Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila.

Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. We previously discovered that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust (Hampel et al., 2017; Seeds et al., 2014). Here, we identify nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming.

Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila.

Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. We previously discovered that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust (Hampel et al., 2017; Seeds et al., 2014). Here, we identify nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming.

Retinoic acid treatment recruits macrophages and increases axonal regeneration after optic nerve injury in the frog Rana pipiens.

Retinoic acid (RA) plays major roles during nervous system development, and during regeneration of the adult nervous system. We have previously shown that components of the RA signaling pathway are upregulated after optic nerve injury, and that exogenous application of all-trans retinoic acid (ATRA) greatly increases the survival of axotomized retinal ganglion cells (RGCs). The objective of the present study is to investigate the effects of ATRA application on the macrophages in the optic nerve after injury, and to determine whether this affects axonal regeneration. The optic nerve was crushed and treated with PBS, ATRA and/or clodronate-loaded liposomes. Nerves were examined at one and two weeks after axotomy with light microscopy, immunocytochemistry and electron microscopy. ATRA application to the optic nerve caused transient increases in the number of macrophages and microglia one week after injury. The macrophages are consistently labeled with M2-type markers, and have considerable phagocytic activity. ATRA increased ultrastructural features of ongoing phagocytic activity in macrophages at one and two weeks. ATRA treatment also significantly increased the numbers of regenerating GAP-43-labeled axons. Clodronate liposome treatment depleted macrophage numbers by 80%, completely eliminated the ATRA-mediated increase in axonal regeneration, and clodronate treatment alone decreased axonal numbers by 30%. These results suggest that the success of axon regeneration is partially dependent on the presence of debris-phagocytosing macrophages, and that the increases in regeneration caused by ATRA are in part due to their increased numbers. Further studies will examine whether macrophage depletion affects RGC survival.

A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age.

There have been relatively few studies of how central synapses age in adult Drosophila melanogaster. In this study we investigate the aging of the synaptic inputs to the Giant Fiber (GF) from auditory Johnston's Organ neurons (JONs). In previously published experiments an indirect assay of this synaptic connection was used; here we describe a new, more direct assay, which allows reliable detection of the GF action potential in the neck connective, and long term recording of its responses to sound. Genetic poisoning using diphtheria toxin expressed in the GF with R68A06-GAL4 was used to confirm that this signal indeed arose from the GF and not from other descending neurons. As before, the sound-evoked action potentials (SEPs) in the antennal nerve were recorded via an electrode inserted at the base of the antenna. It was noted that an action potential in the GF elicited an antennal twitch, which in turn evoked a mechanosensory response from the JONs in the absence of sound. We then used these extracellular recording techniques in males and female of different ages to quantify the response of the JONs to a brief sound impulse, and also to measure the strength of the connection between the JONs and the GF. At no age was there any significant difference between males and females, for any of the parameters measured. The sensitivity of the JONs to a sound impulse approximately doubled between 1 d and 10 d after eclosion, which corresponds to the period when most mating is taking place. Subsequently JON sensitivity decreased with age, being approximately half as sensitive at 20 d and one-third as sensitive at 50 d, as compared to 10 d. However, the strength of the connection between the auditory input and the GF itself remained unchanged with age, although it did show some variability that could mask any small changes.

Application of CNTF or FGF-2 increases the number of M2-like macrophages after optic nerve injury in adult Rana pipiens.

We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 µm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances.

Shaking-B misexpression increases the formation of gap junctions but not chemical synapses between auditory sensory neurons and the giant fiber of Drosophila melanogaster.

