Whole-brain optical access in small adult vertebrates with two- and three-photon microscopy

Although optical microscopy has allowed us to study the entire brain in early developmental stages, access to the brains of live, adult vertebrates has been limited. Danionella, a genus of miniature, transparent fish closely related to zebrafish has been introduced as a neuroscience model to study the entire adult vertebrate brain. However, the extent of optically accessible depth in these animals has not been quantitatively characterized. Here, we show that two- and three-photon microscopy can be used to access the entire depth of the adult wild type Danionella dracula brain without any modifications to the animal other than mechanical stabilization. Three-photon microscopy provides high signal to background ratio and optical sectioning through the deepest part of the brain. While vasculature can be observed with two-photon microscopy, the deeper regions have low contrast. We show that multiphoton microscopy is ideal for readily penetrating the entire adult brain within the geometry of these animals' head structures and without the need for pigment removal. With multiphoton microscopy enabling optical access to the entire adult brain and a repertoire of methods that allow observation of the larval brain, Danionella provides a model system for readily studying the entire brain over the lifetime of a vertebrate.

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Functional analysis of enhancer elements regulating the expression of the Drosophila homeodomain transcription factor DRx by gene targeting

Hereditas ◽

10.1186/s41065-021-00210-z ◽

2021 ◽

Vol 158 (1) ◽

Author(s):

Christine Klöppel ◽

Kirsten Hildebrandt ◽

Dieter Kolb ◽

Nora Fürst ◽

Isabelle Bley ◽

...

Keyword(s):

Transcription Factor ◽

Brain Development ◽

Gene Targeting ◽

Developmental Stages ◽

Expression Patterns ◽

Adult Brain ◽

Central Complex ◽

Larval Brain ◽

Endogenous Expression ◽

The Brain

Abstract Background The Drosophila brain is an ideal model system to study stem cells, here called neuroblasts, and the generation of neural lineages. Many transcriptional activators are involved in formation of the brain during the development of Drosophila melanogaster. The transcription factor Drosophila Retinal homeobox (DRx), a member of the 57B homeobox gene cluster, is also one of these factors for brain development. Results In this study a detailed expression analysis of DRx in different developmental stages was conducted. We show that DRx is expressed in the embryonic brain in the protocerebrum, in the larval brain in the DM and DL lineages, the medulla and the lobula complex and in the central complex of the adult brain. We generated a DRx enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the endogenous expression of DRx. With the help of eight existing enhancer-Gal4 strains and one made by our group, we mapped various enhancers necessary for the expression of DRx during all stages of brain development from the embryo to the adult. We made an analysis of some larger enhancer regions by gene targeting. Deletion of three of these enhancers showing the most prominent expression patterns in the brain resulted in specific temporal and spatial loss of DRx expression in defined brain structures. Conclusion Our data show that DRx is expressed in specific neuroblasts and defined neural lineages and suggest that DRx is another important factor for Drosophila brain development.

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Regulatory modules mediating the complex neural expression patterns of the homeobrain gene during Drosophila brain development

Hereditas ◽

10.1186/s41065-021-00218-5 ◽

2022 ◽

Vol 159 (1) ◽

Author(s):

Kirsten Hildebrandt ◽

Dieter Kolb ◽

Christine Klöppel ◽

Petra Kaspar ◽

Fabienne Wittling ◽

...

Keyword(s):

