**4.3 Nigrostriatal lesion reduces DA uptake but increases DA transport**

The reduction in the levels of tissue DA and DOPAC appeared soon after the first injection. The DOPAC/DA ratio, reflecting TH protein activity (Lavielle et al., 1978; Bannon & Roth, 1983; Zetterström et al., 1988; Nissbrandt et al., 1989; Soares da Silva & Garret, 1990), and the amount of TH protein in the tissue remained unchanged during the first 6 weeks. This reduction in intraterminal DA stores that is not associated with changes in DA synthesis is consistent with the observations that we report below of increased basal DA release during the very early phases of denervation (Dentresangle et al., 2001; Dzahini et al., 2010).

After six and eight injections, both the tissue DA and DOPAC continued to decrease and tissue levels of TH protein began to decline. In contrast, the DOPAC/DA ratio increased, confirming a delay in increased DA synthesis. This could be the result of decreased intraterminal DA. TH activity is under the control of cytoplasmic amine levels through an end-product regulatory process (Ames et al., 1978; Mann and Gordon, 1979; Zigmond et al., 1989; Fillenz, 1993). Therefore increased TH activity, which is often reported after partial lesion of the DA pathway (Zigmond et al., 1990) could be, at least in part, a consequence of reduced intraterminal DA pools. The simultaneous reduction in the amount of tissue TH protein may occur in one of two ways: (1) a reduction in the number of DA terminals, or (2) increased protein turnover associated with the increased catalytic activity (Vrana and Roskoski, 1983; Lavergne et al., 1994).

This set of results is consistent with the hypothesis that the preservation of extracellular DA after partial destruction of the nigro-striatal DA pathway is due to activation of basal DA release. However, as already mentioned, this is unlikely to be the result of electrical activation of DA neurons in the SNc. Thus, alterations in DA transport at the level of the DA terminals in the STR are more likely.

The amplitude of the denervation produced by eight injections of the toxin was evaluated initially. The amount of TH protein and the binding of transport sites are considered to be valid indices of DA pathway denervation (van Horne et al., 1992; Maloteau, 1998). In the present experiment, tissue TH protein levels were reduced to 63% of the levels found in controls, and the *B*max of [3H]GBR12935 was reduced to 60.4% of the value found in shamoperated animals. Thus, about 40% of DA terminals may have degenerated after eight injections of the toxin.

Given this 40% reduction in the number of binding sites in the synaptosomal preparation of lesioned rats, the kinetic parameters of the DA uptake process are apparently paradoxical. First, the global DA uptake was only reduced by 11% (insignificant decrease), and when adjusted according to the number of sites (reduced by 40%), it was actually increased (146% of controls). This observation suggests an increase in the efficiency of the transport process, compensating for the reduction in the population of DA terminals. Second, the *Vmax*, which

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

The reduction in the levels of tissue DA and DOPAC appeared soon after the first injection. The DOPAC/DA ratio, reflecting TH protein activity (Lavielle et al., 1978; Bannon & Roth, 1983; Zetterström et al., 1988; Nissbrandt et al., 1989; Soares da Silva & Garret, 1990), and the amount of TH protein in the tissue remained unchanged during the first 6 weeks. This reduction in intraterminal DA stores that is not associated with changes in DA synthesis is consistent with the observations that we report below of increased basal DA release during

After six and eight injections, both the tissue DA and DOPAC continued to decrease and tissue levels of TH protein began to decline. In contrast, the DOPAC/DA ratio increased, confirming a delay in increased DA synthesis. This could be the result of decreased intraterminal DA. TH activity is under the control of cytoplasmic amine levels through an end-product regulatory process (Ames et al., 1978; Mann and Gordon, 1979; Zigmond et al., 1989; Fillenz, 1993). Therefore increased TH activity, which is often reported after partial lesion of the DA pathway (Zigmond et al., 1990) could be, at least in part, a consequence of reduced intraterminal DA pools. The simultaneous reduction in the amount of tissue TH protein may occur in one of two ways: (1) a reduction in the number of DA terminals, or (2) increased protein turnover associated with the increased catalytic activity (Vrana and

This set of results is consistent with the hypothesis that the preservation of extracellular DA after partial destruction of the nigro-striatal DA pathway is due to activation of basal DA release. However, as already mentioned, this is unlikely to be the result of electrical activation of DA neurons in the SNc. Thus, alterations in DA transport at the level of the DA

