Engrailed Alters the Specificity of Synaptic Connections ofDrosophilaAuditory Neurons with the Giant Fiber

Abstract

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, RNAimediated 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.

Active Mechanisms of Vibration Encoding and Frequency Filtering in Central Mechanosensory Neurons

“…However, it seems likely that JONs form both electrical and chemical synapses onto B1 cells, because the very earliest part of the B1 step response (<10 ms from step onset) was affected by both the shakB mutation and the nicotinic antagonists. This conclusion is also consistent with anatomical data indicating that JONs form both gap junctions and chemical synapses with central neurons (Pezier et al, 2014; Pezier et al, 2016; Sivan-Loukianova and Eberl, 2005). …”

Effects of gap junction misexpression on synapses between auditory sensory neurons and the giant fiber ofDrosophila melanogaster

“…In the innexin study we observed that overexpression of the 'correct' ShakB isoform, ShakB(N+16), in a subset of JONs induces ectopic synaptic coupling, whereas expression of the 'wrong' isoform, ShakB(N), abolishes it entirely [16]. In an earlier paper we had shown that ectopic synaptic coupling could also be induced by overexpression of the transcription factor Engrailed (En) in those same sensory neuronsin that case we showed that it was accompanied by the de novo formation of chemical synapses, along with an increase in postsynaptic dendritic branching [15]. Taken together, these results suggested the possibility that both En and ShakB(N+16) are similarly able to alter the specificity of the JON-GF synaptic connection, by causing the formation of new mixed electrical and chemical synapses between inappropriate synaptic partners.…”

“…To process other flies, nervous systems were dissected in PBS, fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature and rinsed in several changes of PBS. After removal, nervous systems were processed for antibody labeling, and cleared and mounted as previously described [15]. nc82 (anti-Bruchpilot) antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB) and used at a dilution of 1/20.…”

“…Confocal image stacks were imported into Fiji image analysis software [25,26], where they were 3D-rotated to a standard position and adjusted for optimal contrast. The large artefactual Brp-sh-Strawberry agglomerations noted in [15] were automatically removed from the red channel by applying a minimum filter (3 pixels) followed Image quantification was similar to that described previously [15]. Using Fiji, the channels were Autothresholded using the Intermodes method for Brp, the Default method for the DA488 and NB signals, and the RenyiEntropy method for the ShakB signal.…”

Insect Hearing

“…The different neuronal subpopulations of JO, which vary in their respective sensitivities to oscillatory (e.g., sound) or stationary (e.g., wind/gravity) stimulation have initially been defi ned anatomically by their distinct target zones within the fl y's brain . The homeodomain transcription factor Engrailed (En) is a suffi cient, and most likely necessary, requirement for the subpopulation-specifi c axonal targeting of JO auditory neurons to the GFN (Pézier and Blagburn 2013 ;Pézier et al 2014 ). Misexpression of En in En-negative JO-Ns causes them to form ectopic chemical and electrical synapses with the GFN; RNAi-mediated knockdown of En expression in En-positive JONs, in turn, reduces the strengths of JO-N to GFN synaptic connections.…”