Cholinergic activity induces astrocyte Ca²⁺ elevations and LTP in CA3-CA1 synapses in the hippocampus in vivo.

<p>(A) Schematic drawing of the experimental approach used to monitor Ca²⁺ levels in hippocampal astrocytes in vivo; representative images of astrocytes labeled with sulforhodamine 101 (SR101) and loaded with Fluo-4-AM; corresponding merge image; and image of Fluo-4-loaded astrocytes displaying regions of interests. Scale bar, 20 µm. (B) Fluorescence traces of Ca²⁺ levels in regions of interests in astrocytes showed in (A) evoked by tail pinch sensory stimulation (horizontal bars) in control and in the presence of atropine. (C) Proportion of astrocytes responding to sensory stimulation in control (66 astrocytes from <i>n</i> = 8 rats), atropine (32 astrocytes from <i>n</i> = 4 rats), and MCPG (15 astrocytes from <i>n</i> = 3 rats). (D) Schematic drawing of the in vivo experimental approach showing the stimulating electrode in the Schaffer collaterals (SC) and the extracellular recording electrode of fEPSPs placed in the hippocampal CA1 region, and a representative trace of a field potential showing hippocampal theta rhythm activity (bottom) during tail pinch sensory stimulation. Right, Relative fEPSP slope (from basal values) versus time. Zero time corresponds to the onset of stimulation (as in all other figures). Inset: mean fEPSPs before and 60 min after stimulation. (E) Average relative changes of fEPSP evoked 60 min after sensory stimulation in control (<i>n</i> = 7), atropine (<i>n</i> = 6), and MCPG (<i>n</i> = 6). (F) Schematic drawing showing the additional stimulating electrode in the medial septum nucleus. Right, Relative fEPSP slope (from basal values) versus time. Zero time corresponds to the onset of stimulation that lasted 90.7 s (horizontal bar). Inset: mean fEPSPs before and 60 min after septum stimulation. (G) Average relative changes of fEPSP evoked 60 min after stimulation in control (<i>n</i> = 9), atropine (<i>n</i> = 6), and MCPG (<i>n</i> = 6). ***<i>p</i><0.001. Data are presented as means ± s.e.m (as in all other figures).</p

Cholinergic-induced hippocampal LTP requires astrocyte Ca²⁺ elevations.

<p>(A) Fluorescence image showing dialysis of sulforhodamine B into the astrocytic network after loading a single astrocyte with the dye (1 mg/ml) through the whole-cell recording pipette. Scale bar, 40 µm. (B) Schematic drawing depicting BAPTA or GDPβS dialysis into the astrocytic network from the recorded astrocyte. (C) Pseudocolor images representing fluorescence intensities of fluo-4- and BAPTA-filled astrocytes before (basal) and during alveus stimulation. Scale bar, 20 µm. (D) Astrocyte Ca²⁺ spike probability in control, BAPTA-, and GDPβS-loaded astrocytes. (E) Average relative changes of maximum astrocyte Ca²⁺ spike probability (from basal values) during alveus stimulation in control (100 astrocytes from <i>n</i> = 11 slices), BAPTA- (96 astrocytes from <i>n</i> = 10 slices), and GDPβS-loaded astrocytes (76 astrocytes from <i>n</i> = 10 slices). (F) Mean EPSCs (<i>n</i> = 10 consecutive EPSCs) before and 60 min after alveus TBS in a slice with BAPTA-loaded astrocytes. (G) Relative EPSC amplitudes versus time in control and BAPTA- and GDPβS-loaded astrocytes. (H) Average relative changes of EPSC amplitudes evoked 60 min after alveus TBS in control (<i>n</i> = 8), BAPTA- (<i>n</i> = 7), and GDPβS-loaded astrocytes (<i>n</i> = 5). In (D) and (G), zero time corresponds to the onset of stimulation that lasted 90.7 s (horizontal bars). **<i>p</i><0.01, ***<i>p</i><0.001. Data are presented as means ± s.e.m.</p

Cholinergic-induced hippocampal LTP is altered in IP3R2^−/− mice.

