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In JoVE (1)
Other Publications (16)
- Differentiation; Research in Biological Diversity
- Trends in Neurosciences
- Proceedings of the National Academy of Sciences of the United States of America
- The Journal of Physiology
- Experimental Neurology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature Protocols
- Developmental Neurobiology
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- PLoS Biology
Articles by Kurt Haas in JoVE
Single Cell Electroporation in vivo within the Intact Developing Brain
D. Sesath Hewapathirane1,2, Kurt Haas1,2
1Brain Research Centre, University of British Columbia - UBC, 2Department of Cellular and Physiological Sciences, University of British Columbia - UBC
Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.
Other articles by Kurt Haas on PubMed
Differentiation; Research in Biological Diversity. Jun, 2002 | Pubmed ID: 12147134
Electroporation is becoming more popular as a technique for transfecting neurons within intact tissues. One of the advantages of electroporation over other transfection techniques is the ability to precisely target an area for transfection. Here we highlight this advantage by describing methods to restrict transfection to either a single cell, clusters of cells, or to include large portions of the brain of the intact Xenopus tadpole. Electroporation is also an effective means of gene delivery in the retina. We have developed these techniques to examine the effects of regulated gene expression on various neuronal properties, including structural plasticity and synaptic transmission. Restriction of transfection to individual cells aids in imaging of neuronal morphology, while bulk cell transfection allows examination of the affects of gene expression on populations of cells by biochemical assays, imaging, and electrophysiological recording.
Nature. Oct, 2002 | Pubmed ID: 12368855
Previous studies suggest that neuronal activity may guide the development of synaptic connections in the central nervous system through mechanisms involving glutamate receptors and GTPase-dependent modulation of the actin cytoskeleton. Here we demonstrate by in vivo time-lapse imaging of optic tectal cells in Xenopus laevis tadpoles that enhanced visual activity driven by a light stimulus promotes dendritic arbor growth. The stimulus-induced dendritic arbor growth requires glutamate-receptor-mediated synaptic transmission, decreased RhoA activity and increased Rac and Cdc42 activity. The results delineate a role for Rho GTPases in the structural plasticity driven by visual stimulation in vivo.
Diabetes. Dec, 2003 | Pubmed ID: 14633862
Heterozygous mutations in the coding sequence of the serpentine melanocortin 4 receptor (MC4R) are the most frequent genetic cause of severe human obesity. Since haploinsufficiency has been proposed as a causal mechanism of obesity associated with these mutations, reduction in gene transcription caused by mutations in the transcriptionally essential regions of the MC4R promoter may also be a cause of severe obesity in humans. To test this hypothesis we defined the minimal promoter region of the human MC4R and evaluated the extent of genetic variation in this region compared with the coding region in two cohorts of severely obese subjects. 5'RACE followed by functional promoter analysis in multiple cell lines indicates that an 80-bp region is essential for the transcriptional activity of the MC4R promoter. Systematic screening of 431 obese children and adults for mutations in the coding sequence and the minimal core promoter of MC4R reveals that genetic variation in the transcriptionally essential region of the MC4R promoter is not a significant cause of severe obesity in humans.
Proceedings of the National Academy of Sciences of the United States of America. Aug, 2006 | Pubmed ID: 16882725
The size and shape of neuronal dendritic arbors affect the number and type of synaptic inputs, as well as the complexity and function of brain circuits. In the intact brain, dendritic arbor growth and the development of excitatory glutamatergic synapse are concurrent. Consequently, it has been difficult to resolve whether synaptic inputs drive dendritic arbor development. Here, we test the role of AMPA receptor (AMPAR)-mediated glutamatergic transmission in dendrite growth by expressing peptides corresponding to the intracellular C-terminal domains of AMPAR subunits GluR1 (GluR1Ct) and GluR2 (GluR2Ct) in optic tectal neurons of the Xenopus retinotectal system. These peptides significantly reduce AMPAR synaptic transmission in transfected neurons while leaving visual system circuitry intact. Daily in vivo imaging over 5 days revealed that GluR1Ct or GluR2Ct expression dramatically impaired dendrite growth, resulting in less complex arbors than controls. Time-lapse images collected at 2-h intervals over 6 h show that both GluR1Ct and GluR2Ct decrease branch lifetimes. Ultrastructural analysis indicates that synapses formed onto neurons expressing the GluRCt are less mature than synapses onto control neurons. These data suggest that the failure to form complex arbors is due to reduced stabilization of new synapses and dendritic branches. Although visual stimulation increases dendritic arbor growth rates in control tectal neurons, a weak postsynaptic response to visual experience in GluRCt-expressing cells leads to retraction of branches. These results indicate that AMPAR-mediated transmission underlies experience-dependent dendritic arbor growth by stabilizing branches, and support a competition-based model for dendrite growth.
