A newly identified trigeminal brain pathway in a night-migratory bird could be dedicated to transmitting magnetic map information.

Night-migratory songbirds can use geomagnetic information to navigate over thousands of kilometres with great precision. A crucial part of the magnetic 'map' information used by night-migratory songbirds is conveyed via the ophthalmic branches of the trigeminal nerves to the trigeminal brainstem complex, where magnetic-driven neuronal activation has been observed. However, it is not known how this information reaches the forebrain for further processing. Here, we show that the magnetically activated region in the trigeminal brainstem of migratory Eurasian blackcaps (Sylvia atricapilla) represents a morphologically distinctive neuronal population with an exclusive and previously undescribed projection to the telencephalic frontal nidopallium. This projection is clearly different from the known trigeminal somatosensory pathway that we also confirmed both by neuronal tracing and by a thorough morphometric analysis of projecting neurons. The new pathway we identified here represents part of a brain circuit that-based on the known nidopallial connectivities in birds-could potentially transmit magnetic 'map' information to key multisensory integration centres in the brain known to be critically involved in spatial memory formation, cognition and/or controlling executive behaviour, such as navigation, in birds.

The Neural Basis of Long-Distance Navigation in Birds.

Migratory birds can navigate over tens of thousands of kilometers with an accuracy unobtainable for human navigators. To do so, they use their brains. In this review, we address how birds sense navigation- and orientation-relevant cues and where in their brains each individual cue is processed. When little is currently known, we make educated predictions as to which brain regions could be involved. We ask where and how multisensory navigational information is integrated and suggest that the hippocampus could interact with structures that represent maps and compass information to compute and constantly control navigational goals and directions. We also suggest that the caudolateral nidopallium could be involved in weighing conflicting pieces of information against each other, making decisions, and helping the animal respond to unexpected situations. Considering the gaps in current knowledge, some of our suggestions may be wrong. However, our main aim is to stimulate further research in this fascinating field.

Visual but not trigeminal mediation of magnetic compass information in a migratory bird.

Magnetic compass information has a key role in bird orientation, but the physiological mechanisms enabling birds to sense the Earth's magnetic field remain one of the unresolved mysteries in biology. Two biophysical mechanisms have become established as the most promising magnetodetection candidates. The iron-mineral-based hypothesis suggests that magnetic information is detected by magnetoreceptors in the upper beak and transmitted through the ophthalmic branch of the trigeminal nerve to the brain. The light-dependent hypothesis suggests that magnetic field direction is sensed by radical pair-forming photopigments in the eyes and that this visual signal is processed in cluster N, a specialized, night-time active, light-processing forebrain region. Here we report that European robins with bilateral lesions of cluster N are unable to show oriented magnetic-compass-guided behaviour but are able to perform sun compass and star compass orientation behaviour. In contrast, bilateral section of the ophthalmic branch of the trigeminal nerve in European robins did not influence the birds' ability to use their magnetic compass for orientation. These data show that cluster N is required for magnetic compass orientation in this species and indicate that it may be specifically involved in processing of magnetic compass information. Furthermore, the data strongly suggest that a vision-mediated mechanism underlies the magnetic compass in this migratory songbird, and that the putative iron-mineral-based receptors in the upper beak connected to the brain by the trigeminal nerve are neither necessary nor sufficient for magnetic compass orientation in European robins.

Magnetic field changes activate the trigeminal brainstem complex in a migratory bird.

The upper beak of birds, which contains putative magnetosensory ferro-magnetic structures, is innervated by the ophthalmic branch of the trigeminal nerve (V1). However, because of the absence of replicable neurobiological evidence, a general acceptance of the involvement of the trigeminal nerve in magnetoreception is lacking in birds. Using an antibody to ZENK protein to indicate neuronal activation, we here document reliable magnetic activation of neurons in and near the principal (PrV) and spinal tract (SpV) nuclei of the trigeminal brainstem complex, which represent the two brain regions known to receive primary input from the trigeminal nerve. Significantly more neurons were activated in PrV and in medial SpV when European robins (Erithacus rubecula) experienced a magnetic field changing every 30 seconds for a period of 3 h (CMF) than when robins experienced a compensated, zero magnetic field condition (ZMF). No such differences in numbers of activated neurons were found in comparison structures. Under CMF conditions, sectioning of V1 significantly reduced the number of activated neurons in and near PrV and medial SpV, but not in lateral SpV or in the optic tectum. Tract tracing of V1 showed spatial proximity and regional overlap of V1 nerve endings and ZENK-positive (activated) neurons in SpV, and partly in PrV, under CMF conditions. Together, these results suggest that magnetic field changes activate neurons in and near the trigeminal brainstem complex and that V1 is necessary for this activation. We therefore suggest that V1 transmits magnetic information to the brain in this migratory passerine bird.

