Figure 6

Primary dopaminergic neurons in EVM cultures from protein kinase Cδ knockout (PKCδ −/−) mice are protected against tumor necrosis factor (TNF) toxicity. Primary mouse EVM neuronal cultures were treated with recombinant TNF (30 ng/ml) in supplement-free neurobasal medium. TNF was re-added 24 h later for a total treatment time of 48 h. PKCδ wild-type (PKCδ +/+) and knockout (PKCδ −/−) EVM cultures were treated in parallel. (A) Immunocytochemistry for tyrosine hydroxylase (TH)-positive neurons. Dopaminergic neurons were identified by TH staining (green) and microtubule-associated protein 2 (MAP-2) (red) was used as a pan-neuronal marker. Images were acquired at 20 × magnification. Representative fields are shown for each treatment. In the TNF-treated group, extensive loss of neurites and degeneration of dopaminergic neurons is evident in cultures from PKCδ wild-type (+/+) mice (top panel) whereas cultures from PKCδ knockout (−/−) mice had visible branched neurites, more numerous cell bodies and were resistant to degeneration induced by TNF (lower panel). (B,C) TH-positive neuron counts and dopamine uptake assays. (B) Dopaminergic neurons were identified by tyrosine hydroxylase labeling, and the number of TH-positive neurons from 12 random fields per well were counted at 20 × magnification for each treatment group. (C) The uptake of tritiated [³ H] dopamine was determined in cultures from PKCδ wild-type (WT) and knockout mice. The values for non-specific uptake of dopamine obtained in the presence of mazindol were subtracted as background and the data expressed as percent control. Data represent the group mean ± SEM, n = 4 to 6 per group and experiments were repeated three times. ***P <0.001 indicates a significant difference between control and TNF-treated groups in PKCδ wild type cultures. # (P <0.05) and ## (P <0.01) indicate a significant difference between TNF-treated groups in PKCδ wild-type and PKCδ knockout (−/−) cultures.