Article Figures & Data
Figures
- Figure 1.
The development of inhibitory neurotransmission is dynamically altered in the Fmr1−/y mutant BLA. A, Recordings of sIPSCs recorded from BLA excitatory neurons. B–D, Histograms of pooled data for sIPSC biophysical parameters: amplitude (B), frequency (C), and decay constant (D). Mean ± SEM, sample sizes, and p values (Student's two-tailed t test vs control) are given in Table 1. *p < 0.05; **p < 0.01. E–G, Histograms of pooled data for mIPSC biophysical parameters: amplitude (E), frequency (F), and decay constant (G). Mean ± SEM, sample sizes, and p values are given in Table 1. Note that dynamic fluctuations in inhibitory function are observed over development.
- Figure 2.
Dynamic changes in the levels of synaptic GABA concentration at inhibitory synapses in the Fmr1−/y BLA. The ability of TPMPA to attenuate the amplitude of mIPSCs is inversely proportional to the concentration of GABA localized to the synapse. A, Averages of >150 mIPSC events recorded in the absence (black sweeps for control; gray sweeps for Fmr1−/y) or presence (red traces) of 100 mm TPMPA. B, Histogram of pooled data of changes in mIPSC amplitude in the presence of TPMPA. Note that TPMPA has a significantly greater efficacy at P10 (*p < 0.05) and P21 (**p < 0.01) but not at P14 or P16 in Fmr1−/y. Mean ± SEM change in mIPSC amplitude, sample sizes, and p values are given in Table 2.
- Figure 3.
Developmental increase of GABA transporter function and expression. A, B, Averages of >150 sIPSC events normalized for amplitude before (black and gray traces for control and Fmr1−/y, respectively) and after (red traces) the application of 1 μm NO-711. A, At P10, no significant differences in decay constant were observed in either genotype in the presence of NO-711. B, NO-711 significantly increased the decay constants of both control and Fmr1−/y mutant sIPSCs at P21. C, NO-711 enhancement as a measure of the percentage difference in decay constant before and after treatment with NO-711. Note that NO-711 significantly increased the decay constant of P21 Fmr1−/y sIPSCs to a greater degree than in control sIPSCs (*p < 0.05). All decay constant and NO-711 enhancement values ± SEM, sample sizes, and p values are listed in Table 2. Immunohistochemical detection of GAT1 expression in control (D) and Fmr1−/y KOs (E). LA, Lateral amygdala; PirC, piriform cortex. Scale bars, 250 μm.
- Figure 4.
GABAAR subunit-selective pharmacology of sIPSCs is disrupted in the Fmr1−/y mutant BLA. A, Sensitivity to the α1 GABAAR agonist zolpidem is reduced in Fmr1−/y mutants at postnatal ages. sIPSCs were recorded in the presence of 200 nm zolpidem, an α1 GABAAR agonist that acts to lengthen the decay constant. “Zolpidem Enhancement” measures the percentage difference between the decay constant in the presence of zolpidem and that measured in the absence of the drug. Control responses to zolpidem increased steadily throughout development, consistent with the developmentally regulated increase in α1 GABAARs (black line). In contrast, sIPSCs from Fmr1−/y mutant principal neurons showed no increase in zolpidem sensitivity (gray line). *p < 0.05; **p < 0.01. B, The developmental profile of clonazapam-induced pharmacological changes is also disrupted. Overall, clonazepam sensitivity decreases throughout development in control, consistent with the developmental decrease in α3-containing GABAARs. Note that this trend is disrupted in Fmr1−/y. *p < 0.05; **p < 0.01. All values ± SEM, sample sizes, and p values are given in Table 2.
- Figure 5.
mIPSC quantal analysis reveals smaller postsynaptic GABAAR patch sizes in Fmr1−/y inhibitory synapses. A, B, Representative mIPSC amplitude histograms for control (A) and Fmr1−/y (B) at P10, P14, P16, and P21. Dashed lines demarcate the least-squares best-fit function. Underlying Gaussian functions are shown in colored lines. C, Summary of the number of deconvolved Gaussians across cells for control and Fmr1−/y at each corresponding age. Note that the average number of Gaussians was only significantly different between genotypes at P10 (*p < 0.05) and P21 (**p < 0.01).
- Figure 6.
Peak-scaled nonstationary noise analysis suggests significantly higher unit currents and lower numbers of GABAARs in Fmr1−/y at P10 and P21 but not at P14 or P16. A, B, Current variance (σ2) and mean current (Im) plots for control (A) and Fmr1−/y (B) mIPSCs at P21. Solid lines indicate the best fit of the parabolic function σ2 = iIm − Im2/N. C, D, Mean estimated values for unit current (i; C) and the number of GABAARs (N; D) over development. **p < 0.01, Kolmorogov–Smirnoff test.
- Figure 7.