Prussian blue technique is prone to yield false negative results in magnetoreception research.

Perls's Prussian blue staining technique has been used in magnetoreception research to screen tissues for iron-rich structures as proxies for putative magnetoreceptor structures based on magnetic particles. However, seemingly promising structural candidates in the upper beak of birds detected with Prussian blue turned out to be either irreproducible or located in non-neuronal cells, which has spurred a controversy that has not been settled yet. Here we identify possible pitfalls in the previous works and apply the Prussian blue technique to tissues implicated in magnetic-particle-based magnetoreception, in an effort to reassess its suitability for staining single-domain magnetite, i.e., the proposed magnetic substrate for the interaction with the external magnetic field. In the upper beak of night-migratory songbirds, we found staining products in great numbers, but not remotely associated with fiber terminals of the traced ophthalmic branch of the trigeminal nerve. Surprisingly, staining products were absent from the lamina propria in the olfactory rosette of rainbow trout where candidate magnetoreceptor structures were identified with different techniques earlier. Critically, magnetosome chains in whole cells of magnetotactic bacteria remained unstained. The failure to label single-domain magnetite in positive control samples is a serious limitation of the technique and suggests that two most influential but antipodal studies conducted previously stood little chances of obtaining correct positive results under the assumption that magnetosome-like particles were present in the tissues. Nonetheless, the staining technique appears suitable to identify tissue contamination with iron-rich fine dust trapped in epithelia already in vivo.

In Search for the Avian Trigeminal Magnetic Sensor: Distribution of Peripheral and Central Terminals of Ophthalmic Sensory Neurons in the Night-Migratory Eurasian Blackcap (Sylvia atricapilla).

In night-migratory songbirds, neurobiological and behavioral evidence suggest the existence of a magnetic sense associated with the ophthalmic branch of the trigeminal nerve (V1), possibly providing magnetic positional information. Curiously, neither the unequivocal existence, structural nature, nor the exact location of any sensory structure has been revealed to date. Here, we used neuronal tract tracing to map both the innervation fields in the upper beak and the detailed trigeminal brainstem terminations of the medial and lateral V1 subbranches in the night-migratory Eurasian Blackcap (Sylvia atricapilla). The medial V1 subbranch takes its course along the ventral part of the upper beak to innervate subepidermal layers and the mucosa of the nasal cavity, whereas the lateral V1 subbranch runs along dorsolateral levels until the nostrils to innervate mainly the skin of the upper beak. In the trigeminal brainstem, medial V1 terminals innervate both the dorsal part and the ventral, magnetically activated part of the principal sensory trigeminal brainstem nuclei (PrV). In contrast, the lateral V1 subbranch innervates only a small part of the ventral PrV. The spinal sensory trigeminal brainstem nuclei (SpV) receive topographically ordered projections. The medial V1 subbranch mainly innervates rostral and medial parts of SpV, whereas the lateral V1 subbranch mainly innervates the lateral and caudal parts of SpV. The present findings could provide valuable information for further analysis of the trigeminal magnetic sense of birds.

Morphology, biochemistry and connectivity of Cluster N and the hippocampal formation in a migratory bird.

The exceptional navigational capabilities of migrating birds are based on the perception and integration of a variety of natural orientation cues. The "Wulst" in the forebrain of night-migratory songbirds contains a brain area named "Cluster N", which is involved in processing directional navigational information derived from the Earth´s magnetic field. Cluster N is medially joined by the hippocampal formation, known to retrieve and utilise navigational information. To investigate the connectivity and neurochemical characteristics of Cluster N and the hippocampal formation of migratory birds, we performed morphological and histochemical analyses based on the expression of calbindin, calretinin, parvalbumin, glutamate receptor type 1 and early growth response protein-1 in the night-migratory Garden warbler (Sylvia borin) and mapped their mutual connections using neuronal tract tracing. The resulting expression patterns revealed regionally restricted neurochemical features, which mapped well onto the hippocampal and hyperpallial substructures known from other avian species. Magnetic field-induced neuronal activation covered caudal parts of the hyperpallium and the medially adjacent hippocampal dorsomedial/dorsolateral subdivisions. Neuronal tract tracings revealed connections between Cluster N and the hippocampal formation with the vast majority originating from the densocellular hyperpallium, either directly or indirectly via the area corticoidea dorsolateralis. Our data indicate that the densocellular hyperpallium could represent a central relay for the transmission of magnetic compass information to the hippocampal formation where it might be integrated with other navigational cues in night-migratory songbirds.