The amplitude of the denervation produced by eight injections of the toxin was evaluated initially. The amount of TH protein and the binding of transport sites are considered to be valid indices of DA pathway denervation (van Horne et al., 1992; Maloteau, 1998). In the present experiment, tissue TH protein levels were reduced to 63% of the levels found in controls, and the *B*max of [3H]GBR12935 was reduced to 60.4% of the value found in shamoperated animals. Thus, about 40% of DA terminals may have degenerated after eight

Given this 40% reduction in the number of binding sites in the synaptosomal preparation of lesioned rats, the kinetic parameters of the DA uptake process are apparently paradoxical. First, the global DA uptake was only reduced by 11% (insignificant decrease), and when adjusted according to the number of sites (reduced by 40%), it was actually increased (146% of controls). This observation suggests an increase in the efficiency of the transport process, compensating for the reduction in the population of DA terminals. Second, the *Vmax*, which

the very early phases of denervation (Dentresangle et al., 2001; Dzahini et al., 2010).

**4.3 Nigrostriatal lesion reduces DA uptake but increases DA transport** 

*K*m values were 0.78, 0.13 and 0.27 µM respectively.

Roskoski, 1983; Lavergne et al., 1994).

terminals in the STR are more likely.

injections of the toxin.

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

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

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

#### **5.1 Experimental observations**

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.

Normal and Physio-Pathological Striatal Dopamine Homeostasis 259

(0.42±0.06 µM) and ASP (0.47±0.05 µM) were in the range of previously reported values. Importantly, DA was in the nano molar range in the extracellular space whereas metabolites and amino acids were in the micro molar range. Three weeks following the lesion in the lateral part of the SN, an increase in DA was detected in the medial STR (29.2±0.28 nM). Simultaneously an increase in GLU was observed (24.4 ± 0.75 µM). In contrast, the DA metabolites, DOPAC (10.8±0.10 µM) and HVA*ext* (8.86±0.90 µM), remained unchanged. Likewise, GABA (0.63±0.17 µM) and ASP (0.47±0.05 µM) were not significantly altered.

Fig. 12. Tissue DA and amino acid in the striatum 3 weeks following lesioning of the SN

Treatment with chronic memantine, amantadine or riluzol seemed to counteract the tonic increase in DA induced following the lesion that was observed via voltammetry and confirmed using microdialysis (Fig. 12). Only a moderate increase in DA persisted following chronic amantadine administration (12.4 ± 1.26 nM versus 7.28 ± 0.36 nM in controls). The DA concentration remained at basal levels following treatment with memantine and riluzol (Fig. 12). Acute treatments with the same substances were less efficient at counteracting the increase in DA but the same trends were apparent (15.1 nM, 24.1 nM and 24.4 nM respectively for memantine, amantadine and riluzol versus 29.2 nM in the controls, Dzahini et al., 2010). The GLU antagonists did not interact with the DA metabolites DOPAC and HVA. The results are

presented in Fig. 12 as percentages of the values obtained in lesioned rats.

using 6-OHDA

Treatment of lesioned rats with GLUantagonists

Fig. 11. Lateral lesion of the SN and its consequences for striatal DA denervation and release

Cartography of the STR using voltammetry

The STR was stereotaxically explored in one coronal plane (8.2 mm anterior to the ear axe) to localize biochemical changes *in vivo*. Figure 11 shows the extracellular concentrations of DA expressed in nM and as the ratio (in %) between values obtained in lesioned and control animals in each homologous striatal area for each subregion explored. In the lesioned animals, DA measured in the intact median region (lat. 2) showed a notable increase (352%, p< 0.001) in comparison with control values. This effect was less pronounced in the intermediate and lateral striatal areas (lat. 3–4) but was nevertheless present. In the most completely DA-deafferented striatal regions, DA levels did not differ significantly from control values, being only slightly higher. In contrast, the extracellular level of DOPAC decreased enormously (26% of control values; p< 0.001) in the lateral part of the STR in comparison with values detected in the homologous striatal region of the control group. In the medial region DOPAC concentrations remained poorly affected (Dzahini et al., 2010).