The Regulation of Dendritic Arbor Development and Plasticity by Glutamatergic Synaptic Input: a Review of the Synaptotrophic Hypothesis
The Journal of Physiology. Mar, 2008 | Pubmed ID: 18202093
The synaptotropic hypothesis, which states that synaptic inputs control the elaboration of dendritic (and axonal) arbors was articulated by Vaughn in 1989. Today the role of synaptic inputs in controlling neuronal structural development remains an area of intense research activity. Several recent studies have applied modern molecular genetic, imaging and electrophysiological methods to this question and now provide strong evidence that maturation of excitatory synaptic inputs is required for the development of neuronal structure in the intact brain. Here we critically review data concerning the hypothesis with the expectation that understanding the circumstances when the data do and do not support the hypothesis will be most valuable. The synaptotrophic hypothesis contributes at both conceptual and mechanistic levels to our understanding of how relatively minor changes in levels or function of synaptic proteins may have profound effects on circuit development and plasticity.
Experimental Neurology. Jun, 2008 | Pubmed ID: 18402939
The immature brain is exceptionally susceptible to seizures. However, it remains unclear whether seizures occurring during development affect critical processes underlying neural circuit formation, leading to long-term functional consequences. Here we characterize a novel in vivo model system of developmental seizures based on the transparent albino Xenopus laevis tadpole, which allows direct examination of seizure activity, and seizure-induced effects on neuronal development within the intact unanesthetized brain. Pentylenetetrazol (PTZ), kainic acid, bicuculline, picrotoxin, 4-aminopyridine, and pilocarpine were tested for their ability to induce behavioral seizures in freely swimming tadpoles when bath applied. All six chemoconvulsants consistently induced similar patterns of abnormal behavior in a dose-dependent manner, characterized by convulsive clonus-like motor patterns with periods of behavioral arrest. Extracellular field recordings demonstrated rhythmic synchronous epileptiform electrographic responses induced by convulsants irrespective of mechanism of action, that could be terminated by the anti-epileptic drug valproate. PTZ-induced seizures were further characterized using in vivo two-photon fluorescence imaging of neuronal calcium dynamics, in unanesthetized immobilized tadpoles. Imaging of calcium dynamics during PTZ-induced seizures revealed waves of neural activity propagating through large populations of neurons within the brain. Analysis of single-cell responses demonstrated distinct synchronized high-amplitude calcium spikes not observed under baseline conditions. Similar to other developmental seizure models, prolonged seizures failed to induce marked neuronal death within the brain, detected by cellular propidium iodide incorporation in vivo or TUNEL labeling. This novel developmental seizure model system has distinct advantages for controlled seizure induction, and direct visualization of both seizure activity and seizure-induced effects on individual developing neurons within the intact unanesthetized brain. Such a system is necessary to address important questions relating to the long-term impact of common perinatal seizures on developing neural circuits.
PKM Zeta Restricts Dendritic Arbor Growth by Filopodial and Branch Stabilization Within the Intact and Awake Developing Brain
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2009 | Pubmed ID: 19793981
The molecular mechanisms underlying activity-dependent neural circuit growth and plasticity during early brain development remain poorly understood. Protein kinase Mzeta (PKMz), an endogenous constitutively active kinase associated with late-phase long-term synaptic potentiation and memory in the mature brain, is expressed in the embryonic Xenopus retinotectal system with heightened levels during peak periods of dendrite growth and synaptogenesis. In vivo rapid time-lapse imaging of actively growing tectal neurons and comprehensive three-dimensional tracking of dynamic dendritic growth behavior finds that altered PKMz activity affects morphologic stabilization. Exogenous expression of PKMz within single neurons stabilizes dendritic filopodia by increasing dendritic filopodial lifetimes and decreasing filopodial additions, eliminations, and motility, whereas long-term in vivo imaging demonstrates restricted expansion of the dendritic arbor. Alternatively, blocking endogenous PKMz activity in individual growing tectal neurons with an inhibitory peptide (zeta-inhibitory peptide) destabilizes dendritic filopodia and over long periods promotes excessive arbor expansion. Furthermore, inhibiting endogenous PKMz throughout the tectum decreases colocalization of immunostained presynaptic and postsynaptic markers, SNAP-25 and PSD-95, respectively, suggesting impaired synapse maintenance. Together, these results implicate PKMz activity in restricting dendritic arborization during embryonic brain circuit development through synaptotropic stabilization of dynamic processes.
