**3. Regulatory mechanisms**

246 Neuroscience – Dealing with Frontiers

observations on psychostimulants and amphetamine-like substances (Butcher et al., 1988; Fisher and Cho, 1979; Leviel, 2001; Levi and Raiteri, 1993; Nash and Brodkin, 1991). In this process, transmitter molecules are transported outside the cell, most likely via an exchangediffusion process. This mode of transfer has been extensively studied and various mathematical models have been proposed over the course of the last four decades (Chubb et al., 1972; Stein, 1990, for a review; Thoenen et al., 1969; Wheeler et al., 1993; Ziance and

During the 1980s, several studies were devoted to the regulatory mechanism of DA release in the STR and the substantia nigra pars reticulate (SNr). Particular attention was given to the control of striatal release, which is known to be regulated by GLU. It appeared that, in addition to a spike-mediated DA efflux, GLU-induced amine overflow could occur independently of any sodium or calcium transfer. This would mean that some of the extracellular DA, the NMDA-mediated DA that is released, is not dependent on the firing of DA neurons (Carter et al*.*, 1988; Cheramy et al*.*, 1986; Giorguieff et al*.*, 1977; Jhamandas and Marien, 1987). The NMDA-mediated release of DA even remained unchanged when the nigrostriatal pathway was blocked with lesions or tetrodotoxin (Keefe et al*.*, 1992, 1993). Recently, using voltammetry associated with electrochemical detection, the same type of observation was again reported (Borland and Michael, 2004). Such observations had already led Anthony Grace to propose, at the beginning of the 1990s, that DA could be released in the STR following two different types of mechanism (Grace, 1991): first, the firingdependent exocytosis of quantal amounts of amine previously stored in specific pools, and,

More recently, Falkenburger et al. (2001) confirmed the existence of DA-RT in slices from rat SNc. This explained at least part of the dendritic release of DA. The presence of the somatodendritic release of DA from the nigrostriatal cells has been known for many years (Cheramy et al., 1981) but the mechanism of this release was, and remains, an open question. As in the experiment conducted by Olivier and co-workers, the demonstration was based on the use of DAT inhibitors (GBR 12909, GBR 12935) that were able to block the GLU-induced release of DA. In a more recent study (Opazo et al*.*, 2010), the voltammetric current corresponding to outward moving DA was increased by adding 1- Aminocyclopentane-trans 1,3 dicarboxylic acid (*trans* ACPD), an activator of the GLU group I mGLUR, to rat nigral slices. This confirmed the role of GLU in this type of DA release. It was also demonstrated that protein kinase C (PKC) activation produced a robust increase in external DA levels that was reversibly inhibited by GBR 12935, a well-known DAT inhibitor. These observations confirmed the identities of the two DA releasing processes. These observations were also made *in vitro* in an engineered neuroblastoma cell line (SH-SY5Y

The two release processes act simultaneously in both the SNc and the dSTR. As mentioned above, and given the various morphologies of DA neurons (Arluison et al*.*, 1978; Gauthier et al., 1999; Gerfen et al., 1987; Prensa et al., 2000), the possibility that some types of DA neurons could be specialized in one or the other of the release modes cannot be excluded. For instance, it is noteworthy that mesencephalic DA neurons from the ventral tegmental area poorly express DAT (Blanchard et al., 1994; Ciliax et al*.*, 1999; Mengual and Pickel, 2004; Nirenberg et al*.*, 1996; Sesack et al., 1998). The dual release function of DA neurons may not be equally distributed throughout the whole population of DA cells. The term

Rutledge, 1972).

simultaneously, NMDA-mediated release.

cells) stably expressing DAT (Opazo et al., 2010).

