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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.

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.

A diverse range of species is known to rely on the Earth’s magnetic field for spatial information. Vidal-Gadea et al. claimed that C. elegans are magneto-sensitive, exploiting the magnetic field to guide their burrowing behavior [1]. Our attempts to replicate their findings were unsuccessful [2], which Vidal-Gadea attributed to the satiety of the animals and the environment in which they were raised. Here, we report our repeated experiments, having adopted several suggestions proposed by Vidal-Gadea et al. [3]. We find that shortening the length of the behavioral assay and raising the animals in a Faraday cage does not result in magnetotactic behavior. We reluctantly conclude that the assays employed by Vidal-Gadea are not robust or C. elegans are not magneto-sensitive.

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 C. elegans possesses a magnetic sense that guides their vertical movement in soil. In making this claim they relied on three different behavioural 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.

The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022-12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8-106 fAm(2)) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation.

In their recent manuscript Xie and colleagues argue that pigeon ISCA1 and CRY4 act as a magnetic protein biocompass asserting that they have unambiguously proved that these two proteins interact and form a complex with unique biophysical features. We do not think the evidence presented in their manuscript supports this conclusion. Firstly, we show that for all of their experiments Xie and colleagues employed the incorrect sequence of CRY4, which did not include the functionally critical C-terminal tail. Secondly, we demonstrate that both CRY4 and ISCA1 are broadly expressed in all pigeon tissues, lacking the spatially restricted expression pattern that would one would except from a specialised sensory molecule. We conclude that ISCA1 and CRY4, even if they do interact in vivo, are highly unlikely to form a functional protein biocompass.