**2. Modes of ACh signaling and their roles in behavior**

#### **2.1 Tonic ACh signaling**

Tonic signaling (sometimes referred to as volume transmission) represents the traditional conceptualization of cholinergic transmission in the central nervous system. In this form of signaling, ACh functions as a slow-acting neuromodulator that exerts its effects on timescales of several seconds to minutes or even hours. Neuromodulatory ACh originating from the basal forebrain has been shown to be involved in functions such as the transition between arousal states, REM sleep, and exploratory behavior [2, 12–15]. Tonic signaling tends to lack both spatial and temporal precision.

Early evidence suggested that "ACh efflux" was not only involved in global arousal states but also directly involved in attentional processes and reward-based learning [16–18]. An early study using *in vivo* microdialysis measured ACh efflux in the medial prefrontal cortex (mPFC) during a 5-choice serial reaction time task (5-CSRTT) and found that in male rats that were well trained on this task, there was increased ACh efflux, an effect that was attenuated in rats not previously trained [19]. Interestingly, they found that ACh efflux was positively correlated with the number of trials completed during the session in which the cue light was illuminated for 5 seconds every trial, compared to 0.5 or 0.25 seconds. The authors contribute that this rise is dependent on the fact that the longer stimulus may elongate the saliency of the food reward and novelty of the task, which has been shown to be dependent on ACh [18, 20]. However, a follow-up study demonstrated that the increase in ACh efflux was due to the performance of the task alone, as rats whose reinforcement was yoked to another subject's performance rather than their own did not show the same increase in ACh efflux, despite also receiving the same pattern of rewards and being exposed to the cue stimuli [21]. Later literature would go on to implicate attentional control of behavior, particularly cue detection, as a cognitive function dependent on phasic ACh signaling, not general ACh tone [9, 22, 23].

Understanding of tonic cholinergic signaling came mostly from studies using *in vivo* microdialysis, in which cholinesterase activity is typically inhibited to ensure a

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

sufficient concentration of ACh for electrochemical detection. This leads to a potential misunderstanding of cholinergic activity in the frontal cortex, with some suggesting that ACh "tone" may even be a methodological artifact [4]. One of the reasons for such a conclusion stems from the seemingly hyperefficient action of AChE, which is the main enzyme involved in the hydrolysis of ACh in the synaptic cleft and therefore the termination of its action. However, it has been shown that a sufficient volume of ACh is able to transiently inhibit AChE [24, 25], leading to further increases in ACh that can potentially spill over into the extracellular fluid and increase cholinergic tone. Such a mechanism would involve a positive feedback loop in which significant quantities of ACh release can inhibit AChE, further increasing the concentration of extracellular ACh. However, this inhibition may be short-lived [24, 25], perhaps on the timescale of milliseconds, and therefore may cause small "pulses" of spillover without hindering fast synaptic phasic signaling at the point of inhibition.

However, the possibility that visual attention tasks require both intact tonic and phasic ACh signaling in the mPFC cannot be ruled out, and it is possible that performance on these tasks requires an alert attentional state governed by tonic signaling, which has been shown to mediate shifts in attention, as well as phasic signaling to encode for specific behavioral epochs and serve as an indicator of cue presentation. Such a mechanism would have been missed by the microdialysis approaches. Thus, the possibility that task performance is reliant on phasic signaling, while behavioral engagement is dependent on the ambient tonic signaling that is perpetually present in the cortex cannot be ruled out and the delineation of the two is a potential area of future research.

#### **2.2 Phasic ACh signaling**

Contrasted with tonic ACh signaling, phasic signaling involves much faster time scale circuit-specific release. Phasic signaling is thought to be wired, meaning that it involves the release of ACh from a single cell that innervates a single cortical cell in a highly specific manner [4]. Unlike tonic signaling, it does not involve spillover from the synapse into the extracellular fluid, rather it is constrained to the synapse. This type of signaling occurs on a much faster timescale—likely that of only hundreds of milliseconds. This type of signaling has been implicated in cortical control of attention, specifically cue detection—the cognitive process needed to determine whether a cue that signals a reward is present or not [6].

Based on work using mice, it has been demonstrated that projections originating in the nucleus basalis of Meynert (NbM) and substantia innominata (SI), which form the NbM complex, send their axons to the mPFC and are necessary for cue detection [6]. Particularly, it is thought that these projections are involved in the shift between vigilance and cue detection [6]. This is further demonstrated by the fact that disruption of the mPFC's cholinergic innervation impairs cue detection, while disrupting projections to the other targets of the NbM, such as the motor cortex, yields no effect on this task [23]. Phasic ACh signaling in this circuit is likely the causal mediator of cue detection, as it has been shown that optogenetic stimulation of the NbM during a cue detection task improved performance during cued trials and increased the false alarm rate during non-cued trials, suggesting that millisecond timescale cholinergic signaling originating in the NbM is involved directly in the encoding of the representation of the cue in the prefrontal cortex in mice [9].

