Spiking activity in the visual thalamus is coupled to pupil dynamics across temporal scales.

The processing of sensory information, even at early stages, is influenced by the internal state of the animal. Internal states, such as arousal, are often characterized by relating neural activity to a single "level" of arousal, defined by a behavioral indicator such as pupil size. In this study, we expand the understanding of arousal-related modulations in sensory systems by uncovering multiple timescales of pupil dynamics and their relationship to neural activity. Specifically, we observed a robust coupling between spiking activity in the mouse dorsolateral geniculate nucleus (dLGN) of the thalamus and pupil dynamics across timescales spanning a few seconds to several minutes. Throughout all these timescales, 2 distinct spiking modes-individual tonic spikes and tightly clustered bursts of spikes-preferred opposite phases of pupil dynamics. This multi-scale coupling reveals modulations distinct from those captured by pupil size per se, locomotion, and eye movements. Furthermore, coupling persisted even during viewing of a naturalistic movie, where it contributed to differences in the encoding of visual information. We conclude that dLGN spiking activity is under the simultaneous influence of multiple arousal-related processes associated with pupil dynamics occurring over a broad range of timescales.

Exploring the feasibility of pupillometry training and perceptions of potential use for intracranial pressure monitoring in Uganda: A mixed methods study.

INTRODUCTION: Traumatic brain injury (TBI) accounts for the majority of Uganda's neurosurgical disease burden; however, invasive intracranial pressure (ICP) monitoring is infrequently used. Noninvasive monitoring could change the care of patients in such a setting through quick detection of elevated ICP. PURPOSE: Given the novelty of pupillometry in Uganda, this mixed methods study assessed the feasibility of pupillometry for noninvasive ICP monitoring for patients with TBI. METHODS: Twenty-two healthcare workers in Kampala, Uganda received education on pupillometry, practiced using the device on healthy volunteers, and completed interviews discussing pupillometry and its implementation. Interviews were assessed with qualitative analysis, while quantitative analysis evaluated learning time, measurement time, and accuracy of measurements by participants compared to a trainer's measurements. RESULTS: Most participants (79%) reported a positive perception of pupillometry. Participants described the value of pupillometry in the care of patients during examination, monitoring, and intervention delivery. Commonly discussed concerns included pupillometry's cost, understanding, and maintenance needs. Perceived implementation challenges included device availability and contraindications for use. Participants suggested offering continued education and engaging hospital leadership as implementation strategies. During training, the average learning time was 13.5 minutes (IQR 3.5), and the measurement time was 50.6 seconds (IQR 11.8). Paired t-tests to evaluate accuracy showed no statistically significant difference in comparison measurements. CONCLUSION: Pupillometry was considered acceptable for noninvasive ICP monitoring of patients with TBI, and pupillometer use was shown to be feasible during training. However, key concerns would need to be addressed during implementation to aid device utilization.

Post-Saccadic Oscillations of the Pupil and Lens Reduce Fixation Stability in Retinitis Pigmentosa and Age-Related Macular Degeneration.

Purpose: Post-saccadic oscillations (PSOs) reflect movements of gaze that result from motion of the pupil and lens relative to the eyeball rather than eyeball rotations. Here, we analyzed the characteristics of PSOs in subjects with age-related macular degeneration (AMD), retinitis pigmentosa (RP), and normal vision (NV). Our aim was to assess the differences in PSOs between people with vision loss and healthy controls because PSOs affect retinal image stability after each saccade. Methods: Participants completed a horizontal saccade task and their gaze was measured using a pupil-based eye tracker. Oscillations occurring in the 80 to 200 ms post-saccadic period were described with a damped oscillation model. We compared the amplitude, decay time constant, and frequency of the PSOs for the three different groups. We also examined the correlation between these PSO parameters and the amplitude, peak velocity, and final deceleration of the preceding saccades. Results: Subjects with vision loss (AMD, n = 6, and RP, n = 5) had larger oscillation amplitudes, longer decay constants, and lower frequencies than subjects with NV (n = 7). The oscillation amplitudes increased with increases in saccade deceleration in all three groups. The other PSO parameters, however, did not show consistent correlations with either saccade amplitude or peak velocity. Conclusions: Post-saccadic fixation stability in AMD and RP is reduced due to abnormal PSOs. The differences with respect to NV are not due to differences in saccade kinematics, suggesting that anatomic and neuronal variations affect the suspension of the iris and the lens in the patients' eyes.

