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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).

Background Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings. Results A well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments. Conclusions The structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.

Activity of an innervated tissue can be modulated based on an acquired biomarker through feedback loops. How to convert this biomarker into a meaningful stimulation pattern is still a topic of intensive research. In this article, we present a simple closed-loop mechanism to control the mean angle of a locust’s leg in real time by modulating the frequency of the stimulation on its extensor motor nerve. The nerve is interfaced with a custom-designed cuff electrode and the feedback loop is implemented online with a proportional control algorithm, which runs solely on a microcontroller without the need of an external computer. The results show that the system can be controlled with a single-input, single-output feedback loop. The model described in this article can serve as a primer for young researchers to learn about neural control in biological systems before applying these concepts in advanced systems. We expect that the approach can be advanced to achieve control over more complex movements by increasing the number of recorded biomarkers and selective stimulation units.

Knowledge about body motion kinematics and underlying muscle contraction dynamics usually derives from electromyographic (EMG) recordings. However, acquisition of such signals in snakes is challenging because electrodes either attached to or implanted beneath the skin may unintentionally be removed by force or friction caused from undulatory motion, thus severely impeding chronic EMG recordings. Here, we present a reliable method for stable subdermal implantation of up to eight bipolar electrodes above the target muscles. The mechanical stability of the inserted electrodes and the overnight coverage of the snake body with a “sleeping bag” ensured the recording of reliable and robust chronic EMG activity. The utility of the technique was verified by daily acquisition of high signal-to-noise activity from all target sites over four consecutive days during stimulus-evoked postural reactions in Amazon tree boas and Western diamondback rattlesnakes. The successful demonstration of the chronic recording suggests that this technique can improve acute experiments by enabling the collection of larger data sets from single individuals.

Genetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. 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 for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons.Significance statementGenome engineering of animals is crucial for translational medicine and the study of genetic traits. Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, providing the basis for in vivo genome editing. We demonstrated the functionality of this system by successful genome editing in chicken and porcine cells and tissues. These animals facilitate organ specific in vivo genome editing in both species without laborious germ line modifications, which will reduce the number of animals needed for genetic studies. They also provide a new tool for functional genomics, developmental biology and numerous other applications in biomedical and agricultural science.

The midbrain is a subcortical area involved in central functions such as integrating sensory modalities, movement initiation and bottom-up and top-down attention. In chicken, the midbrain roof is termed optic tectum (TeO) and consists of 15 layers with distinct in- and output regions. Visual input targets the superficial layers, while auditory input terminates in deeper layers. It has been shown that ablation of sensory epithelia leads to changes in the cellular patterning and structural organization of the sensory pathways. For the tectum, ablation of the eye anlagen was shown to affect retinorecipient neurons. While the gross morphology remained intact after enucleation, the shape of dendritic endings was changed presumably due to missing presynaptic input during synaptic pruning. We investigated the effect of deafferentation in a multisensory cell type, the Shepherd's crook neuron (SCN) in the TeO. SCNs have distinct dendritic branches in retinorecipient layers (superficial layers 1 to 5 and 7) and in layers where auditory input terminates. To assess whether removal of a single sensory input only affects the dendrites recipient for that input, we removed the eye anlagen and retrogradely labeled SCNs later in embryogenesis to visualize the morphology in lesioned and non-lesioned embryos. We found no changes in the gross morphology or in the basal dendrites, but an altered growth of the fine structures at the apical dendrite of SCNs in the retinorecipient layers. Our data indicate that the neuronal morphology of SCNs is mostly predefined before retinal innervation affect the fine structure.

Summary The estimation of one’s distance to a potential threat is essential for any animal’s survival. Rattlesnakes inform about their presence by generating acoustic broadband rattling sounds. 1 Rattlesnakes generate their acoustic signals by clashing a series of keratinous segments onto each other, which are located at the tip of their tails. 1 , 2 , 3 Each tail shake results in a broadband sound pulse that merges into a continuous acoustic signal with fast-repeating tail shakes. This acoustic display is readily recognized by other animals 4 , 5 and serves as an aposematic threat and warning display, likely to avoid being preyed upon. 1 , 6 The spectral properties of the rattling sound 1 , 3 and its dependence on the morphology and size of the rattle have been investigated for decades 7 , 8 , 9 and carry relevant information for different receivers, including ground squirrels that encounter rattlesnakes regularly. 10 , 11 Combining visual looming stimuli with acoustic measurements, we show that rattlesnakes increase their rattling rate (up to about 40 Hz) with decreasing distance of a potential threat, reminiscent of the acoustic signals of sensors while parking a car. Rattlesnakes then abruptly switch to a higher and less variable rate of 60–100 Hz. In a virtual reality experiment, we show that this behavior systematically affects distance judgments by humans: the abrupt switch in rattling rate generates a sudden, strong percept of decreased distance which, together with the low-frequency rattling, acts as a remarkable interspecies communication signal. Video abstract Download : Download video (68MB)

The midbrain is an important subcortical area involved in distinct functions such as multimodal integration, movement initiation, bottom-up, and top-down attention. Our group is particularly interested in cellular computation of multisensory integration. We focus on the visual part of the avian midbrain, the optic tectum (TeO, counterpart to mammalian superior colliculus). This area has a layered structure with the great advantage of distinct input and output regions. In chicken, the TeO is organized in 15 layers where visual input targets the superficial layers while auditory input terminates in deeper layers. One specific cell type, the Shepherd's crook neuron (SCN), extends dendrites in both input regions. The characteristic feature of these neurons is the axon origin at the apical dendrite. The molecular identity of this characteristic region and thus, the site of action potential generation are of particular importance to understand signal flow and cellular computation in this neuron. We present immunohistochemical data of structural proteins (NF200, Ankyrin G, and Myelin) and ion channels (Pan-Nav , Nav 1.6, and Kv 3.1b). NF200 is strongly expressed in the axon. Ankyrin G is mainly expressed at the axon initial segment (AIS). Myelination starts after the AIS as well as the distribution of Nav channels on the axon. The subtype Nav 1.6 has a high density in this region. Kv 3.1b is restricted to the soma, the primary neurite and the axon branch. The distribution of functional molecules in SCNs provides insight into the information flow and the integration of sensory modalities in the TeO of the avian midbrain.

