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First published online 2 August 2005
doi: 10.1242/jcs.02515

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Synaptic transmission onto hippocampal glial cells with hGFAP promoter activity

¹ Experimental Neurobiology, Department of Neurosurgery, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
² Bogomoletz Institute of Physiology, Bogomoletz St. 4, 01024 Kiev, Ukraine
³ Cellular Neurosciences, Max Delbrück Center for Molecular Medicine, Berlin, Robert Rössle Str., Germany

View larger version (187K): [in a new window]   Fig. 1. Morphological evidence of synapse-like structures between hGFAP/EGFP-positive glial cells and neurons. Glial cells in the hippocampal CA1 area of hGFAP/EGFP transgenic mice were immunolabelled by anti-GFP antibodies and visualized by HRP-reaction (A), silver intensified immunogold reaction (B) and post-embedding labelling with immunogold particles (C,D). EGFP-positive profiles in A are visible by dense, black peroxidase reaction product. In B, glial profiles (outlined by arrowheads) are labelled by black silver grains. EGFP-positive presumed GluR cells in A and B are in contact with synaptic nerve terminals. Areas of glial-neuron contact are magnified in the inserts (delineated by dashed rectangles). Scale bar in A, B=0.2 µm and 0.1 µm in inserts. Synapse-like structures in (C,D) display typical synaptic terminals with synaptic vesicles inside the neuronal `partner' and post-synaptic densities (arrowheads) in the labelled EGFP-positive glial profiles. Circles are intended to mark the 12 nm gold particles. There are also mitochondria (mit) and endoplasmic reticulum (black arrow head) in the labelled profiles. Note unspecific gold labelling on mitochondria in (C,D). Scale bars in C,D=0.25 µm.

View larger version (99K): [in a new window]   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 (Vrest=–83 mV, Ri=78 M, Cm=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; Vrest=–84 mV, Ri=3 M, Cm=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). (B1-3) 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 (Vrest=–84 mV, Ri=72 M, Cm=29 pF). In this cell, post-recording analysis did not detect S100ß immunoreactivity [S100ß, red (r); TRITC dextran, green (g)]. (B2) Another GluR cell (Vrest=–83 mV, Ri=270 M, Cm=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 (Vrest=–86 mV, Ri=5.1 M, Cm=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.

View larger version (27K): [in a new window]   Fig. 3. Repetitive sub-threshold Schaffer-collateral stimulation (100 microseconds, 1 Hz) revealed postsynaptic depolarisations of unequally distributed amplitudes in GluR-type glial cells. Extracellular field potentials and membrane potentials of a GluR cell were simultaneously recorded in a p11 mouse (Vrest=–80 mV, Ri=400 M, Cm=30 pF). (A) Typical pairs of recording traces are given. Single stimulation pulses caused stable dendritic field potentials (A1,2 upper traces, C upper trace) and time-correlated glial depolarizations of up to 7.3 mV (A1, lower trace) or glial failures (A2, lower trace). Spontaneous glial depolarisations were observed in a few cases (A3 arrow, 0.9 mV). (B) The average of 119 successively recorded pairs of traces is depicted with different time scaling. (B1) Field potentials showed only synaptic potentials (449±65 µV) without postsynaptic population spikes, as visualized at higher time resolution. (B2) The corresponding glial ePSPs averaged out at 2.4±1.8 mV. (C) The time course of field potential amplitudes (crosses, upper trace) and GluR cell ePSPs is plotted. While field potentials remained almost unchanged, the glial responses represented a mixture of failures and depolarisations over the 2 minutes recording period (circles, lower panel). (D) The amplitudes of the glial ePSPs were clearly non-Gaussian distributed (D1). The noise amplitude histogram was received from analysing baseline recorded at resting potential (1 second, corresponding to 3106 points) and fitting to a Gaussian function (D2). The Gaussian fit displayed a half width of 124±5 µV and peaked at –80.7±0.09 mV. For clarity, the centre was scaled to 0 mV. All data in this figure were obtained from the same GluR cell, [Cl–]i was always 135 mM.

View larger version (33K): [in a new window]   Fig. 4. Schaffer-collateral stimulation reveals two types of glial ePSCs with different inactivation kinetics. (A,B) Sub-threshold field potentials (top traces) were elicited applying a paired pulse protocol (150 microseconds, 150 µA, 50 milliseconds interval, every 20 seconds) while recording a GluR cell in the voltage-clamp mode (–80 mV, bottom traces). Typical examples of rapidly (A, =2.4 milliseconds, arrow) and slowly (B, =33 milliseconds, arrowhead) inactivating glial ePSCs are shown. (C) Fast and slowly decaying ePSCs occurred in the same individual GluR cell. 120 single sub-threshold pulses (100 microseconds, 150 µA) were applied every 3 seconds while currents were recorded at –80 mV. The top left trace represents the total average of all responses. Subsequently, current traces were sorted according to inactivation time constants. The left panel shows averages of pooled responses, while the right panel gives typical original traces. The upper pair of traces shows fast inactivating currents (fast=3.4 milliseconds, n=16), the next summarizes traces with slowly inactivating currents (tslow=34.7 milliseconds, n=48), followed by responses with biphasic inactivation (fast=4.6 milliseconds, slow=30.3 milliseconds, n=16). The bottom pair shows failure traces (n=35). Arrows denote fast responses, arrowheads denote slowly decaying responses. [Cl–]i was always 135 mM.

