Model organisms and systems in neuroethology: one hundred years of history and a look into the future.

The Journal of Comparative Physiology lived up to its name in the last 100 years by including more than 1500 different taxa in almost 10,000 publications. Seventeen phyla of the animal kingdom were represented. The honeybee (Apis mellifera) is the taxon with most publications, followed by locust (Locusta migratoria), crayfishes (Cambarus spp.), and fruitfly (Drosophila melanogaster). The representation of species in this journal in the past, thus, differs much from the 13 model systems as named by the National Institutes of Health (USA). We mention major accomplishments of research on species with specific adaptations, specialist animals, for example, the quantitative description of the processes underlying the axon potential in squid (Loligo forbesii) and the isolation of the first receptor channel in the electric eel (Electrophorus electricus) and electric ray (Torpedo spp.). Future neuroethological work should make the recent genetic and technological developments available for specialist animals. There are many research questions left that may be answered with high yield in specialists and some questions that can only be answered in specialists. Moreover, the adaptations of animals that occupy specific ecological niches often lend themselves to biomimetic applications. We go into some depth in explaining our thoughts in the research of motion vision in insects, sound localization in barn owls, and electroreception in weakly electric fish.

Optocollic responses in adult barn owls (Tyto furcata).

Barn owls, like primates, have frontally oriented eyes, which allow for a large binocular overlap. While owls have similar binocular vision and visual-search strategies as primates, it is less clear whether reflexive visual behavior also resembles that of primates or is more similar to that of closer related, but lateral-eyed bird species. Test cases are visual responses driven by wide-field movement: the optokinetic, optocollic, and optomotor responses, mediated by eye, head and body movements, respectively. Adult primates have a so-called symmetric horizontal response: they show the same following behavior, if the stimulus, presented to one eye only, moves in the nasal-to-temporal direction or in the temporal-to-nasal direction. By contrast, lateral-eyed birds have an asymmetric response, responding better to temporal-to-nasal movement than to nasal-to-temporal movement. We show here that the horizontal optocollic response of adult barn owls is less asymmetric than that in the chicken for all velocities tested. Moreover, the response is symmetric for low velocities (< 20 deg/s), and similar to that of primates. The response becomes moderately asymmetric for middle-range velocities (20-40 deg/s). A definitive statement for the complex situation for higher velocities (> 40 deg/s) is not possible.

Development of the horizontal optocollic reflex in juvenile barn owls (Tyto furcata pratincola).

Adult barn owls and primates possess an almost symmetric monocular rotational horizontal optocollic reflex. In primates, the reflex is initially asymmetric and becomes symmetric with time after birth. The condition in barn owls has not been studied so far. Here, we present data on the development of this reflex in this bird. We tested juvenile barn owls from the time before they open their eyes after hatching to the time they reach adult feather length. Wide-field visual patterns served as stimuli. They were presented at different rotational speeds in binocular and monocular settings. The binocular horizontal optocollic responses of juvenile barn owls were symmetric and adult-like on the first day that the birds responded to the stimulus. The monocular responses showed different rates of development in respect to stimulus velocity and stimulus direction. For velocities up to 20 deg/s, the monocular reflex was also adult-like on the first day that the birds responded to the stimulus. An initially higher asymmetry for 30 deg/s compared to adults disappeared within about two weeks. The development at even higher velocities remained unclear.

Sound localization in barn owls studied with manipulated head-related transfer functions: beyond broadband interaural time and level differences.

Interaural time and level differences are important cues for sound localization. We wondered whether the broadband information contained in these two cues could fully explain the behavior of barn owls and responses of midbrain neurons in these birds. To tackle this problem, we developed a novel approach based on head-related transfer functions. These filters contain the complete information present at the eardrum. We selected positions in space characterized by equal broadband interaural time and level differences. Stimulation from such positions provides reduced information to the owl. We show that barn owls are able to discriminate between such positions. In many cases, but not all, the owls may have used spectral components of interaural level differences that exceeded the known behavioral resolution and variability for discrimination. Alternatively, the birds may have used template matching. Likewise, neurons in the optic tectum of the barn owl, a nucleus involved in sensorimotor integration, contained more information than is available in the broadband interaural time and level differences. Thus, these data show that more information is available and used by barn owls for sound localization than carried by broadband interaural time and level differences.

