**3. The contribution of acetylcholine receptors in modulating tonic and phasic activity**

#### **3.1 Nicotinic receptors**

Nicotinic ACh receptors (nAChR) in the cortex have been shown to be essential for a number of cognitive functions, such a top-down attentional control of behavior and general working memory [36, 37]. Given their fast-acting, ionotropic responses, nAChRs are thought to be involved in phasic ACh signaling. Furthermore, given their selective laminar expression throughout different regions of the cortex, it stands to reason that nAChRs would be selectively expressed in circuits where fast, wired ACh transmission is the norm, and in such a distribution that promotes spatially restrained and functionally heterogeneous responses to ACh signaling from the basal forebrain.

#### *Modes of Acetylcholine Signaling in the Prefrontal Cortex: Implications for Cholinergic… DOI: http://dx.doi.org/10.5772/intechopen.110462*

While there is evidence to suggest a role for the utilization of these receptors in phasic signaling, the distinction between the role of nAChR in the two modes of cholinergic neurotransmission seems to be much more nuanced.

Much of the evidence surrounding the role of nAChRs in fast synaptic ACh signaling is electrophysiological. It has been shown that the activation of nAChRs evokes short-latency depolarizing postsynaptic potentials in mouse neocortical pyramidal neurons [38], which has been suggested to function as laminar selectivity relative to muscarinic function. Furthermore, nAChR activation throughout the cortex seems to be layer specific, as Poorthuis et al. [39] showed that nAChR activation leads to inhibition of pyramidal cells in the mPFC in layers II and III, but enhanced excitability of layers V and VI. Interestingly, their data suggest that the response to ACh in this region is dependent on different subunits depending on the layer of the cortex. They demonstrated that pyramidal neurons in layer VI exhibit slow inward currents in the presence of ACh that are absent in β2 subunit deficient mice and were blocked by an antagonist for β2 containing nAChRs and were only occasionally accompanied by α7 mediated currents. Meanwhile, layer V mPFC pyramidal cells showed an attenuated response to ACh in α7 deficient mice as well as following the application of an α7 antagonist, but not in β2 deficient mice, suggesting differential expression of these subunits by layer. They also performed two-photon imaging in cortical pyramidal cells of each layer in β2 and α7 deficient mice and found that nAChR-induced neuronal activation is dependent on the β2 subunit when ACh changes in concentration slowly, suggesting that not only is the β2 subunit utilized during tonic signaling, but also potentially reveals an interesting anatomical phenomena in which layer VI, in which β2 containing nAChRs are most involved in, is involved in tonic signaling, while layer V may not be [39].

The idea that nicotinic receptors are involved in tonic ACh signaling is an interesting one as well, in that it would stand as evidence against the assumption nAChRs mediate phasic signaling exclusively, and that tonic signaling is mostly reliant on muscarinic receptors. In addition to its presumed role in the detection of ambient ACh, β2 subunit deletion has been shown to impair attentional performance in mice [40], suggesting a role of such receptor subtypes in phasic ACh signaling, or at least furthering the idea that attentional performance requires some changes in ambient ACh levels. Interestingly enough, the deletion of this subunit can also upregulate muscarinic receptor excitability to compensate in layer VI of the mPFC, which was also seen following the deletion of the α5 subunit as well [41]. The previously mentioned study by Poorthuis and colleagues [39] notably blocked muscarinic receptors to isolate neuronal responses due to nicotinic activation, meaning that such an increase in excitation was masked, and cholinergic tone that would have been detected primarily by muscarinic receptors may have instead bound to nicotinic receptors in layer VI. An alternative explanation is that such a mechanism may allow for further priming of the cortex to ACh signaling to make up for the reduction caused by subunit deletion, allowing for phasic-dependent cognitive processes to attempt to bounce back even in the presence of less cholinergic signaling.

In addition to the β2 subunit, it seems the α5 nAChR subunit is necessary for attention as well, as deletion of the Chrna5 gene, which encodes for the α5 subunit, impairs attention [42]. It was also demonstrated that the loss of Chrna5 delays cholinergic excitation, such that mPFC layer VI pyramidal cells from Chrna5 deficient mice show attenuated onset kinetics but unaffected response magnitude during optogenetic stimulation of cholinergic afferents *in vitro* and that enhancing nicotinic receptor affinity pharmacologically can actually restore the typical response to optogenetic

stimulation [8]. The authors suggest that α5 subunit containing nAChRs in the PFC may be necessary to define a critical window for cue detection, such that a delay to their onset results in the animal being unable to properly integrate detected stimuli *via* corticothalamic connections and ends up missing the cue altogether. Another interesting factor that may play into the α5 subunit's role in phasic ACh signaling in cue detection is its relative rarity, in that its expression is much less prevalent than subunits such as β2 [43], and therefore may be more likely to facilitate spatially constrained wired transmission. However, this is yet to be investigated and represents a considerable gap in our understanding of the role of the α5 subunit in attention.