View larger version (29K): [in a new window]   Fig. 5. Pharmacological identification of GABAA receptor-mediated ePSCs in GluR cells. (A,B) Averaged sub-threshold field potentials (top traces) and whole-cell currents (–80 mV, bottom) are depicted, which were recorded in response to 120 single stimulation pulses (150 µs, 10 second intervals) in the presence of NBQX (10 µM; A) or bicuculline (10 µM; B). Note the almost complete block of glial inward currents by bicuculline. [Cl–]i was always 135 mM.

View larger version (34K): [in a new window]   Fig. 6. Properties of postsynaptic GABAA receptor currents in hippocampal GluR cells. Data were taken in the presence of NBQX (10 µM) and D-APV (25 µM). (A) Membrane currents were evoked as described in Fig. 2A. (B) Paired-pulse stimulation (150 µs, 29 V, 50 milliseconds delay, 10 seconds interstimulus interval; 37 double pulses) evoked slowly decaying PSCs (17 milliseconds). Currents were sorted according to stimulation success, and averaged. In 8 cases, both pulses evoked glial PSCs (upper left trace; amplitudes 3.0 and 2.4 pA) while 14 paired pulses produced double failures (right, top). Seven stimulation pairs induced responses upon the first (left, bottom), another 8 upon the second pulse (right, bottom). The total failure rate was 58%. (C) Short (150 µs) stimulation pulses evoked glial PSCs, which had opposite directions at –80 mV and 0 mV (averaged responses of 120 and 30 single traces, respectively). (D) Spontaneous GABA-mediated PSCs occurred rarely (about 1 event per minute; arrow). (E) Kinetics of the GABA-mediated glial sPSC labelled by an arrow in (D) at higher time resolution. (F) Increasing [K+]o to 10 mM significantly increased the frequency glial GABA sPSCs (50 events per minute). (G) Analysis of averaged sPSCs (187 events) revealed an amplitude of 3.2 pA, a desensitization time constant of 19.5 milliseconds, a rise time of 1.9 milliseconds (10-90% time to peak), and a half width of 14.6 milliseconds. With the exception of (B), all recordings in this figure were obtained from the same individual cell; [Cl–]i was always 27 mM.

View larger version (33K): [in a new window]   Fig. 7. Quantal analysis of GABA-mediated glial ePSCs suggests a small number of release sites. (A) Averaged response (120 stimuli, 150 microseconds) in the presence of NBQX (10 µM) and D-APV (25 µM). Peak amplitude (4.8 pA) was reached 5.4 milliseconds after the stimulus. (B) ePSC amplitudes were plotted against time (left), and the cumulative fraction was calculated (right). (C) Eight successively recorded exemplary traces document the presence of failures and ePSCs in GluR cells. (D) The ePSC amplitude histogram resembles a binomial distribution, characteristic of a small number of release sites. The individual ePSC amplitudes were determined as the difference between the averaged currents measured between 4.9 and 5.9 milliseconds after stimulation, and the average of 1 milliseconds corresponding baseline current. Inset depicts the amplitude histogram of current noise, taken from baseline traces before stimulation (–100 milliseconds to 0 milliseconds in C). Evaluation of 600 milliseconds baseline noise (7373 points) revealed a Gaussian distribution around 0 pA with a half width of 1.03 pA. (E) shows the superposition of ePSC amplitude histograms of 4 GluR cells (interstimulus interval, 10 seconds). [Cl–]i was always 27 mM.

View larger version (34K): [in a new window]   Fig. 8. GluR cells also receive glutamatergic input. Spontaneous (A1-A5) and evoked responses (B1,B2) were obtained from the same individual cell at p 10 (Vrest=–86 mV, Ri=230 M, Cm=73 pF). (A1-A4) After application of bicuculline (10 µM), only the fast events persisted. (A2) represents mean sPSCs with slow (top, 5.6 pA, =21.6 milliseconds, n=2) and rapid kinetics (bottom, 8.6 pA, =4.4 milliseconds, n=2) at higher resolution, taken from trace in (A1) before bicuculline application (i.e. under control condition). The sPSC in (A3) was taken from the trace in (A1) during bicuculline application and displayed on a fast time scale and larger current scale. (A4) shows the mean of 29 sPSCs taken from the trace in (A1) during bicuculline at higher resolution [5.7±1.8 pA, =3.6 milliseconds, rise time 1.1 milliseconds (10-90%), half width 4.8 milliseconds]. (A5) represents the continuation of the trace in bicuculline shown in (A1), 15 minutes later. Note the presence of spontaneous, bicuculline-insensitive activity before and shortly after additional wash-in of NBQX (10 µM) and D-APV (25 µM). Two minutes after co-application of bicuculline with NBQX and APV, spontaneous activity completely disappeared. (B1) In the same cell, near-field stimulation evoked bicuculline-resistant ePSCs with kinetics similar to the sPSCs (A3,A4). Paired pulses (n=119; 150 microseconds, 8V, 50 milliseconds delay, 1 second interstimulus interval) were applied, and currents were sorted and averaged as described in Fig. 6B. In 32 cases, both pulses evoked glial PSCs (upper left trace; amplitudes 6.7 and 6.9 pA; =1.5 milliseconds) while 29 paired pulses produced double failures (right, top). 21 stimulation pairs induced responses upon the first (left, bottom), another 37 upon the second pulse (right, bottom). The total failure rate was 49%. (B2) Co-application of bicuculline with NBQX (10 µM) and D-APV (25 µM) led to a complete block of ePSCs. [Cl–]i was always 27 mM.