Disrupted Choline Clearance and Sustained Acetylcholine Release In Vivo by a Common Choline Transporter Coding Variant Associated with Poor Attentional Control in Humans.

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild-type (WT) mice, CHT-mediated clearance of choline in male and female mice expressing one or two Val89 alleles was reduced by over 80% in cortex and over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.SIGNIFICANCE STATEMENT Acetylcholine (ACh) signaling depends on the functional capacity of the neuronal choline transporter (CHT). Previous research demonstrated that humans expressing the common CHT coding variant Val89 exhibit attentional vulnerabilities and attenuated fronto-cortical activation during attention. Here, we find that mice engineered to express the Val89 variant exhibit reduced CHT-mediated choline clearance and a diminished capacity to sustain ACh release. Additionally, Val89 mice lack cognitive flexibility in response to an attentional challenge. These findings provide a mechanistic and cognitive framework for interpreting the attentional phenotype associated with the human Val89 variant and establish a model that permits a more invasive interrogation of CNS effects as well as the development of therapeutic strategies for those, including Val89 carriers, with presynaptic cholinergic perturbations.

α4ß2* Nicotinic Cholinergic Receptor Target Engagement in Parkinson Disease Gait-Balance Disorders.

OBJECTIVE: Attentional deficits following degeneration of brain cholinergic systems contribute to gait-balance deficits in Parkinson disease (PD). As a step toward assessing whether α4ß2* nicotinic acetylcholine receptor (nAChR) stimulation improves gait-balance function, we assessed target engagement of the α4ß2* nAChR partial agonist varenicline. METHODS: Nondemented PD participants with cholinergic deficits were identified with [18 F]fluoroethoxybenzovesamicol positron emission tomography (PET). α4ß2* nAChR occupancy after subacute oral varenicline treatment was measured with [18 F]flubatine PET. With a dose selected from the nAChR occupancy experiment, varenicline effects on gait, balance, and cognition were assessed in a double-masked placebo-controlled crossover study. Primary endpoints were normal pace gait speed and a measure of postural stability. RESULTS: Varenicline doses (0.25mg per day, 0.25mg twice daily [b.i.d.], 0.5mg b.i.d., and 1.0mg b.i.d.) produced 60 to 70% receptor occupancy. We selected 0.5mg orally b.i.d for the crossover study. Thirty-three participants completed the crossover study with excellent tolerability. Varenicline had no significant impact on the postural stability measure and caused slower normal pace gait speed. Varenicline narrowed the difference in normal pace gait speed between dual task and no dual task gait conditions, reduced dual task cost, and improved sustained attention test performance. We obtained identical conclusions in 28 participants with treatment compliance confirmed by plasma varenicline measurements. INTERPRETATION: Varenicline occupied α4ß2* nicotinic acetylcholine receptors, was tolerated well, enhanced attention, and altered gait performance. These results are consistent with target engagement. α4ß2* agonists may be worth further evaluation for mitigation of gait and balance disorders in PD. ANN NEUROL 2021;90:130-142.

Theta-gamma coupling emerges from spatially heterogeneous cholinergic neuromodulation.

Theta and gamma rhythms and their cross-frequency coupling play critical roles in perception, attention, learning, and memory. Available data suggest that forebrain acetylcholine (ACh) signaling promotes theta-gamma coupling, although the mechanism has not been identified. Recent evidence suggests that cholinergic signaling is both temporally and spatially constrained, in contrast to the traditional notion of slow, spatially homogeneous, and diffuse neuromodulation. Here, we find that spatially constrained cholinergic stimulation can generate theta-modulated gamma rhythms. Using biophysically-based excitatory-inhibitory (E-I) neural network models, we simulate the effects of ACh on neural excitability by varying the conductance of a muscarinic receptor-regulated K+ current. In E-I networks with local excitatory connectivity and global inhibitory connectivity, we demonstrate that theta-gamma-coupled firing patterns emerge in ACh modulated network regions. Stable gamma-modulated firing arises within regions with high ACh signaling, while theta or mixed theta-gamma activity occurs at the peripheries of these regions. High gamma activity also alternates between different high-ACh regions, at theta frequency. Our results are the first to indicate a causal role for spatially heterogenous ACh signaling in the emergence of localized theta-gamma rhythmicity. Our findings also provide novel insights into mechanisms by which ACh signaling supports the brain region-specific attentional processing of sensory information.

