**5. ACh in brain disorders: circuit dysfunction**

Reductions in markers for the cholinergic phenotype, such as the expression of ChAT, the enzyme necessary for ACh synthesis, are present throughout the basal forebrain following both AD and AUD, a consequence that is considered one of the hallmarks of both of these disorders in particular. These cholinergic deficits are present in two of the main basal forebrain circuits, the projections from the NbM complex (NbM, HDB, SI) to the cortical mantle, and the projections from the MS/DB to the hippocampus. In both disorders, there is a suppression in the basal forebrain cholinergic phenotype. In the case of AUD, there is evidence from animal models to suggest that these deficits may not be permanent and can be rescued *via* the use of voluntary wheel running exercise [64], the actions of neurotrophins [65], and the

AChE inhibitor galantamine [66]. In contrast, chronic treadmill exercise or voluntary wheel running has been shown to attenuate age-related reduction of cholinergic fibers in the cortex and hippocampus and improve some learning and memory outcomes but has minor effects on the number of ChAT [47] positive neurons [67–69]. However, exercise has been shown to improve ACh levels in the hippocampus in an Aβ1–42 peptide rat model [70]. This does suggest that deficits caused by both alcohol-related brain damage and AD may involve a reduction in functional cholinergic neurons, leading to reductions in overall ACh signaling, which can be rescued. It, therefore, seems likely that in both of these disorders there would be a disruption in either, or both, tonic and phasic ACh signaling in the brain.

The drivers of the selective neuropathological vulnerability of cholinergic neurons, across brain disorders, are their large size and extensive projections, which require high metabolic expenditures and trophic factors to maintain the considerable cytoskeletal surface, as well as the machinery for axonal transport over long distances. These morphological properties of cholinergic neurons increase their vulnerability to oxidative stress, neuroinflammation, and altered energy homeostasis that occurs during aging and disease states [71, 72].

Human clinical data have long supported the role of chronic heavy alcohol use leading to premature brain aging, as well as a risk for the development of dementia, including AD [73–77]. Furthermore, alcohol consumption has been linked to an increased risk of dementia in individuals with a genetic predisposition to AD [78, 79]. Recent data from preclinical studies demonstrate that the consequences of adult or developmental EtOH exposure resemble advanced brain aging or produces accelerated AD-related pathology in transgenic models with AD-related transgenes [80–83]. Advanced aging, AD, and AUD have some overlapping neuropathological sequelae: upregulation of proinflammatory markers, suppressed hippocampal neurogenesis, suppression of basal forebrain cholinergic phenotype, and altered pro- and mature NT levels—as well as a change in the ratio of Trk to p75NTRs [84–88]. A common pathway for cholinergic dysfunction in AD and AUD is the disruption of neurotrophins and their receptors (see **Figure 2**), which may drive additive effects of AD and AUD pathology.

#### **5.1 Alzheimer's disease and cholinergic dysfunction**

The increase in AChE activity in AD has been known for some time, as many of the drugs currently available for the treatment of this disease target this enzyme and inhibit its activity [89–91]. Cholinesterase inhibition has been shown to increase cognitive performance on the Stroop task in human patients with AD, with the degree of inhibition directly correlating with performance [92]. In AD, AChE inhibitors prolong ACh action, as well as increase the uptake of NGF to improve cholinergic neuronal survival [93]. Furthermore, cholinesterase inhibitor therapy in AD improves cognitive performance by increasing the activation of frontal cortical circuits as determined by fMRI studies [94, 95]. However, only a subset of patients with AD is effectively responsive to AChE inhibitors, and cholinergic basal forebrain integrity is a key predictor of treatment success [96].

As mentioned previously, it is likely that AChE is needed for both tonic and phasic ACh signaling, but is a more immediate causal effector of phasic signaling. It is likely that tonic signaling is required to ready the cortex for phasic signaling, and AChE inhibition may also work to increase ACh tone and facilitate cognitive performance. However, there may be a degree of specificity for phasic signaling when it comes to this modulation. Should the hypothesis that phasic signaling is more immediately

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

#### **Figure 2.**

*Changes in neurotrophins and their receptors that modulate cholinergic cell survival. Both Alzheimer's disease (AD) and alcohol use disorder (AUD) alter the expression and ratio of pro-to-mature nerve growth factor (NGF) and brain-derived growth factor (BDNF), as well as the tropomyosin kinase receptors for NGF (TrkA), BDNF (TrkB), and p75 receptor. The pro-forms of neurotrophins are potent ligands for p75NTR and can induce cell death or loss of cholinergic phenotype when coupled with sortilin. Thus, AUD likely accelerates the pathological sequela of AD. \*Created with BioRender.com*

dependent on AChE than tonic signaling be supported, then modulation of AChE should have more of an effect on phasic-dependent processes. However, since tonic signaling is likely needed to modulate phasic signaling and *vice versa*, disentangling these two may be difficult experimentally, since the effects that AChE inhibition may have on tonic signaling may occur after that of phasic signaling, but still on a relatively short pharmacological timescale.