The avian centrifugal visual system, which projects from the brain to the retina, has been intensively studied in several Neognathous birds that have a distinct isthmo-optic nucleus (ION). However, birds of the order Palaeognathae seem to lack a proper ION in histologically stained brain sections. We had previously reported in the palaeognathous Chilean Tinamou (Nothoprocta perdicaria) that intraocular injections of Cholera Toxin B subunit retrogradely label a considerable number of neurons, which form a diffuse isthmo-optic complex (IOC). In order to better understand how this IOC-based centrifugal visual system is organized, we have studied its major components by means of in vivo and in vitro tracing experiments. Our results show that the IOC, though structurally less organized than an ION, possesses a dense core region consisting of multipolar neurons. It receives afferents from neurons in L10a of the optic tectum, which are distributed with a wider interneuronal spacing than in Neognathae. The tecto-IOC terminals are delicate and divergent, unlike the prominent convergent tecto-ION terminals in Neognathae. The centrifugal IOC terminals in the retina are exclusively divergent, resembling the terminals from “ectopic” centrifugal neurons in Neognathae. We conclude that the Tinamou's IOC participates in a comparable general IOC-retina-TeO-IOC circuitry as the neognathous ION. However, the connections between the components are structurally different and their divergent character suggests a lower spatial resolution. Our findings call for further comparative studies in a broad range of species for advancing our understanding of the evolution, plasticity and functional roles of the avian centrifugal visual system.

Echolocating bats evolved a sophisticated biosonar imaging system that allows for a life in dim-light habitats. However, especially for far-range operations such as homing, bats can support biosonar by vision. Large eyes and a retina that mainly consists of rods are assumed to be the optical adjustments that enable bats to use visual information at low light levels. In addition to optical mechanisms, many nocturnal animals evolved neural adaptations such as elongated integration times or enlarged spatial sampling areas to further increase the sensitivity of their visual system by temporal or spatial summation of visual information. The neural mechanisms that underlie the visual capabilities of echolocating bats have, however, so far not been investigated. To shed light on spatial and temporal response characteristics of visual neurons in an echolocating bat, Phyllostomus discolor, we recorded extracellular multiunit activity in the retino-recipient superficial layers of the superior colliculus (SC). We discovered that response latencies of these neurons were generally in the mammalian range, whereas neural spatial sampling areas were unusually large compared to those measured in the SC of other mammals. From this we suggest that echolocating bats likely use spatial but not temporal summation of visual input to improve visual performance under dim-light conditions. Furthermore, we hypothesize that bats compensate for the loss of visual spatial precision, which is a byproduct of spatial summation, by integration of spatial information provided by both the visual and the biosonar systems. Given that knowledge about neural adaptations to dim-light vision is mainly based on studies done in non-mammalian species, our novel data provide a valuable contribution to the field and demonstrate the suitability of echolocating bats as a nocturnal animal model to study the neurophysiological aspects of dim-light vision.

Visual information in birds is to great extent processed in the optic tectum (TeO), a prominent laminated midbrain structure. Retinal input enters the TeO in its superficial layers, while output is limited to intermediate and deeper layers. In addition to visual information, the TeO receives multimodal input from the auditory and somatosensory pathway. The TeO gives rise to a major ascending tectofugal projection where neurons of tectal layer 13 project to the thalamic nucleus rotundus, which then projects to the entopallium. A second tectofugal projection system, called the accessory pathway, has however not been studied as thoroughly. Again, cells of tectal layer 13 form an ascending projection that targets a nucleus known as either the caudal part of the nucleus dorsolateralis posterior of the thalamus (DLPc) or nucleus uveaformis (Uva). This nucleus is known for multimodal integration and receives additional input from the lateral pontine nucleus (PL), which in turn receives projections from layer 8–15 of the TeO. Here, we studied a particular cell type afferent to the PL that consists of radially oriented neurons in layer 9. We characterized these neurons with respect to their anatomy, their retinal input, and the modulation of retinal input by local circuits. We found that comparable to other radial neurons in the tectum, cells of layer 9 have columnar dendritic fields and reach up to layer 2. Sholl analysis demonstrated that dendritic arborization concentrates on retinorecipient layers 2 and 4, with additional arborization in layers 9 and 10. All neurons recorded in layer 9 received retinal input via glutamatergic synapses. We analyzed the influence of modulatory circuits of the TeO by application of antagonists to γ-aminobutyric acid (GABA) and acetylcholine (ACh). Our data show that the neurons of layer 9 are integrated in a network under strong GABAergic inhibition, which is controlled by local cholinergic activation. Output to the PL and to the accessory tectofugal pathway thus appears to be under strict control of local tectal networks, the relevance of which for multimodal integration is discussed.

Lugaro cells are inhibitory interneurons found in the upper granular layer of the cerebellar cortex, just below or within the Purkinje cell layer. They are characterized by (1) a fusiform soma oriented in the parasagittal plane, (2) two pairs of dendrites emanating from opposite ends of the soma, (3) innervation from Purkinje cell collaterals, and (4) an axon that projects into the molecular layer akin to granular cell parallel fibers. Lugaro cells have been described in mammals, but not in other vertebrate classes, save one report in teleost fish. Here, we propose the existence of Lugaro cells in the avian cerebellum based on the morphological characteristics and connectivity described above. Immunohistochemical staining against the calcium binding protein secretagogin (SCGN) revealed Lugaro-like cells in the pigeon cerebellum. Some SCGN-labeled cells exhibit fusiform somata and dendrites parallel to the Purkinje cell layer in the parasagittal plane, as well as long axons that project into the molecular layer and travel alongside parallel fibers in the coronal plane. While mammalian Lugaro cells are known to express calretinin, the SCGN-labeled cells in the pigeon do not. SCGN-labeled cells also express glutamic acid decarboxylase, confirming their inhibitory function. Calbindin labeling revealed Purkinje cell terminals surrounding the SCGN-expressing cells. Our results suggest that Lugaro cells are more widespread among vertebrates than previously thought and may be a characteristic of the cerebellum of all vertebrates.

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).