Morphology of the "prefrontal" nidopallium caudolaterale in the long-distance night-migratory Eurasian blackcap (Sylvia atricapilla).

Migrating birds have developed remarkable navigational capabilities to successfully master biannual journeys between their breeding and wintering grounds. To reach their intended destination, they need to calculate navigational goals from a large variety of natural directional and positional cues to set a meaningful motor output command. One brain area, which has been associated with such executive functions, is the nidopallium caudolaterale (NCL), which, due to its striking similarities in terms of neurochemistry, connectivity and function, is considered analogous to the mammalian prefrontal cortex. To establish a baseline for further analyses elucidating the neuronal correlates underlying avian navigation, we performed quantitative and qualitative analyses of dopaminergic fibres in the brains of long-distance night-migratory Eurasian blackcaps (Sylvia atricapilla). We identified four regions in the caudal telencephalon, each of which was characterized by its specific dopaminergic innervation pattern. At least three of them presumably constitute subareas of the NCL in Eurasian blackcaps and could thus be involved in integrating navigational input from different sensory systems. The observed heterogeneity and parcellation of the NCL subcompartments in this migratory species could be a consequence of the special demands related to navigation.

Night-time neuronal activation of Cluster N in a day- and night-migrating songbird.

Magnetic compass orientation in a night-migratory songbird requires that Cluster N, a cluster of forebrain regions, is functional. Cluster N, which receives input from the eyes via the thalamofugal pathway, shows high neuronal activity in night-migrants performing magnetic compass-guided behaviour at night, whereas no activation is observed during the day, and covering up the birds' eyes strongly reduces neuronal activation. These findings suggest that Cluster N processes light-dependent magnetic compass information in night-migrating songbirds. The aim of this study was to test if Cluster N is active during daytime migration. We used behavioural molecular mapping based on ZENK activation to investigate if Cluster N is active in the meadow pipit (Anthus pratensis), a day- and night-migratory species. We found that Cluster N of meadow pipits shows high neuronal activity under dim-light at night, but not under full room-light conditions during the day. These data suggest that, in day- and night-migratory meadow pipits, the light-dependent magnetic compass, which requires an active Cluster N, may only be used during night-time, whereas another magnetosensory mechanism and/or other reference system(s), like the sun or polarized light, may be used as primary orientation cues during the day.

Calcium-binding proteins label functional streams of the visual system in a songbird.

The vertebrate nervous system has been shown to contain high concentrations of intracellular calcium-binding proteins, each of them with a restricted expression pattern in specific brain regions and specific neuronal subpopulations. Using immunohistochemical staining techniques, we analyzed the expression pattern of calbindin, calretinin and parvalbumin in visual brain areas of a songbird species, the zebra finch (Taeniopyga guttata). Here we show that the analyzed proteins are expressed in a complementary fashion within different brain substructures generally corresponding to functional subpathways of the avian visual system. In detail, calbindin is expressed in the brain structures that belong to the thalamofugal pathway, whereas parvalbumin-positive neurons are found in the brain structures that are part of the tectofugal visual pathway. Originally, the expression of calcium-binding proteins has been associated with specific morphological or neurochemical criteria of neurons. Our results suggest that their expression pattern also indicates a functional segregation of brain substructures linked to vision in the zebra finch brain. As the selective labeling of functional streams has also been shown for the visual system in mammalian species, function-selective expression of calcium-binding proteins might be a general feature of vertebrates.

Magnetic map navigation in a migratory songbird requires trigeminal input.

Recently, virtual magnetic displacement experiments have shown that magnetic cues are indeed important for determining position in migratory birds; but which sensory system(s) do they use to detect the magnetic map cues? Here, we show that Eurasian reed warblers need trigeminal input to detect that they have been virtually magnetically displaced. Birds with bilaterally ablated ophthalmic branches of the trigeminal nerves were not able to re-orient towards their conspecific breeding grounds after a virtual magnetic displacement, exactly like they were not able to compensate for a real physical displacement. In contrast, sham-operated reed warblers re-oriented after the virtual displacement, like intact controls did in the past. Our results show that trigeminally mediated sensory information is necessary for the correct function of the reed warblers' magnetic positioning system.

