The Vertical System and the VS1 Neuron
We have recently described the cells composing the Drosophila Vertical System in structural detail [11]. Our conclusion was that there are six VS cells in each Drosophila lobula plate, and that they bear a close structural resemblance to the well characterized VS neurons in blowflies and house flies [14, 16–19]. Each cell has a complex elaboration of dendrites in the lobula plate with axons that travel medially and terminate near the esophagus.
Because different neurons of the Vertical System have different characteristic structures and levels of complexity [11], it was necessary to select a single type of VS neuron for quantitative analyses. We have restricted our quantitative analyses to the VS1 neuron because it is unambiguously recognizable, highly stereotyped, and has the most complex dendrites of any VS neuron [11]. The VS1 dendrite is characterized by a main dendritic shaft that produces one or a few dorsally projecting branches before sweeping ventrally. As the main shaft extends ventrally, it continues to produce smaller branches that combine to form a narrow band covering the medial part of the lobula plate (Fig. 1A).
In order to define quantitatively some aspects of the dendrites' structure, we first obtained three-dimensional confocal images of VS1 dendritic trees, and then traced the dendrites to produce three-dimensional computer diagrams of the dendrites. From these tracings, we measured dendritic branching complexity based on the total number of branch points found in the dendrites of single VS1 cells. We also used the tracings to determine the combined length of all of the dendritic branches for each cell.
The Effect of Input Deprivation on VS1 Structure
Given the high stereotypy of VS1 dendrites, we used VS1 to study the effects that sensory experience may have on the development of dendrites in the Drosophila visual system. We compared the dendrites of flies raised on a 12 hours light, 12 hours dark cycle (12L:12D) to those flies raised in constant darkness (24D) from larvae to at least 48 hours after eclosion. This period of darkness spans from before the development of adult visual structures to after the critical period for visual system plasticity [20].
VS1 cells appeared to be unchanged by dark rearing. The overall shape of the dendrites and the field that they cover was the same as in 12L:12D animals (Fig 1A,1B) and the complexity of the dendritic trees was unchanged in dark-reared animals (Table 1). The finer structures of the dendrites were also grossly similar in the visually deprived flies (Fig 2A,2B) and there was no significant difference between the two groups for spine number or spine density (Table 1). Given no apparent effects of dark rearing on the dendrites of the VS1 neuron, we looked at the axons to see whether the output from these cells might be affected by a lack of visual experience. As for the dendrites, the axons appeared to be similar in visually deprived flies and normal flies (Fig 3A,3B). Quantitatively, there was no difference in axon complexity between the two groups (Table 1). These results showing no changes in axon or dendrite morphology are consistent with a recent study showing normal physiological function of these neurons in dark-reared blowflies [21].
Apart from the possibility that VS1 dendritic development is independent of synaptic activity, it is possible that spontaneous activity generated in visual circuits is sufficient to promote VS1 dendritic development. However, the neural elements that directly innervate VS dendrites are not fully characterized. To determine whether spontaneous activity from the eye plays a role in the normal formation of higher order visual dendrites, we made use of a GMR-hid transgene (the Glass Multimer Reporter driving expression of the head involution defective gene) that causes expression of a cell death protein specifically in the eye-imaginal disc as soon as photoreceptors are born. This system has been shown be efficient in killing photoreceptors at early stages of their development [22, 23]. Although covering a similar dendritic field (Fig. 1C), we found that dendritic trees of VS1 cells in GMR-hid flies showed a slight but significant decrease in dendritic length and branching complexity as compared to wild-type 12L:12D flies (Table 1). The dendritic structures of these VS1 cells were slightly abnormal in some cases (Fig 1C), but these defects were mild, and inconsistent. There were no significant differences seen in GMR-hid flies for any of the other parameters that we studied, including spine number, spine density (Fig 2C and Table 1). Further analyses revealed no significant changes in the average length of individual dendritic segments or in the branching order structure for VS1 dendrites in GMR-hid flies (data not shown).
It is well known that in Drosophila the development of second order neurons is dependent on photoreceptor axon innervation (reviewed in [24]), and indeed we noted a reduction in the size of the lamina in GMR-hid flies (data not shown). One explanation for the reduction in VS1 dendrite length in GMR-hid flies is that the entire lobula plate may be smaller, as previously described for eye-ablated animals [24]. If this were true then a VS1 cell could innervate its lobula plate normally, but would have less total dendritic length. To test this possibility, we measured the length of the dorsal-ventral axis on the dendritic trees for wild-type 12L:12D flies versus GMR-hid flies. We saw a slight decrease in the length of this axis from 112.7 μm in wild-type (n = 10) to 102.5 μm in GMR-hid flies (n = 5, t-test p = 0.111), indicating that the decrease in dendritic length may be due at least in part to a decrease in the size of the dendritic field in these animals potentially caused by reduction of lower order visual neurons. Further supporting the idea that these neurons are essentially normal is the fact that the axon termini in GMR-hid flies were unaltered in appearance and complexity as compared to control and dark-reared flies (Fig. 3C and Table 1).
Past work in cockroach has shown a more dramatic role for input in the structure of efferent dendrites. Mizrahi and Libersat deprived cockroach sensory giant interneurons of input through direct deafferentation, and observed an average reduction of 55% in a variety of measures of dendrite complexity [25]. These more severe effects are likely due to the fact that the cockroach neurons were directly deprived of their presynaptic partners, while the VS1 neurons analyzed in this study were affected only indirectly. These combined results imply that the structural complexity of these large dendritic systems rests heavily on the presence of presynaptic partners, and less on input to the system on the whole or activity in upstream circuitry.