Relevance of Electrical Light on Circadian, Neuroendocrine, and Neurobehavioral Regulation in Laboratory Animal Facilities

Abstract

Light is a key extrinsic factor to be considered in operations and design of animal room facilities. Over the past four decades, many studies on typical laboratory animal populations have demonstrated impacts on neuroendocrine, neurobehavioral, and circadian physiology. These effects are regulated independently from the defined physiology for the visual system. The range of physiological responses that oscillate with the 24 hour rhythm of the day include sleep and wakefulness, body temperature, hormonal secretion, and a wide range of other physiological parameters. Melatonin has been the chief neuroendocrine hormone studied, but acute light-induced effects on corticosterone as well as other hormones have also been observed. Within the last two decades, a new photosensory system in the mammalian eye has been discovered. A small set of retinal ganglion cells, previously thought to function as a visual output neuron, have been shown to be directly photosensitive and act differently from the classic photoreceptors of the visual system. Understanding the effects of light on mammalian physiology and behavior must take into account how the classical visual photoreceptors and the newly discovered ipRGC photoreceptor systems interact. Scientists and facility managers need to appreciate lighting impacts on circadian, neuroendocrine, and neurobehavioral regulation in order to improve lighting of laboratory facilities to foster optimum health and well-being of animals.

Introduction

Light is a key component to life on earth. The sun’s irradiance provides the biosphere with warmth and energy for photosynthesis, as well as signals that all living organisms use to link themselves to the natural day/night rhythm generated by the rotation of the planet [1]. Light supports vision and allows organisms to navigate their surroundings, but it also operates at a level below consciousness, controlling the daily hormonal rhythms that regulate homeostasis.

Throughout nature, a range of physiological responses oscillates with the 24-hour rhythm of the day, ie, a circadian rhythm. The circadian timing system controls daily rhythms such as sleep and wakefulness, body temperature, hormonal secretion, and a wide range of other physiological parameters. These inherent rhythms persist even when the organism’s environment remains in a constant state. This indicates that these rhythms are under the control of endogenous oscillators or the circadian timing system, which allows the organism to anticipate and prepare for the profound changes in its natural environment at dawn and dusk. Although light is the primary stimulus for regulation of the circadian system [2], other external stimuli such as the timing of sound, temperature, and social cues may also influence circadian physiology [3,4].

Light is a key extrinsic factor to be considered in operations and design of animal room facilities. Over the past 4 decades, many studies on typical laboratory animal populations have demonstrated impacts on neuroendocrine, neurobehavioral, and circadian physiology [5]. These effects, for the most part, are regulated independently from the defined physiology for the visual system [6,7]. Selected studies have been identified and are listed in Table 1 for reference. The research areas and endpoints are varied but represent important contributions to the influence of light on laboratory animal biology and health.

Table 1.

