Influenza A viruses represent a significant threat to human health and animal welfare. Viral hemagglutinin cleavage by host proteases is essential for viral infectivity. The transmembrane serine protease 2 (TMPRSS2) is responsible for the posttranslational modification of the viral surface protein of low pathogenic avian Influenza viruses (LPAIVs), representing a promising target for the restriction of viral replication. This study used gene targeting and chemical inhibition of TMPRSS2-related proteases to evaluate the suitability of chicken cells and organ cultures for investigating host proteases. We generated TMPRSS2 knockout (KO) chicken DF-1 cells and tested their susceptibility to H1N1. We implemented tracheal organ cultures (TOCs) and lung cultures (LCs) to assess the efficacy of Camostat mesylate (CAM) during infection with H9N2 and H6N8. Both methods, genetic modification and chemical inhibition, led to significantly lower virus replication compared to the control groups. Furthermore, chemical inhibition of host proteases prolonged the ciliary activity in TOCs and the survival of LCs upon virus infection. Our data demonstrate the importance of TMPRSS2-related proteases as a promising target for limiting avian influenza infections and reveal the usefulness of TOCs and LCs for evaluating therapeutics.

BACKGROUND:Luminal proteases have been implicated in epithelial barrier dysfunction and visceral hypersensitivity in irritable bowel syndrome (IBS), yet their impact on the enteric nervous system (ENS), the principal regulator of gastrointestinal function, remains unknown. OBJECTIVE:To investigate whether faecal mediators differentially activate enteric neurons across IBS subtypes and whether proteolytic and proteomic profiles explain neuronal phenotypes. DESIGN:The effects of faecal supernatants (FSN) from 21 IBS-D (diarrhoea-predominant), 9 IBS-C (constipation-predominant) and 18 healthy control (HC) patients recruited across centres in three countries on guinea pig distal colon submucous plexus neurons were assessed using a neuroimaging technique. Faecal proteolytic activities and proteomic profiles were analysed. RESULTS:IBS-D and IBS-C supernatants evoked significantly stronger neuronal activation than HC, demonstrating that FSN directly modulate ENS. In IBS-D, but not IBS-C, effects were mediated by serine and cysteine proteases and PAR-1. Proteome analysis revealed a significant difference in 47 proteins between IBS-D and HC, including several immunoglobulin components, underlying the role of microinflammation in IBS-D. A combination of amylases, trypsin-2 and an immunoglobulin protein demonstrated high diagnostic performance to distinguish IBS-D from HC. CONCLUSION:These findings uncover a previously unrecognised luminal-ENS axis in IBS and reveal fundamentally different pathological mechanisms between IBS-D and IBS-C. IBS-D is characterised by proteases and PAR-1-dependent neuronal activation and a distinct immune-enriched faecal proteome, whereas mediators in IBS-C act independently of these factors. These findings establish a functional link between faecal protease activity, ENS signalling and molecular biomarkers, highlighting new therapeutic and diagnostic avenues for subtype-specific management of IBS.

Cold exposure stimulates the sympathetic nervous system (SNS) to activate brown fat thermogenesis and maintain optimal body temperature, while simultaneously triggering compensatory hyperphagia to restore energy balance. The mechanisms coordinating energy expenditure and intake, however, remain unclear. Here, we reveal that the hypothalamic-pituitary-adrenal (HPA) axis plays a dual role in this process: endogenous adrenocorticotropic hormone (ACTH) directly stimulates the melanocortin-2 receptor (MC2R) in brown adipocytes to promote thermogenesis, whereas glucocorticoids drive cold-induced hyperphagia and act permissively to enhance ACTH-mediated energy expenditure. These findings uncover previously unrecognized functions of the HPA axis and a delicate hormonal interplay that orchestrates energy homeostasis during cold stress. Targeting these pathways may offer novel strategies to mitigate hyperphagic responses associated with increased energy expenditure, with potential implications for obesity treatment. ![Figure][1] Highlights ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, DFG-TRR 333/1-450149205, DFG-Emmy Noether program-441904031, KL 973/20-1, #532683878, SCHN 1696/1-1, #532683878, FI 1546/8-1, #532683878 European Research Council, ERC starting grant: 101078516 National Natural Science Foundation of China (NSFC), 32330012 [1]: pending:yes

We used diverse methods to characterize the role of avian lateral spiriform nucleus (SpL) in basal ganglia motor function. Connectivity analysis showed that SpL receives input from globus pallidus (GP), and the intrapeduncular nucleus (INP) located ventromedial to GP, whose neurons express numerous striatal markers. SpL-projecting GP neurons were large and aspiny, while SpL-projecting INP neurons were medium sized and spiny. Connectivity analysis further showed that SpL receives inputs from subthalamic nucleus (STN) and substantia nigra pars reticulata (SNr), and that the SNr also receives inputs from GP, INP, and STN. Neurochemical analysis showed that SpL neurons express ENK, GAD, and a variety of pallidal neuron markers, and receive GABAergic terminals, some of which also contain DARPP32, consistent with GP pallidal and INP striatal inputs. Connectivity and neurochemical analysis showed that the SpL input to tectum prominently ends on GABAA receptor-enriched tectobulbar neurons. Behavioral studies showed that lesions of SpL impair visuomotor behaviors involving tracking and pecking moving targets. Our results suggest that SpL modulates brainstem-projecting tectobulbar neurons in a manner comparable to the demonstrated influence of GP internus on motor thalamus and of SNr on tectobulbar neurons in mammals. Given published data in amphibians and reptiles, it seems likely the SpL circuit represents a major direct pathway-type circuit by which the basal ganglia exerts its motor influence in nonmammalian tetrapods. The present studies also show that avian striatum is divided into three spatially segregated territories with differing connectivity, a medial striato-nigral territory, a dorsolateral striato-GP territory, and the ventrolateral INP motor territory.