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.

Experienced migratory songbirds do not display goal-ward orientation after release following a cross-continental displacement: an automated telemetry study.

The ability to navigate implies that animals have the capability to compensate for geographical displacement and return to their initial goal or target. Although some species are capable of adjusting their direction after displacement, the environmental cues used to achieve this remain elusive. Two possible cues are geomagnetic parameters (magnetic map hypothesis) or atmospheric odour-forming gradients (olfactory map hypothesis). In this study, we examined both of these hypotheses by surgically deactivating either the magnetic or olfactory sensory systems in experienced white-throated sparrows (Zonotrichia albicollis) captured in southern Ontario, Canada, during spring migration. Treated, sham-treated, and intact birds were then displaced 2,200 km west to Saskatchewan, Canada. Tracking their initial post-displacement migration using an array of automated VHF receiving towers, we found no evidence in any of the groups for compensatory directional response towards their expected breeding grounds. Our results suggest that white-throated sparrows may fall back to a simple constant-vector orientation strategy instead of performing true navigation after they have been geographically displaced to an unfamiliar area during spring migration. Such a basic strategy may be more common than currently thought in experienced migratory birds and its occurrence could be determined by habitat preferences or range size.

Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.

Targeting axons to specific fiber tracts in vivo by altering cadherin expression.

In brain development, neurons have to be connected with specific postsynaptic neurons to establish functional neuronal circuits. Cadherins are cell adhesion molecules, which mark developing neuronal circuits. Each member of this class of molecules is expressed only on a restricted set of fiber fascicles that connect gray matter structures to form functional neural circuits. In view of their expression patterns, cadherins have been postulated to play a functional role in the proper establishment of fiber connections. We chose the chicken optic tectum to analyze the instructive potential of cadherins in axonal pathfinding. Three tectofugal pathways, the tectothalamic, tectobulbar, and tectoisthmic tracts, exit the dorsal mesencephalon via the brachium of the superior colliculus, a large fiber structure, which can be divided in specific subtracts that are characterized by the selective expression of N-cadherin, cadherin-7, cadherin-6B, or R-cadherin. By using in vivo electroporation, we overexpressed each of the cadherins in tectal projection neurons between embryonic days 6 and 11. Cotransfection with green fluorescent protein expression plasmid allowed us to assess the pathway choice, which the transgenic axons had made. Quantification based on confocal laser scanning microscopic images revealed that transgenic axons selectively fasciculated with tectofugal tracts specified by the matching type of cadherin. This is the first direct evidence that cadherins mediate differential axonal pathfinding in vivo, possibly by a preferentially homotypic adhesive mechanism.

Cadherin expression coincides with birth dating patterns in patchy compartments of the developing chicken telencephalon.

Some gray matter regions of the vertebrate brain, e.g., the mammalian striatum, are organized into clusters of functionally similar neurons ("patches") that are surrounded by a gray matter matrix. A similar type of compartmentation recently has been found also in the avian telencephalon, based on two sorts of evidence. First, a birth dating study showed that, in some telencephalic areas, cells born at the same time form "isochronic" cell clusters, which differ in their birth dating pattern from their surrounding tissue. Second, patchy expression was found for different members of the cadherin family of adhesion molecules (R-cadherin, N-cadherin, and/or cadherin-7). The relation between these two findings has remained unclear. Here, we demonstrate a general spatial coincidence between the birth dating patterns and the heterogeneities in cadherin expression. This coincidence is found in areas of the avian ventral and lateral pallium (ventral hyperstriatum, neostriatum, and ectostriatum) and in a part of the archistriatum, which is of pallial origin. For example, in the neostriatum, a part of the ventral pallium, both the cadherin-7-positive patches and their R-cadherin-positive surroundings are born between embryonic day 5 and 7, but the cadherin-7-positive cells are born during a shorter time period than the R-cadherin-positive cells. We propose that the patchy gray matter architecture observed in the mammalian striatum and avian pallium are regulated by a common type of morphogenetic mechanism. This mechanism possibly involves the differential expression of adhesive factors, such as cadherins.

Patch/matrix patterns of gray matter differentiation in the telencephalon of chicken and mouse.

