Article Figures & Data
Figures
- Figure 1.
Action potential firing rate of neurons homeostatically stabilized after depolarization-induced increase. A, One minute traces show action potentials from representative untreated control and 7 mm [K+]-treated neurons before, during, and after acute depolarization with 7 mm [K+] external solution. B, The action potential firing rate increased in response to acute depolarization and was smaller in 7 mm [K+]-treated neurons than in controls. Loose patch recordings were obtained from untreated control neurons and from neurons that had been grown in medium treated with an elevated 7 mm [K+]. C, D, One minute traces from representative untreated control and 7 mm [K+]-treated neurons (C) show similar action potential firing rates (D). Recordings from 7 mm [K+]-treated neurons were performed in external medium with 7 mm [K+], whereas those from control neurons were in standard external medium containing 2.5 mm [K+]. *p < 0.05.
- Figure 2.
High activity treatment increased amplitude and frequency of mIPSCs. A, Trace of averaged mIPSCs from a representative HA-treated neuron had a larger amplitude than that from an untreated neuron (inset, normalized traces). B, One minute recordings from representative HA-treated and untreated neurons. C, Normalized mIPSC amplitude distribution histograms from representative HA-treated and untreated neurons revealed a shift toward higher amplitude events after HA treatment. D, Cumulative probability plot of mIPSC frequency from representative HA-treated and untreated neurons demonstrating a shift toward higher frequency of events after HA treatment.
- Figure 3.
Firing rate of HA-treated neurons increases when inhibitory transmission is reduced to control levels. A, Traces of averaged mIPSCs from representative neurons after 48 h of HA treatment (left, center) along with a trace of averaged mIPSCs from an untreated control neuron (right). Application of 0.1 μm bicuculline in the external solution during a whole-cell recording reduced the amplitude of mIPSCs of an HA-treated neuron to control levels (center). B, The firing rate of HA-treated neurons significantly increases when inhibitory transmission is reduced to the level of untreated controls using 0.1 μm bicuculline.
- Figure 4.
mIPSC amplitude increased before frequency increased during HA treatment. A, Traces of averaged mIPSCs from representative neurons after 6, 12, 24, and 48 h of HA treatment, and from untreated control neuron. B, After 12 h of HA treatment, mIPSC amplitude was larger than in untreated controls. Larger amplitudes are also observed after 24 and 48 h of HA treatment. C, Increased mIPSC frequency occurred at 48 h of HA treatment (see Table 1 for details). *p < 0.05; **p < 0.005.
- Figure 5.
GABAR γ2 subunit surface expression increased in HA-treated neurons while total expression remained unchanged. A, B, A biotinylation assay was used to label surface GABARs containing the γ2 subunit, and both surface and total γ2 subunit protein expression were measured by Western blot. Blots were reprobed with an actin antibody. A representative blot shows total expression of the γ2 subunit was similar in both HA-treated and control sample (A), whereas surface expression of the γ2 subunit was significantly higher in HA-treated samples (B). C, Total expression of the γ2 subunit in HA-treated samples expressed as a percentage fraction of control γ2 subunit expression. D, Surface expression of the γ2 subunit in HA-treated samples expressed as a percentage fraction of control γ2 subunit expression. *p < 0.05.
- Figure 6.
Both postsynaptic and presynaptic elements of GABAergic synapses were larger after 48 h of HA treatment. A–E, Clusters of GABAR immunoreactivity on the surface of untreated control neurons (A, B) and HA-treated neurons (C, D). γ2 clusters were larger after 48 h of HA treatment (E). F–J, GAD-65 immunoreactivity in untreated control neurons (F, G) and in HA-treated neurons (H, I). GAD-65 puncta were larger after 48 h of HA treatment (J). K–O, γ2 clusters colocalized with GAD-65 puncta in untreated neurons (K) (magnified inset, L) and in HA-treated neurons (M) (magnified inset, N). Colocalized puncta were larger after 48 h of HA treatment (O). Scale bar, 20 μm. **p < 0.005.
- Figure 7.
