A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity

Pende, Marko; Vadiwala, Karim; Schmidbaur, Hannah; Stockinger, Alexander; Murawala, Prayag; Saghafi, Saiedeh; Dekens, Marcus P. S.; Becker, Klaus; Revilla-i-Domingo, Roger; Papadopoulos, Sofia-Christina; Zurl, Martin; Pasierbek, Paweł; Simakov, Oleg; Tanaka, Elly M.; Raible, Florian; Dodt, Hans‐Ulrich

doi:10.1126/sciadv.aba0365

Cited by 78 publications

(86 citation statements)

References 85 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The protocol was adapted from [ 66 ] with the following details: Adult fish (>2 months old) were anesthetized in fish water containing 0.2% tricaine and decapitated; the brain was dissected and fixed in ice-cold 4% PFA overnight. After washes in 1× PBS at RT, the brains were incubated in pre-chilled acetone O/N at −20°C, rehydrated in 1× PBS at RT, and digested with 10 μg/ml Proteinase K (Merck #1245680100) for 10–12 min at RT, followed by 2× glycine washes (2 mg/ml) (Roth #3908.3).…”

Section: Methodsmentioning

confidence: 99%

TMT-Opsins differentially modulate medaka brain function in a context-dependent manner

et al. 2021

View full text Add to dashboard Cite

Vertebrate behavior is strongly influenced by light. Light receptors, encoded by functional opsin proteins, are present inside the vertebrate brain and peripheral tissues. This expression feature is present from fishes to human and appears to be particularly prominent in diurnal vertebrates. Despite their conserved widespread occurrence, the nonvisual functions of opsins are still largely enigmatic. This is even more apparent when considering the high number of opsins. Teleosts possess around 40 opsin genes, present from young developmental stages to adulthood. Many of these opsins have been shown to function as light receptors. This raises the question of whether this large number might mainly reflect functional redundancy or rather maximally enables teleosts to optimally use the complex light information present under water. We focus on tmt-opsin1b and tmt-opsin2, c-opsins with ancestral-type sequence features, conserved across several vertebrate phyla, expressed with partly similar expression in non-rod, non-cone, non-retinal-ganglion-cell brain tissues and with a similar spectral sensitivity. The characterization of the single mutants revealed age- and light-dependent behavioral changes, as well as an impact on the levels of the preprohormone sst1b and the voltage-gated sodium channel subunit scn12aa. The amount of daytime rest is affected independently of the eyes, pineal organ, and circadian clock in tmt-opsin1b mutants. We further focused on daytime behavior and the molecular changes in tmt-opsin1b/2 double mutants, and found that—despite their similar expression and spectral features—these opsins interact in part nonadditively. Specifically, double mutants complement molecular and behavioral phenotypes observed in single mutants in a partly age-dependent fashion. Our work provides a starting point to disentangle the highly complex interactions of vertebrate nonvisual opsins, suggesting that tmt-opsin-expressing cells together with other visual and nonvisual opsins provide detailed light information to the organism for behavioral fine-tuning. This work also provides a stepping stone to unravel how vertebrate species with conserved opsins, but living in different ecological niches, respond to similar light cues and how human-generated artificial light might impact on behavioral processes in natural environments.

show abstract

Section: Methodsmentioning

confidence: 99%

TMT-Opsins differentially modulate medaka brain function in a context-dependent manner

et al. 2021

View full text Add to dashboard Cite

show abstract

“…Light absorption is a minor cause of tissue opacity in mammals, as very few chromophores can absorb electromagnetic waves in the visible range; hence, light absorption can be almost disregarded in tissues that are not rich in heme groups or melanin ( Inyushin et al., 2019 ). It is important to notice that this is not the case of invertebrates or several animal species, in which pigments such as ommochromes or pterins constitute a significant cause of opacity, and for which particular depigmentation clearing protocols have been specifically developed, such as DEpigmEntation-Plus-Clearing (DEEP-Clear) ( Pende et al., 2020 ) or FlyClear ( Pende et al., 2018 ), which can also eliminate heme groups and melanin. But in most studied mammals whatsoever, lipids and RI mismatch are the major factors that lead light to be scattered instead of traveling straight through the tissue, causing blurring and opacity.…”

Section: Introductionmentioning

confidence: 99%

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

et al. 2020

View full text Add to dashboard Cite

Summary Three-dimensional (3D) optical imaging techniques can expand our knowledge about physiological and pathological processes that cannot be fully understood with 2D approaches. Standard diagnostic tests frequently are not sufficient to unequivocally determine the presence of a pathological condition. Whole-organ optical imaging requires tissue transparency, which can be achieved by using tissue clearing procedures enabling deeper image acquisition and therefore making possible the analysis of large-scale biological tissue samples. Here, we review currently available clearing agents, methods, and their application in imaging of physiological or pathological conditions in different animal and human organs. We also compare different optical tissue clearing methods discussing their advantages and disadvantages and review the use of different 3D imaging techniques for the visualization and image acquisition of cleared tissues. The use of optical tissue clearing resources for large-scale biological tissues 3D imaging paves the way for future applications in translational and clinical research.

show abstract

“…However, the retinal pigment epithelium, the interface between the retinal photoreceptors and the choroid, is highly melanized (darkly pigmented), which remains a challenge for standard clearing methods. Specialized clearing techniques including EyeCi ( 63 ), DEEP-Clear ( 64 ), and EyeDISCO ( 65 ) were developed to address this issue and enable light sheet imaging of retinas within intact eyeballs. However, to the best of our knowledge these techniques have not yet been used for retinal microglia studies.…”

Section: Techniques For Imaging Microglia In Situmentioning

confidence: 99%

Tools and Approaches for Studying Microglia In vivo

Eme‐Scolan

Dando

2020

Front. Immunol.

View full text Add to dashboard Cite

Microglia are specialized resident macrophages of the central nervous system (CNS) that have important functions during neurodevelopment, homeostasis and disease. This mini-review provides an overview of the current tools and approaches for studying microglia in vivo. We focus on tools for labeling microglia, highlighting the advantages and limitations of microglia markers/antibodies and reporter mice. We also discuss techniques for imaging microglia in situ, including in vivo live imaging of brain and retinal microglia. Finally, we review microglia depletion approaches and their use to investigate microglial function in CNS homeostasis and disease.

show abstract

A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity

Cited by 78 publications

References 85 publications

TMT-Opsins differentially modulate medaka brain function in a context-dependent manner

TMT-Opsins differentially modulate medaka brain function in a context-dependent manner

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Tools and Approaches for Studying Microglia In vivo

Contact Info

Product

Resources

About