V1-specific knockdown of PSD-95 expression after the phase for OD-plasticity (beyond P 110;

The same mice that were tested in the visual water task before and after virus injections with AAV – shlc or AAV – sh95 (> P 110), were also tested for OD-plasticity using optical imaging of intrinsic signals. In standard cage raised WT mice, OD-plasticity is gone after P 110. Hence, it was of interest to examine whether a visual cortex restricted knockdown of PSD-95 after the phase for OD-plasticity would also restore juvenile-like OD-plasticity as shown before in PSD- 95 knockdown mice < P110.

Therefore, optical imaging of intrinsic signals was performed in control injected mice (AAV – shlc) and PSD-95 knockdown mice (AAV – sh95) after 4 days of MD (P 182 - 215) and after 7 days of MD (P 185 – 218) in the same animals.

After 4 days of MD, control injected (AAV - shlc) animals showed a stronger visual cortical

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region of V1 (Figure 40 A). The ocular dominance indices were positive indicating contralateral dominance and the representative 2-dimensional color coded ocular dominance maps in the binocular part of the left V1 showed warm, red colors (Figure 40 A). This did not change after 7 days of MD after which the binocular region of the V1 was still dominated by inputs coming from the contralateral eye and showed a higher cortical activation after contra eye stimulation than after ipsi eye stimulation. Similarly, the ocular dominance indices were positive and the 2-dimensional color coded ocular dominance maps in the binocular part of the left V1 showed warm, red colors indicating contralateral dominance (Figure 40 B).

In contrast to that, WT mice with a visual cortex restricted knockdown of PSD-95 in both hemispheres showed stronger visual cortical activation after visual stimulation of the ipsi eye than after contra eye stimulation after 4 days of MD (Figure 40 C) as well as after 7 days of MD (Figure 40 D). Hence, demonstrating OD-plasticity and ocular dominance of the ipsilateral eye in the binocular region of V1. The ocular dominance indices were negative and the 2- dimensional color coded ocular dominance maps showed cold, blue and green colors and the histogram of (C-I/C+I) was shifted to the left (Figure 40 C, D).

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Figure 40: Representative examples of ocular dominance maps and indices in WT mice injected with a control virus (AAV - shlc) or a knockdown of PSD-95 (AAV – sh95) in the visual cortex after 4 and after 7 days of MD (> P 110) obtained with optical imaging of intrinsic signals in vivo. Optical imaging maps of contralateral and ipsilateral cortical responses to visual stimulation of either eye with a horizontal moving bar (elevation) of 20°

are displayed from (A, B) a control mouse (AAV - shlc) and (C, D) a mouse with a visual cortex restricted knockdown of PSD-95 after 4 and 7 days of MD, respectively. Grayscale coded response magnitude maps (top rows, expressed as fractional change in reflectance x 10^-4) with black circles indicating monocular deprivation of the eye and white circles indicating that the respective eye remained open. Color-coded phase maps of retinotopy (bottom rows), histograms of the OD-scores (top right of panels) and color-coded OD-maps (right bottom including the average ODI value), are illustrated. (A) After 4 days as well as after 7 days of MD (B) in WT control mice, the activity patches evoked by stimulation of the contra eye were always darker than those evoked by ipsi eye stimulation. The average ODI was always positive and warm, red colors prevailed in the 2-dimensional OD-map, indicating contralateral dominance. In contrast to that, (C) 4 days and (D) 7 days of MD in WT mice with a knockdown of PSD-95 induced an OD-shift towards zero. Activity patches evoked by stimulation of the contralateral (contra) eye were equally strong to that after ipsi eye stimulation, the ODI histogram shifted to the left, the average ODI was negative and cold, blue colors prevailed in the 2-dimensional OD-map. Hence, indicating ipsilateral dominance and ocular dominance plasticity.

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These data reveal that even a late knockdown of PSD-95 renders mice plastic in the VC after 4 days of MD (ODI: -0.05 ± 0.04, n = 6) as well as after 7 days of MD (ODI: 0.04 ± 0.04, n = 6; p

= 0.1; t-test). In contrast, age-matched controls (AAV - shlc) were still dominated by sensory inputs coming from the contralateral eye and values were significantly different from mice with a PSD-95 KD after either 4 days of MD (ODI: 0.27 ± 0.03, n = 5; p < 0.001; t-test) and also after 7 days of MD (ODI: 0.23 ± 0.03, n = 3; p < 0.05; t-test) (Figure 41).

Figure 41: Ocular dominance indices and maximum response magnitude of WT mice with injections of either AAV - shlc (control, n = 5) or AAV – sh95 (knockdown of PSD-95, n= 7) in the visual cortex of both hemispheres after 4 and 7 days of MD. (A) Positive ODI values indicate dominance of the contralateral eye, whereas negative ODI values indicate ipsilateral dominance. Half-filled circles represent ODI values of individual animals after 4 or 7 days of MD; means are marked by thick horizontal lines and the standard deviation is illustrated with small horizontal lines. Control mice (AAV - shlc, green) after 4 and 7 days of MD showed ocular dominance of the contralateral eye and positive ODI, whereas mice with a knockdown of PSD-95 (blue) showed a strong OD-shift towards zero which was significantly different from control injected mice after 4 days (p < 0.001, t-test) and also after 7 days of MD (p < 0.05, t-test). (B) Cortical response amplitudes in the visual cortex expressed as a change

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in reflectance x 10^-4 after visual stimulation of the contralateral (C) or ipsilateral (I) eye in control mice (green) and PSD-95 knockdown mice (blue) after 4 or 7 days of MD. In control animals, cortical activation after visual stimulation of the contralateral eye was significantly higher than after ipsilateral eye stimulation (p < 0.05, paired t-test), reflecting the dominance of the contralateral eye in mice. In WT mice with a knockdown of PSD-95 after 4 and 7 days of MD, the response amplitudes of the two eyes were no longer significantly different after 4 days (p = 0.9, paired t-test) and after 7 days of MD (p = 0.7, paired t-test).

These results indicate that a lack of PSD-95 only in the visual cortex is already sufficient to induce OD-plasticity in WT mice. The results suggest that PSD-95 is regulating the maturation of excitatory synapses and is required for the maturation and the stabilization of cortical networks. A knockdown of PSD-95 contralateral to the deprived eye was already sufficient to trigger juvenile-like OD-plasticity even after the CP (Huang et al., 2015) and beyond the phase for OD-plasticity above P 110 (unpublished data).

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3.9. AAV transduction analysis of V1-specific PSD-95 knockdown and control mice (> P