Forebrain Cholinergic Signaling: Wired and Phasic, Not Tonic, and Causing Behavior.

Conceptualizations of cholinergic signaling as primarily spatially diffuse and slow-acting are based largely on measures of extracellular brain ACh levels that require several minutes to generate a single data point. In addition, most such studies inhibited the highly potent catalytic enzyme for ACh, AChE, to facilitate measurement of ACh. Absent such inhibition, AChE limits the presence of ambient ACh and thus renders it unlikely that ACh influences target regions via slow changes in extracellular ACh concentrations. We describe an alternative view by which forebrain signaling in cortex driving cognition is largely phasic (milliseconds to perhaps seconds), and unlikely to be volume-transmitted. This alternative is supported by new evidence from real-time amperometric recordings of cholinergic signaling indicating a specific function of rapid, phasic, transient cholinergic signaling in attentional contexts. Previous neurochemical evidence may be reinterpreted in terms of integrated phasic cholinergic activity that mediates specific behavioral and cognitive operations; this reinterpretation fits well with recent computational models. Optogenetic studies support a causal relationship between cholinergic transients and behavior. This occurs in part via transient-evoked muscarinic receptor-mediated high-frequency oscillations in cortical regions. Such oscillations outlast cholinergic transients and thus link transient ACh signaling with more sustained postsynaptic activity patterns to support relatively persistent attentional biases. Reconceptualizing cholinergic function as spatially specific, phasic, and modulating specific cognitive operations is theoretically powerful and may lead to pharmacologic treatments more effective than those based on traditional views.Dual Perspectives Companion Paper: Diverse Spatiotemporal Scales of Cholinergic Signaling in the Neocortex, by Anita A. Disney and Michael J. Higley.

Complex Movement Control in a Rat Model of Parkinsonian Falls: Bidirectional Control by Striatal Cholinergic Interneurons.

Older persons and, more severely, persons with Parkinson's disease (PD) exhibit gait dysfunction, postural instability and a propensity for falls. These dopamine (DA) replacement-resistant symptoms are associated with losses of basal forebrain and striatal cholinergic neurons, suggesting that falls reflect disruption of the corticostriatal transfer of movement-related cues and their striatal integration with movement sequencing. To advance a rodent model of the complex movement deficits of Parkinsonian fallers, here we first demonstrated that male and female rats with dual cortical cholinergic and striatal DA losses (DL rats) exhibit cued turning deficits, modeling the turning deficits seen in these patients. As striatal cholinergic interneurons (ChIs) are positioned to integrate movement cues with gait, and as ChI loss has been associated with falls in PD, we next used this task, as well as a previously established task used to reveal heightened fall rates in DL rats, to broadly test the role of ChIs. Chemogenetic inhibition of ChIs in otherwise intact male and female rats caused cued turning deficits and elevated fall rates. Spontaneous turning was unaffected. Furthermore, chemogenetic stimulation of ChIs in DL rats reduced fall rates and restored cued turning performance. Stimulation of ChIs was relatively more effective in rats with viral transfection spaces situated lateral to the DA depletion areas in the dorsomedial striatum. These results indicate that striatal ChIs are essential for the control of complex movements, and they suggest a therapeutic potential of stimulation of ChIs to restore gait and balance, and to prevent falls in PD.SIGNIFICANCE STATEMENT In persons with Parkinson's disease, gait dysfunction and the associated risk for falls do not benefit from dopamine replacement therapy and often result in long-term hospitalization and nursing home placement. Here, we first validated a new task to demonstrate impairments in cued turning behavior in rodents modeling the cholinergic-dopaminergic losses observed in Parkinsonian fallers. We then demonstrated the essential role of striatal cholinergic interneurons for turning behavior as well as for traversing dynamic surfaces and avoiding falls. Stimulation of these interneurons in the rat model rescued turning performance and reduced fall rates. Our findings indicate the feasibility of investigating the neuronal circuitry underling complex movement control in rodents, and that striatal cholinergic interneurons are an essential node of such circuitry.

