Article Figures & Data
Figures
- Fig. 2.
GABA immunoreactivity in cortical cultures.A, MAP2-positive neurons from cortical cultures after 7 d in vitro, viewed with fluorescein filters.B, Same field of view as in A, using rhodamine filters to show GABA-positive neurons. C, GABA-positive multipolar neuron. D, GABA-positive bipolar neuron. Scale bars: A, B, 10 μm; C, D, 25 μm.
- Fig. 1.
Spontaneous activity of cortical neurons in culture. A, Control, A whole-cell recording from a cortical pyramidal neuron after 5 d in vitro. Depolarizing synaptic potentials (arrow) could be detected, which periodically brought the neuron over threshold to fire overshooting action potentials. B, This activity was completely abolished by addition of 0.1 μm TTX to the perfusate.
- Fig. 3.
The effects of activity blockade and neurotrophins on the percentage of GABA-positive neurons in cortical cultures.A, Cultures were treated with 0.1 μm TTX, either alone (TTX) or in the presence of 25 ng/ml BDNF (TTX + BDNF), 50 ng/ml NGF (TTX + NGF), or 25 ng/ml NT3 (TTX + NT3) for 2 d; *significantly different from control, p< 0.01. B, The effects of different doses of BDNF on the ability of TTX to reduce the percentage of GABA-positive neurons were determined. TTX (0.1 μm) was applied in the presence of the indicated concentration of BDNF for 2 d and the percentage of GABA-positive neurons determined. For each condition inA and B, the ratio of GABA-positive to GABA-negative neurons was determined, and these values are expressed as a percent of the values obtained for control cultures (control = 100%, dashed line).
- Fig. 4.
The effects of Trk receptor blockade on the percentage of GABA-positive neurons. K252a, a blocker of Trk receptor signaling, was applied for 2 d at the indicated concentration (10, 50, or 200 nm) and the percentage of GABA-positive neurons determined. The effect of BDNF (25 ng/ml) and TTX (0.1 μm) in the presence of K252a (200 nm) for 2 d was also determined (TTX+BDNF+K252a). Numbers are expressed as a percentage of the value obtained for control cultures (control = 100%,dashed line); *significantly different from control,p < 0.05; **significantly different from control,p < 0.001.
- Fig. 5.
The activity-dependent reduction in the percentage of GABA-positive neurons is reversible. Cultures were treated with TTX (0.1 μm) for 2 d. Cultures were then fixed, processed, and counted immediately (TTX); washed for 2 d before fixation (TTX/WASH); or washed and BDNF (25 ng/ml) added for 2 d before fixation. Numbers are expressed as a percentage of the value obtained for control cultures (control = 100%, dashed line); *significantly different from control, p < 0.05; **significantly different from control, p < 0.01.
- Fig. 6.
Activity blockade reduces inhibition onto pyramidal neurons. A, Representative voltage-clamp recordings of spontaneous IPSCs from pyramidal neurons grown in control medium (CONTROL) in medium supplemented with 0.1 μm TTX for 2 d (TTX) or in medium supplemented with TTX and BDNF (25 ng/ml) for 2 d.B, Frequency of spontaneous IPSCs from pyramidal neurons grown under the conditions indicated in A(n = 8 neurons in each condition).C, Total inhibitory current integrated over time, from the same population as in B; *significantly different from control (Student’s t test, p< 0.04); **p < 0.01.
- Fig. 7.
Activity blockade increases pyramidal neuron firing rates. A, Representative current-clamp recordings of spontaneous firing from pyramidal neurons grown in control medium (CONTROL), in medium supplemented with 0.1 μm TTX for 2 d (TTX), or in medium supplemented with TTX and BDNF (25 ng/ml) for 2 d (TTX + BDNF). B, Average spike frequency of pyramidal neurons grown for 2 d under the conditions described inA or in TTX + 50 ng/ml NGF (TTX + NGF) or TTX + 25 ng/ml NT3 (TTX + NT3); *significantly different from TTX (Student’s t test,p < 0.01).
Tables
Condition Neuronal density (% control) TTX (14) 102 ± 13 TTX + BDNF (11) 127 ± 16 TTX + NGF (6) 130 ± 13 BDNF (9) 98 ± 14 K252a (9) 102 ± 8 TTX + WASH (6) 97 ± 16 TTX + WASH + BDNF (3) 96 ± 6 Neuronal density in cultures grown for 2 d under the different experimental conditions. Drug dosages are as reported in Results. Values are mean ± SEM for the number of cultures indicated in parentheses.
Pyramidal morphology Membrane potential (mV) Input resistance (GΩ) Control (13) −63.4 ± 1.3 0.97 ± 0.05 TTX (14) −63.6 ± 0.8 1.02 ± 0.02 TTX + BDNF (10) −62.4 ± 1.5 0.99 ± 0.06 Bipolar morphology Control (11) −62.6 ± 1.1 1.11 ± 0.05 TTX (11) −60.9 ± 1.5 1.19 ± 0.07 Resting potentials and input resistances of pyramidal and bipolar neurons grown for 2 d under the different experimental conditions. Values were measured under conditions where synaptic transmission was blocked with CNQX and bicuculline. Values are mean ± SEM for the number of neurons indicated in parentheses.
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