Inhibition of Electrical Activity by Retroviral Infection with Kir2.1 Transgenes Disrupts Electrical Differentiation of Motoneurons

BackgroundIn the adult nervous system, GABA acts as a major inhibitory neurotransmitter; however, at early stages of neurodevelopment, GABA receptor activation leads to membrane depolarization and accumulation of [Ca2+]i. The role of excitatory GABAergic neurotransmission in the development of the nervous system is not fully understood. In this study, we investigated the role of excitatory GABA-driven activity in regulating the dendritic morphology and network function in the developing chicken spinal cord.ResultsBoth bicuculline, a GABA receptor antagonist, and muscimol, a GABA agonist, inhibit the generation of spontaneous network activity in the isolated spinal cord at E8 or E10, indicating that altering GABA receptor activation disrupts the generation of spontaneous network activity in the chicken spinal cord. Treatment of chicken embryos with bicuculline or muscimol between E5 and E8 (or between E8 and E10), inhibits the dendritic outgrowth of motoneurons when compared to vehicle-treated embryos. The inhibitory effect of bicuculline or muscimol on the dendritic morphology of motoneurons was likely due to inhibition of GABA-driven network activity since a similar effect was also observed following reduction of network activity by Kir2.1 overexpression in the spinal cord. The inhibitory effect of bicuculline or muscimol was not caused by an adverse effect on cell survival. Surprisingly, chronic treatment of chicken embryos with bicuculline or muscimol has no effect on the shape and duration of the episodes of spontaneous activity, suggesting that maturation of network activity is not altered by disruption of the dendritic outgrowth of motoneurons.ConclusionsTaken together, these findings indicate that excitatory GABA receptor activation regulates the maturation of dendritic morphology in the developing spinal cord by an activity-dependent mechanism. However, inhibition of dendritic outgrowth caused by disruption of GABA-driven activity does not alter the maturation of spontaneous electrical activity generated by spinal cord networks, suggesting that compensatory mechanisms can reverse any adverse effect of dendritic morphology on network function.

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“…[2]. Briefly, pathogen-free eggs were obtained from SPAFAS (Charles River Laboratories, Wilmington, MA, USA) and incubated at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…[2]. Briefly, lumbar segments L1 to L6 were removed at E10 and fixed in Zamboni's fixative (4% paraformaldehyde plus 15% picric acid in 0.1 M PBS) at 4°C overnight, washed three times in PBS, and equilibrated in 30% sucrose/PBS overnight.…”

Section: Methodsmentioning

confidence: 99%

Pharmacological manipulation of GABA-driven activity in ovo disrupts the development of dendritic morphology but not the maturation of spinal cord network activity

2010

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“…Viral infection of chicken embryos was performed as previously described by Yoon et al [15]. Pathogen-free eggs were obtained from SPAFAS (Charles River Laboratories, Wilmington, MA) and incubated at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Downregulation of GluA2 AMPA Receptor Subunits Reduces the Dendritic Arborization of Developing Spinal Motoneurons

Yoon

White

et al. 2012

PLoS ONE

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AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca2+ ions. Although Ca2+-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca2+-permeable AMPA receptors at E6 but the Ca2+ permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca2+ regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca2+ permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development.

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“…We transfected RGCs with a cDNA plasmid encoding a human inward rectifying potassium channel, hKir2.1, which causes membrane hyperpolarization and has been used extensively to quench the excitability of neurons (Hua et al, 2005;Yoon et al, 2008). In particular, Kir2.1 was used to demonstrate the importance of neural activity in Xenopus spinal cord neuron differentiation (Borodinsky et al, 2004;Marek et al, 2010), and zebrafish RGC axon arbour growth (Hua et al, 2005).…”

Section: 2mentioning

confidence: 99%

Neural activity and branching of embryonic retinal ganglion cell dendrites

Hocking

Pollock

Johnston

et al. 2012

Mechanisms of Development

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The shape of a neuron's dendritic arbor is critical for its function as it determines the number of inputs the neuron can receive and how those inputs are processed. During development, a neuron initiates primary dendrites that branch to form a simple arbor. Subsequently, growth occurs by a process that combines the extension and retraction of existing dendrites, and the addition of new branches. The loss and addition of the fine terminal branches of retinal ganglion cells (RGCs) is dependent on afferent inputs from its synaptic partners, the amacrine and bipolar cells. It is unknown, however, whether neural activity regulates the initiation of primary dendrites and their initial branching. To investigate this, Xenopus laevis RGCs developing in vivo were made to express either a delayed rectifier type voltage-gated potassium (KV) channel, Xenopus Kv1.1, or a human inward rectifying channel, Kir2.1, shown previously to modulate the electrical activity of Xenopus spinal cord neurons. Misexpression of either potassium channel increased the number of branch points and the total length of all the branches. As a result, the total dendritic arbor was bigger than for control green fluorescent protein-expressing RGCs and those ectopically expressing a highly related mutant non-functional Kv1.1 channel. Our data indicate that membrane excitability regulates the earliest differentiation of RGC dendritic arbors.

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Inhibition of Electrical Activity by Retroviral Infection with Kir2.1 Transgenes Disrupts Electrical Differentiation of Motoneurons

Cited by 14 publications

References 53 publications

Pharmacological manipulation of GABA-driven activity in ovo disrupts the development of dendritic morphology but not the maturation of spinal cord network activity

Pharmacological manipulation of GABA-driven activity in ovo disrupts the development of dendritic morphology but not the maturation of spinal cord network activity

Downregulation of GluA2 AMPA Receptor Subunits Reduces the Dendritic Arborization of Developing Spinal Motoneurons

Neural activity and branching of embryonic retinal ganglion cell dendrites

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