Comparison of biophysical parameters for sIPSCs in conditional KO and rescue animals to control and full KO animals. Histograms of pooled data of sIPSCs recorded from P10 (A), P14 (B), and P21 (C) time points. Amplitude, frequency, and decay constant of sIPSCs recorded from control (black bars), full KO (Fmr1−/y; gray bars), control animals with cre driver (Dlx5/6Cre; purple bars), conditional KOs (Dlx5/6Cre;Fmr1cKO; red bars), and conditional rescues (Dlx5/6Cre;Fmr1cON; green bars) cells. Sensitivity to zolpidem assessment as a measure of percentage enhancement indicates that Dlx5/6Cre;Fmr1cKO BLA principal neurons show no preference at all ages. *p < 0.05; **p < 0.001; ***p < 0.001, significantly different from control.
- Figure 8.
Summary of altered developmental inhibitory synaptic processes in FXS. The top row depicts development across the four postnatal time points in the wild-type BLA, and the bottom row shows the case of FXS. Our results suggest that at least five processes are dynamically disrupted in the development of inhibitory neurotransmission in the FXS BLA. First, changes in IPSC frequency suggest that there may be alterations in the number of synapses throughout development, with more synapses becoming active at P14 and P16. Second, because the frequency of inhibitory synaptic inputs is elevated beyond control levels at P14–P16 only when action potentials are not blocked, BLA interneurons in Fmr1−/y may have higher firing rates. Third, presynaptic mechanisms governing the amount of GABA in the synapse are transiently upregulated, restoring the amount of synaptic GABA to control levels at P14–P16. Fourth, there is a severe defect in the process of GABAAR maturation in the FXS BLA, in which mature α1 GABAARs fail to become functional, whereas immature α3 GABAARs are retained at mature ages. Finally, the number of GABAARs increases to control levels at P14–P16. However, these compensatory changes fail to be maintained, and inhibitory defects are again present in the mature FXS BLA.
Tables
Genotype (n sIPSC/n mIPSC) sIPSCs mIPSCs Amplitude (mean ± SEM pA) Frequency (mean ± SEM Hz) Decay constant (mean ± SEM ms) Amplitude (mean ± SEM pA) Frequency (mean ± SEM Hz) Decay constant (mean ± SEM ms) P10 Control (24/5) 54.9 ± 3.1 1.1 ± 0.1 34.2 ± 1.3 43.1 ± 4.2 1.1 ± 0.2 29.5 ± 3.0 Fmr1−/y (24/8) 38.4 ± 3.6 0.65 ± 0.7 41.8 ± 2.7 30.9 ± 2.2 0.77 ± 0.2 30.6 ± 1.3 p = 0.0006 p = 0.0092 p = 0.012 p = 0.0095 p = 0.0097 p = 0.815 P14 Control (30/7) 38.3 ± 1.8 1.4 ± 0.2 35.2 ± 1.4 33.3 ± 0.2 1.8 ± 0.2 24.1 ± 1.1 Fmr1−/y (14/9) 53.3 ± 7.1 3.1 ± 0.6 34.2 ± 1.4 32.3 ± 3.2 1.8 ± 0.2 23.0 ± 0.8 p = 0.0093 p = 0.0010 p = 0.689 p = 0.831 p = 0.889 p = 0.401 P16 Control (20/9) 46.7 ± 2.1 1.1 ± 0.09 27.7 ± 1.0 24.7 ± 1.0 2.2 ± 0.2 20.7 ± 1.2 Fmr1−/y (17/8) 44.4 ± 3.0 2.0 ± 0.3 28.0 ± 1.0 28.1 ± 1.9 2.2 ± 0.3 20.1 ± 1.3 p = 0.517 p = 0.0036 p = 0.831 p = 0.126 p = 0.919 p = 0.710 P21 Control (20/6) 52.1 ± 4.9 3.5 ± 0.4 19.6 ± 0.9 19.0 ± 1.2 4.3 ± 0.6 17.8 ± 0.7 Fmr1−/y (21/6) 37.1 ± 3.0 2.5 ± 0.2 27.8 ± 0.8 13.5 ± 1.2 2.0 ± 0.1 17.8 ± 1.0 p = 0.012 p = 0.031 p = 2.2 × 10−8 p = 0.010 p = 0.001 p = 0.900 Summary of sIPSC/mIPSC amplitude, frequency, and decay values. The number of cells (n) is listed in the first column with control cells and Fmr1−/y mutant cells. The values (mean ± SEM) for sIPSC/mIPSC amplitude, frequency, and decay constant are listed for P10, P14, P16, and P21 time points. A Student's two-tailed t test was performed, and p values are listed below the averaged value for each time point. p < 0.05 are shown in bold.
- Table 2.