Likely, a balance between ACh and AChE may be critical to the phasic ACh signaling needed for sustained attention. Thus, disorders such as AD disrupt attentional processes by dysregulation of AChE, leading to dysfunction of phasic ACh signaling in the cortex. It has been shown that presenilin-1 (PS1), the catalytic component of the γ secretase complex and therefore the formation of amyloid β (Aβ) [97, 98] affects the processing of PRiMA, as was shown by the use of using hamster ovarian cells transfected with AChET and a PS1 conditional knockout mouse. They showed that γ secretase inhibition led to an increase in both PRiMA and AChET and that G4 AChE was increased in the membrane rafts in the PFC of PS1 KO mice, representing a potential mechanism by which AD pathology imparts alterations to AChE function [99]. Furthermore, PS1 transgenic mice show increased AChE activity throughout the entirety of the basal forebrain, prior to any behavioral deficits, suggesting that AChE dysfunction may be one of the first steps in the cascade of pathology associated with AD [100]. This increase in AChE activity is typically the therapeutic target of cholinesterase-inhibiting drugs such as donepezil, one of the most widely used drugs for the treatment of AD.

Romberg et al. [101] tested 3xTgAD mice, a strain with APPswe, PS1M146V, and tauP301L mutations to recapitulate some of the major features of AD in humans, on a 5-choice serial reaction time task (5-CSRTT) and found that while the transgenic mice were able to match wild-type controls in performance early on in the task, they would later begin to show impairments across the duration of the task, specifically as stimulus duration was reduced, signifying deficits in sustaining attention. These deficits were rescued *via* the administration of donepezil, demonstrating that inhibiting the alterations to typical AChE activity was sufficient to restore similar functioning to wild-type mice, suggesting that this particular task is reliant on AChE to modulate cortical cholinergic activity [101]. It is likely that AD, through its upregulation of AChE activity, perturbs the frontocortical circuitry necessary for attention by disrupting phasic ACh signaling. Similarly, donepezil was able to reduce scopolamineinduced omissions during the 5-CSRTT, demonstrating that inhibiting AChE can compensate for muscarinic receptor antagonism during this task [102].

Nicotinic receptors have been heavily implicated in the pathogenesis of AD, which likely disrupts nicotinic-dependent signaling in the cortex, and as discussed above has been linked to phasic signaling. This dysfunction likely originates with the SK family of Ca2+ sensitive K+ channels, as treatment with a selective agonist of these channels improves the nAChR function [103]. Dysfunction with such circuitry is evident by the fact that impairments in sustained attention have been shown using a mouse model of AD [104], demonstrating likely deficits with phasic cholinergic deficits, which, as mentioned before, is at least partially dependent on the utilization of nAChRs in the prefrontal cortex [42, 48]. Additionally, there is some evidence to suggest a link between Aβ pathology and nicotinic dysfunction, as it has been shown that Aβ prevents nicotine-induced inhibitory signaling, but not excitatory signaling, in PFC pyramidal neurons *in vitro* [105], suggesting that Alzheimer's disease pathology may be disrupting the balance of excitation and inhibition necessary for phasic ACh signaling in the PFC. The link between Aβ and nAChR function is further supported by data showing that infusion of Aβ increased α-bungarotoxin autoradiography binding to the α7 nAChR in the frontal cortex exclusively in animals that received weekly attentional stimulation, suggesting that α7 nAChR functionality may be impaired by Aβ, but regular activation of attentional circuitry can activate compensational mechanisms in both the cortex to attempt to restore regular functioning [106].

Similarly, within the cortex itself, there are deficits to muscarinic receptors as well, as M1 mAChR's are being considered as a potential target for treatment [107]. However, work using radioligand labeling of human participants with AD has demonstrated no changes in M1 labeling in the cortex in AD, only in the dentate gyrus [108]. It is possible that despite no changes in M1 expression in the cortex, there are still deficits to its typical function, as postmortem analysis of the brains of individuals with AD showed reduced G-protein coupling of the M1 receptor in the cortex, despite no change in its density [109], mirroring the radioligand results from Scarr et al. [108]. It is possible that impaired function of the M1 receptor in AD serves as an indicator of dysfunctional tonic ACh signaling in the cortex, but as mentioned previously, the relationship between muscarinic receptors and tonic signaling is not one-to-one, and there is likely some contribution of nicotinic receptors to tonic signaling dependent behaviors as well.

