|Research in my laboratory currently centers on two areas:
- the activity-driven refinement of retinotopic connections in the visual system
- the role of myosin light chain kinase in regulating actin-myosin based growth cone motility.
Activity-driven refinement of retinotectal projection in zebrafish:
Visual activity, acting via NMDA receptors, refines developing retinotectal maps by shaping retinal arbors. Initially, retinal axons emit many transient side branches along the shaft (resembling a "bottlebrush"), but some branches are stabilized and branch further to give a mature arbor. When MK801 blocks NMDA receptors, dynamic rates of branch addition and deletion increase twofold, as if this prevents release of a stabilizing signal. Ca ++ entry through NMDARs is known to activate phospholipase A2 (cPLA2, a PSD protein) to release arachidonic acid (AA), a putative retrograde signal (Bliss & Collingridge, Nature 1993). AA released by a second enzyme (DAG lipase) mediates L1, NCAM, N-cadherin and FGF stimulation of axon growth via protein kinase C (PKC) activation and GAP-43 phosphorylation to stabilize F-actin (Meiri et al. J Neurosci.1998). We propose that postsynaptic cPLA2 releases AA as a retrograde signal to tap into this presynaptic growth control mechanism.
We previously reported that blocking either presynaptic PKC or DAG lipase causes increased branch turnover like MK801, but additionally causes arbors to remain immature due to interruption of the growth pathway. We have blocked AA release from cPLA2 by injecting into tectal ventricle either a selective pharmacological inhibitor (AACOCF3) or an antisense oligo to suppress expression. Both methods increased branch turnover without effect on arbor maturation (below), a result more similar to that of blocking NMDA receptors. Scrambled oligos had no effect. Injection of the antisense oligo into eye to suppress only presynaptic cPLA2 produced no effect, suggesting a role for postsynaptic cPLA2. After MK801 treatment, exogenous AA reversed the increase in dynamic rates.
Finally, we are using the fluorogenic cPLA2 substrate PED6 (Mol. Probes) to show that trains of spikes (driven by strobe illumination) activate tectal cPLA2. The results implicate the cPLA2-AA-PKC-GAP43 pathway as a part of an F-actin based mechanism of synaptic stabilization.
We are now using DNA constructs to express GAP-43 in retinal ganglion cells as GFP(green fluorescent protein) fusion proteins and assessing the effects on retinal arbor morphology (below). Injection into fertilized eggs results in scattered GFP expression in neurons, including ganglion cells in the eye (A), and their axons can be seen exiting in the optic nerve head and forming arbors in brain (arrows in B and C). GAP43-GFP fusion proteins can also be expressed in axons and observed in time-lapse at half hour intervals (D1 through 4). This results in arbors that are abnormal in many ways. They have thicker transient branches (more F-actin content), fail to concentrate branches into one focus and change more radically over short periods of time.
Molecular mechanisms regulating growth cone motility:
Both actin and myosin are present in growth cones within the lamellipodia and filopodia, and actin-myosin force generation elsewhere is controlled by myosin light chain kinase (MLCK) via the phosphorylation of the myosin light chain. We have shown that pharmacological inhibitors of MLCK stop growth cone motility, collapse lamellipodia and filopodia, and deplete filamentous actin. More recently we have cloned the MLCK gene from goldfish, shown for the first time that it is expressed in neurons with in situ hybridization, and also that it is prominently upregulated in retinal ganglion cells during the regeneration of their axons. We have made inhibitory peptides coupled to fatty acid that can cross the membrane and inhibit MLCK, and they verified the results produced by the pharmacological inhibitors. Similarly constructed peptide inhibitors of protein kinase C do not produce these effects. We can also produce many of the same effects with the myosin ATPase inhibitor, butanedione monoxime. Finally we have made a peptide specific polyclonal antibody for localization of MLCK.
Growing axon with multiple growth cones.