It has already been shown that the extracellular level of calcium (Ca++) ions has an important influence on neurotransmitter release (Katz and Miledi, 1970; Augustine et al., 1987; Smith and Augustine, 1988; Mulkey and Zucker, 1991) and on enzyme activities (Kapatos and Zigmond, 1982; El Mestikawy et al., 1983; Haycock, 1993). Since transient increases in Ca++ within nerve terminals are spatially restricted (Lipscombe et al., 1988; Meldolesi et al., 1988; Miller, 1991) it may be hypothesized that Ca++ independently affects multiple processes within the different ultrastructural compartments of the nerve terminal. The metabolism and release of DA in the STR constitute an appropriate experimental model with which to investigate the potentially distinct effects of Ca++. In the dopaminergic neurons DA synthesis depends on the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in its synthetic pathway (Levitt et al., 1965). This enzyme is activated by phosphorylation under the control of various protein kinases, including two Ca++-dependent ones, the Ca++-phospholipid-dependent protein kinases (PKC) and the Ca++-calmodulin-dependent protein kinase (PKII) (El Mestikawy et al., 1983; Albert et al., 1984; Zigmond et al., 1989; Haycock, 1993).

Below we will present further data about the dual control exerted by Ca++ ions on DA synthesis and on DA-releasing processes. The apparent challenge between synthesis and release in the effects of Ca++ is coming from the fact that DA-RT acts on cytoplasmic DA , unlike exocytosis, which affects a previously vesicularysed stored pool of DA. It can thus be hypothesizd that DA-RT should be more sensitive to those treatments affecting synthesis.

#### **3.1 Synthesis, calcium ions and DA release – Experimental report**

We describe here the only method known to evaluate DA synthesis and release simultaneously *in vivo* in anesthetized rats. The method is based on the measurement of the specific activity (DAsa) of the DA released during continuous superfusion of the tissue with the tritiated DA precursor, [3H]tyrosine. It was previously observed that the neosynthesized amine is released preferentially to an oldly stored pool. Taking advantage of this metabolic specificity, it can be postulated that: 1) increased DA*sa* during higher levels of release reflects enhanced synthesis and 2) decreased DA*sa* associated with increased levels of release reveals the involvement of stored amine (vesicular) and suggests reduced or insufficient synthesis. The DOPAC efflux is also considered to be a robust index of DA synthesis (Zetterström et al., 1988; Leviel et al., 1989). In other words, any treatment reducing synthesis should reduce cytoplasmic DA*sa* and the DOPAC efflux. In contrast, any treatment activating synthesis should elevate DA*sa* and the DOPAC efflux.

Normal and Physio-Pathological Striatal Dopamine Homeostasis 249

Adding the Ca++ ionophore A23187 (1 µmol/l) to the superfusing fluid for 20 min had no effect on the spontaneous release of DA or DAsa (Fig. 7). However, an increase in DOPAC

It is possible to evaluate the effect of the calcium ionophore A23187 on DA synthesis '*in vivo*'. A high dose of amphetamine can be superfused to produce the maximum overflow of the intraterminal DA. Thus 1 hour after administering 20 min of 1 mmol/l amphetamine, the total intraterminal DA had increased, particularly the DA*sa* (Olivier et al., 1999). This

**Time (Min.)**

Fig. 7. Superfusion with the Ca ionophore A23187 increases DA synthesis and DOPAC

The above observations confirm that Ca++ ions are involved in different steps of DA metabolism, including not only the processes of release (Augustine et al., 1987) but also synthesis (Kapatos and Zigmond, 1982; El Mestikawy et al., 1983; Haycock et al., 1984). We have not directly measured the enzymatic activity of TH, but alterations in synthesis were revealed by the dynamic variations in DA*sa* and the [3H]DOPAC efflux. Indeed the DAsa can be used as an index of DA synthesis (Herdon et al., 1985) since the newly synthesized DA has a higher specific activity than the stored amine (Leviel et al., 1989). [3H]DOPAC might also be considered as a good index of amine synthesis according to many authors (Herdon

Superfusion with calcium ionophore (A23187): The forced entry of calcium

(25%) was associated with a 50% increase in the [3H]DOPAC concentration.

demonstrates a large increase in synthesis during this period of time.