Make a Left Turn: Cortico-Striatal Circuitry Mediating the Attentional Control of Complex Movements.

BACKGROUND: In movement disorders such as Parkinson's disease (PD), cholinergic signaling is disrupted by the loss of basal forebrain cholinergic neurons, as well as aberrant activity in striatal cholinergic interneurons (ChIs). Several lines of evidence suggest that gait imbalance, a key disabling symptom of PD, may be driven by alterations in high-level frontal cortical and cortico-striatal processing more typically associated with cognitive dysfunction. METHODS: Here we describe the corticostriatal circuitry that mediates the cognitive-motor interactions underlying such complex movement control. The ability to navigate dynamic, obstacle-rich environments requires the continuous integration of information about the environment with movement selection and sequencing. The cortical-attentional processing of extero- and interoceptive cues requires modulation by cholinergic activity to guide striatal movement control. Cue-derived information is "transferred" to striatal circuitry primarily via fronto-striatal glutamatergic projections. RESULT: Evidence from parkinsonian fallers and from a rodent model reproducing the dual cholinergic-dopaminergic losses observed in these patients supports the main hypotheses derived from this neuronal circuitry-guided conceptualization of parkinsonian falls. Furthermore, in the striatum, ChIs constitute a particularly critical node for the integration of cortical with midbrain dopaminergic afferents and thus for cues to control movements. CONCLUSION: Procholinergic treatments that enhance or rescue cortical and striatal mechanisms may improve complex movement control in parkinsonian fallers and perhaps also in older persons suffering from gait disorders and a propensity for falls. © 2021 International Parkinson and Movement Disorder Society.

Phasic cholinergic signaling promotes emergence of local gamma rhythms in excitatory-inhibitory networks.

Recent experimental results have shown that the detection of cues in behavioral attention tasks relies on transient increases of acetylcholine (ACh) release in frontal cortex and cholinergically driven oscillatory activity in the gamma frequency band (Howe et al. Journal of Neuroscience, 2017, 37, 3215). The cue-induced gamma rhythmic activity requires stimulation of M1 muscarinic receptors. Using biophysical computational modeling, we show that a network of excitatory (E) and inhibitory (I) neurons that initially displays asynchronous firing can generate transient gamma oscillatory activity in response to simulated brief pulses of ACh. ACh effects are simulated as transient modulation of the conductance of an M-type K+ current which is blocked by activation of muscarinic receptors and has significant effects on neuronal excitability. The ACh-induced effects on the M current conductance, gKs , change network dynamics to promote the emergence of network gamma rhythmicity through a Pyramidal-Interneuronal Network Gamma mechanism. Depending on connectivity strengths between and among E and I cells, gamma activity decays with the simulated gKs transient modulation or is sustained in the network after the gKs transient has completely dissipated. We investigated the sensitivity of the emergent gamma activity to synaptic strengths, external noise and simulated levels of gKs modulation. To address recent experimental findings that cholinergic signaling is likely spatially focused and dynamic, we show that localized gKs modulation can induce transient changes of cellular excitability in local subnetworks, subsequently causing population-specific gamma oscillations. These results highlight dynamical mechanisms underlying localization of ACh-driven responses and suggest that spatially localized, cholinergically induced gamma may contribute to selectivity in the processing of competing external stimuli, as occurs in attentional tasks.

The cortical cholinergic system contributes to the top-down control of distraction: Evidence from patients with Parkinson's disease.

