Cerebellar Inputs in the American Alligator (Alligator mississippiensis).

Crocodilians (alligators, crocodiles, and gharials) are the closet living relatives to birds and, as such, represent a key clade to understand the evolution of the avian brain. However, many aspects of crocodilian neurobiology remain unknown. In this paper, we address an important knowledge gap as there are no published studies of cerebellar connections in any crocodilian species. We used injections of retrograde tracers into the cerebellum of the American alligator (Alligator mississippiensis) to describe for the first time the origin of climbing and mossy fiber inputs. We found that inputs to the cerebellum in the American alligator are similar to those of other nonavian reptiles and birds. Retrograde labeled cells were found in the spinal cord, inferior olive, reticular formation, vestibular and cerebellar nuclei, as well as in nucleus ruber and surrounding tegmentum. Additionally, we found no retrogradely labeled cells in the anterior rhombencephalon which suggest that, like other nonavian reptiles, crocodilians may lack pontine nuclei. Similar to birds and other nonavian reptiles, we found inputs to the cerebellum from the pretectal nucleus lentiformis mesencephali. Additionally, we found retrogradely labeled neurons in two nuclei in the pretectum: the nucleus circularis and the interstitial nucleus of the posterior commissure. These pretectal projections have not been described in any other nonavian reptile to date, but they do resemble projections from the nucleus spiriformis medialis of birds. Our results show that many inputs to the cerebellum are highly conserved among sauropsids and that extensive pretectal inputs to the cerebellum are not exclusive to the avian brain. Finally, we suggest that the pontine nuclei of birds are an evolutionary novelty that may have evolved after the last common ancestor between birds and crocodilians, and may represent an intriguing case of convergent evolution with mammals.

Two Types of Auditory Spatial Receptive Fields in Different Parts of the Chicken's Midbrain.

The optic tectum (OT) is an avian midbrain structure involved in the integration of visual and auditory stimuli. Studies in the barn owl, an auditory specialist, have shown that spatial auditory information is topographically represented in the OT. Little is known about how auditory space is represented in the midbrain of birds with generalist hearing, i.e., most of avian species lacking peripheral adaptations such as facial ruffs or asymmetric ears. Thus, we conducted in vivo extracellular recordings of single neurons in the OT and in the external portion of the formatio reticularis lateralis (FRLx), a brain structure located between the inferior colliculus (IC) and the OT, in anaesthetized chickens of either sex. We found that most of the auditory spatial receptive fields (aSRFs) were spatially confined both in azimuth and elevation, divided into two main classes: round aSRFs, mainly present in the OT, and annular aSRFs, with a ring-like shape around the interaural axis, mainly present in the FRLx. Our data further indicate that interaural time difference (ITD) and interaural level difference (ILD) play a role in the formation of both aSRF classes. These results suggest that, unlike mammals and owls which have a congruent representation of visual and auditory space in the OT, generalist birds separate the computation of auditory space in two different midbrain structures. We hypothesize that the FRLx-annular aSRFs define the distance of a sound source from the axis of the lateral visual fovea, whereas the OT-round aSRFs are involved in multimodal integration of the stimulus around the lateral fovea.SIGNIFICANCE STATEMENT Previous studies implied that auditory spatial receptive fields (aSRFs) in the midbrain of generalist birds are only confined along azimuth. Interestingly, we found SRFs s in the chicken to be confined along both azimuth and elevation. Moreover, the auditory receptive fields are arranged in a concentric manner around the overlapping interaural and visual axes. These data suggest that in generalist birds, which mainly rely on vision, the auditory system mainly serves to align auditory stimuli with the visual axis, while auditory specialized birds like the barn owl compute sound sources more precisely and integrate sound positions in the multimodal space map of the optic tectum (OT).

AP-2δ Expression Kinetics in Multimodal Networks in the Developing Chicken Midbrain.

