The application of optogenetic tools to the analysis of central cholinergic signaling using ChAT-Cre lines in either mice (Kalmbach et al., 2012; Huang and Zeng, 2013) or rats (Witten et al., 2011; see Figure 1) allows selective activation and silencing of cholinergic neurons and axonal projections, both in vitro and in vivo. Using this approach, several studies have now shown that ACh signaling occurs through direct, fast synaptic transmission—as well as over longer time scales consistent with more diffuse transmission—in the cortex (Letzkus et al., 2011; Arroyo et al., 2012, 2014; Bennett et al., 2012; Kimura et al., 2014). Activating channelrhodopsin (ChR2) in fibers from the BF elicited a barrage of inhibitory synaptic inputs to layer (L) 2/3 pyramidal cells, which depended on nAChR activation (Arroyo et al., 2012, 2014; Bennett et al., 2012; Kimura et al., 2014). Pyramidal neurons in L2/3 apparently do not express nAChRs themselves, but L2/3 interneurons do (Poorthuis et al., 2013). Activation of BF fibers produced cell type-specific responses in cortical interneurons. L1 and L2/3 LS neurons exhibited both a fast and a slow response, while L2/3 ChAT bipolar neurons exhibited only a slow response. Activation of L2/3 interneurons by ACh via both nicotinic and muscarinic receptors depressed pyramidal neuron firing thereby curtailing visual responses (Kimura et al., 2014). ACh-induced excitatory postsynaptic currents were generated by a mixed population of nAChRs (Arroyo et al., 2012). In addition to a slow dihydro-β-erythroidine (DHβE) sensitive non-α7*-mediated current, a fast component of excitatory post-synaptic potentials (EPSCs) was abolished by methyllycaconitine (MLA) in both L1 and 2/3 interneurons but not in ChAT+ cells. Comparing the reported time course of the inhibitory barrage received by L2/3 pyramidal neurons upon light-induced ACh release with the time course of the two different EPSC components, suggested that L2/3 pyramidal neuron inhibition is more likely dependent on the slow component, rather than the fast component of cholinergic activation. This was confirmed by bath application of DhβE, which prevented the inhibitory drive onto pyramidal cells. In a follow-up study, the same authors found a large trial-to trial variability of the fast component of the ACh-induced current components, indicative of direct synaptic transmission which they propose is mediated by synaptic α7*-containing (α7*) receptors. This was confirmed by lack of effect of AChE inhibitors on the amplitude or kinetics of this fast current component (Bennett et al., 2012; Arroyo et al., 2014). The slow component showed much less trial-to-trial variability and was sensitive to manipulation of AChE activity. From this, the authors conclude that the slow, non-α7* component involves diffusion of ACh over some distance, and arises from the effects of ACh on extra synaptic α4β2* nAChRs, while the faster nAChR EPSCs are mediated by direct transmission via synaptic or peri-synaptic α7* AChRs (Arroyo et al., 2012, 2014). These experiments demonstrate that in superficial layers of the somatosensory, visual and auditory cortex, L1 and L2/3 interneurons receive both direct and diffuse cholinergic inputs, that enable the cholinergic system to manipulate neocortical processing on a millisecond time scale as well as on slower time scales (Arroyo et al., 2012, 2014; Kimura et al., 2014).

On a network level, BF stimulation in anesthetized animals results in a desynchronized state of field potentials (Goard and Dan, 2009; Kalmbach et al., 2012; reviewed in Bloem et al., 2014) and neuronal firing in the BF is correlated with a reduction in low frequency, and an increase of high frequency, oscillations in the cortex (Duque et al., 2000; Manns et al., 2000). Since the frequency band activity is related to the state of arousal and the extent of cortical activation (Uhlhaas et al., 2008; Wang, 2010; Deco and Thiele, 2011; Cachope et al., 2012), this supports the idea that ACh acts as a neuromodulator involved in setting the state of arousal. Mechanistically, it was shown that ACh activated mAChRs on cortical pyramidal neurons (Gulledge et al., 2009), thereby shifting firing modes from bursting to tonic and changing low frequency high amplitude oscillatory activity to high frequency low amplitude activity on a network level (Metherate et al., 1992).

