Cognitive maps and the magnetic sense in vertebrates.

Navigation requires a network of neurons processing inputs from internally generated cues and external landmarks. Most studies on the neuronal basis of navigation in vertebrates have focused on rats and mice and the canonical senses vision, hearing, olfaction, and somatosensation. Some animals have evolved the ability to sense the Earth's magnetic field and use it for orientation. It can be expected that in these animals magnetic cues are integrated with other sensory cues in the cognitive map. We provide an overview of the behavioral evidence and brain regions involved in magnetic sensing in support of this idea, hoping that this will guide future experiments.

Cryptochromes in mammals: a magnetoreception misconception?

Cryptochromes are flavoproteins related to photolyases that are widespread throughout the plant and animal kingdom. They govern blue light-dependent growth in plants, control circadian rhythms in a light-dependent manner in invertebrates, and play a central part in the circadian clock in vertebrates. In addition, cryptochromes might function as receptors that allow animals to sense the Earth's magnetic field. As cryptochromes are also present in mammals including humans, the possibility of a magnetosensitive protein is exciting. Here we attempt to provide a concise overview of cryptochromes in mammals. We briefly review their canonical role in the circadian rhythm from the molecular level to physiology, behaviour and diseases. We then discuss their disputed light sensitivity and proposed role in the magnetic sense in mammals, providing three mechanistic hypotheses. Specifically, mammalian cryptochromes could form light-induced radical pairs in particular cellular milieus, act as magnetoreceptors in darkness, or as secondary players in a magnetoreception signalling cascade. Future research can test these hypotheses to investigate if the role of mammalian cryptochromes extends beyond the circadian clock.

Evaluating the power of a recent method for comparing two circular distributions: an alternative to the Watson U2 test.

Some data are collected on circular (rather than linear) scales. Often researchers are interested in comparing two samples of such circular data to test the hypothesis that they came from the same underlying population. Recently, we compared 18 statistical approaches to testing such a hypothesis, and recommended two as particularly effective. A very recent publication introduced a novel statistical approach that was claimed to outperform the methods that we had indicated were highest performing. However, the evidence base for this claim was limited. Here we perform simulation studies to offer a more detailed comparison of the new "Angular Randomisation Test" (ART) with existing tests. We expand previous evaluations in two ways: exploring small and medium sized samples, and exploring a range of different shapes for the underlying distribution(s). We find that the ART controls type I error rates at the nominal level. The ART had greater power than established methods in detecting a difference in underlying distribution caused by a shift around the circle. Its performance advantage in this case was strongest when samples where small and unbalanced in size. When the difference between underlying unimodal distributions was in shape rather than central tendency, then the ART was at least as good (and sometimes considerably more powerful) than the established methods, except when distributions samples were small and uneven in size, and the smaller sample came from a more concentrated underlying distribution. In such cases its power could be markedly inferior to established alternatives. The ART was also inferior to alternatives in dealing with axially distributed data. We conclude that under widely-encountered circumstances the ART test can be recommended for its simplicity of implementation, but researchers should be aware of situations where it cannot be recommended.

The multivariate analysis of variance as a powerful approach for circular data.

BACKGROUND: A broad range of scientific studies involve taking measurements on a circular, rather than linear, scale (often variables related to times or orientations). For linear measures there is a well-established statistical toolkit based on linear modelling to explore the associations between this focal variable and potentially several explanatory factors and covariates. In contrast, statistical testing of circular data is much simpler, often involving either testing whether variation in the focal measurements departs from circular uniformity, or whether a single explanatory factor with two levels is supported. METHODS: We use simulations and example data sets to investigate the usefulness of a MANOVA approach for circular data in comparison to commonly used statistical tests. RESULTS: Here we demonstrate that a MANOVA approach based on the sines and cosines of the circular data is as powerful as the most-commonly used tests when testing deviation from a uniform distribution, while additionally offering extension to multi-factorial modelling that these conventional circular statistical tests do not. CONCLUSIONS: The herein presented MANOVA approach offers a substantial broadening of the scientific questions that can be addressed statistically using circular data.

Electrophysiology and the magnetic sense: a guide to best practice.