Cocaine- and amphetamine-regulated transcript peptides (CARTp) are neuropeptides that act as neurotransmitters in the brain of vertebrates. The expression of CARTp has been characterized in teleosts, amphibians, and several mammalian species, but comparative data in reptiles and birds are nonexistent. In this study, we show the distribution of immunoreactivity against CART peptides (CARTp-ir) in the brains of two bird species: the pigeon (Columba livia) and zebra finch (Taeniopygia guttata). We found CARTp-ir cells and terminals in the brains of both, but no major differences between the two species. As in mammals, teleost fish, and amphibians, CARTp-ir terminals and cells were abundant in subpallial regions, particularly the striatum and nucleus accumbens. We also found CARTp-ir cells and terminals in the hypothalamus, and a large number of CARTp-ir terminals in the substantia nigra, ventral tegmental area, periaqueductal gray, parabrachial nucleus, and dorsal vagal complex. However, in contrast to other vertebrates, CARTp-ir was not found in the olfactory bulb. In addition there was almost no CARTp-ir in the pallium or the hippocampal formation, and little CARTp-ir in the cerebellum. The conserved expression of CARTp in the subpallium, hypothalamus, and dorsal vagal complex of birds suggests that some of the functions of CARTp, such as regulation of food intake and interactions with the social control network and mesolimbic reward system, are conserved among vertebrates. J. Comp. Neurol. 524:3747-3773, 2016. © 2016 Wiley Periodicals, Inc.

Background: The epithelial layer of the nasal mucosa is the first barrier for drug permeation during intranasal drug delivery. With increasing interest for intranasal pathways, adequate in vitro models are required. Here, porcine olfactory (OEPC) and respiratory (REPC) primary cells were characterised against the nasal tumour cell line RPMI 2650. Methods: Culture conditions for primary cells from porcine nasal mucosa were optimized and the cells characterised via light microscope, RT-PCR and immunofluorescence. Epithelial barrier function was analysed via transepithelial electrical resistance (TEER), and FITC-dextran was used as model substance for transepithelial permeation. Beating cilia necessary for mucociliary clearance were studied by immunoreactivity against acetylated tubulin. Results: OEPC and REPC barrier models differ in TEER, transepithelial permeation and MUC5AC levels. In contrast, RPMI 2650 displayed lower levels of MUC5AC, cilia markers and TEER, and higher FITC-dextran flux rates. Conclusion: To screen pharmaceutical formulations for intranasal delivery in vitro, translational mucosal models are needed. Here, a novel and comprehensive characterisation of OEPC and REPC against RPMI 2650 is presented. The established primary models display an appropriate model for nasal mucosa with secreted MUC5AC, beating cilia and a functional epithelial barrier, which is suitable for long-term evaluation of sustained release dosage forms.

Echolocating bats are known to fly and forage in complete darkness using the echoes of their actively emitted calls to navigate and to detect prey. However, under dim light conditions many bats can also rely on vision. Many flying animals have been shown to navigate by optic flow information, and recently, bats were shown to exploit echo-acoustic flow to navigate through dark habitats. Here we show for the bat Phyllostomus discolor that in lighted habitats where self-motion induced optic flow is strong, optic and echo-acoustic flow interact in their efficiency to guide navigation. Echo-acoustic flow showed a surprisingly strong effect compared to optic flow. We thus demonstrate multimodal interaction between two far-ranging spatial senses, vision and echolocation, available in this combination almost exclusively for bats and toothed whales. Our results highlight the importance of merging information from different sensory systems in a sensory-specialist animal to successfully navigate and hunt under difficult conditions.

ispartof: FRONTIERS IN NEURAL CIRCUITS vol:13 ispartof: location:Switzerland status: published

Echolocating bats are known to fly and forage in complete darkness, using the echoes of their actively emitted calls to navigate and to detect prey. However, under dim light conditions many bats can also rely on vision. Many flying animals have been shown to navigate by optic flow information and, recently, bats were shown to exploit echo-acoustic flow to navigate through dark habitats. Here, we show for the bat

The optic tectum (TeO), or superior colliculus, is a multisensory midbrain center that organizes spatially orienting responses to relevant stimuli. To define the stimulus with the highest priority at each moment, a network of reciprocal connections between the TeO and the isthmi promotes competition between concurrent tectal inputs. In the avian midbrain, the neurons mediating enhancement and suppression of tectal inputs are located in separate isthmic nuclei, facilitating the analysis of the neural processes that mediate competition. A specific subset of radial neurons in the intermediate tectal layers relay retinal inputs to the isthmi, but at present it is unclear whether separate neurons innervate individual nuclei or a single neural type sends a common input to several of them. In this study, we used in vitro neural tracing and cell-filling experiments in chickens to show that single neurons innervate, via axon collaterals, the three nuclei that comprise the isthmotectal network. This demonstrates that the input signals representing the strength of the incoming stimuli are simultaneously relayed to the mechanisms promoting both enhancement and suppression of the input signals. By performing in vivo recordings in anesthetized chicks, we also show that this common input generates synchrony between both antagonistic mechanisms, demonstrating that activity enhancement and suppression are closely coordinated. From a computational point of view, these results suggest that these tectal neurons constitute integrative nodes that combine inputs from different sources to drive in parallel several concurrent neural processes, each performing complementary functions within the network through different firing patterns and connectivity.

Pitvipers have a specialized sensory system in the upper jaw to detect infrared (IR) radiation. The bilateral pit organs resemble simple pinhole cameras that map IR objects onto the sensory epithelium as blurred representations of the environment. Trigeminal afferents transmit information about changing temperature patterns as neuronal spike discharge in a topographic manner to the hindbrain nucleus of the lateral descending trigeminal tract (LTTD). A presumed, yet so far unknown neuronal connectivity within this central nucleus exerts a synaptic computation that constrains the relatively large receptive field of primary afferent fibers. Here, we used intracellular recordings of LTTD neurons in isolated rattlesnake brains to decipher the spatio-temporal pattern of excitatory and inhibitory responses following electrical stimulation of single and multiple peripheral pit organ-innervating nerve branches. The responses of individual neurons consisted of complex spike sequences that derived from spatially and temporally specific interactions between excitatory and inhibitory synaptic inputs from the same as well as from adjacent peripheral nerve terminal areas. This pattern complies with a central excitation that is flanked by a delayed lateral inhibition, thereby enhancing the contrast of IR sensory input, functionally reminiscent of the computations for contrast enhancement in the peripheral visual system.

