GBL administration could be induced by the persistent reduction of extracellular DA secondary to the GBL-induced inhibition of DA cell firing rate (Walters et al., 1973; Bannon et al., 1981, 1982). This hypothesis explains the similar effect observed for GBL (which decreases the presynaptic DA receptor stimulation as a consequence of an extracellular DA concentration decrease) and haloperidol (which decreases the presynaptic DA receptor stimulation as a consequence of a direct blockade) administration.
Type IIR. Some previous electrochemical studies have suggested a regional diversity for striatal DA transmission (May and Wightman, 1989; Peters and Michael, 2000), but, as far as we know, this is the first article reporting striatal regions that respond to the MFB stimulation with a decrease of extracellular DA (type IIR). The finding of type IIR increases in some experimental conditions (use of bipolar short-lasting electrical stimuli, highly sensitive pretreated electrodes and electrochemical methods, and short carbon fibers that reduce the probability of recording adjacent fountain and drain regions simultaneously) and decreases in others (use of long-lasting high-frequency stimuli that increase the type I component of type IR/IIR, recording in nonperipheral regions of the striatum where the finding of type IIR is more common, and administration at the beginning of experiments of repetitive stimuli with short interstimulus). Although a mixture of all these factors can markedly reduce the probability of finding drain regions, we cannot explain why type IIR has never been previously reported. We believe that this response has probably been observed in other laboratories, but it was never systematically studied because its biological explanation and significance are less evident than for type IR.
At the beginning, we found evidence suggesting that type IIR was not produced by collateral nondopaminergic associated phenomena such as electrical artifact, modifications in other transmitters, or modifications in pH or oxygen. Type IIR vanished after decreasing the oxidation potential (Fig. 2G), which shows that it is not a nonspecific artifact induced by MFB stimulation. The amperometric conditions used also suggest that type IIR is not the consequence of changes in other neurotransmitters such as serotonin [the oxidation potential used does not oxidate serotonin (Fig. 1D), and the position of MFB electrodes made the stimulation of serotonergic ascending fibers very improbable] and GABA (GABA is not directly detectable with present methods) released from nondopaminergic mesostriatal neurons. Another two possible confusing variables are oxygen and pH, which can be detected by carbon fiber electrodes and change in the striatum after MFB stimulation (Zimmerman and Wightman, 1991; Zimmerman et al., 1992; Cramer et al., 1997; Venton et al., 2003). DA oxidation and oxygen reduction occur at different potentials (+0.3 and -1.4 V, respectively) (Zimmerman et al., 1992; Venton et al., 2003) and show a different kinetic response (Venton et al., 2003), ruling out the oxygen involvement on type II. Previous (Venton et al., 2003) and present data show a partial overlapping between pH and DA signals when studied with FCV. However, this was not the case for amperometry. Thus, in vitro studies showed DA and pH amperometric waves that did not overlap at the oxidation potential used in in vivo studies (+200 mV). In addition, acetazolamide, a drug that decreases the pH response to MFB stimulation but not the DA response (Venton et al., 2003), did not change type IIR. The pH changes previously observed with FCV after MFB stimulation are slower (began 2 s after stimulation) and longer lasting (>30 s) than type IIR studied here (which generally began during stimulation and persisted for only a few seconds). Thus, in vitro (no pH wave at +200-mV oxidation potential) and in vivo (no type IIR modification after acetazolamide administration and a different kinetic for type IIR to that reported for pH response) studies do not support pH modifications as the basis for type IIR.
On the other hand, we found evidence showing that, similarly to that previously reported for type IR, type IIR is produced by a short-lasting modification of the DA concentration. Thus, type IIR changed after administration of drugs that modify DA synapse (cocaine, haloperidol, and AMPT) and DA cell firing activity (GBL). Data obtained after DA synthesis inhibition were particularly significant. Type IIR markedly decreased or completely vanished after inhibiting tyrosine hydroxylase activity with AMPT, showing that this response needs endogenous catecholamines. Under these circumstances, type IIR was quickly restored by the local perfusion of DA in the striatum, showing that this is the catecholamine involved in type IIR. Contrary to the DA increase associated with type IR (Chergui et al., 1994; Dugast et al., 1994; Gonon, 1997), type IIR is caused by a transitory decrease in the extracellular DA concentration. Thus, the DA cell firing rate activation can increase (Wightman and Zimmerman, 1990; Gonon, 1997; Wu et al., 2001) or decrease extracellular DA, with the striatal region under study being the factor determining one response or the other.
