Traumatic Brain Injury Induces Microglial and Caspase3 Activation in the Retina.

Traumatic brain injury (TBI) is among the main causes of sudden death after head trauma. These injuries can result in severe degeneration and neuronal cell death in the CNS, including the retina, which is a crucial part of the brain responsible for perceiving and transmitting visual information. The long-term effects of mild-repetitive TBI (rmTBI) are far less studied thus far, even though damage induced by repetitive injuries occurring in the brain is more common, especially amongst athletes. rmTBI can also have a detrimental effect on the retina and the pathophysiology of these injuries is likely to differ from severe TBI (sTBI) retinal injury. Here, we show how rmTBI and sTBI can differentially affect the retina. Our results indicate an increase in the number of activated microglial cells and Caspase3-positive cells in the retina in both traumatic models, suggesting a rise in the level of inflammation and cell death after TBI. The pattern of microglial activation appears distributed and widespread but differs amongst the various retinal layers. sTBI induced microglial activation in both the superficial and deep retinal layers. In contrast to sTBI, no significant change occurred following the repetitive mild injury in the superficial layer, only the deep layer (spanning from the inner nuclear layer to the outer plexiform layer) shows microglial activation. This difference suggests that alternate response mechanisms play a role in the case of the different TBI incidents. The Caspase3 activation pattern showed a uniform increase in both the superficial and deep layers of the retina. This suggests a different action in the course of the disease in sTBI and rmTBI models and points to the need for new diagnostic procedures. Our present results suggest that the retina might serve as such a model of head injuries since the retinal tissue reacts to both forms of TBI and is the most accessible part of the human brain.

Converging Evidence on D-Amino Acid Oxidase-Dependent Enhancement of Hippocampal Firing Activity and Passive Avoidance Learning in Rats.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptor activation requires the binding of a co-agonist on the glycine-binding site. D-serine is the main endogenous co-agonist of NMDA receptors, and its availability significantly depends on the activity of the metabolic enzyme D-amino acid oxidase (DAAO). Inhibition of DAAO increases the brain levels of D-serine and modulates a variety of physiological functions, including cognitive behavior. METHODS: Here, we examined the effects of a novel 4-hydroxypyridazin-3(2H)-one derivative DAAO inhibitor, Compound 30 (CPD30), on passive avoidance learning and on neuronal firing activity in rats. RESULTS: D-serine administration was applied as reference, which increased cognitive performance and enhanced hippocampal firing activity and responsiveness to NMDA after both local and systemic application. Similarly to D-serine, CPD30 (0.1 mg/kg) effectively reversed MK-801-induced memory impairment in the passive avoidance test. Furthermore, local iontophoretic application of CPD30 in the vicinity of hippocampal pyramidal neurons significantly increased firing rate and enhanced their responses to locally applied NMDA. CPD30 also enhanced hippocampal firing activity after systemic administration. In 0.1- to 1.0-mg/kg doses, CPD30 increased spontaneous and NMDA-evoked firing activity of the neurons. Effects of CPD30 on NMDA responsiveness emerged faster (at 10 minutes post-injection) when a 1.0-mg/kg dose was applied compared with the onset of the effects of 0.1 mg/kg CPD30 (at 30 minutes post-injection). CONCLUSIONS: The present results confirm that the inhibition of DAAO enzyme is an effective strategy for cognitive enhancement. Our findings further facilitate the understanding of the cellular mechanisms underlying the behavioral effects of DAAO inhibition in the mammalian brain.

Dissociating cholinergic influence on alertness and temporal attention in primates in a simple reaction time paradigm.

The ability to promptly respond to behaviourally relevant events depends on both general alertness and phasic changes in attentional state driven by temporal expectations. Using a variable foreperiod simple reaction time (RT) task in four adult male rhesus macaques, we investigated the role of the cholinergic system in alertness and temporal expectation. Foreperiod effects on RT reflect temporal expectation, while alertness is quantified as overall response speed. We measured these RT parameters under vehicle treatment and systemic administration of the muscarinic receptor antagonist scopolamine. We also investigated whether and to what extent the effects of scopolamine were reversed by donepezil, a cholinesterase inhibitor widely used for the treatment of dementia. In the control condition, RT showed a continuous decrease as the foreperiod duration increased, which clearly indicated the effect of temporal expectation on RT. This foreperiod effect was mainly detectable on the faster tail of the RT distribution and was eliminated by scopolamine. Furthermore, scopolamine treatment slowed down the average RT. Donepezil treatment was efficient on the slower tail of the RT distribution and improved scopolamine-induced impairments only on the average RT reflecting a general beneficial effect on alertness without any improvement in temporal expectation. The present results highlight the role of the cholinergic system in temporal expectation and alertness in primates and help delineate the efficacy and scope of donepezil and other cholinomimetic agents as cognitive enhancers in present and future clinical practice.

