scholarly journals Multifunctional Cre-dependent transgenic mice for high-precision all-optical interrogation of neural circuits

2021 ◽  
Author(s):  
Hayley A Bounds ◽  
Masato Sadahiro ◽  
William D Hendricks ◽  
Marta Gajowa ◽  
Ian Antón Oldenburg ◽  
...  
Keyword(s):  
Neural Circuits ◽  
Physiological Activity ◽  
Large Cell ◽  
Optical Systems ◽  
Mammalian Brain ◽  
Neural Ensemble ◽  
Activity Measurements ◽  
All Optical

Causally relating the detailed structure and function of neural circuits to behavior requires the ability to precisely and simultaneously write-in and read-out neural activity. All optical systems that combine two photon (2p) calcium imaging and targeted photostimulation provide such an approach, but require co-expression of an activity indicator, such as GCaMP, and an optogenetic actuator, ideally a potent soma-targeted opsin. In the mammalian brain, such co-expression has so far been achieved by viral transduction, which is invasive and can produce variable, focal, and sometimes toxic overexpression. To overcome this challenge, we developed and validated a Cre-reporter mouse ("Ai203") that conditionally expresses a soma-targeted opsin, ChroME, fused to GCaMP7s. 1p or 2p illumination of expressing neurons in vitro and in vivo produces powerful, precise activation comparable to viral expression of ChroME. The soma-targeted GCaMP7s provides sensitive activity measurements for tracking physiological activity, and the soma-targeted ChroME provides powerful control over neural ensemble activity with holographic optogenetics. We further demonstrate the use of the Ai203 reporter line in 1p optogenetic manipulation of performance on a cortex-dependent visual task and in 2p synaptic connectivity mapping. This new transgenic line could thus greatly facilitate the study of neural circuits by providing a flexible, convenient, and stable tool for all-optical access to large, cell-type specific neural populations throughout the nervous system.

Opioids and testicular activity in the frog, Rana esculenta

1993 ◽  
Vol 137 (1) ◽  
pp. 49-NP ◽  
Author(s):  
F. Facchinetti ◽  
A. R. Genazzani ◽  
M. Vallarino ◽  
M. Pestarino ◽  
A. Polzonetti-Magni ◽  
...  
Keyword(s):  
Physiological Activity ◽  
Mammalian Species ◽  
Rana Esculenta ◽  
Cell Degeneration ◽  
Efferent System ◽  
Naltrexone Treatment ◽  
Nucleus Infundibularis ◽  
The Brain

ABSTRACT The presence and activity of brain, pituitary and testicular β-endorphin (β-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. β(1–31) and (1–27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, β-EP(1–31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. β-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for β-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally. Journal of Endocrinology (1993) 137, 49–57

scholarly journals Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor

1996 ◽  
Vol 316 (3) ◽  
pp. 703-707 ◽  
Author(s):  
Ralf BIRKENHÄGER ◽  
Bernard SCHNEPPE ◽  
Wolfgang RÖCKL ◽  
Jörg WILTING ◽  
Herbert A. WEICH ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial Cells ◽  
Physiological Activity ◽  
Expression System ◽  
Vascular Endothelial ◽  
Placenta Growth Factor ◽  
Splice Forms

Vascular endothilial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PlGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PlGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

scholarly journals Structure of the Lifeact–F-actin complex

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000925 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Hypervariable Region ◽  
Binding Pocket ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
D Loop ◽  
Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

scholarly journals SPON1 Can Reduce Amyloid Beta and Reverse Cognitive Impairment and Memory Dysfunction in Alzheimer’s Disease Mouse Model

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1275
Author(s):  
Soo Yong Park ◽  
Joo Yeong Kang ◽  
Taehee Lee ◽  
Donggyu Nam ◽  
Chang-Jin Jeon ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Model ◽  
Amyloid Precursor Protein ◽  
Amyloid Beta ◽  
Physiological Activity ◽  
Precursor Protein ◽  
Age Related

Alzheimer’s disease (AD) is a complex, age-related neurodegenerative disease that is the most common form of dementia. However, the cure for AD has not yet been founded. The accumulation of amyloid beta (Aβ) is considered to be a hallmark of AD. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), also known as beta secretase is the initiating enzyme in the amyloidogenic pathway. Blocking BACE1 could reduce the amount of Aβ, but this would also prohibit the other functions of BACE1 in brain physiological activity. SPONDIN1 (SPON1) is known to bind to the BACE1 binding site of the amyloid precursor protein (APP) and blocks the initiating amyloidogenesis. Here, we show the effect of SPON1 in Aβ reduction in vitro in neural cells and in an in vivo AD mouse model. We engineered mouse induced neural stem cells (iNSCs) to express Spon1. iNSCs harboring mouse Spon1 secreted SPON1 protein and reduced the quantity of Aβ when co-cultured with Aβ-secreting Neuro 2a cells. The human SPON1 gene itself also reduced Aβ in HEK 293T cells expressing the human APP transgene with AD-linked mutations through lentiviral-mediated delivery. We also demonstrated that injecting SPON1 reduced the amount of Aβ and ameliorated cognitive dysfunction and memory impairment in 5xFAD mice expressing human APP and PSEN1 transgenes with five AD-linked mutations.

