Shared and differential features of Robo3 expression pattern in amniotes

In Bilaterians, commissural neurons project their axons across the midline of the nervous system to target neurons on the opposite side. In mammals, midline crossing at the level of the hindbrain and spinal cord requires the Robo3 receptor which is transiently expressed by all commissural neurons. Unlike other Robo receptors, mammalian Robo3 receptors do not bind Slit ligands and promote midline crossing. Surprisingly, not much is known about Robo3 distribution and mechanism of action in other vertebrate species. Here, we have use whole-mount immunostaining, tissue clearing and light-sheet fluorescent microscopy to study Robo3 expression pattern in multiple embryonic tissue from diverse representatives of amniotes at distinct stages, including squamate (African house snake), birds (chicken, duck, pigeon, ostrich, emu and zebra finch), early postnatal marsupial mammals (fat-tailed dunnart), and eutherian mammals (mouse and human). The analysis of this rich and unique repertoire of amniote specimen reveals conserved features of Robo3 expression in midbrain, hindbrain and spinal cord commissural circuits, which together with subtle but meaningful modifications could account for species-specific evolution of sensory-motor and cognitive capacities. Our results also highlight important differences of precerebellar nuclei development across amniotes. This article is protected by copyright. All rights reserved

Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains

Fertility in mammals is controlled by hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH). These neurons differentiate in the olfactory placodes during embryogenesis and migrate from the nose to the hypothalamus before birth. Information regarding this process in humans is sparse. Here, we adapted new tissue-clearing and whole-mount immunohistochemical techniques to entire human embryos/fetuses to meticulously study this system during the first trimester of gestation in the largest series of human fetuses examined to date. Combining these cutting-edge techniques with conventional immunohistochemistry, we provide the first chronological and quantitative analysis of GnRH neuron origins, differentiation and migration, as well as a 3D atlas of their distribution in the fetal brain. We reveal not only that the number of GnRH-immunoreactive neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioral and physiological processes

Neuroscience in the third dimension: shedding new light on the brain with tissue clearing

Abstract For centuries analyses of tissues have depended on sectioning methods. Recent developments of tissue clearing techniques have now opened a segway from studying tissues in 2 dimensions to 3 dimensions. This particular advantage echoes heavily in the field of neuroscience, where in the last several years there has been an active shift towards understanding the complex orchestration of neural circuits. In the past five years, many tissue-clearing protocols have spawned. This is due to varying strength of each clearing protocol to specific applications. However, two main protocols have shown their applicability to a vast number of applications and thus are exponentially being used by a growing number of laboratories. In this review, we focus specifically on two major tissue-clearing method families, derived from the 3DISCO and the CLARITY clearing protocols. Moreover, we provide a “hands-on” description of each tissue clearing protocol and the steps to look out for when deciding to choose a specific tissue clearing protocol. Lastly, we provide perspectives for the development of tissue clearing protocols into the research community in the fields of embryology and cancer

Observation of the intraspecific interactions of cats in multi-cat households

The growing popularity of the domestic cat (Felis silvestris catus) as a pet, results in an increasing number of multi-cat households. Cohousing cats may or may not socially bond. In the latter case, tension between the cats may lead to social stress and inter-cat conflicts. Until now, research investigating intraspecific behaviour between cats inside the private home context is sparse. In the current study, cohousing cats were studied using an owner questionnaire as well as video observations of the cats in their home context. The aim was to discriminate behavioural displays between bonded and non-bonded cats. Forty-two households, each owning 2 cats, were included in the study. Cat pairs were attributed to bonded or non-bonded groups based on the owners’ description. Video observations were carried out with The Observer XT 15 (Noldus) by using a feline ethogram based on literature and video observations. Statistical analysis in R consisted of mixed models and conditional inference trees. Both mixed models (p = 0.006) and conditional inference tree (p = 0.006) indicated that wrestling behaviour was more often present in bonded cats. According to the conditional inference tree (p=0,008), non-bonded cats more often show social staring behaviour during feeding moments. In conclusion, discrepancies in social behaviour were found between bonded and non-bonded cats. Further research is needed to get a better understanding of social dynamics of cohousing cats

Tridimensional Visualization and Analysis of Early Human Development

International audienceGenerating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease

What caregivers don’t tell you ... A comparison between survey responses and home videos of cat-cat interactions