Past animal and human studies robustly report that the cholinergic system plays an essential role in both top-down and bottom-up attentional control, as well as other aspects of cognition (see Ballinger et al., 2016 for a recent review). However, current understanding of how two major cholinergic pathways in the human brain (the basal forebrain-cortical pathway, and the brainstem pedunculopontine-thalamic pathway) contribute to specific cognitive functions remains somewhat limited. To address this issue, we examine how individual variation in the integrity of striatal-dopaminergic, thalamic-cholinergic, and cortical-cholinergic pathways (measured using Positron Emission Tomography in patients with Parkinson's disease) was associated with individual variation in the initial goal-directed focus of attention, the ability to sustain attentional performance over time, and the ability to avoid distraction from a highly-salient, but irrelevant, environmental stimulus. Compared to healthy controls, PD patients performed similarly in the precision of attention-dependent judgments of duration, and in sustaining attention over time. However, PD patients' performance was strikingly more impaired by the distractor. More critically, regression analyses indicated that only cortical-cholinergic integrity, not thalamic-cholinergic or striatal-dopaminergic integrity, made a specific contribution to the ability to resist distraction after controlling for the other variables. These results demonstrate that the basal forebrain cortical cholinergic system serves a specific role in executing top-down control to resist external distraction.

Cortical cholinergic signaling controls the detection of cues.

The cortical cholinergic input system has been described as a neuromodulator system that influences broadly defined behavioral and brain states. The discovery of phasic, trial-based increases in extracellular choline (transients), resulting from the hydrolysis of newly released acetylcholine (ACh), in the cortex of animals reporting the presence of cues suggests that ACh may have a more specialized role in cognitive processes. Here we expressed channelrhodopsin or halorhodopsin in basal forebrain cholinergic neurons of mice with optic fibers directed into this region and prefrontal cortex. Cholinergic transients, evoked in accordance with photostimulation parameters determined in vivo, were generated in mice performing a task necessitating the reporting of cue and noncue events. Generating cholinergic transients in conjunction with cues enhanced cue detection rates. Moreover, generating transients in noncued trials, where cholinergic transients normally are not observed, increased the number of invalid claims for cues. Enhancing hits and generating false alarms both scaled with stimulation intensity. Suppression of endogenous cholinergic activity during cued trials reduced hit rates. Cholinergic transients may be essential for synchronizing cortical neuronal output driven by salient cues and executing cue-guided responses.

The hot 'n' cold of cue-induced drug relapse.

Environmental cues associated with rewards can acquire motivational properties. However, there is considerable variation in the extent to which a reward cue gains motivational control over behavior, depending on the individual and the form of the cue. When a discrete cue is paired with food reward, it acquires greater control over motivated behavior in some rats (sign-trackers, STs) than others (goal-trackers, GTs) as indicated by the propensity to approach the cue, the willingness to work to obtain it, and its ability to reinstate reward-seeking behavior. Here, we review studies that employ this ST/GT animal model to investigate characteristics of individuals that are especially susceptible to reward cue-elicited behavior and the involvement of dopamine and acetylcholine neuromodulator systems in the susceptibility to cue-induced drug relapse. First, we discuss individual differences in the attribution of incentive salience to different forms of reward cues and the involvement of the mesolimbic dopamine system. We then discuss individual differences in cognitive/attentional control and the contributions of the cholinergic system in processing reward cues. It is suggested that in STs a propensity to attribute motivational properties to a drug cue is combined with poor attentional control in the face of these cues, making them particularly vulnerable to transition from casual/experimental patterns of drug use to addiction and to cue-induced relapse.

Diverse Roads to Relapse: A Discriminative Cue Signaling Cocaine Availability Is More Effective in Renewing Cocaine Seeking in Goal Trackers Than Sign Trackers and Depends on Basal Forebrain Cholinergic Activity.

