Geometry and kinetics of dopaminergic transmission in the rat striatum and in mice lacking the dopamine transporter

L.°E Agnati, I~. Fuxe, C. Nicholson and E. Sykovfi (Eds.) Progress in Brain Research, Vo1125 © 2000 Elsevier Science BV. All rights reserved.

CHAPTER

16

Geometry and kinetics of dopaminergic transmission in the rat striatum and in mice lacking the dopamine transporter F. Gonon L*, J.B.

Burie 2, M. Jaber, M. Benoit-Marand 1, B. Dumartin 1 and B. Bloch I

CNRS UMR 5541, and 2 CNRS UMR 5466 UFR M.L 2S, Universit# Victor Segalen Bordeaux 2, France

Introduction In the mid 80s Agnati et al. (1986) proposed that intercellular communication in the central nervous system can be grouped into two broad classes based on spatio-temporal characteristics: wiring transmission (WT) and volume transmission (VT) (for a recent review of this concept see Zoli et al., 1998). "WT was defined as a mode for intercellular communication which occurs via a relatively constrained cellular chain (wire), while VT was defined as the three-dimensional diffusion of a signal in the extracellular fluid volume for a distance larger than the synaptic cleft" (Zoli et al., 1998). Dopaminergic transmission in the striatum (including the ventral part, i.e. the nucleus accumbens) has been extensively studied since it is involved in major pathologies (Parkinson, schizophrenia, drug addiction). During the first symposium devoted to 'Volume Transmission in the Brain' in 1989 we favored the view that dopaminergic transmission in the striatum is of the WT type. In fact, in the light of the data available at that time, we proposed that the direct dopaminergic transmission in the striatum occurs inside the synaptic cleft (Gonon et al., 1991). The main functional argument supporting this thesis was that

the extrasynaptic extracellular dopamine concentration, as measured by microdialysis or with electrochemical techniques, was too low to stimulate dopaminergic receptors of the D1 type (Gonon and Buda, 1985; Gonon, 1988; Gonon et al., 1991). However, we also pointed out that some other aspects of dopaminergic transmission such as autoregulation of dopamine release via presynaptic D2 autoreceptors exhibit the characteristics of VT (Gonon et al., 1991; Suaud-Chagny et al., 1991). Since this first symposium, several important observations have been reported concerning dopaminergic transmission in the striatum; all these data support the view that this transmission occurs outside the synaptic cleft. Therefore, according to the definition given by Zoli et al. (1998) dopaminergic transmission must be considered of the VT type. In this chapter these data are reviewed and the spatiotemporal characteristics of dopaminergic transmission in the striatum are discussed in the light of a comparison between normal animals and mice lacking the dopamine transporter.

Geometry of dopaminergic transmission in normal rodents

Localization of dopaminergic receptors D1 receptors

*Corresponding author. Tel.: 33 557 57 15 40; Fax: 33 556 98 61 83; e-mail: Francois.Gonon@umr5541 .u-bordeaux2.fr

In the striatum the most abundant dopaminergic receptors are D1 and D2 receptors (Missale et al.,

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1998). The immunocytochemical localization of these receptors has been studied at the subcellular level by several distinct groups. Most striatal D1 receptors are located in dendrites and "D1 immunoreactive axon terminals are exceedingly rare" (Hersch et al., 1995). In the striatum most dopaminergic terminals form symmetrical synaptic contact on the neck of the dendritic spine (Pickel et al., 1981; Freund et al., 1984). The vast majority of the postsynaptic D1 receptors are not located in front of these dopaminergic synapses but are distributed along the dendritic membranes with a higher density in the peripheral zone of asymmetrical synapses formed by glutamatergic terminals on the head of dendritic spines (Fig. 1) (Levey et al., 1993; Yung et al., 1995; Hersh et al., 1995; Caill6 et al., 1996). D2 receptors In contrast to D1 receptors, presynaptic D2 receptors have been identified in many axon terminals in

the striatum. Most of the terminals labeled with D2 immunoreactivity have been identified as dopaminergic and these observations provide an anatomical basis regarding the role of presynaptic D2 receptors on autoregulation of dopamine release (Levey et al., 1993; Sesack et al., 1994; Yung et al., 1995; Hersh et al., 1995). The subcellular localization of postsynaptic D2 receptors on striatal dendrites has been less firmly established than that of D1 receptors. Nevertheless, similar to D1 receptors, most D2 receptors located on the internal surface of the dendritic membranes were not found in front of dopaminergic terminals but outside these synapses (Levey et al., 1993; Yung et al., 1995; Hersh et al., 1995). These observations strongly suggest that dopamine released at the level of the symmetric contacts formed by dopaminergic terminals must diffuse in the extracellular space to reach the postsynaptic striatal receptors (Levey et al., 1993; Yung et al., 1995; Hersh et al., 1995; Caill6 et al., 1996; Pickel et al., 1996).