Single Mild Traumatic Brain Injury Induces Persistent Disruption of the Blood-Brain Barrier, Neuroinflammation and Cognitive Decline in Hypertensive Rats.

Traumatic brain injury (TBI) induces blood-brain barrier (BBB) disruption, which contributes to secondary injury of brain tissue and development of chronic cognitive decline. However, single mild (m)TBI, the most frequent form of brain trauma disrupts the BBB only transiently. We hypothesized, that co-morbid conditions exacerbate persistent BBB disruption after mTBI leading to long term cognitive dysfunction. Since hypertension is the most important cerebrovascular risk factor in populations prone to mild brain trauma, we induced mTBI in normotensive Wistar and spontaneously hypertensive rats (SHR) and we assessed BBB permeability, extravasation of blood-borne substances, neuroinflammation and cognitive function two weeks after trauma. We found that mTBI induced a significant BBB disruption two weeks after trauma in SHRs but not in normotensive Wistar rats, which was associated with a significant accumulation of fibrin and increased neuronal expression of inflammatory cytokines TNFα, IL-1ß and IL-6 in the cortex and hippocampus. SHRs showed impaired learning and memory two weeks after mild TBI, whereas cognitive function of normotensive Wistar rats remained intact. Future studies should establish the mechanisms through which hypertension and mild TBI interact to promote persistent BBB disruption, neuroinflammation and cognitive decline to provide neuroprotection and improve cognitive function in patients with mTBI.

Discrete coding of stimulus value, reward expectation, and reward prediction error in the dorsal striatum.

To investigate how the striatum integrates sensory information with reward information for behavioral guidance, we recorded single-unit activity in the dorsal striatum of head-fixed rats participating in a probabilistic Pavlovian conditioning task with auditory conditioned stimuli (CSs) in which reward probability was fixed for each CS but parametrically varied across CSs. We found that the activity of many neurons was linearly correlated with the reward probability indicated by the CSs. The recorded neurons could be classified according to their firing patterns into functional subtypes coding reward probability in different forms such as stimulus value, reward expectation, and reward prediction error. These results suggest that several functional subgroups of dorsal striatal neurons represent different kinds of information formed through extensive prior exposure to CS-reward contingencies.

Prediction of economic choice by primate amygdala neurons.

The amygdala is a key structure of the brain's reward system. Existing theories view its role in decision-making as restricted to an early valuation stage that provides input to decision mechanisms in downstream brain structures. However, the extent to which the amygdala itself codes information about economic choices is unclear. Here, we report that individual neurons in the primate amygdala predict behavioral choices in an economic decision task. We recorded the activity of amygdala neurons while monkeys chose between saving liquid reward with interest and spending the accumulated reward. In addition to known value-related responses, we found that activity in a group of amygdala neurons predicted the monkeys' upcoming save-spend choices with an average accuracy of 78%. This choice-predictive activity occurred early in trials, even before information about specific actions associated with save-spend choices was available. For a substantial number of neurons, choice-differential activity was specific for free, internally generated economic choices and not observed in a control task involving forced imperative choices. A subgroup of choice-predictive neurons did not show relationships to value, movement direction, or visual stimulus features. Choice-predictive activity in some amygdala neurons was preceded by transient periods of value coding, suggesting value-to-choice transitions and resembling decision processes in other brain systems. These findings suggest that the amygdala might play an active role in economic decisions. Current views of amygdala function should be extended to incorporate a role in decision-making beyond valuation.

Intrinsic anticipatory motives in non-human primate food consumption behavior.

