Due to the efficient coupling between endogenous muscarinic receptors and GIRK channels, we found that firing of individual CHIs resulted in monosynaptic spontaneous inhibitory post-synaptic currents (IPSCs) in MSNs. These results indicate that muscarinic receptors in striatal output neurons reliably decode CHI firing. eTOC blurb Mamaligas and Ford examine the activation of muscarinic M4-receptors on direct-pathway medium spiny neurons in the striatum. By measuring synaptic currents through overexpressed GIRK channels, they find that cholinergic interneuron firing evokes transient spontaneous events through these metabotropic receptors. Introduction Cholinergic interneurons (CHIs) are the major source of ACh in the striatum (Bolam et al., 1984; Kawaguchi, 1993; Lim et al., 2014; Wilson et al., 1990). While these cells represent a small population of striatal neurons (1 C 2%), their broad arborizations and tiled distribution provide dense ACh innervation throughout the striatum. CHI firing is tightly locked to rewarding cues and associated stimuli that occur during reward-based learning tasks (Aosaki et al., 1994; Atallah et al., 2014; Morris et al., 2004). Together these findings suggest that as a result of short-term depression, the extent of ACh release can be modulated in response to these patterns of firing. Discussion M4-receptors are the most highly expressed class of muscarinic receptors in the striatum (Bernard et al., 1992; Yan et al., 2001). These inhibitory receptors are localized primarily in dMSNs (Bernard et al., 1992; Yan et al., 2001), where they decrease excitability (Howe and Surmeier, 1995) and oppose dopamine D1-receptors induction of glutamate long-term plasticity (Shen et al., 2015). Increasing M4-receptor activity in vivo rescues L-DOPA induced impairments in synaptic plasticity and L-DOPA induced dyskinesia in animal models (Shen et al., 2015). As animals lacking M4-receptors selectively in dMSNs Polygalasaponin F exhibit increased locomotor activity and behavioral sensitization to psychostimulants (Jeon et al., 2010) muscarinic GPCR signaling through these receptors is an important regulator of the striatonigral pathway. Despite the importance of these receptors in regulating striatal function, it has remained unclear how the release of ACh drives the activation of muscarinic receptors on MSNs. Using GIRK2 channels as a readout of muscarinic activation, our results show that muscarinic receptors on dMSNs receive independent, phasic synaptic inputs from CHIs. The firing of a single action potential in CHIs evoked unitary GIRK-mediated IPSCs in post-synaptic dMSNs. Our results also indicate that the physiological and anatomical characteristics of CHIs allow for consistently depressed ACh release during CHI tonic firing. Despite this depression, muscarinic M4-receptors reliably encode ACh release evoked by physiological CHI firing patterns without failure. As a result of the depression of ACh release during physiological firing of CHIs, MSNs increase their dynamic range of receptor activation, potentially allowing for differential behavioral responses in response to influential stimuli. We found that in the absence of GIRK, muscarinic receptors also could rapidly regulate dMSN output locally in the striatum through an Polygalasaponin F inhibition of axon collateral Polygalasaponin F synapses. Thus, the firing of CHIs may be endogenously encoded in striatal circuits through the transient inhibition of local circuits. Although CHIs only comprise a small percentage of neurons in the striatum, their tiled distribution and extensive arborizations position them to modulate a large MSN population. Cholinergic terminals have been found to make sparse synaptic connections (~3% synaptic incidence) with MSN dendrites, occurring primarily at symmetrical synapses (Bolam et al., 1984; Contant et al., 1996; Descarries and Mechawar, 2000). Despite this low connectivity, monosynaptic rabies-tracing studies that have mapped CHI inputs to MSNs have found CHIs are extensively connected with multiple dMSNs (Salin et al., 2009). Interestingly, the connections formed between CHIs and MSNs using rabies stands in contrast to similar studies that have examined SNc dopamine inputs onto MSNs (Wall et al., 2013). While SNc terminals provide abundant innervation to the striatum, monosynaptic rabies tracing approaches have found that only a small proportion of the total dopaminergic inputs are labeled (Wall et al., 2013). One possibility for the increased monosynaptic Rabbit Polyclonal to CLIC6 rabies labeling of cholinergic synapses may be that CHI terminals span shorter distances to MSN dendrites than DA synapses. The symmetrical synapses formed between CHIs and MSNs where M4-receptors have been found (Hersch et al., 1994), may therefore be the cholinergic synapses labeled with trans-synaptic rabies mapping. We found that CHI firing resulted in consistent amplitude muscarinic IPSCs. The low failure rate, low coefficient of variation and high probability of release of IPSCs suggest that each CHI action potential drives release of transmitter from many active release sites. The large number of active terminals likely allows for the overall stable amplitude of muscarinic events. This may be similar to other synapses at which multiple release sites and a high probability of release contribute to.