The synapse between auditory Johnston's Organ neurons (JONs) and the giant fiber (GF) of Drosophila is structurally mixed, being composed of cholinergic chemical synapses and Neurobiotin- (NB) permeable gap junctions, which consist of the innexin Shaking-B (ShakB). Previous observations showed that misexpression of one ShakB isoform, ShakB(N+16), in a subset of JONs that do not normally form gap junctions results in their de novo dye coupling to the GF. Misexpression of the transcription factor Engrailed (En) in these neurons also has this effect, and in addition causes the formation of new chemical synapses. These results, along with earlier studies suggesting that gap junctions are required for the development of some chemical synapses, led to the hypothesis that ShakB would, like En, have an instructive effect on the distribution of mixed chemical/electrical contacts. To test this, we first confirmed quantitatively that ShakB(N+16) misexpression increased the dye-coupling of JONs with the GF, indicating the formation of ectopic gap junctions. Conversely, expression of the 'incorrect' isoform, ShakB(N), abolished dye coupling. Immunocytochemistry of the ShakB protein showed that ShakB(N+16) increased gap junctional plaques in JON axons but ShakB(N) did not. To test our hypothesis, fluorescently-labeled presynaptic active zone protein (Brp) was expressed in JONs and the changes in its distribution on the GF dendrites was assayed with confocal microscopy in animals with misexpression of ShakB(N+16), ShakB(N) or, as a positive control, En. Using different methods of image analysis, we confirmed our previous result that En misexpression increased the chemical synapses with the GF and the amount of GF medial dendrite branching. However, contrary to our hypothesis, misexpression of ShakB did not increase these parameters. Immunostaining showed no association between presynaptic active zones and the new ShakB plaques, further evidence against the hypothesis. We conclude that both subsets of JON form chemical synapses onto the GF dendrites but only one population forms gap junctions, comprised of ShakB(N+16). Misexpression of this isoform in all JONs does not instruct the formation of new mixed chemical/electrical synapses, but results in the insertion of new gap junctions, presumably at the sites of existing chemical synaptic contacts with the GF.

Exogenous Modulation of Retinoic Acid Signaling Affects Adult RGC Survival in the Frog Visual System after Optic Nerve Injury.

After lesions to the mammalian optic nerve, the great majority of retinal ganglion cells (RGCs) die before their axons have even had a chance to regenerate. Frog RGCs, on the other hand, suffer only an approximately 50% cell loss, and we have previously investigated the mechanisms by which the application of growth factors can increase their survival rate. Retinoic acid (RA) is a vitamin A-derived lipophilic molecule that plays major roles during development of the nervous system. The RA signaling pathway is also present in parts of the adult nervous system, and components of it are upregulated after injury in peripheral nerves but not in the CNS. Here we investigate whether RA signaling affects long-term RGC survival at 6 weeks after axotomy. Intraocular injection of all-trans retinoic acid (ATRA), the retinoic acid receptor (RAR) type-α agonist AM80, the RARß agonist CD2314, or the RARγ agonist CD1530, returned axotomized RGC numbers to almost normal levels. On the other hand, inhibition of RA synthesis with disulfiram, or of RAR receptors with the pan-RAR antagonist Ro-41-5253, or the RARß antagonist LE135E, greatly reduced the survival of the axotomized neurons. Axotomy elicited a strong activation of the MAPK, STAT3 and AKT pathways; this activation was prevented by disulfiram or by RAR antagonists. Finally, addition of exogenous ATRA stimulated the activation of the first two of these pathways. Future experiments will investigate whether these strong survival-promoting effects of RA are mediated via the upregulation of neurotrophins.

Optic nerve injury upregulates retinoic acid signaling in the adult frog visual system.

Retinoic acid (RA) is important during development, in neuronal plasticity, and also in peripheral nervous system regeneration. Here we use the frog visual system as a model to investigate the changes in RA signaling that take place after axonal injury to the central nervous system. Immunocytochemistry was used to localize different components of RA signaling within sections of the retina and optic tectum, namely, the synthetic enzyme retinaldehyde dehydrogenase (RALDH), the RA binding proteins CRABPI and II, the retinoic acid receptors RARα, ß and γ, and finally the catabolic enzyme CYP26A1. The levels of these proteins were quantified in extracts of retina and tectum using Western blotting. Animals were studied at 1 week, 3 weeks and 6 weeks after optic nerve transection. At the latter time point the RGC axons were re-entering the optic tectum. All the components of RA signaling were present at low to moderate levels in retinas and tecta of control, unoperated animals. In retina, soon after optic nerve injury there was a large increase in RALDH, some increase in the CRABPs, and a large increase in RGC RARß and (expression. These increases continued as the RGC axons were regenerating, with the addition of later RARα expression at 6 weeks. At no stage did CYP26A1 expression significantly change. In the tectum the levels of RALDH increased after axotomy and during regrowth of axons (3 weeks), then decreased at 6 weeks, at which time the levels of CYP26A1 increased. Axotomy did not cause an immediate increase in tectal RAR levels but RARα and RARß increased after 3 weeks and RARγ only after 6 weeks. These results are consistent with RA signaling playing an important role in the survival and regeneration of frog RGCs.

Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila melanogaster. The purpose of this study was to identify which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakB(N+16) isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. However, the identity of the innexin that forms the presynaptic hemichannels in the JONs remains unknown. We used electrophysiology, immunocytochemistry and dye injection, along with presynaptically-driven RNA interference, to investigate this question. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakB(L) or ShakB(N) isoforms alone did not reduce the synaptic strength, implying that it is ShakB(N+16) that is required in the presynaptic neurons. Overexpression of ShakB(N+16) in JONs caused the formation of ectopic dye coupling, whereas ShakB(N) prevented it altogether, supporting this conclusion and also suggesting that gap junction proteins may have an instructive role in synaptic target choice.

Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber.

We show that a subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster, which express the transcription factors Engrailed (En) and Invected (Inv), form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. We then show that misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. We also introduce a simple electrophysiological recording paradigm for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity.

Ciliary neurotrophic factor and fibroblast growth factor increase the speed and number of regenerating axons after optic nerve injury in adult Rana pipiens.

Neurotrophins such as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) and growth factors such as fibroblast growth factor (FGF-2) play important roles in neuronal survival and in axonal outgrowth during development. However, whether they can modulate regeneration after optic nerve injury in the adult animal is less clear. The present study investigates the effects of application of these neurotrophic factors on the speed, number, and distribution of regenerating axons in the frog Rana pipiens after optic nerve crush. Optic nerves were crushed and the factors, or phosphate-buffered saline, were applied to the stump or intraocularly. The nerves were examined at different times after axotomy, using anterograde labeling with biotin dextran amine and antibody against growth-associated protein 43. We measured the length, number, and distribution of axons projecting beyond the lesion site. Untreated regenerating axons show an increase in elongation rate over 3 weeks. CNTF more than doubles this rate, FGF-2 increases it, and BDNF has little effect. In contrast, the numbers of regenerating axons that have reached 200 µm at 2 weeks were more than doubled by FGF-2, increased by CNTF, and barely affected by BDNF. The regenerating axons were preferentially distributed in the periphery of the nerve; although the numbers of axons were increased by neurotrophic factor application, this overall distribution was substantially unaffected.

Auditory responses of engrailed and invected-expressing Johnston's Organ neurons in Drosophila melanogaster.

The roles of the transcription factor Engrailed (En), and its paralogue Invected (Inv), in adult Drosophila Johnston's Organ sensory neurons are unknown. We used en-GAL4 driven CD8-GFP and antibody staining to characterize these neurons in the pedicel (second antennal segment). The majority of En and Inv-expressing Johnston's Organ neurons (En-JONs) are located in the ventral part of the posterior group of JONs, with only a few in the medial group. Anatomical classification of En-JON axon projections shows they are mainly type A and E, with a few type B. Extracellular recording of sound-evoked potentials (SEPs) from the antennal nerve was used along with Kir2.1 silencing to assess the contribution that En-JONs make to the auditory response to pure-tone sound stimuli. Silencing En-JONs reduces the SEP amplitude at the onset of the stimulus by about half at 100, 200 and 400 Hz, and also reduces the steady-state response to 200 Hz. En-JONs respond to 82 dB and 92 dB sounds but not 98 dB. Despite their asymmetrical distribution in the Johnston's Organ they respond equally strongly to both directions of movement of the arista. This implies that individual neurons are excited in both directions, a conclusion supported by reanalysis of the morphology of the pedicel-funicular joint. Other methods of silencing the JONs were also used: RNAi against the voltage-gated Na⁺ channel encoded by the para gene, expression of attenuated diphtheria toxin, and expression of a modified influenza toxin M2(H37A). Only the latter was found to be more effective than Kir2.1. Three additional JON subsets were characterized using Flylight GAL4 lines. inv-GAL4 88B12 and Gycß100B-GAL4 12G03 express in different subsets of A group neurons and CG12484-GAL4 91G04 is expressed in B neurons. All three contribute to the auditory response to 200 Hz tones.

Changes in fibroblast growth factor-2 and FGF receptors in the frog visual system during optic nerve regeneration.

We have previously shown that application of fibroblast growth factor-2 (FGF-2) to cut optic nerve axons enhances retinal ganglion cell (RGC) survival in the adult frog visual system. These actions are mediated via activation of its high affinity receptor FGFR1, enhanced BDNF and TrkB expression, increased CREB phosphorylation, and by promoting MAPK and PKA signaling pathways. The role of endogenous FGF-2 in this system is less well understood. In this study, we determine the distribution of FGF-2 and its receptors in normal animals and in animals at different times after optic nerve cut. Immunohistochemistry and Western blot analysis were conducted using specific antibodies against FGF-2 and its receptors in control retinas and optic tecta, and after one, three, and six weeks post nerve injury. FGF-2 was transiently increased in the retina while it was reduced in the optic tectum just one week after optic nerve transection. Axotomy induced a prolonged upregulation of FGFR1 and FGFR3 in both retina and tectum. FGFR4 levels decreased in the retina shortly after axotomy, whereas a significant increase was detected in the optic tectum. FGFR2 distribution was not affected by the optic nerve lesion. Changes in the presence of these proteins after axotomy suggest a potential role during regeneration.

