Atypical electrophysiological and behavioral responses to diazepam in a leading mouse model of Down syndrome

Figures

• 

  Figure 1. Diazepam induces significantly different changes on the cortical activity spectral profile of control and Ts65Dn mice. (a) Freely moving mice implanted with EEG electrodes were connected through a cable to the recording unit. The experiment consisted of a 30-min baseline recording, followed by a 90-min post-dosing recording, in which mice were acutely treated with 1, 3, or 10 mg/kg diazepam (i.p.). (b–d) Grouped scatter plots of the first two canonical variables for all mice analyzed are shown in (b–d) for 1-, 3-, and 10-mg/kg doses of diazepam, respectively. Colored points represent mice and are labelled by cluster group. (e) Can1 discriminated the control mice group from the Ts65Dn mice group at 1-, 3-, and 10-mg/kg doses. (f) Can2 differentiated control and Ts65Dn mice groups at 1- and 3-mg/kg doses. (g) The centroid of control mice group treated with 10 mg/kg diazepam was at the highest level of the dendrogram’s hierarchical tree, whereas the centroids of Ts65Dn mice groups treated with 3- and 10-mg/kg doses had the shortest distance among all centroids. (h–j) PSD obtained from baseline and post-dosing recordings were compared for each dose in each genotype (black asterisks…

• 

  Figure 2. Ts65Dn mice show altered beta-band neuronal activity responses when compared to control euploid mice. (a) Diazepam at 1-mg/kg dose significantly increased beta-2 band power in control but not in Ts65Dn mice. (b) Diazepam-treated control mice showed an increase in the power of the beta-2 mean frequency compared to its baseline condition and diazepam-treated Ts65Dn mice. (c) Diazepam-treated control mice also showed a lower beta-1 band power than its baseline condition and diazepam-treated Ts65Dn mice. Number of mice: Ct baseline (N = 6), Ct 1 mg/kg diazepam (N = 6), Ts baseline (N = 7), and Ts 1 mg/kg diazepam (N = 7). Data represent mean ± SEM. Statistical significance is expressed as * and ** for P < 0.05 and P < 0.01, respectively.

• 

  Figure 3. Diazepam-treated Ts65Dn mice show genotype-dependent differences in neural synchrony. (a, b) Histograms of phase differences between cortical and hippocampal EEG recordings, which were computed by dividing the phase-spectrum (0–360°) into equal-sized 100 bins (3.6°/bin). (a) Control and Ts65Dn mice showed similar phase-lag distributions during baseline recordings. (b) Diazepam at 1-mg/kg dose narrowed the peak of phase-difference distribution in control mice as the number of phase-lag estimates close to zero increased, whereas phase lag distribution of diazepam-treated Ts65Dn mice exhibited an almost uniform distribution. (c) Diazepam-treated control mice showed an increase in the pairwise coherence in the beta-2 frequency range (19.40–23.20 Hz; grey asterisks) compared to their pre-injection baseline. (d) Diazepam-treated Ts65Dn mice showed no significant changes in coherent activity compared to their pre-injection baseline. Significant differences in pairwise coherence at 20.60–23.40 Hz and 43.20–44.60 Hz frequency ranges were detected between diazepam-treated control and Ts65Dn mice (black asterisks in c). Number of mice: Ct baseline (N = 6), Ct 1 mg/kg diazepam (N = 6), Ts…

• 

  Figure 4. Ts65Dn and control mice display a similar dose-dependent susceptibility to picrotoxin-induced seizures, which can be effectively prevented by diazepam. (a) Video-EEG was recorded from control and Ts65Dn mice that were i.p. injected with picrotoxin at 2- (control, N = 14; Ts65Dn, N = 15) and 4-mg/kg doses (control, N = 11; Ts65Dn, N = 14). The experiment consisted of a 30-min baseline recording, followed by a 90-min post-dosing recording. (b) The administration of 2 mg/kg picrotoxin evoked convulsive seizures (CS) in approximately 64% and 47% of control and Ts65Dn mice, respectively. All mice of both genotypes exhibited CS when treated with 4 mg/kg picrotoxin. (c, d) Picrotoxin produced a dose-dependent effect on seizure severity score (c) and latency for CS onset (d) in both control and Ts65Dn mice, the highest dosage of picrotoxin (4 mg/kg) induced more severe seizures within a shorter period of time. (e) Survival curves for control and Ts65Dn mice treated with 4 mg/kg picrotoxin were not significantly different. (f) We then acutely pretreated control and Ts65Dn mice with 3 mg/kg diazepam (i.p.) before seizure-induction by 2 mg/kg picrotoxin. The experiment consisted of a 30-min…

• 

  Figure 5. The magnitude of CA1 TBS-induced LTP is depressed by GABA and diazepam in control- but not in Ts65Dn-derived slices. (a, b) The calculated mean fEPSP slopes of TBS-induced LTP for control- (a) and Ts65Dn- (b) derived slices. Arrow indicates LTP induction, and representative traces show synaptic response during baseline (time 1) and at end of recording (time 2). The magnification of the last 10 min of recording is also shown. Scale bars represent 1 mV (horizontal) and 10 ms (vertical). (c, d) Summary graph of the LTP data from control- (c) and Ts65D- (d) derived slices (mean fEPSP slope during the last 10 min of recording). Both GABA and diazepam reduced the mean level of TBS-induced LTP in control-derived slices, whereas neither GABA nor diazepam affected the mean level of TBS-induced LTP in Ts65Dn-derived slices. (e) Normalized data show a greater effect of inhibition on LTP in control slices at 200 μM GABA and 10 μM diazepam than in those derived from 48 Ts65Dn mice. Number of slices (one slice per mouse for each condition): Ct aCSF (N = 12), Ct 200 μM GABA (N = 12), Ct 1 μM diazepam (N = 12), Ct 10 μM diazepam (N = 12), Ts aCSF (N = 12), Ts 200 μM GABA (N = 12), Ts 1 μM…

• 

  Figure 6. Ts65Dn mice show altered sensitivity to diazepam modulation of anxiety-like behavioral responses compared to control euploid mice. (a) Control and Ts65Dn mice were i.p. injected with either diazepam at 1-mg/kg dose (control, N = 16; Ts65Dn, N = 14) or saline (control, N = 16; Ts65Dn, N = 14) 30 min before testing in the EPM. (b) Representative trial trajectories of saline- and diazepam-treated mice exposed to EPM task. (c, d) Diazepam-treated control mice showed an increase in both the number of open arms entries (c) and the percentage of time spent in open arms (d) than vehicle-treated control mice. (e) Diazepam-treated control mice also showed an increase in total arms entries than vehicle-treated control mice. Regarding ethological parameters, (f) diazepam at 1-mg/kg dose decreased the percentage of stretched attend posture (SAP) in control mice compared with vehicle-treated control mice. Vehicle-treated Ts65Dn mice had significantly less protected SAP than vehicle-treated control mice. (g) Diazepam-treated control mice showed an increase in the percentage of unprotected SAP in control mice than vehicle-treated control mice. (h) Diazepam at 1-mg/kg dose also decreased the…