Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system.

SUMMARY Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila’s preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons, called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8, and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8’s dendritic field, but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s->Dm8->Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ~16 R7 signals for transfer to the lobula, a higher visual center.

Summary Background In the fly’s visual motion pathways, two cell types - T4 and T5 - are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF-channel from L1’s partner, L2, to T5 are so far not known however. Results Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron L3, providing a candidate substrate for L3’s combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9’s input synapses are spatially segregated on T5’s dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express ChAT. Conclusions We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.

SUMMARY How CNS neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns, and innervated by medulla neurons dendrites and photoreceptor axons. Here we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive ~16 R7 photoreceptor inputs. R7- and R8-derived Activin/TGF-β selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.

The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs. The flies’ optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad spectrum photoreceptors R1-R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate UV spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.

Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. Here we show that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, we show that mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled(ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Ablhave an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization.

In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye’s narrow-spectrum photoreceptors, R7 and R8, and their downstream neurons, Tm5a/b/c and Tm20, in the second optic neuropil, or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GRASP (GFP reconstitution across synaptic partners) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identified anatomically the visual projection neurons LT11 and LC14, and the lobula intrinsic neurons Li3 and Li4, as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses revealed that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons while LT11 is postsynaptic to the chromatic Tm neurons with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we developed and applied a “two-tag” double labeling method to label LT11’s dendrites and the mitochondria in Tm5c’s presynaptic terminals. Serial electron microscopic reconstruction confirmed that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals.

In vertebrates, including the mammals, the chromosomal DNAs are modified by C methylation at a limited number of CpG dinucleotides, resulting in m 5 CpG, methylated at position 5 of the C residues. This methylation at CpG has been implicated in the regulation of a number of genetic activities during mammalian cell differentiation and embryo development. These activities include tissue-specific gene transcription, X chromosome inactivation, genomic imprinting, cellular defense against viral agents, and tumorigenesis (references 1, 2, 11, 13, 15, and 34 and references therein). The level of m 5 CpG in the vertebrate cells is presumably balanced by the combined actions of DNA cytosine (C-5) methyltransferases (CpG MTases) (2, 26) and DNA demethylases (3).It is generally accepted that regulation of the different cellular activities by DNA methylation is modulated mainly through the m 5 CpG residues. Indeed, m 5 CpG could be recognized by a specific class of proteins that consist of the methylated-DNA binding domain (MBD) (21). These "MBD proteins," in particular MeCP2 and MBD2/3, could preferentially bind to m 5 CpG-containing DNA, recruit histone deacetylase (HDAC)-containing complexes, and thus cause chromatin condensation in the vicinity of m 5 CpG (12,22,23,33). This is very likely one major, but not the only, scheme involved in the functioning of vertebrate DNA methylation.Unlike the vertebrates, several invertebrate species, including Drosophila melanogaster (27, 31), do not have apparent DNA methylation in their genomes. Nor has CpG MTase been reported to occur in these invertebrate animals. It was thus surprising to find mammalian CpG MTase-related proteins as well as MBD proteins expressed in Drosophila. A Drosophila protein, DmMTR1, is characteristically similar to the vertebrate maintenance CpG MTase, dnmt1, in several aspects (9). In particular, DmMTR1 molecules are located outside the nuclei during interphase of the syncytial Drosophila embryos, as is dnmt1 in the mouse blastocyst (19). However, DmMTR1 molecules appear to be rapidly transported into and then out of the nuclei again, as the embryos undergo the mitotic waves (9). Immunofluorescence data further indicate that DmMTR1 molecules "paint" the whole set of condensed Drosophila chromosomes throughout the mitotic phase, suggesting that it may play an essential role in the cell cycle-regulated condensation of the Drosophila chromosomes. In addition to DmMTR1, another Drosophila polypeptide, DmMT2, exhibiting high sequence homology to mammalian dnmt2, a relatively short homolog of dnmt1 but without detectable methylation activities (25), has been identified through a database search (9, 30). Expression of DmMT2 in Drosophila is developmentally regulated (9).Interestingly, several recent reports have also noted the identification of an MBD protein encoded by the Drosophila genome (9,30,33). Like the mammalian MBD (12,22,23,33), the Drosophila MBD protein, which was termed the "Drosophila MBD-like sequence" (33) or dMBD2/3 (30), interacts with HDAC in...