Future-oriented behavior is regarded as a cornerstone of human cognition. One key phenomenon through which future orientation can be studied is the delay of gratification, when consumption of an immediate reward is withstood to achieve a larger reward later. The delays used in animal delay of gratification paradigms are rather short to be considered relevant for studying human-like future orientation. Here, for the first time, we show that rhesus macaques exhibit human-relevant future orientation downregulating their operant food consumption in anticipation of a nutritionally equivalent but more palatable food with an unprecedentedly long delay of approximately 2.5 h. Importantly, this behavior is not a result of conditioning but intrinsic to the animals. Our results show that the cognitive time horizon of primates, when tested in ecologically valid foraging-like experiments, extends much further into the future than previously considered, opening up new avenues for translational biomedical research.

Increased brain cytokine level associated impairment of vigilance and memory in aged rats can be alleviated by alpha7 nicotinic acetylcholine receptor agonist treatment.

Age-related neurocognitive disorders are common problems in developed societies. Aging not only affects memory processes, but may also disturb attention, vigilance, and other executive functions. In the present study, we aimed to investigate age-related cognitive deficits in rats and associated molecular alterations in the brain. We also aimed to test the effects of the alpha7 nicotinic acetylcholine receptor (nAChR) agonist PHA-543613 on memory as well as on the sustained attention and vigilance of aged rats. Short- and long-term spatial memories of the rats were tested using the Morris water maze (MWM) task. To measure attention and vigilance, we designed a rat version of the psychomotor vigilance task (PVT) that is frequently used in human clinical examinations. At the end of the behavioral experiments, mRNA and protein expression of alpha7 nAChRs, cytokines, and brain-derived neurotrophic factor (BDNF) were quantitatively measured in the hippocampus, frontal cortex, striatum, and cerebellum. Aged rats showed marked cognitive deficits in both the MWM and the PVT. The deficit was accompanied by increased IL-1beta and TNFalpha mRNA expression and decreased BDNF protein expression in the hippocampus. PHA-543613 significantly improved the reaction time of aged rats in the PVT, especially for unexpectedly appearing stimuli, while only slightly (non-significantly) alleviating spatial memory deficits in the MWM. These results indicate that targeting alpha7 nAChRs may be an effective strategy for the amelioration of attention and vigilance deficits in age-related neurocognitive disorders.

Chemogenetic inhibition of the lateral hypothalamus effectively reduces food intake in rats in a translational proof-of-concept study.

Despite the therapeutic potential of chemogenetics, the method lacks comprehensive preclinical validation, hindering its progression to human clinical trials. We aimed to validate a robust but simple in vivo efficacy assay in rats which could support chemogenetic drug discovery by providing a quick, simple and reliable animal model. Key methodological parameters such as adeno-associated virus (AAV) serotype, actuator drug, dose, and application routes were investigated by measuring the food-intake-reducing effect of chemogenetic inhibition of the lateral hypothalamus (LH) by hM4D(Gi) designer receptor stimulation. Subcutaneous deschloroclozapine in rats transfected with AAV9 resulted in a substantial reduction of food-intake, comparable to the efficacy of exenatide. We estimated that the effect of deschloroclozapine lasts 1-3 h post-administration. AAV5, oral administration of deschloroclozapine, and clozapine-N-oxide were also effective but with slightly less potency. The strongest effect on food-intake occurred within the first 30 min after re-feeding, suggesting this as the optimal experimental endpoint. This study demonstrates that general chemogenetic silencing of the LH can be utilized as an optimal, fast and reliable in vivo experimental model for conducting preclinical proof-of-concept studies in order to validate the in vivo effectiveness of novel chemogenetic treatments. We also hypothesize based on our results that universal LH silencing with existing and human translatable genetic neuroengineering techniques might be a viable strategy to affect food intake and influence obesity.

Effect of a single 30 min UMTS mobile phone-like exposure on the thermal pain threshold of young healthy volunteers.

