Pharmacological Inhibition of PKC-Alpha Activity Reduces Synaptic Activity in Bipolar Cell Terminals of Goldfish Retinae as Monitored by FM1-43 Imaging PKC Staining Using Phosphospecific Antibodies

Abstract

Abstract: : Purpose:To study the impact of PKC activity on synaptic activity and structural changes of bipolar cell terminals. We investigated the distribution of activated aPKC immunoreactivity (IR) and vesicle endocytosis in axon terminals of Mb bipolar cells in-vitro after pharmacological manipulation of aPKC activity. Methods:Isolated dark adapted retinae were cultured for 30 min in Ringer containing 2.3 nM of an isoform specific PKC antagonist (Gö6976). Aldehyde fixed tissue was then prepared for immunohistochemistry. We used an antiserum against the phosphorylated form of aPKC (paPKC). paPKC- IR in Mb terminals was examined with confocal microscopy. Semiquantitative analysis of paPKC-IR was performed in small areas (0,95 µm2) directly beneath the membrane of the terminal (membrane compartment, MC) and in the centre of the terminal (cytoplasmatic compartment, CC). The synaptic activity of Mb cells was monitored in living slice preparations treated with the Gö6971 (2.3 nM) and the styryl dye FM1-43 (5µM) and analysed with confocal microscopy. Results: Immunfluorescence label for activated aPKC was preferentially localised at the MC of dark adapted Mb terminals in control groups. Incubation with Gö6976 in the dark lead to a diffuse staining of paPKC-IR within the terminal. Semiquantitative analysis of paPKC-IR showed that the Gö6976 effect was statistically significant when measuring the ratio (MC/CC) of paPKC-IR in control groups (MC/CC: 2.5±0.45, mean±SEM; n=60 terminals from 4 retinae) compared to Gö6976 treated groups (MC/CC: 1.2±0.3, mean±SEM; n=60 terminals from 4 retinae). Slice preparations treated with Ringer or K+-Ringer and FM1-43 showed a distinct labelling of Mb terminals, whereas fewer labelled terminals were visible in preparations treated with Ringer in the presence of Gö6976.<br Conclusions: Our data suggests that the manipulation of PKC activity influences the distribution of activated aPKC-IR in Mb terminals. The preferential localisation of the activated enzyme at the MC and the reduced FM1-43 labeling after PKC-inhibition indicates that the molecular machinery for synaptic vesicle cycling and for synaptic transmission seems to be under the control of activated aPKC.

Keywords: bipolar cells • retina: proximal(bipolar, amacrine, and gangli • synapse