Behavioural Brain Research

The Elevated Plus Maze (EPM) offers a standard set up for understanding anxiety, unconditioned risk-avoidance in rodents. The animal shows a preference for enclosed safe arms and avoids risky, open arms that evoke anxiety due to elevated platform (unconditioned response to elevation). A few rodent studies aiming to understand sex-skewed representation in anxiety disorders use the maze and report that more females compared to males show high levels of anxiety and risk-avoidance on an elevated platform. Ethograms derived from examining animal behaviour in the EPM provide precise measures of behaviour of interest, enabling objective assessment of anxiety and risk behaviour. We report two new parameters that might be critical for quantitative analysis of EPM task as a measure of anxiety with reference to sex-differences in risk-taking: (a) spatial preference for arms (open & closed) (b) temporal shift in arm preference in task trials. We first report results from the conventionally derived measures confirming that males spent more time in the open arms indicating low anxiety and higher risk-taking. Preferences for the two open and two closed arms were non-uniform for males and females; male showed stronger preference for one of the two risky open arms, and females showed a stronger preference for one of the two safe closed arms. Temporal analyses indicated that males spent more time in open arms in 3 out of 6 time bins (time blocks), and females spent more time in the closed arms in 5 out of 6 time bins (time blocks), however, counter-intuitively, females showed larger increase in time spent in open arms in the last phase potentially indicating greater regulation of anxiety and increased risk-taking. Inclusion of spatial and temporal parameters in EPM studies might improve our understanding of cognitive and biological sex-differences pertaining to anxiety, and risky behaviour.

The growing interest in ontogenetic studies of learning and memory, along with early-life perturbations, has led to the use of younger rodents as a key biological variable in many investigations. This development prompts an important question about whether procedures and apparatuses designed for studying learning and memory in adults should be simply adapted for use with younger and smaller rodents. The current study examined how arena size affects novel object recognition (NOR) performance in juvenile and adult rats. A commonly used larger arena reliably detected novel object preference in adults but not in juveniles. Adjusting the arena size based on average weight differences between age groups improved the consistency of NOR performance in juvenile rats. Sex differences were observed: adult males performed reliably across all arena sizes, whereas adult females required larger arenas to demonstrate effective NOR performance. These findings highlight the importance of tailoring arena dimensions to developmental stage and sex for accurate cognitive assessment. Specifically, they support the use of weight-based arena scaling as a methodological approach in developmental neurobehavioral research and emphasize the importance of careful design when studying female rodents. Future studies should explore similar environmental adjustments for other behavioral tests in juvenile and female populations.

Sex differences have been documented in threat conditioning, but research into potential sex differences in avoidance paradigms, particularly active avoidance, remains limited. This research gap is particularly concerning given that women are disproportionately affected by stress- and anxiety-related disorders, characterized by maladaptive avoidance. Yet, preclinical research has historically focused on male subjects, limiting our understanding of the neurobiological mechanisms underlying sex differences in threat responses. To address this, we investigated sex-specific strategies in adult Long Evans rats (10 female, 9 male) using a modified platform-mediated avoidance (PMA) task that created a high-conflict choice between reward-seeking and safety. Behavior was tracked over 25 days, with analyses focusing on a stable performance phase (days 20-25) objectively defined using change point analysis. The study design included an initial cohort and a replication cohort to ensure the findings robustness. Females consistently prioritized safety, spending significantly more time foregoing reward to avoid foot shock, while males engaged in more persistent reward-seeking despite the risk of shock. This difference was not driven by differential reward motivation. Furthermore, female strategies were not significantly modulated by the estrous cycle. Thus, male and female rats employ fundamentally different strategies to resolve approach-avoidance conflict: females adopt a robust, safety-first strategy, while males demonstrate a risk-prone, reward-oriented approach. These findings highlight the importance of considering biological factors underlying threat responses, suggesting that characterizing these neural mechanisms may guide more targeted interventions for anxiety and trauma-related disorders.

