Voltage Dependence of the Calcium Signal

<div><p>(A) Acutely isolated DUM neurons are maintained in calcium-free saline containing TTX and TEA, and clamped to a holding potential of −60 mV followed by command potentials of −50, −40, −30, and 0 mV. The amplitudes of the resulting intracellular calcium signals change as a function of the command potential.</p> <p>(B) Percent change (means and standard deviations from three neurons) in intracellular calcium levels (<i>y</i>-axis) plotted as a function of command voltage (<i>x</i>-axis).</p></div

Voltage-Induced Intracellular Calcium Signals Depend on G-protein Activation

<div><p>Acutely isolated neurons are loaded with GTPγS (100 μM), and a voltage step from −90 mV to 0 mV is repeated 30 times in calcium-containing saline to minimize availability of native G-protein.</p> <p>(A) In calcium-containing saline depolarization causes calcium inward current accompanied by an intracellular calcium signal.</p> <p>(B) Exposing the GTPγS-loaded cell for 3 min to zero-calcium saline causes a complete loss of membrane currents and the calcium signal.</p> <p>(C) Switching back to calcium-containing saline for 3 min restores the original current and the calcium signal.</p></div

Voltage-Induced Intracellular Calcium Signals That Occur in the Absence of Extracellular Calcium Do Not Depend on RYRs

<div><p>(A) In dantrolene-loaded, acutely isolated DUM neurons in TEA- and TTX-containing saline, a voltage step from −90 mV holding to 0 mV test potential causes a calcium inward and a calcium-activated potassium outward current accompanied by an intracellular calcium signal.</p> <p>(B) After 3 min in calcium-free saline, a slow elevation in intracellular calcium occurs in response to the same voltage step as in (A), although RYRs are blocked by intracellular application of dantrolene (100 nM).</p> <p>(C) Switching back to calcium-containing extracellular saline restores membrane currents and intracellular calcium signals as observed in (A).</p></div

Voltage-Induced Intracellular Calcium Signals Depend on IP3R and PLC Activation

<div><p>(A) (i) In heparin-loaded (500 nM), acutely isolated DUM neurons in TEA- and TTX-containing saline, a voltage step from −90 mV to 0 mV causes a calcium inward and a calcium-activated potassium outward current accompanied by an intracellular calcium signal. (ii) Exposing the heparin-loaded cell for 3 min to zero-calcium saline causes a complete loss of all membrane currents and the calcium signal. (iii) Switching back to calcium-containing saline for 3 min restores the original currents and the calcium signal.</p> <p>(B) Same representative experiment as in (A), but the neuron is loaded with the PLC blocker U73122. Pharmacological block of IP3Rs and of PLC both abolish voltage-induced calcium signals in the absence of calcium inward current.</p></div

Bursts of Action Potentials Induce Intracellular Calcium Elevations in the Absence of Calcium Influx

<div><p>(A) In normal extracellular saline, a burst of action potentials (lower trace) induced by current injection of 0.5 nA is accompanied by a large elevation in intracellular calcium (upper trace).</p> <p>(B) Action potential shape and frequency are altered after 3 min in zero-calcium extracellular saline, but bursts are still accompanied by an elevation in intracellular calcium, although the calcium signal is slower and of lower amplitude as compared to (A).</p> <p>(C) Switching back to normal saline for 3 min restores signals as observed in (A).</p></div

Heterologous expression of MECP2 causes dendritic defects in Drosophila motoneurons.

<p>(A) Schematic drawing of location of MN1-5 in the <i>Drosophila</i> nervous system and their innervation of the dorsal longitudinal flight muscle (DLM) fibers. MN5 is depicted in green, and MN5 dendritic projection in the dorsal mesothoracic neuromere is demarked by a dotted green line (B) Overview of MN5 structure in a representative control animal. A geometric reconstruction of MN5 dendritic structure is superimposed on the projection view. (C) Overview of MN5 dendritic structure following targeted expression of full-length human MECP2. Geometric reconstruction superimposed on projection image of MN5. (D) Double staining of MN5 (green) and anti-MECP2 immunolabeling (magenta) shows MECP2 localization to MN5 nucleus (white) and some other nuclei of neurons with <i>C380-GAL4/UAS-MECP2</i> expression. (E) Same as (D) but anti-MECP2 immunolabeling only to show that no MECP2 protein was detected through MN5 processes. Inset depicts anti-MECP2 immunostaining in a representative single optical section through MN5 soma and primary neurite. MN5 outline is demarked by white line, and white arrow demarks MN5 nucleus. MECP2 protein could not be detected in any part of MN5 except the nucleus. (F) Quantitative metric measures of dendritic structure in MN5 from controls (gray bars) and in MN5 with MECP2 expression (magenta). Values are normalized to mean control values (dotted line). Arrows indicate statistical significant differences (Students T-test, p≤0.01). Error bars indicate standard deviation. (G and H) Mean number of dendritic branches (G) and mean dendritic radius in controls (gray squares) and following MECP2 expression (magenta circles) over branch order. Error bars indicate standard deviation. Axis in (H) is clipped at branch order 41 because only few dendrites of higher branch orders exist (see G).</p

MECP2-induced motoneuron defects result in specific motor behavioral deficiencies.

