Complex analytic Néron models for arbitrary families of intermediate Jacobians

Given a family of intermediate Jacobians (for a polarizable variation of integral Hodge structure of odd weight) on a Zariski-open subset of a complex manifold, we construct an analytic space that naturally extends the family. Its two main properties are: (a) the horizontal and holomorphic sections are precisely the admissible normal functions without singularities; (b) the graph of any admissible normal function has an analytic closure inside our space. As a consequence, we obtain a new proof for the zero locus conjecture of M. Green and P. Griffiths. The construction uses filtered D-modules and M. Saito’s theory of mixed Hodge modules; it is functorial, and does not require normal crossing or unipotent monodromy assumption

Time course of the SR101-staining procedure.

<p>(<b>A–E′</b>) Images show 2-photon time-lapse recording of SR101-stainings and unstaining during washout in acute slices (40 µm, 21 images, 0.5 min⁻¹). <b>A–E</b> show the results from the ventrolateral medulla (VLM) and <b>A′–E′</b> from hippocampus. <b>A, A′:</b> Maximum intensity projections of the EGFP-fluorescence of the astrocytes. <b>B–E, B′–E′:</b> Images show maximum intensity projections of the SR101-labeling at 4 different time points in the hippocampus and in the VLM. Scale bars: 40 µm. <b>F–H:</b> Analysis of the time course of SR101-staining. Arrows underneath the traces represent the time points according to the images in A–E and A′–E′, respectively. Data (mean ± SEM) is derived from 3–5 cells per slice (6 slices in hippocampus and 5 in the VLM). <b>F:</b> Note that the SR101-staining of hippocampal astrocytes (red) is much stronger than the staining in the VLM astrocytes (green). <b>G:</b> In the VLM additionally EGFP-negative cells are stained while SR101 is applied (blue trace) but the fluorescence is disappearing during the washout. <b>H:</b> Normalized time course of the staining of astrocytes (red in the hippocampus; green in the VLM) and EGFP-negative cells in the VLM (blue).</p

Rhythmic inward currents in astrocytes of the pre-Bötzinger Complex (preBötC).

<p>(<b>A</b>) To identify astrocytes a CCD-image was taken and the astrocyte, identified by its (green) fluorescence in the center of the image was whole-cell recorded in voltage-clamp mode showing (<b>B</b>) respiratory-rhythmic inward currents that were partly obscured by the noise (V_hold = -70 mV; upper trace). The integrated preBötC-field potential (preBötC ∫), recorded in parallel, is shown in the lower trace. <b>(C)</b> Cycle triggered averaging of inward currents was performed, using preBötC-field potentials as triggers to allow the measurement of the amplitude of the respiratory rhythmic current (I_resp,A). (<b>D–F</b>) Input resistance of the astrocytes remains unchanged during astrocytic inward currents: Panels (<b>D</b>) and (<b>E</b>) show whole-cell recordings taken from a fluorescent preBötC astrocyte. (<b>D</b>) Current traces recorded in response to the voltage step protocol, show in the insert, identified this astrocyte as passive. (<b>E</b>) In the presence of bicuculline (20 µM), large amplitude preBötC field potentials were accompanied by large inward currents (asterisks) in the astrocytes (D). Hyperpolarizing voltage steps (−10 mV) were applied to the astrocyte to measure membrane input resistance (R_in), which did not change in association with inward current transients (<b>F</b>; n = 3).</p

Effect of probenecid on SR101-labeling.

<p><b><i>A:</i></b> Overlay of astroglial EGFP-fluorescence and SR101-fluorescence in the VLM after staining in CTRL conditions. <b>B:</b> Overlay of astroglial EGFP- and SR101-fluorescence after staining with Probenecid (Prob, 1 mM). <b>C,D:</b> Statistical comparison shows no change of astroglial SR101-labeling in the VLM. <b>E:</b> Overlay of astroglial EGFP- and SR101-fluorescence in the hippocampus after staining in CTRL conditions. <b>F:</b> Overlay of astroglial EGFP- and SR101-fluorescence after staining with Probenecid present in the staining solution shows a reduction of astroglial SR101-labeling in hippocampal astrocytes. <b>G,H:</b> Statistical comparison reveals a significant reduction of the intensity (G) as well as the percentage of EGFP-positive astrocytes that were also labeled (H). Scale bars: 40 µm.</p

Comparison of SR101-staining in different ages.

<p><b>A–C:</b> staining in the ventrolateral medulla (VLM) with 1 µM SR101 for 20 min and 10 min wash out. EGFP-fluorescence is not shown. The SR101 labeling was poor in all three tested ages. <b>D–F:</b> In the <i>stratum radiatum</i> of the hippocampal CA1 region SR101-labeling of juvenile (P33) an adult mice (P99) was very similar as compared to neonatal mice.</p

SR101 in neurons of the ventrolateral medulla.