Magnetoreception, sensing the Earth's magnetic field, is used by many species in orientation and navigation. While this is established on the behavioural level, there is a severe lack in knowledge on the underlying neuronal mechanisms of this sense. A powerful technique to study the neuronal processing of magnetic cues is electrophysiology but, thus far, few studies have adopted this technique. Why is this the case? A fundamental problem is the introduction of electromagnetic noise (induction) caused by the magnetic stimuli, within electrophysiological recordings which, if too large, prevents feasible separation of neuronal signals from the induction artefacts. Here, we address the concerns surrounding the use of electromagnetic coils within electrophysiology experiments and assess whether these would prevent viable electrophysiological recordings within a generated magnetic field. We present calculations of the induced voltages in typical experimental situations and compare them against the neuronal signals measured with different electrophysiological techniques. Finally, we provide guidelines that should help limit and account for possible induction artefacts. In conclusion, if great care is taken, viable electrophysiological recordings from magnetoreceptive cells are achievable and promise to provide new insights on the neuronal basis of the magnetic sense.

Quantum magnetic imaging of iron organelles within the pigeon cochlea.

The ability of pigeons to sense geomagnetic fields has been conclusively established despite a notable lack of determination of the underlying biophysical mechanisms. Quasi-spherical iron organelles previously termed "cuticulosomes" in the cochlea of pigeons have potential relevance to magnetoreception due to their location and iron composition; however, data regarding the magnetic susceptibility of these structures are currently limited. Here quantum magnetic imaging techniques are applied to characterize the magnetic properties of individual iron cuticulosomes in situ. The stray magnetic fields emanating from cuticulosomes are mapped and compared to a detailed analytical model to provide an estimate of the magnetic susceptibility of the individual particles. The images reveal the presence of superparamagnetic and ferrimagnetic domains within individual cuticulosomes and magnetic susceptibilities within the range 0.029 to 0.22. These results provide insights into the elusive physiological roles of cuticulosomes. The susceptibilities measured are not consistent with a torque-based model of magnetoreception, placing iron storage and stereocilia stabilization as the two leading putative cuticulosome functions. This work establishes quantum magnetic imaging as an important tool to complement the existing array of techniques used to screen for potential magnetic particle-based magnetoreceptor candidates.

Advice on comparing two independent samples of circular data in biology.

Many biological variables are recorded on a circular scale and therefore need different statistical treatment. A common question that is asked of such circular data involves comparison between two groups: Are the populations from which the two samples are drawn differently distributed around the circle? We compared 18 tests for such situations (by simulation) in terms of both abilities to control Type-I error rate near the nominal value, and statistical power. We found that only eight tests offered good control of Type-I error in all our simulated situations. Of these eight, we were able to identify the Watson's U2 test and a MANOVA approach, based on trigonometric functions of the data, as offering the best power in the overwhelming majority of our test circumstances. There was often little to choose between these tests in terms of power, and no situation where either of the remaining six tests offered substantially better power than either of these. Hence, we recommend the routine use of either Watson's U2 test or MANOVA approach when comparing two samples of circular data.

The expression, localisation and interactome of pigeon CRY2.

Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.

Neuronal circuits and the magnetic sense: central questions.

Magnetoreception is the ability to sense the Earth's magnetic field, which is used for orientation and navigation. Behavioural experiments have shown that it is employed by many species across all vertebrate classes; however, our understanding of how magnetic information is processed and integrated within the central nervous system is limited. In this Commentary, we review the progress in birds and rodents, highlighting the role of the vestibular and trigeminal systems as well as that of the hippocampus. We reflect on the strengths and weaknesses of the methodologies currently at our disposal, the utility of emerging technologies and identify questions that we feel are critical for the advancement of the field. We expect that magnetic circuits are likely to share anatomical motifs with other senses, which culminates in the formation of spatial maps in telencephalic areas of the brain. Specifically, we predict the existence of spatial cells that encode defined components of the Earth's magnetic field.

Eyes are essential for magnetoreception in a mammal.