Fluid control protocols are widely used in neuroscience to motivate laboratory macaques to engage with behavioural tasks. Despite strong evidence that the physiology of the animals is not compromised by such protocols, fluid control remains controversial due to its potential impact on the psychological well-being of the animals. To address this concern, we investigated the effect of fluid control on the affective state of 23 socially-housed adult macaques (10 females) engaged in neuroscience experiments. The protocol involved up to five consecutive days of fluid control per week, followed by a minimum of two days with unrestricted fluid access. The affective state of the animals was primarily assessed by quantifying the frequency of pharmacologically-validated behavioural indicators of high-arousal negative affect (self-scratching, body shaking, self-grooming). The analysis was subsequently extended to validated behavioural indicators of low-arousal negative affect (Inactive not alert) and other behaviours suspected of indicate high-arousal negative affect but lacking proper validation (pacing, yawning). In total, 700 hours of video footage spanning up to seven years of intermittent fluid control per animal were analysed. Despite this extensive dataset, the study found no significant impact of fluid control on average, or any evidence of habituation or sensitization over the years on any of the affective state indicators. Additional results indicate that these null results are not due to a lack of sensitivity, supporting the view that fluid control, as implemented in this study, does not have an adverse impact on the psychological well-being of laboratory macaques. We argue that macaque welfare will be best served by focusing future refinement on other procedures.

The rapid serial visual presentation (RSVP) task and continuous performance tasks (CPT) are used to assess attentional impairments in patients with psychiatric and neurological conditions. This study developed a novel touchscreen task for rats based on the structure of a human RSVP task and used pharmacological manipulations to investigate their effects on different performance measures. Normal animals were trained to respond to a target image and withhold responding to distractor images presented within a continuous sequence. In a second version of the task a false-alarm image was included so performance could be assessed relative to two types of non-target distractors. The effects of acute administration of the stimulant and non-stimulant treatments for ADHD (amphetamine and atomoxetine) were tested in both tasks. Methylphenidate, ketamine and nicotine were tested in the first task only. Amphetamine made animals more impulsive and decreased overall accuracy but increased accuracy when the target was presented early in the image sequence. Atomoxetine improved accuracy overall with a specific reduction in false-alarm responses and a shift in the attentional curve reflecting improved accuracy for targets later in the image sequence. However, atomoxetine also slowed responding and increased omissions. Ketamine, nicotine and methylphenidate had no specific effects at the doses tested. These results suggest that stimulant versus non-stimulant treatments have different effects on attention and impulsive behaviour in this rat version of an RSVP task. These results also suggest that RSVP-like tasks have the potential to be used to study attention in rodents.

BackgroundEvidence from the fields of evolutionary biology and neuroscience supports the theory that spatial cognition and social cognition share neural mechanisms. Rodent models are widely used to study either spatial or social cognition, but few studies have explored the interactions between the spatial and social cognitive domains due to the lack of appropriate paradigms. New methodOur study introduces the Vertical Maze (VM), a novel behavioral apparatus designed to measure multiple aspects of spatial and social cognition. The VM features a standard 3-chamber maze positioned above multilevel columns allowing for the presentation of conspecifics at varying spatial distances and familiarity levels. This arrangement enables conspecifics to serve as discriminative stimuli for both social and spatial spontaneous and goal-oriented assessments. The three-dimensional design of the VM allows rats to use multisensory cues to judge distance, direction, and social identity of conspecifics. ResultsIn the present study, we found that rats can 1) discriminate the spatial distance of conspecifics located below them in an operant conditioning task, and 2) discriminate social novelty when conspecifics are presented below at the near, middle, and far distances in a spontaneous exploration task. Critically, it was necessary for rats to explore all levels of the maze to perform these discriminations. Comparison with existing methodsThis new method advances the field by permitting the presentation of social information (conspecifics) at different spatial distances. The use of conspecifics to serve as stimuli for both social and spatial discriminations allows more direct comparison of behavioral measures across these information domains. Importantly, the presentation of conspecifics as stimuli below the 3-chamber level of the maze engages auditory, visual, and olfactory systems, encouraging a robust multisensory representation of conspecifics presented at a distance. ConclusionsOur results confirm that the VM is an effective tool for studying both spatial and social cognition, facilitating the development of novel automated tasks in these areas. This new method opens new avenues for investigating the neural and cognitive foundations of spatial and social behavior, as well as for exploring the possibility of shared mechanisms across these cognitive domains.

