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In JoVE (2)
- अर्ध - मोटी मस्तिष्क स्लाइसें में एक उच्च संकल्प प्रतिदीप्ति इमेजिंग के लिए रैपिड दृष्टिकोण
- मस्तिष्क के ऊतकों के जीर्ण Optogenetic उत्तेजना के लिए फाइबर ऑप्टिक आरोपण
Other Publications (18)
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Development (Cambridge, England)
- Genes & Development
- Developmental Biology
- Genes & Development
- Neuron
- Nature Methods
- Journal of Neurophysiology
- Pediatric Blood & Cancer
- Nature
- Journal of Neurophysiology
- Cancer Cell
- Frontiers in Neuroscience
- Current Opinion in Neurobiology
- PloS One
- Nature Communications
- Stem Cells (Dayton, Ohio)
- Neuron
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Articles by Benjamin R. Arenkiel in JoVE
JoVE Neuroscience
अर्ध - मोटी मस्तिष्क स्लाइसें में एक उच्च संकल्प प्रतिदीप्ति इमेजिंग के लिए रैपिड दृष्टिकोण
1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Precisionary Instruments Inc., 3Departments of Molecular & Human Genetics and Neuroscience, Baylor College of Medicine (BCM), 4Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital
यहाँ हम अर्द्ध मोटी मस्तिष्क स्लाइसें में छवि fluorescently लेबल की कोशिकाओं के लिए एक तेजी से और सरल विधि का वर्णन. हम फिक्सिंग, टुकड़ा करने की क्रिया, और ऑप्टिकली मस्तिष्क के ऊतकों समाशोधन का वर्णन कैसे मानक epifluorescent या confocal इमेजिंग बरकरार तंत्रिका ऊतक के भीतर और व्यक्तिगत कोशिकाओं neuronal नेटवर्क कल्पना करने के लिए इस्तेमाल किया जा सकता है.
JoVE Neuroscience
मस्तिष्क के ऊतकों के जीर्ण Optogenetic उत्तेजना के लिए फाइबर ऑप्टिक आरोपण
1Department of Molecular & Human Genetics, Baylor College of Medicine (BCM), 2Department of Neuroscience, Baylor College of Medicine (BCM), 3Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital
optogenetics के विकास अब ठीक आनुवंशिक रूप से परिभाषित न्यूरॉन्स और सर्किट को प्रोत्साहित करने का मतलब है, दोनों प्रदान करता है
Other articles by Benjamin R. Arenkiel on PubMed
Hoxb1 Neural Crest Preferentially Form Glia of the PNS
The vertebrate cranial neural crest cells give rise to many complex derivatives of the head, neck, and face, including neuronal and glial cells that act in concert for proper development of the anterior-peripheral nervous system. Several genes have been implicated in the processes of neural crest specification, migration, and differentiation; among these are the hox gene clusters. To determine the fates of hox-expressing cranial neural crest, we describe the results of a genetic lineage analysis by using the Cre/loxP system to drive the activation of different ROSA26 reporter alleles under the regulation of the hoxb1 locus. By targeting the 3' untranslated region of the hoxb1 gene, we have preserved endogenous gene activity and have been able to accurately follow the fates of the cells derived from the hoxb1 expression domain. Emphasis was placed on identifying the cell and tissue types that arise from the rhombomere 4-derived neural crest. Our results demonstrate that, in addition to forming much of the cartilage, bones, and muscle of the ears and neck, a significant population of rhombomere 4-derived neural crest is fated to generate the glial component of the seventh cranial nerve.
Ablation of Specific Expression Domains Reveals Discrete Functions of Ectoderm- and Endoderm-derived FGF8 During Cardiovascular and Pharyngeal Development
Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. We postulate that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.
