Modified intrinsic optical signals indicate a protective capacity of PSD-95 loss

3.1.4.1. Loss of PSD-95 attenuates the increase of tissue light reflectance during HSD

The protective effect of PSD-95 KO against hypoxia, as so far indicated by delayed HSD onset, was also observed on the level of HSD-accompanying change in tissue light reflectance (IOS). The IOS displayed a characteristic profile with a brief, weak decrease in tissue light reflectance (visible in Figure 8A and C), followed by a sharp and pronounced increase, which slowly declined after resubmission of oxygen. Frequently, a secondary increase during recovery was visible, which was likely caused by “undershoot” of the cell volume as cells recover from the preceding swelling (Figure 8, arrowheads) (Andrew et al., 1999; Somjen, 2001). Furthermore, dendritic beading has been shown to occur in the brain following ischemia and might as well account for the increase in light scattering (Hori & Carpenter, 1994).

As seen in Figure 8A and B, the increase in reflectance was markedly attenuated in PSD-95 KO mice when oxygen was resupplied 1 or 2 minutes upon HSD onset. The same tendency was present for longer hypoxic periods (4 min, Figure 8C). Furthermore, PSD-93/95 DKO slices showed a somewhat lower reflectance change for early reoxygenation (1 minute) and a slowly developing, moderate secondary increase during recovery, which was absent in the other experimental groups. In contrast to affected intensities of tissue reflectance change, the time course seemed to be unaltered among the genotypes.

In detail, by comparing the maximal tissue reflection (Figure 8D), control slices demonstrated 20.40 ± 1.58% increase upon hypoxia for the 1 minute condition, whereas this signal was substantially reduced to 11.23 ± 1.52% in the absence of PSD-95 [F3,55 = 7.617, p < 0.001;

PSD-95 KO vs. WT, p < 0.001]. The KO of PSD-93 as well as PSD-93 and PSD-95 simultaneously did not have significant effects as compared to WT; however, reflectance increases were stronger than in PSD-95 KO mice [PSD-93 KO: 19.52 ± 1.51%; PSD-93/95 DKO: 16.46 ± 1.46%; PSD-95 KO vs. PSD-93 KO, p < 0.001; PSD-95 KO vs. PSD-93/95 DKO, p = 0.016].

I obtained similar results for IOS intensities when the brain tissue was reoxygenated 2 minutes after HSD onset. WT hippocampi showed an increase of 19.68 ± 1.33%, whereas light reflectance augmented by only 14.82 ± 1.65% in PSD-95 KO mice [PSD-93 KO: 20.82 ± 1.39%; PSD-93/95 DKO: 20.01 ± 1.27%; F3,58 = 3.720, p = 0.016; PSD-95 KO vs. WT, p = 0.020; PSD-95 KO vs. PSD-93 KO, p = 0.003; PSD-95 KO vs. PSD-93/95 DKO, p = 0.015].

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Regarding the immense prolonged and stressful hypoxic period up to 4 minutes, no genotype differences were evident [WT: 18.47 ± 1.40%; PSD-95 KO: 16.74 ± 1.84%; PSD-93 KO:

21.23 ± 1.72%; PSD-93/95 DKO: 21.21 ± 1.65%; F3,48 = 1.755, p = 0.168]. Nevertheless, tissue light reflectance recovered much slower and remained quite high after reoxygenation, especially in case of PSD-93 KO and PSD-93/95 DKO mice. Strikingly, the second increase was intensified and prolonged in all slices, irrespective of genotype (Figure 8C). All these observations point to a diminished or even absent recovery of neuronal tissue after the extensive time period of oxygen lack. This aspect, I will further examine in section 3.1.5.3 by assessing synaptic recovery via recording of evoked responses.

In conclusion, when slices were reoxygenated within less than 4 minutes, the HSD-associated increase in tissue light reflectance was strongly attenuated in the absence of PSD-95. This effect was seen in slices reoxygenated either 1 or 2 minutes after HSD onset and thus replicable. In line with my previous findings on HSD onset, these data point to a protective effect of the PSD-95 loss against hypoxia-induced network dysfunction. Strikingly, this effect was abolished by simultaneous deletion of PSD-93.

