CIDNP Research Articles

Is Cryptochrome a Primary Sensor of Extremely Low Frequency Magnetic Fields in Childhood Leukemia?

Extremely low frequency magnetic fields (ELF MF) are classified as possibly carcinogenic to humans, but the biophysical mechanisms of a causal relationship remain unclear. A cryptochrome-based radical pair mechanism (RPM) has been invoked as the primary MF sensor in animal magnetoreception to explain effects from MF strengths in the nT range. Model studies of the RPM in aprotic solution require cryogenic temperatures and MFs in the µT range to elicit marginal responses, implying that physiological responses evoked by radical pairs in biological milieu are unlikely. We explore ideas about how signals from transient radical pairs in cryptochrome might be transduced and amplified in mutagenic responses. The cryptochrome-based RPM involves blue light activation of the flavin adenine dinucleotide (FAD) cofactor followed by electron transfer (ET) from a conserved triad of tryptophan residues. However, intramolecular ET involving additional conserved aromatic residues in cryptochrome likely extends beyond the canonical triad. Further, ascorbate, which is present at millimolar concentrations in leukocytes, is likely to transfer an electron to the ultimate amino acid radical formed during one-electron reduction of FAD. The ascorbyl radical has been proposed as a potential radical pair partner in the RPM. The flavin semiquinone radical may be reoxidized to the resting state via ET to O2 to form O2•−. Increased levels of the ascorbyl radical and O2•− would contribute to oxidative stress pathways in the cell. Oxidative stress responses in cancer are thought to be mediated in part by the mitogen activated protein kinase (MAPK) signaling pathway. We have presented evidence for alteration of MAPK activation in response to ELF MF. Acknowledgement: This project has received funding from the European Union's Seventh Programme for research, technological development, and demonstration under grant agreement No. 282891.

Photocurrent enhancements in a porphyrin-viologen linked compound under plasmonic and magnetic fields

Zinc-porphyrin( ZnP )–viologen(V2+) linked compound containing six methylene group ( ZnP (6) V )–silver nanoparticle ( AgNP ) composite films was fabricated by combining electrostatic layer-by-layer adsorption and the Langmuir–Blodgett method. The incident photo to photocurrent efficiency (IPCE) values of the ZnP (6) V – AgNP composite films are higher than those of the ZnP (6) V films and much higher than those of ZnP derivative films without V2+moiety as a reference. The large increase in the IPCE values of the ZnP (6) V – AgNP composite films likely comes from a combination of localized surface plasmon resonance (LSPR) from AgNPs and photoinduced intramolecular electron-transfer upon linking to a V2+moiety. The photocurrents of the ZnP (6) V – AgNP composite films and the ZnP (6) V films increase upon application of a magnetic field. Magnetic field effects (MFEs) were clearly observed for both ZnP (6) V – AgNP composite films and the ZnP (6) V films. Photocurrents increase with magnetic field under low magnetic fields (B ≤ 150–300 mT) and are constant under high magnetic fields (B > 150–300 mT). MFEs can be explained by a radical pair mechanism. The magnitude of the MFEs in the ZnP (6) V – AgNP composite films is higher than that in the ZnP (6) V films. A remarkable increase in photocurrent for the ZnP (6) V – AgNP composite films was observed because of LSPR from the AgNPs in the presence of a magnetic field when compared with the ZnP (6) V films in the absence of a magnetic field.

Modulation of the rate of reversible electron transfer in oxidized tryptophan and tyrosine containing peptides in acidic aqueous solution.

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) was used to investigate reversible intramolecular electron transfer (IET) in short-lived oxidized peptides, which had different structures and contained tryptophan and tyrosine residues, in an acidic aqueous solution with a pH below the pKa of the tryptophanyl cation radical. The CIDNP kinetic data were obtained at the microsecond scale and were analyzed in detail to calculate the rate constants for electron transfer in both directions: from the tyrosine residue to the tryptophanyl cation radical, kf, and from the tryptophan residue to the neutral tyrosyl radical, kr. The charge of the terminal amino group and the presence of glycine and proline spacers were shown to strongly affect the rate constants of the reaction under study. Among these functional groups, the presence and the location of the positive charge on the amino group in close proximity to the cationic indolyl radical had the strongest effect on the rate constant of the forward IET from the tyrosine residue to the tryptophanyl radical cation, kf. This effect was manifested as an increase of 2 orders of magnitude in kf for a change in the linkage order between residues in the dipeptide: kf = 4 × 10(3) s(-1) for the oxidized Tyr-Trp increased to kf = 5.5 × 10(5) s(-1) in oxidized Trp-Tyr. The reverse rate constant for IET was less sensitive to the amino group charge. Moreover, the presence of glycine or proline spacers in the peptides with a tryptophan residue at the N-terminus not only reduced the IET rate constant but also shifted the equilibrium of the IET in the reaction under study toward the formation of tyrosyl radicals with respect to the peptide Trp-Tyr. That is, the glycine or proline spacers affected the difference in the reduction potential of the tryptophanyl and tyrosyl radicals.