scholarly journals Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro

2020 ◽  
Vol 14 ◽  
Author(s):  
Kevin Dorgans ◽  
Bernd Kuhn ◽  
Marylka Yoe Uusisaari
Keyword(s):  
Inferior Olive ◽  
Brain Slices ◽  
Brain Regions ◽  
Mammalian Brain ◽  
Wide Field ◽  
Voltage Imaging ◽  
Subthreshold Oscillations ◽  
Cellular Resolution

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy

2019 ◽  
Vol 56 (8) ◽  
pp. 548-556 ◽  
Author(s):  
Malte Lenders ◽  
Franciska Stappers ◽  
Christoph Niemietz ◽  
Boris Schmitz ◽  
Michel Boutin ◽  
...  
Keyword(s):  
Fabry Disease ◽  
Cell Model ◽  
White Blood Cells ◽  
Disease Patient ◽  
Patient Specific ◽  
Specific Cell ◽  
Missense Mutations ◽  
Activity Measurements

BackgroundPatients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD.MethodsSince current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy.ResultsUnder treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells.ConclusionWe conclude that repeated AGAL activity measurements in patients’ white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

scholarly journals A Novel JAK1 Mutant Breast Implant-Associated Anaplastic Large Cell Lymphoma Patient-Derived Xenograft Fostering Pre-Clinical Discoveries

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1603 ◽  
Author(s):  
Danilo Fiore ◽  
Luca Vincenzo Cappelli ◽  
Paul Zumbo ◽  
Jude M. Phillips ◽  
Zhaoqi Liu ◽  
...  
Keyword(s):  
Cell Lymphoma ◽  
Breast Implant ◽  
Large Cell ◽  
Tumor Xenograft ◽  
Sensitivity Threshold ◽  
Primary Lymphoma ◽  
Molecular Features ◽  
Primary Sample

Breast implant-associated lymphoma (BIA-ALCL) has recently been recognized as an independent peripheral T-cell lymphoma (PTCL) entity. In this study, we generated the first BIA-ALCL patient-derived tumor xenograft (PDTX) model (IL89) and a matching continuous cell line (IL89_CL#3488) to discover potential vulnerabilities and druggable targets. We characterized IL89 and IL89_CL#3488, both phenotypically and genotypically, and demonstrated that they closely resemble the matching human primary lymphoma. The tumor content underwent significant enrichment along passages, as confirmed by the increased variant allele frequency (VAF) of mutations. Known aberrations (JAK1 and KMT2C) were identified, together with novel hits, including PDGFB, PDGFRA, and SETBP1. A deep sequencing approach allowed the detection of mutations below the Whole Exome Sequencing (WES) sensitivity threshold, including JAK1G1097D, in the primary sample. RNA sequencing confirmed the expression of a signature of differentially expressed genes in BIA-ALCL. Next, we tested IL89’s sensitivity to the JAK inhibitor ruxolitinib and observed a potent anti-tumor effect, both in vitro and in vivo. We also implemented a high-throughput drug screening approach to identify compounds associated with increased responses in the presence of ruxolitinib. In conclusion, these new IL89 BIA-ALCL models closely recapitulate the primary correspondent lymphoma and represent an informative platform for dissecting the molecular features of BIA-ALCL and performing pre-clinical drug discovery studies, fostering the development of new precision medicine approaches.

The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity

2011 ◽  
Vol 35 (2) ◽  
pp. 188-196 ◽  
Author(s):  
R. Vargas ◽  
I. þ. Jóhannesdóttir ◽  
B. Sigurgeirsson ◽  
H. þorsteinsson ◽  
K. Æ. Karlsson
Keyword(s):  
Neural Development ◽  
Neural Circuits ◽  
Brain Slices ◽  
Functional Roles ◽  
Zebrafish Brain ◽  
Vitro Model ◽  
Immunohistochemical Techniques ◽  
Neuronal Groups

Recently, the zebrafish ( Danio rerio ) has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits. Information about the techniques used to obtain a workable preparation is not readily available. Here, we describe a complete protocol for obtaining in vitro and in vivo zebrafish brain preparations. In addition, we performed extracellular recordings in the whole brain, brain slices, and immobilized nonanesthetized larval zebrafish to evaluate the viability of the tissue. Each type of preparation can be used to detect spontaneous activity, to determine patterns of activity in specific brain areas with unknown functions, or to assess the functional roles of different neuronal groups during brain development in zebrafish. The technique described offers a guide that will provide innovative and broad opportunities to beginner students and researchers who are interested in the functional analysis of neuronal activity, plasticity, and neural development in the zebrafish brain.

scholarly journals Micro-optical Components for Bioimaging on Tissues, Cells and Subcellular Structures

Micromachines ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 405 ◽  
Author(s):  
Hui Yang ◽  
Yi Zhang ◽  
Sihui Chen ◽  
Rui Hao
Keyword(s):  
Imaging Techniques ◽  
Point Of Care ◽  
Biological Information ◽  
Optical Systems ◽  
Optical Components ◽  
Integrated Optical ◽  
Fast Development ◽  
Near Future

Bioimaging generally indicates imaging techniques that acquire biological information from living forms. Among different imaging techniques, optical microscopy plays a predominant role in observing tissues, cells and biomolecules. Along with the fast development of microtechnology, developing miniaturized and integrated optical imaging systems has become essential to provide new imaging solutions for point-of-care applications. In this review, we will introduce the basic micro-optical components and their fabrication technologies first, and further emphasize the development of integrated optical systems for in vitro and in vivo bioimaging, respectively. We will conclude by giving our perspectives on micro-optical components for bioimaging applications in the near future.

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