Chapter 7 General discussion
B.3 S UPPLEMENTAL D ISCUSSION
receptor (Conn et al., 2009; Jones et al., 2012; Dean and Scarr, 2020; Foster et al., 2021). Subtype- specific muscarinic PAMs have no intrinsic activity at their respective receptor subtype, but act to boost normal cholinergic signaling thereby conserving the spatial and temporal endogenous ACh signaling and avoiding overstimulation of peripheral ACh receptors and subsequent adverse side effects (Foster and Conn, 2017; Gould et al., 2018; Rook et al., 2018b). Our study thus provides an important benchmark for the development of new drugs that aim to enhance multiple cognitive domains while minimizing side effects.
B.3.2 Cholinergic receptor expression profiles
Interpreting the cognitive-behavioral effects of donepezil is facilitated by considering the distributions of cholinergic subreceptors. Cholinergic receptors are divided into two broad families of metabotropic muscarinic receptors and ionotropic nicotinic receptors. Nicotinic receptors are made up of subunits with relevant ones being alpha4/beta2-containing nicotinic receptors and alpha7-containing nicotinic receptors. The alpha4/beta2 receptors can be found in the cortex and striatum, while the alpha7 receptors are more abundantly found in the cortex than the striatum while both are found at much higher concentrations in the hippocampus (Breese et al., 1997; Quik et al., 2000; Hillmer et al., 2011). The expression of M1, M2 and M4 muscarinic receptors, muscarinic subtypes most commonly found in the brain, is high within the cortex and striatum among other regions (Levey et al., 1991; Hersch et al., 1994). Despite their comparably high expression in the PFC and striatum, studies suggest that the striatum has a particularly high muscarinic binding potential (Tsukada et al., 2004b) and respond stronger to muscarinic ACh receptor activation compared with the PFC (Thorn et al., 2019). We speculate that these brain area specific neuromodulatory profiles underly the observed dose specific improvements of cognitive flexibility and attentional control of interference.
There is evidence that suggests the M5 receptor subtype is expressed on midbrain dopaminergic neurons and regulates dopamine release (Foster et al., 2014) alongside other cholinergic receptors (Zhang and Sulzer, 2012; Cachope and Cheer, 2014). The role that ACh plays in dopaminergic release is thus a potential confound when using systemic, non-specific
cholinergic agonists and AChE inhibitors such as donepezil and may contribute to the behavioral effects we observe.
The widely accepted use of scopolamine to induce cognitive deficits in monkeys (and rodents) in order to model dementia symptoms may suggest that donepezil’s mechanism of action is muscarinic in nature (see Table B1 and B2) and thus muscarinic (M1 receptors particularly) are a common target of pharmacological intervention for Alzheimer’s disease (Verma et al., 2018).
However, Alzheimer’s disease is associated with a loss of cortical alpha4 and (to a lesser degree) alpha7 nicotinic receptor subunits (Court et al., 2001) and chronic donepezil administration is associated with nicotinic receptor upregulation, which suggests a role for nicotinic modulation beyond the involvement of muscarinic receptor action (Kume et al., 2005). Studies directly comparing donepezil with nicotinic agonists find some overlapping results and some differences (Luine et al., 2002).
As discussed in the main text, different dosing regimes may exert behavioral effects more strongly through nicotinic or muscarinic mechanisms and although previous studies have attempted to dissociate their relative contributions (Mirza and Stolerman, 2000), this should be expanded through further studies utilizing tasks with different cognitive demands. It is for example possible that each task may be represented by a non-parabolic function resulting from the interaction of the various cholinergic receptors and their dynamics (Figure B5). Such an interaction may potentially explain the lack of enhancement observed at our medium (0.1 mg/kg) donepezil dose in our visual search task.