Selected Studies on Influences of Light on Animal Biology and Health

Species	Research Areas/Impacts	Reference
Danio rerio	Effects of lighting conditions and wavelengths during early development for ontogeny of daily gene expression rhythms	(86)
Danio rerio	Deep brain photoreceptors and directly light-entrainable circadian pacemakers	(87)
Danio rerio	Sound and light effects on spatial distribution and swimming behavior	(88)
Danio rerio	Quantitative responses of adult zebrafish to changes in ambient illumination	(89)
Danio rerio	Gene expression in response to sleep deprivation and light and dark periods	(90)
Danio rerio	Characterized light-induced gene transcription in zebrafish at several organizational levels	(91)
Drosophila melanogaster	Effects of disconnected visual system mutations and disruption of circadian rhythms	(92)
Drosophila melanogaster	Light-induced degradation of proteins and entrainment of circadian clock	(93)
Eylais extendens	Impact of temperature and periods of light on hatching of larvae	(94)
Gallus Gallus domesticus	Light-emitting diode light vs fluorescent light on growing performance, activity levels, and well-being	(95)
Gallus gallus	Effects of monochromatic light on immune response of broilers	(96)
Homo sapiens	Capacity of different irradiances of monochromatic light to reduce plasma melatonin in normal humans	(97)
Homo sapiens	Action spectrum of melatonin suppression	(98)
Homo sapiens	Bright light can reset human circadian Pacemaker that controls daily variations in function	(99)
Homo sapiens	Light and night and breast cancer	(100)
Homo sapiens	Correlation of seasonal clock changes and medical leaves due to ulcerative colitis and Crohn’s disease	(101)
Homo sapiens	Phase response curves in response to single bright light exposure	(102)
Homo sapiens	Bright light suppresses melatonin secretion in humans	(103)
Homo sapiens	Novel short-wavelength photopigment in light-induced melatonin suppression	(104)
Homo sapiens	Testing whether blue-enriched polychromatic light has increased efficacy for melatonin suppression, circadian phase-shifting, and alertness	(105)
Homo sapiens	Circadian resetting response in humans is wavelength dependent	(106)
Macaca mulatta	Exposure to light or melatonin shifts phase of intrinsic circadian rhythms, dependent on timing of stimulus	(107)
Macaca mulatta	Distinct population of intrinsically photosensitive melanopsin-expressing ganglion cells in retina	(64)
Mesocricetus auratus	Effects of different light spectra on suppression of pineal melatonin content	(24)
Mesocricetus auratus	Spectral sensitivity of photorecepters mediating entrainment of mammalian circadian rhythms	(23)
Mesocricetus auratus	Photic sensitivity for circadian response to light varies with photoperiod	(108)
Mus	Distribution of ipRGC subtypes across mouse retina with anatomical and functional specialization	(109)
Mus	Blue-enriched LED light promotes homeostatic regulation of health and well-being	(84)
Mus	Melanopsin and rod–cone photoreceptive systems account for all major accessory visual functions	(56)
Mus	Phase of peripheral clocks responds to a photoperiodic lights-off signal as well as feeding and adrenal entrainment stimuli	(110)
Mus	Behavioral and molecular consequences of light and dark phase testing	(111)
Mus	Effects of feeding during light or dark period in mice kept on long or short days	(112)
Nauphoeta cinerea	Photoperiod-dependent release of suppression pheromone	(113)
Onchidium reevesii	Effects of circadian rhythms and light dark cycle masking on daily transcriptional oscillations of long- term memory	(114)
Rattus	Blue light exposure and prostate cancer metabolic signaling and proliferative activities	(83)
Rattus	Phototransduction in ganglion cells as primary photoreceptors	(42)
Rattus	Effects of light at night on pineal gland serotonin levels	(15)
Rattus	Effects of varying ambient light intensity on phase and amplitude of urinary melatonin rhythms	(72)
Rattus	Effects of light and color intensity of body temperature entrainment	(21)
Rattus norvegicus	Retinal photopigment that mediates pineal response to light in rats	(19)
Rattus norvegicus	Filtering narrow bandwidth of light from nocturnal lighting may efficiently attenuate overall disruption of circadian endocrine rhythms	(115)
Rattus norvegicus	Effects of blue light on neuroendocrine, metabolic, and physiological parameters of health in rats	(116)
Saimiri sciureus	Circadian rhythms of locomotor activity under conditions of self-controlled light-dark cycles	(117)

The primary neuroendocrine hormone affected by light is melatonin. Melatonin, or N-acetyl-5-methoxytryptamine, is an indoleamine hormone synthesized and released by the pineal gland during the hours of darkness in most species studied to date. Melatonin was first isolated by dermatologist Aaron Lerner, who was interested in skin pigmentation issues and the ability of pineal extracts to lighten frog skin [8,9]. Numerous physiological functions are associated with melatonin, chief among them circadian rhythm regulation and seasonal reproduction [10]. Other physiological roles of melatonin include protection of tissues from oxidative stress [11] and oncostatic effects [12].