AP-2 is a family of transcription factors involved in many aspects of development, cell differentiation, and regulation of cell growth and death. AP-2δ is a member of this group and specific gene expression patterns are required in the adult mouse brain for the development of parts of the inferior colliculus (IC), as well as the cortex, dorsal thalamus, and superior colliculus. The midbrain is one of the central areas in the brain where multimodal integration, i.e., integration of information from different senses, occurs. Previous data showed that AP-2δ-deficient mice are viable but due to increased apoptosis at the end of embryogenesis, lack part of the posterior midbrain. Despite the absence of the IC in AP-2δ-deficient mice, these animals retain at least some higher auditory functions. Neuronal responses to tones in the neocortex suggest an alternative auditory pathway that bypasses the IC. While sufficient data are available in mammals, little is known about AP-2δ in chickens, an avian model for the localization of sounds and the development of auditory circuits in the brain. Here, we identified and localized AP-2δ expression in the chicken midbrain during embryogenesis. Our data confirmed the presence of AP-2δ in the inferior colliculus and optic tectum (TeO), specifically in shepherd's crook neurons, which are an essential component of the midbrain isthmic network and involved in multimodal integration. AP-2δ expression in the chicken midbrain may be related to the integration of both auditory and visual afferents in these neurons. In the future, these insights may allow for a more detailed study of circuitry and computational rules of auditory and multimodal networks.

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Zebrin Expression in the Cerebellum of Two Crocodilian Species.

While in birds and mammals the cerebellum is a highly convoluted structure that consists of numerous transverse lobules, in most amphibians and reptiles it consists of only a single unfolded sheet. Orthogonal to the lobules, the cerebellum is comprised of sagittal zones that are revealed in the pattern of afferent inputs, the projection patterns of Purkinje cells, and Purkinje cell response properties, among other features. The expression of several molecular markers, such as aldolase C, is also parasagittally organized. Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme expressed in the cerebellar Purkinje cells of the vertebrate cerebellum. In birds, mammals, and some lizards (Ctenophoresspp.), ZII is expressed in a heterogenous fashion of alternating sagittal bands of high (ZII+) and low (ZII-) expression Purkinje cells. In contrast, turtles and snakes express ZII homogenously (ZII+) in their cerebella, but the pattern in crocodilians is unknown. Here, we examined the expression of ZII in two crocodilian species (Crocodylus niloticus and Alligator mississippiensis) to help determine the evolutionary origin of striped ZII expression in vertebrates. We expected crocodilians to express ZII in a striped (ZII+/ZII-) manner because of their close phylogenetic relationship to birds and their larger and more folded cerebellum compared to that of snakes and turtles. Contrary to our prediction, all Purkinje cells in the crocodilian cerebellum had a generally homogenous expression of ZII (ZII+) rather than clear ZII+/- stripes. Our results suggest that either ZII stripes were lost in three groups (snakes, turtles, and crocodilians) or ZII stripes evolved independently three times (lizards, birds, and mammals).

Neural Maps of Interaural Time Difference in the American Alligator: A Stable Feature in Modern Archosaurs.

Detection of interaural time differences (ITDs) is crucial for sound localization in most vertebrates. The current view is that optimal computational strategies of ITD detection depend mainly on head size and available frequencies, although evolutionary history should also be taken into consideration. In archosaurs, which include birds and crocodiles, the brainstem nucleus laminaris (NL) developed into the critical structure for ITD detection. In birds, ITDs are mapped in an orderly array or place code, whereas in the mammalian medial superior olive, the analog of NL, maps are not found. As yet, in crocodilians, topographical representations have not been identified. However, nontopographic representations of ITD cannot be excluded due to different anatomical and ethological features of birds and crocodiles. Therefore, we measured ITD-dependent responses in the NL of anesthetized American alligators of either sex and identified the location of the recording sites by lesions made after recording. The measured extracellular field potentials, or neurophonics, were strongly ITD tuned, and their preferred ITDs correlated with the position in NL. As in birds, delay lines, which compensate for external time differences, formed maps of ITD. The broad distributions of best ITDs within narrow frequency bands were not consistent with an optimal coding model. We conclude that the available acoustic cues and the architecture of the acoustic system in early archosaurs led to a stable and similar organization in today's birds and crocodiles, although physical features, such as internally coupled ears, head size, or shape, and audible frequency range, vary among the two groups.SIGNIFICANCE STATEMENT Interaural time difference (ITD) is an important cue for sound localization, and the optimal strategies for encoding ITD in neuronal populations are the subject of ongoing debate. We show that alligators form maps of ITD very similar to birds, suggesting that their common archosaur ancestor reached a stable coding solution different from mammals. Mammals and diapsids evolved tympanic hearing independently, and local optima can be reached in evolution that are not considered by global optimal coding models. Thus, the presence of ITD maps in the brainstem may reflect a local optimum in evolutionary development. Our results underline the importance of comparative animal studies and show that optimal models must be viewed in the light of evolutionary processes.

