Long-term, high-resolution in vivo calcium imaging in pigeons.

In vivo 2-photon calcium imaging has led to fundamental advances in our understanding of sensory circuits in mammalian species. In contrast, few studies have exploited this methodology in birds, with investigators primarily relying on histological and electrophysiological techniques. Here, we report the development of in vivo 2-photon calcium imaging in awake pigeons. We show that the genetically encoded calcium indicator GCaMP6s, delivered by the adeno-associated virus rAAV2/7, allows high-quality, stable, and long-term imaging of neuronal populations at single-cell and single-dendrite resolution in the pigeon forebrain. We demonstrate the utility of our setup by investigating the processing of colors in the visual Wulst, the avian homolog of the visual cortex. We report that neurons in the Wulst are color selective and display diverse response profiles to light of different wavelengths. This technology provides a powerful tool to decipher the operating principles that underlie sensory encoding in birds.

MAPping tubulin mutations.

Microtubules are filamentous structures that play a critical role in a diverse array of cellular functions including, mitosis, nuclear translocation, trafficking of organelles and cell shape. They are composed of α/ß-tubulin heterodimers which are encoded by a large multigene family that has been implicated in an umbrella of disease states collectively known as the tubulinopathies. De novo mutations in different tubulin genes are known to cause lissencephaly, microcephaly, polymicrogyria, motor neuron disease, and female infertility. The diverse clinical features associated with these maladies have been attributed to the expression pattern of individual tubulin genes, as well as their distinct Functional repertoire. Recent studies, however, have highlighted the impact of tubulin mutations on microtubule-associated proteins (MAPs). MAPs can be classified according to their effect on microtubules and include polymer stabilizers (e.g., tau, MAP2, doublecortin), destabilizers (e.g., spastin, katanin), plus-end binding proteins (e.g., EB1-3, XMAP215, CLASPs) and motor proteins (e.g., dyneins, kinesins). In this review we analyse mutation-specific disease mechanisms that influence MAP binding and their phenotypic consequences, and discuss methods by which we can exploit genetic variation to identify novel MAPs.

Codon modification of Tuba1a alters mRNA levels and causes a severe neurodevelopmental phenotype in mice.

The tubulinopathies are an umbrella of rare diseases that result from mutations in tubulin genes and are frequently characterised by severe brain malformations. The characteristics of a given disease reflect the expression pattern of the transcript, the function of a given tubulin gene, and the role microtubules play in a particular cell type. Mouse models have proved to be valuable tools that have provided insight into the molecular and cellular mechanisms that underlie the disease state. In this manuscript we compare two Tuba1a mouse models, both of which express wild-type TUBA1A protein but employ different codon usage. We show that modification of the Tuba1a mRNA sequence results in homozygous lethality and a severe neurodevelopmental phenotype. This is associated with a decrease in the number of post-mitotic neurons, PAX6 positive progenitors, and an increase in the number of apoptotic cells. We attribute this to a decrease in the stability of the modified Tuba1a transcript, and the absence of compensation by the other neurogenic tubulins. Our findings highlight the importance of maintaining the wild-type coding sequence when engineering mouse lines and the impact of synonymous genetic variation.

Myths in magnetosensation.

The ability to detect magnetic fields is a sensory modality that is used by many animals to navigate. While first postulated in the 1800s, for decades, it was considered a biological myth. A series of elegant behavioral experiments in the 1960s and 1970s showed conclusively that the sense is real; however, the underlying mechanism(s) remained unresolved. Consequently, this has given rise to a series of beliefs that are critically analyzed in this manuscript. We address six assertions: (1) Magnetoreception does not exist; (2) It has to be magnetite; (3) Birds have a conserved six loci magnetic sense system in their upper beak; (4) It has to be cryptochrome; (5) MagR is a protein biocompass; and (6) The electromagnetic induction hypothesis is dead. In advancing counter-arguments for these beliefs, we hope to stimulate debate, new ideas, and the design of well-controlled experiments that can aid our understanding of this fascinating biological phenomenon.

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.

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.

A proteomic survey of microtubule-associated proteins in a R402H TUBA1A mutant mouse.

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs.

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.

A high sensitivity ZENK monoclonal antibody to map neuronal activity in Aves.

The transcription factor ZENK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. It has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. To address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZENK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain.

Why (and how) we should publish negative data.

Negative data and refutations are a crucial element of the scientific process. But it needs solid arguments to convince editors and reviewers to publish negative results.

A Putative Mechanism for Magnetoreception by Electromagnetic Induction in the Pigeon Inner Ear.

A diverse array of vertebrate species employs the Earth's magnetic field to assist navigation. Despite compelling behavioral evidence that a magnetic sense exists, the location of the primary sensory cells and the underlying molecular mechanisms remain unknown [1]. To date, most research has focused on a light-dependent radical-pair-based concept and a system that is proposed to rely on biogenic magnetite (Fe3O4) [2, 3]. Here, we explore an overlooked hypothesis that predicts that animals detect magnetic fields by electromagnetic induction within the semicircular canals of the inner ear [4]. Employing an assay that relies on the neuronal activity marker C-FOS, we confirm that magnetic exposure results in activation of the caudal vestibular nuclei in pigeons that is independent of light [5]. We show experimentally and by physical calculations that magnetic stimulation can induce electric fields in the pigeon semicircular canals that are within the physiological range of known electroreceptive systems. Drawing on this finding, we report the presence of a splice isoform of a voltage-gated calcium channel (CaV1.3) in the pigeon inner ear that has been shown to mediate electroreception in skates and sharks [6]. We propose that pigeons detect magnetic fields by electromagnetic induction within the semicircular canals that is dependent on the presence of apically located voltage-gated cation channels in a population of electrosensory hair cells.