The mammalian striatum, a subpallial area, consists of two compartments (patches/striosomes and matrix) that differ in their neuronal birth dates, connectivity, neurochemistry, and molecular make-up. For example, members of the cadherin family of adhesion molecules (cadherin-8 and OL-protocadherin) are differentially expressed by the striosomes and the striatal matrix. A patch/matrix type of organization also has recently been found in the ventral hyperstriatum and the neostriatum of the chicken pallium, where cell clusters of similar birthdates ("isochronic" clusters) are surrounded by a matrix of cells that are born at a different time. Immunostaining with antibodies against cadherins reveals a similar arrangement of cell clusters. In the avian neostriatum, cadherin-7-positive cell clusters ("islands") are surrounded by a matrix of cells that express R-cadherin. The islands coincide, at least in part, with the isochronic cell clusters, as shown by pulse-labeling with bromodeoxyuridine. Likewise, isochronic clusters of the hyperstriatum ventrale relate to patchy heterogeneities in the cadherin-7 immunoreactivity pattern. Cadherins are known to mediate the aggregation and sorting of cells during development in many organs. Their differential expression by isochronic cell populations in the mammal subpallium and avian pallium suggests a common morphogenetic mechanism that regulates the formation of the patch/matrix patterns in these regions.

Magnetic field-driven induction of ZENK in the trigeminal system of pigeons (Columba livia).

Magnetoreception remains one of the few unsolved mysteries in sensory biology. The upper beak, which is innervated by the ophthalmic branch of the trigeminal nerve (V1), has been suggested to contain magnetic sensors based on ferromagnetic structures. Recently, its existence in pigeons has been seriously challenged by studies suggesting that the previously described iron-accumulations are macrophages, not magnetosensitive nerve endings. This raised the fundamental question of whether V1 is involved in magnetoreception in pigeons at all. We exposed pigeons to either a constantly changing magnetic field (CMF), to a zero magnetic field providing no magnetic information, or to CMF conditions after V1 was cut bilaterally. Using immediate early genes as a marker of neuronal responsiveness, we report that the trigeminal brainstem nuclei of pigeons, which receive V1 input, are activated under CMF conditions and that this neuronal activation disappears if the magnetic stimuli are removed or if V1 is cut. Our data suggest that the trigeminal system in pigeons is involved in processing magnetic field information and that V1 transmits this information from currently unknown, V1-associated magnetosensors to the brain.

Migratory Reed Warblers Need Intact Trigeminal Nerves to Correct for a 1,000 km Eastward Displacement.

Several studies have shown that experienced night-migratory songbirds can determine their position, but it has remained a mystery which cues and sensory mechanisms they use, in particular, those used to determine longitude (east-west position). One potential solution would be to use a magnetic map or signpost mechanism like the one documented in sea turtles. Night-migratory songbirds have a magnetic compass in their eyes and a second magnetic sense with unknown biological function involving the ophthalmic branch of the trigeminal nerve (V1). Could V1 be involved in determining east-west position? We displaced 57 Eurasian reed warblers (Acrocephalus scirpaceus) with or without sectioned V1. Sham operated birds corrected their orientation towards the breeding area after displacement like the untreated controls did. In contrast, V1-sectioned birds did not correct for the displacement. They oriented in the same direction after the displacement as they had done at the capture site. Thus, an intact ophthalmic branch of the trigeminal nerve is necessary for detecting the 1,000 km eastward displacement in this night-migratory songbird. Our results suggest that V1 carries map-related information used in a large-scale map or signpost sense that the reed warblers needed to determine their approximate geographical position and/or an east-west coordinate.

An iron-rich organelle in the cuticular plate of avian hair cells.

Hair cells reside in specialized epithelia in the inner ear of vertebrates, mediating the detection of sound, motion, and gravity. The transduction of these stimuli into a neuronal impulse requires the deflection of stereocilia, which are stabilized by the actin-rich cuticular plate. Recent electrophysiological studies have implicated the vestibular system in pigeon magnetosensation. Here we report the discovery of a single iron-rich organelle that resides in the cuticular plate of cochlear and vestibular hair cells in the pigeon. Transmission electron microscopy, coupled with elemental analysis, has shown that this structure is composed of ferritin-like granules, is approximately 300-600 nm in diameter, is spherical, and in some instances is membrane-bound and/or organized in a paracrystalline array. This organelle is found in hair cells in a wide variety of avian species, but not in rodents or in humans. This structure may function as (1) a store of excess iron, (2) a stabilizer of stereocilia, or (3) a mediator of magnetic detection. Given the specific subcellular location, elemental composition, and evolutionary conservation, we propose that this structure is an integral component of the sensory apparatus in birds.