Several groups of mammals use the Earth's magnetic field for orientation, but their magnetosensory organ remains unknown. The Ansell's mole-rat (Fukomys anselli, Bathyergidae, Rodentia) is a microphthalmic subterranean rodent with innate magnetic orientation behaviour. Previous studies on this species proposed that its magnetoreceptors are located in the eye. To test this hypothesis, we assessed magnetic orientation in mole-rats after the surgical removal of their eyes compared to untreated controls. Initially, we demonstrate that this enucleation does not lead to changes in routine behaviours, including locomotion, feeding and socializing. We then studied magnetic compass orientation by employing a well-established nest-building assay under four magnetic field alignments. In line with previous studies, control animals exhibited a significant preference to build nests in magnetic southeast. By contrast, enucleated mole-rats built nests in random magnetic orientations, suggesting an impairment of their magnetic sense. The results provide robust support for the hypothesis that mole-rats perceive magnetic fields with their minute eyes, probably relying on magnetite-based receptors in the cornea.

The biophysical, molecular, and anatomical landscape of pigeon CRY4: A candidate light-based quantal magnetosensor.

The biophysical and molecular mechanisms that enable animals to detect magnetic fields are unknown. It has been proposed that birds have a light-dependent magnetic compass that relies on the formation of radical pairs within cryptochrome molecules. Using spectroscopic methods, we show that pigeon cryptochrome clCRY4 is photoreduced efficiently and forms long-lived spin-correlated radical pairs via a tetrad of tryptophan residues. We report that clCRY4 is broadly and stably expressed within the retina but enriched at synapses in the outer plexiform layer in a repetitive manner. A proteomic survey for retinal-specific clCRY4 interactors identified molecules that are involved in receptor signaling, including glutamate receptor-interacting protein 2, which colocalizes with clCRY4. Our data support a model whereby clCRY4 acts as an ultraviolet-blue photoreceptor and/or a light-dependent magnetosensor by modulating glutamatergic synapses between horizontal cells and cones.

Grouped circular data in biology: advice for effectively implementing statistical procedures.

ABSTRACT: The most common statistical procedure with a sample of circular data is to test the null hypothesis that points are spread uniformly around the circle without a preferred direction. An array of tests for this has been developed. However, these tests were designed for continuously distributed data, whereas often (e.g. due to limited precision of measurement techniques) collected data is aggregated into a set of discrete values (e.g. rounded to the nearest degree). This disparity can cause an uncontrolled increase in type I error rate, an effect that is particularly problematic for tests that are based on the distribution of arc lengths between adjacent points (such as the Rao spacing test). Here, we demonstrate that an easy-to-apply modification can correct this problem, and we recommend this modification when using any test, other than the Rayleigh test, of circular uniformity on aggregated data. We provide R functions for this modification for several commonly used tests. In addition, we tested the power of a recently proposed test, the Gini test. However, we concluded that it lacks sufficient increase in power to replace any of the tests already in common use. In conclusion, using any of the standard circular tests (except the Rayleigh test) without modifications on rounded/aggregated data, especially with larger sample sizes, will increase the proportion of false-positive results-but we demonstrate that a simple and general modification avoids this problem. SIGNIFICANCE STATEMENT: Circular data are widespread across biological disciplines, e.g. in orientation studies or circadian rhythms. Often these data are rounded to the nearest 1-10 degrees. We have shown previously that this leads to an inflation of false-positive results when testing whether the data is significantly different from a random distribution using the Rao test. Here we present a modification that avoids this increase in false-positives for rounded data while retaining statistical power for a variety of tests. In sum, we provide comprehensive guidance on how best to test for departure from uniformity in non-continuous data.

Model selection versus traditional hypothesis testing in circular statistics: a simulation study.

Many studies in biology involve data measured on a circular scale. Such data require different statistical treatment from those measured on linear scales. The most common statistical exploration of circular data involves testing the null hypothesis that the data show no aggregation and are instead uniformly distributed over the whole circle. The most common means of performing this type of investigation is with a Rayleigh test. An alternative might be to compare the fit of the uniform distribution model to alternative models. Such model-fitting approaches have become a standard technique with linear data, and their greater application to circular data has been recently advocated. Here we present simulation data that demonstrate that such model-based inference can offer very similar performance to the best traditional tests, but only if adjustment is made in order to control type I error rate.

