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

<p>Abstract</p> <p>Background</p> <p>In 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.</p> <p>Results</p> <p>Both 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.</p> <p>Conclusions</p> <p>Taken 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.</p

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

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca2+-dependent K+ channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K+ currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na+ channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons

Effect of GluA2 RNAi on the dendritic morphology of E6 chicken lumbar motoneurons.

<p><b><i>A–D</i></b><i>)</i> Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi construct has no effect on the dendritic complexity of E6 motoneurons including dendritic arbor/cell (<b><i>A</i></b>), number of primary dendrites (<b><i>B</i></b>), number of branches (<i>C</i>), and number of ends (<b><i>D</i></b>). <b><i>E–F</i></b>) Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi construct has no effect on the cell body perimeter (<b><i>E</i></b>) but causes a significant reduction in soma area (<b><i>F</i></b>). <b><i>G</i></b>) Comparison of E6 dendritic morphology as a function of dendritic order in control embryos or embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi construct.</p

Changes in the Ca²⁺-permeability of AMPA receptors evoked by infection of chicken embryos with an RCASBP(B)-RFP-GluA2 RNAi contruct.

<p><b><i>A</i></b><i>)</i> Typical whole-cell currents in E11 motoneurons evoked by kainate application in 10 mM Ca²⁺/Na⁺-free external solution, at four different holding potentials ranging from −80 to +40 mV. Notice that in motoneurons infected with the RCASB(B)-RFP-GluA2 RNAi viral construct there is a significant increase in the inward Ca²⁺ currents evoked by kainate (empty arrow) at a holding potential of −80 mV. <b>B</b>) Plot of the reversal potential of kainate-evoked currents obtained in E11 motoneurons isolated from non-infected embryos (control) or embryos infected with RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi viral particles. <b>C</b>) Plot showing the relative permeability of Ca²⁺ to that of Cs⁺ (P_Ca2+/P_Cs+) in E11 motoneurons isolated from non-infected embryos or embryos infected with RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi viral particles. The ratio of P_Ca2+/P_Cs+ was calculated according to the extended GHK constant field equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049879#s2" target="_blank">Methods</a>). Infection of chicken embryos with an RCASBP(B)-RFP-GluA2 RNAi construct results in a significant increase in the Ca²⁺ permeability of kainate-activated channels compared with control, RCASBP(B)-RFP or RCASBP(B)-RFP-scrambled RNAi-infected embryos (*p≤0.05 vs. control, **p≤0.05 vs. RCASBP(B)-RFP, ***p≤0.05 vs. RCASBP(B)-RFP-scrambled RNAi).</p

Effect of GluA2 RNAi on the dendritic morphology of E11 chicken lumbar motoneurons.

<p><i>A</i><b><i>–D</i></b><i>)</i> Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi construct causes a significant decrease in the dendritic complexity of E11 motoneurons. There is a significant reduction in the overall length of the dendritic arbor/cell (<b><i>A</i></b>), number of primary dendrites (<b><i>B</i></b>), number of branch points (<b><i>C</i></b>), and number of ends (<b><i>D</i></b>). <b><i>E–F</i></b><i>)</i> Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi construct has no effect on the cell body morphology of E11 motoneurons including cell body perimeter (<b><i>E</i></b>) and cell body area (<b><i>F</i></b>). <b><i>G</i></b>) Comparison of E11 dendritic morphology as a function of dendritic order in control embryos and RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi–infected embryos. Downregulation of GluA2 expression causes a significant reduction of the number of proximal dendrites (2^nd and 3^rd dendritic orders) and no effect on higher order dendrites (4^th–8^th dendritic orders) when compared to control or RCASBP(B)-RFP-scrambled RNAi-infected embryos. * p<0.05 vs. control; ** p<0.05 vs. RCASBP(B)-RFP-scrambled RNAi. ns denotes no significant differences between the groups as indicated by one-way ANOVA.</p

Effect of GluA2 RNAi on motoneurons survival.

