Analyses of gene expressions: In order to ensure maximum reliability and validity of results from experiments downstream of RNA isolation, the nucleic acid integrity was

measured through MOPS/FA gel electrophoresis. With the exception of one sample from the liver of an animal treated for 49 hrs (which was not used for further analyses) the integrity of all RNA samples was considered more than sufficient. An exemplary gel shows clear bands for 18S and 28S rRNAs, whereby the 28S band is larger than the 18S band (Fig. 3.2_15).

Fig. 3.2_15: Exemplary MOPS/FA gel showing the integrity of extracted RNAs

Since it was not possible to record and quantify parameters of behavior which could identify the development of a schizophrenia-like phenotype (e.g. prepulse inhibition, latent inhibition, vertical activity or rearing behavior), animals were observed by blind raters and could always be accurately identified as control or experimental animals respectively.

Additionally, the expression of serine racemase mRNA was examined after 1 hour of treatment as a metabolic internal positive control, since it has been shown that MK-801 leads to an increase in serine racemase expression. Using two different pairs of primers it was shown that animals treated with MK-801 did indeed have a markedly higher expression of serine racemase compared to vehicle-treated controls (Fig. 3.2_16).

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Fig. 3.2_16: RT-PCRs for serine racemase using two different primer pairs as metabolic internal positive control of MK-801 treatment

The application of equal amounts of RNA for first strand synthesis was controlled through parallel analyses of a housekeeping gene, 28S rDNA, which encodes for 28S rRNA.

After 1 hour of treatment with MK-801 increases in gene expression in the forebrain were observed for serine racemase and D-aspartate oxidase and to a small extent for PEX14.

Due to the non-quantitative nature of gel electrophoresis, especially the latter finding should be interpreted with care. No differences were observed in catalase, Nrf2 or any of the liver samples (Fig. 3.2_17).

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Fig. 3.2_17: RT-PCRs of selected genes from forebrain and liver of animals treated with MK-801 or a vehicle for 1 hour

Housekeeping controls for the expression profile of the 28S rDNA-gene show that equal amounts of RNA were used for first strand synthesis and that subsequently equal amounts of amplified cDNA were loaded onto the gel (Fig. 3.2_18).

Expression profiles of serine racemase are somewhat ambivalent. Even though, as expected, signal intensity is higher after 25 and 73 hrs in the MK-801-treated group, the expression appears to also increase after 49 and 73 hrs in the vehicle-treated animals.

Especially after 49 hrs the intensity appears to be higher in the vehicle- rather than the MK-801-treated animals.

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Fig. 3.2_18: RT-PCRs of serine racemase and 28S rDNA (housekeeping gene) from the forebrain of animals treated for 25, 49 or 73 hrs with MK-801 or a vehicle as controls for the MK-801 treatment and the use of equal amounts of RNA for first strand synthesis as well as amplified cDNA for gel loading

Expression profiles of MAOA and COMT genes are similar in that there appears to be a slight decrease in MK-801-treated animals after 25 hrs followed by an upregulation of gene expression after 49 hrs. The major difference between these two genes shows up after 73 hrs, where MAOA expression does not appear to be altered between groups, whereas COMT expression seems slightly decreased. The expression patter of MAOB, however, differs markedly from that of the other two degrading enzymes in that levels decrease in both groups from 25 over 49 to 73 hrs of treatment. Additionally the expression in the MK-801-treated animals is lower in comparison to controls at all given time points.

Fig. 3.2_19: RT-PCRs of the genes coding for dopamine metabolizing enzymes from the forebrain of animals treated for 25, 49 or 73 hrs with MK-801 or a vehicle

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Both antioxidant enzyme expression levels appear to increase after treatment with MK-801 (Fig. 3.2_20). In the case of catalase there appears to be no difference detectable between vehicle treated groups (25, 49 or 73 hrs), but in the appropriate MK-801 treated brains the signal intensity is always higher, with the most pronounced increase found after 49 hrs. The differences in SOD2 expression levels are more complex. After 25 hrs the difference between control and MK-801 brains is minimal, but after 49 and 73 hrs it becomes apparent that SOD2 expression is induced in animals treated with MK-801.

Interestingly, however, the levels in the 73 hrs group are lower both in vehicle and MK-801 treated animals compared to the 49 hrs group, even though the expression is still relatively higher after MK-801 treatment. A possible explanation herefore could be that the increase in expression after 49 hrs creates a surplus of enzyme, wherefore not as many new mRNA copies need to be transcribed. Alternatively the stability of the mRNA could be altered, whereby each strand could serve as template for the translation of relatively more protein copies. In any case, the amount of enzyme present, as examined through immunolabeling, is equal in all vehicle-treated groups and always relatively higher in the MK-801-treated animals (see below). Unfortunately this explanation does not account for the reduction in band intensity in the vehicle treated animals after 73 hrs.

Fig. 3.2_20: RT-PCRs of the genes coding for the antioxidant enzymes catalase and SOD2 from the forebrain of animals treated for 25, 49 or 73 hrs with MK-801 or a vehicle

Marker proteins of peroxisomes (Pex14p) and astrocytes (GFAP) show differential expression patterns. While treatment with MK-801 appears to have little to no effect on the expression of the PEX14 gene it appears to lead to an activation of astrocytes as shown through the rapid increase in GFAP expression after 25 hrs of treatment (Fig. 3.2_21). The

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bands of all three vehicle-treated groups are relatively similar, whereas the expression of the gene in the MK-801 groups increases dramatically, but is then reduced in the 49 and 72 hrs groups. Since GFAP is a cytoskeleton protein, which can be seen in higher abundance in MK-801-treated animals in the morphological analyses as well, an increase in gene expression early after treatment with MK-801 would lead to higher protein content in the astrocytes.

Therefore an ongoing increased level of expression should not be necessary. The reduction of mRNA copies in the animals treated with MK-801 for 49 and 73 hrs could therefore be explained by the fact that the early increase after 25 hrs might be sufficient over a longer time, thereby negating the necessity for further increases in gene expression.

Fig. 3.2_21: RT-PCRs of the genes coding for peroxisomal (PEX14) and astrocyte (GFAP) marker proteins from the forebrain of animals treated for 25, 49 or 73 hrs with MK-801 or a vehicle

3.2.4.2 Morphological analyses: Immunolabeling of catalase in the hippocampus of vehicle-