Brain Development ◽

Gene Targeting ◽

Developmental Stages ◽

Expression Patterns ◽

Regulatory Elements ◽

Adult Brain ◽

Specific Cell ◽

Larval Brain ◽

Developmental Defects ◽

The Brain

Abstract Background The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp). All three genes encode transcription factors with important functions in brain development. Hbn mutants are embryonic lethal and characterized by a reduction in the anterior protocerebrum, including the mushroom bodies, and a loss of the supraoesophageal brain commissure. Results In this study we conducted a detailed expression analysis of Hbn in later developmental stages. In the larval brain, Hbn is expressed in all type II lineages and the optic lobes, including the medulla and lobula plug. The gene is expressed in the cortex of the medulla and the lobula rim in the adult brain. We generated a new hbnKOGal4 enhancer trap strain by reintegrating Gal4 in the hbn locus through gene targeting, which reflects the complete hbn expression during development. Eight different enhancer-Gal4 strains covering 12 kb upstream of hbn, the two large introns and 5 kb downstream of the gene, were established and hbn expression was investigated. We characterized several enhancers that drive expression in specific areas of the brain throughout development, from embryo to the adulthood. Finally, we generated deletions of four of these enhancer regions through gene targeting and analysed their effects on the expression and function of hbn. Conclusion The complex expression of Hbn in the developing brain is regulated by several specific enhancers within the hbn locus. Each enhancer fragment drives hbn expression in several specific cell lineages, and with largely overlapping patterns, suggesting the presence of shadow enhancers and enhancer redundancy. Specific enhancer deletion strains generated by gene targeting display developmental defects in the brain. This analysis opens an avenue for a deeper analysis of hbn regulatory elements in the future.

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Intravital multiphoton microscopy of dynamic renal processes

AJP Renal Physiology ◽

10.1152/ajprenal.00473.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. F1084-F1089 ◽

Cited By ~ 118

Author(s):

Bruce A. Molitoris ◽

Ruben M. Sandoval

Keyword(s):

Proximal Tubule ◽

Multiphoton Microscopy ◽

Field Of View ◽

Glomerular Permeability ◽

Structural Alterations ◽

Two Photon Microscopy ◽

Two Photon ◽

Dynamic Events ◽

Subcellular Resolution ◽

Functioning Kidney

Recent advances in microscopy and optics, computer sciences, and the available fluorophores used to label molecules of interest have empowered investigators to utilize intravital two-photon microscopy to study the dynamic events within the functioning kidney. This emerging technique enables investigators to follow functional and structural alterations with subcellular resolution within the same field of view over seconds to weeks. This approach invigorates the validity of data and facilitates analysis and interpretation as trends are more readily determined when one is more closely monitoring indicative physiological parameters. Therefore, in this review we emphasize how specific approaches will enable studies into glomerular permeability, proximal tubule endocytosis, and microvascular function within the kidney. We attempt to show how visual data can be quantified, thus allowing enhanced understanding of the process under study. Finally, emphasis is given to the possible future opportunities of this technology and its present limitations.

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Extended depth of focus multiphoton microscopy via incoherent pulse splitting

10.1101/2020.03.27.012260 ◽

2020 ◽

Author(s):

Bingying Chen ◽

Tonmoy Chakraborty ◽

Stephan Daetwyler ◽

James D. Manton ◽

Kevin Dean ◽

...

Keyword(s):

Multiphoton Microscopy ◽

Laser Pulses ◽

Low Complexity ◽

Phase Mask ◽

Light Sources ◽

Depth Of Focus ◽

Ultrafast Laser Pulses ◽

Two Photon Microscopy ◽

Two Photon ◽

Extended Depth Of Focus

AbstractWe present a phase mask that can be easily added to any multi-photon raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point-spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.

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

10.1101/698480 ◽

2019 ◽

Author(s):

Bruno M. Fontinha ◽

Theresa Zekoll ◽

Mariam Al-Rawi ◽

Miguel Gallach ◽

Florian Reithofer ◽

...