<p><b><i>A</i></b><i>) Islet1/2</i>-staining in the lumbar spinal cord of E11 chicken embryos. Only <i>Islet1/2</i>-positive neurons in the motoneurons pool were counted (circled area). <i>Islet1/2</i>-positive interneurons located in dorsal and medial portions of the spinal cord were not included in our measurements. <b><i>B</i></b><i>)</i> Total number of <i>Islet1/2</i>-positive neurons in the lumbar spinal cord of E11 chicken embryos in control (non-infected) and in RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi–infected embryos. Infection of chicken embryos with the RCASBP(B)-RFP-GluA2 RNAi has no overall effect on motoneuron survival when compared with control or RCASBP(B)-RFP-scrambled RNAi-infected embryos.</p

Effect of GluA2 downregulation on the maturation of spontaneous network activity in the chicken spinal cord.

<p><b><i>A–C</i></b><i>)</i> Typical example of an episode of spontaneous activity generated by the spinal cord of control (non-infected, <b><i>A</i></b>), RCASBP(B)-RFP-scrambled RNAi (<b><i>B</i></b>) or RCASBP(B)-RFP-GluA2 RNAi (<b><i>C</i></b>)-infected embryos at E11. The overall shape of each episode remains the same under each treatment condition. <b><i>D–E</i></b>) Downregulation of GluA2 expression did not alter the episode duration <i>(D)</i> and inter-episode interval <i>(</i><b><i>E</i></b><i>)</i> in E11 spinal cords.</p

Typical Neurolucida drawing of DiI-labeled motoneurons, traced from control (non-infected) or RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi-infected chicken embryos.

<p>The dendritic morphology of the motoneurons was assessed at E6 (<i>A</i>) or E11 (<i>B</i>). There are little changes in the architecture of the dendritic tree of E6 motoneurons following infection with the RCASBP(B)-RFP-GluA2 RNAi vital construct when compared with control or RCASBP(B)-RFP-scrambled RNAi-infected embryos. However, there is a significant reduction in the complexity of the dendritic tree of E11 motoneurons following infection with the RCASBP(B)-RFP-GluA2 RNAi viral construct (the dendritic tree appears less complex and with fewer branches).</p

Effect of RCASBP(B)-RFP-GluA2 RNAi infection on chicken embryos.

<p><b><i>A–B</i></b><i>)</i> Expression of red fluorescence protein (RFP) transgene in the lumbar spinal cord of E6 (<b><i>A</i></b>) and E11 (<b><i>B</i></b>) chicken embryos following retroviral infection with an RCASBP(B)-RFP-GluA2 RNAi construct. Embryos infected with the RCASBP(B)-RFP-GluA2 RNAi construct show strong fluorescent labeling throughout the whole spinal cord cross section. cc = central canal, nc = notochord. <b><i>C</i></b><i>)</i> Averaged number of labeled neurons for the RCASBP(B) viral protein p27 gag as a percent of the total number of neuron labeled with the motoneuron marker <i>Islet1/2</i> in chicken embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP- GluA2 RNAi construct. Lumbar ventral neurons were isolated from E6, E8 or E11 chicken embryos and immunolabeled with p27 gag and <i>Islet1/2</i> in order to assess the extent of viral infection of spinal motoneurons. Notice that ≥60% of infected cells also tested positive for the motoneuron marker <i>Islet1/2</i> at all ages tested. <b><i>D</i></b><i>)</i> Representative example of Western Blot data collected from the E6 and E11 ventral spinal cords from control chicken embryos (non-infected) or embryos infected with an RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi constructs. The anti-GluA2 antibody detected a band with a relative molecular weight of ∼102 kD. To normalize for changes in protein loading in each well, membranes were reprobed for β-actin (∼42 kD). Infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct causes a significant reduction in GluA2 expression at E11 as determined by immunoblot analysis. <b><i>E</i></b><i>)</i> Expression of GluA2 protein as a function of β-actin in chicken ventral spinal cords. The age-dependent increase in GluA2 protein expression between E6 and E11 chicken spinal cords was reversed by infection of chicken embryos with an RCASBP(B)-RFP- GluA2 RNAi construct. In these experiments, RCASBP(B)-RFP-scrambled RNAi or RCASBP(B)-RFP-GluA2 RNAi viral particles were injected into the developing neural tube at E2 (approximately 36 hr after incubation). Embryos were allowed to develop up to E11 before tissue isolation and processing.</p