BackgroundThe 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 FindingsWe 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/SignificanceWe 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.
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.
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.
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.
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.
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 preexposure 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.
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.
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.
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