The mechanisms that underlie the development of the nervous system are numerous and diverse. Over the past several decades, research has begun to give us a sense of the importance of both preprogrammed, invariant mechanisms for neural development, and also programs for development that depend on experience and the electrical activity of the developing neurons themselves. The fact that certain types of neurons develop their basic morphologies even when isolated in culture from other cells provides a simple but powerful argument for the importance of cell autonomous mechanisms in the establishment of neuronal structure [1, 2]. These and numerous other experiments have provided overwhelming evidence that neurons possess endogenous, activity independent programs that account for important aspects of their development.

On the other hand, neurons deprived of contact with or activity from their normal synaptic partners seldom attain a fully mature structure. For example, Purkinje cells deprived of their efferent projections in Weaver mice have dendritic arborizations that do not extend normally [3]. Additionally, the structures of neurons can be affected by an absence of activity from their efferent partners, even if those partners are present. This phenomenon of activity dependent neuronal development has been studied particularly effectively in the visual systems of various model systems. For instance, Xenopus tectal neurons have simple dendrites until the axons of their efferent partners in the retina arrive to innervate them. At this time, the dendrites elaborate into their mature structures. A pharmacological block of certain activity from the efferent neurons prevents the elaboration of the tectal dendrites, indicating that activity plays a critical role in the processes by which these tectal neurons attain their mature morphology [4].

These and other studies have established a role for environmental input and synaptic activity in the development and maturation of dendrites, but the importance of these inputs varies among different types of neurons and different model systems (reviewed in [5–7]). Moreover, many of the mechanistic details of activity-dependent dendrite development remain elusive. Genetically advantageous model systems like Drosophila and C. elegans offer tools such as the genetic ablation of targeted cell types and temporally and spatially controlled blockades of neural activity. This line of research has been restricted by the fact that the best dendrites for study (those that are highly complex and stereotyped) have been described in vertebrates, which are not easily amenable to genetic manipulation. Neurons described in Drosophila and C. elegans have been simple, highly variable, or both. One exception is provided by the multiple dendrite (MD) neurons in the Drosophila embryonic peripheral nervous system. The dendrites of these neurons, which are fairly complex and highly stereotyped, have yielded important information about the genetic program controlling dendritic development [8, 9]. However, the MD neurons are primary sensory neurons, and as such, their dendrites are not postsynaptic to axonal input. This limits their usefulness in studies of synapse formation and activity-dependent dendritic development.

In an effort to facilitate dendritic studies, we have used the MARCM system to label individual neurons [10] and have recently characterized the six neurons of the Vertical System (VS) in the lobula plate of the Drosophila optic lobe [11]. As they serve as integrators of visual information from wide areas of the eye [12], VS neurons have highly complex dendritic trees that sweep over a large part of the lobula plate. In addition, their dendritic trees are highly stereotyped from animal to animal and are individually identifiable [11]. They also exhibit easily distinguishable spine-like structures [13] that have been shown in blowfly to be enriched for post-synaptic densities [14] allowing for potential analogies to be drawn to dendritic spines in vertebrate neurons. Also like vertebrate dendritic spines, whose actin-mediated changes in morphology may be important for connective plasticity (reviewed in [15]), the spines in VS cells are rich in actin [13], supporting the idea that they could serve similar functions in the insect VS neurons as in numerous types of neurons in vertebrates.

Vertical System neurons' extraordinary dendritic complexity and consistent structure from animal to animal should make them well suited for quantitative analyses. In the current study, we test the role of activity in the development of one particular VS neuron, VS1. We use two approaches to interfere with input into VS1: dark rearing, and genetic ablation of the eyes, and compare the VS1 dendrites in these flies to wild type light reared flies.

In conclusion, these experiments show no role for experience in the development of these complex high order visual processing neurons. Dark-reared and visually experienced flies possessed VS1 neurons with similar dendritic morphology, complexity, and spine structures, and similar axon morphology and complexity. Flies lacking photoreceptors showed similar neural structures, with only a slight reduction of dendritic branching complexity and overall dendritic length. Taken together, it seems likely that the dendritic morphogenesis of VS1 (and likely other VS neurons) is largely determined by internal genetic programs combined perhaps with the local environment in the lobula plate.

Drosophila melanogaster were grown on standard media at 25°C. During clonal analysis, larvae that hatched over a two-hour interval were moved to plastic vials containing approximately 10 ml of food. Larvae were kept at a concentration of 80 per vial. Mitotic recombination was induced via heat shock (40 min in a 37°C water bath, 30 min at room temperature, 40 min in 37°C water bath) at two and three days after hatching. Adult female flies between 2 and 5 days after eclosure were dissected, fixed, and stained as described [26]. Genotypes used are hs-flp / + ; FRT ^{G 13}, tubP-GAL80 /FRT ^{G 13}, UAS-mCD8-GFP ; GAL4-3A / + or hs-flp, UAS-mCD8-GFP / + ; tubP-GAL80, FRT ^2A/GAL4-3A, FRT ^2Afor wild type flies (12L:12D and 24D), and hs-flp, UAS-mCD8-GFP / + ; GMR-hid / + ; tubP-GAL80, FRT ^2A/GAL4-3A, FRT ^2Afor flies with ablated eyes.

Dark-reared flies were moved to dark boxes at 25°C three days after larval hatching, before adult photoreceptors are born and before any connections are made. Adult heads were removed under a safelight and fixed prior to dissection.

A Bio-Rad MRC 1024 laser scanning confocal microscope and the Laser Sharp image collection program were used. Images were prepared using Adobe Photoshop. Three-dimensional traces of the dendrites were produced from confocal stacks using MicroBrightField Neurolucida software (as in [11]). Briefly, dendritic branches were traced such that turning points, branch points, and endpoints were specified in X, Y, and Z positions within the confocal stack. MicroBrightField NeuroExplorer software was then used to give quantitative measures. Branching complexity was measured as the total number of branch points in the dendritic tree, and total length for these traces was defined as the combined length of all of the branch segments (as in [13]). In quantifying spines, we included terminal segments from 1–3 μm long, since these were the structures with the most dense postsynaptic terminals as previously described using electron microscopy [14]. Our efforts to develop a postsynaptic marker as a means of quantifying spines were unsuccessful. Structures longer than 3 μm were treated as dendritic branches.