In addition, there is evidence to suggest that the basal forebrain is involved in the formation of stimulus associations in mice. Tu et al. [11] found that cholinergic signaling during associative conditioning can affect the strength of the association formed in a time-dependent manner. Although the NbM is commonly cited as the primary source of cholinergic projections to the prelimbic cortex (PrL), and injection of retrograde tracer into the PrL revealed that the greatest source of cholinergic innervation originated in the horizontal diagonal band (HDB). Therefore, Tu et al. [11] targeted the PrL projections originating in the HDB for optogenetic manipulation and found that stimulation during the unconditioned stimulus impaired associative learning while inhibition facilitated it. Furthermore, they found that optogenetic stimulation during the conditioned stimulus did not affect the strength of associative learning, but inhibition lead to impaired learning. This was further accompanied by fiber photometry data that show that the level of excitation of the PrL correlates with the strength of the memory, such that ACh signaling during the unconditioned stimulus strengthened across sessions. These data suggest that phasic signaling is uniquely sensitive to timing, such that the functional role of the cholinergic projections from the HDB to the PrL region involves specifically timed excitation, further lending evidence for the role of ACh in encoding specific stimulus representations [11].

One of the ways that cholinergic signaling can be achieved on this timescale is through the actions of acetylcholinesterase (AChE), which is an incredibly effective hydrolytic enzyme, making the local regulation of AChE as one of the ways in which a degree of heterogeneity is introduced between tonic and phasic ACh signaling. Local expression of AChE may contribute to some of the anatomical heterogeneity between tonic and phasic signaling and makes it likely that phasic signaling would occur, due to its potent catalytic action. However, this has not been investigated thoroughly and represents a future area of inquiry. As cholinesterase activity is likely one of the most important regulators of spatially and temporally restricted ACh signaling in the prefrontal cortex, our understanding of its spatial distribution in the cortex is of paramount importance.

### **2.3 Tonic vs. phasic signaling: differing viewpoints on the distinction**

The presence of such a highly potent catalytic mechanism such as the hydrolysis of ACh by AChE has led some to suggest that tonic ACh signaling in the forebrain is unlikely to affect behavior at all. In this view, the fact that the rate-limiting step of ACh hydrolysis is the diffusion of ACh into the synapse, and not the hydrolytic action of AChE itself, is evidence to suggest that ACh is unlikely to travel distances beyond the synapse and therefore changes in extracellular ACh concentration is unlikely to be a contributor to behavioral events [4]. However, others believe that the tonic/phasic distinction is an oversimplification and that ACh signaling most likely has both fast and slow components that both contribute to behavior. In this view, cholinergic signaling varies as a function of anatomy, receptor subtypes, and ACh hydrolysis—and therefore the concept of ACh tone may still have some functional role in behavior [26].

While the exact middle ground between these two viewpoints is yet to be determined, one study demonstrated both relevant tonic and phasic ACh signaling simultaneously in the PFC and dorsal hippocampus of mice in an attempt to differentiate between their functions. Using electrochemical choline biosensors, Teles-Grilo Ruivo et al. [15] demonstrated that tonic ACh signaling during sleep was highest exclusively during REM sleep that preceded wakefulness. They also demonstrated that tonic signaling was highest as the animal approached a reward in a randomized forced alternation T-maze. Additionally, they found that phasic ACh was associated with the presentation of the reward, with phasic signaling showing response halfwidths

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

significantly shorter than during tonic signaling. Importantly, they demonstrated that both tonic and phasic signaling were highly coordinated between the PFC and the dorsal hippocampus, suggesting that not only does such a distinction in modes of ACh transmission exist in the cortex, but likely can be extended to the hippocampus as well. Should this be the case, the drivers of the distinction are likely to be driven by ubiquitous mechanisms such as cholinesterase activity, or some intrinsic property of basal forebrain anatomy. Ruivo et al. [15] suggest that their results demonstrate that tonic ACh signaling, especially during REM sleep, maybe prepare the mPFC and hippocampus for subsequent alertness and later attentional demands.

Additionally, there is electrophysiological evidence to suggest a dichotomy in cholinergic signaling in the basal forebrain. Unal et al. [27] demonstrated that there are two distinct populations of basal forebrain cholinergic neurons that are distinguished by their electrophysiological properties. Early firing neurons were more excitable, quicker firing, and had more pronounced refractory periods following firing, while later firing neurons were less excitable but more sustained in their ACh release. The authors suggested early firing cells may be involved in phasic signaling and therefore be important for attention, while the late firing cells may be involved in tonic signaling and therefore more important for global arousal states [27]. This suggests that the dichotomy has its roots in electrophysiological correlates.

