M1-selective muscarinic allosteric modulation enhances cognitive flexibility and effective salience in nonhuman primates

significance Muscarinic receptors mediate the procognitive effects of acetylcholine, but it has remained unclear whether they differentially affect the cognitive subfunctions of attentional filtering, set shifting, and learning. To clarify the functional specificity of M1 mAChRs, we assessed these diverse functions using a recently developed, highly selective M1 PAM. This M1 PAM caused domain-specific cognitive improvement of flexible learning and extradimensional set shifting, reduced perseverations and enhanced target recognition during learning without altering attentional filtering functions. These domain-specific improvements contrasted to effects of a nonselective acetylcholinesterase inhibitor that primarily enhanced attention and caused dose-limiting adverse side effects. These results demonstrate domain-specific improvements in cognitive flexibility suggesting M1 PAMs are versatile compounds for treating cognitive deficits in schizophrenia and Alzheimer’s disease.

Given the results of the LMEM and the maximal effect size with the first third of blocks in the FRL (Fig. S1A), all analyses for the FRL task used only the first third of blocks to capture the period where VU0453595 had its strongest effect on performance.
Effect sizes were reported as either eta squared values when referring to ANOVA results or Cohen's d when appropriate (i.e. when post-hoc analysis showed a significant effect at a single dose). The Cohen's d was computed by directly comparing vehicle to the significant dose using this formula:

Visual search task
In the first VS block, target detection times across distractor conditions were not different with VU0453595 relative to vehicle control (F(3,944) = 1.67, n.s.; h 2 = .004), with the exception of faster target detection times in the second VS block at the 3 mg/kg dose (experimental condition main effect: F(3,944) = 3.67, p = .01; h 2 = .008; Tukey's, p < .05). With regards to performance, in the VS block at the end of the session there were no significant effects, while in the first VS block there was a significant main effect of compound (F(3,944) = 3.80, p = .01; h 2 = .010) with reduced accuracy at 3 mg/kg dose (Tukey's, p = .04) irrespective of the number of distractors.
Despite the lack of set size effects, the raw target detection times were overall significantly faster with the 1 mg/kg dose in the second block (F(3,708) = 4.67, p = .003; h 2 = .018; Tukey's, p = .02) with more improvement with high target-distractor similarity (cohen's d = -.447) than low target-distractor similarity (cohen's d = -.427) (Fig. 3E). There was also a general reduction in performance during the first block (F(3,708) = 2.84, p = 0.04; h 2 = 0.011) (Fig. 3F). We also tested if there was a speed-accuracy trade-off at any dose of VU0453595 during either the first or second VS block. We found no significant changes in the speed-accuracy relationship between vehicle and any of the administered doses of VU0453595 ( Figure S2).

Characterization of adverse cholinergic side effects
Each subject was observed during their consumption of VU0453595 stirred into a strawberry yogurt and honey vehicle (20 g total) placed in a small paper cup. Of the 4 subjects, 1 placed the entire cup immediately in their mouth while the other 3 consumed all of the paper cup's content before either ripping it and licking it clean or eating parts of the paper cup alongside the yogurt. None of the subjects had any day where they spilled an observable volume of yogurt. A modified Irwin test, which measures cholinergic effects on the autonomic and somatomotor systems, was applied to all subjects twice daily (Table S1). Subjects were observed throughout the experiment through a video monitoring system ( Figure 1A) and formally evaluated for the modified Irwin test once, ~110 minutes after administration (immediately before start of task), and a second time, immediately after subjects finished all behavioral tasks for their session (< 2 hours after the first assessment). Ratings of 0, 1 or 2 were assigned to each item on the test reflecting no change, a slight change, or a significant change respectively. During the first 1 mg/kg dose of VU0453595, 4/4 monkeys experienced slight changes in arousal while 2/4 monkeys experienced slight unrest and 1/4 experienced a slight increase in yawning. No other symptoms were observed beyond the first 1 mg/kg dosing. At the 3 mg/kg dose, 4/4 monkeys experienced slight unrest, 3/4 monkeys experienced slight changes in arousal and a single monkey experienced vasodilation (redness of the face). Most of the symptoms observed at the 3 mg/kg dose also occurred during the first 3 mg/kg dosing event. Only a single monkey had any symptoms post-task completion, once at the 1 mg/kg dose and once at the 3 mg/kg dose, both instances involved a slight change in arousal. No changes were observed at the 0.3 mg/kg dose.

Possible M1 agonism
Although in vitro data suggests little to no agonistic properties of VU0453595 (21), we cannot completely rule out the possibility that the inverted-U shaped responses observed with this compound may be due, in part, to ago-PAM activity at the highest dose tested in vivo. This would suggest that the endogenous signaling at the M1 mAChR supporting cognitive flexibility is sensitive to exogenous intervention. The possible agonism of M1 PAMs such as VU0453595 in vivo will be the subject of future studies.

Possible contributions of M1 potentiation of memory or motivation/effort control
The current study dissociated the relative importance of an M1 PAM (VU0453595) for cognitive flexibility and attentional filtering and contrasted these effects to those of donepezil ( Table 1). The functional dissociation of the effects highlights the importance of a multi-task paradigm for understanding ligand actions on behavior (3,45,52) and supports efforts to develop multi-task batteries covering a wide range of cognitive domains (1, 45,[52][53][54]. While our study tested already multiple markers of cognitive flexibility and attention, it was not yet incorporating tests of domains that M1 modulating compounds might also affect. For example, scopolamine challenges have long suggested that M1 mAChRs in the medial temporal lobe support longer-term memory processes (55)(56)(57), making it possible that M1 mAChR modulation might have positive consequences in this domain.
Motivation and the ability to control effort are other domains that we did not test and which some studies have suggested to be modulated by mAChRs. The task we deployed varied cognitive load which inevitably increases difficulty and the amount of effort subjects needed to exert. Although we did not control for motivational factors explicitly, visual inspection suggested it was not modulated by VU0453595 because the learning improvements were somewhat more pronounced at lower than higher load in the learning task and did not vary with increasing distractor difficulty (target-distractor similarity) in the search task. These findings resonate with the results of a scopolamine challenge study in NHP that found no effects of increasing difficulty in a stimuluslocation association learning task (58). However, when testing for a memory load effect with a visuo-spatial paired associate task, Taffe and colleagues (59) found that scopolamine reduced performance particularly when 3 or 4 stimulus-object associations needed to be learned and retrieved but not when 1 or 2 associations were involved. Such a memory load differs from the cognitive load that we imposed by increasing the number of distracting features in the learning task and from the perceptual load that we varied with increasing target distractor similarity. However, it will be important to identify in future studies which motivation or load dependent processes are modulated specifically by M1 selective mAChR modulation.
(increased delta, theta and alpha power) 1 per dose N/A TAK-071: alone it decreased alpha power @ 3 mg/kg and theta + alpha power @ 1 mg/kg. It lead to partial rescue of alpha power @ 1 mg/kg (delta lowered but n.s.). It also lead to partial rescue of alpha and delta power @ 3 mg/kg (delta lowered but n.s.) Donepezil: alone it increased alpha power @ 0.3 mg/kg. It also lead to partial rescue of delta, theta and alpha power. Xanomeline: partial rescue of delta, theta and alpha power (all trends; n.s.