Light is an environmental stimulus of primary importance to all forms of life, playing a fundamental role in the regulation of circadian rhythms and influencing a wide range of physiological and behavioral functions in living organisms (1, 2). However, with the increasing use of electronic devices, human exposure to light has increased significantly. For many people, this means that overall exposure to light from electronic devices can now total 8–10 h a day versus 3–5 h in earlier decades (3). The evening period is particularly critical because many people now extend their device use well into the hours before sleep, which significantly increases exposure to blue-enriched light (4–9).

Blue light is a high energy electromagnetic radiation in the wavelength range of 380–500 nm, known to be able to penetrate ocular tissues and reach the retina and deeper brain regions (10). Modern electronic devices such as smartphones, tablets, and laptops emit blue light with spectral peaks between 445 and 455 nm. Peak irradiance at 450 nm reaches up to 0.00526 mW/cm² on laptops and 0.00102 mW/cm² on smartphones under typical usage conditions—levels that, while below sunlight intensity, raise concerns about cumulative exposure and its long-term effects on ocular and neural health (11). In humans, prolonged exposure to blue light has been associated with retinal phototoxicity, circadian rhythm disruption, and neurodegeneration (12–14). Evidence from experimental and clinical studies suggests that even moderate levels of artificial light exposure (100–1,000 lux), especially in the evening, can suppress melatonin production, disrupt sleep and other biological processes controlled by the body’s circadian clock, and cause adverse effects, including mental disorders such as depression, anxiety, bipolar disorder and self-mutilation (15), and metabolic disorders such as cardiovascular disease and cancer (4–9, 16).

Although direct evidence in humans remains limited, these findings in animal models underscore the need to better understand the biological consequences of chronic blue light exposure. Prolonged exposure to blue light can induce early puberty in male rats, suppress spermatogenesis, and impair testicular integrity (17), highlighting the broader physiological effects of light exposure. Studies in both cell culture and animal models have also shown that blue light can accelerate aging, significantly reduce lifespan, and promote molecular stress responses in neural tissues, including oxidative stress and DNA damage (9, 14, 18).

The main photoreceptor shared between invertebrates and mammals are opsins, photosensitive membrane proteins associated with a chromophore, that change conformation from a resting state to a signaling state during the phototransduction process after the absorption of a photon (19, 20). In humans, rod photoreceptors contain rhodopsin (OPN2), which is responsible for twilight vision, while cone photoreceptors contain opsins (OPN1), which are responsible for daytime vision and can be divided into four subgroups according to their absorption spectra: long (LW or red), short 1 (SW1 or UV/violet), short 2 (SW2 or blue) and medium (MW) (20, 21). In addition to the visual opsins, animals also express non-visual opsins such as melanopsins (Opn4); encephalopsins or panopsins (Opn3); neuropsins (Opn5) and the retinal photoisomerase G protein-coupled receptor (RGR) (20, 22–24). These non-visual opsins are present in several structures besides the retina, including the brain, testicles, liver, skin, spinal cord and lungs, suggesting an extra-retinal photomodulation (22). Drosophila has seven opsin genes (Rh1, Rh2, Rh3, Rh4, Rh5, Rh6 and Rh7) (20, 25), which encode their corresponding proteins (rhodopsins). Rh1’s predominant expression in the outer photoreceptors occurs directly when exposed to environmental light (26). In contrast, Rh7 is expressed in the central brain, particularly in circadian pacemaker neurons, and has been proposed to mediate non-visual responses to light (27). However, its role in light-induced neurodegeneration remains poorly understood.

Studies in animals with ablated eyes or retinal degeneration show that physiological responses to light—such as pupillary reflexes, adjustments in circadian rhythms, early mortality and brain neurodegeneration (remain when the light source is removed), raising questions about the mechanisms of light perception in the organism (9, 18, 22, 28–30).

Advancements in the field have revealed the involvement of non-visual opsins in a variety of physiological functions, including the regulation of circadian rhythms (31), hormonal secretion (32), thermoregulation (33), and neuronal activity (34). For instance, Opn4 plays a central role in circadian entrainment through light detection in retinal ganglion cells; however, its expression in other tissues suggests the presence of additional functions (35). A similar observation has been made with Opn3 and Opn5, which have been implicated in metabolic regulation and neuroendocrine signaling (33, 36). The precise phototransduction mechanisms of these opsins in extra-retinal tissues remain to be elucidated; however, experimental models have demonstrated that these proteins can respond to light stimuli, even in the absence of ocular input, thereby supporting the hypothesis of peripheral light sensing (37). However, the mechanisms through which these non-visual opsins, particularly in invertebrate models, contribute to the physiological effects of light exposure, especially in the context of neurodegeneration, remain to be elucidated.