Pupil diameter as an indicator of sound pair familiarity after statistically structured auditory sequence.

Inspired by recent findings in the visual domain, we investigated whether the stimulus-evoked pupil dilation reflects temporal statistical regularities in sequences of auditory stimuli. We conducted two preregistered pupillometry experiments (experiment 1, n = 30, 21 females; experiment 2, n = 31, 22 females). In both experiments, human participants listened to sequences of spoken vowels in two conditions. In the first condition, the stimuli were presented in a random order and, in the second condition, the same stimuli were presented in a sequence structured in pairs. The second experiment replicated the first experiment with a modified timing and number of stimuli presented and without participants being informed about any sequence structure. The sound-evoked pupil dilation during a subsequent familiarity task indicated that participants learned the auditory vowel pairs of the structured condition. However, pupil diameter during the structured sequence did not differ according to the statistical regularity of the pair structure. This contrasts with similar visual studies, emphasizing the susceptibility of pupil effects during statistically structured sequences to experimental design settings in the auditory domain. In sum, our findings suggest that pupil diameter may serve as an indicator of sound pair familiarity but does not invariably respond to task-irrelevant transition probabilities of auditory sequences.

Anticipating noxious stimulation rather than afferent nociceptive input may evoke pupil asymmetry.

Unilateral nociceptive stimulation is associated with subtle signs of pupil asymmetry that may reflect lateralized activity in the locus coeruleus. To explore drivers of this pupil asymmetry, electrical stimuli, delivered alone or 200 ms before or after an acoustic startle stimulus, were administered to one ankle under four experimental conditions: with or without a 1.6 s anticipatory period, or while the forearm ipsilateral or contralateral to the electrical stimulus was heated tonically to induce moderate pain (15 healthy participants in each condition). Pupil diameter was measured at the start of each trial, at stimulus delivery, and each second for 5 s after stimulus delivery. At the start of the first trial, the pupil ipsilateral to the side on which electric shocks were later delivered was larger than the contralateral pupil. Both pupils dilated robustly during the anticipatory period and dilated further during single- and dual-stimulus trials. However, pupil asymmetry persisted throughout the experiment. Tonically-applied forearm heat-pain modulated the pupillary response to phasic electrical stimuli, with a slight trend for dilatation to be greater contralateral to the forearm being heated. Together, these findings suggest that focusing anxiously on the expected site of noxious stimulation was associated with dilatation of the ipsilateral pupil whereas phasic nociceptive stimuli and psychological arousal triggered bilateral pupillary dilatation. It was concluded that preparatory cognitive activity rather than phasic afferent nociceptive input is associated with pupillary signs of lateralized activity in the locus coeruleus.

Does vibrotactile stimulation of the auricular vagus nerve enhance working memory? A behavioral and physiological investigation.

BACKGROUND: Working memory is essential to a wide range of cognitive functions and activities. Transcutaneous auricular vagus nerve stimulation (taVNS) is a promising method to improve working memory performance. However, the feasibility and scalability of electrical stimulation are constrained by several limitations, such as auricular discomfort and inconsistent electrical contact. OBJECTIVE: We aimed to develop a novel and practical method, vibrotactile taVNS, to improve working memory. Further, we investigated its effects on arousal, measured by skin conductance and pupil diameter. METHOD: This study included 20 healthy participants. Behavioral response, skin conductance, and eye tracking data were concurrently recorded while the participants performed N-back tasks under three conditions: vibrotactile taVNS delivered to the cymba concha, earlobe (sham control), and no stimulation (baseline control). RESULTS: In 4-back tasks, which demand maximal working memory capacity, active vibrotactile taVNS significantly improved the performance metric d' compared to the baseline but not to the sham. Moreover, we found that the reduction rate of d' with increasing task difficulty was significantly smaller during vibrotactile taVNS sessions than in both baseline and sham conditions. Arousal, measured as skin conductance and pupil diameter, declined over the course of the tasks. Vibrotactile taVNS rescued this arousal decline, leading to arousal levels corresponding to optimal working memory levels. Moreover, pupil diameter and skin conductance level were higher during high-cognitive-load tasks when vibrotactile taVNS was delivered to the concha compared to baseline and sham. CONCLUSION: Our findings suggest that vibrotactile taVNS modulates the arousal pathway and could be a potential intervention for enhancing working memory.

Pupillary responses as a biomarker of cognitive effort and the impact of task difficulty on reward processing in schizophrenia.

