**4. The role of acetylcholinesterase in tonic and phasic ACh signaling**

One of the ways in which tonic and phasic ACh signaling are regulated is through the actions of AChE, the catalytic enzyme which hydrolyzes ACh and is thought to be the main determinant of the duration of cholinergic signaling. As stated before, AChE can hydrolyze ACh nearly as fast as the cell can release it, and thus the high catalytic activity of this enzyme is evidence itself of the presence of phasic ACh signaling in the prefrontal cortex. However, it has also been hypothesized that this is one of the reasons as to why tonic ACh signaling is unlikely to directly contribute to behavior at all. Sarter and Lustig [4] state that the fact that AChE is such an efficient hydrolyzer of ACh makes it unlikely for synaptic spillover to occur, and therefore tonic ACh as measured by *in vivo* microdialysis may be a methodological artifact due to the cholinesterase inhibitors perfused into the brain during the process. However, it has been shown that inhibition of AChE by an excess of ACh may occur *via* the formation of a ternary complex [25], which represents one mechanism by which high-ACh concentration can overwhelm AChE and spill over into the extracellular fluid. Therefore, a

central role of potentially heterogeneous utilization of AChE during tonic and phasic ACh signaling distinction must be considered.

In the brain, AChE is exclusively expressed as the tailed AChET variant. This subunit is able to form an amphiphilic tetramer known as G4 AChE, which is facilitated by and tethered to proline-rich membrane anchor (PRiMA), anchoring AChE to the presynaptic membrane [56, 57]. Acetylcholinesterase is co-expressed with markers of cholinergic neurons such as choline acetyltransferase (ChAT) *in vivo* [57], suggesting that AChE is likely expressed in cholinergic cells before undergoing tetramerization *via* PRiMA in the endoplasmic reticulum and being transported down the axon [58]. PRiMA likely links tailed G4 AChE to the presynaptic membrane *via* membrane rafts [59]. Interestingly, PRiMA shows robust co-expression with M1 muscarinic receptors, which are located postsynaptically, suggesting that AChE may originate primarily from cholinergic axons but also intrinsically from neurons in the cortex [60]. Additionally, this co-expression may have interesting functional implications, however, it may simply be due to the ubiquity of expression of the M1 receptor in basal forebrain target regions. To date, no studies have looked to colocalize PRiMA with nicotinic receptors, making any potential differences in PRiMA receptors between neurons expressing the two receptor types is unknown but represents a future area of inquiry.

Experimental manipulations to inhibit the endogenous action of AChE have been shown to cause attentional impairments, such as during a five-choice serial reaction time task in healthy rats [61], suggesting that inhibition of AChE in healthy subjects impairs behaviors likely dependent on phasic signaling. AChE knockout mice have been shown to exhibit a variety of motor deficits due to the role of peripheral AChE in muscle contraction and thermoregulation [62]. Farar et al. [63] characterized PRiMA KO mice on a number of motor and behavioral measures. They found that despite only very subtle motor impairments on the rotarod test and the wire task, these mice had a nearly 200–300-fold increase in extracellular ACh concentration in the striatum as measured *via in vivo* microdialysis during anesthesia, with none of the thermoregulatory impairments seen in mice with a traditional AChE knockout. Additionally, these mice were not impaired on the Morris water maze. Interestingly, they found that the M1 muscarinic receptor was heavily downregulated across all areas of the brain measured, including by approximately 40% in the cortex, with no such decrease in the α7 nAChR or β2-containing nAChRs. One interpretation of these results laid out by the authors is that these data serve as evidence for the hypothesis that AChE is primarily involved in regulating the extracellular ACh concentration, not terminating synaptic transmission [63].

Should that be the case, then it is possible that PRiMA knockout mice may have deficits in phasic ACh signaling, but not tonic signaling. Thus, the hypothesis may be the inverse of what was proposed by these authors, that AChE is primarily utilized at the cholinergic synapses of the prefrontal cortex to rapidly terminate ACh signaling, while ACh "tone" represents ACh that escapes this mechanism and is, therefore, less active during this form of signaling. This may represent a potential mechanism by which attentional and other cognitive impairments occur in disease states that alter AChE and disrupt the balance between ACh release and hydrolysis, such as Alzheimer's disease, while leaving other functions that are dependent on tonic signaling unimpaired until later in the disease.

A likely mechanism for which AChE is inhibited at the synapse, as represented in **Figure 1** is as such: an overabundance of ACh in the synaptic cleft during phasic signaling may lead to inhibition of AChE which allows acetylcholine to spill out of

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

#### **Figure 1.**

*Proposed mechanism of the regulation of phasic and tonic acetylcholine (ACh) signaling in the prefrontal cortex. A. Phasic acetylcholine signaling by the terminals of early firing basal forebrain neurons at cholinergic synapses leads to the activation of both postsynaptic nicotinic and muscarinic receptors. The termination of cholinergic signaling is mediated by presynaptic membrane-bound acetylcholinesterase (AChE), which hydrolyzes ACh. B. High levels of phasic firing, such as during high attentional demand, lead to an abundance of ACh at the synapse, inhibiting AChE activity and further increasing the concentration of ACh. C. Synaptic spillover of ACh into the extracellular fluid occurs due to transient inhibition of AChE. D. There is also the release of ACh from the terminals of late-firing basal forebrain cholinergic neurons, which further increases ACh tone. Extracellular ACh is still hydrolyzed by membrane-bound AChE. E. Extracellular ACh travels to distal muscarinic and nicotinic receptors within the prefrontal cortex, modulating global arousal states. \*created with BioRender.com*

the synapse and into the extracellular fluid. This ambient ACh would still be under the regulation of AChE, but the enzyme's location on the synaptic membrane would make it more difficult for extracellular acetylcholine to be hydrolyzed, allowing it to accumulate in the extracellular space. Therefore, AChE still regulates ACh tone, albeit mostly indirectly, through its regulation of phasic ACh signaling. Tonic ACh may also be released by non-synaptic terminals, perhaps by specialized basal forebrain cholinergic cells [27, 28], and it is the combination of these two mechanisms that are responsible for ambient ACh fluctuations.
