Article Figures & Data
Figures
- Fig. 1.
Confocal fluorescence image of the retinal surface of the eyecup labeled with Calcium Green-1. Astrocytes (arrows) and Müller cell endfeet (dim, diffuse fluorescence) were labeled. Calcium waves were initiated by advance of a micropipette (left). Extracellular activity of single neurons within the ganglion cell layer was recorded with a metal microelectrode (right). Calcium Green-1 fluorescence was measured within a 15 μm diameter region near the microelectrode tip (open circle). Scale bar, 50 μm.
- Fig. 2.
Peristimulus time histograms of the light-driven activity of an ON (A), an ON–OFF (B), and an OFF (C) neuron, each representative of one of the three response classes observed.Open and closed bars above the histograms indicate periods of light ON and OFF, respectively. Activity was recorded during control periods, before initiation of Ca2+ waves.
- Fig. 3.
Modulation of neuronal activity in an ON neuron. Cell activity is plotted as a spike display in trace 1(vertical lines represent single action potentials) and as a frequency plot in trace 2 (abscissa represents the running average of spike frequency, i.e., a leaky integrator with a linear decay to 0% at 10 sec; 0 frequency is indicated at left end of the trace). Trace 3, Calcium Green-1 fluorescence in a circular region near the recording microelectrode tip. Trace 4, light stimulus, indicating periods of light ON (open segments) and OFF (closed segments). Arrow indicates initiation of the Ca2+ wave. Open and closed bars above trace 1 indicate control and test periods, respectively, during which average spike frequency was measured. Neuron spike frequency and glial cell fluorescence increase concurrently several seconds after initiation of the glial Ca2+ wave. Calibration bars: trace 2(spike frequency), three spikes/sec; trace 3 (Calcium Green-1 fluorescence), 20% ΔF/F; time, 10 sec.
- Fig. 4.
Excitatory and inhibitory modulation of neuronal activity. A frequency plot of cell activity (top trace) and glial cell Calcium Green-1 fluorescence (bottom trace) are shown for each trial. A, Excitatory modulation in an ON neuron. Spike frequency increases in those trials (1 and 3) in which [Ca2+]i in neighboring glial cells also increases. B, Inhibitory modulation in an ON–OFF neuron. Spike frequency decreases when glial [Ca2+]i increases. The peristimulus time histograms of cells in A and B are shown in Figure 2, A and B, respectively. Calibration bars are the same as in Figure 3.
- Fig. 5.
Correlation between neuronal modulation and Ca2+ increases. A, Relation between the magnitude of inhibition of neuronal activity and the amplitude of the Ca2+ wave. Points represent single trials from all cells showing significant inhibitory modulation.Points at the left of the plot (ΔF/F = 0) represent trials in which the Ca2+ wave failed to reach the neuron. Least-squares fit, R2 = 0.42.B, Relation between the time to the peak of neuron modulation and the time to the peak of the Ca2+wave. Trials in which the inhibition of activity was 25% or greater are included. The discrete Ca2+ wave times reflect the 2.55 sec acquisition period of fluorescence images. Least-squares fit, slope = 0.97, intercept = 3.6 sec,R2 = 0.30.
- Fig. 6.
Effect of thapsigargin on the modulation of neuronal activity. Thapsigargin (3.0 μm) reduces the glial Ca2+ increase and the inhibition of activity in an OFF neuron. Calibration bars for trials in Figures 6 and 7: spike frequency, two spikes/sec; Calcium Green-1 fluorescence, 20% ΔF/F; time, 10 sec. A frequency plot of neuronal activity and Calcium Green-1 fluorescence are shown in each trial.
- Fig. 7.
Effect of neurotransmitter antagonists on neuronal modulation. A, Bicuculline (5 μm) and strychnine (1 μm), applied together, abolish the inhibition of activity in an ON–OFF neuron without attenuating the glial Ca2+ increase. B, NBQX (10 μm) and D-AP7 (200 μm) together reduce the inhibition of activity in an ON–OFF neuron without reducing the glial Ca2+ increase. As illustrated, light responses of neurons often changed during drug application and recovery.
Tables
Neuronal type ON ON–OFF OFF Number of cells with significant modulation Inhibition 7 of 24 9 of 15 9 of 14 Excitation 5 of 24 0 of 15 0 of 14 Inhibition Excitation Inhibition Inhibition Magnitude of cell modulation No Ca2+wavea +3 ± 5% (30) −1 ± 3% (20) +1 ± 2% (39) −1 ± 1% (36) Large Ca2+ waveb −35 ± 3% (35) +27 ± 5% (27) −25 ± 3% (35) −25 ± 2% (44) Number of cells. For each cell tested, modulation was judged significant if the modulation in those trials when a Ca2+wave reached the cell (ΔF/F Ca2+signal > 22%) was significantly different than when a Ca2+ wave failed to reach the cell (ΔF/F < 1%) (p < 0.05).
Magnitude of cell modulation. Shown are the average modulation values for trials in cells with statistically significant modulation. Separate means are given for trials when a Ca2+ wave reached the cell and did not reach the cell. For ON neurons, separate means are given for cells with excitatory and inhibitory modulation.
F1-a Trials when the ΔF/FCa2+ signal was <1%.
F1-1002 Trials when ΔF/F was >22%.
- Table 2.
Effect of neurotransmitter antagonists on the inhibitory modulation of neuronal activity
Condition Control Antagonist Recovery Inhibitory neurotransmitter antagonists Bicuculline (5 μm) −31 ± 5% (15) −13 ± 3% (17)* −26 ± 3% (16)* Strychnine (1 μm) −28 ± 5% (20) −8 ± 3% (17)* −25 ± 4% (19)* Bicuculline + strychnine −28 ± 4% (28) 0 ± 1% (23)* −20 ± 2% (32)* Glutamate antagonists NBQX (10 μm) −25 ± 5% (22) −6 ± 3% (22)* −15 ± 2% (23)* D-AP7 (200 μm) −24 ± 5% (21) −12 ± 4% (18)** −22 ± 5% (14) NBQX + D-AP7 −18 ± 2% (19) −2 ± 2% (19)* −12 ± 3% (18)* Shown are the average modulation values for trials when a Ca2+ wave reached the neuron. Only neurons with significant inhibitory modulation were tested. Antagonists were applied for 15 min before testing. Recovery followed a 60 min washout.
* p < 0.01, antagonist versus control, recovery versus antagonist.
** p < 0.05.
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