ERK and CREB activation in neurons and distinct vimentin-positive cells by Rolipram/BDNF co-treatment.

<p>Projections of collapsed confocal scans showed double-stainings with pERK (A, G) or pCREB (D, J) antibodies and antibodies directed against neuron-specific 200 kDa neurofilament (B, E) or vimentin (H, K) in spiral ganglion cell cultures pre-treated with BDNF (47 h, 30 min) and exposed to Rolipram for 30 min. Nuclei were stained with DAPI. Arrows point to an intense labelling of pERK (C, F) or pCREB (I, L) in the somata of stained SGN. Additionally, pERK (C, F) and pCREB (I, J) immunosignals were present in distinct vimentin-positive cells with a flattened (filled arrowhead) or spindle-shaped (unfilled arrowhead) morphology. Asterisks mark cells with a star-like morphology with a weak reactivity for pERK. Scale bar: 70 μm in C, 75 μm in F, I, L).</p

Primary antibodies and their dilutions used for immunofluorescence analysis.

<p>Primary antibodies and their dilutions used for immunofluorescence analysis.</p

ERK and CREB activation in neurons and distinct vimentin-positive cells by Rolipram/BDNF co-treatment.

<p>Projections of collapsed confocal scans showed double-stainings with pERK (A, G) or pCREB (D, J) antibodies and antibodies directed against neuron-specific 200 kDa neurofilament (B, E) or vimentin (H, K) in spiral ganglion cell cultures pre-treated with BDNF (47 h, 30 min) and exposed to Rolipram for 30 min. Nuclei were stained with DAPI. Arrows point to an intense labelling of pERK (C, F) or pCREB (I, L) in the somata of stained SGN. Additionally, pERK (C, F) and pCREB (I, J) immunosignals were present in distinct vimentin-positive cells with a flattened (filled arrowhead) or spindle-shaped (unfilled arrowhead) morphology. Asterisks mark cells with a star-like morphology with a weak reactivity for pERK. Scale bar: 70 μm in C, 75 μm in F, I, L).</p

Rolipram and BDNF application induced the activation of ERK and CREB.

<p>Immunofluorescence staining of activated ERK (pERK; A–D) and activated CREB (pCREB; E–H) in spiral ganglion cell cultures incubated in medium for 48 h (A, E) or exposed to Rolipram (R; B, F) or BDNF (C, G) for 30 min after a pre-incubation of (47 h, 30 min) in medium. D, H shows the pERK and pCREB immunoreactivity in cells pre-treated with BDNF (47 h, 30 min) and additionally exposed to Rolipram for 30 min (D, H). Photographs were taken under the same conditions in which exposure time and intensity was adjusted to the untreated control group (A, E). Application of Rolipram (B, F) or BDNF (C, G) induced the activation of ERK and CREB. Stronger pERK and pCREB immunoreactivity was observed after co-treatment with Rolipram and BDNF (D, H). BDNF: brain-derived neurotrophic factor; R: Rolipram. Scale bar: 50 μm (A–H).</p

Rolipram treatment alone and in combination with BDNF improved the survival of SGN.

<p>The neuronal survival was assessed by the amount of surviving neurons after a cultivation period of 48(% neuronal survival). Rolipram (R) applied at a concentration of 0.1 nM enhanced the survival of SGN after serum deprivation and resulted in a significant higher survival rate when compared to the untreated group and to the positive control (treated with BDNF [50 ng/ml]). Elevated Rolipram concentrations (1, 3, 10 nM) did not result in an increased neuronal survival compared to the untreated control group. Co-treatment with Rolipram and BDNF caused an additional increase of neuronal survival independently of varying Rolipram concentrations. Values are given as mean ± SEM; N = 4; n = 3; One-way ANOVA with Tukey's multiple post hoc test was used to compare means: *p<0.05; **p<0.01; ***p<0.001. Reference of the significance is marked by the thick bar. N quotes the number of independent experiments; n gives the number of repetitions of each condition within one experiment.</p

BDNF release from spiral ganglion cells is increased after co-treatment with Rolipram and BDNF.

