**2. The dual DA release process**

The extracellular DA concentration in the caudate nucleus could be relevant to something other than the electrical activity of dopaminergic cells. Increased DA release was even observed in the STR under experimental conditions that decreased cell firing in the substantia nigra (SNc, Romo et al., 1986). Since the first proposal by Raiteri et al. (1977) that DA could be released by a calcium-independent and carrier-mediated process, numerous authors have claimed that the firing-dependent release of DA does not explain all the reported alterations in the extracellular DA concentration and that an additional release mechanism must exist (Liang and Rutledge, 1982; Parker and Cubeddu, 1986; Vizi and Labos, 1991). In 1991, A. Grace gathered relevant data and proposed the presence of two modes of release of the amine in the STR (Fig 1), one 'phasic', taking place within the millisecond range, and controlled by The firing running on DA axons, and a 'tonic' mode, taking place over minutes or hours and mainly dependent on various direct influences on the DA terminals including that of Glutamate (GLU). Using differential pulse amperometry (DPA) simultaneously associated with local superfusion and HPLC detection we observed this dual release of DA in response to axonal stimulation and the application of GLU agonists (Fig. 2; Olivier et al., 1995).

Fig. 1. The dual release of DA through vesicles exocytose or DA transport (TrP).

#### **2.1 Spontaneous and evoked DA release**

242 Neuroscience – Dealing with Frontiers

The two first paragraphs will be devoted to the duality of normal DA neurotransmission. It is however crucial to understand how the two different mechanisms of release (tonic and phasic) could maintain their equilibrium in pathological situations. Numerous animal models of neurodegeneration have been created. To date, only situations mimicking the early phases of parkinsonian neurodegeneration have been used to explore this equilibrium. Original results will also be presented here. These will provide new information on the regulatory modulation of the tonic and phasic release occurring during the initial pre-

The extracellular DA concentration in the caudate nucleus could be relevant to something other than the electrical activity of dopaminergic cells. Increased DA release was even observed in the STR under experimental conditions that decreased cell firing in the substantia nigra (SNc, Romo et al., 1986). Since the first proposal by Raiteri et al. (1977) that DA could be released by a calcium-independent and carrier-mediated process, numerous authors have claimed that the firing-dependent release of DA does not explain all the reported alterations in the extracellular DA concentration and that an additional release mechanism must exist (Liang and Rutledge, 1982; Parker and Cubeddu, 1986; Vizi and Labos, 1991). In 1991, A. Grace gathered relevant data and proposed the presence of two modes of release of the amine in the STR (Fig 1), one 'phasic', taking place within the millisecond range, and controlled by The firing running on DA axons, and a 'tonic' mode, taking place over minutes or hours and mainly dependent on various direct influences on the DA terminals including that of Glutamate (GLU). Using differential pulse amperometry (DPA) simultaneously associated with local superfusion and HPLC detection we observed this dual release of DA in response to

axonal stimulation and the application of GLU agonists (Fig. 2; Olivier et al., 1995).

Amphetamine

Fig. 1. The dual release of DA through vesicles exocytose or DA transport (TrP).

**TrP**

**TYR**

**DA**

**Vesicles**

**Phasic DA release**

**Firing dependent Calcium dependent**

**Exocytose**

**Quantic**

symptomatic compensatory phases of Parkinson's disease.

**2. The dual DA release process** 

**Tonic DA Release**

**Firing independent**

**GLU dependent**

**Reverse transport (DA-RT)**

**Ca++ Channel independent Na+ gradient dependent**

Dual release can be demonstrated by the simultaneous use of two probes. DPA uses a carbon fiber electrode to detect the DA released following stimulation of the DA axons in the Medial Forebrain Bundle (MFB) in the lateral hypothalamus. Every 4 minutes, a 20 second stimulation-train pulse produces a large increase in the amperometric signal in the STR that is strictly restricted to the stimulation period (Fig. 2, Olivier et al., 1995). The amplitude of the peaks that occur is roughly constant over time. As discussed elsewhere by Gonon (1988) and Suaud-Chagny et al. (1992), the increase in the oxidation current under these conditions is likely to be mainly due to the DA released, despite the fact that some of this increase could be due to dihydroxyphenyl acetic acid (DOPAC), the first DA metabolite. Simulation of the MFB alters the differential oxidation current but is ineffective in terms of the amount of regional extracellular basal DA measured through a cannula close to the probe used for DPA. The DA collected through the superfusion system originates from a region considerably larger than the carbon fiber. The carbon fiber is only sensitive to events occurring in the range of µm from its suface whereas the superfusing probe collects diffusing molecules from within several hundreds of microns. Differences also arise from the duration of the measurements. Stimulation of the MFB only lasts for a short period of the collection time (here 0.08%). Given this short period of time, any increase in the release of the amine remains undetectable in the superfusing fluid. The superfusing methods are particularly useful for detecting 'regional' fluctuations whereas voltammetry reports local events.

