Rapid and efficient electroporation-based gene transfer into primary dissociated neurons

Dityateva, Galina; Hammond, Martin S. L.; Thiel, Corinna; Ruonala, Mika O.; Delling, Markus; Siebenkotten, Gregor; Nix, Michael; Dityatev, Alexander

doi:10.1016/s0165-0270(03)00202-4

Cited by 42 publications

(48 citation statements)

References 23 publications

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“…Chick tectal neurons from E7 embryos were prepared as described (30) with minor modifications. The tecta were dissected free of meningeal tissue, cut into small pieces, and incubated in cell dissociation buffer (Sigma) containing 0.002% Trypsin for 15 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

Porten

Seliger

Schneider

et al. 2010

Journal of Biological Chemistry

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Clustering or overexpression of the transmembrane form of the extracellular matrix proteoglycan agrin in neurons results in the formation of numerous highly motile filopodia-like processes extending from axons and dendrites. Here we show that similar processes can be induced by overexpression of transmembrane-agrin in several non-neuronal cell lines. Mapping of the process-inducing activity in neurons and non-neuronal cells demonstrates that the cytoplasmic part of transmembrane agrin is dispensable and that the extracellular region is necessary for process formation. Site-directed mutagenesis reveals an essential role for the loop between ␤-sheets 3 and 4 within the Kazal subdomain of the seventh follistatin-like domain of TM-agrin. An aspartic acid residue within this loop is critical for process formation. The seventh follistatin-like domain could be functionally replaced by the first and sixth but not by the eighth follistatin-like domain, demonstrating a functional redundancy among some follistatin-like domains of agrin. Moreover, a critical distance of the seventh follistatin-like domain to the plasma membrane appears to be required for process formation. These results demonstrate that different regions within the agrin protein are responsible for synapse formation at the neuromuscular junction and for process formation in central nervous system neurons and suggest a role for agrin's follistatin-like domains in the developing central nervous system.Agrin is a proteoglycan with a molecular mass of Ͼ500 kDa that is expressed in many tissues (1, 2). The function of agrin is best characterized in skeletal muscle where it is a key organizer during formation, maintenance, and regeneration of the neuromuscular junction (2-4). Accordingly, mice with an inactivation of the agrn gene die at birth due to non-functional neuromuscular junctions and consequent respiratory failure (5).Little is known about the role of agrin in tissues other than skeletal muscle, in particular in the central nervous system (for review see Refs. 1, 2, 6). Although neurons from mice with a targeted deletion of the agrn gene form synaptic specializations in vitro and in vivo (7,8), the acute suppression of agrin expression or function by antisense oligonucleotides or antibodies influences the formation and function of interneuronal synapses (9, 10). Likewise, brains of agrin-deficient mice, whose perinatal death was prevented by the re-expression of agrin in motor neurons, have a severely reduced number of pre-and postsynaptic specializations as well as functional deficits at excitatory synapses in the CNS 3 (11). Although these data are consistent with a role of agrin during CNS synaptogenesis, the precise function of agrin during CNS development remains unclear.Agrin has been cloned from several species, and the sequences are highly homologous. The agrin cDNAs predict a number of domains with similarity to other extracellular matrix proteins, including four EGF-like repeats and three domains with similarity to globular domain of the lamini...

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Section: Methodsmentioning

confidence: 99%

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

Porten

Seliger

Schneider

et al. 2010

Journal of Biological Chemistry

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“…Cultures were maintained at 37°C in a humidified incubator gassed with 5% CO 2 . Cells were transiently transfected either by electroporation with Nucleofector I electroporator (Amaxa, Cologne, Germany) (Dityateva et al, 2003) or by using Lipofectamine2000 reagent (Invitrogen) according to the protocol of the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

5-HT₇Receptor Is Coupled to Gα Subunits of Heterotrimeric G12-Protein to Regulate Gene Transcription and Neuronal Morphology

Kvachnina¹,

Liu²,

Dityatev³

et al. 2005

J. Neurosci.

180

220

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The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT 4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT 7 receptor. Expression of 5-HT 7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT 7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT 7 receptors significantly increased neurite length, whereas stimulation of 5-HT 4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT 7 R/G12 and 5-HT 4 R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

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“…Primary murine hippocampal neuronal cultures from 1-to 3-day-old C57BL6/J mice pups were prepared as described previously (Dityateva et al, 2003). On day in vitro 8 (DIV 8), primary hippocampal neurons were transfected with a control pSUPER-Mamm-X/scrambled shRNA plasmid (2 mg per well) or were cotransfected with two plasmids encoding shRNA to silence the expression of 5HT 7 receptor.…”

Section: Whole-cell Recordings From Hippocampal Culturesmentioning

confidence: 99%

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking

Renner

Zeug

Woehler

et al. 2012

Journal of Cell Science

Self Cite

166

157

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SummarySerotonin receptors 5-HT 1A and 5-HT 7 are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT 1A and 5-HT 7 receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT 1A or 5-HT 7 receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT 7 -5-HT 7 homodimers, followed by the 5-HT 7 -5-HT 1A heterodimers and 5-HT 1A -5-HT 1A homodimers. Functionally, heterodimerization decreases 5-HT 1A -receptor-mediated activation of G i protein without affecting 5-HT 7 -receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT 1A receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT 1A receptor internalization and also enhances the ability of the 5-HT 1A receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT 7 receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT 1A -5-HT 7 heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

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Rapid and efficient electroporation-based gene transfer into primary dissociated neurons

Cited by 42 publications

References 23 publications

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

5-HT₇Receptor Is Coupled to Gα Subunits of Heterotrimeric G12-Protein to Regulate Gene Transcription and Neuronal Morphology

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking

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Rapid and efficient electroporation-based gene transfer into primary dissociated neurons

Cited by 42 publications

References 23 publications

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

5-HT7Receptor Is Coupled to Gα Subunits of Heterotrimeric G12-Protein to Regulate Gene Transcription and Neuronal Morphology

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking

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5-HT₇Receptor Is Coupled to Gα Subunits of Heterotrimeric G12-Protein to Regulate Gene Transcription and Neuronal Morphology