Combination of Interaural Level and Time Difference in Azimuthal Sound Localization in Owls.

A function of the auditory system is to accurately determine the location of a sound source. The main cues for sound location are interaural time (ITD) and level (ILD) differences. Humans use both ITD and ILD to determine the azimuth. Thus far, the conception of sound localization in barn owls was that their facial ruff and asymmetrical ears generate a two-dimensional grid of ITD for azimuth and ILD for elevation. We show that barn owls also use ILD for azimuthal sound localization when ITDs are ambiguous. For high-frequency narrowband sounds, midbrain neurons can signal multiple locations, leading to the perception of an auditory illusion called a phantom source. Owls respond to such an illusory percept by orienting toward it instead of the true source. Acoustical measurements close to the eardrum reveal a small ILD component that changes with azimuth, suggesting that ITD and ILD information could be combined to eliminate the illusion. Our behavioral data confirm that perception was robust against ambiguities if ITD and ILD information was combined. Electrophysiological recordings of ILD sensitivity in the owl's midbrain support the behavioral findings indicating that rival brain hemispheres drive the decision to orient to either true or phantom sources. Thus, the basis for disambiguation, and reliable detection of sound source azimuth, relies on similar cues across species as similar response to combinations of ILD and narrowband ITD has been observed in humans.

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.

Influence of double stimulation on sound-localization behavior in barn owls.

Barn owls do not immediately approach a source after they hear a sound, but wait for a second sound before they strike. This represents a gain in striking behavior by avoiding responses to random incidents. However, the first stimulus is also expected to change the threshold for perceiving the subsequent second sound, thus possibly introducing some costs. We mimicked this situation in a behavioral double-stimulus paradigm utilizing saccadic head turns of owls. The first stimulus served as an adapter, was presented in frontal space, and did not elicit a head turn. The second stimulus, emitted from a peripheral source, elicited the head turn. The time interval between both stimuli was varied. Data obtained with double stimulation were compared with data collected with a single stimulus from the same positions as the second stimulus in the double-stimulus paradigm. Sound-localization performance was quantified by the response latency, accuracy, and precision of the head turns. Response latency was increased with double stimuli, while accuracy and precision were decreased. The effect depended on the inter-stimulus interval. These results suggest that waiting for a second stimulus may indeed impose costs on sound localization by adaptation and this reduces the gain obtained by waiting for a second stimulus.

Neuroethology of prey capture in the barn owl (Tyto alba L.).

Barn owls are a model system for studying prey capture. These animals can catch mice by hearing alone, but use vision whenever light conditions allow this. The silent flight, the frontally oriented eyes, and the facial ruffs are specializations that evolved to optimize prey capture. The auditory system is characterized by high absolute sensitivity, a use of interaural time difference for azimuthal sound-localization over almost the total hearing range up to at least 9 kHz, and the use of interaural level difference for elevational sound localization in the upper frequency range. Response latencies towards auditory targets were shortened by covert attention, while overt attention helped to orient towards salient visual objects. However, only 20% of the fixation movements could be explained by the saliency of the fixated objects, suggesting a top-down control of attention. In a visual-search experiment the birds turned earlier and more often towards and spent more time at salient objects. The visual system also exhibits high absolute sensitivity, while the spatial resolution is not particularly high. Last but not least, head movements may be classified as fixations, translations, and rotations combined with translations. These motion primitives may be combined to complex head-movement patterns. With the expected easy availability of genetic techniques for specialists in the near future and the possibility to apply the findings in biomimetic devices prey capture in barn owls will remain an exciting field in the future.