GABAR immunoreactivity increase precedes presynaptic GAD-65 increase during HA treatment. A, γ2 subunit-containing GABAR clusters, GAD-65 puncta, and colocalized puncta in neuronal dendrites. B, γ2 clusters were larger than controls at 6 h of HA treatment, GAD-65 were larger at 24 h of HA treatment, and colocalized puncta were larger at 12 h of HA treatment. All increases in puncta size were maintained at subsequent time points through the 48 h treatment. Scale bar, 10 μm. *p < 0.05; **p < 0.005.
- Figure 8.
High activity attenuated the decrease of synaptic strength after brefeldin A treatment. A, Averaged trace from a representative neuron after 90 min of brefeldin A (BFA) treatment superimposed on an average trace from a representative untreated neuron. The mean of median amplitudes for brefeldin A-treated neurons was smaller than untreated neurons. B, Average trace from a representative HA-treated neuron after 90 min of brefeldin A treatment superimposed on an average trace from a representative HA-treated neuron that was not incubated with brefeldin A. The mean of median amplitudes for HA-treated neurons was smaller in those treated with brefeldin A. C, γ2 subunit-containing GABAR clusters in dendrites from untreated and brefeldin A-treated neurons. γ2 clusters in neurons were smaller after 90 min of brefeldin A treatment. D, γ2 subunit-containing GABAR clusters in dendrites from HA-treated neurons under untreated and brefeldin A-treated conditions. In HA-treated neurons the brefeldin A treatment did not result in a decrease in cluster size. Scale bar, 10 μm.
- Figure 9.
GABARs were internalized more slowly in HA-treated neurons. A, An antibody-feeding technique was used to measure the rate of internalization of GABARs containing a labeled γ2 subunit. Representative neurons from 0, 5, 10, 20, 30, and 40 min of incubation time show more rapid loss of fluorescence from the surface of untreated neurons than from HA-treated neurons. B, The half-life of receptors on the surface was larger in the HA-treated neurons. Scale bar, 20 μm.
Tables
Amplitude (pA) Frequency (Hz) 10–90% Rise time τw Control (n = 18) 42.5 ± 1.4 0.3 ± 0.05 1.5 ± 0.06 61.2 ± 3.6 6 h (n = 7) 40.2 ± 3.8 0.4 ± 0.1 1.7 ± 0.11 46.5 ± 5.2 12 h (n = 11) 52.8 ± 2.2* 0.3 ± 0.05 1.4 ± 0.09 57.9 ± 6.3 24 h (n = 9) 65.4 ± 2.8** 0.4 ± 0.1 1.1 ± 0.10 57.3 ± 6.4 48 h (n = 11) 66.1 ± 4.4** 0.6 ± 0.1** 1.4 ± 0.19 37.7 ± 7.5* ↵*p < 0.05, **p < 0.005, ANOVA with post hoc Tukey's multiple-comparison test, n = number of neurons.
γ2 GAD-65 Colocalized Control (n = 48) 18.3 ± 0.7 57.0 ± 1.5 28.1 ± 1.2 6 h (n = 39) 22.1 ± 0.5** 53.9 ± 1.3 29.2 ± 0.9 12 h (n = 39) 21.7 ± 0.5** 54.9 ± 1.1 32.7 ± 1.3* 24 h (n = 66) 23.4 ± 0.4** 70.4 ± 1.5** 35.5 ± 0.6** 48 h (n = 45) 22.1 ± 0.5** 76.2 ± 2.1** 34.3 ± 1.0** ↵*p < 0.05, **p < 0.005, ANOVA with post hoc Tukey's multiple-comparison test. Units = μm2, n = number of neurons.
γ2 GAD-65 Colocalized Control (n = 48) 2.5 ± 0.1 1.6 ± 0.1 0.8 ± 0.1 6 h (n = 39) 2.7 ± 0.1 1.8 ± 0.1 0.8 ± 0.1 12 h (n = 39) 2.3 ± 0.1 1.6 ± 0.1 0.8 ± 0.1 24 h (n = 66) 2.9 ± 0.2 1.7 ± 0.1 0.9 ± 0.1 48 h (n = 45) 2.9 ± 0.1 1.5 ± 0.1 0.9 ± 0.1 *p < 0.05, **p < 0.005, ANOVA with post hoc Tukey's multiple-comparison test. Units = number per 10 μm2, n = number of neurons.