Fig. 1. Double detection of D1 receptors (by immunogold) and tyrosine hydroxylase immunoreactivity (by immunoperoxidase) at the electron microscopic level in the rat striatum. A D1 positive dendrite (d) gives rise to a D1 positive spine (s). A tyrosine hydroxylase positive profile is apposed to the dendritic shaft (arrow head). An unlabelled terminal (uT) forms an asymmetrical synapse on the head of the spine. Scale bar : 0.2 txm (from Caill6 et al., 1996).

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Extrasynaptic dopaminergic receptors are functional Extrasynaptic D2 receptors are functional According to in vivo estimates by microdialysis (Justice, 1993) or by electrochemical monitoring (Gonon and Buda, 1985), the basal extracelluar dopamine concentration in the rat striatum is in the range of 5 to 20 nM. Since D2 receptors are thought to exhibit a nanomolar affinity for dopamine (Kebabian and Calne, 1979; Creese, 1982; Missale et al., 1998), extrasynaptic D2 receptors can be stimulated by dopamine diffusing in the extracellular fluid. Indeed, striatopallidal neurons specifically express D2 receptors and the expression of immediate early genes (e.g. c-fos) is selectively enhanced in these neurons when the tonic influence of the basal extracellular level is blocked by D2 antagonists (Robertson et al., 1992) or by an acute decrease in the extracellular dopamine level (Cole and Difiglia, 1994; Svenningsson et al., 1999). Extrasynaptic D1 receptors are functional D1 receptors exhibit at least a 10 times lower affinity for dopamine than D2 receptors (Kebabian and Calne, 1979; Creese, 1982; Missale et al., 1998). Indeed, stimulation of adenylate cyclase activity by dopamine in the striatum is mediated by D1 receptors and requires a dopamine concentration of at least 0.3 p~M (Kebabian et al., 1972; Kelly and Nahorski, 1987). This high level of extracellular dopamine concentration can be reached either after stimulation of dopamine release by amphetamine (Kuczenski et al., 1991) or, according to earlier electrochemical studies, by electrical stimulation of the dopaminergic pathway at frequencies which exceed the physiological discharge rate of dopaminergic neurons (Ewing et al., 1983; Gonon and Buda, 1985). Therefore, we proposed that the stimulation of extrasynaptic D1 receptors by dopamine in physiological conditions was unlikely (Gonon and Buda, 1985; Gonon et al., 1991). More recent measurement of the rapid variations in the extracellular dopamine concentration by

means of fast electrochemical techniques showed that even electrical stimulation with a single pulse evoked a transient increase in the extracellular dopamine concentration which reached 0.2 IxM (Garris et al., 1994a; Dugast et al., 1994; Gonon, 1997). In the light of these recent data it appears now that extrasynaptic D1 receptors can actually be stimulated by dopamine in physiological conditions. Additional arguments supporting this view have been put forward by comparing the transient changes in the extracellular dopamine concentration and the postsynaptic electrophysiological responses mediated by D1 receptors (Gonon, 1997). We showed a positive correlation between the amplitude of the evoked dopamine overflow in the extracellular fluid and that of the postsynaptic response. Moreover, the postsynaptic response triggered by the evoked dopamine release was compared to that induced in the same neurons by the administration of a D1 agonist, SKF 82958. Since we were able to estimate the extracellular concentration of this drug, we compared its in vivo potency to that of dopamine and we found a potency ratio consistent with the much better affinity of this drug for D1 receptors (Andersen and Jansen, 1990). These data strongly suggest that extrasynaptic D 1 receptors can be stimulated by the released dopamine in physiological conditions.