Negative symptoms of schizophrenia robustly predict functional outcomes but remain relatively resistant to available treatments. Better measures of negative symptoms, especially motivational deficits, are needed to better understand these symptoms and improve treatment development. Recent research shows promise in linking behavioral effort tasks to motivational negative symptoms, reward processing deficits, and defeatist attitudes, but few studies account for individual or group (patient v. control) differences in cognitive ability to perform the tasks. Individuals with poorer abilities might be less motivated to perform tasks because they find them more difficult to perform. This study used a personalized digit span task to control task difficulty while measuring task effort via pupillary responses (greater dilation indicates greater cognitive effort) at varying monetary rewards ($1 & $2). Participants with schizophrenia (N = 34) and healthy controls (N = 41) performed a digit span task with personalized max span lengths and easy (max- 2 digits) and overload (max+ 2 digits) conditions. Consistent with many studies, pupillary responses (cognitive effort) increased with greater difficulty until exceeding capacity. A similar pattern of reward responsivity was seen in both groups, such that greater reward increased dilation (effort) comparably for both groups when difficulty was within capacity. Neither patients nor controls exerted increased effort for greater reward when difficulty exceeded capacity. In patients, positive relationships were found between pupil dilation and defeatist performance beliefs if task difficulty was within capacity; a relationship that reversed if the task was too difficult. The findings demonstrate the importance of accounting for cognitive capacity and task difficulty when evaluating motivation and reward sensitivity and illustrate the utility of pupillary responses as an objective measure of effort in schizophrenia.

Pupil Response in Visual Tracking Tasks: The Impacts of Task Load, Familiarity, and Gaze Position.

Pupil size is a significant biosignal for human behavior monitoring and can reveal much underlying information. This study explored the effects of task load, task familiarity, and gaze position on pupil response during learning a visual tracking task. We hypothesized that pupil size would increase with task load, up to a certain level before decreasing, decrease with task familiarity, and increase more when focusing on areas preceding the target than other areas. Fifteen participants were recruited for an arrow tracking learning task with incremental task load. Pupil size data were collected using a Tobii Pro Nano eye tracker. A 2 × 3 × 5 three-way factorial repeated measures ANOVA was conducted using R (version 4.2.1) to evaluate the main and interactive effects of key variables on adjusted pupil size. The association between individuals' cognitive load, assessed by NASA-TLX, and pupil size was further analyzed using a linear mixed-effect model. We found that task repetition resulted in a reduction in pupil size; however, this effect was found to diminish as the task load increased. The main effect of task load approached statistical significance, but different trends were observed in trial 1 and trial 2. No significant difference in pupil size was detected among the three gaze positions. The relationship between pupil size and cognitive load overall followed an inverted U curve. Our study showed how pupil size changes as a function of task load, task familiarity, and gaze scanning. This finding provides sensory evidence that could improve educational outcomes.

Implementation of a High-Accuracy Neural Network-Based Pupil Detection System for Real-Time and Real-World Applications.

In this paper, the implementation of a new pupil detection system based on artificial intelligence techniques suitable for real-time and real-word applications is presented. The proposed AI-based pupil detection system uses a classifier implemented with slim-type neural networks, with its classes being defined according to the possible positions of the pupil within the eye image. In order to reduce the complexity of the neural network, a new parallel architecture is used in which two independent classifiers deliver the pupil center coordinates. The training, testing, and validation of the proposed system were performed using almost 40,000 eye images with a resolution of 320 × 240 pixels and coming from 20 different databases, with a high degree of generality. The experimental results show a detection rate of 96.29% at five pixels with a standard deviation of 3.38 pixels for all eye images from all databases and a processing speed of 100 frames/s. These results indicate both high accuracy and high processing speed, and they allow us to use the proposed solution for different real-time applications in variable and non-uniform lighting conditions, in fields such as assistive technology to communicate with neuromotor-disabled patients by using eye typing, in computer gaming, and in the automotive industry for increasing traffic safety by monitoring the driver's cognitive state.

Bidirectional brain-body interactions during natural story listening.

