C-kinase manipulations disrupt activity-driven retinotopic sharpening in regenerating goldfish retinotectal...

Regenerating optic axons initially branch over a wide area in tectum to form a crude retinotopic map. The map is sharpened, and retinotopically appropriate synapses are stabilized via NMDA receptors that detect, via summation of EPSPs, the coincident activity of neighboring ganglion cells that make synapses onto common tectal cells. Sharpening shares a number of properties with long-term potentiation (LTP) in hippocampus. This study tested whether protein kinase C (PKC) activation is necessary for sharpening as it is for LTP. Intraocular (IO) or intracranial (IC) injections of kinase inhibitors or activators were made every other day from 19 to 37 days postcrush (sensitive period), and the projections formed were later recorded. Retinotopic sharpening was prevented by IC injection of the following agents: (1) general kinase inhibitors sphingosine and H7 (100-200 microM in fluid above brain), (2) active but not inactive phorbols (TPA, 1 microM), and (3) calphostin C (1 microM), a specific and irreversible PKC inhibitor. The mature projection on the opposite tectum, however, when examined was not unsharpened. Lack of sharpening was reflected in multiunit fields at each tectal point that averaged 27 degrees-30 degrees versus 11 degrees in Ringers and inactive phorbol control regenerates. Intraocular injections of either TPA (1 microM), or calphostin C (1 microM) also prevented sharpening (26 degrees and 32 degrees multiunit fields), suggesting action on PKC axonally transported to the presynaptic terminals. Calphostin C had no noticeable effect on the firing patterns of retinal ganglion cells. The endogenous activator of PKC, arachidonic acid (AA), disrupted sharpening at 20 microM or higher (IC injection, 32 degrees multiunit fields), while a control fatty acid, elaidic acid, had no effect. Although AA at 5 microM showed no effect, and diacylglycerol at 5 microM exhibited only small effects, together they produced a large synergistic effect (32 degrees multiunit fields). Such synergy mirrors the synergy in the activation of several isoforms of PKC. Actual concentrations in the extradural fluid around the brain were assayed via injections of 3H-AA. Levels fell about sixfold after a day and by an additional fivefold the second day before the next injection. The results confirm that activity-driven retinotopic sharpening is very sensitive to manipulations of kinases, especially PKC.