Other studies have looked at the effect of ACh on the direction of the flow of information in the cortex. Again, these studies have been performed in sensory areas because in these regions, neuronal responses could be related to sensory stimulation. In this regard it is reasonably well established that ACh is directly involved in the enhancement of feed-forward thalamic input into the sensory cortical areas (see Bloem et al., 2014, for a review). In L4 of visual cortex, ACh increases the gain and reliability of neuronal responses (Goard and Dan, 2009; Soma et al., 2012, 2013), an effect that is mediated by heteromeric nAChRs (Roberts et al., 2005; Disney et al., 2007). A similar effect of ACh is observed in the barrel cortex (Oldford and Castro-Alamancos, 2003).

In L2/3, the picture is more complex. In general, cholinergic modulation reduces firing rate in these layers by increasing GABAergic inhibition through mAChRs and nAChRs (Disney et al., 2012; Alitto and Dan, 2013; Soma et al., 2013; Kimura et al., 2014). The ACh modulation in firing rates was associated with enhancement of the reliability of encoding and modulation by stimuli presented (Goard and Dan, 2009; Soma et al., 2013).

The cortical depression associated with whisker trimming is accompanied by an increase of heteromeric nAChRs on interneurons in L2/3 and blockade of these nAChRs can prevent the cortical depression. These observations support the contention that heteromeric nAChRs in L2/3 are required for regulating the input- dependent responsiveness of the somatosensory cortex (Brown et al., 2012a).

Intra-cortical projections that connect superficial layers between different cortical columns are also inhibited by ACh through activation of mAChRs (Kimura and Baughman, 1997). Based on this finding and the reduced activity in the superficial layers, it has been suggested that ACh reduces horizontal processing through cortico-cortical interactions (Hasselmo and Giocomo, 2006). Indeed it has been observed in slices, and in vivo animal experiments as well as in humans, that the spatial spread of excitation in response to stimuli is reduced in the presence of elevated levels of ACh (Kimura et al., 1999; Silver et al., 2008). Such a modulation of excitation could have a sharpening effect on tuning curves of receptive fields and on discrimination of sensory stimuli (Roberts et al., 2005; Thiele et al., 2012). The combined effects of ACh—e.g., reduction of lateral interactions and increased sensitivity to thalamic inputs, would be expected to increase network sensitivity to incoming information and enhance signal to noise. A similar selective gain-control effect of ACh is observed with enhanced attention (Briggs et al., 2013) and could be one of the core mechanisms through which ACh modulates selective attention (Hasselmo and Giocomo, 2006; Deco and Thiele, 2011; Hasselmo and Sarter, 2011).

The functional impact of ACh on the deeper L5 and 6 is less well understood. It is clear that deep layer pyramidal and interneurons are modulated by both nAChRs and mAChRs (Gulledge et al., 2007; Kassam et al., 2008; Poorthuis et al., 2013). ACh is associated with both response suppression and response facilitation, although the net effect of endogenous cholinergic signaling is not clear (Soma et al., 2013). In L1, most (if not all) interneurons contain α7* and /or non-α7* nAChRs (Christophe et al., 2002; Alitto and Dan, 2013). Since these neurons inhibit both L1-3 interneurons and L2/3 pyramidal cells, the effect of cholinergic L1 activation appears to be complex with both net inhibition as well as disinhibition of pyramidal cells in deeper layers, and it is likely dependent on the source and extent of ACh release in L1 (Letzkus et al., 2011; Bennett et al., 2012; Cruikshank et al., 2012; Jiang et al., 2013; Arroyo et al., 2014).

Thalamic inputs to L5 neurons are strongly regulated by nicotinic receptor activation (Lambe et al., 2003; Couey et al., 2007; Poorthuis et al., 2013). Whether these are targeted by direct cholinergic inputs is not known. However, within the thalamic reticular nucleus, neurons receive biphasic fast cholinergic inputs mediated by non-α7* nAChRs and mAChRs (Sun et al., 2013).

