Normal visual acuity and orientation discrimination before and after a selective knockdown of

It was investigated whether a knockdown of PSD-95 in the VC of mice (P 0) would result in a similar phenotype as observed in mice with a global knockout of the protein. PSD-95 KO mice exhibit normal visual acuity (Huang et al., 2015; see: Figure 38 A) but impaired orientation discrimination (Figure 38 B) which was tested in the visual water task (Götze and Wenzel;

Löwel laboratory, Göttingen).

WT mice (~P 60) that were either transduced with AAV – sh95 (PSD-95 knockdown) or AAV –

Results

is a behavioral task based on reinforcement learning (Figure 37 A). Both visual acuity and orientation discrimination was examined.

Mice with a knockdown of PSD-95 in both VC showed normal visual acuity and had similar values to that of control injected (AAV - shlc) mice (AAV - sh95: 0.51 ± 0.01 cycles/degree (cyc/deg); n = 4; AAV - shlc: 0.54 ± 0.02; n = 5; p = 0.23; t-test). The values were comparable to that of WT and PSD-95 KO mice (PSD-95 KO: 0.51 ± 0.03 cyc/deg, n = 8; p = 0.95, t-test; WT:

0.56 ± 0.04 cyc/deg, n = 8, p = 0.4; t-test) (Figure 38 B).

Likewise, orientation discrimination was similar in PSD-95 knockdown and control injected mice (AAV - sh95: 15 ± 3.3°; n = 5; AAV - shlc: 14.6 ± 1.3; n = 4; p = 0.94; t-test; Figure 37 C).

Figure 37: Maximum spatial frequency threshold and minimum angle difference of WT mice with VC restricted injections of AAV – shlc (control) or AAV – sh95 (knockdown of PSD-95) at P0 – P2; determined in the visual water task (Prusky, 2000). Maximally perceived spatial frequency (visual acuity) and orientation discrimination of WT mice injected with AAV - shlc (control; n = 4) or a knockdown of PSD-95 (AAV - sh95; n = 5) in the visual cortex. (A) Schematic overview of the visual water task which allows testing of both visual acuity and orientation discrimination of mice. (B) To determine visual acuity, mice are trained to swim towards a monitor screen displaying a stationary, sinusoidal grating of black and white bars, while isoluminent gray is displayed on the reference screen. Visual acuity of mice with a knockdown of PSD-95 in the visual cortex was similar to that of control mice (p = 0.23, t-test). (C) To test orientation discrimination mice are trained to distinguish a horizontal grating from a vertical grating. In front of the stimulus screen, a hidden platform is placed underneath the water surface serving as a reward. Orientation discrimination was not impaired and similar in both groups (p = 0.94, t- test). Each data point indicates the mean of 3 values obtained from each animal. Bars represent mean ± SEM.

Results

Orientation discrimination of PSD-95 knockdown and control injected mice was comparable to values from that of WT mice (WT: 21 ± 3°; n = 7; p = 0.2; t-test) (Figure 38 A). However, they were significantly different from values obtained from PSD-95 KO mice (PSD-95 KO: 51 ± 4.6°; n = 11; p < 0.001; t-test) (Figure 38 B). PSD-95 KD mice showed significantly better orientation discrimination than ubiquitous PSD-95 KO mice which were clearly impaired (PSD- 95 KO: 51.2 ± 4.6°; n = 11; p < 0.001; t-test) (Figure 38 B).

Figure 38: Maximum spatial frequency threshold and minimum angle difference of WT, PSD-95 KO, WT AAV – shlc and PSD-95 KD mice determined in the visual water task (Prusky, 2000). (A) Maximally perceived spatial frequency (visual acuity) of WT mice (n = 8), PSD-95 KO mice (n = 8) and WT mice injected with AAV - shlc (control; n

= 4) or a knockdown of PSD-95 (AAV - sh95; n = 5) in the visual cortex. Visual acuity (cyc/deg) was similar in all tested groups (p > 0.05; t-test). (B) Orientation discrimination of WT mice (n = 7), PSD-95 KO mice (n = 11) and WT mice injected with AAV - shlc (control; n = 4) or a knockdown of PSD-95 (AAV - sh95; n = 5) in the visual cortex. Orientation discrimination was clearly impaired in PSD-95 KO mice compared to that of WT mice (p <

