Fig 1.
The Drosophila giant fiber escape circuit.
(A) Diagram of the anatomy of one half of the system, it being duplicated contralaterally. In the brain, the giant fiber (GF) receives synaptic inputs onto its dendritic branches from the mechanosensory Johnston’s organ neurons (JONs) and from polysynaptic visual pathways (ret: retina, lam: lamina, med: medulla, ColA: lobular ColA interneurons). It also forms synaptic connections with the giant commissural interneurons (GCI). The GF axon descends to the thoracic ganglion where it forms electrical and chemical synapses with the tergotrochanteral motorneuron (TTMn) of the cylindrical tergotrochanteral (jump) muscle (TTM) and the peripherally-synapsing interneuron (PSI) which innervates the dorsal longitudinal motorneurons (DLMn) of the dorsolongitudinal flight muscles (DLM). (B) Wiring diagram of the circuit. Chemical synapses are denoted by triangles, colored to represent the transmitter where it is known. Electrical synapses are denoted by double bars, colored to represent which innexin is involved, with arrows indicating putative rectifying or non-rectifying junctions. The synapse investigated in the current study is indicated with a question mark. Evidence compiled from several publications [5,7,9–11].
Fig 2.
Effects of Innexin knockdown on the amplitudes of sound-evoked potentials.
(A) Sample traces of sound-evoked potentials (SEPs) recorded from the base of the antenna, in response to 200 Hz sound pulses. As the sound level is increased (dB), the SEP amplitude also increases. The first two SEPs in the train are numbered 1 and 2. (B-I) Amplitudes of SEP1 and SEP2 in animals with RNA interference constructs targeting different innexins, compared to controls (JO15-GAL4/+ in B and peb-GAL4 > Dcr-2 in C-I). Paired t-tests or Mann-Whitney tests (with a Bonferroni correction for 3 comparisons) were used to determine significant differences from control. These are indicated with blue asterisks and a light blue background tint. (B) JO15-GAL4 driving ogre-RNAi (short hairpin, so Dcr-2 not required). The JO15-GAL4 driver was used instead of peb-GAL4, which proved lethal. Both SEPs were reduced at higher sound intensities. (C) peb-GAL4 driving Inx2-RNAi (short hairpin). A reduction was seen in SEP2 at low intensity. (D) peb-GAL4 driving Inx3-RNAi and UAS-Dcr-2. Both SEPs were reduced for low or medium sounds. (E) peb-GAL4 driving RNAi of zpg (Inx4) and UAS-Dcr-2. No changes in SEPs were seen. (F) peb-GAL4 driving Inx5-RNAi and UAS-Dcr-2. No changes in SEPs were seen. (G) peb-GAL4 driving Inx6-RNAi (short hairpin). Reduction was seen in both SEPs for low and medium sounds. (H) peb-GAL4 driving Inx7-RNAi and UAS-Dcr-2. No changes in SEPs were seen. (I) peb-GAL4 driving shakB-RNAi (Bloomington short hairpin). A small increase was seen in SEP2 for loud sounds. For full genotypes see S1 Table.
Fig 3.
The strength of the synapse between JONs and the GF can be monitored through indirect recording of the GF system.
(A) Electrophysiological recording set up. A low-voltage stimulus across the eyes (red electrodes) was used to stimulate the visual pathway. A 200-Hz sine-wave sound stimulus (green) delivered with a speaker was used to stimulate the JONs. Sound-evoked potentials (SEPs) were recorded from the antennal nerve with an electrode (dark green) inserted at the base of the antenna. The output of the GF pathway was recorded from the TTM (black electrode) and the DLM (blue electrode). (B1-4) Averages of 10 recording traces. (B1) A suprathreshold voltage stimulus depolarizes the GF above threshold, resulting in action potentials in both DLM and TTM in all 10 trials (10/10). (B2) For subsequent experiments, the voltage stimulus was adjusted so that the GF was just under its threshold for action potentials (subthreshold). Note the large stimulus artifacts in the antennal nerve traces. (B3) A 90 dB sound stimulus summates with the subthreshold voltage stimulus to activate the GF. The GF fires an action potential in 9 of 10 traces, making the averaged amplitudes of the TTM and DLM spikes slightly lower than those in B1. The delay of the voltage stimulus was adjusted in order to measure the GF response to SEP1. (B4) The stimulus delay was increased to measure the response to SEP2. The GF response occurs in only 4 of 10 traces, despite the larger SEP2 amplitude, making the averaged amplitude even smaller. SEP1 is obscured by the stimulus artefact. (C) Box-whisker plots of the GF’s response to SEPs of increasing amplitudes (a measure of the strength of the synapse between JONs and GF), SEPs of different amplitudes being achieved by altering the sound intensity. An increase in amplitude of SEP1 gives an increase in GF spike probability. SEP2 has significantly less excitatory effect on GF, particularly at large amplitudes. The flies are genotype peb-GAL4; UAS-Dcr-2. Numbers of animals are indicated along the top. Statistically significant differences between SEP2 versus SEP1 (Mann-Whitney test, Bonferroni p-correction for 5 comparisons) are indicated with blue asterisks and a light blue background tint. For full genotype, see S1 Table.
