Fig. 2. Post-recording analysis of hGFAP/EGFP-positive cells in the hippocampus. (A₁) The morphology of a GluR cell was visualized by Texas Red dextran-filling during whole cell recording. Subsequent confocal analysis and 2D projection of 32 optical sections (total depth 21 µm) allowed us to resolve details of cellular process arborization. Note the typical nodules appearing as dots all along the fine processes. The current pattern of this GluR-type glial cell is given in the middle panel. Current responses were evoked by de- and hyperpolarizing the membrane between +20 and –160 mV (holding potential –80 mV), and capacitive artefacts were compensated offline (V_rest=–83 mV, R_i=78 M, C_m=37 pF). This cell showed sPSPs and ePSPs sensitive to NBQX and bicuculline. Post-recording immunostaining and triple fluorescence confocal analysis were applied to check for NG2 immunoreactivity. The middle panel shows the three separated colour channels of one confocal plane. To improve visibility, Texas Red dextran labelling of the recorded cell is given in green (g), NG2 immunoreactivity in red (r), and EGFP expression in blue (b). Note that the EGFP fluorescence remaining post-recording was only 16% compared to surrounding cells (b). The superimposed RGB picture (right panel) shows the membrane-associated distribution of NG2 immunoreactivity of the recorded GluR cell (yellow details). (A₂) In contrast to GluR cells, hGFAP/EGFP-positive GluT type astrocytes predominantly expressed time- and voltage-independent currents (middle panel, stimulus protocol as in A₁) and displayed a different morphology (left panel, see text for details; V_rest=–84 mV, R_i=3 M, C_m=71 pF). The cell did not generate sPSCs. The EGFP fluorescence intensity determined post-recording reached 53% of that measured in adjacent cells (b). The cell was NG2-negative (middle panel (r) and right panel). (B_1-3) Analogue to (A), GluR and GluT cells were tested post-recording for S100ß immunoreactivity. The cells were recorded for exactly 1 minute (see text). (B₁, left) 2D projection of a GluR cell after TRITC dextran-filling (16 optical sections, total depth 8.4 µm) revealed a typical morphology with thin, wide spanning, nodule-containing processes. (B₁, middle) Artefact-compensated current pattern of the GluR cell (V_rest=–84 mV, R_i=72 M, C_m=29 pF). In this cell, post-recording analysis did not detect S100ß immunoreactivity [S100ß, red (r); TRITC dextran, green (g)]. (B₂) Another GluR cell (V_rest=–83 mV, R_i=270 M, C_m=24 pF) showed post-recording S100ß labelling. (B₃) Analysis of a GluT cell. Projection of EGFP fluorescence (left, 32 optical sections, total depth 19.5 µm) revealed its characteristic morphology. (B₃, middle) Current pattern of the GluT cell (V_rest=–86 mV, R_i=5.1 M, C_m=61 pF). The cell was filled with Texas Red dextran (g) during recording, and post-recording confocal analysis detected S100ß immunoreactivity [71% fluorescence intensity compared with surrounding S100ß-positive cells (r)]. Scale bars in morphological pictures represent 10 µm; for current patterns, 1 nA and 10 milliseconds, respectively.