Unraveling the Complex Role of Orexin in Pupil Dilation and Arousal


The orexin (hypocretin) system, located in the lateral hypothalamus (LH), is known to have extensive projections throughout the brain, particularly to areas involved in arousal and reward.

These projections regulate various physiological processes, including sleep-wake cycles, autonomic function, feeding behavior, and exploration.

Pupil dilation, a widely used measure of arousal and autonomic function in human experiments, has been shown to be influenced by the orexin network, along with other neuromodulatory systems such as the locus coeruleus (LC) noradrenergic system, cholinergic, and serotonergic systems.

However, the precise role of orexin in modulating pupil dilation and the interplay between arousal and reward representations in individual orexin neurons remain largely unexplored.

Causal Link between Orexin and Pupil Dilation

To investigate the direct effect of orexin on pupil dilation, researchers conducted experiments using optogenetic stimulation of LH orexin neurons while tracking pupil diameter in anesthetized mice. The results showed that stimulation of orexin neurons led to rapid and frequency-dependent pupil dilation, which returned to baseline levels after the stimulation ended. These findings provided strong evidence for a causal link between orexin neurotransmission and pupil size modulation.

Role of Orexin Neurotransmission in Pupil Dilation

To isolate the specific contribution of orexin neurotransmission to pupil dilation, the researchers used an orexin receptor antagonist called almorexant (ALM) during the optogenetic stimulation experiment. They found that blocking orexin receptors with ALM reduced both baseline pupil dilation and the extent of dilation during orexin cell optostimulation.

Interestingly, the initial rapid dilation at the onset of optostimulation was similar in ALM-injected mice compared to control mice but diverged after a few seconds, indicating that fast transmitters released by orexin cells were responsible for the initial dilation. These results firmly established the role of orexin neurotransmission in controlling pupil size.

Effects of Orexin Neuron Ablation on Pupil Modulation

To further investigate the involvement of orexin neurons in pupil control, researchers selectively ablated these neurons using a diphtheria toxin receptor (DTR) mouse model. Contrary to recent reports suggesting a role for orexin cells in light-induced pupil constriction, the study found no significant difference in light-induced constriction between orexin-cell-ablated and control mice.

However, during resting epochs, orexin-cell-ablated mice exhibited significantly reduced pupil size compared to control mice. Interestingly, during running, the differences in pupil size between the two groups were not significant. These findings indicate that orexin neurons are essential for maintaining normal pupil size, especially during resting periods.

Encoding of Arousal, Reward, and Movement in Individual Orexin Neurons

The activity of orexin neurons is known to be influenced by various factors, including reward and locomotion. To investigate the coding of pupil size, reward, and locomotion in individual orexin neurons, researchers used volumetric two-photon imaging in the LH of behaving mice.

They found that pupil size closely followed orexin cell activity, with some cells positively correlated (pupil ON cells) and others negatively correlated (pupil OFF cells) with pupil size.

These different cell types were pooled for further analysis as no significant differences were found in how they coded for other variables. The study also identified cells whose activation was associated with reward consumption. By employing an encoding model based on multivariate linear regressions, researchers determined that pupil size and reward were distributed across orexin neurons.

While some cells coded for both pupil and reward, others represented only one of these variables, with a larger proportion of cells coding exclusively for pupil size. These findings highlight the dual role of orexin neurons in encoding both arousal and reward, with variable contributions from individual cells.

Comparisons with Noradrenergic System and LC-Orexin Interactions

In addition to exploring the role of orexin in pupil modulation, the study also compared the tracking of pupil size with orexin neuron activity and the activity of LC noradrenaline neurons using population-level fiber photometry in behaving mice. The results revealed strong correlations between pupil size and the activity of both orexin and LC noradrenaline neurons, suggesting that both systems play a similar role in modulating pupil dilation.

Furthermore, investigations into LC-Orexin interactions indicated that while orexin cell activation led to pupil dilation, antagonism of orexin receptors did not affect pupil dilation evoked by LC noradrenaline cell stimulation. On the other hand, manipulations suppressing LC noradrenaline cell function abolished the effect of orexin cell body stimulation on pupil dilation.

These findings suggest that there is bidirectional signaling between the LC and orexin systems, with orexin cell→LC signaling influencing the impact of orexin cells on pupil dilation.

Implications and Future Directions

The study provides compelling evidence for the involvement of the orexinergic system in the control of pupil dilation. It demonstrates both tonic and phasic effects of orexin cell activity on pupil size and reveals the role of LC as a mediator of this effect. Orexin neurotransmission, possibly through the release of fast transmitters like glutamate, was found to be crucial for rapid-onset effects on pupil dilation.