Despite new insights as to how rapidly ACh levels may rise and fall in prefrontal cortex during cue detection (Sarter et al., 2014), there is still no direct demonstration of the cellular and synaptic mechanisms by which ACh controls attentional behaviors. Hints emerge from the optogenetic data on the disinhibitory circuit mechanisms in superficial layers of sensory areas (Letzkus et al., 2011; Arroyo et al., 2014), but the architecture of the somatosensory cortex differs substantially from that of prefrontal cortical regions. Indeed, L4 is absent from rodent medial PFC (Uylings et al., 2003), and projections from the mediodorsal thalamus target all layers of mPFC, in contrast to the more discrete segregation of thalamo—cortical input seen in somatosensory areas (Douglas and Martin, 2004; Constantinople and Bruno, 2013). Few studies have appeared that manipulate the cholinergic system using optogenetics during cognitive tasks. In the primary visual cortex (V1) optogenetic stimulation of BF projections improved visual discrimination, a hallmark of visual attention, in a go-no-go task (Pinto et al., 2013). Inhibiting the BF cholinergic projections to the visual cortex with either halo-rhodopsin (NpHR) or archaerhodopsin (Arch) impaired mouse performance on the same tasks (Pinto et al., 2013; Arroyo et al., 2014 for review).

In a recent report of unpublished observations, Sarter et al. (2014) optogenetically manipulated the excitability of BF projections to the PFC in mice performing a sustained attention task (SAT). This would be the first report of optogenetic manipulation of ACh release in the PFC and modulation of attention performance. Using ChAT-Cre mice expressing ChR2 in the BF, the authors report that brief blue light stimulation during cue presentation increases detection of the cue. Optogenetic stimulation of BF fibers in the absence of a cue, which predict the presentation of reward, results in a higher number of false-positive responses in cue detection of ChR2 mice. Inhibition of ACh fibers with NpHR stimulation reduced cue detection (Sarter et al., 2014). Previous studies from the same group have identified transient release of ACh in the mPFC as a modulator of cue-directed attention. In particular, fast ACh release occurred when the cue trial was preceded by an actual or perceived non-cue trial (Howe et al., 2013). Therefore, cholinergic transients may be involved in state-shifting: i.e., in regulating the shift from generalized monitoring to one of cue-directed attention (Sarter et al., 2014). In this sense, the optogenetic increase in false-positive responses, where the animal responds incorrectly to a non-cue trial, might reveal the mechanism by which transient release of ACh in the mPFC determines the transition from cue detection to a behavioral response. Full appreciation of the data underlying these conclusions awaits publication of the primary data referred to in the Sarter review (Sarter et al., 2014).

In addition to the robust modulatory activities of cholinergic signaling in cortex and hippocampus ACh is renowned for its strong regulatory role in subcortical brain regions within the midbrain and striatum. In particular, the core of the brain reward circuitry, comprising the ventral tegmental area (VTA) and the nucleus accumbens (NAc), is strongly modulated by ACh. The main source of ACh to the VTA neurons in the midbrain arises from the brainstem structures LDTg and PPTg, which play a role in acquisition of reward, and reward-related locomotor activity (Corrigall et al., 2002; Champtiaux et al., 2006). The main source of ACh for the NAc/ventral striatum, as well as for the dorsal striatum, is the cholinergic interneurons which comprise less than 2–5% of the total striatal neuron population (Descarries et al., 1997). Notwithstanding the paucity of striatal cholinergic interneurons, ACh signaling is directly involved in the modulation of (1) striatal dopamine (DA) release (Rice and Cragg, 2004; Exley and Cragg, 2008; Wang et al., 2014); (2) local network functionality (Galarraga et al., 1999; Koós and Tepper, 2002); and (3) striatal-dependent behaviors related to reward (Joshua et al., 2008).

The release of DA in striatum is crucial for functions such as motivation, reward and locomotor activity (see Cachope and Cheer, 2014, for a recent review) and cholinergic transmission can drive striatal DA release (Exley and Cragg, 2008). A recent study showed that selective optogenetic activation of accumbal cholinergic interneurons is sufficient to trigger DA release in the same region, and that this effect is independent of the suprathreshold activation of VTA DA neurons per se (Cachope et al., 2012; Threlfell et al., 2012; Wang et al., 2014). As such, the activity of cholinergic interneurons might boost the release of DA to encode aspects of reward-related events. This proposal is in line with studies in which photostimulation of cholinergic interneurons drove striatal DA release via activation of presynaptic nAChRs (Threlfell et al., 2012; Wang et al., 2014). On the other hand, Cachope et al. (2012) reported that the direct effect of cholinergic interneuron activation on DA release was only partially mediated by activation of AChRs. Combining optogenetic manipulation with in vitro pharmacology, revealed the collaboration of both nicotinic (β2*) and muscarinic receptors, together with the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in the enhancement of striatal DA release. Thus, a synergy exists between ACh and glutamate in modulating the activity of the striatal network. The source of glutamate may be the striatal cholinergic interneurons themselves (Gras et al., 2008). Activating ChR2 in cholinergic striatal interneurons triggers postsynaptic responses onto medium spiny neurons (MSNs), the most abundant striatal cell type. Under the stimulation conditions used by Higley et al., the direct postsynaptic responses were blocked by glutamate receptor antagonists alone and were insensitive to AChR blockade (Higley et al., 2011). This suggests that at low levels of stimulation direct control of MSN firing by “cholinergic” interneurons may also rely on fast glutamatergic transmission.