Sensitivity and entanglement in the avian chemical compass.

The radical pair mechanism can help to explain avian orientation and navigation. Some evidence indicates that the intensity of external magnetic fields plays an important role in avian navigation. In this paper, using a two-stage model, we demonstrate that birds could reasonably detect the directions of geomagnetic fields and gradients of these fields using a yield-based chemical compass that is sensitive enough for navigation. Also, we find that the lifetime of entanglement in this proposed compass is angle dependent and long enough to allow adequate electron transfer between molecules.

Photooxidation of Histidine by 3,3′,4,4′-Benzophenone Tetracarboxylic Acid in Aqueous Solution: Time-Resolved and Field-Dependent CIDNP Study

The photooxidation reaction between 3,3′,4,4′-benzophenone tetracarboxylic acid (TCBP) and l-histidine (His) has been investigated in neutral aqueous solution using the technique of chemically induced dynamic nuclear polarization (CIDNP). Relative values of 13C isotropic hyperfine couplings in the TCBP and His radicals were obtained from the 13C-time-resolved CIDNP spectrum, recorded during the photoreaction of TCBP with His at natural abundance of the magnetic isotope 13C. Good agreement was found for the hyperfine coupling constants of the TCBP ketyl radical calculated using methods of density functional theory, and those obtained from the 13C-time-resolved CIDNP spectrum. The mechanism of the quenching reaction of triplet-excited TCBP by His in neutral aqueous solution was established. 1H CIDNP field dependencies for the photoreaction of TCBP with His were obtained and the g-factor of the histidyl radical was found.

Coherent Polarization Transfer Effects Are Crucial for Interpreting Low-Field CIDNP Data

In this work we demonstrate that low-field chemically induced dynamic nuclear polarization (CIDNP) is strongly affected by re-distribution of polarization, which is formed in the course of spin evolution in transient radical pairs, in diamagnetic reaction products. This phenomenon is of importance when the spins of the reaction product are coupled strongly meaning that spin–spin interactions between them are comparable to the differences in their Zeeman interactions with the external magnetic field. In this case, polarization transfer relies on a coherent mechanism; as a consequence, spins can acquire significant polarization even when they have no hyperfine coupling to the electron spins in the radical pairs, i.e., cannot be polarized directly by CIDNP. This is demonstrated by taking CIDNP of n-butylamine as an example: in this case only the α-CH2 protons are polarized directly, which is confirmed by high-field CIDNP, whereas the β-CH2, γ-CH2 and δ-CH3 protons get polarized only indirectly due to the transfer of polarization from the α-CH2 protons. These results show that low-field CIDNP data should be interpreted with care to discriminate between the effects of spin evolution in transient radical pairs and in diamagnetic reaction products.

Multiple re-encounter approach to radical pair reactions and the role of nonlinear master equations.

We formulate a multiple-encounter model of the radical pair mechanism that is based on a random coupling of the radical pair to a minimal model environment. These occasional pulse-like couplings correspond to the radical encounters and give rise to both dephasing and recombination. While this is in agreement with the original model of Haberkorn and its extensions that assume additional dephasing, we show how a nonlinear master equation may be constructed to describe the conditional evolution of the radical pairs prior to the detection of their recombination. We propose a nonlinear master equation for the evolution of an ensemble of independently evolving radical pairs whose nonlinearity depends on the record of the fluorescence signal. We also reformulate Haberkorn's original argument on the physicality of reaction operators using the terminology of quantum optics/open quantum systems. Our model allows one to describe multiple encounters within the exponential model and connects this with the master equation approach. We include hitherto neglected effects of the encounters, such as a separate dephasing in the triplet subspace, and predict potential new effects, such as Grover reflections of radical spins, that may be observed if the strength and time of the encounters can be experimentally controlled.

