. 2018 Oct 1;120(4):2049-2058.
Epub 2018 Aug 15.
Optogenetic Manipulation of Medullary Neurons in the Locust Optic Lobe
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Optogenetic Manipulation of Medullary Neurons in the Locust Optic Lobe
Free PMC article
The locust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts ( Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered excitatory postsynaptic potentials and firing of a postsynaptic, looming-sensitive neuron, the lobula giant movement detector. These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects. NEW & NOTEWORTHY Using Semliki Forest virus, we efficiently delivered channelrhodopsin into neurons of the locust optic lobe. We demonstrate that laser illumination increases the firing of the medullary neurons expressing channelrhodopsin and elicits excitatory postsynaptic potentials and spiking in an identified postsynaptic target neuron, the lobula giant movement detector neuron. This technique allows the manipulation of neuronal activity in locust neural circuits using optogenetics.
LGMD; Semliki Forest virus; locust; medulla; optogenetics.
Semliki Forest Virus (SFV) drives Chop-wide receiver (ChopWR)-Venus expression in medullary neurons of the locust optic lobe.
A: schematic diagram showing the plasmid used to generate SFV A7(74)-based vectors encoding ChopWR-Venus. The viral backbone is derived from pSFV(A774nsP) (Ehrengruber et al. 2003). B: schematic of the locust visual system, showing the location of the lobula giant movement detector (LGMD) in the lobula neuropil of the optic lobe. Its 3 dendritic fields (A–C) are labeled in red. Picrotoxin (PTX) was injected in the dorsal uncrossed bundle (DUB) that mediates inhibition to field C. C: medullary neuron somata expressed ChopWR-Venus in the locust optic lobe (white arrows). D: bundles of transmedullary axons expressing ChopWR-Venus (white arrow) travel toward the lobula neuropil (gray arrow). E and F: double stain of the LGMD excitatory dendritic field (Alexa 594, red) and Venus-labeled transmedullary neuron terminal arbors (green) show close apposition in the lobula. E shows the general structure of the afferents contacting the excitatory dendritic field of the LGMD. F is a close-up showing the organization of afferent axons and presynaptic terminals in the immediate vicinity of dendritic field A. L, lateral; M, medial; D, dorsal; V, ventral. Scale bars are listed in each panel. C–F are from 4 different animals.
Laser stimulation via optic fiber with a diameter of 200 μm activated the medullary neurons (Med) expressing Chop-wide receiver (ChopWR)-Venus and the lobula giant movement detector (LGMD).
A: instantaneous firing rate of medullary neurons expressing ChopWR-Venus was increased during 5 s of 488-nm laser stimulation; top: laser stimulation timing; bottom, black trace is the averaged firing rate across 4 trials (gray traces). Rasters below the instantaneous firing rate (IFR) show the medullary neuronal spikes. B: mean firing rate (gray symbols) of the medullary neurons expressing ChopWR-Venus across 6 locusts (connected black dots) was compared with and without laser stimulation; * P = 0.0156 (one-sided Wilcoxon signed rank test). C: instantaneous firing rate of the LGMD increased during 5 s of 488-nm laser stimulation; black trace is the averaged firing rate across 4 trials (gray traces). Rasters below the IFR show the LGMD spikes. D: mean firing rate of the LGMD across 6 locusts (black dots) was compared with and without laser stimulation; * P = 0.0156. E: LGMD membrane potential ( V m) was depolarized during 5 s of 488-nm laser stimulation in a ChopWR-expressing locust ( top), whereas no depolarization was observed in an untransfected control ( bottom). Black trace represents the averaged V m; gray traces are individual trials (4 trials in the ChopWR-expressing locust and 6 trials in the wild-type locust). F: plot of mean median LGMD V m (± 1 SD) with and without laser stimulation in 6 animals. * P = 0.0156.
Laser stimulation triggers inhibitory inputs to the lobula giant movement detector (LGMD) that can be blocked by the γ-aminobutyric acid A (GABA
A) receptor antagonist picrotoxin (PTX, ≤200 μM; see materials and methods). A: laser stimulation (2 s, 488 nm) via an optic fiber with diameter of 10 μm triggered the firing of the LGMD. Top: laser stimulation timing; middle: LGMD membrane potential (V m) from 1 trial; bottom: the averaged LGMD instantaneous firing rate (IFR; gray trace) across 5 trials (light gray traces). Rasters below are spikes of the LGMD from 5 trials. B: puffing PTX increased laser-triggered firing in the LGMD. Top: laser stimulation timing; middle: LGMD V m from 1 trial; bottom: averaged LGMD IFR (black trace) across 5 trials (light gray traces). Rasters below are spikes of the LGMD from 5 trials. C: firing rate of the LGMD triggered by laser stimulation from 15 trials in 3 locusts (5 per animal) was compared with and without puffing PTX [ P = 3.1 × 10 −5, one-sided Wilcoxon signed rank test (WRST)]. D and E: mean firing rates of the LGMD triggered by laser onset ( D) and offset ( E) from 15 trials in 3 locusts were compared with and without puffing PTX ( P = 3.1 × 10 −5 and 4.3 × 10 −4, one-sided WSRT). Gray symbols indicate mean values across 15 trials in 3 locusts. Connected black dots are single trials.
Firing of the lobula giant movement detector (LGMD) increased and saturated in response to increasing laser power. A, Examples of the LGMD instantaneous firing rate (IFR) in response to laser powers of 2, 8, and 16 mW (from
top to bottom; 1 locust). Laser stimulation timing is shown above the top panel (duration: 5 s). Each panel shows the averaged LGMD IFR (black) from 5 trials (gray traces). Rasters below the LGMD IFR are LGMD spikes from the 5 trials ( P = 0.0312 and 0.125 between groups with laser power at 2 vs. 8, and 8 vs. 16 mW by a one-sided Wilcoxon signed rank test. B: number of LGMD spikes evoked by laser stimulation increased and saturated with increasing power. Gray circles connected by gray dashed line indicate mean value in each group. A Kruskal-Wallis test was used to evaluate the effect of laser power across groups ( P = 0.013). A post hoc signed rank test was used to evaluate the difference between 2 groups (5 locusts, 25 trials for each laser power; P values are on panels; ns, no significant difference).
A laser probe narrowing the region activated by the laser elicited only excitatory postsynaptic potentials (EPSPs) in the lobula giant movement detector (LGMD).
A: schematics of the laser probe ( left) and optic fiber ( right). Dashed lines indicate light path. In the laser probe, the beam exits perpendicular to the shaft, thanks to a mirror. B: laser probe stimulation (5 s, 488 nm) triggered EPSPs in the LGMD. Top: laser stimulation timing; bottom: averaged LGMD membrane potential ( V m; black trace) across 4 trials (gray traces). Middle inset: 2 examples of single EPSPs triggered by laser stimulation. C: comparison of the mean across 3–5 trials of the LGMD V m (±1 SD) for 5 locusts with and without laser stimulation. For each animal, mean V m was higher during laser stimulation and higher during the first 2 s than the last 3 s of laser stimulation. * P = 0.0312 by one-sided Wilcoxon signed rank test.
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Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Channelrhodopsins / genetics*
Channelrhodopsins / metabolism
Excitatory Postsynaptic Potentials
Genetic Vectors / genetics
Protein Engineering / methods
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Semliki forest virus / genetics
Sensory Receptor Cells / metabolism
Sensory Receptor Cells / physiology*