Diffusion of dopamine in the extracellular fluid Dopamine can diffuse outside the synaptic cleft The released dopamine is cleared from the extracellular fluid by reuptake via the dopamine transporter (DAT) (Graefe and Brnish, 1988). The DAT was initially thought to be localized at the level of dopaminergic synapses but recent studies demonstrated DAT labeling on the plasma membrane of dopaminergic terminal fibers near and distant from symmetrical contact formed by dopaminergic terminals (Nirenberg et al., 1996; Pickel et al., 1996). This localization suggests that the released dopamine can diffuse outside the synaptic cleft before clearance by DAT (Nirenberg et al., 1996; Pickel et al., 1996). This view was further supported by functional studies showing that inhibition of DAT drastically

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s l o w e d d o w n the d o p a m i n e c l e a r a n c e but only m o d e r a t e l y e n h a n c e d the m a x i m a l a m p l i t u d e o f the d o p a m i n e overflow e v o k e d in the nucleus a c c u m bens b y a ' p s e u d o - o n e - p u l s e ' stimulation (Garris et al., 1994a) or in the striatum b y a single p u l s e stimulation (Gonon, 1997). T h e s e observations suggest that the r e l e a s e d d o p a m i n e can invade the w h o l e extracellular space to reach the electrode surface b e f o r e m a j o r elimination. Half-life o f the r e l e a s e d d o p a m i n e in the extracellular fluid The m a x i m a l distance to w h i c h d o p a m i n e can diffuse f r o m release sites is limited b y the kinetics o f the d o p a m i n e clearance b y DAT. Fast electroc h e m i c a l m o n i t o r i n g o f the d o p a m i n e overflow e v o k e d b y b r i e f stimulation p r o v i d e s an estimate o f the d o p a m i n e half-life in the striatal extracellular space. This half-life was f o u n d to be in the range o f 60 to 100 m s (Garris and W i g h t m a n , 1994; D u g a s t et al., 1994; S u a u d - C h a g n y et al., 1995; Gonon, 1997). However, as d i s c u s s e d in m o r e detail in Fig. 2, these e x p e r i m e n t a l values r e p r e s e n t an overestimate o f the d o p a m i n e half-life. In fact, we h y p o t h e s i z e that in the living tissue the kinetics o f the d o p a m i n e overflow are faster than those

o b s e r v e d b y c a r b o n fiber electrodes. This is not due to the time resolution o f the e l e c t r o c h e m i c a l technique p e r se (carbon fiber electrode c o u p l e d to continuous a m p e r o m e t r y ) since this time resolution is b e l o w 1 ms as e x e m p l i f i e d b y the electroc h e m i c a l m o n i t o r i n g o f excytotic events (Chow et al., 1996). O u r h y p o t h e s i s is b a s e d on the a s s u m p tion that there is a diffusion zone b e t w e e n the living tissue and the electrode surface (Fig. 2). Diffusion o f d o p a m i n e through this zone d e l a y s and slows d o w n the kinetics o b s e r v e d b y the electrode. W e a t t e m p t e d to take into account this distortion and to calculate the kinetics o f the e v o k e d d o p a m i n e overflow in the living tissue b y deconvolution f o l l o w i n g a p r o c e d u r e a l r e a d y used b y K a w a g o e et al. (1992). A c c o r d i n g to this calculation the dopam i n e half-life m i g h t be in the range o f 2 0 - 3 0 ms in the dorsal striatum (Fig. 2). A n o t h e r a p p r o a c h to estimate the d o p a m i n e halflife has b e e n p r o p o s e d b y Garris et al. (1994a): a s s u m i n g that d o p a m i n e clearance b y DAT can be d e s c r i b e d b y M i c h a e l i s - M e n t e n ' s kinetics, in vivo estimate o f the c o r r e s p o n d i n g p a r a m e t e r s ( K m and V m ) can be used to calculate the d o p a m i n e halflife. T h e s e authors p r o p o s e d a value o f 37 ms for the d o p a m i n e half-life in the nucleus accumbens.