Animal escapology I: theoretical issues and emerging trends in escape trajectories.

Escape responses are used by many animal species as their main defence against predator attacks. Escape success is determined by a number of variables; important are the directionality (the percentage of responses directed away from the threat) and the escape trajectories (ETs) measured relative to the threat. Although logic would suggest that animals should always turn away from a predator, work on various species shows that these away responses occur only approximately 50-90% of the time. A small proportion of towards responses may introduce some unpredictability and may be an adaptive feature of the escape system. Similar issues apply to ETs. Theoretically, an optimal ET can be modelled on the geometry of predator-prey encounters. However, unpredictability (and hence high variability) in trajectories may be necessary for preventing predators from learning a simple escape pattern. This review discusses the emerging trends in escape trajectories, as well as the modulating key factors, such as the surroundings and body design. The main ET patterns identified are: (1) high ET variability within a limited angular sector (mainly 90-180 deg away from the threat; this variability is in some cases based on multiple peaks of ETs), (2) ETs that allow sensory tracking of the threat and (3) ETs towards a shelter. These characteristic features are observed across various taxa and, therefore, their expression may be mainly related to taxon-independent animal design features and to the environmental context in which prey live - for example whether the immediate surroundings of the prey provide potential refuges.

Animal escapology II: escape trajectory case studies.

Escape trajectories (ETs; measured as the angle relative to the direction of the threat) have been studied in many taxa using a variety of methodologies and definitions. Here, we provide a review of methodological issues followed by a survey of ET studies across animal taxa, including insects, crustaceans, molluscs, lizards, fish, amphibians, birds and mammals. Variability in ETs is examined in terms of ecological significance and morpho-physiological constraints. The survey shows that certain escape strategies (single ETs and highly variable ETs within a limited angular sector) are found in most taxa reviewed here, suggesting that at least some of these ET distributions are the result of convergent evolution. High variability in ETs is found to be associated with multiple preferred trajectories in species from all taxa, and is suggested to provide unpredictability in the escape response. Random ETs are relatively rare and may be related to constraints in the manoeuvrability of the prey. Similarly, reports of the effect of refuges in the immediate environment are relatively uncommon, and mainly confined to lizards and mammals. This may be related to the fact that work on ETs carried out in laboratory settings has rarely provided shelters. Although there are a relatively large number of examples in the literature that suggest trends in the distribution of ETs, our understanding of animal escape strategies would benefit from a standardization of the analytical approach in the study of ETs, using circular statistics and related tests, in addition to the generation of large data sets.

Transcriptional control of behavior: engrailed knock-out changes cockroach escape trajectories.

The cerci of the cockroach are covered with identified sensory hairs that detect air movements. The sensory neurons that innervate these hairs synapse with giant interneurons in the terminal ganglion that in turn synapse with interneurons and leg motor neurons in thoracic ganglia. This neural circuit mediates the animal's escape behavior. The transcription factor Engrailed (En) is expressed only in the medially born sensory neurons, which suggested that it could work as a positional determinant of sensory neuron identity. Previously, we used double-stranded RNA interference to abolish En expression and found that the axonal arborization and synaptic outputs of an identified En-positive sensory neuron changed so that it came to resemble a nearby En-negative cell, which was itself unaffected. We thus demonstrated directly that En controls synaptic choice, as well as axon projections. Is escape behavior affected as a result of this miswiring? We showed recently that adult cockroaches keep each escape unpredictable by running along one of a set of preferred escape trajectories (ETs) at fixed angles from the direction of the threatening stimulus. The probability of selecting a particular ET is influenced by wind direction. In this present study, we show that early instar juvenile cockroaches also use those same ETs. En knock-out significantly perturbs the animals' perception of posterior wind, altering the choice of ETs to one more appropriate for anterior wind. This is the first time that it has been shown that knock-out of a transcription factor controlling synaptic connectivity can alter the perception of a directional stimulus.

Escaping away from and towards a threat: the cockroach's strategy for staying alive.

