|
Figure 1. Increased expression of α4 subunit‐containing GABARs in human epilepsy. (A) GABAR 4 subunit protein expression in control and epilepsy cortical tissue. A representative blot from three control, three epilepsy‐ganglioglioma, and three epilepsy‐FCD IIa patients is shown. 30 μg of protein was used for each well and β‐tubulin expression was used as a loading control. Anti‐α4 subunit (1:250 dilution, Novus Biologicals NB300‐194, Littleton, CO) and anti‐β‐tubulin (1:2000, Novus Biologicals NB600‐1514) antibodies were used. (B) The expression of α4 subunits in epilepsy tissue (336.1 ± 42, n = 19) relative to that in control tissue (102.5 ± 12.9, n = 10) ***P < 0.001, unpaired t‐test.
|
|
Figure 2. Diminished neurosteroid modulation of GABARs in human epilepsy. (A) Potentiation of GABA‐evoked currents by increasing concentrations of allopregnanolone recorded from oocytes incorporating control and epilepsy GABARs, n = 22–26 cells expressing receptors isolated from four control and epilepsy tissues each (*P < 0.05 vs. corresponding control, repeated measures two‐way ANOVA followed by Bonferroni post hoc analysis). At 1 μmol/L allopregnanolone, the potentiation was 730 ± 90% in control tissue and 510 ± 50% in epileptic tissue, and at 3 μmol/L allopregnanolone it was 970 ± 80% versus 660 ± 70%. (B) Representative traces of currents evoked by GABA (10 μmol/L) in the presence or absence of allopregnanolone (3 μmol/L) in an oocyte expressing control or epilepsy GABARs.
|
|
Figure 3. Enhanced modulation of GABAR currents by Ro15‐4513 in human epilepsy. (A) Differential effects of Ro15‐4513 on GABA (10 μmol/L)‐evoked currents in oocytes incorporating control and epilepsy GABARs; n = 26–31 cells expressing receptors isolated from four control and epilepsy tissues each. *P < 0.05 versus corresponding control, repeated measures two‐way ANOVA followed by Bonferroni post hoc analysis. (B) Representative traces of currents evoked by GABA (10 μmol/L) in the presence or absence of Ro15‐4513 (1 μmol/L) in an oocyte expressing control or epilepsy GABARs. Ro15‐4513 enhanced the current in oocytes incorporating epilepsy GABARs by 22 ± 5%, whereas it had no effect (−3 ± 5%) on control GABARs.
|
|
Figure 4. Reduced neurosteroid modulation of synaptic currents in HK‐treated neurons. (A) Representative voltage clamp recordings showing the effect of allopregnanolone (10 nmol/L) in cultured dissociated hippocampal neurons either untreated or treated with HK (10 mmol/L, 48 h). (B) Averaged current from representative control and HK‐treated neurons before (black) and after application of allopregnanolone (gray). Bath application of allopregnanolone (10 nmol/L) prolonged the decay from 35 ± 2 msec to 43 ± 3 msec (n = 13, P < 0.0005, paired t‐test) in control cultures. In contrast, the decay was only prolonged from 29 ± 2 msec to 34 ± 2 msec in the HK‐treated neurons (n = 12, P < 0.005, paired t‐test). All values are presented as mean ± SEM. (C) The allopregnanolone modulation of tonic current in HK‐treated and control neurons. The arrows represent the start of bath application of allopregnanolone (10 nmol/L) and picrotoxin (50 μmol/L). Total tonic current in HK‐treated and control cultures was 25 ± 4 pA, n = 5 and 28 ± 3 pA, n = 6 neurons, P > 0.05. Allopregnanolone enhanced the tonic current by 26 ± 5 pA in HK‐treated neurons (n = 14), similar to that in control neurons, 24 ± 3 pA, n = 12, P > 0.05, t‐test.
|
|
Figure 5. Increased surface expression of γ2 and α4 subunits in HK‐treated cultures. (A) Representative western blots showing the surface and total expression of the γ2, α4, and δ subunits in HK‐treated (HK) and control (C) organotypic hippocampal slice cultures. The expression of β‐actin is shown to demonstrate the purity of the surface samples. Mouse monoclonal anti‐γ2 subunit antibody 10F10‐C1‐B8 (2 μg/mL dilution),
49
rabbit polyclonal anti‐α4 subunit antibody (1:400 dilution, Millipore, Billerica, MA), anti‐ subunit antibody (1:1000, Millipore, Billerica, MA), and anti‐β‐actin (1:5000, clone AC‐40, Sigma‐Aldrich) were used. Lane M shows molecular weight marker. (B) Total (T) and surface (S) expression of γ2, α4, and δ subunits in HK‐treated slice cultures. Data represent average and standard error from n = 11 for γ2 subunits, n = 6 for δ subunits and n = 4 for α4 subunits, *P < 0.05. The subunit protein expression in HK‐treated cultures was normalized to that in control cultures which were run in parallel in every experiment. The total γ2 subunit expression in HK‐treated cultures was 179 ± 25% of that in control cultures (P < 0.05, Mann–Whitney test), the δ subunit expression was 91 ± 10% of that in controls (P > 0.05), and that of the α4 subunit was 139 ± 56% of that in controls (P > 0.05). The normalized surface expression (S) of γ2 subunits with HK treatment was 153 ± 11% of that in controls (P < 0.05, Mann–Whitney test, Fig. 4B), the α4 subunit surface expression in HK‐treated neurons was 158 ± 7%, P < 0.05, and the δ surface expression of the subunit in HK‐treated neurons was 121 ± 16%, P > 0.05.
|
|
Figure 6. HK treatment increased co‐localization between γ2 and α4 subunits. (A) Images demonstrating surface immunoreactivity of the γ2 (green) and α4 (red) subunits and their colocalization over dendrites of representative control and HK‐treated neurons. Arrows mark colocalization of the immunoreactive puncta. (B and C) Number of pixels corresponding to the γ2 and α4 subunit immunoreactivity in control and HK‐treated neurons, respectively, n = 16 control neurons from four batches of cultures and n = 26 HK‐treated neurons from 4 batches of cultures, *P < 0.05, **P < 0.005. The average number of γ2 puncta per neuron in controls was 338 ± 84 (n = 16 neurons from 4 replicate cultures), and in HK‐treated neurons it was 654 ± 76 (n = 26 from 4 replicate cultures, P < 0.005, t‐test). The number of α4 subunit puncta was also higher with 170 ± 14 in HK‐treated neurons compared to 97 ± 9 in control neurons (P < 0.05). (D) The number of pixels at which the γ2 and α4 subunit immunoreactivities colocalized, the number of replicates is identical as that in panel B and C, *P < 0.05. The average number of colocalized puncta was 146 ± 13 in control neurons and 395 ± 71 in HK‐treated neurons. (E) Averaged currents illustrating the effect of Ro15‐4513 (300 nmol/L, grey trace) on mIPSCs recorded from a representative control and HK‐treated neuron. The black trace illustrates current before the application of Ro15‐4513. In the control neurons decay shortened from 42 ± 5 msec to 37 ± 5 msec (n = 6 neurons/6 replicate cultures, P < 0.05, paired t‐test), whereas it remained stable in HK‐treated neurons (40 ± 4 msec and 42 ± 6 msec, n = 7 neurons/6 replicate cultures, P > 0.05, paired t‐test). The average change in decay was −5.6 ± 1.5 msec in control neurons and 1.7 ± 1.1 msec in HK‐treated neurons (P < 0.05).
|