0.001, t-test) and significantly different in comparison to PSD-95 knockdown mice (p < 0.001, t-test). WT mice either injected with AAV –shlc (control) or AAV – sh95 (PSD-95 KD) in the visual cortex obtained similar values to that of WT mice (p > 0.05 for all comparisons, t-test). Each data point indicates the mean of 3 values from each animal. Bars represent mean ± SEM. (WT and ubiquitous PSD-95 KO mice were tested by Götze and Wenzel, Löwel laboratory, ENI, Göttingen).

Results

To get better insights if a loss of PSD-95 only in the visual cortex might influence visual capabilities, visual acuity and orientation discrimination was also tested in WT mice before and after a knockdown of PSD-95 in the VWT.

WT mice (P 84 – 111; n = 12) showed normal visual acuity (0.58 ± 0.01 cyc/deg) and orientation discrimination (11.6 ± 0.94°) measured in the VWT (Figure 39) and both visual capabilities did not change after a knockdown of PSD-95 in the visual cortex neither for visual acuity (before injection: 0.57 ± 0.02 cyc/deg; after PSD-95 KD: 0.57 ± 0.02 cyc/deg, n = 7; p = 0.3, t-test) nor for orientation discrimination (before injection: 12.1 ± 1.1°; after PSD-95 KD: 11.2 ± 0.8 °, n = 7; p = 0.6, t-test) (Figure 39). Similarly, control animals injected with AAV - shlc also showed unaltered visual acuity (before injection: 0.58 ± 0.02 cyc/deg; after injection: 0.58 ± 0.04 cyc/deg, n = 5; p = 0.8, t-test) and orientation discrimination (before injection: 11 ± 1.7°; after injection: 9.2 ± 1.3 °, n = 5; p = 0.4; t-test) before and after the injection (Figure 39). PSD-95 KD and control injected animals had also similar visual acuity (p = 0.9; t-test) and orientation discrimination (p = 0.2; t-test). (VWT experiments were done by Lübben (Bachelor student) and Stodieck).

Figure 39: Maximum spatial frequency threshold and minimum angle difference determined in the visual water task in WT mice before and after a knockdown of PSD-95 in the visual cortex. Maximally perceived spatial frequency (visual acuity) and orientation discrimination of WT mice before and after injection with AAV - shlc (control; n = 5) or a knockdown of PSD-95 (AAV - sh95; n = 7) in the visual cortex. (A) Visual acuity of WT mice with a knockdown of PSD-95 in the visual cortex was similar to that of control mice (p = 0.7, t-test) and did

Results

not change after virus injection in the control group (p = 0.8) or in mice after knockdown of PSD-95 (p = 0.3). (B) Likewise, orientation discrimination was not impaired after a visual cortex restricted knockdown of PSD-95 (p = 0.2, t-test) and did not change in the respective groups before and after virus injection in the control group (p = 0.38, t-test) or in the knockdown group (p = 0.55, t-test). Each data point indicates the mean of 3 values obtained from each animal. Bars represent mean ± SEM.

There are two possible explanations why there is a discrepancy between the orientation discrimination of ubiquitous PSD-95 KO mice and mice with a knockdown of PSD-95 only in the visual cortex. Either the transfection efficiency of the virus was not good enough to elicit a behavioral change or there could be other brain regions involved in which PSD-95 is responsible for normal orientation discrimination. However, this seems to be unlikely since testing the transfection efficiency in brain slices revealed no homogenous distribution of the virus in the visual cortex and variation between the different animals could be observed (see Figure 42). Furthermore, our collaborating group under the direction of Prof. Dr. Dr. Schlüter repeated these experiments and could indeed find impaired orientation discrimination in visual cortex restricted PSD-95 knockdown mice (preliminary data).

3.8. V1-specific knockdown of PSD-95 expression after the phase for OD-plasticity