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).