L. D’Angelo

Guidelines for the Care and Welfare of Cephalopods in Research –A consensus based on an initiative by CephRes, FELASA and the Boyd Group:

This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of ∼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.

Anatomical features for an adequate choice of experimental animal model in biomedicine: II. Small laboratory rodents, rabbit, and pig

The anatomical features distinctive to each of the very large array of species used in todays biomedical research must be born in mind when considering the correct choice of animal model(s), particularly when translational research is concerned. In this paper we take into consideration and discuss the most important anatomical and histological features of the commonest species of laboratory rodents (rat, mouse, guinea pig, hamster, and gerbil), rabbit, and pig related to their importance for applied research.

Anatomical features for the adequate choice of experimental animal models in biomedicine: I. Fishes

Fish constitute the oldest and most diverse class of vertebrates, and are widely used in basic research due to a number of advantages (e.g., rapid development ex-utero, large-scale genetic screening of human disease). They represent excellent experimental models for addressing studies on development, morphology, physiology and behavior function in other related species, as well as informative analysis of conservation and diversity. Although less complex, fish share many anatomical and physiological features with mammals, including humans, which make them an important complement to research in mammalian models. In this review we describe and compare the most relevant anatomical features of the most used teleostean species in research, to be taken into consideration when selecting an animal model: zebrafish (Danio rerio), medaka (Oryzias latypes), the turquoise killifish (Nothobranchius furzeri), and goldfish (Carassius auratus). Zebrafish and medaka are the mainstream models for genetic manipulability and studies on developmental biology; the turquoise killifish is an excellent model for aging research; goldfish has been largely employed for neuroendocrine studies.

Orexin and orexin receptor like peptides in the gastroenteric tract of Gallus domesticus: An immunohistochemical survey on presence and distribution.

This study reports the immunohistochemical localization and distribution of orexin A and B-like and their receptors-like peptides in the gastroenteric tract of chicken. The immunoreactivity is distributed in endocrine cells, nerve fibers and neurons, both in the stomach and intestine, and shows a discrete conformity with the data till now reported for Mammals. Our study suggests a possible participation of orexin-like peptides in the modulation of chicken gastroenteric activities and the preservation of their main distribution compared to Mammals. Western blot analysis has confirmed the presence of prepro-orexin and both receptors in the examined tissues. This survey represents the first evidence of the presence of orexin-like peptides in the gastroenteric tract of non mammalian species, and the results could help to better understand the alimentary control and body weight in domestic birds, which are of relevance to determine the productive factors in breeding animals. This study might also serve as a baseline for future experimental studies on the regulation of the gastroenteric functions in non mammalian Vertebrates.

BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations.

Brain derived neurotrophic factor in the retina of the teleost N. furzeri.

BDNF plays an important role in the development and maintenance of visual circuitries in the retina and brain visual centers. In adulthood, BDNF signaling is involved in neural protection and regeneration of retina. In this survey, we investigated the expression of BDNF in the retina of adult Nothobranchius furzeri, a teleost fish employed for age research. After describing the retina of N. furzeri and confirming that the structure is organized in layers as in all vertebrates, we have studied the localization of BDNF mRNA and protein throughout the retinal layers. BDNF mRNA is detectable in all layers, whereas the protein is lacking in the photoreceptors. The occurrence of BDNF provides new insights on its role in the retina, particularly in view of age-related disease of retina.

GDNF family ligand RET receptor in the brain of adult zebrafish

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.

Nerve growth factor in the adult brain of a teleostean model for aging research: Nothobranchius furzeri

Nerve growth factor (NGF) acts on central nervous system neurons, regulating naturally occurring cell death, synaptic connectivity, fiber guidance and dendritic morphology. The dynamically regulated production of NGF beginning in development, extends throughout adult life and aging, exerting numerous roles through a surprising variety of neurons and glial cells. This study analyzes the localization of NGF in the brain of the teleost fish Nothobranchius furzeri, an emerging model for aging research due to its short lifespan. Immunochemical and immunohistochemical experiments were performed by employing an antibody mapping at the N-terminus of the mature chain human origin NGF. Western blot analysis revealed an intense and well defined band of 20 kDa, which corresponds to proNGF of N. furzeri. Immunohistochemistry revealed NGF immunoreactivity (IR) diffused throughout all regions of telencephalon, diencephalon, mesencephalon and rhomboencephalon. It was detected in neurons and in glial cells, the latter mostly lining the mesencephalic and rhomboencephalic ventricles. Particularly in neurons, NGF IR was localized in perikarya and, to a less extent, in fibers. The widespread distribution of proNGF suggests that it might modulate numerous physiological functions in the adult brain of N. furzeri. The present survey constitutes a baseline study to enhance the understanding of the mechanisms underlying the role of NGF during aging processes.

Expression and distribution of leptin and its receptors in the digestive tract of DIO (diet-induced obese) zebrafish

The expression and localization of leptin (A and B) and its receptor family in control and diet-induced obese (DIO) adult male zebrafish gut, after 5-weeks overfeeding, administering Artemia nauplii, as fat-rich food, were investigated. Recently, the obese adult zebrafish was considered an experimental model with pathophysiological pathways similar to mammalian obesity. Currently, there are no reports about leptin in fish obesity, or in a state of altered energy balance. By qRT-PCR, leptin A and leptin B expression levels were significantly higher in DIO zebrafish gut than in the control group (CTRL), and the lowest levels of leptin receptor mRNA appeared in DIO zebrafish gut. The presence of leptin and its receptor proteins in the intestinal tract was detected by western blot analysis in both control and DIO zebrafish. By single immunohistochemical staining, leptin and leptin receptor immunoreactive endocrine cells were identified in the intestinal tract either in DIO or control zebrafish. Moreover, leptin immunopositive enteric nervous system elements were observed in both groups. By double immunohistochemical staining, leptin and its receptor were colocalized especially in DIO zebrafish. Thus, our study represents a starting point in the investigation of a possible involvement of leptin in control of energy homeostasis in control and DIO zebrafish.

Neurotrophin-4 in the brain of adult Nothobranchius furzeri

Neurotrophin-4 (NT-4) is a member of the well-known family of neurotrophins that regulate the development of neuronal networks by participating in neuronal survival and differentiation, the growth of neuronal processes, synaptic development and plasticity, as well as myelination. NT-4 interacts with two distinct receptors: TrkB, high affinity receptor and p75 low-affinity neurotrophin receptor (p75(NTR)). In the present survey, we identified the gene encoding NT-4 in the teleost Nothobranchius furzeri, a model species for aging research. The identified gene shows a similarity of about 72% with medaka, the closest related species. The neuroanatomical localization of NT-4 mRNA is obtained by using an LNA probe. NT-4 mRNA expression is observed in neurons and glial cells of the forebrain and hindbrain, with very low signal found in the midbrain. This survey confirms that NT-4 is expressed in the brain of N. furzeri during adulthood, suggesting that it could also be implicated in the maintenance and regulation of neuronal functions.