Cycles of light and dark, which are perceived through the eyes, entrain suprachiasmatic nuclei (SCN) neural activity, which, in turn, entrains the rhythmic synthesis and secretion of melatonin from the pineal gland. In virtually all species including humans, high levels of melatonin are secreted during the night and low levels are secreted during the day [10]. It should be noted here that many mouse strains are melatonin deficient [13], yet 2 commonly used strains, C3H and CBA, have detectable, high-amplitude rhythms [14].

In addition to entraining melatonin synthesis by the pineal gland, light can acutely suppress melatonin synthesis. Specifically, exposure of the eyes to light during the night can cause a rapid decrease in the high activity of the pineal enzyme aralkylamine N-acetyltransferase and subsequent synthesis of melatonin. The acute light-induced suppression of nocturnal melatonin synthesis was first observed in rats [15] and has been used in numerous studies on mammals, including humans, to help determine the neural and biochemical physiology of melatonin regulation [16,17].

While melatonin has been the chief neuroendocrine hormone studied, acute light-induced effects on corticosterone have been studied. Corticosterone levels have been shown to significantly increase with acute light exposure comparable with major physical stressors such as forced swimming [18]. This observation is particularly important when considering laboratory animal facilities where nocturnal light exposure may occur.

Circadian, Neuroendocrine, and Neurobehavioral Regulation by Light Wavelength

Numerous action spectra studies on neuroendocrine and circadian responses to light utilized polychromatic and, later, monochromatic stimuli that have been studied with examining endpoints of pineal melatonin synthesis, circadian phase shifting, and photoperiodic responses [19–29].

The weakest stimulation of circadian and neuroendocrine responses occurs in the longer wavelengths above 550 nm. Interestingly, however, a study in rodents [30] demonstrated that long-wavelength red light exposure (above 620 nm) at night, if of high enough intensity (8.07 ± 0.95 lux; 3.31 ± 0.38 μW/cm²) and duration (60–90 min), results in the disruption of the circadian organization of neuroendocrine, metabolic, and physiological parameters associated with animal health and well-being.

Ultraviolet wavelengths (300–380 nm) are also capable of eliciting circadian and neuroendocrine responses in some mammalian species. A comparison of fluence-response curves between monochromatic wavelengths of 360 nm and 500 nm in Syrian hamsters indicated that ultraviolet (UV)-A is less potent than the visible spectrum for acute melatonin suppression [25]. A single irradiance exposure of a monochromatic 357-nm pulse suggested that UV-A may be equal to or stronger than a monochromatic 515-nm pulse for phase shifting locomotor rhythms in retinal degenerate rodless and wild-type mice [28]. This observation was consistent with an earlier study where UV cones were shown to be more sensitive than M cones for several behavioral and physiological responses in pigmented house mice (Mus musculus) [32]. UV photoreception has been documented extensively across invertebrate and nonmammalian vertebrate species [31, for review] with the identification of specific UV retinal photoreceptors that support vision in rodents [32,33].

Ultimately, studies using monochromatic light exposures in monkeys and rodents have shown that neuroendocrine, neurobehavioral, and circadian responses are maximally sensitive to blue light (between 459 and 483 nm) [7,34–37]. This sensitivity differs from that of the combined photopic response of classical photoreceptors for vision, which peak in the green part of the spectrum (λ max 555 nm). As discussed below, the concurrent identification and localization of a novel photoreceptor paralleled these observations.

Phototransduction for Circadian, Neuroendocrine, and Neurobehavioral Regulation

Living organisms vary in their capacity to use visible and near-visible electromagnetic energy for survival; however, a fundamental principle shared by all species is their ability to respond to light stimuli. All photobiological responses are mediated by organic molecules that absorb light quanta and then undergo physical-chemical changes. These light-induced changes subsequently evoke broader physiological responses within the organism. This process is termed phototransduction, and the specific organic molecules that absorb light energy to initiate photobiological responses are called chromophores or photopigments. As a rule, these photoactive molecules do not absorb energy equally across the electromagnetic spectrum. Photopigment molecules or molecular complexes have their own characteristic wavelength absorption spectrum that depends on their atomic structure [37–40]. A photopigment’s pattern of wavelength sensitivity, or its absorbance spectrum, is unique to that molecule.

