Experimental design
Dyskinetic rats were randomized to L-DOPA or L-DOPA in combination with a muscarinic ligand in the Day 1 microdialysis session, and treatments were crossed in the Day 2 session. Experimenters were blinded to treatments. Groups were balanced in terms of Day 1 or Day 2 treatments. The effect of muscarinic ligands OFF L-DOPA was investigated in separate groups of rats. In this case, one rat received two different ligands, randomized in Day 1 and Day 2 sessions in a balanced design.
Perfusing muscarinic antagonists through the microdialysis probe allowed to deliver constant amount of compounds in the selected brain areas so to set stable and pharmacologically selective levels, approximating the affinity values for the targeted receptor, in the extracellular space. This was accomplished by taking into account in vitro recovery, which in our microdialysis conditions, with the 3 mm probe implanted in striatum, approximates 10%. We therefore estimated that roughly one tenth of the nominal concentration of the compound in the perfusion Ringer would be delivered to the extracellular space. Considering the poor selectivity of muscarinic receptor antagonists, this approach might represent an advantage over classical microinjection protocols. Telenzepine is an M1 receptor preferential antagonist, with an affinity of 1-3 nM for M1 receptors, a 2-5 fold selectivity for M1 over M4 receptors, and a 5-30 fold selectivity for M1 over M2, M3 and M5 receptors (Doods et al. , 1987; Lazareno et al. , 1990; Tanda et al. , 2007). Telenzepine was perfused through the probe at 100 nM, in order to generate extracellular concentrations of about 10 nM (i.e. 3-10 fold greater than the affinity for M1 receptors). PD-102807 is an M4 preferential receptor antagonist, with 7-28 nM affinity for M4 receptors, a 14-36 fold selectivity for M4 over M3 receptors, and 76-2600 fold selectivity for M4 over M1, M2 and M5 receptors (Bohmeet al. , 2002; Croy et al. , 2016). PD102807 was perfused at 3 µM to generate extracellular concentrations 10-30 greater than its affinity for M4 receptors and much lower than those for M1, M2 and M5 receptors. Tropicamide is considered an M4 preferential receptor antagonist, with an affinity of 15 nM for the M4 receptors (Lazareno et al. , 1993; Lazareno et al. , 1990). However, its selectivity for the M4 receptor over M1, M2 and M3 receptors is only 3-fold (Lazarenoet al. , 1993; Lazareno et al. , 1990). In addition, a more recent study (Croy et al. , 2016) questioned even such small pharmacological preference. Thus, we decided to infuse 100 nM tropicamide to generate concentrations just below the affinity value for the M4 receptor. VU0152100 is an M4 PAM effective in increasing by 70-fold the potency of ACh to stimulate Ca2+ responses at M4 chimeric receptors (Brady et al. , 2008). It does not bind to the M4 orthosteric site nor it potentiates ACh binding to the other muscarinic receptor subtypes, thus appearing very selective for M4 receptors (Brady et al. , 2008). We perfused 100 µM through the probe to generate extracellular levels about 30 times higher than its M4 activity (EC50 380 nM). AFDX-116 is a preferential antagonist of M2 receptors showing nanomolar affinity for M2 receptors (pKi 6.7-7.5), 3-4-fold selectivity over M4 receptors, 4-10 fold selectivity over M1 and >10-fold selectivity over M5 receptors (Billardet al. , 1995; Doods et al. , 1987; Stoll et al. , 2009). The concentration used for reverse dialysis (200 nM) was chosen based on a microdialysis study showing that perfusion of 100 nM AFDX-116 through the probe was capable of increasing by > 70% ACh levels in striatum (Billard et al. , 1995). Based on the affinity values reported above, such concentration is expected to generate extracellular levels slightly below the highest affinity value found in functional studies (pKi 7.5)(Billard et al. , 1995).