Targeted electroporation in Xenopus tadpoles in vivo – from single cells to the entire brain

doi:10.1046/j.1432-0436.2002.700404.x

Differentiation

Volume 70, Issues 4–5, June 2002, Pages 148-154

https://doi.org/10.1046/j.1432-0436.2002.700404.x Get rights and content

Abstract

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.

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Long-term potentiation (LTP) of synaptic transmission is considered to be a cellular counterpart of learning and memory. Activation of postsynaptic NMDA type glutamate receptor (NMDA-R) induces trafficking of AMPA type glutamate receptors (AMPA-R) and other proteins to the synapse in sequential fashion. At the same time, the dendritic spine expands for long-term and modulation of actin underlies this (structural LTP or sLTP). How these changes persist despite constant diffusion and turnover of the component proteins have been the central focus of the current LTP research. Signaling triggered by Ca²⁺-influx via NMDA-R triggers kinase including Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). CaMKII can sustain longer-term biochemical signaling by forming a reciprocally-activating kinase-effector complex with its substrate proteins including Tiam1, thereby regulating persistence of the downstream signaling. Furthermore, activated CaMKII can condense at the synapse through the mechanism of liquid-liquid phase separation (LLPS). This increases the binding capacity at the synapse, thereby contributing to the maintenance of enlarged protein complexes. It may also serve as the synapse tag, which captures newly synthesized proteins.
Proteomic screen reveals diverse protein transport between connected neurons in the visual system
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Intercellular transfer of toxic proteins between neurons is thought to contribute to neurodegenerative disease, but whether direct interneuronal protein transfer occurs in the healthy brain is not clear. To assess the prevalence and identity of transferred proteins and the cellular specificity of transfer, we biotinylated retinal ganglion cell proteins in vivo and examined biotinylated proteins transported through the rodent visual circuit using microscopy, biochemistry, and mass spectrometry. Electron microscopy demonstrated preferential transfer of biotinylated proteins from retinogeniculate inputs to excitatory lateral geniculate nucleus (LGN) neurons compared with GABAergic neurons. An unbiased mass spectrometry-based screen identified ∼200 transneuronally transported proteins (TNTPs) isolated from the visual cortex. The majority of TNTPs are present in neuronal exosomes, and virally expressed TNTPs, including tau and β-synuclein, were detected in isolated exosomes and postsynaptic neurons. Our data demonstrate transfer of diverse endogenous proteins between neurons in the healthy intact brain and suggest that TNTP transport may be mediated by exosomes.
Early Developmental Exposure to Fluoxetine and Citalopram Results in Different Neurodevelopmental Outcomes
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Although selective serotonin reuptake inhibitors are commonly prescribed for prenatal depression, there exists controversy over adverse effects of SSRI use on fetal development. Few studies have adequately isolated outcomes due to SSRI exposure and those due to maternal psychiatric conditions. Here, we directly investigated outcomes of exposure to widely-used SSRIs Fluoxetine and Citalopram on the developing nervous system of Xenopus laevis tadpoles, using an integrative experimental approach. We exposed tadpoles to low doses of Citalopram and Fluoxetine during a critical developmental period and found that different experimental groups displayed opposing behavioral effects. While both groups showed reduced schooling behavior, the Fluoxetine group showed increased seizure susceptibility and reduced startle habituation. In contrast, Citalopram treated tadpoles had decreased seizure susceptibility and increased habituation. Both groups had abnormal dendritic morphology in the optic tectum, a brain area important for behaviors tested. Whole-cell electrophysiological recordings of tectal neurons showed no differences in synaptic function; however, tectal cells from Fluoxetine-treated tadpoles had decreased voltage gated K+ currents while cells in the Citalopram group had increased K+ currents. Both behavioral and electrophysiological findings indicate that cells and circuits in the Fluoxetine treated optic tecta are hyperexcitable, while the Citalopram group exhibits decreased excitability. Taken together, these results show that early developmental exposure to SSRIs is sufficient to induce neurodevelopmental effects, however these effects can be complex and vary depending on the SSRI. This may explain some discrepancies across human studies, and further underscores the importance of serotonergic signaling for the developing nervous system.
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2020, Synapse Development and Maturation: Comprehensive Developmental Neuroscience
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Differentiation

Abstract

References (26)

A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube

Cell

Electroporation-mediated gene transfer in free-swimming embryonic Xenopus laevis

FEBS Lett

Single-cell electroporation for gene transfer in vivo

Neuron

Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo

Biochem Biophys Res Commun

LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons

Cell

Sparking new frontiers: using in vivo electroporation for genetic manipulations

Dev Biol

Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems

Proc Natl Acad Sci USA

Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate

Development

Localized electroporation: a method for targeting expression of genes in avian embryos

Biotechniques

Transient expression of RSVCAT in transgenic zebrafish made by electroporation

Mol Mar Biol Biotechnol

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo

Nat Neurosci

An impulse to the brain-using in vivo electroporation

Nat Neurosci

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development

Development

Cited by (139)

Quantifying neuronal structural changes over time using dynamic morphometrics

Molecular mechanism of hippocampal long-term potentiation – Towards multiscale understanding of learning and memory

Proteomic screen reveals diverse protein transport between connected neurons in the visual system

Early Developmental Exposure to Fluoxetine and Citalopram Results in Different Neurodevelopmental Outcomes

Lhx2/9 and Etv1 Transcription Factors have Complementary roles in Regulating the Expression of Guidance Genes slit1 and sema3a

In vivo imaging of synaptogenesis

REVIEW ARTICLETargeted electroporation in Xenopus tadpoles in vivo – from single cells to the entire brain

Abstract

Cell

FEBS Lett

Neuron

Biochem Biophys Res Commun

Cell

Dev Biol

Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems

Proc Natl Acad Sci USA

Engrailed defines the position of dorsal di-mesencephalic boundary by repressing diencephalic fate

Development

Localized electroporation: a method for targeting expression of genes in avian embryos

Biotechniques

Transient expression of RSVCAT in transgenic zebrafish made by electroporation

Mol Mar Biol Biotechnol

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo

Nat Neurosci

An impulse to the brain-using in vivo electroporation

Nat Neurosci

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development

Development

REVIEW ARTICLE
Targeted electroporation in Xenopus tadpoles in vivo – from single cells to the entire brain