One of the most frequently investigated effects of radiofrequency electromagnetic fields (RF EMFs) on the behavior of complex biological systems is pain sensitivity. Despite the growing body of evidence of EMF-induced changes in pain sensation, there is no currently accepted experimental protocol for such provocation studies for the healthy human population. In the present study, therefore, we tested the effects of third generation Universal Mobile Telecommunications System (UMTS) RF EMF exposure on the thermal pain threshold (TPT) measured on the surface of the fingers of 20 young adult volunteers. The protocol was initially validated with a topical capsaicin treatment. The exposure time was 30 min and the genuine (or sham) signal was applied to the head through a patch antenna, where RF EMF specific absorption rate (SAR) values were controlled and kept constant at a level of 1.75 W/kg. Data were obtained using randomized, placebo-controlled trials in a double-blind manner. Subjective pain ratings were tested blockwise on a visual analogue rating scale (VAS). Compared to the control and sham conditions, the results provide evidence for intact TPT but a reduced desensitization effect between repeated stimulations within the individual blocks of trials, observable only on the contralateral side for the genuine UMTS exposure. Subjective pain perception (VAS) data indicated marginally decreased overall pain ratings in the genuine exposure condition only. The present results provide pioneering information about human pain sensation in relation to RF EMF exposure and thus may contribute to cover the existing gap between safety research and applied biomedical science targeting the potential biological effects of environmental RF EMFs.

No effects of a single 3G UMTS mobile phone exposure on spontaneous EEG activity, ERP correlates, and automatic deviance detection.

Potential effects of a 30 min exposure to third generation (3G) Universal Mobile Telecommunications System (UMTS) mobile phone-like electromagnetic fields (EMFs) were investigated on human brain electrical activity in two experiments. In the first experiment, spontaneous electroencephalography (sEEG) was analyzed (n = 17); in the second experiment, auditory event-related potentials (ERPs) and automatic deviance detection processes reflected by mismatch negativity (MMN) were investigated in a passive oddball paradigm (n = 26). Both sEEG and ERP experiments followed a double-blind protocol where subjects were exposed to either genuine or sham irradiation in two separate sessions. In both experiments, electroencephalograms (EEG) were recorded at midline electrode sites before and after exposure while subjects were watching a silent documentary. Spectral power of sEEG data was analyzed in the delta, theta, alpha, and beta frequency bands. In the ERP experiment, subjects were presented with a random series of standard (90%) and frequency-deviant (10%) tones in a passive binaural oddball paradigm. The amplitude and latency of the P50, N100, P200, MMN, and P3a components were analyzed. We found no measurable effects of a 30 min 3G mobile phone irradiation on the EEG spectral power in any frequency band studied. Also, we found no significant effects of EMF irradiation on the amplitude and latency of any of the ERP components. In summary, the present results do not support the notion that a 30 min unilateral 3G EMF exposure interferes with human sEEG activity, auditory evoked potentials or automatic deviance detection indexed by MMN.

Correction: Enhanced cardiac vagal tone in mental fatigue: Analysis of heart rate variability in Time-on-Task, recovery, and reactivity.

[This corrects the article DOI: 10.1371/journal.pone.0238670.].

Response-related sensorimotor rhythms under scopolamine and MK-801 exposures in the touchscreen visual discrimination test in rats.

The human mu rhythm has been suggested to represent an important function in information processing. Rodent homologue rhythms have been assumed though no study has investigated them from the cognitive aspect yet. As voluntary goal-directed movements induce the desynchronization of mu rhythm, we aimed at exploring whether the response-related brain activity during the touchscreen visual discrimination (VD) task is suitable to detect sensorimotor rhythms and their change under cognitive impairment. Different doses of scopolamine or MK-801 were injected subcutaneously to rats, and epidural electroencephalogram (EEG) was recorded during task performance. Arciform ~ 10 Hz oscillations appeared during visual processing, then two characteristic alpha/beta desynchronization-resynchronization patterns emerged mainly above the sensorimotor areas, serving presumably different motor functions. Beyond causing cognitive impairment, both drugs supressed the touch-related upper alpha (10-15 Hz) reactivity for desynchronization. Reaction time predominantly correlated positively with movement-related alpha and beta power both in normal and impaired conditions. These results support the existence of a mu homologue rodent rhythm whose upper alpha component appeared to be modulated by cholinergic and glutamatergic mechanisms and its power change might indicate a potential EEG correlate of processing speed. The VD task can be utilized for the investigation of sensorimotor rhythms in rats.

Aconitum Alkaloid Songorine Exerts Potent Gamma-Aminobutyric Acid-A Receptor Agonist Action In Vivo and Effectively Decreases Anxiety without Adverse Sedative or Psychomotor Effects in the Rat.