Sensing magnetic directions in birds: radical pair processes involving cryptochrome.

Birds can use the geomagnetic field for compass orientation. Behavioral experiments, mostly with migrating passerines, revealed three characteristics of the avian magnetic compass: (1) it works spontaneously only in a narrow functional window around the intensity of the ambient magnetic field, but can adapt to other intensities, (2) it is an “inclination compass”, not based on the polarity of the magnetic field, but the axial course of the field lines, and (3) it requires short-wavelength light from UV to 565 nm Green. The Radical Pair-Model of magnetoreception can explain these properties by proposing spin-chemical processes in photopigments as underlying mechanism. Applying radio frequency fields, a diagnostic tool for radical pair processes, supports an involvement of a radical pair mechanism in avian magnetoreception: added to the geomagnetic field, they disrupted orientation, presumably by interfering with the receptive processes. Cryptochromes have been suggested as receptor molecules. Cry1a is found in the eyes of birds, where it is located at the membranes of the disks in the outer segments of the UV-cones in chickens and robins. Immuno-histochemical studies show that it is activated by the wavelengths of light that allow magnetic compass orientation in birds.

Spectral editing through laser-flash excitation in two-dimensional photo-CIDNP MAS NMR experiments

In solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) MAS NMR experiments, strong signal enhancement is observed from molecules forming a spin-correlated radical pair in a rigid matrix. Two-dimensional 13C–13C dipolar-assisted rotational resonance (DARR) photo-CIDNP MAS NMR experiments have been applied to obtain exact chemical shift assignments from those cofactors. Under continuous illumination, the signals are enhanced via three-spin mixing (TSM) and differential decay (DD) and their intensity corresponds to the electron spin density in pz orbitals. In multiple-13C labelled samples, spin diffusion leads to propagation of signal enhancement to all 13C spins. Under steady-state conditions, direct signal assignment is possible due to the uniform signal intensity. The original intensities, however, are inaccessible and the information of the local electron spin density is lost. Upon laser-flash illumination, the signal is enhanced via the classical radical pair mechanism (RPM). The obtained intensities are related to isotropic hyperfine interactions aiso and both enhanced absorptive and emissive lines can be observed due to differences in the sign of the local isotropic hyperfine interaction. Exploiting the mechanism of the polarization, selectivity can be increased by the novel time-resolved two-dimensional dipolar-assisted rotational resonance (DARR) MAS NMR experiment which simplifies the signal assignment compared to complex spectra of the same RCs obtained by continuous illumination. Here we present two-dimensional time-resolved photo-CIDNP MAS NMR experiments providing both directly: signal assignment and spectral editing by sign and strength of aiso. Hence, this experiment provides a direct key to the electronic structure of the correlated radical pair.

Oxidation of Purine Nucleotides by Triplet 3,3′,4,4′-Benzophenone Tetracarboxylic Acid in Aqueous Solution: pH-Dependence

The photo-oxidation of purine nucleotides adenosine-5′-monophosphate (AMP) and guanosine-5′-monophosphate (GMP) by 3,3′,4,4′-benzophenone tetracarboxylic acid (TCBP) has been investigated in aqueous solutions using nanosecond laser flash photolysis (LFP) and time-resolved chemically induced dynamic nuclear polarization (CIDNP). The pH dependences of quenching rate constants and of geminate polarization are measured within a wide range of pH values. As a result, the chemical reactivity of reacting species in different protonation states is determined. In acidic solution (pH < 4.9), the quenching rate constant is close to the diffusion-controlled limit: kq = 1.3 × 109 M–1 s–1 (GMP), and kq = 1.2 × 109 M–1 s–1 (AMP), whereas in neutral and basic solutions it is significantly lower: kq = 2.6 × 108 M–1 s–1 (GMP, 4.9 < pH < 9.4), kq = 3.5 × 107 M–1 s–1 (GMP, pH > 9.4), kq = 1.0 × 108 M–1 s–1 (AMP, pH > 6.5). Surprisingly, the strong influence of the protonation state of the phosphoric group on the oxidation of adenosine-5′-monophosphate is revealed: the deprotonation of the AMP phosphoric group (6.5) decreases the quenching rate constant from 5.0 × 108 M–1 s–1 (4.9 < pH < 6.5) to 1.0 × 108 M–1 s–1 (pH > 6.5).