The avian pretectal and ventrothalamic nuclei, encompassing the griseum tectale (GT), n. lentiformis mesencephali (LM), and n. geniculatus lateralis pars ventralis (GLv), are prominent retinorecipient structures related to optic flow operations and visuomotor control. Hence, a close coordination of these neural circuits is to be expected. Yet the connectivity among these nuclei is poorly known. Here, using intracellular labeling and in situ hybridization, we investigated the detailed morphology, connectivity, and neurochemical identity of neurons in these nuclei. Two different cell types exist in the GT: one that generates an axonal projection to the optic tectum (TeO), LM, GLv, and n. intercalatus thalami (ICT), and a second population that only projects to the LM and GLv. In situ hybridization revealed that most neurons in the GT express the vesicular glutamate transporter (VGluT2) mRNA, indicating a glutamatergic identity. In the LM, three morphological cell types were defined, two of which project axons towards dorsal targets. The LM neurons showed strong VGluT2 expression. Finally, the cells located in the GLv project to the TeO, LM, GT, n. principalis precommisuralis (PPC), and ICT. All neurons in the GLv showed strong expression of the vesicular inhibitory amino acid transporter (VIAAT) mRNA, suggesting a GABAergic identity. Our results show that the pretectal and ventrothalamic nuclei are highly interconnected, especially by glutamatergic and GABAergic neurons from the GT and GLv, respectively. This complex morphology and connectivity might be required to organize orienting visuomotor behaviors and coordinate the specific optic flow patterns that they induce. J. Comp. Neurol. 524:2208-2229, 2016. © 2015 Wiley Periodicals, Inc.

The ability to detect motion is crucial for the survival of animals. In the avian optic tectum, motion-sensitive output neurons in the stratum griseum centrale have large dendritic fields and receive direct retinal input at their distal dendrites (bottlebrush endings). It has been hypothesized that the activation of each ending elicits a burst in the neuron. Thus, an object moving across the receptive field would lead to a fixed number of bursts, independent of movement speed. However, experimental confirmation of this hypothesis is still missing. We measured the response of tectal neurons to moving stimuli in vivo and found that in 'fast-bursting' units (~500 Hz within burst), the number of bursts was independent of stimulus speed. These results indicate that the number of bursts might indeed be related to the sequential activation of the bottlebrush endings by visual stimuli.

Most systematic studies of the avian visual system have focused on Neognathous species, leaving virtually unexplored the Palaeognathae, comprised of the flightless ratites and the South American tinamous. We investigated the visual field, the retinal topography, and the pattern of retinal and centrifugal projections in the Chilean tinamou, a small Palaeognath of the family Tinamidae. The tinamou has a panoramic visual field with a small frontal binocular overlap of 20°. The retina possesses three distinct topographic specializations: a horizontal visual streak, a dorsotemporal area, and an area centralis with a shallow fovea. The maximum ganglion cell density is 61,900/ mm(2) , comparable to Falconiformes. This would provide a maximal visual acuity of 14.0 cycles/degree, in spite of relatively small eyes. The central retinal projections generally conform to the characteristic arrangement observed in Neognathae, with well-differentiated contralateral targets and very few ipsilateral fibers. The centrifugal visual system is composed of a considerable number of multipolar centrifugal neurons, resembling the "ectopic" neurons described in Neognathae. They form a diffuse nuclear structure, which may correspond to the ancestral condition shared with other sauropsids. A notable feature is the presence of terminals in deep tectal layers 11-13. These fibers may represent either a novel retinotectal pathway or collateral branches from centrifugal neurons projecting to the retina. Both types of connections have been described in chicken embryos. Our results widen the basis for comparative studies of the vertebrate visual system, stressing the conserved character of the visual projections' pattern within the avian clade.

Pit vipers detect infrared (IR) radiation with loreal pit organs [1] that are connected to the hindbrain by trigeminal nerve fibers [2-4]. The pattern of central afferent termination forms a topographical representation of the sensory periphery within the nucleus of the lateral descending trigeminal tract (LTTD) [4-7]. All LTTD neurons project to another specialized, ipsilateral hindbrain area, the nucleus reticularis caloris (RC) [8-11], before IR signals are integrated with visual signals in the optic tectum [12, 13]. Pit-organ-innervating afferent fibers provoke in individual LTTD neurons a direct, robust spike activity upon peripheral activation [7, 14]. This discharge is truncated by an indirect, delayed synaptic inhibition from afferent fibers of adjacent sensory areas through parallel microcircuitry that converges with afferent fibers onto the same target neurons [7]. Here, we determined the impact of this interaction on IR contrast enhancement and/or motion detection in LTTD and RC neurons using isolated whole-brain preparations of rattlesnakes with intact pit organs. Simulated and real IR source motion provoked weak directional tuning of the discharge in LTTD neurons and RC neurons expressed a strong, motion-direction-differentiating activity. The hierarchically increasing motion sensitivity potentially derives from a direction-specific inhibition or spike frequency adaptation of LTTD neuronal discharge that becomes further pronounced by convergent projections onto individual RC neurons. The emerging signaling pattern complies with contrast enhancement (LTTD) and extraction of movement-related signals (RC), thereby forming a motion detection mechanism that encodes moving IR sources relative to the ambient temperature [14].

The avian centrifugal visual system, which projects from the brain to the retina, has been intensively studied in several Neognathous birds that have a distinct isthmo-optic nucleus (ION). However, birds of the order Palaeognathae seem to lack a proper ION in histologically stained brain sections. We had previously reported in the palaeognathous Chilean Tinamou (Nothoprocta perdicaria) that intraocular injections of Cholera Toxin B subunit retrogradely label a considerable number of neurons, which form a diffuse isthmo-optic complex (IOC). In order to better understand how this IOC-based centrifugal visual system is organized, we have studied its major components by means of in vivo and in vitro tracing experiments. Our results show that the IOC, though structurally less organized than an ION, possesses a dense core region consisting of multipolar neurons. It receives afferents from neurons in L10a of the optic tectum, which are distributed with a wider interneuronal spacing than in Neognathae. The tecto-IOC terminals are delicate and divergent, unlike the prominent convergent tecto-ION terminals in Neognathae. The centrifugal IOC terminals in the retina are exclusively divergent, resembling the terminals from "ectopic" centrifugal neurons in Neognathae. We conclude that the Tinamou's IOC participates in a comparable general IOC-retina-TeO-IOC circuitry as the neognathous ION. However, the connections between the components are structurally different and their divergent character suggests a lower spatial resolution. Our findings call for further comparative studies in a broad range of species for advancing our understanding of the evolution, plasticity and functional roles of the avian centrifugal visual system.