Hoxb1 Functions in Both Motoneurons and in Tissues of the Periphery to Establish and Maintain the Proper Neuronal Circuitry
Formation of neuronal circuits in the head requires the coordinated development of neurons within the central nervous system (CNS) and neural crest-derived peripheral target tissues. Hoxb1, which is expressed throughout rhombomere 4 (r4), has been shown to be required for the specification of facial branchiomotor neuron progenitors that are programmed to innervate the muscles of facial expression. In this study, we have uncovered additional roles for Hoxb1-expressing cells in the formation and maintenance of the VIIth cranial nerve circuitry. By conditionally deleting the Hoxb1 locus in neural crest, we demonstrate that Hoxb1 is also required in r4-derived neural crest to facilitate and maintain formation of the VIIth nerve circuitry. Genetic lineage analysis revealed that a significant population of r4-derived neural crest is fated to generate glia that myelinate the VIIth cranial nerve. Neural crest cultures show that the absence of Hoxb1 function does not appear to affect overall glial progenitor specification, suggesting that a later glial function is critical for maintenance of the VIIth nerve. Taken together, these results suggest that the molecular program governing the development and maintenance of the VIIth cranial nerve is dependent upon Hoxb1, both in the neural crest-derived glia and in the facial branchiomotor neurons.
The Roles of Fgf4 and Fgf8 in Limb Bud Initiation and Outgrowth
Although numerous molecules required for limb bud formation have recently been identified, the molecular pathways that initiate this process and ensure that limb formation occurs at specific axial positions have yet to be fully elucidated. Based on experiments in the chick, Fgf8 expression in the intermediate mesoderm (IM) has been proposed to play a critical role in the initiation of limb bud outgrowth via restriction of Fgf10 expression to the appropriate region of the lateral plate mesoderm. Contrary to the outcome predicted by this model, ablation of Fgf8 expression in the intermediate mesoderm before limb bud initiation had no effect on initial limb bud outgrowth or on the formation of normal limbs. When their expression patterns were first elucidated, both Fgf4 and Fgf8 were proposed to mediate critical functions of the apical ectodermal ridge (AER), which is required for proper limb bud outgrowth. Although mice lacking Fgf4 in the AER have normal limbs, limb development is severely affected in Fgf8 mutants and certain skeletal elements are not produced. By creating mice lacking both Fgf4 and Fgf8 function in the forelimb AER, we show that limb bud mesenchyme fails to survive in the absence of both FGF family members. Thus, Fgf4 is responsible for the partial compensation of distal limb development in the absence of Fgf8. A prolonged period of increased apoptosis, beginning at 10 days of gestation in a proximal-dorsal region of the limb bud, leads to the elimination of enough mesenchymal cells to preclude formation of distal limb structures. Expression of Shh and Fgf10 is nearly abolished in double mutant limb buds. By using a CRE driver expressed in both forelimb and hindlimb ectoderm to inactivate Fgf4 and Fgf8, we have produced mice lacking all limbs, allowing a direct comparison of FGF requirements in the two locations.
Alveolar Rhabdomyosarcomas in Conditional Pax3:Fkhr Mice: Cooperativity of Ink4a/ARF and Trp53 Loss of Function
Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. We report the first mouse model of alveolar rhabdomyosarcoma using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing.
In Vivo Light-induced Activation of Neural Circuitry in Transgenic Mice Expressing Channelrhodopsin-2
Channelrhodopsin-2 (ChR2) is a light-gated, cation-selective ion channel isolated from the green algae Chlamydomonas reinhardtii. Here, we report the generation of transgenic mice that express a ChR2-YFP fusion protein in the CNS for in vivo activation and mapping of neural circuits. Using focal illumination of the cerebral cortex and olfactory bulb, we demonstrate a highly reproducible, light-dependent activation of neurons and precise control of firing frequency in vivo. To test the feasibility of mapping neural circuits, we exploited the circuitry formed between the olfactory bulb and the piriform cortex in anesthetized mice. In the olfactory bulb, individual mitral cells fired action potentials in response to light, and their firing rate was not influenced by costimulated glomeruli. However, in piriform cortex, the activity of target neurons increased as larger areas of the bulb were illuminated to recruit additional glomeruli. These results support a model of olfactory processing that is dependent upon mitral cell convergence and integration onto cortical cells. More broadly, these findings demonstrate a system for precise manipulation of neural activity in the intact mammalian brain with light and illustrate the use of ChR2 mice in exploring functional connectivity of complex neural circuits in vivo.