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Figure 8: Reduced light reflectance response of hippocampal tissue in PSD-95 KO mice after HSD induction. (A-C) Temporal pattern of the reflectance changes aligned to the onset of HSD (at t=0). Oxygen was resupplied 1 min (A), 2 min (B) and 4 min (C) after HSD onset, respectively. Arrowheads indicate a secondary increase in tissue light reflectance. (D) Bar graph depicting the corresponding maximal tissue reflectance rise shown in A-C. PSD-95 KO mice displayed reduced intensity changes as compared to all other genotypes in case of reoxygenation 1 min or 2 min upon HSD onset. Diminished recovery was observed in all brain slices facing the longest hypoxic time period (4 min, C). 1 min: WT, n/m = 13/9; PSD-95 KO, n/m = 16/10, PSD-93 KO, n/m = 15/10; PSD-93/95 DKO, n/m = 15/10. 2 min: WT, n/m = 15/9; PSD-95 KO, n/m = 16/10;

PSD-93 KO, n/m = 17/11; PSD-93/95 DKO, n/m = 14/10. 4 min: WT, n/m = 15/9; PSD-95 KO, n/m

= 16/11; PSD-93 KO, n/m = 17/9; PSD-93/95 DKO, n/m = 14/10. One-way ANOVA followed by post-hoc LSD multiple comparison tests. ***p < 0.001; **p < 0.01; *p < 0.05. All data were given as means ± SEM.

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3.1.4.2. Trend to decelerated HSD wave propagation in PSD-95 KO slices

Further analysis of the IOS provided information about HSD wave characteristics such as its propagation velocity and maximum spread throughout the brain slice.

At light reflectance peak, the relative tissue area being invaded by an HSD episode was calculated with reference to total hippocampal size. In WT mice, 67.92 ± 0.013% of the hippocampus displayed IOS effects, which did not differ significantly from those areas obtained in KO mice [Figure 9A, PSD-95 KO: 64.48 ± 0.017%; PSD-93 KO: 65.88 ± 0.017%;

93-95 DKO: 65.42 ± 0.014%; F3,170 = 0.864, p = 0.461]. Therefore, PSD-95 loss could not protect parts of the hippocampus from the hypoxic, depolarized state by supposably impaired synchronization of neighboring neurons, which could prevent further HSD wave propagation.

In contrast, a trend towards a genotype effect was gained from the analyzed velocities of HSD wave propagation [Figure 9B; F3,175 = 2.508, p = 0.061]. Even though the main effect was not significant, I did an exploratory comparison among the genotypes. In control samples, the velocity averaged in 6.62 ± 0.27 mm/min, which was decelerated to 5.72 ± 0.26 mm/min in the absence of PSD-95, corresponding to a decrease of ~13.6% [PSD-95 KO vs. WT, p = 0.036]. Again, propagation velocities of PSD-93 KO as well as PSD-93/95 DKO were similar to WT levels, but higher compared to PSD-95 KO [PSD-93 KO: 6.57 ± 0.28 mm/min; PSD-93/95 DKO: 6.72 ± 0.22 mm/min; 95 KO vs. 93 KO, p = 0.039; 95 KO vs. PSD-93/95 DKO, p = 0.017; all other p values > 0.05]. These observations corroborate my previous results showing partly dampened hypoxia-induced impairments in PSD-95 KO neuronal networks. Specifically, in this case, by a decelerated transfer of the depolarized and non-functional neuronal state onto the neighboring tissue. The effect of PSD-95 loss was again abrogated by simultaneous knockout of PSD-93.

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Figure 9: The HSD wave proliferated more slowly in the absence of PSD-95. (A) Bar graph illustrating the HSD invaded area as referred to total hippocampus, which was undistinguishable among genotypes. WT, n/m = 41/9; PSD-95 KO, n/m = 46/12; PSD-93 KO, n/m = 44/11; 93-95 DKO, n/m = 43/11. (B) Propagation of the HSD wave front detected in st. radiatum parallel to st.

pyramidale was diminished due to loss of PSD-95 compared to all other genotypes. WT, n/m = 41/9; PSD-95 KO, n/m = 46/12; PSD-93 KO, n/m = 45/11; PSD-93/95 DKO, n/m = 43/11. (A, B) One-way ANOVA followed by post-hoc LSD multiple comparison tests. *p < 0.05. All data were given as means ± SEM.

3.1.5. Partly reduced hypoxia-induced synaptic dysfunction in