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

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

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

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; Wullner *et al.*, 1994).

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 lesions of the nigrostriatal DA pathway in the rat.

Normal and Physio-Pathological Striatal Dopamine Homeostasis 255

slower, reaching 45.5 and 52.7% (median and lateral STR respectively) of the controls after the 8th injection. The tissue DOPAC/DA ratio (0.494 in the control rats) increased significantly only after the 6th injection, reaching a value of 1.4 in the median part of the STR after the 8th injection (Fig. 9). The tissue content of TH (Fig. 9) decreased very slowly and the differences compared with control rats (8.940.60 UTH in the median STR; 9.240.81 UTH in the lateral STR) were only significant after the 8th injection (63 and 84.4% of controls in the median and lateral STR respectively). These alterations were first detected in the medial part of the STR (in the more lesioned part) and 2 weeks later, more laterally. Thus an immediate alteration of tissue DOPAC and DA was observed, followed by a delayed

Binding of [3H]GBR12935 was measured in synaptosomes prepared from rats that had been injected for 8 weeks. The *B*max in the sham-operated group was 3.82 pmole/mg prot. In the lesioned group, *B*max fell to 2.31 pmole/mg prot (60.4%) but returned to 3.42 pmole/mg prot in lesioned animals treated daily with MK801 (89.5% of sham-operated group). The ability of synaptosomes to load [3H]DA was not greatly affected in lesioned animals. The slopes of the [3H]DA accumulation curves were: 0.101 pmole/mg prot/min for the sham-operated animals, 0.089 pmole/mg prot/min for the lesioned group (88.6% of the sham-operated group, n.s.) and 0.098 pmole/mg prot/min for the lesioned animals treated with MK801 (97.1% of the sham-operated group, n.s.) (Fig. 10). However, the ratio between [3H]DA accumulation and [3H]GBR12935 binding (in pmol/pmol of [3H]GBR12935/min, data not shown) was 0.026 (100%) for the sham-operated animals, 0.038 for the lesioned group (146.5%; P<0.05) and 0.028 for the lesioned animals treated with MK801 (108.5% of the

increase in the DOPAC/DA ratio and a late reduction in the amount of TH.


Fig. 10. Lineweaver-Burk plot of [3H]DA capture in striatal synaptosomes


**1/[3HDA] (nM-1)**

**Controls**

**MK801 Treated**

**Lesioned**

Vm Km (Pm/mg/min) (µM)

0.78

0.27

013

2.967

0.382

0.824

sham-operated group, n.s.).

**1/v** 

**(pmole/mgprot/min)**


1/Vm

#### **4.2 Experimental observations**

To address this issue, partial and progressive destruction of the dopaminergic terminals of the STR was achieved and various biochemical parameters were monitored. The injections were not directly located in the striatal tissue but in the lateral ventricle. Recurrent 6-OHDA injections were administered week after week, locally in the rat STR. In these lesioned rats, metabolic alterations of DA were followed along with the kinetic parameters of the uptake process in synaptosomal preparations. These parameters (apparent Km and Vmax) are correlated with the number of uptake sites determined in binding experiments with a ligand of the DAT carrier protein, GBR12935. The possible role of GLU neurotransmission in these alterations was also investigated.

Neither behavioral disturbance nor loss of weight was observed during treatment. Following successive injections, however, progressive biochemical alterations developed in both the median and lateral part of the STR, ipsilaterally to the injected ventricle.

Control values for tissue DA were 54.99.3 ng/mg prot and 45.76.9 ng/mg prot for the median and lateral STR respectively. For tissue DOPAC, the control values were 27.193.6 ng/mg prot and 26.44.11 ng/mg prot in the median and lateral STR respectively. The first changes observed were in tissue DA, which began to decrease after the first two injections, reaching only 23.3 and 29.6% (median and lateral STR respectively) of the values of the sham-injected animals after the 8th injection (Fig. 9). The decrease in tissue DOPAC was

#### **Medial Striatum**

Fig. 9. Tissue DA, DOPAC and TH (left). Mean TH activity indexed by DOPAC/DA ratio (Right).