Genetic Control of Neuronal Activity in Mice Conditionally Expressing TRPV1
Here we describe a knock-in mouse model for Cre-loxP-based conditional expression of TRPV1 in central nervous system neurons. Expression of Cre recombinase using biolistics, lentivirus or genetic intercrosses triggered heterologous expression of TRPV1 in a cell-specific manner. Application of the TRPV1 ligand capsaicin induced strong inward currents, triggered action potentials and activated stereotyped behaviors, allowing cell type-specific chemical genetic control of neuronal activity in vitro and in vivo.
Using Light to Reinstate Respiratory Plasticity
Restoring normal function to damaged or diseased nervous tissue remains a major goal of both basic and clinical neuroscience research. Advances in genetic technologies now allow targeted control of neuronal activity in the mammalian nervous system, providing novel therapeutic avenues to repair or bypass faulty circuits. Here we review recent work published in the Journal of Neuroscience by Alilain et al., demonstrating the use of Channelrhodopsin-2 to restore breathing in rodent models of spinal cord injury.
Bortezomib Reverses a Post-translational Mechanism of Tumorigenesis for Patched1 Haploinsufficiency in Medulloblastoma
Tumor initiation has been attributed to haploinsufficiency at a single locus for a large number of cancers. Patched1 (Ptc1) was one of the first such loci, and Ptc1 haploinsufficiency has been asserted to lead to medulloblastoma and rhabdomyosarcoma in mice.
Molecular Genetics and Imaging Technologies for Circuit-based Neuroanatomy
Brain function emerges from the morphologies, spatial organization and patterns of connectivity established between diverse sets of neurons. Historically, the notion that neuronal structure predicts function stemmed from classic histological staining and neuronal tracing methods. Recent advances in molecular genetics and imaging technologies have begun to reveal previously unattainable details about patterns of functional circuit connectivity and the subcellular organization of synapses in the living brain. This sophisticated molecular and genetic 'toolbox', coupled with new methods in optical and electron microscopy, provides an expanding array of techniques for probing neural anatomy and function.
Adult Neurogenesis Supports Short-term Olfactory Memory
Adult neurogenesis has captivated neuroscientists for decades, with hopes that understanding the programs underlying this phenomenon may provide unique insight toward avenues for brain repair. Interestingly, however, despite intense molecular and cellular investigation, the evolutionary roles and biological functions for ongoing neurogenesis have remained elusive. Here I review recent work published in the Journal of Neuroscience that reveals a functional role for continued neurogenesis toward forming short-term olfactory memories.
Evidence for an Unanticipated Relationship Between Undifferentiated Pleomorphic Sarcoma and Embryonal Rhabdomyosarcoma
Embryonal rhabdomyosarcoma (eRMS) shows the most myodifferentiation among sarcomas, yet the precise cell of origin remains undefined. Using Ptch1, p53 and/or Rb1 conditional mouse models and controlling prenatal or postnatal myogenic cell of origin, we demonstrate that eRMS and undifferentiated pleomorphic sarcoma (UPS) lie in a continuum, with satellite cells predisposed to giving rise to UPS. Conversely, p53 loss in maturing myoblasts gives rise to eRMS, which have the highest myodifferentiation potential. Regardless of origin, Rb1 loss modifies tumor phenotype to mimic UPS. In human sarcomas that lack pathognomic chromosomal translocations, p53 loss of function is prevalent, whereas Shh or Rb1 alterations likely act primarily as modifiers. Thus, sarcoma phenotype is strongly influenced by cell of origin and mutational profile.