Mechanisms to Explain Type IIR. Two factors could be involved in the type IR/type IIR dichotomy observed in different regions of the striatum: anatomical differences in the DA innervation versus DAT density proportion (Ciliax et al., 1995; Gonzalez-Hernandez et al., 2004) and functional differences of mechanisms involved in the DAT activation induced by DA cell spike firing (Meiergerd et al., 1993; Falkenburger et al., 2001; Khoshbouei et al., 2003; Mortensen and Amara, 2003). It has been suggested that the electrochemical heterogeneity of the striatum could be related to the striosoma/matrix architecture of this center (May and Wightman, 1989). However, the functional organization of the striatum (Alexander and DeLong, 1985a,b; Graybiel, 1990), and the heterogeneity for DA innervation (Voorn et al., 1986; Gerfen et al., 1987a,b; Graybiel et al., 1987; Langer et al., 1991; Gerfen, 1992a,b; Joel and Weiner, 2000), DA concentration (Voorn et al., 1986), DA receptor (Gerfen et al., 1990), and DAT density (Graybiel et al., 1993; Gonzalez-Hernandez et al., 2004) do not always fit in with the striosoma/matrix architecture. We are presently trying to establish an electrochemical-morphological relationship between type IIR and local density for DAT and D2 receptors. However, the electrochemical heterogeneity could be more qualitative (functional status) than quantitative (number of molecules). This might be the case for DAT, whose activity can quickly increase during cell hyperpolarization (Sonders et al., 1997; Falkenburger et al., 2001; Khoshbouei et al., 2003) or D2 autoreceptor stimulation (Meiergerd et al., 1993; Cass and Gerhardt, 1994; Mortensen and Amara, 2003). The acute modification of type IIR observed after the spike firing blockade with GBL or the D2 receptor inhibition with haloperidol supports this possibility. The explanation for type IR/IIR could be a mixture of those proposed here for type IR and type IIR. When DA release (more efficient in type IR regions) and DA uptake (more efficient in type IR regions) are balanced in one striatal region, a type IR/IIR could be observed.
Possible Functional Meaning of Fountain-Drain Striatal Matrix. Bearing in mind the physical dimension of the electrode (8 x 100 µm) and the response modifications observed after small changes (200-500 µm) in the brain position of the electrode, present data suggest the existence of an intricate three-dimensional arrangement of fountain-drain regions within the striatum (fountain-drain striatal matrix). Besides other proposed regulatory mechanisms such as synaptic regulation of DA release (Benoit-Marand et al., 2001; Wu et al., 2002) or somatic regulation of the firing rate (Rodriguez et al., 2003a; Rodriguez et al., 2003b), this fountain-drain matrix could provide another way to modulate the striatal action of DA. Considering the ability of DA to diffuse from the synaptic cleft and to cover long distances across the extracellular space (Kawagoe et al., 1992; Agnati et al., 1995; Descarries et al., 1996; Schultz, 1998; Hoistad et al., 2000), the existence of highly effective striatal regions for sequestering DA excesses could prevent the deterioration of striatal activity induced by an excessive DA diffusion (Doucet et al., 1986; Schneider et al., 1994). An early FCV study of the electrochemical heterogeneity of the striatum (May and Wightman, 1989) found some characteristics in the voltammetric response to MFB stimulation (type I responses because no type IIR was reported in this study), suggesting the existence of striatal regions particularly suitable for preventing an excessive DA diffusion ("mass transfer barrier"). Type IIR of drain areas are a suitable substrate for this task. In this regard, disturbances in the fountain-drain matrix distribution may contribute to the deterioration of the therapeutic response generally observed in advanced Parkinson's disease. This is the case of dyskinesias, a motor complication often observed in Parkinson's disease after chronic DA drug administration (Cotzias et al., 1969; Fahn, 2000; Obeso et al., 2000a) and after DA cell implants (Lindvall and Hagell, 2000; Dunnett et al., 2001; Hagell and Brundin, 2001; Winkler et al., 2005). Dyskinesias has been associated with a transitory increase of DA activity in specific striatal regions. Because DAT expression down-regulates in surviving DA neurons (Chinaglia et al., 1992; Miller et al., 1999; Pirker, 2003; Poewe and Scherfler, 2003; Gonzalez-Hernandez et al., 2004), a functional mitigation of drain regions (perhaps together with other factors such as a loss of firing rate auto-regulation) (Rodriguez et al., 2003a) probably facilitates the diffusion (Doucet et al., 1986; Schneider et al., 1994) and accumulation of DA, thus triggering motor complications.
In summary, we report here evidence showing a marked heterogeneity for the DA release and DA uptake in the striatum, where some regions show DA uptake activation during DA cell firing that is more intense than the corresponding DA release. Because these regions probably mitigate DA diffusion throughout the striatum, their disturbance could facilitate motor complications in basal ganglia disorders. Now we are analyzing the possible morphological basis for the fountain-drain matrix and identifying the synaptic mechanisms involved in type IIR.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: DA, dopamine; DAT, dopamine transporter; MFB, medial forebrain bundle; FCV, fast cyclic voltammetry; AMPT, -methyl-L-tyrosine; GBL, -butyrolactone; type IR, amperometric signal increase; type IIR, amperometric signal decrease.
Address correspondence to: Dr. Manuel Rodríguez Díaz, Departamento de Fisiologia, Facultad de Medicina, Universidad de La Laguna, 38320 Tenerife, Canary Islands, Spain. E-mail: mrdiaz{at}ull.es
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