B.3.3 Clinical relevance for aging and aging-related cognitive disorders
With aging and age-related cognitive disorders, several physiological measures are correlated with cognitive decline such as the loss of cholinergic neurons among others (Perry et al., 1978), loss of cholinergic receptors (Court et al., 2001), reduced dendritic density in the PFC (Dumitriu et al., 2010; Arnsten, 2015) and reduced regional cerebral blood flow (Hock et al., 1995). Chronic use of donepezil has been shown to lead to nicotinic receptor upregulation (Kume et al., 2005). Stimulation, within appropriate parameters, of these cholinergic PFC synapses
enhances their efficacy and may protect against age-related loss of these dendrites (Hains et al., 2015). Regional cerebral blood flow is correlated with cognitive deficits in primates (Tsukada et al., 1997, 2000) and has been shown to be rescued in aged but not young monkeys with the use of donepezil (Tsukada et al., 2004b). In general, we believe aging-related changes to the cholinergic system shifts up individuals along their optimal performance curve (as can be visualized in Figure 2.6 or Figure B5) which may require a higher dose of donepezil with increasing age.
Previously, molecular and iontophoretic work has shown that at high doses, certain agonists and compounds may not be beneficial or may even act in a detrimental manner for various cognitive processes (Yang et al., 2013; Vijayraghavan et al., 2018; Galvin et al., 2020b). This is in contrast to studies where often the highest tolerated dose provides the strongest pro-cognitive effects (Cummings et al., 2013). We speculate that the reason deficits are rarely reported in the literature with donepezil is that the dose-limiting side effects prevent the usage of doses far right of the reported inverted-Us where deficits with molecular or iontophoretic methods are observed.
Here we report variability in the optimal dose for different cognitive domains within the same subjects. This suggests that depending on the profile and severity of deficits in different cognitive domains, the optimal dose for addressing different types of deficits may not be the same. Our results suggest that finding the best dose for a patient will benefit from assessing multiple cognitive domains to rule out that beneficial effects of a dose for one domain does not go along with compromised functioning of another domain.
B.3.4 Time-of-testing for drug related changes of task performance
Our finding of a different dose range enhancing flexible learning and visual search is based on behavior in the first third of the experimental session in which the search task always preceded the learning task. One question is therefore whether the conclusions of our study would differ if we had alternated the task order by e.g. randomly presenting part of the FL task in the first 25 min and the visual search task in a ~12 min period after such an initial block of the FL task. However, there are reasons why varying the task order would unlikely change the results and conclusions of our study. First, the FL task learning performance at low dose was facilitated in the first ~25 min of the learning blocks (Figure 2.2C), which suggests that after that time at this low dose the
bioavailability of donepezil was reduced below a level that would enhance the learning task. The pharmacokinetics of donepezil suggest that the bio-available donepezil would be expected to be somewhat higher earlier in time. However, at such higher doses the FL task performance was not improved at any period tested. It is therefore unlikely that the low dose would have provided different results when tested at an earlier time after drug administration at which donepezil concentrations would be expected to be higher. Consistent with this conclusion, the learning performance in the FL-task was stable at the medium and high dose and with the vehicle over the whole session across all three time periods (Figure 2.2C). If the pharmacokinetics would have caused rapid changes at these doses we would have expected an improvement at later time periods of the task at which the administered high dose might have started to wane off. However, we did not see a flexible learning improvement with the medium or high donepezil dose over the ~65 min of FL learning performance. In contrast to the FL task the performance of the visual search was improved at a high dose in the first ~12 min of the session, but less at the low dose. If the search block would be performed around 25 min later (ie.g. after 25 min. of FL performance) the donepezil bioavailability would be expected to be lower. At such lower concentration we would thus expect a somewhat lower visual search improvement. However, we would not have expected an absence of an attentional effect.
In summary, the provided reasoning suggests that our results and conclusions are expected to be similar when we would have alternated the task order, showing task-dependent pro-cognitive donepezil concentrations with a low dose favoring flexible learning improvement and a higher dose improving visual search.