The escape response of the cockroach is a well-studied example of sensorimotor behavior. Cockroaches respond to wind puffs, which may signal a predator attack, by making a swift turn followed by a forward acceleration. We have recently shown that their escape trajectories, measured relative to the position of the threatening stimulus, show preferred directions.1 Previous work has often distinguished between the most common type of escape turn, which begins as a rotation away from the stimulus, and the relatively rare turns initiated towards the stimulus. Here, we analyze these "away" and "towards" responses in light of our recent work on preferred escape trajectories (ETs). We find that the ETs of towards responses show a pattern of frequency distribution similar to that of away responses. The range of the bodyturn angles of towards responses, however, is much smaller than that of away responses, being <30 degrees in most cases, which approximately corresponds to the angular distance between ET peaks. This suggests that cockroaches minimize their turn when making a towards response, which could represent an effective anti-predator behavior that allows cockroaches to reach one of the preferred ETs within a relatively short time.

Cockroaches keep predators guessing by using preferred escape trajectories.

Antipredator behavior is vital for most animals and calls for accurate timing and swift motion. Whereas fast reaction times [1] and predictable, context-dependent escape-initiation distances [2] are common features of most escape systems, previous work has highlighted the need for unpredictability in escape directions, in order to prevent predators from learning a repeated, fixed pattern [3-5]. Ultimate unpredictability would result from random escape trajectories. Although this strategy would deny any predictive power to the predator, it would also result in some escape trajectories toward the threat. Previous work has shown that escape trajectories are in fact generally directed away from the threat, although with a high variability [5-8]. However, the rules governing this variability are largely unknown. Here, we demonstrate that individual cockroaches (Periplaneta americana, a much-studied model prey species [9-14]) keep each escape unpredictable by running along one of a set of preferred trajectories at fixed angles from the direction of the threatening stimulus. These results provide a new paradigm for understanding the behavioral strategies for escape responses, underscoring the need to revisit the neural mechanisms controlling escape directions in the cockroach and similar animal models, and the evolutionary forces driving unpredictable, or "protean"[3], antipredator behavior.

Engrailed expression in subsets of adult Drosophila sensory neurons: an enhancer-trap study.

Engrailed (En) has an important role in neuronal development in vertebrates and invertebrates. In adult Drosophila, although En expression persists throughout adulthood, a detailed description of its expression in sensory neurons has not been made. In this study, en-GAL4 was used to drive UAS-CD8::GFP expression and the projections of sensory neurons were examined with confocal microscopy. En protein expression was confirmed using immunocytochemistry. In the antenna, En is present in subsets of Johnston's organ neurons and of olfactory neurons. En-driven GFP is expressed in axons projecting to 18 identified olfactory glomeruli, originating from basiconic, trichoid and coeloconic sensilla. In most cases both neurons of a sensillum express En. En expression overlaps with that of Acj6, another transcription factor. En-driven GFP is also expressed in a subset of maxillary palp olfactory neurons and in all mechanosensory and gustatory sensilla in the posterior compartment of the labial palps. In the legs and halteres, en-driven GFP is expressed in only a subset of the sensory neurons of different modalities that arise in the posterior compartment. Finally, en-driven GFP is expressed in a single multidendritic sensory neuron in each abdominal segment.

Up-regulation of brain-derived neurotrophic factor by application of fibroblast growth factor-2 to the cut optic nerve is important for long-term survival of retinal ganglion cells.

Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes the survival of retinal ganglion cells (RGCs) in the frog Rana pipiens and results in a rapid up-regulation of brain-derived neurotrophic factor (BDNF) and TrkB synthesis by the RGCs. Here we investigate whether this up-regulation is maintained over the long term and whether it is required for FGF-2's survival effect. At 6 weeks after axotomy and FGF-2 treatment, we found more RGCs immunopositive for BDNF protein and higher intensity of BDNF and TrkB immunostaining, accompanied by increases in BDNF and TrkB mRNA in RGCs. Application of fluorescently labeled siRNA targeted against BDNF to the cut RGC axons showed that it was transported to the cell bodies. Axonal siRNA treatment eliminated the increases in BDNF immunostaining and mRNA that were induced by FGF-2 and had no effect on TrkB mRNA. This reduction in BDNF synthesis by siRNA greatly reduced the long-term survival effect of FGF-2 on RGCs. This, taken together with previous results, suggests that, although FGF-2 may initially activate survival pathways via ERK signaling, its main long-term survival effects are mediated via its up-regulation of BDNF synthesis by the RGCs.