Songorine (SON) is a diterpenoid alkaloid from Aconitum plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats. First, we investigated the effects of microiontophoretically applied SON alone and combined with GABA-receptor agents picrotoxin and saclofen on neuronal firing activity in various brain areas. Next, putative anxiolytic effects of SON (1.0-3.0 mg/kg) were tested against the GABA-receptor positive allosteric modulator reference compound diazepam (1.0-5.0 mg/kg) in the elevated zero maze (EOM). Furthermore, basic cognitive effects were assessed in a rodent version of the psychomotor vigilance task (PVT). Local application of SON predominantly inhibited the firing activity of neurons. This inhibitory effect of SON was successfully blocked by GABA(A)-receptor antagonist picrotoxin but not by GABA(B)-receptor antagonist saclofen. Similar to GABA(A)-receptor positive allosteric modulator diazepam, SON increased the time spent by animals in the open quadrants of the EOM without any signs of adverse psychomotor and cognitive effects observed in the PVT. We showed that, under in vivo conditions, SON acts as a potent GABA(A)-receptor agonist and effectively decreases anxiety without observable side effects. The present findings facilitate the deeper understanding of the mechanism of action and the widespread pharmacological use of diterpene alkaloids in various CNS indications.

Convergent cross-species pro-cognitive effects of RGH-235, a new potent and selective histamine H3 receptor antagonist/inverse agonist.

The histamine H3 receptor is a favourable target for the treatment of cognitive deficits. Here we report the in vitro and in vivo profile of RGH-235, a new potent, selective, and orally active H3 receptor antagonist/inverse agonist developed by Gedeon Richter Plc. Radioligand binding and functional assays were used for in vitro profiling. Procognitive efficacy was investigated in rodent cognitive tests, in models of attention deficit hyperactive disorder (ADHD) and in cognitive tests of high translational value (rat touch screen visual discrimination test, primate fixed-foreperiod visual reaction time task). Results were supported by pharmacokinetic studies, neurotransmitter release, sleep EEG and dipsogenia. RGH-235 displayed high affinity to H3 receptors (Ki = 3.0-9.2 nM, depending on species), without affinity to H1, H2 or H4 receptors and >100 other targets. RGH-235 was an inverse agonist ([35S] GTPγS binding) and antagonist (pERK1/2 ELISA), showing favourable kinetics, inhibition of the imetit-induced dipsogenia and moderate effects on sleep-wake EEG. RGH-235 stimulated neurotransmitter release both in vitro and in vivo. RGH-235 was active in spontaneously hypertensive rats (SHR), generally considered as a model of ADHD, and revealed a robust pro-cognitive profile both in rodent and primate tests (in 0.3-1 mg/kg) and in models of high translational value (e.g. in a rodent touch screen test and in non-human primates). The multiple and convergent procognitive effects of RGH-235 support the view that beneficial cognitive effects can be linked to antagonism/inverse agonism of H3 receptors.

Reward prediction error coding in dorsal striatal neurons.

In the current theory of learning, the reward prediction error (RPE), the difference between expected and received reward, is thought to be a key factor in reward-based learning, working as a teaching signal. The activity of dopamine neurons is known to code RPE, and the release of dopamine is known to modify the strength of synaptic connectivity in the target neurons. A fundamental interest in current neuroscience concerns the origin of RPE signals in the brain. Here, we show that a group of rat striatal neurons show a clear parametric RPE coding similar to that of dopamine neurons when tested under probabilistic pavlovian conditioning. Together with the fact that striatum and dopamine neurons have strong direct and indirect fiber connections, the result suggests that the striatum plays an important role in coding RPE signal by cooperating with dopamine neurons.

Phase entrainment of human delta oscillations can mediate the effects of expectation on reaction speed.

The more we anticipate a response to a predictable stimulus, the faster we react. This empirical observation has been confirmed and quantified by many investigators suggesting that the processing of behaviorally relevant stimuli is facilitated by probability-based confidence of anticipation. However, the exact neural mechanisms underlying this phenomenon are largely unknown. Here we show that performance changes related to different levels of expectancy originate in dynamic modulation of delta oscillation phase. Our results obtained in rhythmic auditory target detection tasks indicated significant entrainment of the EEG delta rhythm to the onset of the target tones with increasing phase synchronization at higher levels of predictability. Reaction times correlated with the phase of the delta band oscillation at target onset. The fastest reactions occurred during the delta phase that most commonly coincided with the target event in the high expectancy conditions. These results suggest that low-frequency oscillations play a functional role in human anticipatory mechanisms, presumably by modulating synchronized rhythmic fluctuations in the excitability of large neuronal populations and by facilitating efficient task-related neuronal communication among brain areas responsible for sensory processing and response execution.