Light entrainment of retinal biorhythms: cryptochrome 2 as candidate photoreceptor in mammals.

The mechanisms that synchronize the biorhythms of the mammalian retina with the light/dark cycle are independent of those synchronizing the rhythms in the central pacemaker, the suprachiasmatic nucleus. The identity of the photoreceptor(s) responsible for the light entrainment of the retina of mammals is still a matter of debate, and recent studies have reported contradictory results in this respect. Here, we suggest that cryptochromes (CRY), in particular CRY 2, are involved in that light entrainment. CRY are highly conserved proteins that are a key component of the cellular circadian clock machinery. In plants and insects, they are responsible for the light entrainment of these biorhythms, mediated by the light response of their flavin cofactor (FAD). In mammals, however, no light-dependent role is currently assumed for CRY in light-exposed tissues, including the retina. It has been reported that FAD influences the function of mammalian CRY 2 and that human CRY 2 responds to light in Drosophila, suggesting that mammalian CRY 2 keeps the ability to respond to light. Here, we hypothesize that CRY 2 plays a role in the light entrainment of retinal biorhythms, at least in diurnal mammals. Indeed, published data shows that the light intensity dependence and the wavelength sensitivity commonly reported for that light entrainment fits the light sensitivity and absorption spectrum of light-responsive CRY. We propose experiments to test our hypothesis and to further explore the still-pending question of the function of CRY 2 in the mammalian retina.

The Hermans-Rasson test as a powerful alternative to the Rayleigh test for circular statistics in biology.

BACKGROUND: Circular data are gathered in diverse fields of science where measured traits are cyclical in nature: such as compass directions or times of day. The most common statistical question asked of a sample of circular data is whether the data seems to be drawn from a uniform distribution or one that is concentrated around one or more preferred directions. The overwhelmingly most-popular test of the null hypothesis of uniformity is the Rayleigh test, even though this test is known to have very low power in some circumstances. Here we present simulation studies evaluating the performance of tests developed as alternatives to the Rayleigh test. RESULTS: The results of our simulations demonstrate that a single test, the Hermans and Rasson test is almost as powerful as the Rayleigh test in unimodal situations (when the Rayleigh test does well) but substantially outperforms the Rayleigh test in multimodal situations. CONCLUSION: We recommend researchers switch to routine use of the new Hermans and Rasson test. We also demonstrate that all available tests have low power to detect departures from uniformity involving more than two concentrated regions: we recommend that where researchers suspect such complex departures that they collect substantially-sized samples and apply another recent test due to Pycke that was designed specifically for such complex cases. We provide clear textual descriptions of how to implement each of these recommended tests and encode them in R functions that we provide.

Risk to pollinators from anthropogenic electro-magnetic radiation (EMR): Evidence and knowledge gaps.

Worldwide urbanisation and use of mobile and wireless technologies (5G, Internet of Things) is leading to the proliferation of anthropogenic electromagnetic radiation (EMR) and campaigning voices continue to call for the risk to human health and wildlife to be recognised. Pollinators provide many benefits to nature and humankind, but face multiple anthropogenic threats. Here, we assess whether artificial light at night (ALAN) and anthropogenic radiofrequency electromagnetic radiation (AREMR), such as used in wireless technologies (4G, 5G) or emitted from power lines, represent an additional and growing threat to pollinators. A lack of high quality scientific studies means that knowledge of the risk to pollinators from anthropogenic EMR is either inconclusive, unresolved, or only partly established. A handful of studies provide evidence that ALAN can alter pollinator communities, pollination and fruit set. Laboratory experiments provide some, albeit variable, evidence that the honey bee Apis mellifera and other invertebrates can detect EMR, potentially using it for orientation or navigation, but they do not provide evidence that AREMR affects insect behaviour in ecosystems. Scientifically robust evidence of AREMR impacts on abundance or diversity of pollinators (or other invertebrates) are limited to a single study reporting positive and negative effects depending on the pollinator group and geographical location. Therefore, whether anthropogenic EMR (ALAN or AREMR) poses a significant threat to insect pollinators and the benefits they provide to ecosystems and humanity remains to be established.