Behavioral Evidence and Neural Correlates of Perceptual Grouping by Motion in the Barn Owl.

Perceiving an object as salient from its surround often requires a preceding process of grouping the object and background elements as perceptual wholes. In humans, motion homogeneity provides a strong cue for grouping, yet it is unknown to what extent this occurs in nonprimate species. To explore this question, we studied the effects of visual motion homogeneity in barn owls of both genders, at the behavioral as well as the neural level. Our data show that the coherency of the background motion modulates the perceived saliency of the target object. An object moving in an odd direction relative to other objects attracted more attention when the other objects moved homogeneously compared with when moved in a variety of directions. A possible neural correlate of this effect may arise in the population activity of the intermediate/deep layers of the optic tectum. In these layers, the neural responses to a moving element in the receptive field were suppressed when additional elements moved in the surround. However, when the surrounding elements all moved in one direction (homogeneously moving), they induced less suppression of the response compared with nonhomogeneously moving elements. Moreover, neural responses were more sensitive to the homogeneity of the background motion than to motion-direction contrasts between the receptive field and the surround. The findings suggest similar principles of saliency-by-motion in an avian species as in humans and show a locus in the optic tectum where the underlying neural circuitry may exist.SIGNIFICANCE STATEMENT A critical task of the visual system is to arrange incoming visual information to a meaningful scene of objects and background. In humans, elements that move homogeneously are grouped perceptually to form a categorical whole object. We discovered a similar principle in the barn owl's visual system, whereby the homogeneity of the motion of elements in the scene allows perceptually distinguishing an object from its surround. The novel findings of these visual effects in an avian species, which lacks neocortical structure, suggest that our basic visual perception shares more universal principles across species than presently thought, and shed light on possible brain mechanisms for perceptual grouping.

Kocuria tytonis sp. nov., isolated from the uropygial gland of an American barn owl (Tyto furcata).

Avian uropygial glands have received increasing attention in recent years, but little is known about micro-organisms in uropygial glands. In this study, we isolated a strain of Gram-stain-positive, non-motile, non-spore-forming cocci, designated 442T, from the uropygial gland of an American barn owl (Tyto furcata) and characterized it using a polyphasic approach. 16S rRNA gene sequence analysis placed the isolate in the genus Kocuria. The G+C content was 70.8 mol%, the major menaquinone was MK-7(H2) and the predominant cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C15 : 0. Phylogenetic analyses based on the 16S rRNA gene identified Kocuria rhizophila DSM 11926T (99.6 % similarity), Kocuria salsicia DSM 24776T (98.7 %), Kocuria varians DSM 20033T (98.3 %) and Kocuria marina DSM 16420T (98.3 %) as the most closely related species. However, average nucleotide identity values below 86 % indicated that the isolate differed from all species hitherto described. Chemotaxonomic analyses and whole-cell protein profiles corroborated these findings. Accordingly, the isolate is considered to be a member of a novel species, for which the name Kocuria tytonis sp. nov. is proposed. The type strain is 442T (=DSM 104130T=LMG 29944T).

Response adaptation in the barn owl's auditory space map.