Fig. 2. Experimental and calculated dopamine overflows evoked in the striatum of one mice before (c. and d.) and 20 min after (b.) inhibition of dopamine uptake by nomifensine (20 mg/kg, s.c.). The release of dopamine was evoked by electrical stimulation of the medial forebrain bundle and the resulting dopamine overflow was continuously monitored by amperometry at + 0.4 V with a carbon fiber electrode (Dugast et al., 1994; Benoit-Marand et al., 2000). Rough experimental recordings are shown as dotted lines. They show a delay of 50 to 70 ms between the stimulation and the maximum of the dopamine overflow. This delay cannnot be accounted for by a diffusion delay inside the living striatal tissue since, due to the high density of dopaminergic terminals, the released dopamine can invade the whole extracellular fluid within 3 ms (see text). As illustrated in a. we hypothesize that the delay we observed on experimental curves is due to a diffusion process in a diffusion zone between the living tissue and the carbon fiber electrode. The temporal distortion caused by this diffusion can be removed by deconvolution if the response function of this diffusion process to a concentration step is known (Press et al., 1991). We assume that the overflow evoked by a single pulse in the presence of uptake inhibition represents a suitable experimental response to a concentration step. In b. this experimental curve was fitted with a function C (t, -r) describing the concentration at the electrode surface after diffusion through a film (thickness: l) in response to a concentration step according to equation 4.17 in Crank (1992): C(t, v)/Cmax= 1 - (4/~r) ~

[(-1)n/(2n+ 1)] x exp[-(2n + 1)2t/'r]

n=0

with r=4/2/D~r 2 (D is the diffusion coefficient for dopamine) The best fit of the experimental curve was obtained for -r= 43 ms and Cm~x= 230 nM. In c. and d. the experimental curve was smoothed (continuous line) using cubic spline interpolation. The calculated dopamine overflow in the living tissue corresponds to the part of the curve for t > 0 which was obtained by deconvolution of the smoothed data using the derivative of the function C (t, "r) normalized to 1 and with "r= 43 ms. Notice that the maximum of the calculated overflow evoked by a single pulse is close to that of the experimental overflow measured after uptake inhibition.

295

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Since the density of DAT per dopaminergic terminals is about two times higher in the dorsal striatum than in the nucleus accumbens, the dopamine halflife is briefer in the former than in the latter region (Suaud-Chagny et al., 1995). Therefore, both estimates of the real dopamine half-life are in excellent agreement.

Kinetics of extrasynaptic receptors stimulation by diffusing dopamine The density of dopaminergic terminals is very high in the striatum and the average distance between release sites is about 4 Ixm (Doucet et al., 1986; Groves et al., 1994). Moreover, at dopamine release sites the probability of release per action potential seems very high in vivo (Garris et al., 1994a).

Diffusion distance of dopamine from release sites a,

The diffusion of dopamine in the extracellular fluid has been carefully studied (Nicholson, 1985, 1995; Rice and Nicholson, 1991). The case of dopamine diffusion in the extracellular fluid following its quantal release by exocytosis of a single vesicle in a synaptic cleft, can be considered to be equivalent to the diffusion resulting from the instantaneous deposition at time zero of a very small amount of dopamine in a point. Nicholson (1985) proposed a simple solution to this problem: the dopamine concentration (C) at a distance r from the release site varies with time (t) according to the following relationship. C = K t -3/2 exp (-r2k2/4Dt) (from equation 13 in Nicholson, (1985)) K is a constant term (see Nicholson, 1985) h is the tortuosity coefficient (X = 1.54 in the striatum, Nicholson, (1995)) D is the diffusion coefficient for dopamine (D = 7.6 x 10-6 cm2/sec, value from Nicholson (1995) at a temperature of 37°C) As illustrated in Fig. 3, this concentration reaches a maximum at a time tmaxgiven by: tm,x= (r2/6)(ke/D) (equation 14 in Nicholson (1985)) tmax= 0.52 x r 2 when tmaxis expressed in ms and r in tzm This relationship can be used to estimate the distance r at which at least 50% of the released dopamine can diffuse before elimination by reuptake. With a dopamine half-life in the dorsal striatum of 25 ms this distance is about 7 ixm (Fig. 3).

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Fig. 3. Diffusion of dopamine from release sites. The drawing in a. summarizes morphological data concerning the localization of the dopaminergic terminals and of the DI receptors in the striatum (see text). Notice that the synaptic cleft represents a minute fraction of the whole extracellular space which occupies 21% of the brain volume (Nicholson, 1985, 1995). The curve in b. represents the dopamine concentration at a distance r= 1.5 &m from the release site resulting from the exocytosis of the content of one dopamine vesicle (i.e. about 3000 dopamine molecules, Pothos et al., 1998). This curve was calculated according to equation 13 of Nicholson (1985) (see text). The relationship between tmax and the diffusion distance r is shown in c. and has been calculated according to equation 14 of Nicholson (1985) (see text).