Narratives can synchronize neural and physiological signals between individuals, but the relationship between these signals, and the underlying mechanism, is unclear. We hypothesized a top-down effect of cognition on arousal and predicted that auditory narratives will drive not only brain signals but also peripheral physiological signals. We find that auditory narratives entrained gaze variation, saccade initiation, pupil size, and heart rate. This is consistent with a top-down effect of cognition on autonomic function. We also hypothesized a bottom-up effect, whereby autonomic physiology affects arousal. Controlled breathing affected pupil size, and heart rate was entrained by controlled saccades. Additionally, fluctuations in heart rate preceded fluctuations of pupil size and brain signals. Gaze variation, pupil size, and heart rate were all associated with anterior-central brain signals. Together, these results suggest bidirectional causal effects between peripheral autonomic function and central brain circuits involved in the control of arousal.

Pupils Dilate More to Harder Vocabulary Words than Easier Ones.

Understanding cognitive effort expended during assessments is essential to improving efficiency, accuracy, and accessibility within these assessments. Pupil dilation is commonly used as a psychophysiological measure of cognitive effort, yet research on its relationship with effort expended specifically during language processing is limited. The present study adds to and expands on this literature by investigating the relationships among pupil dilation, trial difficulty, and accuracy during a vocabulary test. Participants (n = 63, Mage = 19.25) completed a subset of trials from the Peabody Picture Vocabulary Test while seated at an eye-tracker monitor. During each trial, four colored images were presented on the monitor while a word was presented via audio recording. Participants verbally indicated which image they thought represented the target word. Words were categorized into Easy, Medium, and Hard difficulty. Pupil dilation during the Medium and Hard trials was significantly greater than during the Easy trials, though the Medium and Hard trials did not significantly differ from each other. Pupil dilation in comparison to trial accuracy presented a more complex pattern, with comparisons between accurate and inaccurate trials differing depending on the timing of the stimulus presentation. These results present further evidence that pupil dilation increases with cognitive effort associated with vocabulary tests, providing insights that could help refine vocabulary assessments and other related tests of language processing.

The Costs (and Benefits?) of Effortful Listening for Older Adults: Insights from Simultaneous Electrophysiology, Pupillometry, and Memory.

Although the impact of acoustic challenge on speech processing and memory increases as a person ages, older adults may engage in strategies that help them compensate for these demands. In the current preregistered study, older adults (n = 48) listened to sentences-presented in quiet or in noise-that were high constraint with either expected or unexpected endings or were low constraint with unexpected endings. Pupillometry and EEG were simultaneously recorded, and subsequent sentence recognition and word recall were measured. Like young adults in prior work, we found that noise led to increases in pupil size, delayed and reduced ERP responses, and decreased recall for unexpected words. However, in contrast to prior work in young adults where a larger pupillary response predicted a recovery of the N400 at the cost of poorer memory performance in noise, older adults did not show an associated recovery of the N400 despite decreased memory performance. Instead, we found that in quiet, increases in pupil size were associated with delays in N400 onset latencies and increased recognition memory performance. In conclusion, we found that transient variation in pupil-linked arousal predicted trade-offs between real-time lexical processing and memory that emerged at lower levels of task demand in aging. Moreover, with increased acoustic challenge, older adults still exhibited costs associated with transient increases in arousal without the corresponding benefits.

Glossiness perception and its pupillary response.

Recent studies have revealed that pupillary response changes depend on perceptual factors such as subjective brightness caused by optical illusions and luminance. However, the manner in which the perceptual factor that is derived from the glossiness perception of object surfaces affects the pupillary response remains unclear. We investigated the relationship between the glossiness perception and pupillary response through a glossiness rating experiment that included recording the pupil diameter. We prepared general object images (original) and randomized images (shuffled) that comprised the same images with randomized small square regions as stimuli. The image features were controlled by matching the luminance histogram. The observers were asked to rate the perceived glossiness of the stimuli presented for 3,000 ms and the changes in their pupil diameters were recorded. Images with higher glossiness ratings constricted the pupil size more than those with lower glossiness ratings at the peak constriction of the pupillary responses during the stimulus duration. The linear mixed-effects model demonstrated that the glossiness rating, image category (original/shuffled), variance of the luminance histogram, and stimulus area were most effective in predicting the pupillary responses. These results suggest that the illusory brightness obtained by the image regions of high-glossiness objects, such as specular highlights, induce pupil constriction.

The other-race effect of pupil contagion in infancy.