Response adaptation is the change of the firing rate of neurons induced by a preceding stimulus. It can be found in many sensory systems and throughout the auditory pathway. We investigated response adaptation in the external nucleus of the inferior colliculus (ICX) of barn owls ( Tyto furcata), a nocturnal bird of prey and specialist in sound localization. Individual neurons in the ICX represent locations in auditory space by maximally responding to combinations of interaural time and level differences (ITD and ILD). Neuronal responses were recorded extracellularly under ketamine-diazepam anesthesia. Response adaptation was observed in three double stimulation paradigms. In two paradigms, the same binaural parameters for both stimuli were chosen. A variation of the level of the second stimulus yielded a level increase sufficient to compensate for adaptation around 5 dB. Introducing a silent interstimulus interval (ISI) resulted in recovery from adaptation. The time course of recovery was followed by varying the ISI, and full recovery was found after an ISI of 50 ms. In a third paradigm, the ITD of the second stimulus was varied to investigate the representation of ITD under adaptive conditions. We found that adaptation led to an increased precision and improved selectivity while the best ITD was stable. These changes of representation remained for longer ISIs than were needed to recover from response adaptation at the best ITD. Stimuli with non-best ITDs could also induce similar adaptive effects if the neurons responded to these ITDs. NEW & NOTEWORTHY We demonstrate and characterize response adaptation in neurons of the auditory space map in the barn owl's midbrain with acoustic double-stimulation paradigms. An increase of the second level by 5 dB compensated for the observed adaptive effect. Recovery from adaptation was faster than in upstream nuclei of the auditory pathway. Our results also show that response adaptation might improve precision and selectivity in the representation of interaural time difference.

Contribution of action potentials to the extracellular field potential in the nucleus laminaris of barn owl.

Extracellular field potentials (EFP) are widely used to evaluate in vivo neural activity, but identification of multiple sources and their relative contributions is often ambiguous, making the interpretation of the EFP difficult. We have therefore analyzed a model EFP from a simple brainstem circuit with separable pre- and postsynaptic components to determine whether we could isolate its sources. Our previous papers had shown that the barn owl neurophonic largely originates with spikes from input axons and synapses that terminate on the neurons in the nucleus laminaris (NL) (Kuokkanen PT, Wagner H, Ashida G, Carr CE, Kempter R. J Neurophysiol 104: 2274-2290, 2010; Kuokkanen PT, Ashida G, Carr CE, Wagner H, Kempter R. J Neurophysiol 110: 117-130, 2013; McColgan T, Liu J, Kuokkanen PT, Carr CE, Wagner H, Kempter R. eLife 6: e26106, 2017). To determine how much the postsynaptic NL neurons contributed to the neurophonic, we recorded EFP responses in NL in vivo. Power spectral analyses showed that a small spectral component of the evoked response, between 200 and 700 Hz, could be attributed to the NL neurons' spikes, while nucleus magnocellularis (NM) spikes dominate the EFP at frequencies ≳1 kHz. Thus, spikes of NL neurons and NM axons contribute to the EFP in NL in distinct frequency bands. We conclude that if the spectral components of source types are different and if their activities can be selectively modulated, the identification of EFP sources is possible. NEW & NOTEWORTHY Extracellular field potentials (EFPs) generate clinically important signals, but their sources are incompletely understood. As a model, we have analyzed the auditory neurophonic in the barn owl's nucleus laminaris. There the EFP originates predominantly from spiking in the afferent axons, with spectral power ≳1 kHz, while postsynaptic laminaris neurons contribute little. In conclusion, the identification of EFP sources is possible if they have different spectral components and if their activities can be modulated selectively.

Size discrimination in barn owls as compared to humans.

We tested how well barn owls can discriminate objects of different sizes. This ability may be important for the owls when catching prey. We performed a quantitative experiment in the laboratory and trained owls in a task in which the owls had to discriminate whether two rhombi presented simultaneously on a computer monitor were of the same or of different sizes. We obtained full data sets with two experienced owls and one data point with a third owl. For objects being sufficiently larger than the spatial resolution of the barn owl, the angular threshold was related to object size, implying that the discrimination followed Weber's law. The range of Weber fractions we determined was between 0.026 and 0.09. For object sizes close to the spatial resolution, performance degraded. We conducted similar experiments with human subjects. Human thresholds showed the same dependence on object size, albeit down to smaller object sizes. Human performance resulted in a range of Weber fractions extending from 0.025 to 0.036. The differences between owls and humans could be explained by the much higher spatial acuity of humans compared with owls.