**Percent of Controls Specific Activity (Ci/mmole)**

**3.2 The effect of extracellular calcium ions on DA-RT** 

efflux

Fig. 5. Superfusion with CSF from which Ca++ was omitted

Superfusion with calcium-free medium and α-methyl para-tyrosine (α-mpt)

We already know that local superfusion with the calcium channel blockers cadmium and cobalt enhances regional DA release and blocks firing-induced exocytosis. Removing Ca++ ions from the superfusing medium (Ca0) led to a sharp decrease in DA release, stabilizing at around 30% of the basal value after about 80 min. This effect was accompanied by a lowering of the specific activity of DA*sa* and of [3H]DOPAC (Fig. 5). The addition of α-mpt, a TH inhibitor, did not modify the effect of Ca0 treatment: the DA collected in each 20-minute fraction also stabilized at around 30% of the spontaneous values (Fig. 6). These two effects were thus not additive, and the lowering of the DAsa produced by superfusion with Ca0 was of the same amplitude as that observed in the α-mpt treatment. This observation strongly suggests that the main effect of Ca0 could be the inhibition of DA synthesis (Olivier et al., 1999).

Fig. 6. The inhibition of DA synthesis during superfusion in the absence of Ca++

**Specific Activity of the DA released**

We already know that local superfusion with the calcium channel blockers cadmium and cobalt enhances regional DA release and blocks firing-induced exocytosis. Removing Ca++ ions from the superfusing medium (Ca0) led to a sharp decrease in DA release, stabilizing at around 30% of the basal value after about 80 min. This effect was accompanied by a lowering of the specific activity of DA*sa* and of [3H]DOPAC (Fig. 5). The addition of α-mpt, a TH inhibitor, did not modify the effect of Ca0 treatment: the DA collected in each 20-minute fraction also stabilized at around 30% of the spontaneous values (Fig. 6). These two effects were thus not additive, and the lowering of the DAsa produced by superfusion with Ca0 was of the same amplitude as that observed in the α-mpt treatment. This observation strongly suggests that the main effect of Ca0 could be the inhibition of DA synthesis (Olivier et al.,

**Specific Activity of the DA released**

Fig. 6. The inhibition of DA synthesis during superfusion in the absence of Ca++

**(in Ci/mmole)**

**Min. Min.**

Fig. 5. Superfusion with CSF from which Ca++ was omitted

Superfusion with calcium-free medium and α-methyl para-tyrosine (α-mpt)

**(in Ci/mmole)**

**Min. Min.**

**DA Release**

1999).

**DA Release**

**(in percent of controls)**

**(in percent of controls)**

Superfusion with calcium ionophore (A23187): The forced entry of calcium

Adding the Ca++ ionophore A23187 (1 µmol/l) to the superfusing fluid for 20 min had no effect on the spontaneous release of DA or DAsa (Fig. 7). However, an increase in DOPAC (25%) was associated with a 50% increase in the [3H]DOPAC concentration.

It is possible to evaluate the effect of the calcium ionophore A23187 on DA synthesis '*in vivo*'. A high dose of amphetamine can be superfused to produce the maximum overflow of the intraterminal DA. Thus 1 hour after administering 20 min of 1 mmol/l amphetamine, the total intraterminal DA had increased, particularly the DA*sa* (Olivier et al., 1999). This demonstrates a large increase in synthesis during this period of time.

Fig. 7. Superfusion with the Ca ionophore A23187 increases DA synthesis and DOPAC efflux

#### **3.2 The effect of extracellular calcium ions on DA-RT**

The above observations confirm that Ca++ ions are involved in different steps of DA metabolism, including not only the processes of release (Augustine et al., 1987) but also synthesis (Kapatos and Zigmond, 1982; El Mestikawy et al., 1983; Haycock et al., 1984). We have not directly measured the enzymatic activity of TH, but alterations in synthesis were revealed by the dynamic variations in DA*sa* and the [3H]DOPAC efflux. Indeed the DAsa can be used as an index of DA synthesis (Herdon et al., 1985) since the newly synthesized DA has a higher specific activity than the stored amine (Leviel et al., 1989). [3H]DOPAC might also be considered as a good index of amine synthesis according to many authors (Herdon