Stimuli associated with taking drugs are notorious instigators of relapse. There is, however, considerable variation in the motivational properties of such stimuli, both as a function of the individual and the nature of the stimulus. The behavior of some individuals (sign trackers, STs) is especially influenced by cues paired with reward delivery, perhaps because they are prone to process information via dopamine-dependent, cue-driven, incentive salience systems. Other individuals (goal trackers, GTs) are better able to incorporate higher-order contextual information, perhaps because of better executive/attentional control over behavior, which requires frontal cortical cholinergic activity. We hypothesized, therefore, that a cue that "sets the occasion" for drug taking (a discriminative stimulus, DS) would reinstate cocaine seeking more readily in GTs than STs and that this would require intact cholinergic neurotransmission. To test this, male STs and GTs were trained to self-administer cocaine using an intermittent access schedule with periods of cocaine availability and unavailability signaled by a DS+ and a DS-, respectively. Thereafter, half of the rats received an immunotoxic lesion that destroyed 40-50% of basal forebrain cholinergic neurons and later, after extinction training, were tested for the ability of noncontingent presentations of the DS+ to reinstate cocaine seeking behavior. The DS+ was much more effective in reinstating cocaine seeking in GTs than STs and this effect was abolished by cholinergic losses despite the fact that all rats continued to orient to the DS+ We conclude that vulnerability to relapse involves interactions between individual cognitive-motivational biases and the form of the drug cue encountered.SIGNIFICANCE STATEMENT The most predictable outcome of a diagnosis of addiction is a high chance for relapse. When addicts encounter cues previously associated with drug, their attention may be unduly attracted to such cues and these cues can evoke motivational states that instigate and maintain drug-seeking behavior. Although sign-tracking rats were previously demonstrated to exhibit greater relapse vulnerability to Pavlovian drug cues paired with drug delivery, here, we demonstrate that their counterparts, the goal trackers, are more vulnerable if the drug cue acts to signal drug availability and that the forebrain cholinergic system mediates such vulnerability. Given the importance of contextual cues for triggering relapse and the human cognitive-cholinergic capacity for the processing of such cues, goal trackers model essential aspects of relapse vulnerability.

Acetylcholine Release in Prefrontal Cortex Promotes Gamma Oscillations and Theta-Gamma Coupling during Cue Detection.

The capacity for using external cues to guide behavior ("cue detection") constitutes an essential aspect of attention and goal-directed behavior. The cortical cholinergic input system, via phasic increases in prefrontal acetylcholine release, plays an essential role in attention by mediating such cue detection. However, the relationship between cholinergic signaling during cue detection and neural activity dynamics in prefrontal networks remains unclear. Here we combined subsecond measures of cholinergic signaling, neurophysiological recordings, and cholinergic receptor blockade to delineate the cholinergic contributions to prefrontal oscillations during cue detection in rats. We first confirmed that detected cues evoke phasic acetylcholine release. These cholinergic signals were coincident with increased neuronal synchrony across several frequency bands and the emergence of theta-gamma coupling. Muscarinic and nicotinic cholinergic receptors both contributed specifically to gamma synchrony evoked by detected cues, but the effects of blocking the two receptor subtypes were dissociable. Blocking nicotinic receptors primarily attenuated high-gamma oscillations occurring during the earliest phases of the cue detection process, while muscarinic (M1) receptor activity was preferentially involved in the transition from high to low gamma power that followed and corresponded to the mobilization of networks involved in cue-guided decision making. Detected cues also promoted coupling between gamma and theta oscillations, and both nicotinic and muscarinic receptor activity contributed to this process. These results indicate that acetylcholine release coordinates neural oscillations during the process of cue detection.SIGNIFICANCE STATEMENT The capacity of learned cues to direct attention and guide responding ("cue detection") is a key component of goal-directed behavior. Rhythmic neural activity and increases in acetylcholine release in the prefrontal cortex contribute to this process; however, the relationship between these neuronal mechanisms is not well understood. Using a combination of in vivo neurochemistry, neurophysiology, and pharmacological methods, we demonstrate that cue-evoked acetylcholine release, through distinct actions at both nicotinic and muscarinic receptors, triggers a procession of neural oscillations that map onto the multiple stages of cue detection. Our data offer new insights into cholinergic function by revealing the temporally orchestrated changes in prefrontal network synchrony modulated by acetylcholine release during cue detection.

