Low-intensity electromagnetic fields induce human cryptochrome to modulate intracellular reactive oxygen species.

Exposure to man-made electromagnetic fields (EMFs), which increasingly pollute our environment, have consequences for human health about which there is continuing ignorance and debate. Whereas there is considerable ongoing concern about their harmful effects, magnetic fields are at the same time being applied as therapeutic tools in regenerative medicine, oncology, orthopedics, and neurology. This paradox cannot be resolved until the cellular mechanisms underlying such effects are identified. Here, we show by biochemical and imaging experiments that exposure of mammalian cells to weak pulsed electromagnetic fields (PEMFs) stimulates rapid accumulation of reactive oxygen species (ROS), a potentially toxic metabolite with multiple roles in stress response and cellular ageing. Following exposure to PEMF, cell growth is slowed, and ROS-responsive genes are induced. These effects require the presence of cryptochrome, a putative magnetosensor that synthesizes ROS. We conclude that modulation of intracellular ROS via cryptochromes represents a general response to weak EMFs, which can account for either therapeutic or pathological effects depending on exposure. Clinically, our findings provide a rationale to optimize low field magnetic stimulation for novel therapeutic applications while warning against the possibility of harmful synergistic effects with environmental agents that further increase intracellular ROS.

Magnetoreception: activation of avian cryptochrome 1a in various light conditions.

The avian magnetic inclination compass is based on radical pair processes, with cryptochrome (Cry) assumed to form the crucial radical pairs; it requires short-wavelength light from UV to green. Under high-intensity narrow-band lights and when yellow light is added, the magnetic compass is disrupted: migratory birds no longer prefer their migratory direction, but show other orientation responses. The candidate receptor molecule Cry1a is located in the shortwavelength-sensitive SWS1 cone photoreceptors in the retina. The present analysis of avian retinae after the respective illuminations showed that no activated Cry1a was present under 565 nm green light of medium and high intensity, and hardly any under high intensity 502 nm turquoise, whereas we found activated Cry1a at all three tested intensities of 373 nm UV and 424 nm blue light. Activated Cry1a also was found when 590 nm yellow light was added to low-intensity light of the four colors; yet these light combinations result in impaired magnetic orientation. This indicates that the disruption of the magnetic compass does not occur at the receptor level in the retina, but at higher processing stages, where the unnatural, almost monochromatic or bichromatic illumination causes yet unknown responses that interfere with the inclination compass.

Radical-pair-based magnetoreception in birds: radio-frequency experiments and the role of cryptochrome.

The radical-pair hypothesis of magnetoreception has gained a lot of momentum, since the flavoprotein cryptochrome was postulated as a structural candidate to host magnetically sensitive chemical reactions. Here, we first discuss behavioral tests using radio-frequency magnetic fields (0.1-10 MHz) to specifically disturb a radical-pair-based avian magnetic compass sense. While disorienting effects of broadband RF magnetic fields have been replicated independently in two competing labs, the effects of monochromatic RF magnetic fields administered at the electronic Larmor frequency (~1.3 MHz) are disparate. We give technical recommendations for future RF experiments. We then focus on two candidate magnetoreceptor proteins in birds, Cry1a and Cry1b, two splice variants of the same gene (Cry1). Immunohistochemical studies have identified Cry1a in the outer segments of the ultraviolet/violet-sensitive cone photoreceptors and Cry1b in the cytosol of retinal ganglion cells. The identification of the host neurons of these cryptochromes and their subcellular expression patterns presents an important advance, but much work lies ahead to gain some functional understanding. In particular, interaction partners of cryptochrome Cry1a and Cry1b remain to be identified. A candidate partner for Cry4 was previously suggested, but awaits independent replication.

Magnetoreception in birds: I. Immunohistochemical studies concerning the cryptochrome cycle.

