ABSTRACT
Neurons that synthesize GnRH control the reproductive axis and migrate over long distances and through different environments during development. Prior studies provided strong clues for the types of molecules encountered and movements expected along the migratory route. However, our studies provide the first real-time views of the behavior of GnRH neurons in the context of an in vitro preparation that maintains conditions comparable to those in vivo. The live views provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more changes in direction after they enter the brain. Perturbations of guiding fibers distal to moving GnRH neurons in the nasal compartment influenced movement without detectable changes in the fibers in the immediate vicinity of moving GnRH neurons. This suggests that the use of fibers by GnRH neurons for guidance may entail selective signaling in addition to mechanical guidance. These studies establish a model to evaluate the influences of specific molecules that are important for their migration.
Subject(s)
Computer Systems , Gonadotropin-Releasing Hormone/metabolism , Microscopy, Video , Neurons/physiology , Animals , Bicuculline/pharmacology , Brain/embryology , Cell Movement , Cell Shape , Embryo, Mammalian/cytology , Embryo, Mammalian/innervation , GABA Antagonists/pharmacology , GABA-A Receptor Antagonists , Green Fluorescent Proteins , Immunohistochemistry , In Vitro Techniques , Mice , Mice, Transgenic , Neural Pathways/embryology , Neurons/cytology , Neurons/metabolism , Nose/embryology , Tissue FixationABSTRACT
Many studies suggest that migratory guidance cues within the developing brain are diverse across many regions. To better understand the early development and differentiation of select brain regions, an in vitro method was developed using selected inbred and transgenic strains of embryonic mice. In particular, organotypic slices are used to test factors that influence the movements of neurons during brain development. Thick 250 µm slices cut on a vibrating microtome are prepared and maintained in vitro for 0-3 days. Nissl stain analyses often show a uniform distribution of cells in the regions of interest on the day of plating (embryonic days 12-15). After 3 days in vitro, cellular aggregation suggesting nuclear formation or the changing position of cells with a defined phenotype show that reasonably normal cell movements occur in several regions. Movements in vitro that mimic changes in vivo suggest that key factors reside locally within the plane of the slices. Video microscopy studies are used to follow the migration of fluorescently labeled cells in brain slices from mice maintained in serum-free media for 1 to 3 days. Transgenic mice with selective promoter driven expression of fluorescent proteins allow us to view specific cell types (e.g., neurons expressing gonadotropin-releasing hormone). The accessibility of an in vitro system that provides for relatively normal brain development over key brief windows of time allows for the testing of important mechanisms.