Unresponsive Choline Transporter as a Trait Neuromarker and a Causal Mediator of Bottom-Up Attentional Biases.

Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction.SIGNIFICANCE STATEMENT The vulnerability for addiction-like behavior has been associated with psychological traits, such as the propensity to attribute incentive salience to reward cues that is modeled in rats by sign-tracking behavior. Sign-trackers tend to approach and contact cues associated with reward, whereas their counterparts, the goal-trackers, have a preference for approaching the location of the reward. Here, we show that the capacity of presynaptic cholinergic synapses to respond to stimulation by elevating presynaptic choline uptake and releasing acetylcholine is attenuated in sign-trackers. Furthermore, pharmacological inhibition of choline transport induced sign-tracking behavior. Our findings suggest that reduced levels of cholinergic neuromodulation can mediate an attentional bias toward reward-related cues, thereby allowing such cues to exert relatively greater control over behavior.

Distinct Frontoparietal Networks Underlying Attentional Effort and Cognitive Control.

We investigated the brain activity patterns associated with stabilizing performance during challenges to attention. Our findings revealed distinct patterns of frontoparietal activity and functional connectivity associated with increased attentional effort versus preserved performance during challenged attention. Participants performed a visual signal detection task with and without presentation of a perceptual-attention challenge (changing background). The challenge condition increased activation in frontoparietal regions including right mid-dorsal/dorsolateral PFC (RPFC), approximating Brodmann's area 9, and superior parietal cortex. We found that greater behavioral impact of the challenge condition was correlated with greater RPFC activation, suggesting that increased engagement of cognitive control regions is not always sufficient to maintain high levels of performance. Functional connectivity between RPFC and ACC increased during the challenge condition and was also associated with performance declines, suggesting that the level of synchronized engagement of these regions reflects individual differences in attentional effort. Pretask, resting-state RPFC-ACC connectivity did not predict subsequent performance, suggesting that RPFC-ACC connectivity increased dynamically during task performance in response to performance decrement and error feedback. In contrast, functional connectivity between RPFC and superior parietal cortex not only during the task but also during pretask rest was associated with preserved performance in the challenge condition. Together, these data suggest that resting frontoparietal connectivity predicts performance on attention tasks that rely on those same cognitive control networks and that, under challenging conditions, other control regions dynamically couple with this network to initiate the engagement of cognitive control.

Thalamic cholinergic innervation makes a specific bottom-up contribution to signal detection: Evidence from Parkinson's disease patients with defined cholinergic losses.

Successful behavior depends on the ability to detect and respond to relevant cues, especially under challenging conditions. This essential component of attention has been hypothesized to be mediated by multiple neuromodulator systems, but the contributions of individual systems (e.g., cholinergic, dopaminergic) have remained unclear. The present study addresses this issue by leveraging individual variation in regionally-specific cholinergic denervation in Parkinson's disease (PD) patients, while controlling for variation in dopaminergic denervation. Patients whose dopaminergic and cholinergic nerve terminal integrity had been previously assessed using Positron Emission Tomography (Bohnen et al., 2012) and controls were tested in a signal detection task that manipulates attentional-perceptual challenge and has been used extensively in both rodents and humans to investigate the cholinergic system's role in responding to such challenges (Demeter et al., 2008; McGaughy and Sarter, 1995; see Hasselmo and Sarter 2011 for review). In simple correlation analyses, measures of midbrain dopaminergic, and both cortical and thalamic cholinergic innervation all predicted preserved signal detection under challenge. However, regression analyses also controlling for age, disease severity, and other variables showed that the only significant independent neurotransmitter-related predictor over and above the other variables in the model was thalamic cholinergic integrity. Furthermore, thalamic cholinergic innervation exclusively predicted hits, not correct rejections, indicating a specific contribution to bottom-up salience processing. These results help define regionally-specific contributions of cholinergic function to different aspects of attention and behavior.