Cryptochrome 1a, located in the UV/violet-sensitive cones in the avian retina, is discussed as receptor molecule for the magnetic compass of birds. Our previous immunohistochemical studies of chicken retinae with an antiserum that labelled only activated cryptochrome 1a had shown activation of cryptochrome 1a under 373 nm UV, 424 nm blue, 502 nm turquoise and 565 nm green light. Green light, however, does not allow the first step of photoreduction of oxidized cryptochromes to the semiquinone. As the chickens had been kept under 'white' light before, we suggested that there was a supply of the semiquinone present at the beginning of the exposure to green light, which could be further reduced and then re-oxidized. To test this hypothesis, we exposed chickens to various wavelengths (1) for 30 min after being kept in daylight, (2) for 30 min after a 30 min pre-exposure to total darkness, and (3) for 1 h after being kept in daylight. In the first case, we found activated cryptochrome 1a under UV, blue, turquoise and green light; in the second two cases we found activated cryptochrome 1a only under UV to turquoise light, where the complete redox cycle of cryptochrome can run, but not under green light. This observation is in agreement with the hypothesis that activated cryptochrome 1a is found as long as there is some of the semiquinone left, but not when the supply is depleted. It supports the idea that the crucial radical pair for magnetoreception is generated during re-oxidation.

Magnetoreception in birds: II. Behavioural experiments concerning the cryptochrome cycle.

Behavioural tests of the magnetic compass of birds and corresponding immunohistological studies on the activation of retinal cryptochrome 1a, the putative receptor molecule, showed oriented behaviour and activated Cry1a under 373 nm UV, 424 nm blue, 502 nm turquoise and 565 nm green light, although the last wavelength does not allow the first step of photoreduction of cryptochrome to the semiquinone form. The tested birds had been kept under 'white' light before, hence we suggested that there was a supply of semiquinone present at the beginning of the exposure to green light that could be further reduced and then re-oxidized. To test the hypothesis in behavioural experiments, we tested robins, Erithacus rubecula, under various wavelengths (1) after 1 h pre-exposure to total darkness and (2) after 1 h pre-exposure to the same light as used in the test. The birds were oriented under blue and turquoise light, where the full cryptochrome cycle can run, but not under green light. This finding is in agreement with the hypothesis. Orientation under green light appears to be a transient phenomenon until the supply of semiquinone is depleted.

Ontogenetic development of magnetic compass orientation in domestic chickens (Gallus gallus).

Domestic chickens (Gallus gallus) can be trained to search for a social stimulus in a specific magnetic direction, and cryptochrome 1a, found in the retina, has been proposed as a receptor molecule mediating magnetic directions. The present study combines immuno-histochemical and behavioural data to analyse the ontogenetic development of this ability. Newly hatched chicks already have a small amount of cryptochrome 1a in their violet cones; on day 5, the amount of cryptochrome 1a reached the same level as in adult chickens, suggesting that the physical basis for magnetoreception is present. In behavioural tests, however, young chicks 5 to 7 days old failed to show a preference of the training direction; on days 8, 9 and 12, they could be successfully trained to search along a specific magnetic axis. Trained and tested again 1 week later, the chicks that had not shown a directional preference on days 5 to 7 continued to search randomly, while the chicks tested from day 8 onward preferred the correct magnetic axis when tested 1 week later. The observation that the magnetic compass is not functional before day 8 suggests that certain maturation processes in the magnetosensitive system in the brain are not yet complete before that day. The reasons why chicks that have been trained before that day fail to learn the task later remain unclear.

Different responses of two strains of chickens to different training procedures for magnetic directions.

In previous conditioning experiments training domestic chickens to magnetic directions, a brown strain solved the task, whereas a white strain seemed unable to do so (Freire et al. Anim Cogn 11:547-552, 2008). To test whether this was possibly caused by loss of magnetic compass orientation in the white chickens, we analyzed the distribution of cryptochrome 1a, the candidate receptor molecule mediating magnetic compass information, in the retinae of Lohmann Browns and White Leghorns and found no difference between the two strains. Yet, subsequent training experiments replicated the former findings: Lohmann Browns used the magnetic field to find an imprinting stimulus hidden behind the screen in a specific magnetic direction, whereas White Leghorns did not solve the task. However, when we altered the training method by training also in a magnetic field with North shifted to geographic East and including a punishment for incorrect choices, the performance of White Leghorns improved to a significant preference for the expected directions. The Lohmann Browns, on the other hand, seemed frightened and chose randomly. Our results thus demonstrate the crucial role of the training method for conditioning to magnetic stimuli, with differences found even between strains of the same species.