To address this issue, partial and progressive destruction of the dopaminergic terminals of the STR was achieved and various biochemical parameters were monitored. The injections were not directly located in the striatal tissue but in the lateral ventricle. Recurrent 6-OHDA injections were administered week after week, locally in the rat STR. In these lesioned rats, metabolic alterations of DA were followed along with the kinetic parameters of the uptake process in synaptosomal preparations. These parameters (apparent Km and Vmax) are correlated with the number of uptake sites determined in binding experiments with a ligand of the DAT carrier protein, GBR12935. The possible role of GLU neurotransmission in these

Neither behavioral disturbance nor loss of weight was observed during treatment. Following successive injections, however, progressive biochemical alterations developed in

Control values for tissue DA were 54.99.3 ng/mg prot and 45.76.9 ng/mg prot for the median and lateral STR respectively. For tissue DOPAC, the control values were 27.193.6 ng/mg prot and 26.44.11 ng/mg prot in the median and lateral STR respectively. The first changes observed were in tissue DA, which began to decrease after the first two injections, reaching only 23.3 and 29.6% (median and lateral STR respectively) of the values of the sham-injected animals after the 8th injection (Fig. 9). The decrease in tissue DOPAC was

> 0 0,5 1 1,5 2

sham median lateral

**DOPAC/DA ratio**

Fig. 9. Tissue DA, DOPAC and TH (left). Mean TH activity indexed by DOPAC/DA ratio

**B**

2 inj 4 inj 6 inj 8 inj

**Sham Median Lateral**

both the median and lateral part of the STR, ipsilaterally to the injected ventricle.

**A**

**4.2 Experimental observations** 

alterations was also investigated.

**Medial Striatum**

**DA DOPAC TH**

**Lateral Striatum**

**percent of controls**

**percent of controls**

(Right).

2 inj 4 inj 6 inj 8 inj

2 inj 4 inj 6 inj 8 inj

slower, reaching 45.5 and 52.7% (median and lateral STR respectively) of the controls after the 8th injection. The tissue DOPAC/DA ratio (0.494 in the control rats) increased significantly only after the 6th injection, reaching a value of 1.4 in the median part of the STR after the 8th injection (Fig. 9). The tissue content of TH (Fig. 9) decreased very slowly and the differences compared with control rats (8.940.60 UTH in the median STR; 9.240.81 UTH in the lateral STR) were only significant after the 8th injection (63 and 84.4% of controls in the median and lateral STR respectively). These alterations were first detected in the medial part of the STR (in the more lesioned part) and 2 weeks later, more laterally. Thus an immediate alteration of tissue DOPAC and DA was observed, followed by a delayed increase in the DOPAC/DA ratio and a late reduction in the amount of TH.

Binding of [3H]GBR12935 was measured in synaptosomes prepared from rats that had been injected for 8 weeks. The *B*max in the sham-operated group was 3.82 pmole/mg prot. In the lesioned group, *B*max fell to 2.31 pmole/mg prot (60.4%) but returned to 3.42 pmole/mg prot in lesioned animals treated daily with MK801 (89.5% of sham-operated group). The ability of synaptosomes to load [3H]DA was not greatly affected in lesioned animals. The slopes of the [3H]DA accumulation curves were: 0.101 pmole/mg prot/min for the sham-operated animals, 0.089 pmole/mg prot/min for the lesioned group (88.6% of the sham-operated group, n.s.) and 0.098 pmole/mg prot/min for the lesioned animals treated with MK801 (97.1% of the sham-operated group, n.s.) (Fig. 10). However, the ratio between [3H]DA accumulation and [3H]GBR12935 binding (in pmol/pmol of [3H]GBR12935/min, data not shown) was 0.026 (100%) for the sham-operated animals, 0.038 for the lesioned group (146.5%; P<0.05) and 0.028 for the lesioned animals treated with MK801 (108.5% of the sham-operated group, n.s.).