Normal and Physio-Pathological Striatal Dopamine Homeostasis 251

DA. However, the same treatment had no effect on basal DA in the absence of AMPh. These studies demonstrated that AMPh-induced and DAT-mediated currents producing substrate efflux require internal Ca++ release from intracellular stores and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca++ levels (Gnegy et al*.,* 2004). It has also been shown that Ca++/calmodulin-dependent protein kinase-II (CaMKII) plays a key role in the amphetamine-mediated efflux of DA in heterologous cells and dopaminergic neurons. CaMKII binds to the distal C terminus of DAT and co-localizes with DAT in dopaminergic neurons. The distal serines of the N terminus of DAT were phosphorylated by CaMKII *in vitro* and the mutation of these serines eliminated the stimulatory effects of CaMKIIa. A mutation of the DAT C terminus, impairing CaMKIIa binding, also impaired AMPh-induced DA efflux. Thus, it was suggested that the binding of CaMKIIa to the DAT C terminus facilitates phosphorylation of the DAT N terminus, which is responsible for the

A non-amphetaminic effect of CaMKII was also reported. Pseudo-phosphorylation of the hDAT N-terminal serines (S/D mutation), which appear to be phosphorylated by CaMKII, is largely able to evoke the reverse transport of DA even in unclamped cells, i.e. at resting membrane potential. This showed that activation of this enzymatic system (DAT) constitutes a mechanism of release by itself, even in the absence of amphetamine or any exogenous trigger. CaMKII is the most abundant kinase in the brain and could play a role in the regulation of various neurotransmitters (Colbran et al., 2003; Griffith, 2004). It is well known that local increases in Ca++ concentration and the CaMKII complex interact with the transduction of several neuronal signals, whether from DA neurotransmitters or not. Numerous proteins, such as the NR2B subunit of the NMDA receptor, could be involved in tonic release processes. It was recently proposed that tonic hyperactivity of the GLU afferents to the dopaminergic striatal terminals could maintain tonic-activated DA overflow in the rat STR following a 6-OHDA lesion of the SNc (Dzahini et al., 2010), and mechanisms such as those presently known to be evoked could be responsible for the effects observed. The same type of tonic hyperactivity could also be present in the mesencephalic region of the SNc. Sustained hypertonic GLU neurotransmission is often observed and reported in experimental and natural situations of neuro-degeneration, and could maintain secondary

amine overflow without immediate consequences, but cause insidious degeneration.

inward regulatory processes differ from the release mechanism.

**3.3 Intraterminal DA metabolism: From synthesis to release** 

and positively coupled with Ca++ entry.

The main finding of recent reports about the regulatory processes of the DAT protein is the complete dissociation between inward (uptake) and outward (release) efflux. Untill recently DAT was considered to be an exchange diffusion system with a stoichiometric equilibrium linking the two functions. It is now clear that this concept should be revisited and that the

In summary, three steps in DA metabolism occur to determine the extracellular DA concentration: synthesis, exocytosis and reverse transport (fig.8). Directly or indirectly, Ca++ ions modulate each of these. Thus carrier-mediated release (DA-RT) appears to be indirectly and negatively coupled with calcium entry, whereas synthesis and exocytosis are directly

The observations presented here and the numerous reports about DA synthesis, storage and release have enabled the proposal of a general model of the striatal DA terminal in

AMPh-induced dopamine efflux (Fog et al*.*, 2006).

et al., 1985; Soares-Da-Silva, 1987; Zetterström et al., 1988; Leviel et al., 1989; Soares-Da-Silva and Garrett, 1990a, 1990b), who have proposed that extracellular DOPAC originates from an unreleased and recently synthesized pool of dopamine.

Ca++ ions in the regulation of DA synthesis:

Removal of Ca++ from the superfusing fluid induced a large decrease in DA*sa* and in [3H]DOPAC efflux, suggesting reduced DA synthesis. Futhermore the addition of a TH inhibitor, namely α-mpt, to a Ca++-free superfusing fluid did not further decrease the DA*sa* or the [3H]DOPAC, suggesting that the activity of TH is already maximally inhibited by Ca++ removal. TH activity could be inhibited by a rise in DA feedback inhibition resulting from the lowering of Ca++-dependent exocytosis (Leslie et al., 1985; Zucker and Lando, 1986; Westerink et al., 1988). However, the intracellular accumulation of DA should result in increased DOPAC efflux, which was not observed. Conversely, the reduced DA*sa* revealed that even to maintain a low rate of DA release, stored amine (with a low specific activity) has to be involved, showing that in the absence of extracellular calcium ions, DA synthesis is unable to sustain the lower DA release (30% of the control value).

