VU0152100, PD-102807 and tropicamide in striatum
Consistent with previous finding that M4 PAM VU0467154 attenuated LID and aberrant plasticity at striato-nigral MSNs in mice and non-human primates (Shen et al. , 2015), reverse dialysis of VU0152100 in striatum reduced LID and striato-nigral MSNs activation in vivo. Quite unexpectedly, this was also replicated by reverse dialysis of the M4 preferential antagonist PD-102807 (and the putative M4 preferential antagonist tropicamide) in striatum, suggesting that both M4 receptor stimulation and blockade have the same outcome on LID and associated pathways. Since PD-102807 concentrations were chosen to generate extracellular levels (~300 nM) well below the affinity for the M1 receptor (1.3-1.6 µM) (Bohme et al. , 2002; Croy et al. , 2016), it is unlikely that the effects of PD-102807 are due to an interaction with M1 receptors. Rather, the slight differences in the neurochemical patterns of VU0152100 with respect to M4 antagonists would point to an involvement of different sets of striatal M4 receptors. Striatal M4 receptors are expressed presynaptically on ChIs, where they operate as autoreceptors (Bonsi et al. , 2008; Zhang et al. , 2002), and postsynaptically, either on glutamatergic terminals where they inhibit Glu release (Pancani et al. , 2014), or striato-nigral MSNs where they modulate D1 signalling, Ca2+ channels, NMDA currents, and release endocannabinoids that retrogradely modulate DA release (Moehle et al. , 2019). Opposite actions have been attributed to postsynaptic M4 receptors at striato-nigral MSNs. In dissociated striato-nigral MSNs, M4 receptor stimulation enhanced excitability via opening of CaV1 channels, thus facilitating a subsequent stimulation induced by a D1 receptor agonist (Hernandez-Floreset al. , 2015). This is consistent with the increase of L-type Ca2+ currents mediated by M4 receptors in atrial cells (Pemberton et al. , 1995). Conversely, a negative interaction between M4 and D1 receptors on cAMP production, Ca2+ influx at dendritic NMDA receptors, and LTP modulation has been described at striato-nigral MSNs in slices (Shen et al. , 2015). Supporting a postsynaptic action at facilitatory M4 receptors on striato-nigral MSNs (Hernandez-Flores et al. , 2015), PD-102807 and tropicamide, in addition to LID and nigral GABA, also prevented the LID-associated elevation of nigral and striatal Glu, replicating the neurochemical profile of intrastriatal SCH23390, which blocks D1 receptors on striato-nigral MSNs (Mela et al. , 2012). Moreover, AFDX-116 counteracted LID and nigral GABA release inhibition induced by PD-102807, as if the elevation of striatal ACh levels produced by M2 autoreceptor blockade surmounted the postsynaptic M4 blockade and/or functionally counteracted it by overstimulating postsynaptic M1 receptors (Galarraga et al. , 1999). Finally, an fMRI study revealed that systemic M4 PAMs did not affect the D1 agonist-induced changes in cerebral blood volume (CBV), a hemodynamic response reflecting changes in neuronal activity, in striatum while inhibiting the amphetamine-induced CBV changes and DA release in the same area, along with the accompanying hyperlocomotion (Byun et al. , 2014). Thus, the main action of M4 PAMs in striatum has been proposed to be presynaptic, i.e. the modulation of DA release, and not postsynaptic (Moehle et al. , 2019; Moehle et al. , 2017). We can therefore speculate that VU0152100 reduced LID and striato-nigral MSNs activation by inhibiting ectopic DA release from serotonergic terminals, the predominant site of L-DOPA conversion to DA in the DA-depleted striatum (Bastide et al. , 2015), or even by rescuing M4 autoreceptor function in ChIs, which is inhibited due to upregulation of RGS4 proteins (Shenet al. , 2015). The failure of intrastriatal M4 PAM VU0152100 in modulating striatal Glu release would rule out the possibility that the inhibitory control operated by M4 receptors on Glu terminals (Pancani et al. , 2014) contributes to the antidyskinetic effect of VU0152100.