Furthermore, the loss of orexin neurons was shown to affect pupil size during resting periods but not during running, suggesting the involvement of other neural systems in maintaining pupil dilation during locomotion. The research also highlights the distributed coding of pupil size, reward, and locomotion in individual orexin neurons, emphasizing the dichotomy of orexin function in representing both arousal and reward.

The findings have implications for understanding the role of orexin cells in arousal gain control and the exploration-exploitation axis. Orexin neurons may play a role in modulating arousal gain during exploratory behavior, and the influence of reward consumption on orexin neurons could be crucial for transitioning between exploration and exploitation.

Additionally, orexin neurons may act as a centerpiece in mediating transitions during ethologically relevant behaviors such as foraging, where metabolic state signaling via nutrients and hormones could play a significant role. Furthermore, the results have potential applications in diagnosing orexin cell loss in neurological disorders.


The study provides a comprehensive exploration of the orexin system’s involvement in pupil dilation and its interplay with arousal and reward representations. It establishes a causal link between orexin neurotransmission and pupil size and reveals the complex coding properties of individual orexin neurons. The findings contribute to our understanding of the orexin system’s role in arousal, reward, and locomotion, and open avenues for further research into the broader implications of orexin-mediated pupil modulation and its relevance to neurological disorders.

in deep …..

Orexin, also known as hypocretin, is a neuropeptide that regulates arousal, wakefulness, and appetite. It was discovered in 1998 by two independent groups of researchers who named it after its orexigenic (appetite-stimulating) and hypocretin (hypothalamus-secreted) properties . Orexin is produced by a small number of neurons in the lateral hypothalamus, which project widely throughout the central nervous system and modulate various physiological processes .

Orexin exists in two forms, orexin-A and orexin-B, which differ in their amino acid sequence and affinity for the two orexin receptors, OX1R and OX2R. These receptors are G-protein-coupled receptors that activate different intracellular signaling pathways and mediate diverse effects of orexin . Orexin receptors are expressed in various brain regions, such as the cerebral cortex, thalamus, hypothalamus, brainstem, and spinal cord, as well as in peripheral tissues, such as the adrenal gland, pancreas, heart, and blood vessels .

Orexin has been implicated in many physiological functions, especially those related to the regulation of the sleep-wake cycle, feeding behavior, energy homeostasis, neuroendocrine functions, glucose metabolism, stress responses, and reward-seeking and drug addiction . Orexin is essential for maintaining wakefulness and preventing narcolepsy, a disorder characterized by excessive daytime sleepiness and sudden loss of muscle tone (cataplexy). Narcolepsy is caused by a lack of orexin in the brain due to the destruction of orexin-producing neurons or mutations in the orexin or orexin receptor genes .

Orexin also stimulates food intake and influences the preference for high-fat and high-sugar foods. Orexin interacts with other appetite-regulating hormones, such as leptin, ghrelin, insulin, and glucagon-like peptide-1 (GLP-1), to modulate energy balance and body weight . Orexin may also affect glucose metabolism by regulating insulin secretion and sensitivity, as well as glucose uptake and utilization in peripheral tissues .

Orexin is involved in the modulation of neuroendocrine functions by influencing the secretion of various hormones, such as cortisol, growth hormone, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, oxytocin, and vasopressin . Orexin may also affect reproductive functions by regulating sexual behavior and fertility .

Orexin plays a role in the adaptation to stress by activating the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Orexin may also modulate the emotional and behavioral responses to stress by influencing the activity of brain regions involved in fear, anxiety, depression, and social interaction .

Orexin is implicated in the regulation of reward-seeking and drug addiction by modulating the activity of the mesolimbic dopamine system. Orexin may enhance the motivation for natural rewards, such as food and sex, as well as for drugs of abuse, such as cocaine, nicotine, alcohol, and opioids. Orexin may also affect the development of tolerance and withdrawal symptoms to these drugs .

Orexin is a key neuropeptide that regulates many aspects of physiology and behavior that are essential for survival and well-being. Orexin may also be involved in pathological processes of various neurological diseases, such as narcolepsy, depression, ischemic stroke, drug addiction, and Alzheimer’s disease . Therefore, understanding the molecular mechanisms and therapeutic potential of the orexin/receptor system may provide new insights into the prevention and treatment of these diseases.


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