Pharmacological assessment of IPSCs using TPMPA, NO-711, zolpidem, and clonazepam
Control (n)/Fmr1−/y (n) TPMPA efficacy (mean ± SEM %Δ mIPSC amplitude) NO-711 enhancement (mean ± SEM %) Zolpidem enhancement (mean ± SEM %) Clonazepam enhancement (mean ± SEM %) P10 −13.8 ± 2.1 (5)/−27.4 ± 3.4 (7) 2.8 ± 6.1 (9)/5.0 ± 10.6 (4) 6.4 ± 9.1 (5)/16.2 ± 2.7 (6) 54.8 ± 7.8 (11)/13.8 ± 6.3 (10) p = 0.012 p = 0.911 p = 0.230 p = 0.0005 P14 −14.7 ± 4.4 (5)/−15.7 ± 2.0 (7) ND/ND 15.0 ± 4.0 (6)/3.0 ± 0.5 (6) 45.2 ± 7.0 (8)/30.5 ± 3.9 (7) p = 0.834 p = 0.100 p = 0.102 P16 −18.4 ± 3.4 (5)/−22.2 ± 2.8 (5) ND/ND 21.6 ± 2.3 (5)/1.0 ± 11.6 (7) 34.9 ± 4.2 (5)/43.1 ± 12.0 (8) p = 0.401 p = 0.045 p = 0.490 P21 −12.2 ± 2.0 (5)/−27.6 ± 4.7 (6) 27.9 ± 10.8 (5)/57.5 ± 5.2 (7) 50.3 ± 1.7 (8)/11.3 ± 4.5 (8) 2.1 ± 4.8 (12)/ 20.6 ± 7.3 (14) p = 0.018 p = 0.022 p = 0.027 p = 0.048 Pharmacological assessment of IPSCs using TPMPA, NO-711, zolpidem, and clonazepam. TPMPA efficacy is defined as the mean ± SEM of the change in mIPSC amplitude in the presence of 100 μm TPMPA. NO-711 enhancement values (defined as the percentage change in decay constant with drug application) are given for control (roman text) and Fmr1−/y (bold text) at each of the ages tested. Zolpidem and clonazepam enhancement values (defined as the percentage change in decay constant with drug application) are given for wild-type (roman text) and Fmr1−/y (bold text) at each of the ages tested. The number of cells in each group is indicated in parentheses. A Student's two-tailed t test was performed for all conditions. The p values are listed below the averaged value, and p < 0.05 are shown in bold. ND, Not determined.
- Table 3.
Development of sIPSC characteristics in BLA principal neurons from inhibitory cell-specific conditional Fmr1 KO and rescue animals
Genotype (n) Amplitude (mean ± SEM pA) Frequency (mean ± SEM Hz) Decay constant (mean ± SEM ms) Zolpidem enhancement (mean ± SEM %) P10 Dlx5/6Cre (6) 50.2 ± 1.3 1.1 ± 0.09 31.5 ± 1.4 10.7 ± 4.3 Dlx5/6Cre;Fmr1cKO (6) 48.2 ± 2.8 1.4 ± 0.2 34.2 ± 1.0 12.0 ± 5.2 p = 0.55 p = 0.24 p = 0.15 p = 0.67 Dlx5/6Cre;Fmr1cON (5) 35.6 ± 3.7 3.5 ± 0.3 40.1 ± 2.0 4.4 ± 4.1 p = 0.93 p = 0.0005 p = 0.009 p = 0.0522 P14 Dlx5/6Cre (6) 37.7 ± 1.5 1.6 ± 0.1 34.1 ± 1.2 19.5 ± 1.2 Dlx5/6Cre;Fmr1cKO (5) 35.8 ± 3.3 3.0 ± 0.4 32.8 ± 1.3 22.0 ± 3.7 p = 0.616 p = 0.022 p = 0.466 p = 0.674 Dlx5/6Cre;Fmr1cON (8) 42.5 ± 2.7 4.5 ± 0.3 39.7 ± 0.8 5.3 ± 4.4 p = 0.200 p = 0.00002 p = 0.006 p = 0.0521 P21 Dlx5/6Cre (5) 49.1 ± 2.2 3.3 ± 0.2 18.0 ± 0.7 54.5 ± 7.3 Dlx5/6Cre;Fmr1cKO (6) 42.4 ± 2.4 3.2 ± 0.2 19.9 ± 0.7 48.0 ± 4.2 p = 0.058 p = 0.91 p = 0.073 p = 0.46 Dlx5/6Cre;Fmr1cON (9) 33.5 ± 2.4 6.1 ± 0.6 27.8 ± 0.7 5.9 ± 4.4 p = 0.00018 p = 0.0013 p = 1.0 × 10−8 p = 1.0 × 10−4 Development of sIPSC biophysical characteristics in BLA principal neurons from inhibitory cell-specific conditional Fmr1 KO and rescue animals. The mean ± SEM values for amplitude, frequency, decay constant, and zolpidem enhancement are given for sIPSCs recorded from excitatory BLA principal neurons from control Cre driver (Dlx5/6Cre), conditional Fmr1 KO (Dlx5/6Cre;Fmr1cKO), and conditional Fmr1 rescue (Dlx5/6Cre;Fmr1cON) animals. sIPSCs were sampled from animals at ages P10, P14, and P21. Sample sizes for sIPSC amplitude, frequency, and decay constant for each group are given in parentheses in the first column. Student's two-tailed t test performed for conditional Fmr1 KO (Dlx5/6Cre;Fmr1cKO) and conditional Fmr1 rescue (Dlx5/6Cre;Fmr1cON) mice were individually compared with control mice (Dlx5/6Cre). The p values are listed below the averaged value, and p < 0.05 are shown in bold.
