Article Figures & Data
Figures
- Fig. 1.
Bafilomycin A1 reduced the probability of synaptic transmitter release. A,B, Representative traces of evoked EPSCs in control cultures and after incubation with bafilomycin. In many cases, EPSCs were absent in bafilomycin-treated cultures. When present, the amplitude of EPSCs in bafilomycin was smaller than in control, and the number of synaptic failures was increased by bafilomycin. C, Averaged (n = 10) EPSCs in control and bafilomycin. Shown is the same pair of pre-postsynaptic neurons as in B. D,Mean evoked EPSC amplitude in control and bafilomycin-treated cultures (n = 25 and 35 presynaptic neurons, respectively).E, Mean percentage of synaptic failures observed during trials of 10 stimuli delivered at 1 Hz in control and after incubation with bafilomycin (n = 25 and 35 presynaptic neurons, respectively). F, mEPSCs in control and after incubation with bafilomycin. G, Mean frequency of mEPSCs measured during 1–4 min in control and in bafilomycin-treated cells (n = 9). H, Average cumulative probability plots of the mEPSC amplitude in control (n = 9) and after incubation with bafilomycin (n = 8). Cumulative probability plots were calculated in 1 pA bins and were not significantly different between control and bafilomycin-treated cells (Kolmogorov–Smirnov test). Holding potential was −60 mV. Significant differences with respect to control were established by the Student's t test atp < 0.02 (**) and p < 0.001 (***).
- Fig. 2.
Bafilomycin reduced astrocyte-evoked neuronal responses but did not prevent Ca2+ waves in astrocytes. A, Representative whole-cell current neuronal responses to astrocyte stimulation in control (left) and after incubation with bafilomycin (right). Mechanical stimulation of astrocytes is indicated by asterisk. Fast, high-amplitude synaptic currents have been truncated. B, Proportion of mechanically stimulated astrocytes that evoked glutamate-dependent responses in adjacent neurons in control and bafilomycin-treated cultures (n = 3 different cultures).C, Current amplitude recorded in neurons after mechanical stimulation of adjacent astrocytes in control and bafilomycin-treated cells (n = 37 and 45 stimulated astrocytes, respectively). Significant differences with respect to control were established by the Student's t test atp < 0.01 (**) and p < 0.001 (***). D,E, Mechanically induced Ca2+ waves recorded in cultures loaded with the Ca2+ indicator fluo-3 in control conditions and after incubation with bafilomycin. Left to right panels show pseudocolor images representing intensity of fluo-3 emission, taken before, during, and after mechanical stimulation, at the times indicated. Zero time corresponds to the time of astrocyte stimulation. Mechanical stimulation increased the intracellular Ca2+ in the injected cell as well as in neighboring nonstimulated astrocytes.
- Fig. 3.
Microinjection of the light chain of the Botulinum toxin B (BoNT/B) into astrocytes reduces synaptobrevin immunofluorescence. An individual astrocyte in a microisland was microinjected with fluoro-ruby (A) together with the light chain of BoNT/B. Subsequent immunostaining of the preparation with anti-synaptobrevin II showed a reduced immunoreactivity in the microinjected cell (B). Images inA and B were constructed from a stack of 16 successive images (2 μm deep) obtained with laser scanning confocal microscopy. Boxes show regions used to compute average linescan fluorescent intensities (20 lines wide) that are presented in D. In C andD, the normalized intensities of fluoro-ruby and anti-synaptobrevin immunoreactivity are shown as average line scans. In BoNT/B-injected astrocytes (D), the intensity of the anti-synaptobrevin immunoreactivity (green line) is reduced in the region that corresponds to the injected cell (red line), whereas in astrocytes injected with fluoro-ruby alone (C, original micrographs are not shown), it remains constant in comparison to neighboring uninjected cells.
- Fig. 4.
Astrocyte Ca2+ wave propagation was unaffected by injection of BoNT/B. A,Mechanically induced Ca2+ wave recorded in astrocytes loaded with the Ca2+ indicator fluo-4 in control conditions. Panels show images in pseudocolor mode representing intensity of fluo-4 emission, taken before, during, and after mechanical stimulation, at the times indicated. Zero time corresponds to the time of astrocyte stimulation. Mechanical stimulation increases intracellular Ca2+ in the injected cell as well as in neighboring non-stimulated astrocytes.B, Stimulation of a single astrocyte microinjected with fluoro-ruby and BoNT/B (left panel) increases the intracellular Ca2+ in the injected cell as well as in neighboring unstimulated astrocytes. Right panels show pseudocolor images representing intensity of fluo-4 emission at the times indicated in A. Zero time corresponds to the time of astrocyte stimulation. C, As in B but with a single astrocyte microinjected with fluoro-ruby, BoNT/B, and BAPTA (left panel). Mechanical stimulation of the injected cell did not change the fluorescent emission of fluo-4 in either the stimulated or neighboring astrocytes (right panels).
- Fig. 5.
BoNT/B prevents glutamate-mediated SIC in adjacent neurons. A, Left panel, Phase-contrast image of a microisland containing two astrocytes and two neurons (somas indicated by arrows). Right panel, Epifluorescence image of the same microisland after the microinjection of both astrocytes with fluoro-ruby. B, Representative whole-cell currents recorded from neurons in microislands in which the astrocytes were microinjected with either fluoro-ruby (left) or fluoro-ruby and BoNT/B (right). Although mechanically stimulated astrocytes injected with fluoro-ruby alone reliably evoked SIC in adjacent neurons, those injected with BoNT/B failed to evoke SIC. Mechanical stimulation of the astrocyte is indicated by the asterisk. Note the noise increase during the SIC, which is probably caused by the activation of NMDA receptors (Araque et al., 1998a). C, Proportion of mechanically stimulated astrocytes that evoked glutamate-dependent SIC in adjacent neurons. D, Current amplitude recorded in neurons after mechanical stimulation of adjacent astrocytes. Significant differences were established by the Student'st test at p < 0.01 (**).
Tables
- Table 1.
Astrocyte-induced neuronal responses were unaffected by glutamate transporter inhibitors
Control t-PDC Control β-HA Control DHK Sodium Lithium Astrocytes that evoked neuronal responses (%) 61.0 ± 3.9 66.8 ± 8.6 54.8 ± 2.4 63.5 ± 5.4 75.4 ± 4.0 76.4 ± 3.6 74.5 ± 5.5 76.1 ± 3.9 (n = 3) (n= 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3) SIC amplitude (pA) 39.3 ± 10.0 42.9 ± 7.0 30.6 ± 7.7 28.5 ± 3.5 37.1 ± 5.3 43.9 ± 9.1 159.7 ± 25.2 193.5 ± 29.0 (n = 15) (n = 17) (n = 21) (n = 19) (n = 23) (n = 24) (n = 23) (n = 23) Control solutions include 2 mm Mg2+, except for extracellular sodium and lithium in which extracellular Mg2+ was omitted. No significant differences were found between control and test conditions (Student's ttest).