The role of level anti-crossings in nuclear spin hyperpolarization

Nuclear spin hyperpolarization is an important resource for increasing the sensitivity of NMR spectroscopy and MRI. Signal enhancements can be as large as 3–4 orders of magnitude. In hyperpolarization experiments, it is often desirable to transfer the initial polarization to other nuclei of choice, either protons or insensitive nuclei such as 13C and 15N. This situation arises primarily in Chemically Induced Dynamic Nuclear Polarization (CIDNP), Para-Hydrogen Induced Polarization (PHIP), and the related Signal Amplification By Reversible Exchange (SABRE). Here we review the recent literature on polarization transfer mechanisms, in particular focusing on the role of Level Anti-Crossings (LACs) therein.So-called “spontaneous” polarization transfer may occur both at low and high magnetic fields. In addition, transfer of spin polarization can be accomplished by using especially designed pulse sequences. It is now clear that at low field spontaneous polarization transfer is primarily due to coherent spin-state mixing under strong coupling conditions. However, thus far the important role of LACs in this process has not received much attention. At high magnetic field, polarization may be transferred by cross-relaxation effects. Another promising high-field technique is to generate the strong coupling condition by spin locking using strong radio-frequency fields. Here, an analysis of polarization transfer in terms of LACs in the rotating frame is very useful to predict which spin orders are transferred depending on the strength and frequency of the B1 field. Finally, we will examine the role of strong coupling and LACs in magnetic-field dependent nuclear spin relaxation and the related topic of long-lived spin-states.

Photoinduced transformation of iron chelator deferiprone: Possible implications in drug metabolism and toxicity

Abstract Deferiprone (L1) is an effective iron chelating drug which is widely used for the treatment of iron overload diseases. Although L1 itself does not absorb visible light, the absorption spectra of its Fe(III) complexes exhibit intensive absorption bands between 300 and 800 nm. It was found that L1–Fe(III) complex is stable under irradiation with visible light but undergoes fast decomposition under UV irradiation (with quantum yield more than 0.7). Decomposition of L1 has been observed in the presence of Fe(II) in a dark process. Free, non iron bound L1 exhibits both electron accepting and electron donating abilities. Free radicals of L1 were detected by the chemically induced dynamic nuclear polarization (CIDNP) method during irradiation of its aqueous solution in the presence of quinones, amino acids and NADH. It indicates the ability of deferiprone to produce free radicals not only by direct UV irradiation, but also in photosensitized electron transfer reactions with various biomolecules. Phototransformation of L1 was elucidated by using CIDNP, UV–vis, NMR and mass-spectroscopy techniques. The structures of the products of L1 photolysis include 1,2-dihydro-2,3,4-trihydroxy-1,2-dimethylpyridine. It appears that the metabolism of drugs with iron binding properties can be affected by sunlight, as shown by the iron complexes of L1 and such changes may cause phototoxic and pharmacological effects in patients undergoing treatment.

Observation of Magnetic Field Effects on Transient Fluorescence Spectra of Cryptochrome 1 From Homing Pigeons

Cryptochromes are suggested to be involved in the bird magnetoreception based on the radical pair mechanism (RPM), a well established theory of weak magnetic field effects on chemical reactions. Two members of cryptochrome/photolyase family were found to respond to magnetic field, however, no direct responses of bird cryptochrome to magnetic field as weak as the Earth's magnetic field have been obtained so far. In this study, we used transient fluorescence spectroscopy to characterize the weak magnetic field effects of bird cryptochromes. To do this, we cloned the cryptochrome 1 gene (clCRY1) from the retina of homing pigeons (Columba livia), expressed it in insect Sf9 cells and analyzed the transient fluorescence of purified clCRY1 by application of 45-300μT magnetic fields. The flavin adenine dinucleotide (FADox ) and glucose oxidase (GOD) in PBS buffer were set as controls which could be excited by light to generate radicals, but would not be sensitive to magnetic field. We observed that the transient fluorescence spectra of clCRY1 were sensitive to the applied magnetic field at room temperature. Our result provides a new proof of the cryptochrome-based model of avian magnetoreception in vitro.