The classical visual photoreceptors reside in a thin sheet of neural tissue located in the lining of the back of the eye called the retina. This layer extends forward toward the optical components of the eye, which include the cornea, iris, and lens. The retina was thought to be composed of 5 classes of neurons: photoreceptors, horizontal cells, bipolar cells, amacrine cells, and ganglion cells [41]. More recently, a subset of ganglion cells, the intrinsically photosensitive retinal ganglion cells (ipRGCs), have been identified and described [42–44].

A photopigment named melanopsin has been localized both in the retinas of rodents and humans [45]. Melanopsin is found in a subtype of intrinsically photoreceptive retinal ganglion cells or ipRGCs [42–43,46]. Using immunohistochemistry and double labeling studies in mice, ipRGC anatomy has been described, and 5 types of ipRGCs, which have differing locations, dendritic processes, and cell bodies, have been identified in rodents [47]. Similarly, a family of melanopsin-containing ipRGCs has been identified in the human retina [48].

In contrast to rods and cones, melanopsin is thought to act as both a photopigment and a photoisomerase [45]. Following activation of melanopsin by light, a different portion of the light spectrum regenerates the chromophore and restores melanopsin photosensitivity [49,50]. Although it is well-established that melanopsin photopigment is most sensitive to short-wavelength light, recent studies suggest that melanopsin photoisomerase activity may increase this sensitivity by prior exposure to long-wavelength light [51–53]. However, potentiation by long-wavelength (620 nm) light prior to exposure to blue (480 nm) light as measured by ipRGC cell firing rates in mouse retina was not exhibited in a study by Mawad and Van Gelder [54]. Further studies by Emanuel and Do [55] provide evidence that light is distributed across melanopsin in 3 states or tristability, which permits the temporal summation across wavelengths observed in ipRGC signaling.

Abundant evidence shows that the melanopsin-containing ipRGCs provide primary input for circadian and neuroendocrine regulation. It has also been shown, however, that rod and cone photoreceptors still play a role in this physiology. For example, when considering the phase-shifting action spectra in mice lacking rods and cones, a peak wavelength sensitivity of 480 nm was determined [56]; yet in wild-type mice, action spectra revealed that this peak wavelength sensitivity was closer to 500 nm, indicating involvement of rods and cones [57,58]. Melanopsin- and cone-knockout mice show that the classical rod and cone photoreceptors can compensate for the loss of melanopsin and at least partially mediate light-induced circadian, neuroendocrine, and neurobehavioral responses [7,59–61]. By contrast, when melanopsin is knocked out and the classical visual photoreceptors are compromised, animals lose all visual and nonvisual photoreceptive functions of the eye [62,63]. Further, cellular recording studies from nonhuman primate retinas have demonstrated that rod and cone cells can directly activate ipRGCs [64].

Neural Pathways for Circadian and Neurophysiological Regulation

Neural projections from the ipRGCs form the origin of the retinohypothalamic tract (RHT). The mammalian RHT is the primary neural projection to the circadian oscillator for entraining circadian rhythms to environmental light-dark cycles [65]. This pathway acts to convey information about external light conditions from the retina to several areas of the hypothalamus, including the SCN—the primary site of the biological clock or central timekeeper in the brain. The RHT terminates primarily in the ventrolateral area of the SCN. In turn, the SCN transmit information about lighting and circadian time to a diversity of major control regions of the nervous system, including the pineal gland, where the hormone melatonin is synthesized. The RHT has been studied extensively for its role in synchronizing the endogenous oscillator in the SCN with environmental light cues and mediating systemic circadian physiology [65–67].