The above work was later expanded on by Laszlovszky and colleagues [28]. They recorded from cells in mice both *in vivo* and *in vitro* and determined that basal forebrain cholinergic neurons took one of two forms, excitable, burst-firing cells (Burst-BFCN) and less excitable, rhythmic cells (Reg-BFCN). They found Burst-BFCN cells to be more numerous than Reg-BFCN in both the NbM and the horizontal limb of the HDB and consisted of two subtypes, ones with regular inter-spike intervals which they refer to as Burst-BFCN-SB and ones with Poisson-like inter-spike intervals which they refer to as Burst-BFCN-PL. They found that Burst-BFCN cells showed cortical synchronicity and fired bursts of action potentials in response to both reward and punishment during an auditory cue detection task. Reg-BFCN cells, on the other hand, were found to have precise spikes after behavioral outcomes, mainly hits, but not false alarms, correct rejections, or misses. Furthermore, there was distinct anatomical heterogeneity amongst these cell types, with Burst-BFCN found in the anterior basal forebrain and Reg-BFCN cells found in the posterior division [28]. These findings offer a unique viewpoint on the current tonic/phasic debate. They contend that such a divide between tonic and phasic signaling does exist and has anatomical and electrophysiological origins. Such an explanation seems to suggest that tonic ACh signaling has a much greater role in cue detection and cognitive operations than would be suggested by Sarter and Lustig [4].

Recently, basal forebrain cholinergic neurons have also been categorized into neurons that express calbindin-D28K (D28K) protein (ChAT D28K+) and those that do not (ChAT D28K-). The expression of D28K across the basal forebrain nuclei ranges significantly. About 40% of ChAT neurons in the VDB co-stain D28K, relative to 30% in the MS, 16% in the HDB, and less than 2% in the NBM [29]. ChAT+ neurons that also stain for D28K have fewer processes and a lower firing frequency. Interestingly, D28K is a Ca2+ binding protein that may function to protect cells from Ca2+-dependent neurodegeneration [29]. This is supported by data that the D28K protein is decreased in cholinergic neurons as a function of aging and in AD [30, 31]. Data support that cholinergic neurons are a heterogeneous population of cells, and understanding the unique profiles of the subpopulations may lead to a better understanding of critical behavioral processes they are involved in.

The location of cholinergic neurons is also a predictor of differential function. Amongst the cholinergic nuclei that project to the cortex, the HDB (rostral BF), and NBM/SI (caudal BF), there is data to support differential function across the anatomical location. Early literature on the NbM to prefrontal cortex circuit suggests a limited degree of axon collateralization between individual projection neurons, which limits the crosstalk between cortical cells and layers [32]. Thus, these circuits seem to be suited for phasic signaling, such that limited collateralization allows for a degree of spatial specificity that is necessary for wired cholinergic transmission in the prefrontal cortex. However, there exists some heterogeneity across species in terms of the projection targets of the NbM, such that its innervation of the prelimbic and infralimbic cortex has been well characterized in mice, but the data seem a bit more nuanced in the case of rats, such that retrograde viral tracing does not show NbM innervation of the prelimbic or infralimbic cortices in this species, rather the HDB is the key region [33]. Likewise, there is data to suggest that the mouse prelimbic cortex receives its primary cholinergic innervation from the HDB as well [11].

Assessing cholinergic activity across the BF anteroposterior axis *via* calcium imaging and ACh-specific fiber photometry, it has been revealed that the more rostral cholinergic neurons (HDB) are responsive to pupil change, which marks arousal state, reward delivery, and reward omission. In contrast, cholinergic activity in the caudal region (NbM/SI) was more responsive to unconditioned cues, delivery of shock, and cues that predicted shock [34]. It is therefore possible that the projections of the NbM/ SI to the cortex may be important for salient cue detection and that the projections from the HDB to the prefrontal cortex are more critical for appetitive arousal states.

Cortical receptor location within the architectural layers of the cortex may also play a key role in the outcome of ACh signaling. In addition to the aforementioned factors, evidence for tonic and phasic ACh as distinct behaviorally relevant modes of neurotransmission come from the differential roles and actions of muscarinic and nicotinic ACh receptors within the cortex [31]. There is some evidence to suggest that fast nicotinic ACh receptor (nAChR) mediated signaling is essential for phasic ACh signaling in the cortex while muscarinic ACh receptors (mAChR) are involved in slow changes in ACh concentration such as during tonic signaling. However, such a distinction may not be so cut and dry, as there seems to be a degree of muscarinic activity needed to perform a cue detection task [35], which suggests that muscarinic receptor activity may be needed to regulate global arousal states and "prime" the prefrontal cortex for phasic cholinergic signaling.