Effects of partial lesions of the SN and microdialysis results

Voltammetry furnishs excellent stereotaxic localization of biochemical alterations but the use of pargyline to detect DA could produce a bias in the measurements. To better quantify the changes observed with voltammetry, and to extend the measurements to amino acids, the medial spared region was implanted with a microdialysis probe, and superfusates were subjected to HPLC and two analyses, one for catecholamines and the other for amino acids. In sham-operated and untreated rats, the spontaneous extracellular concentrations of DA (7.28±0.36 nM), DOPAC (9.19±1.1 µM), HVA (6.54±0.59 µM), GLU (10.0±0.27 µM), GABA

**4 3 2** mm

Fig. 11. Lateral lesion of the SN and its consequences for striatal DA denervation and release

The STR was stereotaxically explored in one coronal plane (8.2 mm anterior to the ear axe) to localize biochemical changes *in vivo*. Figure 11 shows the extracellular concentrations of DA expressed in nM and as the ratio (in %) between values obtained in lesioned and control animals in each homologous striatal area for each subregion explored. In the lesioned animals, DA measured in the intact median region (lat. 2) showed a notable increase (352%, p< 0.001) in comparison with control values. This effect was less pronounced in the intermediate and lateral striatal areas (lat. 3–4) but was nevertheless present. In the most completely DA-deafferented striatal regions, DA levels did not differ significantly from control values, being only slightly higher. In contrast, the extracellular level of DOPAC decreased enormously (26% of control values; p< 0.001) in the lateral part of the STR in comparison with values detected in the homologous striatal region of the control group. In the medial region DOPAC concentrations remained poorly affected (Dzahini et al., 2010).

Voltammetry furnishs excellent stereotaxic localization of biochemical alterations but the use of pargyline to detect DA could produce a bias in the measurements. To better quantify the changes observed with voltammetry, and to extend the measurements to amino acids, the medial spared region was implanted with a microdialysis probe, and superfusates were subjected to HPLC and two analyses, one for catecholamines and the other for amino acids. In sham-operated and untreated rats, the spontaneous extracellular concentrations of DA (7.28±0.36 nM), DOPAC (9.19±1.1 µM), HVA (6.54±0.59 µM), GLU (10.0±0.27 µM), GABA

**Striatum**

Lesions

**Subs. Nigra**

Lesions

144 134.8 134.4 127.5 133.2

137.6 140.4 138.5 106.5 159.2 151.3 257.6 352.5 251.9 162.3

Cartography of the STR using voltammetry

**-6**

**-7**

mm

Effects of partial lesions of the SN and microdialysis results

**-5**

**-4**

**-3**

(0.42±0.06 µM) and ASP (0.47±0.05 µM) were in the range of previously reported values. Importantly, DA was in the nano molar range in the extracellular space whereas metabolites and amino acids were in the micro molar range. Three weeks following the lesion in the lateral part of the SN, an increase in DA was detected in the medial STR (29.2±0.28 nM). Simultaneously an increase in GLU was observed (24.4 ± 0.75 µM). In contrast, the DA metabolites, DOPAC (10.8±0.10 µM) and HVA*ext* (8.86±0.90 µM), remained unchanged. Likewise, GABA (0.63±0.17 µM) and ASP (0.47±0.05 µM) were not significantly altered.

Fig. 12. Tissue DA and amino acid in the striatum 3 weeks following lesioning of the SN using 6-OHDA

Treatment of lesioned rats with GLUantagonists

Treatment with chronic memantine, amantadine or riluzol seemed to counteract the tonic increase in DA induced following the lesion that was observed via voltammetry and confirmed using microdialysis (Fig. 12). Only a moderate increase in DA persisted following chronic amantadine administration (12.4 ± 1.26 nM versus 7.28 ± 0.36 nM in controls). The DA concentration remained at basal levels following treatment with memantine and riluzol (Fig. 12). Acute treatments with the same substances were less efficient at counteracting the increase in DA but the same trends were apparent (15.1 nM, 24.1 nM and 24.4 nM respectively for memantine, amantadine and riluzol versus 29.2 nM in the controls, Dzahini et al., 2010). The GLU antagonists did not interact with the DA metabolites DOPAC and HVA. The results are presented in Fig. 12 as percentages of the values obtained in lesioned rats.

