Visual cortex restricted knockdown of PSD-93 in WT mice

Since it was shown that a visual cortex restricted knockdown of PSD-95 is sufficient to induce juvenile-like OD-plasticity in WT mice (Huang et al., 2015), it was investigated whether PSD- 93 manipulations selectively only the visual cortex are sufficient to induce an earlier closure of the CP for OD-plasticity in WT mice, as it was seen in PSD-93 KO mice before.

PSD-93 expression was silenced selectively only in the visual cortex of both hemispheres in WT mice (P 0 – P 2) with an adeno-associated virus (AAV) with a short hairpin (sh) RNA targeting PSD-93 (AAV - sh93). Control animals were injected with AAV sh - luciferase (AAV - shlc). Animals were tested between P 28 – P 35 during the late CP after 4 days of MD with optical imaging of intrinsic signals in vivo.

Similar to WT and PSD-93 KO mice in the late CP, PSD-93 knockdown mice and controls without MD showed contralateral dominance with stronger visual cortical activation after contra eye stimulation than after visual stimulation of the ipsi eye. Representative 2- dimensional ocular dominance maps in the left visual cortex of both experimental groups showed warm, red colors indicating contralateral dominance and all control mice exhibited positive ocular dominance indices (Figure 72).

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Figure 72: Representative examples of ocular dominance maps and indices in WT control mice without MD either injected with AAV - shlc (GFP controls) or AAV - sh93 (PSD-93 knockdown) in both visual cortices at P 0 – P 2. 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) two control mice and (C, D) two PSD- 93 knockdown mice without MD, respectively. Grayscale coded response magnitude maps (top rows, expressed as fractional change in reflectance x 10^-4), 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. In both groups, the activity patches evoked by stimulation of the contralateral (contra) eye were always darker than those evoked by ipsilateral eye stimulation. The average ODI was positive and warm, red colors prevailed in the 2-dimensional OD-map, indicating contralateral dominance.

After 4 days of monocular deprivation, control injected mice showed OD-plasticity with weakened cortical activation after visual stimulation of the contra eye, whereas ipsi eye stimulation activated the visual cortex similarly strong as in mice without MD. The representative 2-dimensional ocular dominance maps in the left V1 showed cold, blue colors

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indicating ipsilateral dominance. The histograms were shifted to the left and all mice exhibited negative ocular dominance indices (Figure 73 A, B).

In contrast to control injected mice, this was not the case in PSD-93 knockdown mice. Here, 4 days of MD could not induce OD-plasticity and the cortical activation after visual stimulation of the contra eye remained higher than after visual stimulation of the ipsi eye. Additionally, the 2-dimensional OD-map showed warm, red colors indicating contralateral dominance and all mice exhibited positive ocular dominance indices (Figure 73 C, D).

Figure 73: Representative examples of ocular dominance maps and indices in WT mice after four days of MD either injected with AAV - shlc (GFP controls) or AAV - sh93 (PSD-93 knockdown) in both visual cortices at P 0 – P 2. 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) two control mice and (C, D) two PSD- 93 knockdown mice after 4 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. After 4 days of MD in WT control injected mice, there was an

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OD-shift towards zero. Activity patches evoked by stimulation of the contralateral eye were equally strong to that after ipsi eye stimulation and the histogram shifted to the left. Average ODI were negative and cold, blue colors prevailed in the 2-dimensional OD-map, indicating ipsilateral dominance and hence ocular dominance plasticity. In contrast to that, in PSD-93 knockdown mice, the activity patches evoked by stimulation of the contralateral eye after MD were still darker than those evoked after ipsilateral eye stimulation. The average ODI was always positive and warm, red colors prevailed in the 2-dimensional OD-map, indicating contralateral dominance and no ocular dominance plasticity.

In mice with a visual cortex specific knockdown of PSD-93, 4 days of MD could not induce an OD-shift (ODI: 0.20 ± 0.02; n = 6), whereas in age-matched control mice injected with AAV - shlc, OD-plasticity was present after 4 days of MD (ODI: -0.07 ± 0.02; n = 3, p < 0.001; t-test) (Figure 74 A). This strong OD-shift in control injected WT mice was mediated by a reduction of deprived eye responses (WT shlc late CP contra no MD: 1.88 ± 0.06; n = 3; WT shlc late CP contra 4d MD: 0.81 ± 0.08; n = 3; p < 0.001; t-test), whereas ipsilateral eye evoked responses in V1 remained unchanged (WT shlc late CP ipsi no MD: 1.01 ± 0.01; n = 3; WT shlc late CP ipsi 4d MD: 0.93 ± 0.12; p = 0.6; t-test) (Figure 74 B). In PSD-93 knockdown mice, the visual cortex was still dominated by cortical activation of the contralateral eye after 4 days of MD (WT sh93 late CP contra no MD: 2.0 ± 0.2; n = 3; WT sh93 late CP contra 4d MD: 1.4 ± 0.2; n = 6; p = 0.1;

t-test). Furthermore, the ipsilateral eye evoked responses in V1 did not change after MD in PSD-93 knockdown mice (WT sh93 late CP ipsi no MD: 1.13 ± 0.02; n = 3; WT sh93 late CP ipsi 4d MD: 0.91 ± 0.12; n = 6; p = 0.26; t-test) as shown in Figure 74 B.

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A

B

Figure 74: Ocular dominance indices and average response magnitudes of WT mice injected with a control virus (AAV - shlc; n = 3) or with a knockdown of PSD-93 (AAV - sh93; n = 3) without MD and after 4 days of MD (control, n = 3 / PSD-93 KD, n = 6) during the late CP. (A) Positive ODI values indicate dominance of the contralateral eye, negative ODI values represent ipsilateral dominance. Unicolored circles represent ODI values of individual animals without MD, half-filled circles represent individual animals after 4 days of MD; means are marked by thick horizontal lines and the standard deviation is illustrated with small horizontal lines. WT control injected mice (green) and PSD-93 knockdown mice (red) without MD showed ocular dominance of the contralateral eye and similar ODI values (t-test, p = 0.55). Four days of monocular deprivation in control mice induced a significant OD-shift towards the open eye (t-test, p < 0.001), which was significantly different from values in PSD-93 knockdown mice, which did not show ocular dominance plasticity after MD (t-test, p < 0.001).

(B) Average cortical responses expressed as a change in reflectance x 10^-4 by stimulation of the contralateral (C) or ipsilateral (I) eye in control and PSD-93 knockdown mice without MD and after four days of MD. In all animals without MD, cortical activation after visual stimulation of the contralateral eye was significantly higher than after ipsilateral eye stimulation (control: p < 0.01; PSD-93 KD: p < 0.05; paired t-test), reflecting the dominance of the contralateral eye in mice. In control mice after four days of MD, the response strength of the two eyes were no longer significantly different. The deprived eye responses were significantly reduced compared to that

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of mice without MD (p < 0.001; t-test). In contrast, cortical responses did not change in PSD-93 knockdown mice after MD and remained higher after contralateral eye stimulation in comparison to cortical activation after ipsilateral eye stimulation (p < 0.01; paired t-test).

This result is consistent with the previous observation that 4 days of MD are not sufficient to induce OD-plasticity in PSD-93 deficient mice in the visual cortex beyond P 28. Control injected animals showed the same phenotype as WT mice with a strong OD-shift after 4 days of MD, which was also mediated by a depression of deprived eye responses in V1.

3.20. Visual capabilities measured with the virtual-reality optomotor system in WT