The data analysts were blinded to dose group. al., 1989; Alexander et al., 1990; Graybiel, 2008; Pisani et al., 2005). Dopaminergic inputs to the striatum from the substantia nigra pars compacta (SNc) play an important role in regulating striatal output and BG-influenced behaviors (Beaulieu and Gainetdinov, 2011). In addition, dopamine (DA) signaling in the striatum is dysregulated in multiple movement disorders and psychiatric illnesses (Albin et al., 1989; Goodchild et al., 2013; Wichmann and DeLong, 1996). A major regulator of DA signaling in the BG is acetylcholine (ACh), acting through muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs) (Bonsi et al., 2011; Picciotto et al., 2012). nAChRs are non-selective cation channels that have profound effects on DA release (Cachope and Cheer, 2014), and have been implicated in multiple disorders where DA is disturbed (Crunelle et al., 2010). mAChRs are G protein-coupled receptors that also modulate DA release and signaling (Kruse et al., 2014; Shin et al., 2015; Zhang et al., 2002). For instance, mAChR agonists inhibit DA release and suppress behavioral effects of psychostimulants that act by increasing DA levels (Bendor et al., 2010; Foster et al., 2014; Foster et al., 2016; Starke et al., 1989). Furthermore, disturbances of mAChR signaling have been implicated in many neurologic and neuropsychiatric disorders that are thought to involve changes in DA signaling (Aosaki et al., 2010; Holt et al., 1999; Kataoka et al., 2010; Pisani et al., 2007). Cholinergic interneurons (ChIs) in the striatum are large, aspiny, tonically-active interneurons that are thought to be central to the ability of ACh to modulate the BG (Holt et al., 1999; Pisani et al., 2007; Tanimura et al., 2017). However, recent studies suggest that non-striatal sources of ACh may also regulate BG output. Hindbrain cholinergic nuclei of the pedunculopontine nucleus (PPN) and laterodorsal tegmental nucleus (LDT) project to BG nuclei and brain regions that regulate the BG such as the SNc (Dautan et al., 2014; Saper and Androsterone Loewy, 1982). Optogenetic or electrical activation of projections from either the PPN or LDT can modulate locomotion, reward, and gait (Wen et al., 2015; Xiao et al., 2015), suggesting that cholinergic neurons in the hindbrain may also regulate the BG output. However, the sites of action of these hindbrain cholinergic nuclei and the AChR subtypes they modulate are not well understood. In recent years, M4 has emerged as the primary mAChR subtype responsible for regulating DA signaling in the striatum (Conn et al., 2009), as psychomotor effects of non-selective mAChR agonists are diminished in M4 knockout (KO) mice (Gomeza et al., 2001; Guo et al., 2010). Additionally, selective positive allosteric modulators (PAMs) of M4 decrease amphetamine-induced increases in extracellular DA in the striatum and functional magnetic resonance imaging (fMRI) studies reveal that M4 PAMs induce a profound Androsterone reduction in amphetamine-induced activation of the striatum and other forebrain regions (Byun et al., 2014). Furthermore, M4 PAMs reduce effects of DA-releasing stimulants on locomotor activity and other behavioral responses (Brady et al., 2008; Bubser et al., 2014; Byun et al., 2014; Dencker et al., 2012; CD247 Foster et al., 2016). Recent studies also suggest that M4 PAMs act in part, by inhibition of DA release in the striatum by release of an endocannabinoid from spiny projection neurons (SPNs) and activation Androsterone of presynaptic cannabinoid receptor 2 (CB2) on DA terminals (Foster et al., 2016). While the behavioral effects of M4 PAMs are likely to be partially mediated by inhibition of DA release, M4 is highly expressed in SPNs that express the DA receptor subtype 1 (D1); forming the direct pathway (D1-SPNs) and send inhibitory projections to the substantia nigra pars reticulata (SNr)(Hersch et al., 1994; Levey et al., 1991). Activation of D1-SPNs in the striatum prospects to GABA launch, inhibition of GABAergic SNr projection neurons, disinhibition of the thalamus, leading to excitation of the cortex and facilitating goal-directed movement. Interestingly, D1 activates a Androsterone unique G protein in D1-SPNs, Golf, that couples D1 to activation of adenylyl cyclase (AC), formation of cAMP, and activation of protein kinase A (Corvol et al., 2007; Herve, 2011; Zhuang et al., 2000). This signaling pathway is critical for many of the behavioral actions of amphetamine that are reversed by M4.