297 Therefore, if dopamine is released simultaneously from all release sites, the whole extracellular fluid is invaded by dopamine within less than 3 ms (as estimated from Equation (14) of Nicholson (1985), see above). The kinetics of dopamine diffusion outside the synaptic cleft has been studied in more details by Garris et al. (1994a) and they also found that, 3 ms after release, the dopamine concentration can be considered as uniform throughout the extracellular space. These considerations have two important consequences. First, dopamine diffusion up to a locally uniform extracellular dopamine concentration is much faster than the elimination of dopamine by reuptake. Second, the delay due to diffusion process before complete stimulation of extrasynaptic dopaminergic receptors is short (below 3 ms) and the duration of this stimulation is regulated by reuptake.

Conclusions (1) The striatal dopaminergic transmission is extrasynaptic but local In the striatum dopaminergic transmission undoubtedly occurs outside synaptic clefts; the released dopamine must diffuse in the extracellular space to reach its target but this diffusion process is fast ( < 3 ms). Since dopamine is rapidly cleared by reuptake, the maximal diffusion distance is in the range of 7 to 10 ~zm. Therefore, regional variations regarding dopaminergic innervation (density, probability of release per action potential) would result in heterogeneous dopamine overflows with well defined boundaries. Such regional variations have been actually reported (Garris et al., 1994b). In conclusion, the stfiatal dopaminergic transmission is extrasynaptic but local. (2) Dopaminergic transmission in the striatum must be classified as a VT All recent data discussed above inescapably lead to the conclusion that, contrary to previous proposals (Gonon and Buda, 1985; Gonon et al., 1991; Agnati et al., 1995), dopaminergic transmission in the striatum of normal rodents must be classified as VT. In fact, according to Agnati and Fuxe's definition

(Agnati et al., 1995; Zoli et al., 1998), dopamine fulfills all the criteria of a substance to be recognized as a VT signal: (i) it is released by a cell in a regulated fashion; (ii) it diffuses at relevant concentration in the extracellular fluid for a distance larger than the synaptic cleft; (iii) it is able to activate selective receptors in a number of target cells; (iv) it triggers physiological responses in target cells.

Kinetics of dopaminergic transmission

Discharge activity of dopaminergic neurons In rats and mice, dopaminergic neuronal cell bodies exhibit two kinds of discharge activity: single spikes and bursts of two to six action potentials (Grace and Bunney, 1984; Sanghera et al., 1984). Individual dopaminergic neurons can switch from one pattern to another and sensory stimuli favor the bursting pattern in unrestrained rats (Freeman and Bunney, 1987). In monkeys, dopaminergic neurons respond to an appetitive stimulus by a burst (Mirenowicz and Schultz, 1996). Grace and Bunney (1984) hypothesized that "bursting may be as important as firing frequency in affecting DA release" and this hypothesis was supported by our early studies (Gonon and Buda, 1985; Gonon, 1988). More recently, we showed however that the main mechanism subserving the high extracellular DA level evoked by stimulations mimicking the bursting activity is not facilitation of the DA release per se, but accumulation of the released DA as a result of overcoming of DA uptake (Chergui et al., 1994a). The single spike pattern of activity is due to the intrinsic properties of dopaminergic neurons since it is observed in slices (Sanghera et al., 1984; Grace and Onn, 1989). In contrast, the bursting activity, which is not observed in vitro (Grace and Onn, 1989), is due to excitatory afferents and is mediated by NMDA receptors (Chergui et al., 1993, 1994b). In anesthetized animals the incidence of bursting is low in the vast majority of dopaminergic neurons (Grace and Bunney, 1984; Sanghera et al., 1984). About 80% of the basal extracellular dopamine concentration is due to the release of dopamine

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triggered by the tonic activity of dopaminergic neurons (Gonon and Buda, 1985; Gonon, 1988).