Reducing falls in Parkinson's disease: interactions between donepezil and the 5-HT6 receptor antagonist idalopirdine on falls in a rat model of impaired cognitive control of complex movements.

Falls are a leading cause of death in the elderly and, in a majority of patients with Parkinson's disease (PD), the leading levodopa-insensitive cause of hospitalization and long-term care. Falling in PD has been attributed to degeneration of forebrain cholinergic neurons that, in interaction with striatal dopamine losses, impairs the cognitive control of balance, gait, and movement. We previously established an animal model of these dual cholinergic-dopaminergic losses ("DL rats") and a behavioral test system (Michigan Complex Motor Control Task, MCMCT) to measure falls associated with traversing dynamic surfaces and distractors. Because the combined treatment of the acetylcholinesterase inhibitor donepezil and the 5-HT6 receptor antagonist idalopirdine (Lu AE58054) was reported to exhibit synergistic pro-cholinergic activity in rats and improved cognition in patients with moderate Alzheimer's disease, here we assessed the effects of this treatment on MCMCT performance and attention in DL rats. Compared with the vehicle-treated group, the combined treatment greatly reduced (Cohen's d = 0.96) falls in DL rats when traversing dynamic surfaces and when exposed to a passive distractor. However, falls associated with a dual task distractor and sustained attentional performance did not benefit from this treatment. Analyses of the behavior in fall-prone moments suggested that this treatment improved the efficacy and speed of re-instating forward movement after relatively short stoppages. This treatment may reduce fall propensity in PD patients via maintaining planned movement sequences in working memory and improving the vigor of executing such movements following brief periods of freezing of gait.

'Hot' vs. 'cold' behavioural-cognitive styles: motivational-dopaminergic vs. cognitive-cholinergic processing of a Pavlovian cocaine cue in sign- and goal-tracking rats.

Discrete Pavlovian reward cues acquire more potent incentive motivational properties (incentive salience) in some animals (sign-trackers; STs) compared to others (goal-trackers; GTs). Conversely, GTs appear to be better than STs in processing more complex contextual cues, perhaps reflecting their relatively greater bias for goal-directed cue processing. Here, we investigated the activity of two major prefrontal neuromodulatory input systems, dopamine (DA) and acetylcholine (ACh), in response to a discrete Pavlovian cue that was previously paired with cocaine administration in STs and GTs. Rats underwent Pavlovian training in which light cue presentations were either paired or unpaired with an intravenous cocaine infusion. Following a 10-day abstinence period, prefrontal dialysates were collected in STs and GTs during cue presentations in the absence of cocaine. In STs, the cue previously paired with cocaine significantly increased prefrontal DA levels. DA levels remained elevated over baseline across multiple cue presentation blocks, and DA levels and approaches to the cue were significantly correlated. In STs, ACh levels were unaffected by cue presentations. In contrast, in GTs, presentations of the cocaine cue increased prefrontal ACh, but not DA, levels. GTs oriented towards the cue at rates similar to STs, but they did not approach it and elevated ACh levels did not correlate with conditioned orientation. The results indicate a double dissociation between the role of prefrontal DA and ACh in STs and GTs, and suggest that these phenotypes will be useful for studying the role of neuromodulator systems in mediating opponent behavioural-cognitive styles.

What do phasic cholinergic signals do?