Processing multimodal sensory information is vital for behaving animals in many contexts. The barn owl, an auditory specialist, is a classic model for studying multisensory integration. In the barn owl, spatial auditory information is conveyed to the optic tectum (TeO) by a direct projection from the external nucleus of the inferior colliculus (ICX). In contrast, evidence of an integration of visual and auditory information in auditory generalist avian species is completely lacking. In particular, it is not known whether in auditory generalist species the ICX projects to the TeO at all. Here we use various retrograde and anterograde tracing techniques both in vivo and in vitro, intracellular fillings of neurons in vitro, and whole-cell patch recordings to characterize the connectivity between ICX and TeO in the chicken. We found that there is a direct projection from ICX to the TeO in the chicken, although this is small and only to the deeper layers (layers 13-15) of the TeO. However, we found a relay area interposed among the IC, the TeO, and the isthmic complex that receives strong synaptic input from the ICX and projects broadly upon the intermediate and deep layers of the TeO. This area is an external portion of the formatio reticularis lateralis (FRLx). In addition to the projection to the TeO, cells in FRLx send, via collaterals, descending projections through tectopontine-tectoreticular pathways. This newly described connection from the inferior colliculus to the TeO provides a solid basis for visual-auditory integration in an auditory generalist bird. J. Comp. Neurol. 525:513-534, 2017. © 2016 Wiley Periodicals, Inc.

The avian inferior colliculus (IC), also referred to as the nucleus mesencephalicus lateralis pars dorsalis (MLd), is an auditory midbrain nucleus that converges auditory cues from tonotopically organized brainstem nuclei. This information is relayed onto the optic tectum on the one hand and to nucleus ovoidalis on the other hand. Morphologically, there has been considerable debate about the number and nomenclature of the subnuclei within the IC. Here, we provide morphological characteristics of single cells in five IC subnuclei in chicken. The cellular structure within the IC was studied by whole-cell patch technique and biocytin iontophoresis. In addition, histological staining was performed, to delineate the borders between subnuclei of the IC. We were able to discriminate between 5 subnuclei: the core of the central nucleus (ICCc), the medial and lateral shell of the central nucleus (ICCms and ICCls), the external nucleus (ICX) and the superficial nucleus (ICS) of the IC. Our findings suggest the existence of at least two different morphologies of neurons with two subtypes each. The IC in chicken is a largely homogenous nucleus in terms of neuronal anatomy on a cellular level. However, its compartmentation into diversified subnuclei with different neurophysiological characteristics suggests a complex system to process auditory information. The auditory system in chicken is not as hypertrophied as in specialists such as the barn owl, but appears to have comparable connectivity and cellular morphology.

The mammalian subthalamic nucleus (STN) is a glutamatergic cell group within the indirect pathway of the basal ganglia. It receives input from the external globus pallidus (GP) and in turn projects to the internal GP and the substantia nigra pars reticulata (SNr). While the direct pathway from striatum to SNr is well established in anurans, it is unknown whether they possess an indirect pathway including a STN homologue. The subthalamic region comprises the dorsocaudal suprachiasmatic nucleus (dcSC), the posterior entopeduncular nucleus (EP), and the ventral part of the ventral thalamus (vVM/VL). In the fire-bellied toad Bombina orientalis we investigated whether one of these areas match the criteria established for the mammalian STN. We delineated the SNr in the midbrain tegmentum by labeling the striatonigral terminal field by means of GABA-, substance P-, and enkephalin immunohistochemistry and striatal tracer injections. Subsequently, we used double fluorescence tracing with injections into the SNr and GP to stain different parts of the indirect pathway. Confocal laser scan analysis revealed that dcSC, EP, and vVM/VL contain retrogradely labeled neurons projecting to the SNr, contacted by anterogradely labeled terminals arising in the GP. Immunohistochemical stainings with antibodies against glutamate and the glutamate transporters EAAC1 and vGluT2 demonstrated that the investigated nuclei contain glutamatergic neurons. Our results suggest that all regions in the subthalamic region fulfill our morphological criteria, except the connection back to the GP. An indirect basal ganglia pathway seems to be present in anuran amphibians, although we cannot exclusively delineate an STN homologue.

We present a method to identify specific sub-populations of auditory neurons in a mixed primary cell culture of the chicken brainstem, allowing the study of individual neurons with a known identity in vitro. To label specific afferent cell types, we injected retrograde tracers (dextrans coupled to fluorescent dyes) into either the mid-line or the superior olivary nuclei (SON) of the isolated chicken brainstem in vitro. Mid-line injections resulted in stable labelling of neurons of the nucleus magnocellularis (NM), whereas injections into the SON retrogradely labelled neurons of the nucleus laminaris (NL). The fluorescent label survives the dissociation procedure and is detectable for at least 1 week in vitro. Only about 0.1% of all cells in vitro are pre-labelled. The auditory identity of the pre-labelled neurons was confirmed with calretinin immunocytochemistry and electrophysiological recordings, where the cells had typical firing patterns of auditory brainstem neurons. In the future, this method can be combined with single cell PCR to match nuclear origin, firing patterns and the expression of functional molecules in vitro.

Flying animals need to react fast to rapid changes in their environment. Visually guided animals use optic flow, generated by their movement through structured environments. Nocturnal bats cannot make use of optic flow, but rely mostly on echolocation. Here we show that bats exploit echo-acoustic flow to negotiate flight through narrow passages. Specifically, bats' flight between lateral structures is significantly affected by the echo-acoustic salience of those structures, independent of their physical distance. This is true although echolocation, unlike vision, provides explicit distance cues. Moreover, the bats reduced the echolocation sound levels in stronger flow, likely to compensate for the increased summary target strength of the lateral reflectors. However, bats did not reduce flight velocity under stronger echo-acoustic flow. Our results demonstrate that sensory flow is a ubiquitous principle for flight guidance, independent of the fundamentally different peripheral representation of flow across the senses of vision and echolocation.

Proteomic analyses of zebra finch (Taeniopygia guttata) optic tectum resulted in identification of 176 proteins. In the Swissprot database, only 52 proteins were identified as bird homologs and only 71 proteins were identified in songbird transcriptome databases, reflecting a lack of completeness in the T. guttata genomic sequence. Analysis in Kyoto encyclopedia of genes and genome (KEGG) pathway database found that identified proteins most frequently belong to glucose, pyruvate, glyoxylate, dicarboxylate, alanine, and aspartate metabolism pathways. A number of identified proteins have been previously reported to exist in the avian optic tectum. The immunohistochemical localization of selected proteins showed their distribution in similar laminae of the owl (Tyto alba) and chicken (Gallus gallus) tectum. Immunohistochemical analysis of identified proteins can provide clues about cell types and circuit layout of the avian optic tectum in general. As the optic tectum of nonmammals is homologous to the superior colliculus of mammals, the analysis of the tectal and collicular proteome may provide clues about conserved cell and circuit layout, circuit function, and evolution.