<p>(A) Pseudocolor images representing fluorescence intensities of pyramidal neurons filled with fuo-4 through the recording pipette before (basal) and during alveus TBS in wildtype (top) and IP₃R2^−/− mice (bottom). Scale bar, 20 µm. (B) Pseudocolor images representing fluorescence intensities of fluo-4-filled astrocytes before (basal) and during alveus TBS in wildtype (top) and IP₃R2^−/− mice (bottom). Scale bar, 15 µm. (C) Proportion of responding neurons and astrocytes to ACh application and alveus TBS in wildtype and IP₃R2^−/− mice (6 and 10 neurons from <i>n</i> = 6 and 10 slices for each stimulus in wildtype and IP₃R2^−/− mice, respectively; for ACh: 111 and 157 astrocytes from <i>n</i> = 6 and 15 slices; for TBS: 81 and 64 astrocytes from <i>n</i> = 9 and 10 slices, in wildtype and IP₃R2^−/− mice, respectively). (D) Astrocyte Ca²⁺ spike probability in wildtype and IP₃R2^−/− mice (81 and 64 astrocytes from <i>n</i> = 9 and 10 slices, respectively). (E) Average relative changes of maximum astrocyte Ca²⁺ spike probability (from basal values) during alveus stimulation in control (81 astrocytes from <i>n</i> = 9 slices), atropine (25 astrocytes from <i>n</i> = 4 slices), and MCPG (40 astrocytes from <i>n</i> = 5 slices) in wildtype mice and control IP₃R2^−/− mice (64 astrocytes from <i>n</i> = 10 slices). (F) Relative EPSC amplitudes versus time in slices from wildtype (<i>n</i> = 8) and IP₃R2^−/− (<i>n</i> = 8) mice. (G) Average relative changes of EPSC amplitudes evoked 60 min after alveus TBS in slices from wildtype mice in control (<i>n</i> = 8), atropine (<i>n</i> = 4), and MCPG (<i>n</i> = 5), and from IP₃R2^−/− mice (<i>n</i> = 8). (H) Relative mean fEPSP slope versus time in <i>in vivo</i> wildtype (<i>n</i> = 6) and IP₃R2^−/− mice (<i>n</i> = 4) before and after sensory stimulation. (I) Average relative changes of the mean fEPSP slope evoked 60 min after sensory stimulation in wildtype (<i>n</i> = 6) and IP₃R2^−/− mice (<i>n</i> = 6). In (D), (F), and (H), zero time corresponds to the onset of stimulation (horizontal bars). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Cholinergic activity in hippocampal slices induces astrocyte Ca²⁺ elevations and LTP in CA3-CA1 synapses.

<p>(A) Schematic drawing showing the stimulating electrodes (alveus and SC) and the whole-cell recording electrode (CA1 pyramidal neuron) in hippocampal slices, and a representative postsynaptic response (bottom) to one train of alveus TBS (action potentials evoked by TBS were truncated). (B) Pseudocolor images representing fluorescence intensities of fluo-4-filled astrocytes before and during alveus stimulation. Scale bar, 40 µm. (C) Astrocyte Ca²⁺ spike probability versus time. (D) Average relative changes of maximum astrocyte Ca²⁺ spike probability (from basal values) during alveus stimulation in control (132 astrocytes from <i>n</i> = 13 slices), atropine (94 astrocytes from <i>n</i> = 10 slices), and MCPG (71 astrocytes from <i>n</i> = 7 slices). (E) Mean EPSCs before and 60 min after alveus stimulation. (F) Relative EPSC amplitudes (from basal values) versus time. (G) Average relative changes of EPSC amplitudes evoked 60 min after stimulation in control (<i>n</i> = 13), atropine (<i>n</i> = 10), and MCPG (<i>n</i> = 12). In (C) and (F), zero time corresponds to the onset of stimulation that lasted 90.7 s (horizontal bars). *<i>p</i><0.05, ***<i>p</i><0.001. Data are presented as means ± s.e.m.</p

Cholinergic-induced hippocampal LTP depends on mild postsynaptic depolarizations.

<p>(A) Left, CA1 pyramidal neuron response to a train of alveus TBS recorded in current-clamp conditions. Center, mean EPSCs (<i>n</i> = 10 consecutive EPSCs) before and 60 min after alveus TBS. Right, relative EPSC amplitudes versus time (<i>n</i> = 6). Zero time corresponds to the onset of alveus TBS that lasted 90.7 s (horizontal bar). (B, C, and D) as in (A), but in QX-314-loaded neuron recorded in current-clamp conditions (<i>n</i> = 7), in voltage-clamp conditions at a holding potential of −70 mV (<i>n</i> = 5) and −30 mV (<i>n</i> = 5), respectively. (E) Representative neuronal responses to application of ACh in current- (CC) and voltage-clamp (VC) conditions at a holding potential of −70 mV. (F) Relative EPSC amplitudes versus time in CC and VC before and after ACh application (arrow). (G) Average relative changes of EPSC amplitudes evoked 60 min after ACh application in CC and VC (<i>n</i> = 10 and 10, respectively). ***<i>p</i><0.001.</p

CCL2⁺ cells are GFAP⁺ astrocytes in human glioma.