Animal models such as the Drosophila melanogaster fly have been used to investigate the role of different opsins due to functional homology with human photoreceptive cells (9, 38). of which we highlight two main ones: rhodopsin 1 (Rh1), encoded by the ninaE gene, present in the outer photoreceptor cells of the eye and orthologous to human OPN4 (39), and rhodopsin 7 (Rh7), present in the central brain in a subset of circadian pacemaker neurons (25, 39). Previous studies have shown that excessive activation of Rh1 by high-intensity or prolonged light exposure can lead to retinal degeneration in Drosophila, characterized by photoreceptor cell death and structural damage (40).

In this investigation, Rh1 and Rh7 were selected as the subjects of study due to their status as the most well-characterized visual and non-visual opsins, respectively, in the Drosophila model. Rh1 is the most abundantly expressed rhodopsin in the retina and is essential for phototransduction in the outer photoreceptor cells. Conversely, Rh7 is uniquely expressed in the brain, particularly in neurons involved in circadian regulation, and is considered the only known non-visual opsin in Drosophila. This makes Rh7 a compelling candidate for evaluating extra-retinal light effects on the nervous system.

In this study, the primary objective was to evaluate the role of visual and non-visual opsins in the central nervous system of Drosophila melanogaster following exposure to blue light. To this end, we employed genetically modified flies lacking Rh1 and Rh7, and we conducted a comprehensive analysis of DNA damage and vacuole formation. These phenomena are well-established indicators of neurodegeneration, and our study sought to elucidate the underlying mechanisms through which these light-sensitive proteins influence the brain’s response to electromagnetic radiation. The study yielded direct evidence for the differential contribution of visual (Rh1) and non-visual (Rh7) opsins to the negative effects of blue light exposure on the nervous system, underscoring their potential as targets in neuroprotection strategies.

The experimental workflow in Figure 1 illustrates the key steps of this study, from fly stock maintenance and experimental group assignment to outcome measurements in Drosophila melanogaster, including DNA damage and vacuole quantification analyses (Figure 1).

The relevance of studying neurodegeneration in Drosophila melanogaster has increased in recent years, as several studies have shown that excessive light exposure, especially at blue wavelength range (~450–490 nm), can induce neuron death. In this work, we investigated the effects of blue light exposure in Drosophila melanogaster flies with loss of function of visual and non-visual opsins. Our analyses show that ablation of different opsins (Rh1, a light-sensitive pigment, and Rh7, typically associated with non-visual functions) leads to distinct profiles of DNA damage (γ-H2Av) and vacuole formation in the brain of Drosophila. These findings support the hypotheses that each opsin contributes to photosensitivity and intracellular signaling in unique ways, culminating in distinct patterns of neurodegeneration under light exposure (25, 38). The Rh1¹ strains showed increased DNA damage under blue light, compared to w¹¹¹⁸ and Rh7¹, supporting the notion that blocking the classical phototransduction pathway enhances DNA damage. The same pattern is observed across genotypes with respect to vacuole-associated neurodegeneration. The formation of neurodegeneration vacuoles in Drosophila indicates neuronal tissue loss and permanent brain damage (50), which can be assessed through the quantification of actin and synaptic protein filaments.

Quantitative analysis of Figures 6, 8 revealed an increase in γ-H2Av-positive cells and a modest rise in brain vacuolization in Rh1¹ flies compared to w¹¹¹⁸ controls. Compared to Rh7¹ flies, the Rh1¹ strain also showed a trend toward higher numbers of γ-H2Av-positive cells and slightly more vacuolization. These results suggest that the absence of the visual opsin Rh1¹ may increase susceptibility to blue light-induced neurodegeneration, supporting a potential role of Rh1¹ in mediating light-induced neurodegeneration in the central nervous system. Although the Rh7-ablated flies showed less damage under blue light than the Rh1-ablated ones, it was still greater than the w¹¹¹⁸ control.

Based on Figure 7, we identified and listed the Drosophila brain regions in which the vacuoles were observed. The analysis of the brain of Drosophila on the Codex platform (54) allowed us to highlight potential areas that are more vulnerable to neurodegeneration (Figure 9). The input regions represent the connections that transport information from other areas or sensory neurons to the brain. Output regions extend axonal connections from the neuropil to motor neurons or other brain areas.

Interestingly, vacuole was not equally distributed throughout the brain, but concentrated in specific regions, such as some ventrolateral regions of the protocerebrum (AVLP—Figure 9D, PVLP—Figure 9E) and periesophageal neuropils (WED—Figure 9F, PLP—Figure 9G), according to our volumetric analysis. Previous studies have linked the occurrence of vacuolization in specific regions of the Drosophila brain to local dysfunction of neurons and glial cells, which can culminate in severe functional impairment (55). When we cross-referenced our vacuole localization findings with the data available on the Codex platform for these regions (such as AVLP, PVLP, WED, PLP), we found an enrichment of excitatory (acetylcholine, glutamate) and inhibitory (GABA) neurotransmitter inputs and outputs, suggesting that these areas may be particularly vulnerable to oxidative stress when light-dependent processes—whether visual or non-visual—are compromised. This is consistent with studies showing that an imbalance between excitatory and inhibitory signaling can exacerbate degenerative processes. There is also evidence that excessive glutamatergic signaling contributes to neurodegenerative disorders in mammals, including Alzheimer’s disease (56). Furthermore, dysregulation of the excitatory/inhibitory balance, in part mediated by GABA, may accelerate neuronal degeneration and potentially contribute to cognitive and motor deficits.