Avoidance learning involves associating a behavioral response with the omission of an expected threat, but the neural mechanisms that drive this learning remain unclear. Research on fear extinction points to a critical role for opioid receptors in the ventrolateral periaqueductal gray (vlPAG) in computing the aversive prediction error signal that is generated when there is a difference between expected and actual aversive events. Based on these fear extinction findings, we hypothesized that vlPAG opioid signaling might also support the early stages of avoidance learning. To test this, 15 Wistar rats (7 females, 8 males) received intra-vlPAG infusions of either naloxone hydrochloride (n = 7, 2.5 {micro}g/0.5 {micro}l), a non-selective opioid receptor antagonist, or vehicle (n = 8, 0.5 {micro}l), immediately before the first and second session of two-way active avoidance training. No infusions were given before the third session, to examine avoidance performance under continued, drug-free acquisition. For the fourth and final session, drug conditions were reversed to examine the acute effect of naloxone on already established avoidance performance. Our results indicated that intra-vlPAG naloxone did not impair avoidance acquisition, nor its performance. These findings suggest that opioid signaling in the vlPAG may not be essential for the initial learning or expression of two-way active avoidance.

The study aimed to investigate to what extent blockade of muscarinic receptors affects the speed of endogenous versus exogenous attentional shift times, and how it affects learning induced improvements of attention shift times. Subjects viewed an array of 10 moving clocks and reported the time a clock indicated when cued. Target clocks were indicated by peripheral or central cues, including conditions of pre-cuing. This allowed assessing shift times when attention was pre-allocated, when peripheral cues triggered exogenous attention shifts, and when central cues triggered endogenous attention shifts. In study 1, each subject participated in 2 sessions (scopolamine/placebo), whereby the order of drug intake was counterbalanced across subjects, and subjects were blinded to conditions. Scopolamine/placebo was administered before a psychophysical experiment was conducted. In study 2, the effect of muscarinic blockade on learning induced improvements of attention shift times was investigated. Here scopolamine/placebo was administered immediately after the first (of two) psychophysical sessions, whereby a given subject either received scopolamine or placebo pills. Confirming previous results, we show that pre-cuing resulted in the fastest shift times, followed by exogenous cuing, with endogenous attentional shifts being slowest. Scopolamine application increased attentional shift times across all 3 conditions compared to placebo, but in a dose dependent manner. Additionally, blockade of muscarinic receptors immediately after the first session reduced learning dependent improvement of attention shift times. These results demonstrate that muscarinic receptors play an important role in attention shifting, and they contribute to learning of attention shifting.

We sought to delineate neural mechanisms underlying the effects of controlled breathing in humans, such as in meditation or breathwork, which can reduce depression, anxiety, stress, and pain. Thus, we developed a murine model, where breathing frequency in awake mice can be substantially slowed. When done for 30 min/day for 4 weeks, these mice had significant reductions in stress-related changes in behavior compared to control mice. We conclude that slow breathing effects on emotional state are present in mice, and which cannot be attributed directly to top-down influences such as volitional or emotional control or placebo effects. Our study paves the way for investigations of the neural mechanisms underlying body-brain interactions related to the effects of controlled breathing as well as a platform for optimization of its therapeutic use for amelioration of ordinary and pathological stress and anxiety in humans.

Conditioned orienting (OR) is a form of cue-directed behavior that is thought to reflect a behavioral phenotype of increased incentive-motivational processing of conditioned stimuli. Previous research has shown that OR also represents a cognitive phenotype that includes impaired attentional function and response inhibition, increased novelty seeking and, in females, resistance to extinction of conditioned place preference (CPP) for the stimulant drug amphetamine (AMP). The following experiments aim to increase our understanding of the OR cognitive phenotype in drug reward processing in male rats. In the first experiment, preference for AMP in males with or without an OR phenotype is assessed using CPP. In the second experiment, our understanding of OR as a predictor of AMP reward response is expanded by measuring potential behavioral mechanisms including affective and motor response to multiple AMP administrations. The results of the two experiments suggest that while the OR phenotype is not a predictor of resistance to extinction of AMP preference in males, OR as a discrete response variable predicts AMP preference after repeated exposures (i.e., AMP challenge). Moreover, we observed that rats with an OR phenotype had a higher positive affective response, measured via ultrasonic vocalizations (USVs) in the 50 kHz range, after multiple AMP administrations. OR scores predicted USVs but did not alter the gross motor response to AMP. Taken together, these results suggest that orienting behavior is a predictor of AMP preference in males, particularly after multiple exposures, and that one behavioral mechanism that could explain this is that male Orienters have a strong positive affective response to AMP.