One further subtype that has been implicated in attention control, and by extension, the role of its dysfunction in cognitive decline, is the α7 nAChR which is composed entirely of α7 subunits. This particular subunit has been highly implicated in the cognitive decline associated with AD [44–46]. It has been shown that an α7 nAChR agonist was sufficient to enhance learning speed but not filter distracting information, which was instead enhanced by an α4/ β2 agonist, with no effect on learning speed [47]. Additionally, it has been shown that performance on the 5-choice continuous performance task, a measure of attention in rodents, was improved following the administration of encenicline, a partial agonist to the α7 nAChR, in poorperforming rats [48]. Due to the fact the dysfunction of α7 has been demonstrated in Alzheimer's disease, it is likely that this particular subtype of receptor is necessary for attentional processes in the cortex, in addition to other processes.

#### **3.2 Muscarinic receptors**

Muscarinic ACh receptors (mAChR) are thought to be mediators of tonic acetylcholine signaling, due to their longer duration of action *via* G-protein coupled mechanisms that require second messenger signaling. mAChRs are present in the prefrontal cortex both presynaptically and postsynaptically [49]. Thus, they would seem to be much more suited as receptors of tonic signaling, and likely their longer-lasting duration of action and the fact that a single muscarinic receptor can have amplified second messenger signaling lends itself to the idea that muscarinic receptors are involved in relatively slow timescale changes in ACh concentration in the prefrontal cortex. Similarly, such a mechanism would be suitable for the detection of relatively small concentrations of ACh present in the extracellular fluid during tonic transmission, as opposed to the much larger concentration present at the cholinergic synapse during phasic signaling [26].

However, there may be a degree of specificity as to which muscarinic receptor subtypes are involved in tonic signaling. It has been shown that the Gq-coupled M1 mAChR, but not the M3 or M5, is essential to the response of pyramidal neurons to tonic ACh [35], suggesting a role for only specific Gq-coupled receptors, not all of them. This is interesting, as the location of M1 receptors on the dendritic shaft and spines of cortical pyramidal cells provides a degree of anatomical evidence for volume transmission in the cortex, likely meaning that the M1 receptor's anatomical distribution is what favors it toward the detection of ambient ACh over long distances due to the fact that cholinergic neurons are not known to make axo-axonic synapses [50]. These findings point to a specialized role of muscarinic receptors in detecting global, seconds-scale changes in ACh concentration indicative of perisynaptic signaling and tonic transmission.

As mentioned previously, muscarinic receptor activation may serve to "prime" the cortex for subsequent phasic signaling in such a way that both forms of signaling

#### *Modes of Acetylcholine Signaling in the Prefrontal Cortex: Implications for Cholinergic… DOI: http://dx.doi.org/10.5772/intechopen.110462*

are necessary for attentional processes to occur. A likely mechanism is *via* increases in cortical pyramidal cell excitability, in which muscarinic receptor activation has been shown to alter *via* the induction of LTD in these cells in the medial prefrontal cortex [51]. This is further supported by findings that mice that are deficient in the inhibitory M2 muscarinic receptor show enhanced attentional performance, despite having impaired object-location learning and spontaneous recognition memory [52], suggesting that while the Gq-coupled muscarinic receptors, such as M1, play an important role in cortical excitability, the activation of Gi-coupled receptors such as M2 may be detrimental to attentional performance. It seems likely that muscarinic receptor activation is needed for cue detection and sustained attentional processes, as a non-specific muscarinic receptor antagonist is sufficient to impair attentional performance on a two-choice visual discrimination task in mice [53]. Likewise, M1 receptor antagonism impairs performance on a divided attention task in rats, unlike antagonism of the M4 receptor which did not [54]. Additionally, the M1-positive allosteric modulator TAK-071 has been shown to rescue attentional performance in rats with basal forebrain cholinergic cell loss [55]. Such studies suggest that while muscarinic receptors may play a role in attentional processes, their role in phasic ACh signaling remains unclear.

While it seems that muscarinic receptors are needed for attentional processes due to their effects on global arousal states, the dichotomy of tonic signaling being dependent solely on muscarinic receptors while phasic signaling is dependent solely on nicotinic receptors is evidently an oversimplification. Likely, the mechanism by which attentional control occurs is reliant on both nAChRs and mAChRs, such that mAChRs are needed to orient the organism toward the object of their attention, while nAChRs, at least the α7 and α5 subtypes, are involved in the response of cortical neurons to short timescale phasic ACh release. Thus, both nAChRs and mAChRs seem necessary for phasic ACh signaling and its reliant cognitive operations such as cue detection. However, mAChRs on their own may be mediators of tonic ACh signaling, and thus a degree of tonic ACh efflux is needed to ready the prefrontal cortex for an orientation toward a specific stimulus. However, more research is needed to delineate the contributions of each receptor system to tonic and phasic signaling in the prefrontal cortex, though the way to do so does not seem immediately straightforward.