Visual search in barn owls: Task difficulty and saccadic behavior.

How do we find what we are looking for? A target can be in plain view, but it may be detected only after extensive search. During a search we make directed attentional deployments like saccades to segment the scene until we detect the target. Depending on difficulty, the search may be fast with few attentional deployments or slow with many, shorter deployments. Here we study visual search in barn owls by tracking their overt attentional deployments-that is, their head movements-with a camera. We conducted a low-contrast feature search, a high-contrast orientation conjunction search, and a low-contrast orientation conjunction search, each with set sizes varying from 16 to 64 items. The barn owls were able to learn all of these tasks and showed serial search behavior. In a subsequent step, we analyzed how search behavior of owls changes with search complexity. We compared the search mechanisms in these three serial searches with results from pop-out searches our group had reported earlier. Saccade amplitude shortened and fixation duration increased in difficult searches. Also, in conjunction search saccades were guided toward items with shared target features. These data suggest that during visual search, barn owls utilize mechanisms similar to those that humans use.

Responses to Pop-Out Stimuli in the Barn Owl's Optic Tectum Can Emerge through Stimulus-Specific Adaptation.

UNLABELLED: Here, we studied neural correlates of orientation-contrast-based saliency in the optic tectum (OT) of barn owls. Neural responses in the intermediate/deep layers of the OT were recorded from lightly anesthetized owls confronted with arrays of bars in which one bar (the target) was orthogonal to the remaining bars (the distractors). Responses to target bars were compared with responses to distractor bars in the receptive field (RF). Initially, no orientation-contrast sensitivity was observed. However, if the position of the target bar in the array was randomly shuffled across trials so that it occasionally appeared in the RF, then such sensitivity emerged. The effect started to become significant after three or four positional changes of the target bar and strengthened with additional trials. Our data further suggest that this effect arises due to specific adaptation to the stimulus in the RF combined with suppression from the surround. By jittering the position of the bar inside the RF across trials, we demonstrate that the adaptation has two components, one position specific and one orientation specific. The findings give rise to the hypothesis that barn owls, by active scanning of the scene, can induce adaptation of the tectal circuitry to the common orientation and thus achieve a "pop-out" of rare orientations. Such a model is consistent with several behavioral observations in owls and may be relevant to other visual features and species. SIGNIFICANCE STATEMENT: Natural scenes are often characterized by a dominant orientation, such as the scenery of a pine forest or the sand dunes in a windy desert. Therefore, orientation that contrasts the regularity of the scene is perceived salient for many animals as a means to break camouflage. By actively moving the scene between each trial, we show here that neurons in the retinotopic map of the barn owl's optic tectum specifically adapt to the common orientation, giving rise to preferential representation of odd orientations. Based on this, we suggest a new mechanism for orientation-based camouflage breaking that links active scanning of scenes with neural adaptation. This mechanism may be relevant to pop-out in other species and visual features.

Envelope contributions to the representation of interaural time difference in the forebrain of barn owls.

Birds and mammals use the interaural time difference (ITD) for azimuthal sound localization. While barn owls can use the ITD of the stimulus carrier frequency over nearly their entire hearing range, mammals have to utilize the ITD of the stimulus envelope to extend the upper frequency limit of ITD-based sound localization. ITD is computed and processed in a dedicated neural circuit that consists of two pathways. In the barn owl, ITD representation is more complex in the forebrain than in the midbrain pathway because of the combination of two inputs that represent different ITDs. We speculated that one of the two inputs includes an envelope contribution. To estimate the envelope contribution, we recorded ITD response functions for correlated and anticorrelated noise stimuli in the barn owl's auditory arcopallium. Our findings indicate that barn owls, like mammals, represent both carrier and envelope ITDs of overlapping frequency ranges, supporting the hypothesis that carrier and envelope ITD-based localization are complementary beyond a mere extension of the upper frequency limit.NEW & NOTEWORTHY The results presented in this study show for the first time that the barn owl is able to extract and represent the interaural time difference (ITD) information conveyed by the envelope of a broadband acoustic signal. Like mammals, the barn owl extracts the ITD of the envelope and the carrier of a signal from the same frequency range. These results are of general interest, since they reinforce a trend found in neural signal processing across different species.

