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Not so fast: giant interneurons control precise movements of antennal scales during escape behavior of crayfish

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Abstract

High-speed video recordings of escape responses in freely behaving crayfish revealed precisely coordinated movements of conspicuous head appendages, the antennal scales, during tail-flips that are produced by giant interneurons. For tail-flips that are generated by the medial giants (MG) in response to frontal attacks, the scales started to extend immediately after stimulation and extension was completed before the animal began to propel backwards. For tail-flips that are elicited by caudal stimuli and controlled by the lateral giants (LG), scale extensions began with significant delay after the tail-flip movement was initiated, and full extension of the scales coincided with full flexion of the tail. When we used implanted electrodes and stimulated the giant neurons directly, we observed the same patterns of scale extensions and corresponding timing. In addition, single action potentials of MG and LG neurons evoked with intracellular current injections in minimally restrained preparations were sufficient to activate scale extensions with similar delays as seen in freely behaving animals. Our results suggest that the giant interneurons, which have been assumed to be part of hardwired reflex circuits that lead to caudal motor outputs and stereotyped behavior, are also responsible for activating a pair of antennal scales with high temporal precision.

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Acknowledgements

We would like to thank Dr. David Yager who graciously allowed us to use his high-speed video system for this project. We would also like to thank former lab members David Rotstein, Flor Orellana-Diaz, and Ashley Whiteman who contributed to the development of this project and helped with some of the initial experiments. Lastly, we would like to thank current lab members Lucy Venuti, Tawen Ho, and Norma Pena Flores for constructive discussions and their feedback on the initial draft of the manuscript. All experiments were conducted in accordance with animal care guidelines at the University of Maryland, College Park.

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Correspondence to Jens Herberholz.

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Movie S4: Electrical stimulation of the ventral nerve cord with implanted electrodes elicits an LG tail-flip. The movement of the antennal scales and body parallels the observation made with natural stimuli. Due to the dual camera views (from the top and from the front via a mirror), the timing between scale extension and tail flexion can be observed (WMV 3468 kb)

Supplemental Figure S1: Screenshots of single frames showing the synchronized video frames and corresponding physiological traces displayed in Photron Motion Tools for each experimental set. A) The exact time point when scale extension started during an LG tail-flip evoked with a poke to the tail. The window below the video frame shows the recordings of the bath electrodes and the alignment of the cursor with the starting point of the large neuro-muscular field potential that identifies the firing of the LG neuron. Some weak activity has been recorded before the LG neuron fires. B) The time point when the scales were half-way extended during an MG tail-flip evoked with implanted electrodes. The window below the video frame shows the cursor has moved to a position several milliseconds past the signal triggered by the stimulator that activates the implanted electrodes. C) The time point of full extension of the scales during an MG tail-flip elicited with current injection. The window below the video frame shows the position of the cursor past the signal provided by the intracellular amplifier that triggers the current injection. The traces to the right show the current injection and electrophysiological trace that recorded the MG action potential with extracellular electrodes in the ventral nerve cord rostral from the injection site. A current pulse of 300 nA was injected for 2 ms into the MG (JPEG 65 kb)

All videos were recorded at 2000 f/s except Movie S4, which was recorded at 1000 f/s. Videos recorded at 2000 f/s are slowed down ~ 80 × during playback, and they were slowed down further (4 ×) when prepared in Windows Live Movie Maker. The video recorded at 1000 f/s is slowed down 40 × during playback and was slowed down further (8 ×) when prepared in Windows Live Movie Maker. Thus, all movies will appear at the same playback speed. 10 s of video compares to approximately 30 ms in real time. Original video recordings were trimmed and a title page and captions for stimulus onset (for movies showing current injections) were added. No other adjustments were made. Movie S1: A poke to the head elicits an MG tail-flip. The movement of the antennal scales begins quickly after the stimulus, and extension of both scales is almost complete before the animal starts to move backward (WMV 1575 kb)

Movie S2: A poke to the tail elicits an LG tail-flip. The movement of the antennal scales begins with considerable delay after the animal started to move, and extension of both scales is completed around the time when the tail is fully flexed and the animal moves upward (WMV 2347 kb)

Movie S3: Electrical stimulation of the brain connective with implanted electrodes elicits an MG tail-flip. The movement of the antennal scales and body parallels the observation made with natural stimuli (WMV 1325 kb)

Movie S4: Electrical stimulation of the ventral nerve cord with implanted electrodes elicits an LG tail-flip. The movement of the antennal scales and body parallels the observation made with natural stimuli. Due to the dual camera views (from the top and from the front via a mirror), the timing between scale extension and tail flexion can be observed (WMV 3468 kb)

Movie S5: Stimulation of a single MG neuron in the abdominal nerve cord with suprathreshold current elicits an MG tail-flip. Since the animal is pinned down, only weak body movements can be observed. However, the movement and timing of the antennal scales parallel the observation made with natural and electrical stimuli. The time of current injection into the MG neuron is illustrated by the word STIM appearing over the preparation (WMV 1146 kb)

Movie S6: Stimulation of a single LG neuron in the abdominal nerve cord with suprathreshold current elicits an LG tail-flip. Since the animal is pinned down, only weak body movements can be observed. However, the movement and timing of the antennal scales parallel the observation made with natural and electrical stimuli. The time of current injection into the LG neuron is illustrated by the word STIM appearing over the preparation (WMV 685 kb)

Movie S7: Stimulation of a single MG neuron in the abdominal nerve cord with suprathreshold current elicits an MG tail-flip. The left scale of the animal was moved out of its folded position with a fine brush. Upon current injection and MG activation, the scale extends from its current position. The contralateral scale starts moving at the same time from its folded position. The time of current injection into the MG neuron is illustrated by the word STIM appearing over the preparation (WMV 796 kb)

Movie S8: Stimulation of a single LG neuron in the abdominal nerve cord with suprathreshold current elicits an LG tail-flip. The right scale of the animal was moved out of its folded position with a fine brush. Upon current injection and LG activation, the scale first moves back towards the animal’s midline (i.e., retracts) before it extends from its folded position. The retracted scale and contralateral scale start extending at similar times. The time of current injection into the LG neuron is illustrated by the word STIM appearing over the preparation (WMV 899 kb)

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Herberholz, J., Swierzbinski, M.E., Widjaja, A. et al. Not so fast: giant interneurons control precise movements of antennal scales during escape behavior of crayfish. J Comp Physiol A 205, 687–698 (2019) doi:10.1007/s00359-019-01356-y

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Keywords

  • Crayfish
  • Tail-flip
  • Giant interneurons
  • Antennal scales
  • Sensory-motor integration