Although the RHT has its densest projection in or around the SCN, this pathway has diffuse projections to other areas, including the preoptic nuclei, anterior and lateral hypothalamic areas, retrochiasmatic area, dorsal hypothalamic nuclei, the intergeniculate leaflets, and the midbrain pretectal area [65,67]. The ventrolateral preoptic nucleus is known to be integral in the sleep/arousal state. Additionally, projections to the intergeniculate leaflet from the retina are involved in regulation of circadian phase shifting and other integration of photoperiodic information. Projections to the ventral subparaventricular zone are thought to be involved in the circadian and photic modulation of sleep and locomotor activity. The pretectal area receives projections from the RHT, which contributes to the control of the pupillary light response. In summary, these projections, which are relatively separate from areas of the brain that are involved in supporting vision, are thought to form an irradiance detection system providing photic information to several brain regions controlling numerous physiological functions [66]. Figure 1 provides a simplified illustration of the neural anatomy that support vision and circadian, neuroendocrine, and neurobehavioral responses [66,68].

Figure 1.

The diagram above is a highly simplified schematic of the neuroanatomy responsible for mediating both the sensory capacity of the visual system and the nonvisual regulation of circadian, neuroendocrine, and neurobehavioral functions. IGL = intergeniculate leaflets; ipRGCs = intrinsically photosensitive retinal ganglion cells; POT = primary optic tract; PTA = pretectal areas; RHT = retinohypothalamic tract; SCN = suprachiasmatic nuclei; VLPO = ventrolateral preoptic nuclei; vSPZ = ventral subparaventricular zones. This figure is modeled after an illustration in [85] and adapted from a diagram in [67].

Circadian, Neuroendocrine, and Neurobehavioral Regulation by Light Intensity

Threshold sensitivities to light response vary among mammalian species. There is a known dose-response effect of irradiance and suppression of melatonin in the pineal gland of hamsters [25,69]. Hamsters and other nocturnal rodents are very sensitive to light for suppression of pineal melatonin—as much as 150 times more sensitive than humans [70]. Recent analysis for threshold sensitivities to differing biological responses for a number of laboratory rodents revealed that under certain light sources, as little as 0.01 lux may be sufficient to elicit a biological response [71].

A seminal study by Lynch and colleagues [72] showed that with adaptation, rats could respond to a dim stimulus as either a light or dark signal in terms of circadian regulation depending on what light intensity was available for the rest of the day. For 17 days, 1 group was exposed to alternating 12-hour periods of dim light and total darkness (dim to dark), while a second group was exposed alternately to dim light and bright light (dim to bright). Both groups were then exposed to constant dim light for 15 days, returned to their original lighting conditions, and 18 days later, one-half of each group was killed at the midpoint of the dim light phase and the other one-half 12 hours later. Both groups excreted melatonin into their urine when exposed to daily cycles in light intensity: the dim to bright rats excreted 69% of the total daily urinary melatonin output during the dim light phase, while the dim to dark group of rats excreted 70% during the dark phase [72].

Additionally, studies in animal models show high sensitivity to phase shifting by light after exposure to extended periods of darkness [73,74]. In fact, maintaining absolute darkness during the animal’s dark phase is essential. As little as 0.2 lux of light has been shown to alter circadian rhythms [75] as well as cause increases in oncogenesis via melatonin suppression in rats with as little as 0.25 lux [76].

Ocular and Physiological Elements That Mediate the Biological Effects of Light

In the light regulation of biological effects, there are 2 main sets of elements: (1) physical/biological stimulus processing, and (2) sensory/neural signal processing. The physical and biological stimulus-processing elements involved are the light source physics, the conscious and reflex behavior of the animal relative to the light source, and the transduction of light to the retina through the pupil and ocular media. The sensory/neural signal processing is initiated as photons are absorbed by retinal photopigments and neural signals are generated. Factors influencing this physiology include:

1. Wavelength sensitivity of the photoreceptors;

2. Distribution of the photoreceptors;

3. State of photoreceptor adaptation; and

4. Ability of the central nervous system to integrate photic stimuli over space and time.

Each of these elements determines the effectiveness of an environmental photic stimulus for regulating the mammalian physiology.