Neurons in the auditory hindbrain pathway of the chicken are physiologically and morphologically highly specialized. It remains unclear to what extent independent differentiation vs. activity-dependent mechanisms determines the development of this system. To address this question we established a primary culture system of the early auditory hindbrain neurons. Primary cultures of neurons from nucleus magnocellularis and nucleus laminaris were prepared from embryonic day 6.5 chicken. These cells developed in culture under serum-free conditions for up to 15 days. Immunocytochemical staining and whole-cell patch recordings were used to characterize the development of the neurons. A stable expression of the calcium-binding protein calretinin, which serves as a characteristic marker of the auditory pathway, was found at all stages. A voltage-gated potassium channel (Kv3.1b) with a specific function in the auditory system was also expressed after about 1 week in culture. Electrophysiological recordings showed a general maturation of the neuronal phenotype as reflected by an increase in the mean resting membrane potential, a decrease in the mean input resistance as well as a maturation of action potential parameters. Four groups of neurons that generate action potentials could be distinguished. One of these showed the phasic firing pattern of auditory brainstem neurons known from slice preparations. In older cultures we demonstrated functional synaptogenesis in vitro by recording postsynaptic activity elicited by extracellular stimulation and styryl dye loading of vesicles. Thus, isolated neurons from the auditory region of the avian brainstem differentiate to specific neuronal subtypes and autonomously develop synaptic connections in vitro.

The avian pretectal and ventrothalamic nuclei, encompassing the griseum tectale (GT), n. lentiformis mesencephali (LM), and n. geniculatus lateralis pars ventralis (GLv), are prominent retinorecipient structures related to optic flow operations and visuomotor control. Hence, a close coordination of these neural circuits is to be expected. Yet the connectivity among these nuclei is poorly known. Here, using intracellular labeling and in situ hybridization, we investigated the detailed morphology, connectivity, and neurochemical identity of neurons in these nuclei. Two different cell types exist in the GT: one that generates an axonal projection to the optic tectum (TeO), LM, GLv, and n. intercalatus thalami (ICT), and a second population that only projects to the LM and GLv. In situ hybridization revealed that most neurons in the GT express the vesicular glutamate transporter (VGluT2) mRNA, indicating a glutamatergic identity. In the LM, three morphological cell types were defined, two of which project axons towards dorsal targets. The LM neurons showed strong VGluT2 expression. Finally, the cells located in the GLv project to the TeO, LM, GT, n. principalis precommisuralis (PPC), and ICT. All neurons in the GLv showed strong expression of the vesicular inhibitory amino acid transporter (VIAAT) mRNA, suggesting a GABAergic identity. Our results show that the pretectal and ventrothalamic nuclei are highly interconnected, especially by glutamatergic and GABAergic neurons from the GT and GLv, respectively. This complex morphology and connectivity might be required to organize orienting visuomotor behaviors and coordinate the specific optic flow patterns that they induce. J. Comp. Neurol. 524:2208-2229, 2016. © 2015 Wiley Periodicals, Inc.

Cocaine- and amphetamine-regulated transcript peptides (CARTp) are neuropeptides that act as neurotransmitters in the brain of vertebrates. The expression of CARTp has been characterized in teleosts, amphibians, and several mammalian species, but comparative data in reptiles and birds are nonexistent. In this study, we show the distribution of immunoreactivity against CART peptides (CARTp-ir) in the brains of two bird species: the pigeon (Columba livia) and zebra finch (Taeniopygia guttata). We found CARTp-ir cells and terminals in the brains of both, but no major differences between the two species. As in mammals, teleost fish, and amphibians, CARTp-ir terminals and cells were abundant in subpallial regions, particularly the striatum and nucleus accumbens. We also found CARTp-ir cells and terminals in the hypothalamus, and a large number of CARTp-ir terminals in the substantia nigra, ventral tegmental area, periaqueductal gray, parabrachial nucleus, and dorsal vagal complex. However, in contrast to other vertebrates, CARTp-ir was not found in the olfactory bulb. In addition there was almost no CARTp-ir in the pallium or the hippocampal formation, and little CARTp-ir in the cerebellum. The conserved expression of CARTp in the subpallium, hypothalamus, and dorsal vagal complex of birds suggests that some of the functions of CARTp, such as regulation of food intake and interactions with the social control network and mesolimbic reward system, are conserved among vertebrates. J. Comp. Neurol. 524:3747-3773, 2016. © 2016 Wiley Periodicals, Inc.

In everyday life we constantly perceive and discriminate between a large variety of sensory inputs, the vast majority of which consist of more than one modality. We performed two experiments to investigate whether chickens use the information present in multimodal signals. To test whether audiovisual stimuli are better detected than visual or acoustic stimuli alone, we first measured the detection threshold with a staircase paradigm. We found that chickens were able to detect weaker stimuli using audiovisual stimuli. Next, we tested whether the multimodal nature of a stimulus also increases the discrimination between two stimuli by measuring the smallest difference that the animals could still distinguish from each other. We found that chickens can discriminate smaller differences using audiovisual stimuli in comparison to visual stimuli alone, but not in comparison to acoustic stimuli alone. Thus, even in a relatively unspecialized species such as the chicken, the benefits of multimodal integration are exploited for sensory processing.

The avian retino-tecto-rotundal pathway plays a central role in motion analysis and features complex connectivity. Yet, the relation between the pathway's structural arrangement and motion computation has remained elusive. For an important type of tectal wide-field neuron, the stratum griseum centrale type I (SGC-I) neuron, we quantified its structure and found a spatially sparse but extensive sampling of the retinal projection. A computational investigation revealed that these structural properties enhance the neuron's sensitivity to change, a behaviorally important stimulus attribute, while preserving information about the stimulus location in the SGC-I population activity. Furthermore, the SGC-I neurons project with an interdigitating topography to the nucleus rotundus, where the direction of motion is computed. We showed that, for accurate direction-of-motion estimation, the interdigitating projection of tectal wide-field neurons requires a two-stage rotundal algorithm, where the second rotundal stage estimates the direction of motion from the change in the relative stimulus position represented in the first stage.