Transmission mediated by D1 receptors In anesthetized rats, when dopaminergic neurons of the ventral tegmental area (VTA) are forced to fire in bursts by local injection of NMDA, the extracellular dopamine concentration in the nucleus accumbens is enhanced by almost one order of magnitude (i.e. up to 150 nM) (Suaud-Chagny et al., 1992). This enhanced dopamine concentration induces a prominent increase in the discharge activity of a subpopulation of target neurons and this excitation is mediated by D 1 receptors (Gonon and Sundstrom, 1996). When the dopamine release is evoked by an electrical stimulation mimicking one burst (i.e. 4 pulses at 15 Hz, whole duration: 200 ms), an excitatory response can be recorded in a subpopulation of striatal neurons (Gonon, 1997). This response is mediated by D1 receptors, lasts for 0.5 s and is delayed as compared to the evoked dopamine overflow: it starts 0.3 s after the beginning of the stimulation train, i.e. when the released dopamine has been almost completely eliminated by reuptake (Gonon, 1997). This delayed excitatory response fully develops when D1 receptors are stimulated by dopamine for a sufficient duration (i.e. by a burst) since single pulse stimulations are much less effective (Gonon, 1997). All the characteristics of this delayed excitatory response are very similar to those observed in vitro concerning effects mediated by receptors which are coupled with Gproteins (Cole and Nicoll, 1984; Surprenant and Williams, 1987; Isaacson et al., 1993; Batchelor et al., 1994). These data and other studies (Chergui et al., 1996, 1997) strongly suggest that stimulation of D1 receptors by the dopamine release which is evoked by bursts, facilitates the discharge activity of target neurons. The time course of this postsynaptic response is delayed and prolonged as compared to that of the presynaptic signal (i.e. the dopamine overflow) and these kinetics are governed by that of the intracellular G-protein-mediated messenger systems in the target neurons. The diffusion delay which is due to the extrasynaptic localization of D 1 receptors plays a negligible role in the whole time course of dopaminergic transmission.

Transmission mediated by D2 receptors Several studies showed that the expression of early genes (e.g. c-fos) in striatopallidal neurons is tonically inhibited, via D2 receptors, by the basal extracellular dopamine level (Robertson et al., 1992; Cole and Difiglia, 1994; Svenningsson et al., 1999). This view is in line with the tonic D2mediated attenuation of cortical excitation in nucleus accumbens reported by O'Donnell and Grace (1994). Transient inhibition of the tonic discharge activity of dopaminergic neurons induces a parallel decrease in the extracellular dopamine concentration (Suaud-Chagny et al., 1992). Unfortunately, the postsynaptic response to transient changes in the extracellular dopamine concentration mediated by D2 receptors in striatopallidal neurons has not yet been investigated. However, such phasic responses are likely to occur. In fact, the tonic activity of dopaminergic neurons is interrupted by brief (200 ms) silent periods which seem to code errors in the temporal prediction of reward (Hollerman and Schultz, 1998). These silent periods might represent the relevant phasic signal as regards the transmission mediated by D2 receptors.

Conclusion: the temporal properties of the dopamine signal are those of a WT signal The classification of intercellular communication in the brain either as WT or VT has been associated with differential properties. The WT was associated with a brief transmission delay (ms), a time scale in the range of ms to s and a phasic biological effect whereas the VT was associated with a prolonged delay (s to min.), a time scale in the range of s to min. and with a tonic biological effect (Zoli et al., 1998). The data discussed above clearly show that dopaminergic transmission in the striatum exhibits properties associated with WT rather than with VT. Indeed, the fact that dopamine is a VT signal does not imply a prolonged delay: the delay due to dopamine diffusion outside the synaptic cleft is below 3 ms. In other words the onset of the dopamine signal is below 3 ms and its offset (i.e. dopamine clearance by reuptake) occurs within less than 50 ms. Considering together both pre- and

299 postsynaptic aspects of dopaminergic transmission the whole time scale is still below one second and it must be emphasized again that this duration is governed by the kinetics of the intracellular Gprotein-mediated messenger system in target neurons. Thus, the spatial characteristics, which lead to the classification of dopamine as a VT signal, play a negligible role in the time scale of dopaminergic transmission. Last, but not least, the dopaminergic transmission mediated by D1 receptors is solely activated by brief bursts. Although the postsynaptic response is delayed and prolonged, it is closely timely locked to the presynaptic signal. This exclusively phasic transmission might play a major role in learning since it is involved in the coding of salient stimuli. These characteristics might be also those of the transmission mediated by D2 receptors. In conclusion, the temporal properties of dopaminergic transmission in the striatum are those which have been associated with a WT by Zoli et al. (1998).