#### **5.2 Alcohol use disorder and cholinergic dysfunction**

The cholinergic pathology in AUD is similar to that of AD, with some overlap in the effects of binge ethanol exposure during adolescence and age-related cognitive decline [110]. It is therefore likely that alcohol-related damage to the basal forebrain

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

leads to dysfunction of tonic and phasic ACh signaling as well. Acute ethanol exposure in rats has been shown to lead to deficits in sustained attention, demonstrating that phasic ACh signaling in the cortex is likely dysregulated during intoxication [111], though the impairments due to acute ethanol likely have to do with the depressive effect of ethanol throughout the entirety of the brain. However, exposure to binge levels of ethanol in adolescence has been shown to lead to deficits that persist across the lifespan [112–114]. Adolescent intermittent ethanol (AIE) exposure has been shown to lead to reductions in ChAT immunostaining in the NbM in adulthood, an effect exclusive to rats that are exposed to ethanol in adolescence, but not adulthood [115]. This loss of ChAT has been shown to be rescued by galantamine, an AChE inhibitor [66]. Likely, this type of alcohol exposure leads to an upregulation of cholinesterase activity, leading to overactive hydrolysis of ACh that is making it difficult for precisely time-locked phasic ACh signaling to occur, similar to AD.

However, there is some evidence to suggest that overactive AChE is leading to some of these deficits in ways beyond its disruption of phasic ACh signaling. It has been shown that overactive AChE induces apoptosis in both living mice and cell cultures exposed to ethanol [116], suggesting that it is possible that ethanol may overstimulate AChE activity and lead to an apoptotic cascade. However, it is unclear how this relates to the rescue of alcohol-related deficits to cholinergic phenotype, as it has been shown that ChAT cells in the basal forebrain are not dead following exposure to binge levels of ethanol in adolescence, but rather are entering a quiescent state that can be rescued either *via* neurotrophins [65], cholinesterase inhibiting drugs [66], or voluntary wheel running exercise [64]. The relation between the quiescent state these cells take, and the apoptotic mechanism described is yet to be ascertained.

Similarly, AIE has been shown to have effects on tonic ACh signaling in the prefrontal cortex. Adolescent alcohol exposure has been shown to attenuate behaviorally relevant acetylcholine efflux in the PFC during a spontaneous alternation task, which was accompanied by reductions in ChAT in the NbM and the medial septum/diagonal band (MS/DB), suggesting that AIE disrupts innervation of the PFC by the basal forebrain, leading to a reduction in cholinergic tone [117]. Likely, ACh tone is needed during this task to induce a state of general arousal in which the animal is actively attenuating to extra-maze cues to determine the arms of the maze it has visited already and to avoid visiting the same arms consecutively [13]. Fernandez and Savage [112] also demonstrated parallel behavioral impairments, as rats exposed to AIE showed deficits on operant attention set-shifting task, a task that has been shown to be dependent on the mPFC [118]. The exact role of phasic ACh signaling during this task is yet to be investigated, but it is possible that the detection of a visual cue to indicate which of the two levers indicates reward is dependent on a similar mechanism to cue detection in the sustained attention task, and the fact that AIE rats are impaired on the shift from spatial side reinforcement to a visual cue determining reinforcement suggests that these two tasks may be dependent on similar mechanisms. However, more work is needed to determine whether phasic acetylcholine signaling is required for attention.

Other models of alcohol-related brain damage have shown similar effects on the cholinergic system and the prefrontal cortex. For example, adult rats either fed a pyrithiamine deficient diet (PTD), given access to ethanol in their drinking bottles (CET), or a combination of both (PTD-CET) were shown to have impaired spontaneous alternation behavior and reduced ACh efflux in the mPFC during this task, which was accompanied by a reduced latency to lever press during a set shift during operant attention set shifting despite no impairment in performance [119]. This suggests that

there are tonic ACh signaling deficits in this model, as demonstrated by the decreased ACh efflux, as well as possible phasic signaling deficits, as the increased amount of time needed to make a lever press in PTD, CET, and PTD-CET animals suggests that the time course of ACh signaling in PFC is being disrupted. The deficits seen during spontaneous alternation during PTD have been shown to be rescued by the AChE inhibitor tacrine [120], suggesting a role of AChE overexpression in the pathology seen in this disorder, suggesting that overactive AChE, mirroring what is seen in AD, is responsible for the tonic ACh deficits. Presumably, these deficits would extend to phasic ACh-dependent processes, but they were not tested here. The fact that cholinesterase inhibition has such an effect on tonic signaling is interesting, but not surprising, as while the mechanism proposed within this review suggests that AChE is more important for phasic signaling, it is nevertheless required to regulate ACh tone as well, exerting its actions both directly by hydrolyzing extracellular ACh and indirectly by regulating phasic signaling and synaptic spillover.

Alcohol-related brain damage and AD seem to converge on the cholinergic system, and the projections from the basal forebrain to the prefrontal cortex seem to be a set of circuits that show particular vulnerability to perturbations. Dysfunction of these circuits likely has effects on both tonic and phasic ACh signaling simultaneously, and it seems that, at the moment, it would be conceptually difficult to investigate an experimental manipulation that would affect one type of signaling but not the other. However, it may be possible to determine whether the time course of the impairments seen in these two types of signaling differ, such that perhaps phasic signaling is first affected by the early cholinergic deficits seen in AD, which later expands to tonic signaling deficits later on. This remains to be determined and represents a future direction for research into modes of ACh signaling.