Fig 4.
Effects of Innexin knockdown with RNAi on the strength of the JON-GF connection.
Box-whisker plots of the probability of GF spiking in response to SEP1 and SEP2. Controls are shown in gray and correspond to JO15-GAL4/+ in A and peb-GAL4 > Dcr-2 in B-H. Experimental RNAi data are shown in pink. Ogre-RNAi (A) is driven by JO15-GAL4; all the other innexin RNAis are driven by peb-GAL4 with or without UAS-Dcr-2 depending on the RNAi construct. The numbers of animals are indicated along the top. Statistically significant differences between RNAi versus control (Mann-Whitney test, Bonferroni correction for 6 comparisons) are indicated with blue asterisks and a light blue background tint. (A) RNAi of ogre (Inx1). The GF responds significantly more strongly to SEP1 with amplitudes 0.3–0.4 mV. (B) RNAi of Inx2. There is no significant change in the response of the GF. (C) For RNAi of Inx3 there is no significant change in the response of the GF. (D) RNAi of zpg (Inx4). There is no significant change in the response of the GF. (E) For RNAi of Inx5 there is no significant change in the response of the GF. (F) RNAi of Inx6. The GF responds significantly more strongly to SEP1 of 0–0.3 mV amplitudes and to SEP2 of 0–0.4 mV amplitudes. (G) RNAi of Inx7. There is no significant change in the response of the GF. (H) RNAi of shakB (Inx8), using the Bloomington HMC04895 line. Only for this innexin is there a significant decrease in the response of the GF, to SEP1 of greater than 0.2 mV and SEP2 greater than 0.4 mV. For full genotypes see S1 Table.
Fig 5.
ShakB knockdown with different RNAi lines and drivers.
Box-whisker plots of the probability of GF spiking in response to SEP1 and SEP2. Controls are shown in gray, experimental data in pink. Statistically significant differences between RNAi versus control (Mann-Whitney test, Bonferroni p-correction for 6 comparisons) are indicated with blue asterisks and a light blue background tint. (A) The Vienna GD12666 line (shakB-RNAiV) strongly decreases the GF response to both SEPs. (B) The same line, driven by JO15-GAL4 (without Dcr-2) in auditory JONs alone, is also effective at inhibiting the GF response. (C) The JF02603 line, which targets shakB(n) (shakB(n)-RNAi) has no effect on the JON-GF connection. (D) The JF02604 line, which targets shakB(l) (shakB(l)-RNAi) also has no effect on the JON-GF connection. For full genotypes see S1 Table.
Fig 6.
ShakB RNAi driven in the GF alters transmission at its output synapses.
(A) Dorsal and (B) frontal views of a complete confocal stack of the GF in the brain, stained by CD8::GFP driven by the R79H05-GAL4 driver. Scale bar is 20 μm. (C) Circuit diagram of the outputs of the GF, indicating the recording electrodes in the DLM (blue) and TTM (black) muscles. The GF is stimulated directly by high voltage across the eyes. The approximate latency between this stimulus and the muscles responses is indicated. (D) Example traces illustrating the increased latency in an animal where shakB-RNAi is driven by R79H05-GAL4. (E) Examples of traces showing how shakB-RNAi in the GF decreases the ability of DLM to follow the stimuli at 50Hz. (F) Examples of traces showing how shakB-RNAi in the GF decreases the ability of both DLM and TTM to follow the stimuli at 200Hz. (G-I) Box-whisker plots showing significant effects of shakB-RNAi on the latency (G) and following frequency (H, I) of the muscle responses (Mann-Whitney test). For full genotypes see S1 Table.
Fig 7.
ShakB RNAi prevents NB coupling of JONs with GF.