A follow-up of this study was conducted looking at the connections between ACh interneurons and other local interneurons. including the PV+ interneurons, that also contact MSNs directly (Koós and Tepper, 1999; Gittis et al., 2010). Activating dorsal striatal ACh interneurons triggers the co-release of ACh and glutamate on PV+ interneurons, activating slow non-α7* nAChR currents, and both AMPA and N-methyl-D-aspartate (NMDA) receptors (Nelson et al., 2014). ACh and glutamate co-release was dependent on the presence of the vesicular glutamate transporter VGLUT3. This transporter is also involved in enhancing the vesicular loading of ACh and is important for di-synaptic inhibition of MSNs after PV+ excitation, a common feature in striatal information processing (Gras et al., 2008).

Activation of ChR2 in cholinergic interneurons in striatum also triggered GABA-A receptor-mediated postsynaptic currents in MSNs both in vivo and in vitro (Witten et al., 2010). Optogenetic stimulation of striatal cholinergic interneurons activated di-synaptic inhibitory responses in MSNs in vitro (Nelson et al., 2014). This effect was still present when PV+ neurons were ablated leading the authors to suggest that the di-synaptic inhibition of MSNs might be mediated by GABA release from DA terminals, that are studded with β2* nAChRs and targeted by cholinergic interneuronal projections. Thus, striatal network activity could be orchestrated by cholinergic interneurons through simultaneous regulation of DA and GABA release in the same striatal area.

Optogenetic studies have helped to define the role of striatal cholinergic interneurons in multiple aspects of motor control, associative learning and reward (see Jiang et al., 2014, for review; Exley and Cragg, 2008). During reward-related events, cholinergic interneurons initially increase their firing activity, and then pause, after which they start firing in a third phase of elevated activity (e.g., Morris et al., 2004). Most likely, these phasic activity periods support DA release from VTA projections, with the nAChR mediated component being independent of VTA action potential firing (e.g., see Wang et al., 2014). By combining pharmacological, optogenetic and electrophysiological techniques, Straub et al. (2014) recently suggested reward coding resides in the pause in striatal ACh interneuron activity that results from the direct effect of nigrostriatal DA projections via D2 dopamine receptors on cholinergic interneurons. The authors did not identify which neurotransmitter is involved in the rebound phase. Other studies have suggested that the pause of ACh interneuron firing may be caused by a GABA component. Activating VTA GABA neurons that project to the striatum with ChR2 and recording activity on ACh interneurons resulted in a pause of ACh interneuron firing (Van Bockstaele and Pickel, 1995; Tan et al., 2012; Van Zessen et al., 2012). Interestingly this effect was only observed in striatal ACh interneurons, sparing the other cell population and it was insensitive to DA receptor blockade. Behavioral studies have confirmed that GABA mediated inhibition of cholinergic interneurons is a requisite component of stimulus-outcome association under relevant learning conditions, pinpointing the pivotal role of ACh interneurons in goal directed behaviors (Brown et al., 2012b). In addition, Witten et al. (2010) have reported that cholinergic interneuron silencing by NpHR stimulation reduced cocaine preference in behaving mice.

Taken together, these findings support the idea that cholinergic interneurons play a crucial role in the modulation of striatal activity, and striatal-dependent behavior. Recent anatomical studies have also underscored the potential importance of direct projections from the brainstem (PPTg and LDTg) to striatal cells (Dautan et al., 2014). Hence, it will be interesting to learn how cooperation between these different elements of the cholinergic system modulates striatal activity.