Keyword(s):

Developmental Stages ◽

Fine Tuning ◽

Ecological Niches ◽

Natural Environments ◽

Time Behavior ◽

Dependent Manner ◽

Peripheral Tissues ◽

Similar Expression ◽

Vertebrate Brain ◽

Starting Point

AbstractVertebrate 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 non-visual 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, if 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-RGCs brain tissues and 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 day-time rest is affected independently of eyes, pineal and the circadian clock in tmt-opsin1b mutants. We further focused on day-time behavior and the molecular changes in tmt-opsin1b/2 double mutants, and revealed that – despite their similar expression and spectral features– these Opsins interact in part non-additively. Specifically, double mutants complement molecular and (age-dependently) behavioral phenotypes observed in single mutants.Our work provides a starting point to disentangle the highly complex interactions of vertebrate non-visual Opsins, suggesting that tmt-opsin-expressing cells together with other visual and non-visual 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 in different ecological niches respond to similar light cues and how human generated artificial light might impact on behavioral processes in natural environments.

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EPCO-20. RELATING GLIOBLASTOMA HETEROGENEITY TO HUMAN FETAL GLIAL DEVELOPMENT THROUGH HIGH RESOLUTION SINGLE NUCLEI TRANSCRIPTOMICS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.299 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii73-ii73

Author(s):

Zarmeen Mussa ◽

Susana Ramos ◽

Elisa Nabel ◽

Kimaada Allette ◽

Ammar Hamid ◽

...

Keyword(s):

Stem Cell ◽

Regulatory Networks ◽

Developmental Stages ◽

Cell Types ◽

Prenatal Development ◽

Adult Brain ◽

Stem Cell Population ◽

Cortical Plate ◽

Spatiotemporal Resolution ◽

Germinal Matrix

Abstract Glioblastoma (GBM) is thought to be driven by a therapy-resistant cancer stem cell population that recapitulates developmental phenotypes. Direct comparisons of GBM to glial states during human fetal development are limited due to paucity of data from late prenatal gestation, when gliogenesis is thought to occur. Here, we generated a comprehensive single nuclei RNA sequencing (snRNAseq) dataset of approximately 200,000 nuclei taken from the germinal matrix and the cortical plate of 16 fetal postmortem samples, ranging from 17 to 41 gestational weeks, enabling high spatiotemporal resolution of late neurogenesis and early-to-peak gliogenesis. We performed unbiased clustering to identify broad cell types within each sample and integrated all fetal samples to analyze evolving glial states and relationships across two regions and four developmental stages. Subclustering analysis of developing glia from the germinal matrix and cortical plate resolved developmental cell type signatures that are absent in the adult brain. Trajectory inference and pseudo-time analyses reconstructed relationships within these glial lineages and states, identifying a robust common glial progenitor population (GPC) with distinct signature, preceding both oligodendrocyte progenitor cell (OPC) and astrocyte lineage commitment during late prenatal development. We then performed snRNAseq on approximately 30,000 nuclei taken from the core and infiltrating edge of two surgically resected GBM samples with IDH-mutant and IDH-wildtype status and EGFR amplification. Uniform manifold approximation and projection (UMAP) dimensionality reduction revealed distinct neoplastic and non-neoplastic population clusters within each GBM sample. Projecting our previously defined neural stem cell / progenitor signatures onto each GBM UMAP identified notable predominance of the GPC-like developmental signature throughout both GBM tumors with focal minor contributions from the OPC-, transit amplifying-, and astrocyte-like signatures. The high spatial and temporal resolution of the generated roadmap dissolves GBM intratumoral heterogeneity into distinct developmental molecular states driven by potentially targetable regulatory networks.

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

PLoS Biology ◽

10.1371/journal.pbio.3001012 ◽

2021 ◽

Vol 19 (1) ◽

pp. e3001012

Author(s):

Bruno M. Fontinha ◽

Theresa Zekoll ◽

Mariam Al-Rawi ◽

Miguel Gallach ◽

Florian Reithofer ◽

...

Keyword(s):

Developmental Stages ◽

Fine Tuning ◽

Ecological Niches ◽

Natural Environments ◽

Dependent Manner ◽

Peripheral Tissues ◽

Similar Expression ◽

Vertebrate Brain ◽

Complex Interactions ◽

Starting Point

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.

