Article Figures & Data
Figures
- Figure 1.
Glutamate receptor blockers prevented spikes triggered by polysynaptic activity. A, Current-clamp recording from a neuron in a culture pretreated with TTX for 2 d and then washed in normal medium for 20 min. Each trace shows the response to a sequence of three field stimuli (1 ms) at 0.5 s intervals: three trials in the absence of blockers (black) are superimposed on three trials obtained within 2 min of adding 10 μm CNQX and 50 μm dl-APV (red). Holding current was 0 pA. For each trace, the stimulus artifact was subtracted by delivering a pulse with the direction of the electric field reversed, adding the two records, and dividing by two. Secondary spikes triggered by polysynaptic activity (arrowed) only occurred after the first stimulus of the sequence. Addition of GluR blockers suppressed both spontaneous synaptic activity and secondary spikes. B, The responses to the first stimulus in A on an expanded time scale. In the presence of GluR blockers, a single stimulus reliably triggered one, and only one, spike.
- Figure 2.
Homeostatic changes in presynaptic calcium influx detected with SyGCaMP2. A, Fluorescence image of cultured hippocampal neurons expressing SyGCaMP2 (left) and pseudocolored images showing the relative change in fluorescence 0.3 s from the beginning of a train of 3 APs delivered at 20 Hz. B, The response to 3 APs was larger in a culture treated with TTX for 2 d. Note the variability in the amplitude of the presynaptic calcium signal between different boutons. Scale bar, 10 μm. C, Averaged SyGCaMP2 response to 1 AP (left) and 3 APs (right). Control cultures are black (n = 805 synapses), TTX-treated cultures are red (n = 1200), and gabazine-treated cultures are green (n = 386). Error bars are SEM. D, Peak amplitude of the SyGCaMP2 signal plotted as a function of the number of APs delivered at 20 Hz. Line fits have the following slopes: control (black), 0.078 ± 0.003; TTX (red), 0.104 ± 0.003; gabazine (green), 0.063 ± 0.004. Error bars are SEM.
- Figure 3.
Homeostatic changes in evoked vesicle fusion detected with SypHy. A, Fluorescence image of cultured hippocampal neurons expressing SypHy (left) and pseudocolored images showing the relative change in fluorescence in response to 40 APs at 20 Hz (middle) and 1 AP (right). Top row: TTX-treated culture. Bottom row: gabazine-treated culture. Note that the pseudocolor scale has a maximum (white) of 2 for images showing the response to 40 APs, but only 0.5 for the images showing the response to 1 AP. B, Relative change in SypHy fluorescence in response to a train of 40 APs at 20 Hz (left) and just 1 AP (right). Note the different scales. Black: control (n = 945 synapses). Red: TTX-treated cultures (n = 470 synapses). Green: gabazine-treated (n = 793). Bars are SEM. C, The distribution of release probabilities (Pr) in control, gabazine-treated, and TTX-treated cultures. Pr was calculated as the ratio of the peak responses to 1 and 40 APs for each individual synapse. Data were described using a gamma distribution, all with a shape parameter of α = 4. Values of the scale parameter β were as follows: control, 0.0147 ± 0.0006; gabazine, 0.0077 ± 0.0005; TTX, 0.0206 ± 0.0006. D, The distribution of SyGCaMP2 signal per AP. Data were described using a gamma distribution, all with α = 2. The scale parameter β was as follows: control, 0.127 ± 0.004 (n = 667 synapses); TTX, 0.17 ± 0.004 (n = 938); gabazine, 0.103 ± 0.013 (n = 404).
- Figure 4.
Homeostatic changes in evoked vesicle fusion reflect a cooperative action of calcium. The relation between Pr measured with SypHy and presynaptic calcium influx measured with SyGCaMP2 is shown. The curve fitted through the points is a power function of the form Axy, with y = 2.8 ± 0.3. Error bars are SEM.