Normal and Physio-Pathological Striatal Dopamine Homeostasis 261

with the hypothesis of a NMDA-induced mechanism causing stable dopaminergic hypertony. The effects of riluzol strengthen this hypothesis. First proposed to inhibit GLU release (Mantz, 1992), its action has also been attributed to persistent blockade of the sodium current (Del Negro, 2005). In our hands, riluzol reduced the release of GLU in sham-operated animals and

To summarize the present results and referenced data, the most original outcome is that DA release in the STR is not simply a readout of activity in DA neurons that provide a diffuse DA tone in the extracellular space. Rather, DA can be released by at least two different mechanisms with different kinetics, locations and regulatory processes. Based on the data reported here, a working hypothesis can be proposed: that a weak reduction in the DA cell population in the SNc leads to metabolic alterations in the STR that result in further death of DA terminals in this region. The development of this process could follow three major steps (Fig. 13): 1) the activation of diencephalic structures, thalamic or sub-thalamic nuclei that

**Cortex**

**[DA]**


**DA**

**SN**

Fig. 13. A working hypothesis of the mechanism involved in the presymptomatic phase of

project toward cortical regions; 2) in turn, a tonic increase in some of the corticostriatal pathways that we know to be responsible for different controls of the striatal GABA neurons

**Electrical Activity**

**Normal**

**Activated**

**Neurotransmission**

**Glutamate**

**[GLU]**

counteracted the lesion-induced GLU and DA as well (Dzahini et al., 2010).

**Dopamine and Glutamate Hypertony in Parkinson' Disease**

Sub Thalamic Nucleus **?**

PD

Thalamic Nucleus **?**

**6. Conclusions: DA/GLU, a deleterious partnership?** 

The extracellular GLU concentration increased tonically after the lesion (24.4±0.75 µM versus 10.0+0.27 µM in controls). This effect was counteracted by treatment with either chonic or acute memantine (47.1±2.3 µM and 27.1±1.53 µM respectively). Chronic riluzol also counteracted the lesion-induced increase in GLU after chronic application (21.8±0.68 µM) and acute riluzol drastically reduced GLU (0.7±0.05 µM). GLU was also reduced after both chonic and acute amantadine treatment (1.04±0.4 µM and 0.74±0.26 µM respectively). No alterations in GABA or ASP were detected in response to the treatment of lesioned rats with GLU antagonists. The results obtained after chronic treatments are presented in Fig. 12 as the percentage of values obtained in lesioned rats. The results obtained after acute treatments are presented elsewhere (Dzahini et al., 2010).

Substantially, the lesion produced a large increase in DA and GLU in the medial STR that was not accompanied by noticeable alterations in the two DA metabolites or the other two amino acids that were measured. The three treatments with GLU antagonists seemed to affect DA metabolism in the same way, counteracting the lesion-induced increase in DA. These treatments had differential effects on GLU.

Twenty-one days after the lesion, the denervated part of the STR (lateral) exhibited biochemical responses previously described after high levels of DA cell depopulation: a drastic reduction in DOPAC and an unmodified or slightly increased DA (Zigmond et al., 1984; Altar et al., 1987). In contrast, a large increase in DA (two- to three-fold) and a modest alteration in DOPAC was observed using voltammetry and confirmed using microdialysis in the spared part of the STR (medial). In this case, the levels of DA probably resulted from permanent (tonic) enhancement of the spontaneous release of DA. It has been shown that partial lesions leave the stimulation/release ratio unmodified (Dentresangle et al., 2001). A much larger lesion of the DA cell population seems to be required to alter the spontaneous pattern of discharge of nigral DA cells (Hollerman and Grace, 1990; Harden and Grace, 1995) or stimulation-induced DA release (Stachowiak et al., 1987). These observations strongly suggest that after lateral SN lesion, a global process activates tonic DA release by an indirect mechanism acting presynaptically in the STR.

It has been claimed that recovery of the DA in the STR after partial lesions of the SN could be related to alterations in GLU neurotransmission (Calabresi et al., 2000; Emmi et al., 1996; Kashani et al., 2007). However, the mechanism and the location of this alteration remain unknown. We found that 3 weeks after the lesion, GLU was clearly enhanced in the medial STR. When GLU neurotransmission was pharmacologically interrupted by specific GLU blockers (riluzol, memantine and amantadine), a reduction in extracellular GLU activation was observed, with a simultaneous reduction in the tonic DA increase induced by the nigral lesion.