Superfusion without Ca++ in the CSF appears to be able to reduce synthesis. Forced Ca++ entry could have the opposite effect. Indeed a low dose of the Ca++ ionophore A23187 (1 µmol/l) slightly increased [3H]DA and [3H]DOPAC efflux, suggesting an increase in DA synthesis (Fig. 7). Such activation of synthesis was confirmed by the potentiation of the amphetamine-induced [3H]DA release that followed pretreatment with A23187 1 µmol/l (Olivier et al., 1999).

This set of simple data is consistent with the concept that moderate Ca++ entry is positively coupled with DA synthesis and that its absence from the extracellular medium is negatively coupled with DA synthesis (Kapatos and Zigmond, 1982; El Mestikawy et al., 1983; Haycock et al., 1984).

Ca++ ions in the regulation of DA-RT:

Surprisingly, *in vivo* blockade of the Ca++ ion channel with Cd++ ions activates DA-RT from the DA terminals, an effect that was inhibited by further application of GBR 12909, a DAT inhibitor. The mechanism by which Cd++ affects DA-RT is not clear. For a long time, reversal of the Na+ gradient was proposed to reverse DA transport (Roth et al., 1976; Amejddki-Chab et al., 1992; Levi and Raiteri, 1993; Okada et al., 1990). It was also proposed by Olivier et al. (1999) that Cd++ indirectly alters DA-RT via Na+/K+ ATPase activity, a hypothesis also proposed for the action of AMPh (Khoshbouei et al., 2003; Pal et al*.,* 1993).

After applying a Ca++ ionophore, A23187, DA synthesis increased. With the classical model used for DA-RT of an exchange diffusion process, it is clear that the cytosolic DA constitutes the direct substrate for internal DAT-sites, regardless of its affinity. Thus, from these experiments, and despite the fact that DA-RT can be considered as being firingindependent, many observations during the last decade have confirmed indirect calcium action on DA-RT (Fog et al*.*, 2006; Gnegy et al., 2004; Kantor et al., 2001; Page et al., 2004).

Numerous reports highlight the role of Ca++ in the amphetamine-dependent DA-RT. The Ca++ chelator BAPTA-AM reduced amphetamine-induced DA efflux as measured by amperometry. A particularly interesting observation was that superfusion of rat striatal slices with 50 µM BAPTA-AM suppressed the amphetamine-induced release of endogenous

et al., 1985; Soares-Da-Silva, 1987; Zetterström et al., 1988; Leviel et al., 1989; Soares-Da-Silva and Garrett, 1990a, 1990b), who have proposed that extracellular DOPAC originates from

Removal of Ca++ from the superfusing fluid induced a large decrease in DA*sa* and in [3H]DOPAC efflux, suggesting reduced DA synthesis. Futhermore the addition of a TH inhibitor, namely α-mpt, to a Ca++-free superfusing fluid did not further decrease the DA*sa* or the [3H]DOPAC, suggesting that the activity of TH is already maximally inhibited by Ca++ removal. TH activity could be inhibited by a rise in DA feedback inhibition resulting from the lowering of Ca++-dependent exocytosis (Leslie et al., 1985; Zucker and Lando, 1986; Westerink et al., 1988). However, the intracellular accumulation of DA should result in increased DOPAC efflux, which was not observed. Conversely, the reduced DA*sa* revealed that even to maintain a low rate of DA release, stored amine (with a low specific activity) has to be involved, showing that in the absence of extracellular calcium ions, DA synthesis is

Superfusion without Ca++ in the CSF appears to be able to reduce synthesis. Forced Ca++ entry could have the opposite effect. Indeed a low dose of the Ca++ ionophore A23187 (1 µmol/l) slightly increased [3H]DA and [3H]DOPAC efflux, suggesting an increase in DA synthesis (Fig. 7). Such activation of synthesis was confirmed by the potentiation of the amphetamine-induced [3H]DA release that followed pretreatment with A23187 1 µmol/l