<p>(A) Representative extracellular recording of MN5 firing patterns during flight in a control (upper trace) and in fly expressing MECP2 in a subset of neurons, including MN5 (<i>C380-GAL4, UAS-mcd8-GFP; Cha-GAL80/UAS-MECP2</i>; lower trace). Traces above the recordings resemble spike counts. Black arrow demarks start of flight, and black asterisk demarks time point of flight stop in <i>MECP2</i> fly. (B) Average in-flight wing beat frequencies of control (white bar) and <i>MECP2</i> flies (grey bar). Error bars represent standard error. (C) Percentage of control (white bar) and <i>MECP2</i> flies (grey bar) engaging into flight upon a wind stimulus. (D) Numbers of flight bouts performed by control (white bar) and by <i>MECP2</i> flies (grey bar) in response to re-occuring wind stimuli (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031835#s4" target="_blank">methods</a>). Data are presented as median and quartiles. Error bars represent minimum and maximum values. (E and F) Total duration of all consecutive flight bouts (E) and average duration of individual flight bouts (F) in control (white bar) and in <i>MECP2</i> flies (grey bar). Data are presented as median and quartiles. Error bars represent minimum and maximum values. ** demarks p<0.01, Mann and Whitney U-test.</p

Heterologous expression of MECP2 does not affect electrophysiological properties of Drosophila motoneurons.

<p>(A) Comparison of typical MN5 firing responses to 300 pA of somatic current injection in a representative control animal (left trace) and following targeted expression of full-length human <i>MECP2</i> under the control of <i>C380-GAL4</i> (right trace). (B) Voltage dependent potassium currents in MN5 as induced by command voltage steps from a holding potential of −90 mV to 20 mV in increments of 10 mV and with cadmium and TTX in the bath solution to block sodium and calcium inward currents. Traces of control animals (left) and following targeted expression of MECP2 (right) reveal qualitatively similar transient A-type current and sustained delayed rectifier like potassium outward currents. (C) Current/Voltage relationships for A-type (left) and sustained delayed rectifier (right) potassium currents are quantitatively similar in controls (gray diamonds) and following expression of MECP2 (magenta diamonds). Error bars represent standard deviations.</p

Heterologous expression of MECP2 with MBD defects does not affect Drosophila motoneuron dendrite development.

<p>(A) Schematic drawings of full-length human MECP2 (magenta) with intact menthyl-CpG-binding domain (MBD) and intact transcriptional repression domain (TRD). The R106W mutation (red) carries a point mutation (see x) that causes a non-functional MBD. The Δ166 mutation (orange) has a truncated MBD and N-terminus. TRD is intact in all three alleles. Nuclear localization sequences (nls) have been reported in the inter-domain region at residues 174 and 190 and also in the TRD domain between residues 255 and 271, and are intact in all three alleles. (B, D, F) Intracellular labeling of MN5 following R106W expression under the control of <i>C380-GAL4</i> (B) and subsequent geometric reconstruction (F) do not reveal obvious dendrite defects in MN5. (D) MECP2 immunolabeling following targeted R106W expression indicates strict nuclear localization (see also white arrow in B). (C, E, G) Intracellular labeling of MN5 following Δ166 expression under the control of <i>C380-GAL4</i> (C) and subsequent geometric reconstruction (G) do not reveal obvious dendrite defects in MN5. (E) MECP2 immunolabeling following targeted R106W expression indicates strict nuclear localization (see also white arrow in C). (H) Averages of total dendritic length in controls (gray bars), and following expression of full-length MECP2 (magenta), R106W (red), and Δ166 (orange). (I) Average numbers of dendritic branches in controls (gray bars), and following expression of full-length MECP2 (magenta), R106W (red), and Δ166 (orange). In (H) and (I) error bars indicate standard deviation, asterisks demark statistical significance at p≤0.05 (ANOVA with Newman Keuls posthoc test).</p

MECP2-caused dendrite defects are partially ameliorated by a reduction in osa dose.

<p>(A) Projection view of a representative intracellular staining of MN5 in a control animal. (B) Projection view of a representative intracellular staining of MN5 in an <i>osa</i> heterozygous mutant background does not reveal obvious differences in dendritic structure as compared to control. (C) Projection view of a representative intracellular staining of MN5 with heterologous expression of full-length MECP2 in an <i>osa</i> heterozygous mutant background does not show similar dendritic defects as compared to MECP2 expression in a wildtype <i>osa</i> background (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031835#pone-0031835-g001" target="_blank">figures 1C, D</a>). (D) MECP2 immunopositive label (magenta) was restricted to the nucleus (see also white arrow in C). (E) Quantitative metric measures of dendritic structure in MN5 from controls (dark gray bars), MN5 in an <i>osa</i> heterozygous mutant background (light gray bars), from MN5 with MECP2 expression (magenta), and from MN5 with MECP2 expression in an <i>osa</i> heterozygous mutant background. Values are normalized to mean control values. Arrows indicate statistical significance (ANOVA with Newman Keuls posthoc test, p≤0.01). Error bars indicate standard deviation. (F) Mean number of dendritic branches over branch order in controls (gray squares), following MECP2 expression (magenta circles), and following MECP2 expression in an <i>osa</i> heterozygous mutant background (blue). Error bars indicate standard deviation.</p