The Magnetic Compass of Birds: The Role of Cryptochrome.

The geomagnetic field provides directional information for birds. The avian magnetic compass is an inclination compass that uses not the polarity of the magnetic field but the axial course of the field lines and their inclination in space. It works in a flexible functional window, and it requires short-wavelength light. These characteristics result from the underlying sensory mechanism based on radical pair processes in the eyes, with cryptochrome suggested as the receptor molecule. The chromophore of cryptochrome, flavin adenine dinucleotide (FAD), undergoes a photocycle, where radical pairs are formed during photo-reduction as well as during re-oxidation; behavioral data indicate that the latter is crucial for detecting magnetic directions. Five types of cryptochromes are found in the retina of birds: cryptochrome 1a (Cry1a), cryptochrome 1b, cryptochrome 2, cryptochrome 4a, and cryptochrome 4b. Because of its location in the outer segments of the ultraviolet cones with their clear oil droplets, Cry1a appears to be the most likely receptor molecule for magnetic compass information.

Author Correction: Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Light-dependent magnetoreception in birds: the crucial step occurs in the dark.

The Radical Pair Model proposes that the avian magnetic compass is based on spin-chemical processes: since the ratio between the two spin states singlet and triplet of radical pairs depends on their alignment in the magnetic field, it can provide information on magnetic directions. Cryptochromes, blue light-absorbing flavoproteins, with flavin adenine dinucleotide as chromophore, are suggested as molecules forming the radical pairs underlying magnetoreception. When activated by light, cryptochromes undergo a redox cycle, in the course of which radical pairs are generated during photo-reduction as well as during light-independent re-oxidation. This raised the question as to which radical pair is crucial for mediating magnetic directions. Here, we present the results from behavioural experiments with intermittent light and magnetic field pulses that clearly show that magnetoreception is possible in the dark interval, pointing to the radical pair formed during flavin re-oxidation. This differs from the mechanism considered for cryptochrome signalling the presence of light and rules out most current models of an avian magnetic compass based on the radical pair generated during photo-reduction. Using the radical pair formed during re-oxidation may represent a specific adaptation of the avian magnetic compass.

Seasonally Changing Cryptochrome 1b Expression in the Retinal Ganglion Cells of a Migrating Passerine Bird.

Cryptochromes, blue-light absorbing proteins involved in the circadian clock, have been proposed to be the receptor molecules of the avian magnetic compass. In birds, several cryptochromes occur: Cryptochrome 2, Cryptochrome 4 and two splice products of Cryptochrome 1, Cry1a and Cry1b. With an antibody not distinguishing between the two splice products, Cryptochrome 1 had been detected in the retinal ganglion cells of garden warblers during migration. A recent study located Cry1a in the outer segments of UV/V-cones in the retina of domestic chickens and European robins, another migratory species. Here we report the presence of cryptochrome 1b (eCry1b) in retinal ganglion cells and displaced ganglion cells of European Robins, Erithacus rubecula. Immuno-histochemistry at the light microscopic and electron microscopic level showed eCry1b in the cell plasma, free in the cytosol as well as bound to membranes. This is supported by immuno-blotting. However, this applies only to robins in the migratory state. After the end of the migratory phase, the amount of eCry1b was markedly reduced and hardly detectable. In robins, the amount of eCry1b in the retinal ganglion cells varies with season: it appears to be strongly expressed only during the migratory period when the birds show nocturnal migratory restlessness. Since the avian magnetic compass does not seem to be restricted to the migratory phase, this seasonal variation makes a role of eCry1b in magnetoreception rather unlikely. Rather, it could be involved in physiological processes controlling migratory restlessness and thus enabling birds to perform their nocturnal flights.

Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals.

Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.

Magnetoreception in birds: the effect of radio-frequency fields.

