In cultured neurons, the exogenous application of neurotrophins (in homogenous concentrations) alters many features of axonal and dendritic arbors. In vivo, however, release of endogenous neurotrophins from neuronal processes creates spatially heterogeneous neurotrophin distributions. To probe the consequences of such endogenous neurotrophin distribution, we produced 'donor neurons' in ferret cortex brain slices that co-expressed brain-derived neurotrophic factor (BDNF) and red fluorescent protein (RFP). Using two-photon microscopy, we analyzed their effects on 'recipient neurons' that expressed green fluorescent protein (GFP) alone. BDNF released from dendrites and cell bodies acted directly on nearby recipient neurons to increase dendritic branching in a distance-dependent manner. Three-dimensional analysis of donor and recipient dendrites indicated that the BDNF source had to be within 4.5 microm to induce dendritic growth in the recipient neuron. Thus, BDNF released from an individual cell alters the structure of nearby dendrites on an exquisitely local scale.

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.