Pupil contagion refers to the observer's pupil-diameter changes in response to changes in the pupil diameter of others. Recent studies on the other-race effect on pupil contagion have mainly focused on using eye region images as stimuli, revealing the effect in adults but not in infants. To address this research gap, the current study used whole-face images as stimuli to assess the pupil-diameter response of 5-6-month-old and 7-8-month-old infants to changes in the pupil-diameter of both upright and inverted unfamiliar-race faces. The study initially hypothesized that there would be no pupil contagion in either upright or inverted unfamiliar-race faces, based on our previous finding of pupil contagion occurring only in familiar-race faces among 5-6-month-old infants. Notably, the current results indicated that 5-6-month-old infants exhibited pupil contagion in both upright and inverted unfamiliar-race faces, while 7-8-month-old infants showed this effect only in upright unfamiliar-race faces. These results demonstrate that the face inversion effect of pupil contagion does not occur in 5-6-month-old infants, thereby suggesting the presence of the other-race effect in pupil contagion among this age group. Overall, this study provides the first evidence of the other-race effect on infants' pupil contagion using face stimuli.

Multimodal decoding of error processing in a virtual reality flight simulation.

Technological advances in head-mounted displays (HMDs) facilitate the acquisition of physiological data of the user, such as gaze, pupil size, or heart rate. Still, interactions with such systems can be prone to errors, including unintended behavior or unexpected changes in the presented virtual environments. In this study, we investigated if multimodal physiological data can be used to decode error processing, which has been studied, to date, with brain signals only. We examined the feasibility of decoding errors solely with pupil size data and proposed a hybrid decoding approach combining electroencephalographic (EEG) and pupillometric signals. Moreover, we analyzed if hybrid approaches can improve existing EEG-based classification approaches and focused on setups that offer increased usability for practical applications, such as the presented game-like virtual reality flight simulation. Our results indicate that classifiers trained with pupil size data can decode errors above chance. Moreover, hybrid approaches yielded improved performance compared to EEG-based decoders in setups with a reduced number of channels, which is crucial for many out-of-the-lab scenarios. These findings contribute to the development of hybrid brain-computer interfaces, particularly in combination with wearable devices, which allow for easy acquisition of additional physiological data.

Brightness illusions drive a neuronal response in the primary visual cortex under top-down modulation.

Brightness illusions are a powerful tool in studying vision, yet their neural correlates are poorly understood. Based on a human paradigm, we presented illusory drifting gratings to mice. Primary visual cortex (V1) neurons responded to illusory gratings, matching their direction selectivity for real gratings, and they tracked the spatial phase offset between illusory and real gratings. Illusion responses were delayed compared to real gratings, in line with the theory that processing illusions requires feedback from higher visual areas (HVAs). We provide support for this theory by showing a reduced V1 response to illusions, but not real gratings, following HVAs optogenetic inhibition. Finally, we used the pupil response (PR) as an indirect perceptual report and showed that the mouse PR matches the human PR to perceived luminance changes. Our findings resolve debates over whether V1 neurons are involved in processing illusions and highlight the involvement of feedback from HVAs.

Double Dissociation of Spontaneous Alpha-Band Activity and Pupil-Linked Arousal on Additive and Multiplicative Perceptual Gain.

Perception is a probabilistic process dependent on external stimulus properties and one's internal state. However, which internal states influence perception and via what mechanisms remain debated. We studied how spontaneous alpha-band activity (8-13 Hz) and pupil fluctuations impact visual detection and confidence across stimulus contrast levels (i.e., the contrast response function, CRF). In human subjects of both sexes, we found that low prestimulus alpha power induced an "additive" shift in the CRF, whereby stimuli were reported present more frequently at all contrast levels, including contrast of zero (i.e., false alarms). Conversely, prestimulus pupil size had a "multiplicative" effect on detection such that stimuli occurring during large pupil states (putatively corresponding to higher arousal) were perceived more frequently as contrast increased. Signal detection modeling reveals that alpha power changes detection criteria equally across the CRF but not detection sensitivity (d'), whereas pupil-linked arousal modulated sensitivity, particularly for higher contrasts. Interestingly, pupil size and alpha power were positively correlated, meaning that some of the effect of alpha on detection may be mediated by pupil fluctuations. However, pupil-independent alpha still induced an additive shift in the CRF corresponding to a criterion effect. Our data imply that low alpha boosts detection and confidence by an additive factor, rather than by a multiplicative scaling of contrast responses, a profile which captures the effect of pupil-linked arousal. We suggest that alpha power and arousal fluctuations have dissociable effects on behavior. Alpha reflects the baseline level of visual excitability, which can vary independent of arousal.

Evaluating the utility of quantitative pupillometry in a neuro-critical care setting for the monitoring of intracranial pressure: A prospective cohort study.