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. 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. 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.

Sound localization is crucial for the survival and reproduction of animals, including non-auditory specialist animals such as the majority of avian species. The chicken (Gallus gallus) is a well-suited representative of a non-auditory specialist bird and several aspects of its auditory system have been well studied in the last decades. We conducted a behavioral experiment where 3 roosters performed a sound localization task with broad-band noise, using a 2-alternative forced choice paradigm. We determined the minimum audible angle (MAA) as measure for localization acuity. In general, our results compare to previous MAA measurements with hens in Go/NoGo tasks. The chicken has high localization acuity compared to other auditory generalist bird species tested so far. We found that chickens were better at localizing broadband noise with long duration (1 s; MAA = 16°) compared to brief duration (0.1 s; MAA = 26°). Moreover, the interaural difference in time of arrival and level (ITD and ILD, respectively) at these MAAs are comparable to what measured in other non-auditory specialist bird species, indicating that they might be sufficiently broad to be informative for azimuthal sound localization.

We combine the results of three prediction algorithms on a test set of 21 amyloidogenic proteins to predict amyloidogenic determinants. Two prediction algorithms are recently developed prediction algorithms of amyloidogenic stretches in protein sequences, whereas the third is a secondary structure prediction algorithm capable of identifying 'conformational switches' (regions that have both the propensity for alpha-helix and beta-sheet). Surprisingly, the results of prediction agree well and also agree with experimentally investigated amyloidogenic regions. Furthermore, they suggest several previously not identified amino acid stretches as potential amyloidogenic determinants. Most predicted (and experimentally observed) amyloidogenic determinants reside on the protein surface of relevant solved crystal structures. It appears that a consensus prediction algorithm is more objective than individual prediction methods alone.

Multimodal integration facilitates object recognition and response to sensory cues. This depends on spatiotemporal coincidence of sensory information, recruitment of NMDA-type glutamate receptors and inhibitory feedback. Shepherd's crook neurons (SCNs) in the avian optic tectum (TeO) are an ideal model for studying cellular mechanism of multimodal integration. They receive different sensory modalities through spatially segregated dendrites, are important for stimulus selection and have an axon-carrying dendrite (AcD). We performed whole-cell patch-clamp experiments in chicken midbrain slices of both sexes. We emulated visual and auditory inputin vitroby stimulating presynaptic afferents electrically. Simultaneous stimulation enhanced responses inversely depending on stimulation amplitude demonstrating the principle of inverse effectiveness. Contribution of NMDA-type glutamate receptors prolonged postsynaptic events for visual inputs only, causing a strong modality-specific difference in synaptic efficacy. We designed a multicompartment model to study the effect of morphological and physiological parameters on multimodal integration by varying the distance between soma and axonal origin and the amount of NMDA receptor (NMDAR) contribution. These parameters changed the preference of the model for one input channel and adjusted the range of input rates at which multimodal enhancement occurred on naturalistic stimulation. Thus, the unique morphology and synaptic features of SCNs shape the integration of input at different dendrites and generates an enhanced multimodal response.SIGNIFICANCE STATEMENTMultimodal integration improves perception and responses to objects. The underlying cellular mechanism depends on a balance between excitation and inhibition, and NMDA-type glutamate receptors that are involved in the multiplicative nature of enhancement following the principle of inverse effectiveness. Based on a detailed analysis of an identified multimodal cell type in the vertebrate midbrain, we studied the influence of cellular morphology and unimodal synaptic properties on multimodal integration. We can show that the combination of cellular morphology and modality-specific synaptic properties including NMDA receptor (NMDAR) contribution is optimal for nonlinear, multimodal enhancement and determines the dynamic response range of the integrating neuron. Our findings mechanistically explain how synaptic properties and cellular morphology of a midbrain neuron contribute to multimodal enhancement.

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.

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.

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.(1-3) 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(7-9) 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.

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.

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.

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).

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 echoacoustic flow to navigate through dark habitats. Here, we show for the bat Phyllostomus discolor that, in lighted habitats where self-motioninduced optic flow is strong, optic and echo-acoustic flow interact to guide navigation. Echo-acoustic flow showed a surprisingly strong effect compared with optic flow. We thus demonstrate multimodal interaction between two far-ranging spatial senses, vision and echolocation, available in this combination almost exclusively in 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.

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.