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Adult brain antigens demonstrated in chick embryos by fractionated antisera

Development ◽

10.1242/dev.13.1.35 ◽

1965 ◽

Vol 13 (1) ◽

pp. 35-43

Author(s):

Harvey P. Friedman ◽

Byron S. Wenger

Keyword(s):

Total Protein ◽

Developmental Stages ◽

Temporal Patterns ◽

Adult Brain ◽

Specific Inhibition ◽

Chick Embryos ◽

Avian Brain ◽

Antigenic Activity ◽

Early Embryos ◽

Inhibition Of Differentiation

The appearance, during development, of antigens which react with antisera against specific adult antigens has been considered to reflect the synthesis of similar substances in the embryo (Clayton, 1960). Within the recognized limitations of serological specificities, where the adult antigens are characteristic of a particular organ or tissue, the appearance in the embryo of cross-reacting substances may be taken as an indication of differentiation in that tissue. The presence in early embryos of antigens serologically identical to those of adult avian brain has been well established (Burke et al, 1944; Schechtman, 1948; Ebert, 1950; Flickinger, 1958). These investigations, while demonstrating the presence of adult antigens at various developmental stages, were generally not quantitative. For the most part they employed qualitative precipitin tests or specific inhibition of differentiation by antisera. In the present study antigenic activity relative to total protein was determined at different stages of development in order to reveal temporal patterns.

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Adult Neuroplasticity: More Than 40 Years of Research

Neural Plasticity ◽

10.1155/2014/541870 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 73

Author(s):

Eberhard Fuchs ◽

Gabriele Flügge

Keyword(s):

Gonadal Hormones ◽

Adult Brain ◽

Neuron Morphology ◽

Morphological Alterations ◽

Vertebrate Brain ◽

Number Of Factors ◽

History Of ◽

Environmental Stimulation ◽

Aging Change ◽

As Stress

Within the last four decades, our view of the mature vertebrate brain has changed significantly. Today it is generally accepted that the adult brain is far from being fixed. A number of factors such as stress, adrenal and gonadal hormones, neurotransmitters, growth factors, certain drugs, environmental stimulation, learning, and aging change neuronal structures and functions. The processes that these factors may induce are morphological alterations in brain areas, changes in neuron morphology, network alterations including changes in neuronal connectivity, the generation of new neurons (neurogenesis), and neurobiochemical changes. Here we review several aspects of neuroplasticity and discuss the functional implications of the neuroplastic capacities of the adult and differentiated brain with reference to the history of their discovery.

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Impact of the extracellular matrix on plasticity in juvenile and adult brains

e-Neuroforum ◽

10.1515/s13295-015-0021-z ◽

2016 ◽

Vol 22 (1) ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

R. Frischknecht ◽

Max F.K. Happel ◽

Max F.K. Happel

Keyword(s):

Extracellular Matrix ◽

Therapeutic Potential ◽

Adult Brain ◽

Brain Maturation ◽

Structural Stabilization ◽

Vertebrate Brain ◽

Flexible Adaptation ◽

Complex Forms ◽

Higher Cognitive Functions ◽

Activity Dependent

AbstractIn the higher vertebrate brain, the delicate balance between structural stabilization and remodeling of synaptic networks changes over the life span. The juvenile brain is characterized by high structural plasticity. A critical step in brain maturation is the occurrence of the extracellular matrix (ECM) that structurally stabilizes neuronal tissue restricting the potential for neuronal remodeling and regeneration. Current research has only begun to understand how this putative limitation of adult neuronal plasticity might impact on learning-related plasticity, lifelong memory reformation and higher cognitive functions. In this review, we summarize recent evidence that recognizes the ECM and its activity- dependent modulation as a key regulator of learning-related plasticity in the adult brain. Experimental modulation of the ECM in local neuronal circuits further opens short-term windows of activity-dependent reorganization, promoting complex forms of cognitive flexible adaptation of valuable behavioral options. This further bears implications for guided neuroplasticity with regenerative and therapeutic potential.

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