Genetic Approaches to Reveal the Connectivity of Adult-born Neurons
Much has been learned about the environmental and molecular factors that influence the division, migration, and programmed cell death of adult-born neurons in the mammalian brain. However, detailed knowledge of the mechanisms that govern the formation and maintenance of functional circuit connectivity via adult neurogenesis remains elusive. Recent advances in genetic technologies now afford the ability to precisely target discrete brain tissues, neuronal subtypes, and even single neurons for vital reporter expression and controlled activity manipulations. Here, I review current viral tracing methods, heterologous receptor expression systems, and optogenetic technologies that hold promise toward elucidating the wiring diagrams and circuit properties of adult-born neurons.
Chemical Genetics: Receptor-ligand Pairs for Rapid Manipulation of Neuronal Activity
Toward the functional dissection of neuronal circuits, a number of new genetic tools have been developed that enable rapid and reversible manipulation of genetically defined neuronal subtypes in intact mammalian brain circuits. Alongside the breakthrough technology of optogenetics, receptor-ligand pairs provide complementary approaches to modulate neuronal activity using chemical genetics.
Activity-induced Remodeling of Olfactory Bulb Microcircuits Revealed by Monosynaptic Tracing
The continued addition of new neurons to mature olfactory circuits represents a remarkable mode of cellular and structural brain plasticity. However, the anatomical configuration of newly established circuits, the types and numbers of neurons that form new synaptic connections, and the effect of sensory experience on synaptic connectivity in the olfactory bulb remain poorly understood. Using in vivo electroporation and monosynaptic tracing, we show that postnatal-born granule cells form synaptic connections with centrifugal inputs and mitral/tufted cells in the mouse olfactory bulb. In addition, newly born granule cells receive extensive input from local inhibitory short axon cells, a poorly understood cell population. The connectivity of short axon cells shows clustered organization, and their synaptic input onto newborn granule cells dramatically and selectively expands with odor stimulation. Our findings suggest that sensory experience promotes the synaptic integration of new neurons into cell type-specific olfactory circuits.
Transient Activation of Specific Neurons in Mice by Selective Expression of the Capsaicin Receptor
The ability to control the electrical activity of a neuronal subtype is a valuable tool in deciphering the role of discreet cell populations in complex neural circuits. Recent techniques that allow remote control of neurons are either labor intensive and invasive or indirectly coupled to neural electrical potential with low temporal resolution. Here we show the rapid, reversible and direct activation of genetically identified neuronal subpopulations by generating two inducible transgenic mouse models. Confined expression of the capsaicin receptor, TRPV1, allows cell-specific activation after peripheral or oral delivery of ligand in freely moving mice. Capsaicin-induced activation of dopaminergic or serotonergic neurons reversibly alters both physiological and behavioural responses within minutes, and lasts ~10 min. These models showcase a robust and remotely controllable genetic tool that modulates a distinct cell population without the need for invasive and labour-intensive approaches.
Tracing Synaptic Connectivity Onto Embryonic Stem Cell-derived Neurons
Transsynaptic circuit tracing using genetically modified rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it is now possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem cells (ESCs) in vitro, and these properties are under intense investigation toward devising cell replacement therapies. Here, we report the generation of a novel mouse ESC (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ESC-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to synaptically connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest. STEM Cells2012;30:2140-2151.
Imaging Neural Activity Using Thy1-GCaMP Transgenic Mice
The ability to chronically monitor neuronal activity in the living brain is essential for understanding the organization and function of the nervous system. The genetically encoded green fluorescent protein-based calcium sensor GCaMP provides a powerful tool for detecting calcium transients in neuronal somata, processes, and synapses that are triggered by neuronal activities. Here we report the generation and characterization of transgenic mice that express improved GCaMPs in various neuronal subpopulations under the control of the Thy1 promoter. In vitro and in vivo studies show that calcium transients induced by spontaneous and stimulus-evoked neuronal activities can be readily detected at the level of individual cells and synapses in acute brain slices, as well as chronically in awake, behaving animals. These GCaMP transgenic mice allow investigation of activity patterns in defined neuronal populations in the living brain and will greatly facilitate dissecting complex structural and functional relationships of neural networks.