Fig. 2. The effect of MFB stimulation on tonic (up) and phasic down DA release Superfusion in the presence of cadmium ions:

Normal and Physio-Pathological Striatal Dopamine Homeostasis 245

The first evidence that regional extracellular DA and synaptic DA are not governed by the same mechanism is derived from the fact that although they should vary in parallel, that is clearly not the case. Many observations based on voltammetry have confirmed the ability of DA axons transiting through the MFB to carry an electrical potential, since an increase in extracellular DA can occur in response to electrical stimulations of the MFB (see above, Ewing and Wightman, 1984; Ewing et al., 1983; Gonon and Buda, 1985; Kuhr et al*.,* 1986; Stamford et al., 1986). The voltammetric approach can only be used in a very restricted region of the space due to the size of the probes used. When using methods suitable for larger regions (microdialysis, push-pull cannula systems), it became clear that to alter DA levels in the STR, stimulation of the DA afferents required axonal recruitment and stimulation frequencies outside the physiological range (Grace, 1991; Olivier et al., 1995). Indeed, more than 60–70% of DA varicosities were found to be asynaptic, and DA synapses represent only about 1.8–7% of all striatal synapses (Decarries and Mechawar, 2000). Even if DA cell firing can elicit DA release in the STR for a brief period of time, the synaptic DA released may not be the only contributor to extracellular DA levels and would not be expected to be responsible for homeostatic DA control in the STR (Grace, 1991; Levi and Raiteri, 1993; Olivier, 1995). DA concentration is probably only partially monitored by nigral DA firing cells and DA homeostasis is unlikely to be controlled by spill-over of the amine from synaptic events.

It has been known for many years that exocytosis is not the only outward process of the neurotransmitter exchange between neurons and the extracellular space (Stein, 1967). Regarding monoamines, several models in the peripheral nervous system have been proposed that are generally based on exchange diffusion (Bogdanski and Brodie, 1969; Paton 1973a, 1973b). Concerning the release of DA in the central nervous system, DAT was thought to be partly involved in DA release by some authors (Marchi et al., 1985; Olivier et al., 1995; Raiteri et al., 1979) and the mechanism of this carrier-mediated release came from

Fig. 4. The dual effect of GLU receptor blockade

Using DAT to re-visit DA release: DA-RT

**Min. Min.**

To block the voltage-dependent calcium channel, 100 µM cadmium was added to the artificial LCR supplying the cannula. This treatment blocks the stimulation-evoked release of DA detected using DPA (Fig. 3). The same effect can be obtained using 1 mM cobalt (not shown) in place of cadmium and remains unchanged in the presence of 0.5 µM GBR12909, a DAT inhibitor. Surprisingly, in the presence of cadmium or cobalt the amount of DA detected in the superfusing fluid greatly increased during the first 40 minutes of application (Fig. 3). Over this period of time, the DA collected fell below control values. When 0.5 µM GBR12909 was simultaneously added with cadmium, no further increase in the amount of DA was detected in the superfusing fluid.

Fig. 3. Blockade of calcium channels via local application of cadmium

Local superfusion with GLU agonists:

Addition of the GLU agonists NMDA (1 mM) and kainate (0.1 mM) to the superfusing fluid increased the amount of DA collected through the push-pull cannula and reduced the amperometric signal evoked by MFB stimulations (Fig. 4). The reduction in the amperometric signal was not counteracted by the presence of 0.5 µM GBR12909. In contrast the increase in the spontaneous release measured through the cannula in response to NMDA or Kainate application completely disappeared.

#### **2.2 From quantal release to reverse transport, the duality of DA release**

These experimental observations constitute the most simple and direct evidence of the presence of a double mechanism of release of DA from the DA terminals in the STR. A large set of complementary data will now be briefly reviewed.

Can DA homeostasis be regulated by the firing-evoked synaptic release of DA?

To block the voltage-dependent calcium channel, 100 µM cadmium was added to the artificial LCR supplying the cannula. This treatment blocks the stimulation-evoked release of DA detected using DPA (Fig. 3). The same effect can be obtained using 1 mM cobalt (not shown) in place of cadmium and remains unchanged in the presence of 0.5 µM GBR12909, a DAT inhibitor. Surprisingly, in the presence of cadmium or cobalt the amount of DA detected in the superfusing fluid greatly increased during the first 40 minutes of application (Fig. 3). Over this period of time, the DA collected fell below control values. When 0.5 µM GBR12909 was simultaneously added with cadmium, no further increase in the amount of

**Min**. **Min.**

DA was detected in the superfusing fluid.