Humans and other animals generally perceive motion independently of the cues that define the moving object. To understand the underlying mechanisms of this generalization of stimulus attributes, we have examined the cellular properties of avian wide-field tectal neurons that are sensitive to a variety of moving stimuli but not to static stationary stimuli. This in vitro study showed phasic signal transfer at the retinotectal synapse and binary dendritic responses to synaptic inputs that interact in a mutually exclusive manner in the postsynaptic tectal neuron. A model of the tectal circuitry predicts that these two cellular properties mediate sensitivity to a wide range of dynamic spatiotemporal stimuli, including moving stimuli, but not to static stationary stimuli in a tectal neuron. The computation that is independent of stimulus detail is initiated by tectal neurons and is completed by rotundal neurons that integrate outputs from multiple tectal neurons in a directionally selective manner.

The stable and specific locking-in of pre- and postsynaptic membranes in synaptogenesis may be mediated by integral membrane proteins, such as members of the cadherin family. Cadherins are ideal candidate molecules for mediating synaptic specificity because they are differentially expressed in functionally connected brain structures. We studied the expression of four classic cadherins (R-cadherin, N-cadherin, cadherin-6B and cadherin-7) at the synaptic level on the somata and the proximal neurites of identified neuron populations that were traced selectively in the developing chicken visual system. Three major findings were observed. (1) Synapses on somata of shepherd's crook cells of the optic tectum are associated preferentially with one cadherin subtype. (2) In an isthmic nucleus that contains a mixed population of cells expressing different cadherins, somatic synapses tend to express the same cadherin subtype as the rest of the cell. (3) In the oculomotor complex, two cadherin subtypes are expressed only by synapses on the axon hillock. However, another neuron type that projects from the tectum to the isthmic nucleus does not show such selective synaptic cadherin staining. Our findings support the idea that a cadherin-based adhesive mechanism can mediate synaptic specificity.

Zusammenfassung Bei allen Wirbeltieren ist das visuelle Mittelhirn eingebunden in die Raumorientierung und speziell in die Ausrichtung der sensorischen Systeme. Wie autonom aber ist diese Struktur? Bei Fischen, Amphibien, Reptilien und Vögeln wird das optische Tectum als wichtigstes visuelles Zentrum betrachtet, das eine zentrale Funktion bei der gesamten Verhaltenssteuerung hat. Dem Colliculus superior der Säuger wird dagegen nur eine untergeordnete Rolle als ausführende Station unter Kontrolle des Kortex zugewiesen. Diese klassische Sicht auf die Gehirnorganisation der Wirbeltiere ist nach neueren Befunden nicht mehr aufrechtzuerhalten. Wir haben in diesem Artikel verschiedene Befunde zusammengestellt, die bei allen Wirbeltieren auf eine vergleichbare Integration des visuellen Mittelhirns in die Verarbeitung sensorischer Stimuli hinweisen. In vergleichend anatomischen Arbeiten sind identische Projektionen und Zelltypen beschrieben worden. Läsionsexperimente führen zu Ausfallserscheinungen, die nicht mit der klassischen Vorstellung erklärt werden können. Bei Vögeln und bei Säugern werden zunehmend sensorische Verarbeitungsleistungen im Mittelhirn beschrieben, deren Ergebnisse nicht nur zur Auslösung adäquater Orientierungsreaktionen genutzt werden. Funktionelle Untersuchungen deuten vielmehr an, dass die Aktivität an das Vorderhirn weitergegeben wird und dort zu weiteren Wahrnehmungsleistungen oder zu kognitiver Kontrolle beiträgt. Dies deutet eine eigenständige Bedeutung des Mittelhirns in der Verarbeitung an, die weit über die klassische Sicht als Reflexzentrum hinausgeht.

In everyday life we constantly perceive and discriminate between a large variety of sensory inputs, the vast majority of which consist of more than one modality. We performed two experiments to investigate whether chickens use the information present in multimodal signals. To test whether audiovisual stimuli are better detected than visual or acoustic stimuli alone, we first measured the detection threshold with a staircase paradigm. We found that chickens were able to detect weaker stimuli using audiovisual stimuli. Next, we tested whether the multimodal nature of a stimulus also increases the discrimination between two stimuli by measuring the smallest difference that the animals could still distinguish from each other. We found that chickens can discriminate smaller differences using audiovisual stimuli in comparison to visual stimuli alone, but not in comparison to acoustic stimuli alone. Thus, even in a relatively unspecialized species such as the chicken, the benefits of multimodal integration are exploited for sensory processing.

During development of the nervous system, neurotrophic interactions are essential for the synchronization of substructures such as retina and visual midbrain. However, morphological studies of postsynaptic elements after ablation of afferents are sparse. We investigated the effect of uni- and bilateral eye anlagen removal on identified projection neurons in the chicken optic tectum. Without retinal input, neurons in the stratum griseum centrale express their specific cell adhesions molecules, retain large dendritic fields and form specialized dendritic endings; however, the latter are deformed and extend over a much larger area. Our results show that even monosynaptically innervated tectal neurons develop largely independently from trophic retinal inputs and only become dependent on these after synaptic contact with retinal afferents.

Most systematic studies of the avian visual system have focused on Neognathous species, leaving virtually unexplored the Palaeognathae, comprised of the flightless ratites and the South American tinamous. We investigated the visual field, the retinal topography, and the pattern of retinal and centrifugal projections in the Chilean tinamou, a small Palaeognath of the family Tinamidae. The tinamou has a panoramic visual field with a small frontal binocular overlap of 20°. The retina possesses three distinct topographic specializations: a horizontal visual streak, a dorsotemporal area, and an area centralis with a shallow fovea. The maximum ganglion cell density is 61,900/ mm(2) , comparable to Falconiformes. This would provide a maximal visual acuity of 14.0 cycles/degree, in spite of relatively small eyes. The central retinal projections generally conform to the characteristic arrangement observed in Neognathae, with well-differentiated contralateral targets and very few ipsilateral fibers. The centrifugal visual system is composed of a considerable number of multipolar centrifugal neurons, resembling the "ectopic" neurons described in Neognathae. They form a diffuse nuclear structure, which may correspond to the ancestral condition shared with other sauropsids. A notable feature is the presence of terminals in deep tectal layers 11-13. These fibers may represent either a novel retinotectal pathway or collateral branches from centrifugal neurons projecting to the retina. Both types of connections have been described in chicken embryos. Our results widen the basis for comparative studies of the vertebrate visual system, stressing the conserved character of the visual projections' pattern within the avian clade.