Magnetic Field Effects on Geotactic Responses in Drosophila Melanogaster

A wide range of organisms have the ability to sense Earth's magnetic field and use it as a method of orientation. The biophysical mechanism of this sixth sense is not well understood with two mechanisms currently discussed, one based on iron-oxide particles (magnetite mechanism) and, alternatively, a mechanism based on the sensitivity of electron spins (radical-pair mechanism). One of the challenges hindering progress towards conclusive identification of the magnetoreceptors is the lack of experimental assays with genetic models demonstrating sensitivity to the direction of earth-strength magnetic fields.Here, we present results from two assays testing the effect of magnetic fields on geotactic responses of fruit flies, Drosophila Melanogaster. We are investigating Canton-S wild type fruit flies as well as two strains selected for positive ('Lo') and negative ('Hi') geotaxis. Experiments in a vertical nine-level geotaxis Hirsch maze assay show a significant effect of reversing the direction of the magnetic field on the responses of Canton-S strains. Under a double strength Earth field and a zero field, there was no significant effect. We also tested effects of a strong inhomogeneous field and one end of the maze. In a second assay, we began testing the Canton-S strain in a two-armed T-maze to allow for better comparison with previously published assays and we report preliminary results. Effects on geotaxis can potentially pave the way towards identifying a role of cryptochromes, a putative magnetoreceptor in the radical pair mechanism, because Hi and Lo strains have been shown to differ strongly in their cryptochrome expression.

Dominance of the triplet mechanism in the electron spin polarisation of slowly reacting triplets

For the triplet mechanism (TM) of electron spin polarisation in photochemically generated free radicals to dominate, the chemical reaction has to be very fast and capable of competing with the electron spin-lattice relaxation process of the triplet. This is a well-established condition. Here we report a counter example of electron spin polarisation, where this condition appears to be violated, and a dominant spin polarisation characteristic of the TM is observed, even when the triplet reacts very slowly. The system studied is the photoreduction of 4,4'-dihydroxy-benzophenone (DHBP) in 2-propanol producing the DHBP ketyl radical and the (CH3)2C˙OH radical. At room temperature, electron spin polarisation of the time-resolved EPR (TREPR) spectra of this system is dominated by the radical pair mechanism involving ST0 mixing, as the rate of the hydrogen abstraction reaction cannot compete with the spin-lattice relaxation of triplet DHBP. However, with the lowering of temperature, TREPR spectra dominated by a net emissive polarisation are seen, even though the rate of the reaction remains slow. Existence of this single-phase, hyperfine-line independent emissive polarisation due to the TM is established by the simulation of their TREPR spectra at -48 °C. To explain the increased contribution of the TM with the lowering of temperature, we proposed that the presence of two hydroxyl groups of DHBP could form hydrogen bonds with the solvent and steadily increase the micro-viscosity experienced by DHBP appreciably as the temperature was lowered. This would progressively increase the rotational correlation time of triplet DHBP, enabling the TM route to contribute increasingly to the radical generation. TREPR experiments with 4,4'-dimethoxy-benzophenone in 2-propanol, where similar hydrogen bonding is not possible, showed the spectra to be dominated by the ST0-RPM with little contribution of a net emissive signal. This observation qualitatively confirmed our proposal of enhanced micro-viscosity experienced by DHBP in 2-propanol at low temperatures, resulting in the enhanced emissive spin polarisation due to the TM, even though the chemical reaction remains slow.

Magnetic field effects in flavoproteins and related systems.

Within the framework of the radical pair mechanism, magnetic fields may alter the rate and yields of chemical reactions involving spin-correlated radical pairs as intermediates. Such effects have been studied in detail in a variety of chemical systems both experimentally and theoretically. In recent years, there has been growing interest in whether such magnetic field effects (MFEs) also occur in biological systems, a question driven most notably by the increasing body of evidence for the involvement of such effects in the magnetic compass sense of animals. The blue-light photoreceptor cryptochrome is placed at the centre of this debate and photoexcitation of its bound flavin cofactor has indeed been shown to result in the formation of radical pairs. Here, we review studies of MFEs on free flavins in model systems as well as in blue-light photoreceptor proteins and discuss the properties that are crucial in determining the magnetosensitivity of these systems.