Amantadine and memantine were proposed to have some neuroprotective effects after discovering the importance of NMDA receptor-mediated excitotoxicity as a factor underlying neurodegeneration in Parkinson's disease (Greenamyre and O'Brien, 1991). Indeed antagonists of NMDA receptors have been shown to inhibit neurodegeneration of the DA system induced by MPP+ and methamphetamine (Sonsalla et al., 1989; Turski et al., 1991). In our case, the effect of the two GLU antagonists was to counteract the increase in GLU and DA induced by the nigral lesion. Several studies have suggested that NMDAinduced DA release could be the main regulatory mechanism of extracellular DA (Bannon et al., 2001; Leviel, 2001; Mortensen and Amara, 2003), and our observations are consistent

The extracellular GLU concentration increased tonically after the lesion (24.4±0.75 µM versus 10.0+0.27 µM in controls). This effect was counteracted by treatment with either chonic or acute memantine (47.1±2.3 µM and 27.1±1.53 µM respectively). Chronic riluzol also counteracted the lesion-induced increase in GLU after chronic application (21.8±0.68 µM) and acute riluzol drastically reduced GLU (0.7±0.05 µM). GLU was also reduced after both chonic and acute amantadine treatment (1.04±0.4 µM and 0.74±0.26 µM respectively). No alterations in GABA or ASP were detected in response to the treatment of lesioned rats with GLU antagonists. The results obtained after chronic treatments are presented in Fig. 12 as the percentage of values obtained in lesioned rats. The results obtained after acute

Substantially, the lesion produced a large increase in DA and GLU in the medial STR that was not accompanied by noticeable alterations in the two DA metabolites or the other two amino acids that were measured. The three treatments with GLU antagonists seemed to affect DA metabolism in the same way, counteracting the lesion-induced increase in DA.

Twenty-one days after the lesion, the denervated part of the STR (lateral) exhibited biochemical responses previously described after high levels of DA cell depopulation: a drastic reduction in DOPAC and an unmodified or slightly increased DA (Zigmond et al., 1984; Altar et al., 1987). In contrast, a large increase in DA (two- to three-fold) and a modest alteration in DOPAC was observed using voltammetry and confirmed using microdialysis in the spared part of the STR (medial). In this case, the levels of DA probably resulted from permanent (tonic) enhancement of the spontaneous release of DA. It has been shown that partial lesions leave the stimulation/release ratio unmodified (Dentresangle et al., 2001). A much larger lesion of the DA cell population seems to be required to alter the spontaneous pattern of discharge of nigral DA cells (Hollerman and Grace, 1990; Harden and Grace, 1995) or stimulation-induced DA release (Stachowiak et al., 1987). These observations strongly suggest that after lateral SN lesion, a global process activates tonic DA release by an

It has been claimed that recovery of the DA in the STR after partial lesions of the SN could be related to alterations in GLU neurotransmission (Calabresi et al., 2000; Emmi et al., 1996; Kashani et al., 2007). However, the mechanism and the location of this alteration remain unknown. We found that 3 weeks after the lesion, GLU was clearly enhanced in the medial STR. When GLU neurotransmission was pharmacologically interrupted by specific GLU blockers (riluzol, memantine and amantadine), a reduction in extracellular GLU activation was observed, with a simultaneous reduction in the tonic DA increase induced by the nigral lesion. Amantadine and memantine were proposed to have some neuroprotective effects after discovering the importance of NMDA receptor-mediated excitotoxicity as a factor underlying neurodegeneration in Parkinson's disease (Greenamyre and O'Brien, 1991). Indeed antagonists of NMDA receptors have been shown to inhibit neurodegeneration of the DA system induced by MPP+ and methamphetamine (Sonsalla et al., 1989; Turski et al., 1991). In our case, the effect of the two GLU antagonists was to counteract the increase in GLU and DA induced by the nigral lesion. Several studies have suggested that NMDAinduced DA release could be the main regulatory mechanism of extracellular DA (Bannon et al., 2001; Leviel, 2001; Mortensen and Amara, 2003), and our observations are consistent

treatments are presented elsewhere (Dzahini et al., 2010).

These treatments had differential effects on GLU.

indirect mechanism acting presynaptically in the STR.

with the hypothesis of a NMDA-induced mechanism causing stable dopaminergic hypertony. The effects of riluzol strengthen this hypothesis. First proposed to inhibit GLU release (Mantz, 1992), its action has also been attributed to persistent blockade of the sodium current (Del Negro, 2005). In our hands, riluzol reduced the release of GLU in sham-operated animals and counteracted the lesion-induced GLU and DA as well (Dzahini et al., 2010).