Dendritic computation is essential for understanding information processing in single neurons and brain circuits. Optical methods are suited best to investigate function and biophysical properties of cellular compartments at high spatial and temporal resolution. Promising approaches include the use of voltage sensitive dyes, genetically encoded voltage sensors, or hybrid voltage sensors (hVoS) consisting of fluorescent proteins and voltage-dependent quenchers that, so far, are not available in avian neuroscience.We have adapted a hVoS system for a chicken midbrain slice preparation by combining genetically expressed farnesylated eGFP with dipicrylamine (DPA). Depending on the cellular potential, DPA is shifted in the membrane, resulting in quenching of eGFP fluorescence linearly to the membrane potential by Förster resonance electron transfer.In ovo electroporation resulted in labelled neurons throughout the midbrain with a high level of fine structural detail. After application of DPA, we were able to optically record electrically evoked action potentials with high signal-to-noise ratio and high spatio-temporal resolution.Standard methods available for avian neuroscience such as whole-cell patch clamp yield insufficient data for the analysis of dendritic computation in single neurons. The high spatial and temporal resolution of hVoS data overcomes this limitation. The results obtained by our method are comparable to hVoS data published for mammals.With the protocol presented here, it is possible to optically record information processing in single avian neurons at such high spatial and temporal resolution, that cellular and subcellular events can be analysed.

Pit vipers (Crotalinae) have a specific sensory system that detects infrared radiation with bilateral pit organs in the upper jaw. Each pit organ consists of a thin membrane, innervated by three trigeminal nerve branches that project to a specific nucleus in the dorsal hindbrain. The known topographic organization of infrared signals in the optic tectum prompted us to test the implementation of spatiotopically aligned sensory maps through hierarchical neuronal levels from the peripheral epithelium to the first central site in the hindbrain, the nucleus of the lateral descending trigeminal tract (LTTD). The spatial organization of the anatomical connections was revealed in a novel in vitro whole-brain preparation of the western diamondback rattlesnake (Crotalus atrox) that allowed specific application of multiple neuronal tracers to identified pit-organ-supplying trigeminal nerve branches. After adequate survival times, the entire peripheral and central projections of fibers within the pit membrane and the LTTD became visible. This approach revealed a morphological partition of the pit membrane into three well-defined sensory areas with largely separated innervations by the three main branches. The peripheral segregation of infrared afferents in the sensory epithelium was matched by a differential termination of the afferents within different areas of the LTTD, with little overlap. This result demonstrates a topographic organizational principle of the snake infrared system that is implemented by maintaining spatially aligned representations of environmental infrared cues on the sensory epithelium through specific neuronal projections at the level of the first central processing stage, comparable to the visual system.

The anuran torus semicircularis consists of several subnuclei that are part of the ascending auditory pathway as well as audiomotor interface structures. Additionally, recent anatomical studies suggest that the midbrain tegmentum is an integral part of the audiomotor network. To describe the chemoarchitecture of these nuclei, taking into account the toral subdivisions, we investigated the distribution of serotonin, leucine-enkephalin, substance P, tyrosine-hydroxylase, dopamine D2-receptor, parvalbumin, aspartate, GABA, and estrogen-binding protein-immunoreactivity in the midbrain of Bombina orientalis, Discoglossus pictus and Xenopus laevis. In the torus semicircularis, the highest density of immunoreactive fibers and terminals for all transmitters was found in the laminar nucleus. Parvalbumin-like immunoreactivity was highest in the principal nucleus, and D2-receptor-like immunoreactivity was uniformly distributed throughout the torus. In the tegmentum, axons and/or dendrites were stained with all antibodies except estrogen-binding protein. Additionally, heavily stained enkephalin and substance P-immunopositive fiber plexus were found in the lateral and dorsal tegmentum. The immunostainings revealed no qualitative differences between the three species. Immunopositive cell bodies were labeled in several brain areas, the connectivity of which with torus and tegmentum is discussed on the background of functional questions. The putative neuromodulatory innervation of both the laminar nucleus of the torus semicircularis and the tegmentum may be the anatomical basis for the influence of the animal's endogenous state on the behavioral reaction to sensory stimuli. These data corroborate earlier anatomical and physiological findings that the neurons of these nuclei are key elements in the audio-motor interface.

The widefield vertical neurons of the lower stratum griseum superficiale (SGS3) and upper stratum opticum (SO) of the superior colliculus provide an extrageniculate route for visual information to reach the pulvinar. Previous physiological studies indicate that SGS3/SO neurons have large receptive fields and respond to small moving stimuli. We sought to better characterize the dendritic morphology of SGS3/SO neurons with intracellular filling in slice preparations of the ground squirrel superior colliculus. We found that dendrites of widefield vertical cells end in monostratified arrays of spiny terminal specializations called "bottlebrush" dendritic endings. Two major subtypes of neurons are described. Type I neurons have somata restricted to the SGS3 and bottlebrush endings in the most superficial sublayer of the SGS. Type II neurons are found at the base of the SGS and in the upper SO, and have bottlebrush endings arrayed within the middle sublayers of the SGS. Bottlebrush endings may sample and integrate laminated afferents to the superior colliculus, and cellular subtypes may underlie multiple information streams within the tectopulvinar pathway. A similar dendritic morphology and projection pattern can be found in cells of the avian optic tectum that project upon the nucleus rotundus, a thalamic nucleus homologous to the mammalian caudal/inferior pulvinar. Because motion processing is a dominant feature of the avian tectorotundal pathway, the current results suggest that both dendritic morphology and motion processing are conserved features of widefield vertical cells in the tectopulvinar pathway of vertebrates.

In this contribution, it is argued that the prevailing view of the sensory system as an intricate, but passive processor of external information does not capture the full complexity of brain performance. Instead, we try to reinforce a notion that sees the brain as a system embedded within the environment and actively exploring it. We will attempt to emphasize the bidirectional interaction between brain and environment at all levels of processing and at different scales of the system description. This modified approach to the understanding of the brain has profound consequences for experimental investigations, starting with the experimental design and extending into data analysis and interpretation.