Local superfusion with GLU agonists:

Fig. 3. Blockade of calcium channels via local application of cadmium

**2.2 From quantal release to reverse transport, the duality of DA release** 

Can DA homeostasis be regulated by the firing-evoked synaptic release of DA?

NMDA or Kainate application completely disappeared.

set of complementary data will now be briefly reviewed.

Addition of the GLU agonists NMDA (1 mM) and kainate (0.1 mM) to the superfusing fluid increased the amount of DA collected through the push-pull cannula and reduced the amperometric signal evoked by MFB stimulations (Fig. 4). The reduction in the amperometric signal was not counteracted by the presence of 0.5 µM GBR12909. In contrast the increase in the spontaneous release measured through the cannula in response to

These experimental observations constitute the most simple and direct evidence of the presence of a double mechanism of release of DA from the DA terminals in the STR. A large

Fig. 4. The dual effect of GLU receptor blockade

The first evidence that regional extracellular DA and synaptic DA are not governed by the same mechanism is derived from the fact that although they should vary in parallel, that is clearly not the case. Many observations based on voltammetry have confirmed the ability of DA axons transiting through the MFB to carry an electrical potential, since an increase in extracellular DA can occur in response to electrical stimulations of the MFB (see above, Ewing and Wightman, 1984; Ewing et al., 1983; Gonon and Buda, 1985; Kuhr et al*.,* 1986; Stamford et al., 1986). The voltammetric approach can only be used in a very restricted region of the space due to the size of the probes used. When using methods suitable for larger regions (microdialysis, push-pull cannula systems), it became clear that to alter DA levels in the STR, stimulation of the DA afferents required axonal recruitment and stimulation frequencies outside the physiological range (Grace, 1991; Olivier et al., 1995). Indeed, more than 60–70% of DA varicosities were found to be asynaptic, and DA synapses represent only about 1.8–7% of all striatal synapses (Decarries and Mechawar, 2000). Even if DA cell firing can elicit DA release in the STR for a brief period of time, the synaptic DA released may not be the only contributor to extracellular DA levels and would not be expected to be responsible for homeostatic DA control in the STR (Grace, 1991; Levi and Raiteri, 1993; Olivier, 1995). DA concentration is probably only partially monitored by nigral DA firing cells and DA homeostasis is unlikely to be controlled by spill-over of the amine from synaptic events.

#### Using DAT to re-visit DA release: DA-RT

It has been known for many years that exocytosis is not the only outward process of the neurotransmitter exchange between neurons and the extracellular space (Stein, 1967). Regarding monoamines, several models in the peripheral nervous system have been proposed that are generally based on exchange diffusion (Bogdanski and Brodie, 1969; Paton 1973a, 1973b). Concerning the release of DA in the central nervous system, DAT was thought to be partly involved in DA release by some authors (Marchi et al., 1985; Olivier et al., 1995; Raiteri et al., 1979) and the mechanism of this carrier-mediated release came from

Normal and Physio-Pathological Striatal Dopamine Homeostasis 247

'volume transmission', developed in the 1980s (Agnati et al., 1986) and recently used to characterize DA neurotransmission, does not refer to a mode of release but rather to a mode of extracellular transmission. The terms 'phasic release' and 'tonic release' most frequently refer to the pattern of discharge of the DA axons (regular or bursting). The most appropriate terms that have emerged to date to qualify the present types of neurotransmitter release are 'quantal' for the firing-dependent process and 'reverse transport' (DA-RT) or 'lateral release' for the carrier-mediated mechanism. We also use the term 'lateral release' to refer to the non-synaptic localization of this mechanism, due to the extra-synaptic position of the DA

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.,

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.

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

transporter. In this case, 'axial release' could refer to synaptic transmission.

1983; Albert et al., 1984; Zigmond et al., 1989; Haycock, 1993).

should elevate DA*sa* and the DOPAC efflux.

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

**3. Regulatory mechanisms** 

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 Rutledge, 1972).

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, simultaneously, NMDA-mediated release.

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

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 'volume transmission', developed in the 1980s (Agnati et al., 1986) and recently used to characterize DA neurotransmission, does not refer to a mode of release but rather to a mode of extracellular transmission. The terms 'phasic release' and 'tonic release' most frequently refer to the pattern of discharge of the DA axons (regular or bursting). The most appropriate terms that have emerged to date to qualify the present types of neurotransmitter release are 'quantal' for the firing-dependent process and 'reverse transport' (DA-RT) or 'lateral release' for the carrier-mediated mechanism. We also use the term 'lateral release' to refer to the non-synaptic localization of this mechanism, due to the extra-synaptic position of the DA transporter. In this case, 'axial release' could refer to synaptic transmission.