This set of simple data is consistent with the concept that moderate Ca++ entry is positively coupled with DA synthesis and that its absence from the extracellular medium is negatively coupled with DA synthesis (Kapatos and Zigmond, 1982; El Mestikawy et al., 1983; Haycock

Surprisingly, *in vivo* blockade of the Ca++ ion channel with Cd++ ions activates DA-RT from the DA terminals, an effect that was inhibited by further application of GBR 12909, a DAT inhibitor. The mechanism by which Cd++ affects DA-RT is not clear. For a long time, reversal of the Na+ gradient was proposed to reverse DA transport (Roth et al., 1976; Amejddki-Chab et al., 1992; Levi and Raiteri, 1993; Okada et al., 1990). It was also proposed by Olivier et al. (1999) that Cd++ indirectly alters DA-RT via Na+/K+ ATPase activity, a hypothesis also

After applying a Ca++ ionophore, A23187, DA synthesis increased. With the classical model used for DA-RT of an exchange diffusion process, it is clear that the cytosolic DA constitutes the direct substrate for internal DAT-sites, regardless of its affinity. Thus, from these experiments, and despite the fact that DA-RT can be considered as being firingindependent, many observations during the last decade have confirmed indirect calcium action on DA-RT (Fog et al*.*, 2006; Gnegy et al., 2004; Kantor et al., 2001; Page et al., 2004). Numerous reports highlight the role of Ca++ in the amphetamine-dependent DA-RT. The Ca++ chelator BAPTA-AM reduced amphetamine-induced DA efflux as measured by amperometry. A particularly interesting observation was that superfusion of rat striatal slices with 50 µM BAPTA-AM suppressed the amphetamine-induced release of endogenous

proposed for the action of AMPh (Khoshbouei et al., 2003; Pal et al*.,* 1993).

an unreleased and recently synthesized pool of dopamine.

unable to sustain the lower DA release (30% of the control value).

Ca++ ions in the regulation of DA synthesis:

(Olivier et al., 1999).

Ca++ ions in the regulation of DA-RT:

et al., 1984).

DA. However, the same treatment had no effect on basal DA in the absence of AMPh. These studies demonstrated that AMPh-induced and DAT-mediated currents producing substrate efflux require internal Ca++ release from intracellular stores and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca++ levels (Gnegy et al*.,* 2004). It has also been shown that Ca++/calmodulin-dependent protein kinase-II (CaMKII) plays a key role in the amphetamine-mediated efflux of DA in heterologous cells and dopaminergic neurons. CaMKII binds to the distal C terminus of DAT and co-localizes with DAT in dopaminergic neurons. The distal serines of the N terminus of DAT were phosphorylated by CaMKII *in vitro* and the mutation of these serines eliminated the stimulatory effects of CaMKIIa. A mutation of the DAT C terminus, impairing CaMKIIa binding, also impaired AMPh-induced DA efflux. Thus, it was suggested that the binding of CaMKIIa to the DAT C terminus facilitates phosphorylation of the DAT N terminus, which is responsible for the AMPh-induced dopamine efflux (Fog et al*.*, 2006).

A non-amphetaminic effect of CaMKII was also reported. Pseudo-phosphorylation of the hDAT N-terminal serines (S/D mutation), which appear to be phosphorylated by CaMKII, is largely able to evoke the reverse transport of DA even in unclamped cells, i.e. at resting membrane potential. This showed that activation of this enzymatic system (DAT) constitutes a mechanism of release by itself, even in the absence of amphetamine or any exogenous trigger. CaMKII is the most abundant kinase in the brain and could play a role in the regulation of various neurotransmitters (Colbran et al., 2003; Griffith, 2004). It is well known that local increases in Ca++ concentration and the CaMKII complex interact with the transduction of several neuronal signals, whether from DA neurotransmitters or not. Numerous proteins, such as the NR2B subunit of the NMDA receptor, could be involved in tonic release processes. It was recently proposed that tonic hyperactivity of the GLU afferents to the dopaminergic striatal terminals could maintain tonic-activated DA overflow in the rat STR following a 6-OHDA lesion of the SNc (Dzahini et al., 2010), and mechanisms such as those presently known to be evoked could be responsible for the effects observed. The same type of tonic hyperactivity could also be present in the mesencephalic region of the SNc. Sustained hypertonic GLU neurotransmission is often observed and reported in experimental and natural situations of neuro-degeneration, and could maintain secondary amine overflow without immediate consequences, but cause insidious degeneration.