Adolescence is a time of high-risk behavior and increased exploration. This developmental period is marked by a greater probability of initiating drug use and is associated with an increased risk to develop addiction and dependency in adulthood. Human adolescents are predisposed toward an increased likelihood of risk taking behaviors, including drug use or initiation. The purpose of the study was to examine differences in developmental risk-taking behaviors. Adolescent and adult animals were exposed to a novel stimulus in a familiar environment to assess impulsive behaviors, novelty preference and exploratory behaviors. Adolescent animals had greater novelty-induced locomotor activity, greater novelty preference, and showed higher approach and exploratory behaviors compared to preadolescent and adult animals. These data support the notion that adolescents may be predisposed toward sensation seeking and consequently are more likely to engage in risk taking behaviors, such as drug use initiation.

The present study assessed sex differences and the role of ovarian hormones in the behavioral effects of nicotine withdrawal. Study 1 compared physical signs, anxiety-like behavior, and corticosterone levels in male, intact female, and ovariectomized (OVX) female rats during nicotine withdrawal. Estradiol (E2) and progesterone levels were also assessed in intact females that were tested during different phases of the 4-day estrous cycle. Study 2 assessed the role of ovarian hormones in withdrawal by comparing the same measures in OVX rats that received vehicle, E2, or E2+progesterone prior to testing. Briefly, rats received a sham surgery or an ovariectomy procedure. Fifteen days later, rats were prepared with a pump that delivered nicotine for 14 days. On the test day, rats received saline or the nicotinic receptor antagonist, mecamylamine to precipitate withdrawal. Physical signs and anxiety-like behavior were assessed on the elevated plus maze (EPM) and light-dark transfer (LDT) tests. During withdrawal, intact females displayed greater anxiety-like behavior and corticosterone levels as compared to male and OVX rats. Females tested in estrus (when E2 is relatively low) displayed less anxiety-like behavior and corticosterone versus all other phases. Anxiety-like behavior and corticosterone were positively correlated with E2 and negatively correlated with progesterone. Intact females displaying high E2/low progesterone displayed greater anxiety-like behavior and corticosterone as compared to females displaying low E2/high progesterone. Lastly, OVX-E2 rats displayed greater anxiety-like behavior than OVX-E2+progesterone rat. These data suggest that E2 promotes and progesterone reduces anxiety-like behavior produced by withdrawal.

Courtship songs in mice have been investigated to understand the mechanisms and ecological relevance of vocal communication. There is evidence that courtship song characteristics vary between different genotypes, but little is known on whether individuals, even within the same genotype, differ from each other in the composition, complexity, and temporal consistency of their songs. In a first study, we aimed to systematically identify song features typical of different genotypes, by assessing the composition and complexity (i.e., entropy) of the syllabic sequences of male laboratory mice from four different strains (Mus musculus f. domestica: C57BL/6J, BALB/c, DBA/2 and B6D2F1). Mice were individually presented with a swab containing fresh female urine for 5 minutes to elicit courtship songs. The four strains differed not only in the composition but also in the complexity of their syllabic sequences. In a second study, we investigated within-strain individual differences in temporal consistency and recurring motifs (i.e., identical sets of syllables that are repeated within a song), using BALB/c and DBA/2 mice. The same procedure as in the first study was followed, but in addition testing was repeated weekly over three weeks. Both strains showed some level of individual temporal consistency; BALB/c in the overall amount of emitted vocalisations and DBA/2 in the expression of specific syllable types. However, hierarchical cluster analysis revealed remarkable individual variability in how consistent song characteristics were over time. Furthermore, recurring motifs were expressed at varying levels depending on the individual. Taken together, not only genotype but also individuality can affect variability in courtship songs in mice, suggesting the existence of different courtship strategies (e.g., higher song consistency to facilitate individual identification) related to varying levels of behavioural plasticity. HIGHLIGHTSO_LICourtship songs in mice can serve as a model to study vocal communication C_LIO_LIWe explore how genotype and individuality affect courtship songs characteristics C_LIO_LIGenotypes differ in composition and also in complexity of syllabic sequences C_LIO_LIWe find remarkable individual variability in how consistent songs are over time C_LIO_LIResults suggest the existence of variation in male courting behaviour C_LI

The relationships between distinct abilities and their interdependencies during memory tasks and motor learning activities are not clear. An important question is whether being proficient in memory or motor learning tasks also translates into better performance in another, similar task - reflecting potential generalization of motor learning abilities. To investigate the correlation between memory performance and motor learning, we used a combination of behavioral tasks that assess general exploratory behavior, declarative memory, and fine motor learning in female mice. For the exploratory behavior, we used the Open Field task, and assessed declarative memory using the Novel Object Recognition and Y-maze. Motor learning was assessed through the Vertical Pole and Pellet Reaching task. We found a negative correlation between the Vertical Pole task and the motor learning task, where a shorter time for an individual to turn on a pole correlated with an increased number of attempts to reach a sugar pellet in the motor learning task. Additionally, a positive correlation was found between the Y-maze and the motor learning task, where a higher exploration rate indicated a higher success ratio in the pellet reaching task. These results can be used to conduct a pre-study for challenging motor tasks that include pre-behavioral procedures on mice. Our study indicates that both the Vertical Pole and the Y-maze can be suitable predictors of motor learning performance and activity.