Exposure to an inhomogeneous static magnetic field increases thermal pain threshold in healthy human volunteers.

In the present experiment, the effect of a single 30 min inhomogeneous static magnetic field (SMF) exposure on thermal pain threshold (TPT) was examined in 15 young healthy human volunteers. The SMF had a maximum peak-to-peak amplitude of 330 mT with a maximum gradient of 13.2 T/m. In either of two experimental sessions (SMF or SHAM), four blocks of 12 TPT trials were carried out under SMF or SHAM exposure on all fingertips of the dominant hand, excluding the thumb. TPT and visual analog scale (VAS) data were recorded at 0, 15, and 30 min exposure time, and 30 min following exposure. SMF treatment resulted in a statistically significant increase in TPT during the entire exposure duration and diminished within-block thermal habituation, leaving pain perception unchanged. These results indicate that SMF-induced peripheral neuronal or circulatory mechanisms may be involved in the observed TPT increase by setting the pain fibre adaptation potential to higher levels.

Potentiation of cognitive enhancer effects of Alzheimer's disease medication memantine by alpha7 nicotinic acetylcholine receptor agonist PHA-543613 in the Morris water maze task.

RATIONALE: There are controversial pieces of evidence whether combination therapies using memantine and cholinesterase inhibitors are beneficial over their monotreatments. However, results of preclinical studies are promising when memantine is combined with agonists and allosteric modulators of the alpha7 nicotinic acetylcholine receptor (nAChR). OBJECTIVES: Here, we tested the hypothesis that cognitive enhancer effects of memantine can be potentiated through modulating alpha7 nAChRs in a scopolamine-induced amnesia model. METHODS: Monotreatments, as well as co-administrations of selective alpha7 nicotinic acetylcholine receptor agonist PHA-543613 and memantine were tested in the Morris water maze task in rats. The efficacy of the co-administration treatment was observed on different domains of spatial episodic memory. RESULTS: Low dose of memantine (0.1 mg/kg) and PHA-543613 (0.3 mg/kg) successfully reversed scopolamine-induced short-term memory deficits both in monotreatments and in co-administration. When recall of information from long-term memory was tested, pharmacological effects caused by co-administration of subeffective doses of memantine and PHA-543613 exceeded that of their monotreatments. CONCLUSION: Our results further support the evidence of beneficial interactions between memantine and alpha7 nAChR ligands and suggest a prominent role of alpha7 nAChRs in the procognitive effects of memantine.

Delay-dependent cholinergic modulation of visual short-term memory in rhesus macaques.

Cholinergic neuromodulation is known to play a key role in visual working memory (VWM) - keeping relevant stimulus representations available for cognitive processes for short time periods (up to a few minutes). Despite the growing body of evidence on how the neural and cognitive mechanisms of VWM dynamically change over retention time, there is mixed evidence available on cholinergic effects as a function of VWM delay period in non-human primates. Using the delayed matching to sample VWM task in rhesus macaques (N = 6), we aimed to characterize VWM maintenance in terms of performance changes as a function of delay duration (across a wide range of delays from 1 to 76 s). Then, we studied how cholinergic neuromodulation influences VWM maintenance using the muscarinic receptor antagonist scopolamine administered alone as transient amnestic treatment, and in combination with two doses of the acetylcholinesterase inhibitor donepezil, a widely used Alzheimer's medication probing for the reversal of scopolamine-induced impairments. Results indicate that scopolamine-induced impairments of VWM maintenance are delay-dependent and specifically affect the 15-33 s time range, suggesting that scopolamine worsens the normal decay of VWM with the passage of time. Donepezil partially rescued the observed scopolamine-induced impairments of VWM performance. These results provide strong behavioral evidence for the role of increased cholinergic tone and muscarinic neuromodulation in the maintenance of VWM beyond a few seconds, in line with our current knowledge on the role of muscarinic acetylcholine receptors in sustained neural activity during VWM delay periods.