Introduction
The contribution of striatal cholinergic interneurons (ChIs) to striatal
functions and parkinsonism has long been recognized
(Calabresi et al. , 2000;
Pisani et al. , 2007). In the last
decade, the role of striatal ChIs in levodopa (L-DOPA) induced
dyskinesia (LID) has also emerged (Bordiaet al. , 2019; Moehle et
al. , 2019). Dyskinesia is a major side-effect of L-DOPA
pharmacotherapy of Parkinson’s disease (PD), and is clinically
characterized by abnormal involuntary movements (AIMs) of dystonic and
choreic nature (Bastide et al. ,
2015). LID has a complex and partially unknown etiology, but the final
neurobiological event is the sensitization of D1 expressing
striato-nigral GABAergic medium-sized spiny neurons (MSNs) to L-DOPA
(Bastide et al. , 2015). Striatal
ChIs profusely innervate both striato-pallidal and striato-nigral MSNs
and influence their activity either directly or indirectly, via
modulation of dopamine, serotonin, glutamate release from striatal
afferents, and GABA release from striatal interneurons. It was
originally found that ablation or functional inhibition of ChIs
attenuate LID (Ding et al. , 2011;
Won et al. , 2014), although a more
recent optogenetic study revealed that ChIs dually modulate LID,
facilitating it at low firing rates and attenuating it at higher ones
(Bordia et al. , 2016).
Pharmacological studies showed that both muscarinic
(Bordia et al. , 2016;
Chambers et al. , 2019;
Ding et al. , 2011;
Shen et al. , 2015) and nicotinic
(Quik et al. , 2015) receptors are
involved in LID. However, while it has been convincingly demonstrated
that nicotinic receptor blockade (with receptor antagonists) or
desensitization (with receptor agonists) prevents LID
(Quik et al. , 2015), the role of
muscarinic receptors is far from clear
(Bordia et al. , 2019). This might
be due to the different neuronal distribution and functional roles of
muscarinic receptor subtypes. In fact, muscarinic receptors are GPCR
belonging to two major families which couple to Gi/o (M2
and M4 subtypes) or Gq/11 (M1, M3 and M5 subtypes), and
are unevenly distributed on the different populations of striatal
neurons and nerve terminals. In particular, striato-pallidal MSNs
predominantly express M1 receptors, while striato-nigral MSNs express
both M1 and M4 receptors (Bernard et
al. , 1992; Hersch et al. , 1994).
The M4 receptor is considered the main autoreceptor type at striatal
ChIs (Zhang et al. , 2002),
although also M2 autoreceptors contribute to negative auto feed-back
(Bonsi et al. , 2008). Another major
problem in dissecting out the role of endogenous ACh and muscarinic
receptor subtypes in LID is the poor selectivity of muscarinic
antagonists available. Thus, the unselective muscarinic antagonists
dicyclomine and atropine prevented LID in 6-hydroxydopamine (6-OHDA)
hemilesioned mice (Bordia et al. ,
2016; Ding et al. , 2011) whereas
atropine also prevented LID inhibition induced by optogenetic elevation
of ChIs firing rates (Bordia et al. ,
2016). M4 selective positive allosteric modulators (PAMs) reduced LID
in murine and non-human primate models of LID, pointing out a crucial
role for M4 receptors in this movement disorder
(Shen et al. , 2015). In partial
agreement, the putative M4 “preferential” antagonist tropicamide
(Lazareno et al. , 1993;
Lazareno et al. , 1990) worsened
AIMs induced by L-DOPA but attenuated those induced by a D1 receptor
agonist in 6-OHDA hemilesioned rats
(Chambers et al. , 2019),
suggesting the involvement of different sets of M4 receptors. However,
the M4 “selectivity” of tropicamide, if any
(Croy et al. , 2016), is negligible
which calls for a re-evaluation of its effects with relatively more
selective compounds. Finally, the role of ACh in LID might extend beyond
striatal ChIs. In fact, cholinergic inputs from the pedunculopontine
tegmental nucleus (PPN) modulate basal ganglia function
(Xiao et al. , 2016), and D1
signaling at striato-nigral MSNs terminals
(Moehle et al. , 2017). Although a
preliminary report showed that injection of tropicamide in PPN did not
modulate LID expression (Chambers et
al. , 2019), whether M4 receptor stimulation or blockade in SNr shape
LID remains to be established.
In the present study, in vivo dual probe microdialysis was used to
clarify the role of striatal and nigral M1 and M4 receptors in the
modulation of LID and the underlying striato-nigral MSNs activation. A
reverse microdialysis approach was adopted to deliver the M1 and/or M4
preferential antagonists telenzepine, PD-102807, tropicamide as well as
the selective M4 PAM VU0152100 in the dorsolateral striatum or SNr,
concurrently with systemic L-DOPA administration. This approach had the
two-fold advantage of i) targeting muscarinic receptors in specific
brain areas, ii) minimizing the poor selectivity of muscarinic receptor
antagonists by setting pharmacological selective concentrations in the
perfusion Ringer (see Methods). Dyskinetic behavior was monitored
concurrently with GABA release in SNr, a readout of striato-nigral MSNs
activation (Marti et al. , 2012;
Mela et al. , 2012;
Mela et al. , 2007), and glutamate
release in striatum, a possible index of cortico-basal
ganglia-thalamo-cortical loop activation
(Bastide et al. , 2015). The
feasibility of this approach was previously demonstrated showing that
reverse dialysis of the D1 antagonist SCH23390, but not the D2
antagonist raclopride, in striatum prevented LID and its neurochemical
correlates (Mela et al. , 2012).