Neuron. Oct, 2009 | Pubmed ID: 19874791
During embryogenesis, brain neurons receiving the same sensory input may undergo potentiation or depression. While the origin of variable plasticity in vivo is unknown, it plays a key role in shaping dynamic neural circuit refinement. Here, we investigate effects of natural visual stimuli on neuronal firing within the intact, awake, developing brain using calcium imaging of 100 s of central neurons in the Xenopus retinotectal system. We find that specific patterns of visual stimuli shift population responses toward either potentiation or depression in an N-methyl-D-aspartate receptor (NMDA-R)-dependent manner. In agreement with Bienenstock-Cooper-Munro metaplasticity, our results show that functional potentiation or depression can be predicted by individual neurons' specific receptive field properties and historic firing rates. Interestingly, this activity-dependent metaplasticity is itself NMDA-R dependent. Furthermore, network analysis reveals increased correlated firing of neurons that undergo potentiation. These findings implicate metaplasticity as a natural property regulating experience-dependent refinement of nascent embryonic brain circuits.
In Vivo Single-cell Excitability Probing of Neuronal Ensembles in the Intact and Awake Developing Xenopus Brain
Nature Protocols. 2010 | Pubmed ID: 20379139
Sensory experience can elicit long-lasting plasticity of both single neurons and ensemble neural circuit response properties during embryonic development. To investigate their relationship, one must image functional responses of large neuronal populations simultaneously with single-cell resolution. In this protocol, we describe a noninvasive approach to assay functional plasticity of individual neurons and neuronal populations in vivo using targeted infusion of calcium-sensitive dyes, two-photon microscopy and synchronized visual stimuli presentations. This technique allows visualization of approximately 200 neurons while probing visual responses in the optic tectum of awake, immobilized Xenopus laevis tadpoles. The protocol includes visual training paradigms that elicit long-lasting potentiation or depression of functional responses, allowing investigations of population and single-neuron plasticity induced by natural sensory stimuli in the awake, intact, developing brain. Setup time for this protocol, including dye injection and chamber preparation, is approximately 2 h. Excitability probing experiments can then be performed for at least 3 h.
Neurexin-neuroligin Cell Adhesion Complexes Contribute to Synaptotropic Dendritogenesis Via Growth Stabilization Mechanisms in Vivo
Neuron. Sep, 2010 | Pubmed ID: 20869594
Cell adhesion molecules are well characterized for mediating synapse initiation, specification, differentiation, and maturation, yet their contribution to directing dendritic arborization during early brain circuit formation remains unclear. Using two-photon time-lapse imaging of growing neurons within intact and awake embryonic Xenopus brain, we examine roles of β-neurexin (NRX) and neuroligin-1 (NLG1) in dendritic arbor development. Using methods of dynamic morphometrics for comprehensive 3D quantification of rapid dendritogenesis, we find initial trans-synaptic NRX-NLG1 adhesions confer transient morphologic stabilization independent of NMDA receptor activity, whereas persistent stabilization requires NMDA receptor-dependent synapse maturation. Disrupting NRX-NLG1 function destabilizes filopodia while reducing synaptic density and AMPA receptor mEPSC frequency. Altered dynamic growth culminates in reduced dendritic arbor complexity as neurons mature over days. These results expand the synaptotropic model of dendritogenesis to incorporate cell adhesion molecule-mediated morphological stabilization necessary for directing normal dendritic arborization, providing a potential morphological substrate for developmental cognitive impairment associated with cell adhesion molecule mutations.
