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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s).)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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.

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 (Fe 3 O 4 ) [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 (Ca V 1.3) 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., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Evolution has equipped life on our planet with an array of extraordinary senses, but perhaps the least understood is magnetoreception. Despite compelling behavioral evidence that this sense exists, the cells, molecules, and mechanisms that mediate sensory transduction remain unknown. So how could animals detect magnetic fields? We introduce and discuss 3 concepts that attempt to address this question: (1) a mechanically sensitive magnetite-based magnetoreceptor, (2) a light-sensitive chemical-based mechanism, and (3) electromagnetic induction within accessory structures. In discussing the merits and issues with each of these ideas, we draw on existing precepts in sensory biology. We argue that solving this scientific mystery will require the development of new genetic tools in magnetosensitive species, coupled with an interdisciplinary approach that bridges physics, behavior, anatomy, physiology, molecular biology, and genetics.

The quantification of salivary oxytocin (OXT) concentrations emerges as a helpful tool to assess peripheral OXT secretion at baseline and after various challenges in healthy and clinical populations. Both positive social interactions and stress are known to induce OXT secretion, but the relative influence of either of these triggers is not well delineated. Choir singing is an activity known to improve mood and to induce feelings of social closeness, and may therefore be used to investigate the effects of positive social experiences on OXT system activity. We quantified mood and salivary OXT and cortisol (CORT) concentrations before, during, and after both choir and solo singing performed in a randomized order in the same participants (repeated measures). Happiness was increased, and worry and sadness as well as salivary CORT concentrations were reduced, after both choir and solo singing. Surprisingly, salivary OXT concentrations were significantly reduced after choir singing, but did not change in response to solo singing. Salivary OXT concentrations showed high intra-individual stability, whereas salivary CORT concentrations fluctuated between days within participants. The present data indicate that the social experience of choir singing does not induce peripheral OXT secretion, as indicated by unchanged salivary OXT levels. Rather, the reduction of stress/arousal experienced during choir singing may lead to an inhibition of peripheral OXT secretion. These data are important for the interpretation of future reports on salivary OXT concentrations, and emphasize the need to strictly control for stress/arousal when designing similar experiments.

TRPV1 is a polymodally activated cation channel acting as key receptor in nociceptive neurons. Its function is strongly affected by kinase-mediated phosphorylation leading to hyperalgesia and allodynia. We present behavioral and molecular data indicating that TRPV1 is strongly modulated by Cdk5-mediated phosphorylation at position threonine-407(mouse)/T406(rat). Increasing or decreasing Cdk5 activity in genetically engineered mice has severe consequences on TRPV1-mediated pain perception leading to altered capsaicin consumption and sensitivity to heat. To understand the molecular and structural/functional consequences of TRPV1 phosphorylation, we generated various rTRPV1T406 receptor variants to mimic phosphorylated or dephosphorylated receptor protein. We performed detailed functional characterization by means of electrophysiological whole-cell and single-channel recordings as well as Ca(2+)-imaging and challenged recombinant rTRPV1 receptors with capsaicin, low pH, or heat. We found that position T406 is critical for the function of TRPV1 by modulating ligand-sensitivity, activation, and desensitization kinetics as well as voltage-dependence. Based on high resolution structures of TRPV1, we discuss T406 being involved in the molecular transition pathway, its phosphorylation leading to a conformational change and influencing the gating of the receptor. Cdk5-mediated phosphorylation of T406 can be regarded as an important molecular switch modulating TRPV1-related behavior and pain sensitivity.