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

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

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

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

Normal and Physio-Pathological Striatal Dopamine Homeostasis 263

catheter, to a microsyringe. Weekly injections of 10 µl of a solution (0.1% ascorbic acid in 0.9% NaCl) with or without 35 µg of 6-OHDA were given to awake and freely moving animals. Four groups of 12 animals received 2, 4, 6 or 8 weekly injections of the toxin (n=6) or the toxin vehicle only (n=6). A fifth group (n=6) of rats received 8 weekly injections of the

A bipolar electrode was implanted in the medial forebrain bundle (MFB) containing the DA ascending fibers (coordinates: A:5; L:1.3; Ht:2). Every 4 minutes, the MFB was stimulated for 20 seconds by 20 bursts (one per second) of 25 positive square pulses, each lasting 0.5 ms, with a 2 ms interpulse interval (40 Hz theoretical frequency). Each burst was triggered 300

Difference Normal Pulse Voltammetry (DNPV) and Differential Pulse Amperometry (DPA) were performed using treated carbon fiber electrodes produced as described previously (Olivier et al., 1995; Dentresangle et al., 2001). Their active part was the surface of one pyrolytic carbon fiber (SOFICAR, France), 250 µm long and 8 µm in diameter. A stainless steel tweezer fixed on the interaural bar was used as an auxilliary electrode and the Ag/AgCl reference electrode was a silver wire coated with AgCl. This was maintained in contact with the skull by means of a sponge moistened with Phosphate Buffer Saline (PBS) solution (PBS, pH 7.4). The three electrodes were connected to a pulse voltammetric system (Biopulse, SOLEA Tacussel, France). Carbon-fiber electrodes were electrochemically treated as previously described (Gonon et al., 1984). To calibrate the electrodes, voltammograms were recorded *in vitro* in a standard solution of DOPAC or DA and ascorbic acid in PBS. The values of the oxidation potentials and the amplitude of the peaks were stabilized after 4–5 successive scans (10 to 15 min). Their values were 60 mV for ascorbic acid, 60 mV for DOPAC and 90 mV for DA. It had previously been verified that the amplitude of the oxidation peak for these substances is linearly correlated with their concentrations. The oxidation potentials for DA and DOPAC were however too close to be properly differentiated *in vivo* and DA was three orders of magnitude lower than DOPAC. Thus, for adequate detection of DA, DOPAC formation was inhibited by pretreatment with pargyline

Animals were submitted to superfusion of the striatum to analyze extracellular DA, DOPA, HVA, GLU, GABA and ASP (Dzahini et al., 2011). They were implanted with a dialysate probe (250 µm in diameter and 4 mm length, cut off 6000 dalton, CMA, Sweden) in the anterior part of the caudate nucleus (Ant.: 8.4 mm; Lat.: 2.5 mm; H.: 6 mm, same stereotaxic references). The cannula was supplied (1 µl/min) with an artificial CSF (in mM, 145 NaCl,

Push-pull cannulae were used in an experiment devoted to measuring the radioactive form of DA and DOPAC (Leviel et al., 1989, 1990, 1991). The cannulae (1.0 mm outer diameter) were supplied (flow rate: 12.5 µl/min) with an artificial cerebrospinal fluid (CSF) adjusted to pH 7.4 with an O2-CO2 (95:5 v/v) mixture. 3,5-[3H]TYR (50 Ci/mmole, Dositek, France) was

ms after the measurement of DPA (Dentresangle et al., 2001; Dzahini et al., 2010).

toxin and a daily injection of 2 mg/kg MK801 (i.p.).

**7.4 Medial Forebrain Bundle (MFB) stimulation** 

(75 mg/kg, i.p.), an inhibitor of monoamine oxidase.

**7.6 Superfusion procedures (microdialysis & push-pull cannula)** 

2.7 KCl, 1.0 MgCl2, 1.2 CaCl2, 0.45 NaH2PO4, 2.3 Na2HPO4, adjusted to pH 7.4).

**7.5 Voltammetric investigations** 

and DA terminals; 3) indirect activation through GLU afferents to DA terminals of tonic DA release, which initially constitutes autotherapy allowing the behavioral compensation observed in Parkinson's disease. However the toxic effect of DA on the DA terminal (production of H2O2 when present in excess) could constitute a secondary pathological agent, maintaining the degenerative process. Each of the three steps mentioned here require further elucidation and demonstration, but a large set of data in each case is consistent with this hypothesis.

DA homeostasis constitutes one of the functions of the DAT via DAT-RT. This is under the control of many regulatory processes including tonic GLU neurotransmission. Experiments regarding the consequences of partial lesions seem to indicate a role for this GLU-dependent regulation in the tonic DA overflow and in maintaining a permanent excess of extracellular DA. It remains to be determined how local GLU can induce the presynaptic and tonic activation of DA release. This could be achieved directly through GLU receptors located on DA terminals or indirectly through heterologous neurotransmission systems such as GABA or ACH.
