br STAR Methods br Author Contributions

STAR★Methods
Author Contributions
Acknowledgments We thank Antony M. Dean for initial discussions and encouragement concerning ancestral sequence reconstruction. We also thank Natalie K. Goto for constructive criticism concerning the manuscript, Steven M. Sine for providing Apramycin Sulfate receptor subunits, and Chris R. Free for technical assistance. F.J.P.-A. was supported by the Generalitat de Catalunya and the Erasmus Mundus - Action 2 Program. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2016-04801) to C.J.B.d.C.
Introduction Cholinergic neurons in the basal forebrain (BF) and medial septum/diagonal band of Broca (MS-DBB) innervate cortical and subcortical structures, including the prefrontal cortex and hippocampus, respectively (Mesulam et al., 1983). These projections play an important role in attention and memory processes (Hasselmo and Sarter, 2011), likely by desynchronizing neuronal networks to enhance the signal-to-noise ratio for salient information (Chen et al., 2015; Everitt and Robbins, 1997; Fu et al., 2014; Harris and Thiele, 2011; Hasselmo, 2006; Pinto et al., 2013). De-innervation of cholinergic afferents results in attentional deficits and reduced vigilance (McGaughy et al., 2000), and stimulation of cholinergic afferents can also produce reinforcement of behavior triggered by rewarding or aversive stimuli (Hangya et al., 2015; Liu et al., 2015). Acetylcholine release is also critical for switching neuronal networks into high-arousal states that are similarly characterized by less synchronized activity (Saper et al., 2010). However, the precise timing and location of acetylcholine release have remained unclear, leaving open the question of whether cholinergic nuclei function in a coordinated or an independent manner and by what mechanisms and timescales acetylcholine release regulates arousal, attention, or reinforcement learning (Teles-Grilo Ruivo and Mellor, 2013). Microdialysis studies have shown acetylcholine release in neocortex and hippocampus increases during attention, stress, exploration, and locomotion (Pepeu and Giovannini, 2004) and that acetylcholine levels are high during REM sleep but low during slow-wave or non-REM (NREM) sleep (Marrosu et al., 1995). However, the limited temporal resolution of microdialysis prevents detection on a sub-minute timescale that is most relevant to many cognitive processes and, furthermore, leaves open the question of whether fluctuations in acetylcholine are mediated by an increase in non-synchronized release from multiple presynaptic boutons over a period of minutes (tonic release) or highly synchronized release within a few seconds (phasic release) (Sarter et al., 2009). Higher temporal-resolution measurements of cholinergic neuron activity by juxtacellular recording or calcium imaging show low basal firing rates (Lee et al., 2005; Simon et al., 2006) that increase in MS-DBB neurons projecting to the hippocampus during aversive stimuli (Lovett-Barron et al., 2014) or in BF neurons projecting to the neocortex during whisking (Eggermann et al., 2014; Nelson and Mooney, 2016), waking, and REM sleep (Lee et al., 2005). However, juxtacellular recordings or calcium imaging necessarily restrict movement; the duration of recordings; and, therefore, the range of behavioral states tested. Alternative approaches using optogenetic identification of extracellularly recorded cholinergic neuron activity reveal that cholinergic neurons are activated in response to rewarding or aversive cues, suggesting a role in reinforcement of behavior (Hangya et al., 2015), but this method does not distinguish where acetylcholine is subsequently released. To overcome these limitations and investigate the spatiotemporal dynamics of acetylcholine release across a range of behavioral states and brain regions in freely moving animals with sub-second temporal resolution, we made use of electrochemical enzyme-based biosensors (Baker et al., 2015; Bruno et al., 2006a; Parikh et al., 2004, 2007; Zhang et al., 2010). This technique enables the measurement of extracellular levels of acetylcholine not confined to the synaptic cleft and, to date, has only been used to investigate phasic acetylcholine release in the medial prefrontal cortex (mPFC), where it was found to be involved in the processes of cue detection (Parikh et al., 2007).