These two groups were then sorted into intrinsic or afferent. Intrinsic connections are formed by local neurons that synapse within the same area without projecting signals to distant regions. A high percentage of intrinsic connectivity indicates strong self-regulation, with internal circuits responsible for processing information locally. In contrast, afferent connections bring sensory or modulatory information from other parts of the nervous system. Therefore, regions with high afferent connections may be more vulnerable to damage caused by light exposure, as connection loss can lead to more severe dysfunctions. The periesophageal neuropil (Figure 9A) has lower intrinsic connectivity among all the evaluated regions (approximately 72–74%), suggesting greater activation by external afferents associated with Rh7¹ photo sensing.

The formation of vacuoles in the brains of flies possibly affects neurotransmitter balance and, consequently, behavior. The main neurotransmitters listed in the inputs and outputs of Figure 9 are Acetylcholine (ACH), GABA and Glutamate (GLUT), associated with anxious behavior (57), neurodegenerative processes (56), and ageing (58).

The right antennal lobe (Figure 9C) has the highest proportion of afferent connections (~68.4% output and 53.1% input), which may have been compromised by vacuole formation in all groups (Figure 7). Furthermore, regions such as the posterior ventrolateral (PVLP_L—Figure 9E) and posterior lateral protocerebrum (PLP_L—Figure 9E) have high intrinsic connectivity (>96%), indicating more preserved circuits and less vulnerability to external damage. The high number of vacuoles in those regions suggest that areas with greater dependence on external stimuli may be more vulnerable to light-induced neurodegeneration.

The occurrence of vacuolization in areas of high afferent connectivity indicates that the degeneration is affecting circuits that receive external inputs. This affects the sensory response and behavioral modulation of γ-H2Av-positive Rh1¹ flies more prominently.

One hypothesis for the observed differences between the roles of visual and non-visual opsins is that, in situations of excessive blue light, neural pathways mediated by non-visual opsins (such as Rh7) are disturbed. This interaction could disrupt tissue homeostasis, leading to cumulative DNA damage and subsequent vacuolization. The absence of Rh1 would exacerbate this effect, perhaps due to the loss of protective feedback normally mediated by the retina, such as AMP-activated protein kinase (AMPK) (59), which results in increased vacuolization in the affected regions.

This study offers valuable insights, though some limitations must be acknowledged. The limited number of biological replicates reduces statistical power; however, the consistency of results across replicates and methods supports the reliability of our findings.

Also, we employed a continuous exposure paradigm (24 h/day for 20 days), which, while useful to maximize cumulative dose and reveal clear effects, does not capture the potential influence of light–dark cycles, intermittent exposures, or different dose–response regimes. Furthermore, our analyses were focused on γ-H2Av as a marker of DNA damage and vacuolization as a marker of neurodegeneration; other relevant cellular pathways, including oxidative stress, apoptosis, synaptic integrity, and glial responses, were not assessed here. It would also be valuable to assess behavioral outcomes such as locomotor performance, and to explore rescue experiments in which Rh1 or Rh7 expression is restored. These approaches will help refine our understanding of how visual and non-visual opsins differentially modulate the neuronal response to blue light.

Future research should also include more detailed temporal analyses of damage progression throughout development and adulthood of animal models. Additionally, it will be essential to investigate the contribution of specific neural circuits and glial populations in these brain regions to elucidate how the absence of an opsin may activate distinct pathways of neurodegeneration in response to light.

Excessive blue light exposure is increasingly recognized as a public health concern, particularly due to widespread use of digital devices. Although the compound eye of Drosophila differs structurally from the camera-type eye of mammals, both systems rely on opsins as light-sensitive G-protein coupled receptors and share conserved downstream signaling cascades.

This study helps to elucidate the mechanisms by which different opsins modulate the cellular response to blue light and supports the idea that classical photoreceptors Rh1 influence processes beyond the retina. Additionally, our data suggests that non-visual opsins such as Rh7 may also participate in mediating photosensitivity within the brain. We show that visual opsins, specifically Rh1, are key mediators of blue light-induced DNA damage in flies’ brains. This aligns with mammalian studies reporting that excessive blue light can impair mitochondrial function, generate reactive oxygen species, and trigger DNA damage in retinal photoreceptors and ganglion cells, ultimately contributing to neurodegeneration and vision-related disorders such as glaucoma and age-related macular degeneration. Therefore, despite anatomical differences, the molecular vulnerability to high-energy blue light is conserved across species, underscoring the translational relevance of our findings and their potential impact on human health.