Fig 1.
Cortical EEG and spectrogram analysis procedures.
(A) Chronic cortical stainless steel electrodes (a) between right sensorimotor and visual cortex, (b) on the right frontal area, and (c) a ground electrode on the left sensorimotor area, (d) hole for icv injection of CNF1. (B) Digital amplifier-recording system. (C) EEG example tracks registered in awake (upper) and in sleep state. (D) Spectral analysis of 60 seconds of EEG traks from mice in wakefulness (left panel) and sleep state (right panel).
Fig 2.
Frequency-and-time domain analysis procedures and experimental protocol.
(A) Example of EEG (lower part of figure) and the calculated spectrogram (higher part of figure). Please note high frequency power values in the spectrogram, corresponding to series of polyspikes in the EEG. Spectral plot was set at 1–100 Hz (yellow square) and at 100–500 Hz (red square). (B) Enlarged record (total recording time 3s) of a seizure episode recorded in Fig 2A, showing a short episode of spontaneous polyspike-and-wave discharge, which is one of the typical seizure pattern recorded in the adult DBA/2J mouse. (C) Experimental protocol and time course.
Fig 3.
Spectral analysis of SWD and differences in SWDs between saline- and CNF1-treated mice.
(A) Example of a 5 seconds SWD episode track evaluated through spectral analysis. Please note the differences in frequency wave between an SWD EEG track with respect to a control track. (B) Differences in total sum in time (seconds) of SWDs between saline (n = 6) and CNF1-treated mice (n = 6), * for p<0.05. Graphs report means ± SEM.
Fig 4.
Differences in HFOs between saline and CNF1-treated mice.
(A) Two calculated spectrograms from periograms of 600s recorded in a representative pathological mouse (25 week-old mouse) treated with saline or with CNF1 (respectively upper and lower part of the figure). Please note the presence of HFO phenomena in the 25 week-old control mouse, phenomena that are greatly decreased in the treated mouse. (B and C) Data from imaging analysis of frequency-and-time domain in the somatosensory cortex of control vs CNF1-treated mice. (B) Band 1–100 Hz, saline (n = 10) vs CNF1 (n = 10). (C) Band 100–500 Hz saline (n = 10) vs CNF1 (n = 10), ** for p<0.01. Graphs report means ± SEM.
Fig 5.
CNF1 effect on the expression of neuroplasticity markers in cortices of 25 week-old mice.
(A) Representative Western Blot of spinophilin and PSD-95 protein expressions in cortex tissue (saline, n = 6; CNF1, n = 8). The amounts of the above proteins are normalized as a function of α-tubulin (histograms, B and C). CNF1 treatment induces a significant increase of both spinophilin (B) and PSD-95 (C) protein content. ** for p < 0.01 and *** for p < 0.001.
Fig 6.
CNF1 effect on proteins involved in mitochondrial dynamics in cortex tissues from pathological mice.
(A) Representative Western Blot of fission and fusion proteins in cortex tissues from 25 week-old mice (saline, n = 6; CNF1, n = 8). The amounts of the above proteins are normalized as a function of α-tubulin. CNF1 treatment induces a significant decrease of both fission proteins hFis1 (B) and Drp1 (C), while not modifying fusion markers expression Opa1 (D) and Mfn2 (E). * for p < 0.05 and *** for p < 0.001.
Fig 7.
CNF1 effect on Drp1 phosphorylation and energetic content in pathological mouse model (25 week-old) cortices.
(A) Representative Western Blot of Drp1 phosphorylated in Ser637 (pDrp1) in cortex tissue (saline, n = 6; CNF1, n = 8). The amount of pDrp1 is normalized as a function of Drp1, in turn normalized on α-tubulin (histogram, B). Note that CNF1 induces a significant augmentation of pDrp1 expression. (C) Differences in ATP cortex content, measured by a luminometric assay, between controls (n = 10) and CNF1-treated (n = 10) mice. (D) Differences in cytochrome c oxidase activity measured by spectrophotometric assay, between controls (n = 6) and CNF1-treated (n = 6) mice. The graphs report means ± SEM. * for p < 0.05, ** for p < 0.01 and *** for p < 0.001.