INTRODUCTION: Assessment of the pupillary light reflex (PLR) is key in intensive care monitoring of neurosurgical patients, particularly for monitoring intracranial pressure (ICP). Quantitative pupillometry using a handheld pupillometer is a reliable method for PLR assessment. However, many variables are derived from such devices. We therefore aimed to assess the performance of these variables at monitoring ICP. METHODS: Sedated patients admitted to neurocritical care in a tertiary neurosurgical centre with invasive ICP monitoring were included. Hourly measurement of ICP, subjective pupillometry (SP) using a pen torch device, and quantitative pupillometry (QP) using a handheld pupillometer were performed. RESULTS: 561 paired ICP, SP and QP pupillary observations from nine patients were obtained (1122 total pupillary observations). SP and QP had a moderate concordance for pupillary size (κ=0.62). SP performed poorly at detecting pupillary size changes (sensitivity=24%). In 40 (3.6%) observations, SP failed to detect a pupillary response whereas QP did. Moderate correlations with ICP were detected for maximum constriction velocity (MCV), dilation velocity (DV), and percentage change in pupillary diameter (%C). Discriminatory ability at an ICP threshold of >22 mmHg was moderate for MCV (AUC=0.631), DV (AUC=0.616), %C (AUC=0.602), and pupillary maximum size (AUC=0.625). CONCLUSION: QP is superior to SP at monitoring pupillary reactivity and changes to pupillary size. Although effect sizes were moderate to weak across assessed variables, our data indicates MCV and %C as the most sensitive variables for monitoring ICP. Further study is required to validate these findings and to establish normal range cut-offs for clinical use.

Combining Cardiovascular and Pupil Features Using k-Nearest Neighbor Classifiers to Assess Task Demand, Social Context, and Sentence Accuracy During Listening.

In daily life, both acoustic factors and social context can affect listening effort investment. In laboratory settings, information about listening effort has been deduced from pupil and cardiovascular responses independently. The extent to which these measures can jointly predict listening-related factors is unknown. Here we combined pupil and cardiovascular features to predict acoustic and contextual aspects of speech perception. Data were collected from 29 adults (mean  =  64.6 years, SD  =  9.2) with hearing loss. Participants performed a speech perception task at two individualized signal-to-noise ratios (corresponding to 50% and 80% of sentences correct) and in two social contexts (the presence and absence of two observers). Seven features were extracted per trial: baseline pupil size, peak pupil dilation, mean pupil dilation, interbeat interval, blood volume pulse amplitude, pre-ejection period and pulse arrival time. These features were used to train k-nearest neighbor classifiers to predict task demand, social context and sentence accuracy. The k-fold cross validation on the group-level data revealed above-chance classification accuracies: task demand, 64.4%; social context, 78.3%; and sentence accuracy, 55.1%. However, classification accuracies diminished when the classifiers were trained and tested on data from different participants. Individually trained classifiers (one per participant) performed better than group-level classifiers: 71.7% (SD  =  10.2) for task demand, 88.0% (SD  =  7.5) for social context, and 60.0% (SD  =  13.1) for sentence accuracy. We demonstrated that classifiers trained on group-level physiological data to predict aspects of speech perception generalized poorly to novel participants. Individually calibrated classifiers hold more promise for future applications.

Pupillary Responses Reflect Dynamic Changes in Multiple Cognitive Factors During Associative Learning in Primates.

Associative learning involves complex interactions of multiple cognitive factors. While adult subjects can articulate these factors verbally, for model animals such as macaques, we rely on behavioral outputs. In our study, we used pupillary responses as an alternative measure to capture these underlying cognitive changes. We recorded the dynamic changes in the pupils of three male macaques when they learned the associations between visual stimuli and reward sizes under the classical Pavlovian experimental paradigm. We found that during the long-term learning process, the gradual changes in the pupillary response reflect the changes in the cognitive state of the animals. The pupillary response can be explained by a linear combination of components corresponding to multiple cognitive factors. These components reflect the impact of visual stimuli on the pupils, the prediction of reward values associated with the visual stimuli, and the macaques' understanding of the current experimental reward rules. The changing patterns of these factors during interday and intraday learning clearly demonstrate the enhancement of current reward-stimulus association and the weakening of previous reward-stimulus association. Our study shows that the dynamic response of pupils can serve as an objective indicator to characterize the psychological changes of animals, understand their learning process, and provide important tools for exploring animal behavior during the learning process.