In addition to the neuromodulatory role of cholinergic systems, brief, temporally discrete cholinergic release events, or "transients", have been associated with the detection of cues in attention tasks. Here we review four main findings about cholinergic transients during cognitive processing. Cholinergic transients are: (1) associated with the detection of a cue and influenced by cognitive state; (2) not dependent on reward outcome, although the timing of the transient peak co-varies with the temporal relationship between detection and reward delivery; (3) correlated with the mobilization of the cue-evoked response; (4) causal mediators of shifts from monitoring to cue detection. We next discuss some of the key questions concerning the timing and occurrence of transients within the framework of available evidence including: (1) Why does the shift from monitoring to cue detection require a transient? (2) What determines whether a cholinergic transient will be generated? (3) How can cognitive state influence transient occurrence? (4) Why do cholinergic transients peak at around the time of reward delivery? (5) Is there evidence of cholinergic transients in humans? We conclude by outlining future research studies necessary to more fully understand the role of cholinergic transients in mediating cue detection.

Cholinergic capacity mediates prefrontal engagement during challenges to attention: evidence from imaging genetics.

In rodent studies, elevated cholinergic neurotransmission in right prefrontal cortex (PFC) is essential for maintaining attentional performance, especially in challenging conditions. Apparently paralleling the rises in acetylcholine seen in rodent studies, fMRI studies in humans reveal right PFC activation at or near Brodmann's areas 9 (BA 9) increases in response to elevated attentional demand. In the present study, we leveraged human genetic variability in the cholinergic system to test the hypothesis that the cholinergic system contributes to the BA 9 response to attentional demand. Specifically, we scanned (BOLD fMRI) participants with a polymorphism of the choline transporter gene that is thought to limit choline transport capacity (Ile89Val variant of the choline transporter gene SLC5A7, rs1013940) and matched controls while they completed a task previously used to demonstrate demand-related increases in right PFC cholinergic transmission in rats and right PFC activation in humans. As hypothesized, we found that although controls showed the typical pattern of robust BA 9 responses to increased attentional demand, Ile89Val participants did not. Further, pattern analysis of activation within this region significantly predicted participant genotype. Additional exploratory pattern classification analyses suggested that Ile89Val participants differentially recruited orbitofrontal cortex and parahippocampal gyrus to maintain attentional performance to the level of controls. These results contribute to a growing body of translational research clarifying the role of cholinergic signaling in human attention and functional neural measures, and begin to outline the risk and resiliency factors associated with potentially suboptimal cholinergic function with implications for disorders characterized by cholinergic dysregulation.

The presynaptic choline transporter imposes limits on sustained cortical acetylcholine release and attention.

Functional variation in the gene encoding the presynaptic choline transporter (CHT) has been linked to attention-deficit/hyperactivity disorder. Here, we report that a heterozygous deletion in the CHT gene in mice (CHT(+/-)) limits the capacity of cholinergic neurons to sustain acetylcholine (ACh) release and attentional performance. Cortical microdialysis and amperometric methods revealed that, whereas wild-type and CHT(+/-) animals support equivalent basal ACh release and choline clearance, CHT(+/-) animals exhibit a significant inability to elevate extracellular ACh following basal forebrain stimulation, in parallel with a diminished choline clearance capacity following cessation of stimulation. Consistent with these findings, the density of CHTs in cortical synaptosomal plasma membrane-enriched fractions from unstimulated CHT(+/-) animals matched those observed in wild-type animals despite reductions in CHT levels in total extracts, achieved via a redistribution of CHT from vesicle pools. As a consequence, in CHT(+/-) animals, basal forebrain stimulation was unable to mobilize wild-type quantities of CHT to the plasma membrane. In behavioral studies, CHT(+/-) mice were impaired in performing a sustained attention task known to depend on cortical cholinergic activity. In wild-type mice, but not CHT(+/-) mice, attentional performance increased the density of CHTs in the synaptosomal membrane in the right frontal cortex. Basal CHT levels in vesicle-enriched membranes predicted the degree of CHT mobilization as well as individual variations in performance on the sustained attention task. Our findings demonstrate biochemical and physiological alterations that underlie cognitive impairments associated with genetically imposed reductions in choline uptake capacity.