The avian magnetic compass, probably based on radical pair processes, works only in a narrow functional window around the local field strength, with cryptochrome 1a as most likely receptor molecule. Radio-frequency fields in the MHz range have been shown to disrupt the birds' orientation, yet the nature of this interference is still unclear. In an immuno-histological study, we tested whether the radio-frequency fields interfere with the photoreduction of cryptochrome, but this does not seem to be the case. In behavioural studies, birds were not able to adjust to radio-frequency fields like they are able to adjust to static fields outside the normal functional range: neither a 2-h pre-exposure in a 7.0 MHz field, 480 nT, nor a 7-h pre-exposure in a 1.315 MHz field, 15 nT, allowed the birds to regain their orientation ability. This inability to adjust to radio-frequency fields suggests that these fields interfere directly with the primary processes of magnetoreception and therefore disable the avian compass as long as they are present. They do not have lasting adverse after-effects, however, as birds immediately after exposure to a radio-frequency field were able to orient in the local geomagnetic field.

The magnetic compass of domestic chickens.

In a recent paper, we showed that domestic chickens can be trained to search for a social stimulus in specific magnetic directions. Chickens can hardly fly and have only small home ranges, hence their having a functional magnetic compass may seem rather surprising. Yet considering the natural habitat of their ancestors and their lifestyle until recently, the advantages of a magnetic compass become evident.

Magnetoreception: activated cryptochrome 1a concurs with magnetic orientation in birds.

The radical pair model proposes that the avian magnetic compass is based on radical pair processes in the eye, with cryptochrome, a flavoprotein, suggested as receptor molecule. Cryptochrome 1a (Cry1a) is localized at the discs of the outer segments of the UV/violet cones of European robins and chickens. Here, we show the activation characteristics of a bird cryptochrome in vivo under natural conditions. We exposed chickens for 30 min to different light regimes and analysed the amount of Cry1a labelled with an antiserum against an epitope at the C-terminus of this protein. The staining after exposure to sunlight and to darkness indicated that the antiserum labels only an illuminated, activated form of Cry1a. Exposure to narrow-bandwidth lights of various wavelengths revealed activated Cry1a at UV, blue and turquoise light. With green and yellow, the amount of activated Cry1a was reduced, and with red, as in the dark, no activated Cry1a was labelled. Activated Cry1a is thus found at all those wavelengths at which birds can orient using their magnetic inclination compass, supporting the role of Cry1a as receptor molecule. The observation that activated Cry1a and well-oriented behaviour occur at 565 nm green light, a wavelength not absorbed by the fully oxidized form of cryptochrome, suggests that a state other than the previously suggested Trp/FAD radical pair formed during photoreduction is crucial for detecting magnetic directions.

Avian ultraviolet/violet cones as magnetoreceptors: The problem of separating visual and magnetic information.

In a recent paper, we described the localization of cryptochrome 1a in the retina of domestic chickens, Gallus gallus, and European robins, Erithacus rubecula: Cryptochrome 1a was found exclusively along the membranes of the disks in the outer segments of the ultraviolet/violet single cones. Cryptochrome has been suggested to act as receptor molecule for the avian magnetic compass, which would mean that the UV/V cones have a double function: they mediate vision in the short-wavelength range and, at the same time, magnetic directional information. This has important implications and raises a number of questions, in particular, how the two types of input are separated. Here, we point out several possibilities how this could be achieved. 

Avian ultraviolet/violet cones identified as probable magnetoreceptors.

BACKGROUND: The Radical-Pair-Model postulates that the reception of magnetic compass directions in birds is based on spin-chemical reactions in specialized photopigments in the eye, with cryptochromes discussed as candidate molecules. But so far, the exact subcellular characterization of these molecules in the retina remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: We here describe the localization of cryptochrome 1a (Cry1a) in the retina of European robins, Erithacus rubecula, and domestic chickens, Gallus gallus, two species that have been shown to use the magnetic field for compass orientation. In both species, Cry1a is present exclusively in the ultraviolet/violet (UV/V) cones that are distributed across the entire retina. Electron microscopy shows Cry1a in ordered bands along the membrane discs of the outer segment, and cell fractionation reveals Cry1a in the membrane fraction, suggesting the possibility that Cry1a is anchored along membranes. CONCLUSIONS/SIGNIFICANCE: We provide first structural evidence that Cry1a occurs within a sensory structure arranged in a way that fulfils essential requirements of the Radical-Pair-Model. Our findings, identifying the UV/V-cones as probable magnetoreceptors, support the assumption that Cry1a is indeed the receptor molecule mediating information on magnetic directions, and thus provide the Radical-Pair-Model with a profound histological background.