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.

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

Cryptochrome: The magnetosensor with a sinister side?

Over the last three decades, evidence has emerged that low-intensity magnetic fields can influence biological systems. It is now well established that migratory birds have the capacity to detect the Earth's magnetic field; it has been reported that power lines are associated with childhood leukemia and that pulsed magnetic fields increase the production of reactive oxidative species (ROS) in cellular systems. Justifiably, studies in this field have been viewed with skepticism, as the underlying molecular mechanisms are unknown. In the accompanying paper, Sherrard and colleagues report that low-flux pulsed electromagnetic fields (PEMFs) result in aversive behavior in Drosophila larvae and ROS production in cell culture. They further report that these responses require the presence of cryptochrome, a putative magnetoreceptor. If correct, it is conceivable that carcinogenesis associated with power lines, PEMF-induced ROS generation, and animal magnetoreception share a common mechanistic basis.

Lidocaine is a nocebo treatment for trigeminally mediated magnetic orientation in birds.

Even though previously described iron-containing structures in the upper beak of pigeons were almost certainly macrophages, not magnetosensitive neurons, behavioural and neurobiological evidence still supports the involvement of the ophthalmic branch of the trigeminal nerve (V1) in magnetoreception. In previous behavioural studies, inactivation of putative V1-associated magnetoreceptors involved either application of the surface anaesthetic lidocaine to the upper beak or sectioning of V1. Here, we compared the effects of lidocaine treatment, V1 ablations and sham ablations on magnetic field-driven neuronal activation in V1-recipient brain regions in European robins. V1 sectioning led to significantly fewer Egr-1-expressing neurons in the trigeminal brainstem than in the sham-ablated birds, whereas lidocaine treatment had no effect on neuronal activation. Furthermore, Prussian blue staining showed that nearly all iron-containing cells in the subepidermal layer of the upper beak are nucleated and are thus not part of the trigeminal nerve, and iron-containing cells appeared in highly variable numbers at inconsistent locations between individual robins and showed no systematic colocalization with a neuronal marker. Our data suggest that lidocaine treatment has been a nocebo to the birds and a placebo for the experimenters. Currently, the nature and location of any V1-associated magnetosensor remains elusive.

Ectopic otoconial formation in the lagena of the pigeon inner ear.

The vertebrate inner ear contains vestibular receptors with dense crystals of calcium carbonate, the otoconia. The production and maintenance of otoconia is a delicate process, the perturbation of which can lead to severe vestibular dysfunction in humans. The details of these processes are not well understood. Here, we report the discovery of a new otoconial mass in the lagena of adult pigeons that was present in more than 70% of birds. Based on histological, tomographic and elemental analyses, we conclude that the structure likely represents an ectopically-formed otoconial assembly. Given its frequent natural occurrence, we suggest that the pigeon lagena is a valuable model system for investigating misregulated otoconial formation.This article has an associated First Person interview with the first author of the paper.

Publisher Correction: Mutations in Vps15 perturb neuronal migration in mice and are associated with neurodevelopmental disease in humans.

In the supplementary information PDF originally posted, there were discrepancies from the integrated supplementary information that appeared in the HTML; the former has been corrected as follows. In the legend to Supplementary Fig. 2c, "major organs of the mouse" has been changed to "major organs of the adult mouse." In the legend to Supplementary Fig. 6d,h, "At E14.5 Mbe/Mbe mutants have a smaller percentage of Brdu positive cells in bin 3" has been changed to "At E14.5 Mbe/Mbe mutants have a higher percentage of Brdu positive cells in bin 3."

Comment on "Magnetosensitive neurons mediate geomagnetic orientation in Caenorhabditis elegans".

A diverse array of species on the planet employ the Earth's magnetic field as a navigational aid. As the majority of these animals are migratory, their utility to interrogate the molecular and cellular basis of the magnetic sense is limited. Vidal-Gadea and colleagues recently argued that the worm Caenorhabditis elegans possesses a magnetic sense that guides their vertical movement in soil. In making this claim, they relied on three different behavioral assays that involved magnetic stimuli. Here, we set out to replicate their results employing blinded protocols and double wrapped coils that control for heat generation. We find no evidence supporting the existence of a magnetic sense in C. elegans. We further show that the Vidal-Gadea hypothesis is problematic as the adoption of a correction angle and a fixed trajectory relative to the Earth's magnetic inclination does not necessarily result in vertical movement.

Improved Genome Assembly and Annotation for the Rock Pigeon (Columba livia).

The domestic rock pigeon (Columba livia) is among the most widely distributed and phenotypically diverse avian species. C. livia is broadly studied in ecology, genetics, physiology, behavior, and evolutionary biology, and has recently emerged as a model for understanding the molecular basis of anatomical diversity, the magnetic sense, and other key aspects of avian biology. Here we report an update to the C. livia genome reference assembly and gene annotation dataset. Greatly increased scaffold lengths in the updated reference assembly, along with an updated annotation set, provide improved tools for evolutionary and functional genetic studies of the pigeon, and for comparative avian genomics in general.