The main finding of recent reports about the regulatory processes of the DAT protein is the complete dissociation between inward (uptake) and outward (release) efflux. Untill recently DAT was considered to be an exchange diffusion system with a stoichiometric equilibrium linking the two functions. It is now clear that this concept should be revisited and that the inward regulatory processes differ from the release mechanism.

In summary, three steps in DA metabolism occur to determine the extracellular DA concentration: synthesis, exocytosis and reverse transport (fig.8). Directly or indirectly, Ca++ ions modulate each of these. Thus carrier-mediated release (DA-RT) appears to be indirectly and negatively coupled with calcium entry, whereas synthesis and exocytosis are directly and positively coupled with Ca++ entry.

#### **3.3 Intraterminal DA metabolism: From synthesis to release**

The observations presented here and the numerous reports about DA synthesis, storage and release have enabled the proposal of a general model of the striatal DA terminal in

Normal and Physio-Pathological Striatal Dopamine Homeostasis 253

**4.1 Progressive DA denervation of the STR enhances DA transport in synaptosomal** 

Why address the consequences of DA cell degeneration? So far we have described the equilibrium between two mechanisms that release DA, but only one of these is likely to be able to maintain DA homeostasis in the extracellular space. For a long time it was thought that the progressive degeneration of the DA cells of the SN, such as observed in Parkinson'disease (PD), is accompanied by the preservation of the DA concentration in the STR, a region that is innervated by DA axons. Basic observations have ascertained that DA metabolism is activated in spared dopaminergic terminals of the partially denervated STR. Both increased DA synthesis and reduced storage capacity were recurrently reported (see Zigmond et al., 1990 for a review). This was considered to be the possible cause of the unmodified (or even sometimes increased) extracellular DA level in this region (Stackowiak et al., 1987; Altar and Marien, 1989; Espino et al., 1995; Dentresangle et al., 2001; Dzahini et al., 2010). Nevertheless, no changes in either the firing pattern or the efficacy of release were observed in nigral DA cells following a moderate partial lesion (Hollerman and Grace, 1990; Dentresangle et al., 2001). On the contrary, spared DA neurons were described as hypoactive and pre-apoptotic (Pasinetti et al., 1989). This adaptation is unlikely to be the

Extracellular DA is taken up into dopaminergic terminals. In partially lesioned rats, the long-term reduction of this DA uptake may allow more diffusion in tissues (Snyder et al., 1990; van Horn et al., 1992; Gerhardt et al., 1996). This hypothesis is reinforced by the fact that the depolarization of cells bearing the DA transporter reduces their ability to take up extracellular amine (Roth et al., 1976; Zahniser et al., 1998). GLU neurotransmission could be involved in this process. Indeed GLU is responsible for tonic membrane depolarization in CP (Wilson et al., 1995) and GLU neurotransmission is known to be hyperactive following a partial lesion (Lindefors and Ungerstedt, 1990; Samuel *et al.*, 1990; Iwasaki *et al.*, 1992;

Reduced DA uptake due to a reduced number of DA terminals is however unable to account for the maintained dopaminergic function as the extracellular DA concentration should also be correlated with a reduction in the number of release sites. No changes in the number of uptake sites *per neuron* were reported and the binding of the transporter ligands was even considered to be a good index of the terminal depopulation (Maloteaux et al., 1988; van Horne et al., 1992). Thus normal or increased levels of extracellular DA, associated with a reduced number of dopaminergic terminals, imply an increased release/uptake ratio *per terminal*. GLU neurotransmission in the STR may mediate this increase. GLU was reported to increase DA synthesis (Desce et al., 1992; Fillenz, 1993; Castro et al., 1996), to activate DA release (Giorguieff et al., 1977; Cheramy et al., 1986; Leviel et al., 1990; Keefe et al., 1992) and to reduce DA uptake (Lin and Chai, 1998). The mechanism by which basal DA release is altered is however poorly understood. In the absence of any change in DA cell activity, alterations should involve DA-RT. This concept of activation of the DA metabolism through local GLU-dependent activation of DA-RT from the striatal DA terminal in response to the DA cell depopulation is widely debated. The following paragraph addresses this problem by analyzing the results of experiments based on progressive or very partial

