Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: Possible role in activity‐driven sharpening

Abstract

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor-dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a "bottlebrush" morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP-43 and stabilize F-actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO-labeled retinal axons of larval zebrafish with time-lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a "bottlebrush" morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half-hour time-lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor-driven stabilization of developing retinal arbors.

Stabilization of Axon Branch Dynamics by Synaptic Maturation

“…Certain aspects of axon remodeling are believed to involve a retrograde signal generated by postsynaptic activation of NMDA-type glutamate receptors (Cline and Constantine-Paton, 1989;Rajan et al, 1999;Schmidt et al, 2000;Ruthazer et al, 2003) and their downstream signaling cascades (Zou and Cline, 1996;Schmidt et al, 2004). An NMDA receptor-dependent retrograde signal might directly act to induce the stabilization and maturation of the presynaptic terminal, thereby stabilizing its axonal branch and inducing further axonal branch elaboration near that site.…”

Multifunctional Role of Protein Kinase C in Regulating the Formation and Maturation of Specific Synapses

“…Downstream signaling molecules may include small GTPases (Elia et al, 2006;Paradis et al, 2007) and specific kinases or phosphatases (Kaufmann et al, 2002;Patel et al, 2006;Sytnyk et al, 2006) that affect the organization of the cytoskeleton required for the formation of synapses. One kinase that might regulate synaptic adhesion and assembly is PKC (Loeb et al, 1998;Xia et al, 2003;Schmidt et al, 2004;Kolkova et al, 2005). PKC inhibition selectively blocked all growth and varicosity formation from sensory neurons when their axon stumps contacted L7, but not when they contacted L11.…”

In vivo imaging of the zebrafish retinotectal map

“…Applying AA directly to the tectum stabilizes branches and decreases dynamic events to lower than normal levels [294]. If the retrograde signaling process is disrupted, branch activation and elimination events double in frequency and axons do not gain a mature appearance [185,294]. In this way, retrograde signals act as a stop signal that stabilizes the existing branch and prevents further outgrowth after proper…”

“…Some of the effect comes through PKC regulation of adhesion molecules, but AA can also act on actin polymerization directly to stabilize the synaptic structure [294,376]. Applying AA directly to the tectum stabilizes branches and decreases dynamic events to lower than normal levels [294]. If the retrograde signaling process is disrupted, branch activation and elimination events double in frequency and axons do not gain a mature appearance [185,294].…”

“…proteins, including GAP43 and protein kinase C (PKC), which alter cytoskeleton dynamics to cause stabilization of branches and synapses [185]. Some of the effect comes through PKC regulation of adhesion molecules, but AA can also act on actin polymerization directly to stabilize the synaptic structure [294,376]. Applying AA directly to the tectum stabilizes branches and decreases dynamic events to lower than normal levels [294].…”