<p>The concentration of released BDNF from spiral ganglion cells after treatment with Rolipram (R; 48 h and 30 min), BDNF (48 h) or both substances (BDNF 47R 30 min) was measured by ELISA. The supernatant of cells treated with BDNF alone or in combination with Rolipram contained significant higher concentrations of BDNF in comparison to supernatants from untreated cultures or from cells treated only with Rolipram. Application of Rolipram for 30 min to BDNF pre-treated cells resulted in a strong increase of BDNF in the supernatant. All values are given as mean ± SEM. N = 3; n = 3 except for the untreated and Rolipram 48 h group: N = 2, n = 3; One-way ANOVA with Tukey's multiple post hoc test was used to compare means: *p = 0.05; **p = 0.01; ***p = 0.001. Reference of the significance is marked by the thick bar. N gives the number of independent approaches; n gives the number of samples per approach.</p

Cell death rate in <i>rd1</i>Cx36^+/+, <i>rd1</i>Cx36^−/− and wt mice.

<p>Dying cone photoreceptors in vertical sections of wild-type (wt) (A, D), <i>rd1</i>Cx36^+/+ (B, E), <i>rd1</i>Cx36^−/− mouse retina (C, F) at the ages of p21 (A–C) and p30 (D–F) were labeled by TUNEL staining (red); nuclei were stained with DAPI (blue). Sections from wt controls indicate the normal retinal layering (A, D). The bar graph (G) summarizes the quantification of TUNEL-positive cells in the ONL in <i>rd1</i>Cx36^+/+ (grey), <i>rd1</i>Cx36^−/− (white) and wt mice (black). The amount of TUNEL-positive cells in both <i>rd1</i> genotypes was significantly increased when compared to wt mice. Comparisons between same-aged <i>rd1</i> mutants revealed no statistical differences. Values are given as mean ± SD; a t-test was used to compare means: ***, p<0.001. n specifies the number of animals. Scale bars = 20 µm in F (applies to A–F).</p

Deletion of Cx36 did not affect remodeling of ON bipolar cells in <i>Rho</i>^−/− and <i>rd1</i> mice.

<p>Vertical sections of the retina were double-stained for G0α (green), a marker for all ON BC, and PKCα (magenta), a marker for rod BC. In Cx36-expressing <i>Rho</i>^−/− and <i>rd1</i> mutants, rod bipolar cell dendrites sprouted into the ONL (B, C, L, M; long arrow) at the onset of degeneration. With progressing photoreceptor degeneration, all ON bipolar cell dendrites were retracted (D, M). In both disease models, PKCα-positive cell somata were frequently found displaced to the ONL (D, E, M, N, asterisks). Remodeling was similar in Cx36-deficient <i>Rho</i>^−/− (G–J) and <i>rd1</i> littermates (P–R). Nomarski micrographs (A, F, K, O) indicate the retinal layering. Scale bars = 10 µm in J (applies to A–J), in R (applies to K–R).</p

Time course of cone loss in <i>rd1</i>Cx36^+/+ and <i>rd1</i>Cx36^−/− mice.

<p>Quantification of cones in vertical sections of the central retina (defined up to a distance of 1,000 µm from the optic nerve), as indicated in A. B–G show magnifications of the quantified regions in the central ONL of cone arrestin-labeled vertical sections from <i>rd1</i>Cx36^+/+ (B–D) and <i>rd1</i>Cx36^−/− (E–G) mice at different ages. The bar graph (H) displays the quantification of cone arrestin-positive cells per 100 µm length at different ages in <i>rd1</i>Cx36^+/+ (grey), <i>rd1</i>Cx36^−/− (white) and wt mice (black). The number of cone photoreceptors in both transgenic mouse lines was reduced at p21 and p30 compared to same-aged wt controls. There are no statistical differences between Cx36-expressing and Cx36-deficient <i>rd1</i> mice. Values are given as mean ± SD; a t-test was used to compare means: ***, p<0.001. n specifies the number of animals. Scale bars = 200 µm in A; 10 µm in G (applies to B–G).</p

Primers used for mouse genotyping.

*<p>related to primer sequences and/or PCR conditions used.</p