In these animals, peb-GAL4 was used to drive expression of CD8::GFP, and also Dcr-2. (A, B) Diagrammatic views of the brain, showing the pair of GF neurons (magenta) in relation to the antennal afferent projection (green and cyan). (A) Oblique side and (B) dorsal views, arrows indicate anterior (A). The asterisk in B indicates the approximate site of GF axon impalement for dye injection. (C) Enlarged dorsal view of the GF dendrites, showing the anterior large dendrite (den) inserted within a cylinder of JO-A axons, to which they are NB-coupled (and hence appear cyan), and also the lateral (lat) and medial (med) dendrites. The olfactory glomeruli, also labeled by peb-GAL4, are indicated (olf). (D) Dorsal view of a complete confocal stack from a control animal, in which peb-GAL4 drives expression of CD8::GFP (green) and Dcr-2 in JON axons (and other sensory neurons—for clarity, olfactory axons are shown as partially transparent). Neurobiotin (NB: blue) and Lucifer Yellow (LY: red) are injected into the GF (making it appear magenta). NB alone transfers across gap junctions into the green JO-A axons (to appear cyan). Both LY and NB transfer into the commissural interneurons (GCI). (E) Projection from 3–5 horizontal sections through the antennal nerve of a control animal, showing peb-driven GFP and a subset of axons containing NB. (F) Projection of 3 confocal sections taken from the anterior end of the GF dendrite shown in D, showing its insertion within a ring of several GFP-expressing JO-A afferents (A), to which it is NB coupled. (G) Higher magnification view of the NB-coupled A afferents. In the right panel, the green channel is omitted for clarity. (H-J) Animal in which peb-GAL4 drives CD8::GFP, Dcr2 and shakB-RNAi. (H). Section through the nerve, showing absence of NB coupling in the axons. (I) Low- and (J) high-power views of the A group of JON axons surrounding the GF dendrite, showing the absence of NB coupling. For full genotypes see S1 Table. Scale bar in D: 20 μm in D, E, F, H, I; Scale bar in G: 10 μm in G and J.
Fig 8.
ShakB immunostaining and RNAi knockdown.
(A) Dorsal view of a complete confocal stack of a control animal, in which peb-GAL4 drives expression of CD8::GFP (green) and Dcr-2 in JON axons (and other sensory neurons—for clarity, olfactory axons are shown as partially transparent). Neurobiotin (NB: blue) is injected into the GF and transfers across gap junctions into the green JO-A axons (to appear cyan). The red channel shows immunostaining for the gap junctional protein ShakB, which forms plaques in the JON-A axons (arrow) and on the medial GF dendrite (asterisk). Scattered staining is also present in the antennal nerve (arrowheads). (B) Maximum intensity projection of 3 frontal slices, showing peb-driven GFP staining in olfactory glomeruli (olf) and JON axons. The A group of the latter is indicated (dotted oval), showing NB dye transfer (cyan). (C) High power view of the A axons, showing the ShakB plaques overlying the NB staining (magenta), which are seen more clearly in the right panel where the green channel is omitted. (D) Projection of 20 frontal sections through the GF medial dendrites (med) and coupled giant commissural neurons (GCI). ShakB plaques (magenta) are present on the medial GF dendrite (asterisk). (E-G) Experimental animal in which peb-GAL4 drives expression of CD8::GFP, Dcr-2 and shakB RNAi. (E, F) ShakB staining is not present around the NB-filled GF dendrites, nor does NB pass into the A axons (dotted oval). (G) ShakB staining on the GF medial dendrite is unaffected by RNAi in the JONs. For full genotypes see S1 Table. Scale bar in A: 20 μm in A, B, D, E, G; Scale bar in C: 10 μm in C and F.
Fig 9.
Expression of different isoforms of ShakB in JON axons alters dye coupling to the GF.
(A-D), JO15-GAL4 drives expression of UAS-ShakB(N+16) in JO-A and JO-B neurons, along with CD8::GFP to label the axons. LY and NB are injected into the GF and NB is transferred across gap junctions into the JON axons. Some LY also appears to be transferred. (A) is a dorsal view of a 3D reconstruction, (B-D) are Z projections of frontal slices. (B) is a projection of several slices through the nerve, in order to show the NB and LY within the axons. (C) and (D) are low and high power views of slices through the axons in the region of the GF dendrite. (D) The A and B groups of axons are indicated (dotted ovals), along with another group of peripheral A axons that do not dye couple (arrow). (E-H) JO15-GAL4 drives expression of the ShakB(N) isoform and abolishes gap junctional coupling. (F) Projection of sections through the nerve, showing the absence of coupling (compare with B). (H). High power frontal section through the axons in the region of the GF dendrite, showing only the GF filled with both NB and LY. For full genotypes see S1 Table. Scale bar in A: 20 μm in A, B, C, E, F, G; Scale bar in D: 10 μm in D and H.