The establishment of peripheral sensory arbors in the leech: In vivo time-lapse studies reveal a highly dynamic process

Abstract

Pressure-sensitive (P) neurons located in the leech CNS form elaborate terminal arbors in the body wall of the animal during mid-embryogenesis. In the experiments discussed here, arbor development in the target region was studied in intact, unanesthetized leech embryos using time-lapse video microscopy of individual, fluorescently stained P neurons. Analysis of time- lapse recordings made over a period of several days revealed that arbor formation is a very dynamic process. At any particular time, most high-order terminal branches were either extending or retracting, in approximately equal numbers and at very similar rates. Many branches underwent several rounds of extension and retraction every hour. Net arbor growth occurred at a much lower rate than the extension and retraction rates of individual branches. Process retraction sometimes resulted in an apparent change in the topological order of processes. Significantly, the initiation of new branches was restricted to a few locations along the parent process, which were termed 'hot spots.' Moreover, the capacity to generate high-order branches correlated with parent process stability. The target region of the growing P cell arbor in the body wall was subsequently examined using confocal microscopy in fixed preparations. The arbor expanded between the longitudinal and circular muscle layers, a region occupied by small unidentified cells. Simultaneous imaging of the dye-labeled terminal arbor and the surrounding tissue at two different wavelengths suggested that the high-order processes were navigating around these cells, which sometimes forced the growing processes to assume a bent form. These observations suggest that the formation of the P cell arbor can be best described as a 'dynamically unstable' process that is constrained by interactions with its environment.