Distribution of the characteristics of barbs and barbules on barn owl wing feathers.

Owls are known for the development of a silent flight. One conspicuous specialization of owl wings that has been implied in noise reduction and that has been demonstrated to change the aerodynamic behavior of the wing is a soft dorsal wing surface. The soft surface is a result of changes in the shape of feather barbs and barbules in owls compared with other bird species. We hypothesized that as the aerodynamic characteristics of a wing change along its chordwise and spanwise direction, so may the shape of the barbs and barbules. Therefore, we examined in detail the shapes of the barbs and barbules in chordwise and spanwise directions. The results showed changes in the shapes of barbs and barbules at the anterior and distal parts of the wing, but not at more posterior parts. The increased density of hook radiates at the distalmost wing position could serve to stiffen that vane part that is subject to the highest forces. The change of pennulum length in the anterior part of the wing and the uniformity further back could mean that a soft surface may be especially important in regions where flow separation may occur.

Barn owls maximize head rotations by a combination of yawing and rolling in functionally diverse regions of the neck.

Owls are known for their outstanding neck mobility: these birds can rotate their heads more than 270°. The anatomical basis of this extraordinary neck rotation ability is not well understood. We used X-ray fluoroscopy of living owls as well as forced neck rotations in dead specimens and computer tomographic (CT) reconstructions to study how the individual cervical joints contribute to head rotation in barn owls (Tyto furcata pratincola). The X-ray data showed the natural posture of the neck, and the reconstructions of the CT-scans provided the shapes of the individual vertebrae. Joint mobility was analyzed in a spherical coordinate system. The rotational capability was described as rotation about the yaw and roll axes. The analyses suggest a functional division of the cervical spine into several regions. Most importantly, an upper region shows high rolling and yawing capabilities. The mobility of the lower, more horizontally oriented joints of the cervical spine is restricted mainly to the roll axis. These rolling movements lead to lateral bending, effectively resulting in a side shift of the head compared with the trunk during large rotations. The joints in the middle of the cervical spine proved to contribute less to head rotation. The analysis of joint mobility demonstrated how owls might maximize horizontal head rotation by a specific and variable combination of yawing and rolling in functionally diverse regions of the neck.

The DNA sugar backbone 2' deoxyribose determines toll-like receptor 9 activation.

CpG motifs within phosphorothioate (PS)-modified DNA drive Toll-like receptor 9 (TLR9) activation, but the rules governing recognition of natural phosphodiester (PD) DNA are less understood. Here, we showed that the sugar backbone determined DNA recognition by TLR9. Homopolymeric, base-free PD 2' deoxyribose acted as a basal TLR9 agonist as it bound to and activated TLR9. This effect was enhanced by DNA bases, even short of CpG motifs. In contrast, PS-modified 2' deoxyribose homopolymers acted as TLR9 and TLR7 antagonists. They displayed high affinity to both TLRs and did not activate on their own, but they competitively inhibited ligand-TLR interaction and activation. Although addition of random DNA bases to the PS 2' deoxyribose backbone did not alter these effects, CpG motifs transformed TLR9-inhibitory to robust TLR9-stimulatory activity. Our results identified the PD 2' deoxyribose backbone as an important determinant of TLR9 activation by natural DNA, restrict CpG-motif dependency of TLR9 activation to synthetic PS-modified ligands, and define PS-modified 2' deoxyribose as a prime effector of TLR9 and TLR7 inhibition.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

Low frequency eardrum directionality in the barn owl induced by sound transmission through the interaural canal.