Dynamic Morphometrics Reveals Contributions of Dendritic Growth Cones and Filopodia to Dendritogenesis in the Intact and Awake Embryonic Brain
Developmental Neurobiology. Jul, 2011 | Pubmed ID: 21793227
Using in vivo rapid and long-interval two-photon time-lapse imaging of brain neuronal growth within the intact and unanesthetized Xenopus laevis tadpole, we characterize dynamic dendritic growth behaviors of filopodia, branches, and dendritic growth cones (DGCs), and analyze their contribution to persistent arbor morphology. The maturational progression of dynamic dendritogenesis was captured by short-term, 5 min interval, imaging for 1h every day for 5 days, and the contribution of short-term growth to persistent structure was captured by imaging at 5 min intervals for 5h, and at 2h intervals for 10h during the height of arbor growth. We find that filopodia and branch stability increases with neuronal maturation, and while the vast majority of dendritic filopodia rapidly retract, 3-7% of interstitial filopodia transition into persistent branches with lifetimes greater than 90 min. Here, we provide the first characterization of DGC dynamics, including morphology and behavior, in the intact and awake developing vertebrate brain. We find that DGCs occur on all growing branches indicating an essential role in branch elongation, and that DGC morphology correlates with dendritic branch growth behavior and varies with maturation. These results demonstrate that dendritogenesis involves a remarkable amount of continuous remodeling, with distinct roles for filopodia and DGCs across neuronal maturation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2011.
Cold Spring Harbor Protocols. Sep, 2011 | Pubmed ID: 21880812
Single-cell electroporation (SCE) is a versatile technique for delivering electrically charged macromolecules, including DNA, RNA, synthetic oligonucleotides, peptides, dyes, and drugs, to individual cells within intact tissues. Here, we describe methods for SCE of single tectal neurons within the albino Xenopus laevis tadpole for delivery of plasmid DNA-expressing protein fluorophores or fluorescent dye. Individual neurons labeled by this technique can then be imaged in three dimensions (3D) within the intact and living brain using in vivo two-photon microscopy for studies of morphology and growth. The SCE protocol is relatively simple and requires minimal and common laboratory equipment, including a fluorescent stereomicroscope, micropipette puller, and electrical stimulator. Once equipment is set up, learning to label cells with fluorescent dyes is straightforward and usually quickly achieved, because direct visualization with fluorescent microscopy offers immediate feedback of success. The main challenges are positioning the pipette tip in a cell body layer and optimizing pipette tip shape and stimulation parameters. Once fluorescent dye loading has been achieved, transfecting neurons with DNA should follow by using the same pipette tip parameters, but extending the stimulation parameters, because plasmid DNA is larger than dye and requires formation of larger pores. Detectable expression of protein fluorophores from transfected DNA typically takes 6-12 h. SCE for dye loading or DNA transfection of tadpole tectal neurons is highly efficient and can be learned in 1 or 2 d by novice laboratory personnel.
Cold Spring Harbor Protocols. Sep, 2011 | Pubmed ID: 21880813
Single-cell electroporation (SCE) is a versatile technique for delivering electrically charged macromolecules including DNA, RNA, synthetic oligonucleotides, peptides, dyes, and drugs to individual cells within intact tissues. Here, we describe methods for in vivo-targeted electroporation of single tectal neurons within the albino Xenopus laevis tadpole. Focal electroporation is achieved using a pipette electrode filled with a solution of the delivery molecules and with a tip diameter much smaller than the width of the target cell. The small tip allows for localization of an electric field, which restricts pore formation to only the individual cell in direct contact with the tip. Thus, the small tip permits focal delivery of the charged molecules within the pipette into individual cells. Factors affecting the efficiency of SCE, as well as various applications of this technique, are discussed. Particular focus is directed toward combining SCE with in vivo two-photon microscopy for three-dimensional (3D) imaging of neuron growth and cell-autonomous effects of altered protein function.
Functional Clustering Drives Encoding Improvement in a Developing Brain Network During Awake Visual Learning
PLoS Biology. Jan, 2012 | Pubmed ID: 22253571
Sensory experience drives dramatic structural and functional plasticity in developing neurons. However, for single-neuron plasticity to optimally improve whole-network encoding of sensory information, changes must be coordinated between neurons to ensure a full range of stimuli is efficiently represented. Using two-photon calcium imaging to monitor evoked activity in over 100 neurons simultaneously, we investigate network-level changes in the developing Xenopus laevis tectum during visual training with motion stimuli. Training causes stimulus-specific changes in neuronal responses and interactions, resulting in improved population encoding. This plasticity is spatially structured, increasing tuning curve similarity and interactions among nearby neurons, and decreasing interactions among distant neurons. Training does not improve encoding by single clusters of similarly responding neurons, but improves encoding across clusters, indicating coordinated plasticity across the network. NMDA receptor blockade prevents coordinated plasticity, reduces clustering, and abolishes whole-network encoding improvement. We conclude that NMDA receptors support experience-dependent network self-organization, allowing efficient population coding of a diverse range of stimuli.