Circular statistics meets practical limitations: a simulation-based Rao's spacing test for non-continuous data.

BACKGROUND: For data collected on a circular rather than linear scale, a very common procedure is to test whether the underlying distribution appears to deviate from circular uniformity. Rao's spacing test is often used to evaluate the support the data offers for the null hypothesis of uniformity. Here we demonstrate that the traditional version of this test fails to adequately control type I error rate when the data is non-continuous (i.e. is rounded/grouped to a finite number of discrete values, e.g. to the nearest degree, a common situation). To overcome this issue, we provide a numerically-intensive simulation version of the test. METHODS: We use a simulation study to explore the performance of the traditional and our novel variant on Rao's spacing test, both in terms of control of type I error rate and statistical power. RESULTS: When data is measured on a continuous circular scale then both methods offer good control of type I error and similar statistical power. If the data is rounded (even to a relatively fine scale such as to the nearest degree - giving 360 possible values), however, the traditional method produces highly inflated type I error rates, particularly with high sample sizes, that make it inappropriate for application to such data. In contrast, our simulation method retains good control of type I error while offering levels of statistical power similar to the traditional Rao test. CONCLUSIONS: The traditional method of applying Rao's spacing test should be replaced by the simulation-based variant introduced here. The two methods offer similar performance but only the simulation method retains good control of the type I error rate when circular data is rounded to a finite set of values (likely due to limited precision of measuring equipment). Adoption of the simulation variant will substantially improve the reliability of this regularly-used test in the commonplace situation where data values are rounded.

No evidence for a magnetite-based magnetoreceptor in the lagena of pigeons.

It is well established that an array of avian species sense the Earth's magnetic field and use this information for orientation and navigation. While the existence of a magnetic sense can no longer be disputed, the underlying cellular and biophysical basis remains unknown. It has been proposed that pigeons exploit a magnetoreceptor based on magnetite crystals (Fe3O4) that are located within the lagena [1], a sensory epithelium of the inner ear. It has been hypothesised that these magnetic crystals form a bed of otoconia that stimulate hair cells transducing magnetic information into a neuronal impulse. We performed a systematic high-sensitivity screen for iron in the pigeon lagena using synchrotron X-ray fluorescence microscopy coupled with the analysis of serial sections by transmission electron microscopy. We find no evidence for extracellular magnetic otoconia or intracellular magnetite crystals, suggesting that if an inner ear magnetic sensor does exist it relies on a different biophysical mechanism.

Dogs can be trained to find a bar magnet.

Magnetoreception, the ability to sense the Earth's magnetic field (MF), is a widespread phenomenon in the animal kingdom. In 1966, the first report on a magnetosensitive vertebrate, the European robin (Erithacus rubecula), was published. After that, numerous further species of different taxa have been identified to be magnetosensitive as well. Recently, it has been demonstrated that domestic dogs (Canis lupus familiaris) prefer to align their body axis along the North-South axis during territorial marking under calm MF conditions and that they abandon this preference when the Earth's MF is unstable. In a further study conducting a directional two-choice-test, dogs showed a spontaneous preference for the northern direction. Being designated as putatively magnetosensitive and being also known as trainable for diverse choice and search tests, dogs seem to be suitable model animals for a direct test of magnetoreception: learning to find a magnet. Using operant conditioning dogs were trained to identify the MF of a bar magnet in a three-alternative forced-choice experiment. We excluded visual cues and used control trials with food treats to test for the role of olfaction in finding the magnet. While 13 out of 16 dogs detected the magnet significantly above chance level (53-73% success rate), none of the dogs managed to do so in finding the food treat (23-40% success rate). In a replication of the experiment under strictly blinded conditions five out of six dogs detected the magnet above chance level (53-63% success rate). These experiments support the existence of a magnetic sense in domestic dogs. Whether the sense enables dogs to perceive MFs as weak as the Earth's MF, if they use it for orientation, and by which mechanism the fields are perceived remain open questions.

Corrigendum: Low-Light Dependence of the Magnetic Field Effect on Cryptochromes: Possible Relevance to Plant Ecology.

[This corrects the article DOI: 10.3389/fpls.2018.00121.].