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It has been previously published that rin1 mRNA is undetectable in embryonic brain, expressed at extremely low levels at P0 and is still weak at P6 (Dhaka et al., 2003). These findings were supported by a developmental expression profile I did by Western blot (Figure 23). Forebrain lysates of mice ranging in age from E13.5 (lysate from whole head)

48 to adult showed no Rin1 expression before P6. Rin1 knock-out forebrain lysate revealed no signal at the expected size (97kDa). EphA4 expression increases only slightly during development whereas the presynaptic marker Synaptophysin is virtually undetectable before E13.5 but shows a marked increase in expression over time paralleling the establishment of synapses.

Figure 23 Developmental expression profile of Rin1

Mouse forebrain protein samples (E13.5 –P48) were subjected to immunoblotting with the antibodies indicated

I used immunhistochemistry with anti-Rin1 antibody and in-situ hybridisation to further characterise the expression pattern of Rin1 in the adult brain. Coronal sections of adult mouse brain were stained with anti-Rin1 antibody or subjected to in-situ hybridisation with rin1 antisense probe. As can be seen in Figure 24, panel A, most Rin1-immunoreactive neurons in the cortex were found in layers II-III and layerV; weaker staining could be also observed in other layers. Panel B shows individual neurons at a high magnification, taken from the same section as shown in A. Panels C and D show localisation of Rin1 protein and mRNA, respectively, in the hippocampus. We observed that Rin1 not only localises to cell bodies but also to the apical and basal dendrites, especially in the CA1 region of the hippocampus (see Figure 24, C).

Figure 24 Localisation of Rin1protein (A-C) and mRNA (D) in the adult mouse brain

Coronal sections of adult mouse brain, stained with anti Rin1-antibody (A-C) or subjected to in-situ hybridisation (D) with rin1 antisense probe. Scale bars 100µm in A, C, E, 10µm in B.

49 I further set out to determine the specific sublocalisation of Rin1 in neurons in vivo, in particular whether Rin1 is present at synapses as suggested by the stainings performed on cultured hippocampal neurons (Figure 22). To address this question, immunofluorescence stainings were performed on adult brain tissue. For technical reasons, the antibody against PSD-95 could not be used for stainings on tissue sections. Stacks of confocal images were acquired and analysed for localisation of Rin1-positive puncta with regard to the localisation of Synaptophysin-positive puncta. Visual examination of these stainings showed a remarkable degree of Synaptophysin-Rin1 juxtaposition indicative of an at least partly, postsynaptic localisation of Rin1. Therefore, a quantitative analysis was performed in such a way that all Synaptophysin positive puncta in single optical sections were marked with a mask of circles around the spots of staining and counted. Then this mask was overlaid on the merged double staining of both Rin1 and Synaptophysin and the number of circles that also encompassed a juxtaposed Rin1-positive spot was counted. The average number of Synaptophysin-positive puncta in a 512 by 512 pixel quadrant at 63x magnification was 340, a mean of 133 had a juxtaposed Rin1-immunoreactive spot, which corresponds to 39%. For pictures of the staining and the quantitative analysis, please see Figure 25.

Figure 25 Synaptic localisation of Rin1

Rin1 staining in red, Synaptophysin staining in green. (A) Rin1 localisation at synapses in the CA1 region of the hippocampus. Insets show boxed areas in the single and merged channels at high magnification. Images were acquired at 63x magnification with a confocal microscope.

50 Scale bar 50μm. (B) High magnification of a single synaptophysin punctum (left panel, red) and Rin1 punctum (middle panel, green) and the merged image suggesting a synaptic structure. The close juxtaposition of the two puncta is indicative of a partly post-synaptic localisation of Rin1. (C) Average number of puncta counted in 512 by 512 pixel fields of single optical confocal planes at 63x magnification. Error bars represent STDEV. On average, 133 of the mean total 340 Synaptophysin puncta had juxtaposed Rin1 puncta (t-test, 1 tailed, 2 sample equal variance: 0,0005) corresponding to 39%.

2.2.3.1 Rin1 and EphA4 – a comparison of expression patterns

When investigating Rin1 distribution in the adult brain, I found striking similarities in the pattern when comparing it to the expression of Eph-receptors, in particular EphA4. I used in-situ hybridisation on coronal slices of adult mouse brain for the comparison. In the hippocampus, both messages were expressed at high levels throughout all regions, namely CA1-CA3 and dentate gyrus (see Figure 26, B and F). In cingulate cortical neurons their expression patterns were nearly indistinguishable (see Figure 26, C and G). In contrast to the findings of Dhaka et al., I detected a strong signal of rin1 mRNA in the thalamus, distributed in a “salt-and-pepper” fashion strongly reminiscent of ephA4 (see Figure 26, D and H). In the amygdala I could observe differences, since ephA4 message was most pronounced in the lateral nucleus (LA) and weak in the basolateral nucleus (BLA) whereas rin1 was detected at more equal levels throughout these nuclei and also detectable in other regions of the amygdala (see Figure 26, A and E and for a scheme of amygdaloid nuclei, please refer to chapter 1.4, Figure 12).

The similarity of expression patterns of rin1 and epha4 were intriguing and prompted me to further investigate the possible functions of Rin1 in postnatal neurons, possibly downstream of Epha4 signalling.

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Figure 26 Comparison of rin1 and ephA4 expression in the adult mouse brain

Coronal sections of adult mouse brain were subjected to in-situ hybridisation with rin1 and ephA4 antisense riboprobes. Scale bars equal 500μm in A, B, E and F and 100μm in C, D, G and H. Orientation indicated by crosses. (L) lateral, (D) dorsal, (M) medial, (V) ventral.

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