Article Figures & Data
Figures
- Fig. 1.
mIPSCs. a, An example of mIPSCs recorded as inward currents from a holding potential of −80 mV.Inset shows the time course of one event. Calibration: 1 sec for the top trace; 100 msec forinset. b, Distribution of mIPSC amplitudes recorded from neurons grown in control medium (Control; open bars) or in medium supplemented with TTX (Activity-deprived; dashed line) for 2 d before recording. Data were pooled from nine neurons in each condition (30 events per neuron).
- Fig. 2.
Activity blockade reduces mIPSC amplitude.A, Average, The average mIPSC from pyramidal neurons grown in control medium or medium supplemented with TTX for 2 d. Scaled Average, The average mIPSCs were scaled to the same peak value and overlaid. B, Same data set as in A; the average mIPSC amplitude for neurons grown in the two conditions. TTX is significantly different from control (p = 0.02;n = 9 neurons in each condition). C, The reversal potential for mIPSCs from neurons grown under the two conditions. Amplitudes were scaled to the values obtained at −80 mV and averaged across neurons grown under the same condition.
- Fig. 3.
Nonstationary fluctuation analysis of mIPSCs indicates that single-channel conductance is unaffected by activity blockade. A, Representative example of the average mIPSC from a control neuron (smooth line) and an overlay of a peak-scaled individual mIPSC (noisy line), showing the fluctuations about the mean current. B, Fluctuation analysis of the neuron shown in A. Plot of variance in mIPSC current about the mean current at different times after the peak (circles). Line shows parabolic fit of the relationship. C, Estimates of the number of open channels (left) and average single-channel conductance (right) for control and TTX-treated neurons.n = 8 and 7 for control and TTX-treated neurons, respectively.
- Fig. 4.
Immunocytochemical labeling of synapsin, GAD65, and GABAAR, and EGFP transfection. A, Dendrites stained against synapsin (red) and GAD65 (green), with double-labeled puncta shown inyellow. B, Dendrite stained against synapsin (red) and GABAARs (green), with overlap shown inyellow. C, Apical dendritic arbor of a pyramidal neuron transfected with EGFP. D, High-magnification image of the neuron in C, showing EGFP-labeled dendrites and immunocytochemical labeling of synapsin (inred). E, Pyramidal neuron double-labeled for synapsin (red) and GABAAR (green). F, Higher-magnification view of the apical dendrite shown in E. Scale bars:A, B, D, F, 5 μm; C, 25 μm; E, 15 μm.Arrows indicate colabeling of puncta (A,B, F) or synapses made onto the EGFP-filled dendrite (C).
- Fig. 5.
Intensity of Immunofluorescent labeling against synapsin, GAD65, and GABAAR in control (left) and TTX-treated (right) cultures. Minimal image processing was used for these illustrations.Black and white images were photographed with the same exposures and image-processed identically.A1, A2, The intensity of staining of synapsin puncta (red) onto EGFP-labeled dendrites (green) does not differ with TTX treatment.B1, GABAAR (green) and synapsin (red) labeling of a pyramidal apical dendrite, control condition. B2, GABAAR labeling of the dendrite in B1, shown in black andwhite. C1, C2, Staining as in B1 and B2 for a pyramidal neuron from a TTX-treated culture. GABAAR labeling is less intense in TTX-treated cultures. D1, GAD65 staining (red) of EGFP-labeled apical dendrites (green), control condition; D2, GAD65 labeling of the dendrite in D1, shown inblack and white. E1,E2, Staining as in D1 andD2 for a TTX-treated culture. GAD65 staining is also less intense in TTX-treated cultures. Scale bar, 5 μm.
- Fig. 6.
Fluorescent intensity of GABAAR labeling and the density of synaptic GABAAR puncta are reduced by activity blockade. A, Intensity of labeling of puncta for synapsin, GAD65, and GABAAR, from randomly selected dendritic regions (Random dendrites) or from pyramidal apical dendrites (Apical dendrites).B, Length density (number of immunoreactive puncta per unit length of dendrite) of puncta immunoreactive for synapsin, GAD65, or GABAAR. Data are expressed as a percentage of control;dashed line indicates control (100%). *p < 0.03; **p < 0.01.
- Fig. 7.
Total apical dendritic branch length and the area of immunoreactive puncta are not affected by activity blockade.A, Average total length of the apical-like dendrite from EGFP-filled pyramidal neurons grown under control conditions (Con) or in TTX for 2 d before fixation and quantification; n = 21 and 12 for control and TTX-treated neurons, respectively. B, Average area of puncta immunolabeled for GAD65 or for synapsin (Syn), for contacts onto EGFP-filled pyramidal apical dendrites. None of the differences between conditions are statistically significant.
- Fig. 8.
mIPSC amplitude and the fluorescent intensity of synaptic GABAAR labeling are both reduced in a multiplicative manner by activity blockade. a, Cumulative distribution of mIPSC amplitudes from nine control neurons (dashed line; 30 events per neuron) and nine neurons treated with TTX for 2 d before recording (solid line; 30 events per neuron). Scaling down the control distribution by a factor of 0.46 produced a good fit to the TTX-treated distribution. b, Cumulative distribution of the intensity measurements from synaptic GABAAR puncta from control (dashed line; 316 puncta) and neurons treated with TTX for 2 d before recording (solid line; 240 puncta). Scaling down the control distribution by a factor of 0.74 produced a good fit to the TTX-treated distribution.