In rodents, young pups communicate with their parents through harmonic calls and ultrasonic vocalizations (USVs). These forms of communication can improve chances of survival, since pups rely on their parents for thermoregulation, nutrition and protection. The extent to which pups modulate calls in response to their surroundings remains unclear. In this study we examined whether olfactory stimuli influence characteristics of pup calls, and how these calls may be affected by pup sex and litter size, in the California mouse (Peromyscus californicus). Pups were isolated and audio recorded during an initial, 3-minute control period, after which they were exposed for 5 minutes to bedding containing one of 4 olfactory cues: scent from their home cage, scent from the home cage of an unfamiliar family, coyote urine, or no scent. Latency to call, call rate, call duration and call characteristics (e.g. frequency and amplitude) were compared between the control period and olfactory-exposure period as well as among olfactory conditions. Pups from 2-pup litters called more quietly (lower amplitude) when exposed to odor from a predator while pups from 3-pup litters called louder (higher amplitude). Additionally, pups tended to reduce their call rates in response to odors from their home cage, consistent with contact quieting. However, pups tended to increase their rate of calling when exposed to predator urine, in contrast to the expectations of predator-induced vocal suppression. Lastly, male pups produced higher-frequency calls and more USVs than females. These results indicate that a number of pup call characteristics in this species can be influenced by acute olfactory stimuli as well as factors such as litter size and sex. The value of these pup call variations for offspring-parent communication is unclear: whether they elicit different parental responses is unknown and would be an interesting/valuable/informative avenue for future studies.

Psychostimulant drugs are so named because they alter the cardiac, brain and behavioral responses in humans and other animals. Acute food deprivation or chronic food restriction potentiates the stimulatory effects of abused drugs and increases the propensity for relapse to drug seeking in drug-experienced animals. The mechanisms by which hunger affects cardiac and behavioral activities are only beginning to be elucidated. Moreover, changes in motor neuron activities at the single neuron level induced by the stimulants, and their modulation by hunger, remain unknown. Here we investigated how the state of hunger affects responses to d-amphetamine by measuring locomotion, cardiac output, and individual motor neuron activity in zebrafish larvae. We used wild-type larval zebrafish to record behavioral and cardiac responses and the larvae of mnx1:GCaMP transgenic zebrafish to record motor neuron responses. Acute administration of d-amphetamine in sated larvae did not induce a significant change in the motor responses (swimming distances, tail activity), heart rate, or motor neuron firing frequency to the stimulant. However, food deprivation enhanced amphetamine-evoked responses significantly. The results extend the finding that signals arising from food deprivation are a key potentiator of the drug responses induced by d-amphetamine to the zebrafish model. The larval zebrafish is an ideal model to further elucidate this interaction and identify key neuronal substrates that may increase vulnerability to drug reinforcement, drug-seeking and relapse.

Adolescence is a critical developmental period, concerning anatomical, neurochemical and behavioral changes. Moreover, adolescents are more sensitive to the long-term deleterious effects of drug abuse. Binge-like consumption of alcohol and marijuana, along with tobacco smoking, is a dangerous pattern often observed in adolescents during weekends. Nevertheless, the long-term effect of their adolescent co-exposure has not been experimentally investigated yet. Long-Evans adolescent male (n = 20) and female (n = 20) rats from postnatal day 30 (P30) until P60 were daily treated with nicotine (0.3 mg/kg, i.p.), and, on two consecutive binging days per week (for a total of eight times), received an intragastric ethanol solution (3 g/kg) and an intraperitoneal (i.p.) dose of cannabinoid 1/2 receptor agonist WIN55,212-2 (1.2 mg/kg). These rats were tested after treatment discontinuation at >P90 for associative food-rewarded operant learning in the two-lever conditioning chambers for six consecutive days on a fixed ratio 1 (FR1) schedule followed by another six days of daily FR2 schedule testing, after 45 days rest. We found the main effects of sex x treatment interactions in FR1 but not in FR2 experiments. Treated females show attenuated operant responses for food pellets during all FR1 and the FR2 schedule, whilst the treated males show an impairment in FR2 but not in the FR1 schedule. Moreover, the treated females percentage of learners was significantly lower than female controls in FR1 while treated males and females were lower than controls in FR2. Our findings suggest that intermittent adolescent abuse of common drugs, such as alcohol and marijuana, and chronic tobacco exposure can cause significant long-term effects on motivation for natural reinforcers later in adulthood in both sexes. Females appear to be more sensitive to the deleterious effects of adolescent polydrug abuse with both sexes having an increased likelihood of developing lifelong brain alterations.