Time-Resolved Magnetic Field Effects Distinguish Loose Ion Pairs from Exciplexes

We describe the experimental investigation of time-resolved magnetic field effects in exciplex-forming organic donor–acceptor systems. In these systems, the photoexcited acceptor state is predominantly deactivated by bimolecular electron transfer reactions (yielding radical ion pairs) or by direct exciplex formation. The delayed fluorescence emitted by the exciplex is magnetosensitive if the reaction pathway involves loose radical ion pair states. This magnetic field effect results from the coherent interconversion between the electronic singlet and triplet radical ion pair states as described by the radical pair mechanism. By monitoring the changes in the exciplex luminescence intensity when applying external magnetic fields, details of the reaction mechanism can be elucidated. In this work we present results obtained with the fluorophore-quencher pair 9,10-dimethylanthracene/N,N-dimethylaniline (DMA) in solvents of systematically varied permittivity. A simple theoretical model is introduced that allows discriminating the initial state of quenching, viz., the loose ion pair and the exciplex, based on the time-resolved magnetic field effect. The approach is validated by applying it to the isotopologous fluorophore-quencher pairs pyrene/DMA and pyrene-d10/DMA. We detect that both the exciplex and the radical ion pair are formed during the initial quenching stage. Upon increasing the solvent polarity, the relative importance of the distant electron transfer quenching increases. However, even in comparably polar media, the exciplex pathway remains remarkably significant. We discuss our results in relation to recent findings on the involvement of exciplexes in photoinduced electron transfer reactions.

Estimating the hyperfine coupling parameters of the avian compass by comprehensively considering the available experimental results

Migratory birds can utilize the geomagnetic field for orientation and navigation through a widely accepted radical-pair mechanism. Although many theoretical works have been done, the available experimental results have not been fully considered, especially the temporary disorientation induced by the field which is increased by 30% of the geomagnetic field and the disorientation of the very weak resonant field of 15 nT. In this paper, we consider the monotonicity of the singlet yield angular profile as the prerequisite of direction sensitivity, and find that for some optimal values of the hyperfine coupling parameters (that is, the order of 10^{-7}∼10^{-6} meV) the experimental results available so far can be satisfied. We also investigate the effects of two decoherence environments and demonstrate that, in order to satisfy the available experimental results, the decoherence rate should be lower than the recombination rate. Finally, we investigate the effects of the fluctuating magnetic noises and find that the vertical noise destroys the monotonicity of the profile completely, but the parallel noise preserves the monotonicity perfectly and even can enhance the direction sensitivity.

Radical-pair model of magnetoreception with spin–orbit coupling

The mechanism used by migratory birds to orientate themselves using the geomagnetic field is still a mystery in many species. The radical pair mechanism, in which very weak magnetic fields can influence certain types of spin-dependent chemical reactions, leading to biologically observable signals, has recently imposed itself as one of the most promising candidates for certain species. This is thanks both to its extreme sensitivity and its capacity to reproduce results from behavioral studies. Still, in order to gain a directional sensitivity, an anisotropic mechanism is needed. Recent proposals have explored the possibility that such an anisotropy is due to the electron–nucleus hyperfine interaction. In this work we explore a different possibility, in which the anisotropy is due to spin–orbit coupling between the electron spin and its angular momentum. We will show how a spin–orbit coupling-based magnetic compass can have performances comparable with the usually studied nuclear hyperfine based mechanism. Our results could thus help researchers actively looking for candidate biological molecules which may host magnetoreceptive functions, both to describe magnetoreception in birds as well as to develop artificial chemical compass systems.

Avian magnetic compass can be tuned to anomalously low magnetic intensities

The avian magnetic compass works in a fairly narrow functional window around the intensity of the local geomagnetic field, but adjusts to intensities outside this range when birds experience these new intensities for a certain time. In the past, the geomagnetic field has often been much weaker than at present. To find out whether birds can obtain directional information from a weak magnetic field, we studied spontaneous orientation preferences of migratory robins in a 4 µT field (i.e. a field of less than 10 per cent of the local intensity of 47 µT). Birds can adjust to this low intensity: they turned out to be disoriented under 4 µT after a pre-exposure time of 8 h to 4 µT, but were able to orient in this field after a total exposure time of 17 h. This demonstrates a considerable plasticity of the avian magnetic compass. Orientation in the 4 µT field was not affected by local anaesthesia of the upper beak, but was disrupted by a radiofrequency magnetic field of 1.315 MHz, 480 nT, suggesting that a radical-pair mechanism still provides the directional information in the low magnetic field. This is in agreement with the idea that the avian magnetic compass may have developed already in the Mesozoic in the common ancestor of modern birds.