Fig. 10. Lineweaver-Burk plot of [3H]DA capture in striatal synaptosomes

Normal and Physio-Pathological Striatal Dopamine Homeostasis 257

is an index of transport capacity, was only 12% of the value in control rats. This observation shows that a large number of sites, still bound to [3H]GBR12935, had become unable to carry DA. Third, it can be concluded from the low *Km* value that the affinity of DA for the carrier protein had increased. These results strongly suggest that moderate DA denervation of the STR results in a reduction in the number of functional DA transporters able to carry DA, but an increase in their affinity, which is responsible in turn for an increased rate of transport. We will see below that a new way to see the structure of the DAT protein could underline this paradoxical data. Indeed DAT is no longer considered as only an uptake processor but

**5. Presynaptic GLU – Induced activation of DA release in the STR after partial** 

The behavioral and biochemical recovery, after partial unilateral lesion, of the dopaminergic nigrostriatal path has been reported in various species from rodents to primates (Hefti et al., 1985; McCallum et al., 2006; Boulet et al., 2008; Perez et al., 2008). This recovery is thought to result from normalization of the extracellular dopamine (DA*ext*) in the STR that was initially reduced by the lesion (Robinson and Whishaw, 1988; Castaneda et al., 1990; Zigmond et al., 1990; Emmi et al., 1996). However, it remains unclear whether this compensation results from overactive nigral neurons, as is proposed to occur after LevoDOPA treatment (Grace, 2008), from direct preterminal influences in the STR (Dentresangle et al., 2001), or from both. A role for GLU in this phenomenon has been proposed given that the GLU tone increases in various regions of the basal ganglia following SNc lesions, including in the STR (Calabresi et al., 1993; Cepeda et al., 2001; Tang et al., 2001), the SNc (Turski et al., 1991; Bezard et al., 1997) and the subthalamic nucleus (Benazzouz et al., 1993; Amalric et al., 1995; Phillips et al., 2006). This was confirmed by the fact that behavioral and biochemical recovery were inhibited by chronic treatment with GLU receptor (GLU*R*) antagonists including MK801 (see the preceding paragraph) and 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid (CPP) (Emmi et al., 1996). The nature of the mechanisms evoked by GLU remains unclear however, because compensatory mechanisms occur very early or following very partial lesions, at a stage at which the spontaneous activity of midbrain DA neurons was not found to be altered (Hollerman and Grace, 1990) and when the impulse/release ratio in the terminal region appeared unchanged (Dentresangle et al., 2001). To complicate the situation, it is now well known that GLU can enhance DA release independently from the spontaneous electrical activity of the midbrain DA neurons (Leviel et al., 1990; Keefe et al.,

To understand the functional link between DA and GLU during the compensatory process a very partial and laterally located lesion of the SN was produced in rats using 6-OHDA. Three weeks after this lesion, it was possible to compare the metabolic consequences of a large denervation in the lateral part of the STR with those in a medial region spared by the denervation. Cartography of the tonic extracellular DA and DOPAC concentration was performed using *in vivo* voltammetry. In rats with similar lesions medial and lateral microdialysis approaches allowed us to measure the extracellular GLU concentration. The effect of GLU antagonists (amantadine, memantine and riluzol) on neurotransmission and

their neuroprotective action on tonic DA enhancement were also tested.

rather as a dual actor of the DA transport regulating extracellular DA homeostasis.

**nigral lesion** 

1992; Olivier et al., 1995, 1999).

**5.1 Experimental observations** 

The apparent *K*m and *V*max of the [3H]DA transport reaction were calculated by linear regression (Fig. 10). The slopes of the curves obtained for lesioned-only animals and lesioned plus MK801-treated animals were both significantly different from that of shamoperated animals (P<0.05). The values of *V*max were 2.967, 0.382 and 0.824 pmole/mg prot/min respectively for sham-operated, lesioned and lesioned + MK801-injected rats. The *K*m values were 0.78, 0.13 and 0.27 µM respectively.