ing (Mirenowicz and Schultz, 1996). The DAT plays a crucial role in favoring the expression of a burst over the tonic activity. In fact, the dopamine release is completely cleared between pulses at frequency mimicking the tonic activity (4 Hz) whereas it accumulates at burst frequency (15 Hz) (Chergui et al., 1994a). In DAT - / - mice the released dopamine accumulates in the extracellular fluid even at low frequencies (Benoit-Marand et al., 2000). Therefore, despite a very low release per pulse, the basal dopamine level sustained by the tonic activity is five times higher than in normal mice (Jones et al., 1998) but the dopamine overflow evoked by a burst is hardly detectable above this high basal level (Benoit-Marand et al., 2000). Thus, DAT - / - mice are not able to express the bursting activity in terms of phasic changes in extracellular DA and this deficit might be involved in the difficulties these mice experience in spatial learning (Gainetdinov, 1999).

Mice lacking the dopamine transporter

Conclusion: in D A T - / - mice dopaminergic transmission is a pure VT

Spatiotemporal characteristics of dopaminergic transmission

Mice lacking the DAT have been generated by Giros et al. (1996). In these mice the extracellular dopamine level measured in vivo by microdialysis is five times higher than in normal mice despite a whole tissue content of dopamine reduced to 5% of the normal level (Jones et al., 1998). In vitro and in vivo electrochemical studies have shown that the dopamine half-life was prolonged up to the range of several seconds (Giros et al., 1996; Jones et al., 1998; Benoit-Marand et al., 2000) and that the amplitude of the DA release per pulse was reduced to 7% in the striatum (Benoit-Marand et al., 2000). From this prolonged half-life it can be inferred that the maximal diffusion distance in the striatum of DAT - / - mice is about 100 txm. Therefore, the topographical organization of dopaminergic transmission is preserved at the level of the cerebral region (Benoit-Marand et al., 2000). Functional consequences

The bursting activity of dopaminergic neurons is specifically involved in positively reinforced learn-

In mice lacking DAT, the striatal dopamine signal exhibits all the spatial and temporal characteristics associated with a VT signal. In particular, the tonic transmission due to the basal extracellular dopamine level is exacerbated and this is in line with the hyperactive phenotype of these mice, but the phasic dopaminergic transmission triggered by bursts cannot be expressed. The disruption of this phasic function might be involved in the learning deficit of these mice. The comparison between normal and DAT - / - mice underlines the functional importance of the temporal characteristics of dopaminergic transmission in the striatum and the major role of the dopamine transporter in the control of these temporal characteristics. Interestingly, the amplitude and time course of the dopamine overflow in the striatum of mice lacking the dopamine transporter are close to those of the dopamine overflow evoked in the medial prefrontal cortex and in the basal amygdaloid nucleus of normal rats (Garris and Wightman, 1994). These authors pointed out that in both latter regions "extracellular dopamine has a greater possibility of extrasynaptic sites of action" than in the striatum. In other words the

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dopamine signal might be considered as a pure VT signal in these two less densely innervated regions. General conclusion In summary dopaminergic transmission in the striatum must be classified as VT, according to Agnati and Fuxe's definition, but exhibits temporal properties of a WT. This is certainly not a unique case; the same classification is also valid regarding the sympathetic transmission mediated by noradrenaline (Gonon et al., 1993; Stjarne et al., 1994). In more general terms it is likely that many neurotransmissions mediated by G-protein-coupled receptors exhibit spatiotemporal characteristics similar to both aforementioned types of transmission (Hille, 1992). In our opinion, the classification of this type of neurotransmission as a VT might have negative consequences since it might be associated with an inaccurate view of its temporal characteristics. Therefore, we feel that the VT concept should be reconsidered according to one of the three following suggestions. First, in the framework of the present VT definition it must be recognized that many examples of VT are fast and can exert phasic functions. Second, the definition of VT vs. WT might be modified to include perisynaptic neurotransmission inside the WT class. In fact, in our opinion, the characteristics of dopaminergic transmission in the striatum (and similar types of neurotransmission) fit well with the definition given for a WT: "a mode for intercellular communication which occurs via a relatively constrained cellular chain" (Zoli et al., 1998). Or third, intercellular communication in the brain might be classified in three rather than only two classes by considering an intermediate class termed perisynaptic WT. Acknowledgments This work was supported by CNRS, the Universit6 Victor Segalen Bordeaux 2 and La R6gion Aquitaine. References Agnati, L.E, Fuxe, K., Zoli, M., Zini, I., Toffano, G. and Ferraguti, E (1986) A correlation analysis of the regional

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