<p>(A) Confocal images show co-localization of CCL2-expressing cells (red) with GFAP (green) in glioma. Insert shows a detail of a single CCL2-expressing cell (red) co-localizing with GFAP (green) and counterstained with DAPI (blue). Scale bar: 30 µm. (B) Areas of infiltration of CD3⁺ T-cells (green) coincide with the areas of CCL2-expressing cells (red) in glioma. Confocal images show a tumorigenic area, infiltrated with CD3⁺ T-cells (green) in an area with numerous CCL2-expressing cells (red). The immunostaining was combined with a counterstaining with DAPI to detect the nucleus (blue). Scale bar: 50 µm. (C) Examples of characteristic CD3 infiltration in samples of glioma. CD3⁺ T-cells can be observed grouped in BVs (BV, limited by broken red line in 1 and 3) but also infiltrated in the parenchyma (2, 4, blue insert magnified in 5). (D) CCL2 expression correlates with the infiltration of T-cells in the tumor areas. The quantification in serial sections of the number of T-cells, either infiltrated or located in the BV lumen, revealed that the level of infiltration of CD3⁺ T-cells in the parenchyma (green) is positively correlated with the level of CCL2 expression (BV; BV lumen).</p

T cells express CCL2 receptor.

<p>Expression of CCR2 in T-cells in human glioma (top panel) and monkey brain (bottom panel). CD3⁺ T-cells (green) express CCR2 in their surface (magenta). Nucleus is stained with DAPI (blue).</p

T-cells come into contact with CCL2⁺ perivascular astrocytes in the areas of infiltration.

<p>(A) CCL2 is highly expressed in perivascular astrocytes where T-cells infiltrate the brain tumor. Detailed confocal analysis of samples of gliomas revealed that tumorigenic areas show CCL2-expressing astrocytes (red) with infiltration of CD3⁺ T-cells (green). Nuclei are stained with DAPI (blue). (B) Infiltration of T-cells occurs throughout multiple anatomical contacts between T-cells and CCL2-expressing astrocytes. Image B1 shows numerous CD3⁺ T-cells (green) establishing specific contacts with CCL2⁺ cells (red). In pictures B2, 3 and 4, a detail of T-cells (green) in contact with CCL2 perivascular astrocytes. Image B4 shows a specific detail of a CD3/CCL2 contact on the xy axis and the two lateral views on the z axis. (C) Confocal analysis of a CD3⁺ T-cell coming into contact with a CCL2⁺ cell located at the perivascular area of adenoviral injected monkey brain. The three dimensional transparency (Composite) of a stack of images shows a CD3⁺ T-cell (green), in close apposition to the endothelium, marked with Col-IV (red), and contacting a CCL2⁺ cell (magenta). A detail of the CD3-CCL2 contact is also shown in a 0.5 µm optical section on the xy axis and the two lateral views on the z axis. (D) Diagram showing how T-cells may come into contact with CCL2-expressing astrocytes in the edge of BVs suggesting that CCL2⁺ astrocytes contribute to the extravasation of T-cells in the brain parenchyma.</p

Human glioma shows CCL2⁺ cells.

<p>(A) The 14 cases of glioma analyzed show CCL2 expression in the tumorigenic areas. Samples of gliomas were immunostained to detect the expression of CCL2. All 14 cases analyzed showed expression of CCL2 in the neoplasic areas. CCL2-expressing cells are localized in the brain parenchyma itself and their number and intensity increase towards the necrotic areas. CCL2-expressing cells can also be seen around BVs (16–19). In addition, counterstaining with hematoxilin is also shown at BV levels (16′–19′) to corroborate the presence of endothelial nuclei. Insert show a detail of the endothelial nuclei. Scale bar; 1–21: 400 µm, 22–24: 60 µm. (B) Tumor cells present high immunoreactivity for GFAP in tumor areas, demonstrating the typical astrocytic cell type. CCL2⁺ cells show a characteristic astrocytic morphology. Scale bar: 30 µm. (C) BVs in, or close to the putative tumorigenic areas show high immunoreactivity for CCL2 in contrast to normal tissue. Additionally, counterstaining with hematoxilin (HHS) is also shown at the same levels. The insert shows a detail of the endothelial nuclei. Scale bar: 100 µm.</p

Intracerebral blocking of CCL2 attenuates the LPS-mediated T-cell infiltration.

<p>(A) Detail of CD4⁺ and CD8⁺ T-cell immunostaining in the striatum of LPS injected mice. Scale bar: 20 µm. (B) The number of infiltrated T-cells (CD4⁺, CD8⁺ and CD3⁺ T-cells) correlates with the number of CCL2⁺ cells in the areas of LPS injection. (C) Intraparenchymal injection of anti-CCL2 antibodies attenuates the LPS-induced infiltration of lymphocytes in the mouse brain parenchyma. The diagram on the top shows the arrangement of the experiment. Graphs show the density of CD8 and CD4 T-cells in the brain parenchyma surrounding the injection site. (D) Astrocytes express CCL2 after LPS injection in mouse brain. Confocal images of the injected areas show the co-localization of GFAP⁺ astrocytes (green) and CCL2 (red) in mouse brain. DAPI (blue) was used as a nuclear counterstaing. * p<0.05 ANOVA-test.</p