Examining light source geometry relative to the eye is one example of studying the elements of ocular physiology that affect circadian regulation. Often, application of the experimental light stimulus consisted of simply switching on the overhead room light. In some of those studies, experimental illuminances were measured by placing a light meter at desk height aimed directly at the overhead lights. This measurement technique, although standard for characterizing architectural lighting [77]. may not accurately correspond to the corneal illuminance experienced by the animals. Conscious and reflex behavior such as head movement, eye motion, eye blink, and eye closure influences the biological efficacy of a light stimulus. Cage placement location makes a difference as well, as 3–19 times more light is available to top tier cages vs those at the bottom rack locations [78]. Further, cage material and bedding play a massive role in the amount of light available to the animal. Recent studies revealed that the spectral transmittance passing through standard laboratory rodent cages of different colored tints significantly influences circadian physiology and metabolism in commonly used rodent strains [79].

The specific ocular and neural elements that mediate the biological and behavioral effects of light in mammals remains an emergent science, especially in determining the interdependence and variability of these elements [7,80, 81, for reviews].

Lighting and Light Measurement for Biological and Behavioral Regulation

Many scientific reports do not provide any data on animal facility lighting with the exception that the levels are within local guidelines. If light measurements are provided, they are typically reported in lux. This is a photopic measurement of light based on the selective responsiveness of the human visual system and, consequently, is inappropriate to be used as a measure in mammalian studies of circadian and neuroendocrine physiology. Radiometric units are best used as they are based strictly on unweighted power measures [7,80,81].

Lux is often reported in animal studies due primarily to the fact that the meters are readily available and reasonably priced. Fortunately, meters that measure the spectral output or power distribution across the spectrum are increasingly available and affordable for animal researchers. In addition to proper radiometric measures, a detailed reporting of light source type used should be given. Experimental conditions or data comparisons across studies are a challenge without this information. It remains problematic that consistency has been lacking in the methods used for quantifying light between different laboratories studying photic regulation of biological and behavioral responses. A consensus position was developed by many of the laboratories that have studied wavelength regulation of the biological and behavioral effects of light in rodents and other species. The best practices for measuring and reporting experimental light stimuli are outlined, and a freely available web-based Toolbox was provided that permits calculation of the effective irradiance experienced by each of the ipRGC, cone, and rod photoreceptors that, in turn, drive circadian, neuroendocrine, and neurobehavioral effects in rodents [7].

The current state of lighting for animal room facilities is in transition from primarily fluorescent to solid state LED lighting. The investigator must be cognizant of the large differences in the distribution of wavelengths in these light sources, with more light being present in the blue-appearing portion of the visible spectrum (465–485 nm) [77]. It has been demonstrated that solid state lighting can have significant effects on pigmented nude rat circadian physiology, significantly increasing in the amount and duration nighttime melatonin signal [82]. Further recent research has expanded in this area to examine the influence of daytime LED light exposure on neuroendocrine, metabolic, and physiological parameters in 3 strains of commonly used laboratory mice [83]. Overall, the effects are shown to be generally beneficial to the animal’s homeostatic regulation of health well-being, with the physiological differences being substantial in some rodent species. Consequently, laboratory animal facility managers and animal researchers need to closely follow the emerging evidence on the potential effects of changing the sources of electrical lighting in animal facilities. Careful attention should be placed on consistency of results in animal experiments before and after lighting changes for animal housing.

Conclusion

Within the last 2 decades, a new photosensory system in the mammalian eye has been discovered, a truly remarkable achievement in physiology. A small set of retinal ganglion cells, previously thought to function as a visual output neuron, has been shown to be directly photosensitive and act differently from the classic photoreceptors of the visual system. Since the discovery of melanopsin [84] and then the localization of that photopigment to ipRGCs [42,43,85], there has been a dramatic increase in studies utilizing light as an independent variable in animal physiology studies. Understanding the effects of light on mammalian physiology and behavior must take into account how the classical visual photoreceptors and the ipRGCs photoreceptor systems interact. Further, it is important to be mindful that there are species variations in responses to light stimuli. What is proven to be true from one mammalian species may not be true others. Ultimately, advances in understanding phototransduction for circadian, neuroendocrine, and neurobehavioral regulation should lead to improvements in lighting of laboratory facilities that foster optimum health and well-being of animals.