Domestic cats are increasingly popular as companion animals, but behavioural problems are often reported, especially in multicat households. Social tension is a common stressor, so understanding intercat interactions and their dynamics is crucial. Nevertheless, direct research in the home setting is rare. As caregivers witness their cats’ behaviour on a daily basis, they are a potentially important source of information, but might be unreliable and subject to bias. This study examined the reliability of caregiver reporting by comparing survey answers with behaviours observed in home videos collected after the survey was completed. The occurrence of five cat-cat interactions (head rubbing, allogrooming, sleeping in physical contact with each other, tail up greetings and social play) was examined in 42 two-cat households using 210 survey answers and 775 videos. The percentage of false negative survey responses for behaviours observed in the videos was conservatively estimated (cFN) at 8%, with 22.9% of the negative answers being falsely negative (FOR – False Omission Rate) and 77.1% truly negative (NPV – Negative Predictive Value). Broad false negatives (bFN), which included uncertain responses as negative reports, were 9.5% of the survey responses with a FOR of 75% and NPV of 25% in this context. Highest values were obtained for head rubbing (cFN: 10.5%, bFN: 14.3%) and allogrooming (cFN: 9.8%; bFN: 11.9%). When focusing on individual cat caregivers, 14 out of 42 caregivers (33.3%) failed to reliably report the occurrence of at least one of the surveyed cat-cat interactions. For interactions that were seen on camera, 23.8% of caregivers (10/42) responded that their cats did not show these interactions and 9.5% (4/42) reported uncertainty about whether it ever occurred. These results should be considered a lower estimate of the magnitude of errors (false negatives) in caregiver reports, and their implications need to be considered in both research that depends on caregiver report, and clinical assessments within behavioural medicine. Many cat-cat interactions, and in particular head rubbing and allogrooming, will be underreported when relying exclusively on caregiver reporting

A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System

International audienc

Clearing method for 3-dimensional immunofluorescence of osteoarthritic subchondral human bone reveals peripheral cholinergic nerves

International audienceThe cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation. Osteoarthritis (OA) is associated with local low-grade inflammation, but the regulatory mechanisms are unclear. Local Ach release could have anti-inflammatory activity since articular cells express Ach receptors involved in inflammatory responses. Using the 3DISCO clearing protocol that allows whole-sample 3-dimensional (3D) analysis, we cleared human OA cartilage-subchondral bone samples to search for cholinergic nerve fibres able to produce Ach locally. We analysed 3 plugs of knee cartilage and subchondral bone from 3 OA patients undergoing arthroplasty. We found no nerves in the superficial and intermediate articular cartilage layers, as evidenced by the lack of Peripherin staining (a peripheral nerves marker). Conversely, peripheral nerves were found in the deepest layer of cartilage and in subchondral bone. Some nerves in the subchondral bone samples were cholinergic because they coexpressed peripherin and choline acetyltransferase (ChAT), a specific marker of cholinergic nerves. However, no cholinergic nerves were found in the cartilage layers. It is therefore feasible to clear human bone to perform 3D immunofluorescence. Human OA subchondral bone is innervated by cholinergic fibres, which may regulate local inflammation through local Ach release

Involving caregivers in behavioural research: A SWOT analysis of two citizen science research methodologies to study cat-cat interactions at home

Citizen science, which involves engaging the general public in research tasks, is increasingly used in animal behaviour studies. In this review we conducted a SWOT analysis (Strengths, Weaknesses, Opportunities, Threats) to evaluate two methodologies of data collection using citizen science in order to study cat-cat interactions: online survey responses and caregiver-recorded home videos analysed by researcher(s). Using the SWOT-analysis on both methodologies, we listed intrinsic aspects that facilitate (Strengths) or interfere with (Weaknesses) reaching scientific goals, as well as the features that the methodology may be able to capitalise on (Opportunities) or which limit its value (Threats). A major strength of online surveys is the possibility to access caregivers’ specific knowledge of their cats, while sampling bias often is a potential weakness. Opportunities of surveys are the methodology´s flexibility and data collection efficiency, but at the same time suffering from threats related to biases associated with caregiver interpretation of their pet’s behaviour. Strengths of caregiver-recorded videos capturing cats’ behaviour include that they allow expert behavioural observations and scoring in a systematic manner (e.g. using an ethogram) and thus yielding quantitative data (whose reliability can be tested between and within observers). Furthermore, given the ubiquity of smartphones, filming cats is not a burden for most caregivers, and the collected recordings can potentially contain high-quality data that may otherwise be inaccessible, or subject to bias if a researcher had been present in the home environment. Though, caregivers’ influence on and lack of standardisation of the recordings are weaknesses which possibly influence the quality of the collected data. Opportunities include public engagement with science, while possible Threats may be related to privacy of the caregivers participating. In this review we consider in more detail each of the four SWOT components related to each methodology in order to optimise cat behaviour research in the future. The authors suggest strategies for future studies using the research methodologies discussed in this review and give specific recommendations when using caregiver-recorded videos in behavioural studies. Additionally, smart combinations of both online surveys with home videos recorded by caregivers might overcome some limitations of the individual methodologies, and would thus be a potentially stronger approach.</p