Transcriptional control of behaviour: engrailed knockout 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

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.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

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

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

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 Gycβ100B-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

Double-stranded RNA interference shows that Engrailed controls the synaptic specificity of identified sensory neurons.

The transcription factor Engrailed (En) controls the topography of axonal projections by regulating the expression of cell-adhesion molecules [1-4] but it is not known whether it also controls the choice of individual synaptic target cells. In the cercal sensory system of the larval cockroach (Periplaneta americana), small numbers of identified wind-sensitive sensory neurons form highly specific synaptic connections with 14 identified giant interneurons [5,6], and target-cell choice is independent of the pattern of axonal projections [6]. En is a putative positional determinant in the array of cercal sensory neurons [7]. In the present study, double-stranded RNA (dsRNA) interference [8] was used to abolish En expression. This treatment changed the axonal arborisation and synaptic outputs of an identified En-positive sensory neuron so that it came to resemble a nearby En-negative cell, which was itself unaffected. We thus demonstrate directly that En controls synaptic choice, as well as axon projections

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 [3,4,5]. 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 [5,6,7,8]. However, the rules governing this variability are largely unknown. Here, we demonstrate that individual cockroaches (Periplaneta americana, a much-studied model prey species [9,10,11,12,13,14]) 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

Subsets of JONs expressing GMR GAL4 drivers.

<p>mCD8::GFP expression driven by different Flylight <i>GAL4</i> drivers (green). <b>A, B</b>. 30 h APF pupal pedicel, showing <i>inv 88B12</i>> GFP, along with antibodies against En protein (red) and both En and Inv (blue). <b>A</b>. Posterior half, with cluster of large En+ neurons. <b>B</b>. Anterior half, with a single large En+ neuron. <b>C, D</b>. 36 h APF pupal pedicel, showing <i>Gycβ100B 12G03</i>> GFP, along with antibodies against En protein (red) and both En and Inv (blue), and cuticular autofluorescence false-colored orange. <b>C</b>. Posterior half, with several dorsal and ventral neurons, some of the latter are En+. <b>D</b>. Anterior half, with weak expression in a single large En+ neuron. <b>E, F</b>. 72 h APF pupal pedicel, showing <i>CG12484 91G04</i>> GFP, along with cuticular autofluorescence false-colored orange. <b>E</b>. Posterior half, with many dorsal and ventral neurons. <b>F</b>. Anterior half, with several neurons. Dorsal (D), lateral (L), and anterior (A) axes are indicated by arrows. Scale bar: 20 µm.</p