<p><b>A–D:</b> two-photon images from the SR101-labeling procedure of EGFP-labeled inhibitory neurons (GlyT2-EGFP) in the ventral-lateral medulla. <b>A,B:</b> Green EGFP-fluorescence is quickly photo-bleached during the 20 min SR101 application. <b>C:</b> At 20 min, SR101 is increasing in numerous cells, but SR101 could only be found in one EGFP-positive neuron (arrow). <b>D:</b> Overlay of panels A and C. <b>E–G:</b> SR101-labeling of GlyT2-EFGP neurons (<b>E</b>) and SR101-labeled cells (<b>F</b>) in the presence of MK-571 (200 µM). <b>G:</b> merged image from E and F. The arrows point to SR101-labeled glycinergic neurons. Scale bars: 40 µm.</p

Astrocytes do not exhibit rhythmic calcium signals.

<p>(<b>A</b>) Current steps evoked in a EGFP-expressing astrocyte by depolarizing and hyperpolarizing voltage steps (10 mV increments) from a holding potential of −70 mV to potentials between −150 to +30 mV. This type of current responses to voltage steps is typical for a passive astrocyte. Panel (<b>B</b>) shows calcium signals (ΔF/F₀) and membrane current (pA) recorded from the particular astrocyte characterized in panel (A), along with simultaneously recorded field potentials (preBötC ∫). In this example, the fluorometric calcium signals (<b>B, Cc</b>) were obtained with Calcium orange (200 µM) loaded via the recording pipette. Rhythmic current fluctuations are buried in the noise but are unmasked by cycle triggered averaging in (C). No phase-locked astrocytic calcium signal could be detected.</p

Electrophysiological characterization of SR101-positive cells in the hippocampus.

<p><b>A:</b> CCD-camera image of an EGFP-expressing astrocyte using a EGFP-filter, 531/40 nm BP filter. The image was taken after the cell was approached with patch-pipette. <b>B:</b> Image of a whole-cell recorded SR101-loaded <i>stratum radiatum</i> cell that did not express EGFP. The picture was merged from the EGFP-filter image (531/40 nm, green) and the dual band filter image (EGFP/mCherry; F56-019, red). <b>C:</b> Membrane current traces of the astrocytes in (A) in response to the voltage-step protocol shown in <b>D</b>. <b>E:</b> membrane current traces of the SR101-positive EGFP-negative cell in (B). Capacitance artifacts have been truncated. <b>F:</b> Averaged I–V curves from EGFP-positive (EGFP; green) and SR101-positive EGFP-negative (SR101; red) cells. <b>G,H:</b> Statistical comparison of resting membrane potential (G) and membrane resistance (H).</p

Active Sulforhodamine 101 Uptake into Hippocampal Astrocytes

<div><p>Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrolateral medulla. Although carbenoxolone is able to decrease the SR101-labeling of astrocytes in the hippocampus, it is unlikely that SR101 is taken up via gap-junction hemichannels because mefloquine, a blocker for pannexin and connexin hemichannels, was unable to prevent SR101-labeling of hippocampal astrocytes. However, SR101-labeling of the hippocampal astrocytes was significantly reduced by substrates of organic anion transport polypeptides, including estron-3-sulfate and dehydroepiandrosterone sulfate, suggesting that SR101 is actively transported into hippocampal astrocytes.</p> </div

Test for the contribution of MRP-transporters to selective SR101-labeling.

<p><b>A–H:</b> Effect of Mrp1-blocker MK-571 on SR101-labeling in the VLM (<b>A–D</b>) and hippocampus (<b>E–H</b>) using EGFP-expressing astrocytes. <b>A:</b> Overlay of EGFP- and SR101-fluorescence in the VLM after staining in CTRL conditions. <b>B:</b> Overlay of EGFP- and SR101-fluorescence after staining with MK-571 (200 µM) in the staining solution. <b>C, D:</b> Statistical comparison shows no change of SR101-fluorescence intensity of EGFP-positive astrocytes in the VLM (<b>C</b>) but a reduction of the fraction of EGFP-positive astrocytes that were also labeled with SR101. <b>E:</b> Overlay of EGFP- and SR101-fluorescence in the hippocampus after staining in CTRL conditions. <b>F:</b> Overlay of EGFP- and SR101-fluorescence after staining with MK-571 (200 µM) in the staining solution. <b>G, H:</b> Statistical comparison reveals that the intensity of SR101-labeling as well as the fraction of EGFP-positive astrocytes that were also labeled with SR101 was significantly reduced by MK-571 present during staining.</p