IntroductionPhysical exercise has repeatedly been reported to have advantageous effects on brain functions, including learning and memory formation. However, objective tools to measure such effects are often lacking. Eyeblink conditioning is a well-characterised method for studying the neural basis of associative learning. As such, this paradigm has potential as a tool to assess to what extent exercise affects one of the most basic forms of learning. Until recently, however, using this paradigm for testing human subjects in their daily life was technically challenging. As a consequence, no studies have investigated how exercise affects eyeblink conditioning in humans. Here we hypothesize that acute aerobic exercise is associated with improved performance in eyeblink conditioning. Furthermore, we explored whether the effects of exercise differed for people with an active versus a sedentary lifestyle. MethodsWe conducted a case-control study using a smartphone-based platform for conducting neurometric eyeblink conditioning in healthy adults aged between 18 - 40 years (n = 36). Groups were matched on age, sex, and education level. Our primary outcome measures included the amplitude and timing of conditioned eyelid responses over the course of eyeblink training. As a secondary measure, we studied the amplitude of the unconditioned responses. ResultsAcute exercise significantly enhanced the acquisition of conditioned eyelid responses; however, this effect was only true for individuals with an active lifestyle. No statistically significant effects were established for timing of the conditioned responses and amplitude of the unconditioned responses. DiscussionThis study highlights a facilitative role of acute aerobic exercise in associative learning and emphasises the importance of accounting for lifestyle when investigating the acute effects of exercise on brain functioning.

Humans show distinct social behaviours when we recognise social similarity in opponents that are members of the same social group. However, little attention has been paid to the role of social similarity in non-human animals. In Wistar subject rats, the presence of an unfamiliar Wistar rat mitigated stress responses, suggesting the importance of social similarity in this phenomenon. We found that the presence of unfamiliar Sprague-Dawley (SD) or Long-Evans (LE) rats, but not an unfamiliar Fischer 344 (F344) rat, similarly mitigated stress in subject rats. It is therefore possible that the subject rats recognised social similarity to unfamiliar SD and LE rats. In this study, we demonstrated that Wistar subject rats were capable of categorizing unfamiliar rats based on their strain, and that Wistar subjects showed a preference for unfamiliar Wistar, SD, and LE rats over F344 rats. However, the subject rats did not show a preference among Wistar, SD, and LE rats. In addition, the results were not due to an aversion to F344 rats, and preference was not affected when anaesthetised rats were presented to subject rats. The findings suggested that rats recognise social similarity to certain unfamiliar strains of rats.

Several theories propose a close relationship between interval timing and the temporal properties of memory. Systemic administration of scopolamine, a muscarinic acetylcholine receptor antagonist, is known to induce memory deficits and impair temporal prediction. However, existing studies on timing using free-moving animals are challenging to interpret due to the confounding effects of movement on interval timing. In this study, we examined the effects of scopolamine on timing behavior in mice using a head-fixed experimental setup. Mice were trained on a fixed-time schedule task with a peak procedure, where a 10% sucrose solution was delivered every 10 seconds through a spout placed within the licking distance of the mice. Following training, the mice exhibited anticipatory licking behavior in response to the timing of sucrose delivery, indicating that they could predict the reward timing. Systemic administration of scopolamine increased the variability of temporal prediction in a dose-dependent manner but did not affect the mean temporal prediction. Single-trial bout analysis revealed that scopolamine impaired the duration of licking bouts without affecting the total number of licks in peak trials, suggesting that the mice were unable to sustain licking at the spout. Additionally, we assessed the effects of scopolamine on spontaneous locomotor activity and excretion in a free-moving open-field task. Scopolamine injections increased locomotor activity and decreased fecal output. Taken together, these findings suggest that the increased variability in timing behavior induced by scopolamine may be attributed to changes in transitions between behavioral states.