The middle ears of birds are typically connected by interaural cavities that form a cranial canal. Eardrums coupled in this manner may function as pressure difference receivers rather than pressure receivers. Hereby, the eardrum vibrations become inherently directional. The barn owl also has a large interaural canal, but its role in barn owl hearing and specifically in sound localization has been controversial so far. We discuss here existing data and the role of the interaural canal in this species and add a new dataset obtained by laser Doppler vibrometry in a free-field setting. Significant sound transmission across the interaural canal occurred at low frequencies. The sound transmission induces considerable eardrum directionality in a narrow band from 1.5 to 3.5 kHz. This is below the frequency range used by the barn owl for locating prey, but may conceivably be used for locating conspecific callers.

The Role of Conduction Delay in Creating Sensitivity to Interaural Time Differences.

Axons from the nucleus magnocellularis (NM) and their targets in nucleus laminaris (NL) form the circuit responsible for encoding interaural time difference (ITD). In barn owls, NL receives bilateral inputs from NM, such that axons from the ipsilateral NM enter NL dorsally, while contralateral axons enter from the ventral side. These afferents act as delay lines to create maps of ITD in NL. Since delay-line inputs are characterized by a precise latency to auditory stimulation, but the postsynaptic coincidence detectors respond to ongoing phase difference, we asked whether the latencies of a local group of axons were identical, or varied by multiples of the inverse of the frequency they respond to, i.e., to multiples of 2π phase. Intracellular recordings from NM axons were used to measure delay-line latencies in NL. Systematic shifts in conduction delay within NL accounted for the maps of ITD, but recorded latencies of individual inputs at nearby locations could vary by 2π or 4π. Therefore microsecond precision is achieved through sensitivity to phase delays, rather than absolute latencies. We propose that the auditory system "coarsely" matches ipsilateral and contralateral latencies using physical delay lines, so that inputs arrive at NL at about the same time, and then "finely" matches latency modulo 2π to achieve microsecond ITD precision.

Maps of interaural delay in the owl's nucleus laminaris.

Axons from the nucleus magnocellularis form a presynaptic map of interaural time differences (ITDs) in the nucleus laminaris (NL). These inputs generate a field potential that varies systematically with recording position and can be used to measure the map of ITDs. In the barn owl, the representation of best ITD shifts with mediolateral position in NL, so as to form continuous, smoothly overlapping maps of ITD with iso-ITD contours that are not parallel to the NL border. Frontal space (0°) is, however, represented throughout and thus overrepresented with respect to the periphery. Measurements of presynaptic conduction delay, combined with a model of delay line conduction velocity, reveal that conduction delays can account for the mediolateral shifts in the map of ITD.

Estimating characteristic phase and delay from broadband interaural time difference tuning curves.

Characteristic delay and characteristic phase are shape parameters of interaural time difference tuning curves. The standard procedure for the estimation of these parameters is based on the measurement of delay curves measured for tonal stimuli with varying frequencies. Common to all procedures is the detection of a linear behavior of the phase spectrum. Hence a reliable estimate can only be expected if sufficiently many relevant frequencies are tested. Thus, the estimation precision depends on the given bandwidth. Based on a linear model, we develop and implement methods for the estimation of characteristic phase and delay from a single broadband tuning curve. We present two different estimation algorithms, one based on a Fourier-analytic interpretation of characteristic delay and phase, and the other based on mean square error minimization. Estimation precision and robustness of the algorithms are tested on artificially generated data with predetermined characteristic delay and phase values, and on sample data from electrophysiological measurements in birds and in mammals. Increasing the signal-to-noise ratio or the bandwidth increases the estimation accuracy of the algorithms. Frequency band location and strong rectification also affect the estimation accuracy. For realistic bandwidths and signal-to-noise ratios, the minimization algorithm reliably and robustly estimates characteristic delay and phase and is superior to the Fourier-analytic method. Bandwidth-dependent significance thresholds allow to assess whether the estimated characteristic delay and phase values are meaningful shape parameters of a measured tuning curve. These thresholds also indicate the sampling rates needed to obtain reliable estimates from interaural time difference tuning curves.