**4. Evolving DA release with DA cell degeneration** 

result of the electrical hyperactivity of the surviving nigral DA cells.

**preparations** 

Wullner *et al.*, 1994).

lesions of the nigrostriatal DA pathway in the rat.

physiological conditions (Leviel et al., 1989). As mentioned above, components of this model may be attributed to different DA neurons. In the model presented in Fig. 8, a central pool of cytosolic DA constitutes the central point of the functional organization. Three input and four output routes are then described. The major way of supplying this central pool is via DA synthesis. A secondary supply mechanism could involve the intraterminal egress of DA from the vesicular storage pool. The third input comprises DA uptake transport.

The first and major output of cytoplasmic DA involves the formation of DOPAC by the MAO enzyme. The second use of cytoplasmic DA involves terminal overflow through DA-RT. A small proportion of the synthesized DA is vesicularized in a neosynthesized pool constituting the preferentially releasable compartment of the amine, and is released by exocytosis. Some of this vesicularized amine supplies a storage compartment (old stored DA).

This model takes into account the different mechanisms of DA release and DA metabolism that can be independently regulated. For example, reduced exocytotic release (by the blockade of calcium channels) can be accompanied by increased DA-RT, leading to an increase in extracellular amine. Thus even if the depolarization of DA axons is clearly responsible for exocytotic DA release, the preterminal influences could lead to secondary release that is differentially regulated, producing the paradoxical observations that are often reported (Leviel et al., 1989).

Fig. 8. The four-compartments model of intraterminal DA metabolism including dual release

physiological conditions (Leviel et al., 1989). As mentioned above, components of this model may be attributed to different DA neurons. In the model presented in Fig. 8, a central pool of cytosolic DA constitutes the central point of the functional organization. Three input and four output routes are then described. The major way of supplying this central pool is via DA synthesis. A secondary supply mechanism could involve the intraterminal egress of DA

The first and major output of cytoplasmic DA involves the formation of DOPAC by the MAO enzyme. The second use of cytoplasmic DA involves terminal overflow through DA-RT. A small proportion of the synthesized DA is vesicularized in a neosynthesized pool constituting the preferentially releasable compartment of the amine, and is released by exocytosis. Some of

This model takes into account the different mechanisms of DA release and DA metabolism that can be independently regulated. For example, reduced exocytotic release (by the blockade of calcium channels) can be accompanied by increased DA-RT, leading to an increase in extracellular amine. Thus even if the depolarization of DA axons is clearly responsible for exocytotic DA release, the preterminal influences could lead to secondary release that is differentially regulated, producing the paradoxical observations that are often

*Glutamate*

*Prolactine Estradiol*

*Dépol, K+*

*GABA*

*GAP Ca++*

*Nomifensine*


*Amphétamine*

*GBR GLU NMDA*

**Axial Release:Phasic**

**DA ExtraCell.** 

*Ca++*

**Lateral Release:Tonic**

from the vesicular storage pool. The third input comprises DA uptake transport.

this vesicularized amine supplies a storage compartment (old stored DA).

**Exocytose**

Ca

Cyto Sq.

**Reverse Transport DA-RT**

Fig. 8. The four-compartments model of intraterminal DA metabolism including dual

Ca

**Uptake**

reported (Leviel et al., 1989).

**Tyrosine**

Ca

**DOPA**

**TH**

release

**DBH**

**DA**

Ca

**DA**

Free

Ca

**Terminal Cytoplasm**

**DA**

**DOPAC**

**MAO**

**DA**
