Antagonistic Inhibitory Circuits Integrate Visual and Gravitactic Behaviors - PubMed (original) (raw)

Antagonistic Inhibitory Circuits Integrate Visual and Gravitactic Behaviors

Michaela Bostwick et al. Curr Biol. 2020.

Abstract

Larvae of the tunicate Ciona intestinalis possess a central nervous system of 177 neurons. This simplicity has facilitated the generation of a complete synaptic connectome. As chordates and the closest relatives of vertebrates, tunicates promise insight into the organization and evolution of vertebrate nervous systems. Ciona larvae have several sensory systems, including the ocellus and otolith, which are sensitive to light and gravity, respectively. Here, we describe circuitry by which these two are integrated into a complex behavior: the rapid reorientation of the body followed by upward swimming in response to dimming. Significantly, the gravity response causes an orienting behavior consisting of curved swims in downward-facing larvae but only when triggered by dimming. In contrast, the majority of larvae facing upward do not respond to dimming with orientation swims-but instead swim directly upward. Under constant light conditions, the gravity circuit appears to be inoperable, and both upward and downward swims were observed. Using connectomic and neurotransmitter data, we propose a circuit model that can account for these behaviors. The otolith consists of a statocyst cell and projecting excitatory sensory neurons (antenna cells). Postsynaptic to the antenna cells are a group of inhibitory primary interneurons, the antenna relay neurons (antRNs), which then project asymmetrically to the right and left motor units, thereby mediating curved orientation swims. Also projecting to the antRNs are inhibitory photoreceptor relay interneurons. These interneurons appear to antagonize the otolith circuit until they themselves are inhibited by photoreceptors in response to dimming, thus providing a triggering circuit.

Keywords: Ciona; connectome; gravitaxis; multisensory integration; neural circuit.

Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.

Figure 1.. Ciona gravitaxis circuit.

A. Twenty-five hours post-fertilization (hpf) larva expressing Kaede fluorescent protein from an injected plasmid containing the vesicular glutamate transporter (VGLUT) cis-regulatory element. The antenna cells are green. B. Simplified minimal gravitaxis circuit. Cells of similar type and connectivity (circles) and their synaptic connections (lines) were combined based on connectome data from a 21-hpf larva. Cell types includes the two antenna cells (Ant), the eleven antenna relay neurons (Ant RN), the three left and right motoganglion interneurons (MGIN L and MGIN R, respectively), the five left and right motor neurons (MN L and MN R, respectively), and the 18 left and right muscles (Mu L and Mu R, respectively). Likely neurotransmitter types derived from in situ hybridization studies are indicated (Glut: glutamate; GABA: gamma-aminobutyric acid; ACh: acetylcholine), as well as putative excitatory (green), inhibitory (red) and electrical (gap junction) synapses (black). Other abbreviations: OT: otolith cell, BV: brain vesicle; MG: motor ganglion.

Figure 2

Figure 2. Ciona gravitaxis behavior.

A. Imaging set-up for recording gravitaxis behavior. B. Five-second projection images of 21- and 25-hour post fertilization (hpf) larvae swimming in response to the turning off of visible light (lights-off condition; swim traces in white on dark background). The beginning position of larvae is indicated in green and their position after 5 seconds is in red. Up is towards the top of the images. The orange arrow indicates a larva swimming downward and the blue arrow indicates a larva swimming sideways. C. Quantified swim trajectory data for swims under the indicated conditions and developmental ages. Percentages of larvae within the categorized trajectories [(UP, DOWN and SIDE (sideways)] are shown. Values are from the averages of nine videos (± standard deviation), with 20 larvae assessed per video. D. Matrix comparing the compiled swim data from different conditions (numbered as in C) for significant differences. (*: p < 0.05; ***: p<0.005; Wilcoxon Signed Rank Test). E. Quantified swim trajectory data for 25-hpf larvae in response to 6- and 10-fold dimming of visible light. F. lights-off induced swims are more tortuous at 21-hpf than at 25-hpf (***: p <0.005; Wilcoxon Ranksum Test).

Figure 3.

Figure 3.. Reorientation Behavior.

A. Response of stationary up-facing and down-facing larvae to lights-off. The initial position of larvae are shown in green, and swims are projected over time in white. B. Response of swimming larvae to lights-off. Projection images of larvae show the trajectory before lights-off (green) and after (blue). Magenta marks the first frame of lights-off. C. Both upward and downward swimmers accelerate at lights-off. Swim speeds before and after lights-off are indicated (*: p<0.05; ***: p<0.005; T-test).

Figure 4.

Figure 4.. Gravitaxis response is inhibitory.

A. Imaging set-up used to record behavioral response of Ciona larvae to being rotated with respect to gravity. B. Results of rotation experiments. The top row shows up-facing larvae immediately before rotation, immediately after rotation, or after visible light is turned off. The bottom row shows the same conditions, but with down-facing larvae. The results show the percentage of larvae in 10-second windows displaying short, long, or no swims. For comparisons between groups, swim categories were assigned values and assessed with the Wilcoxon Signed Rank Test (*: p<0.05; *** p<0.005). C. Five-second projection images showing swims in control and picrotoxin-treated larvae. Swims appear as white lines. Up is toward the top of the image. D. Quantification of swim trajectories in control and picrotoxin-treated larvae, analyzed as in Figure 2C.

Figure 5.

Figure 5.. Gravitaxis comparisons in 21- and 25-hours post fertilization larvae.

A. Control (DMSO vehicle) and perampanel-treated larvae. Shown are 10-second projections of swims following lights-off. Larvae were assessed at 21- and 25 hours post fertilization (hpf). B. Quantification of swim trajectory in control and perampanel-treated larvae. Shown are the swim trajectories [UP, DOWN or SIDE (sideways)] in five second windows following lights-off. The values are averages from 10 time-lapse videos (20 larvae analyzed per video) plus or minus standard deviations. For comparisons between groups, swim directions were assigned values and assessed with the Wilcoxon Signed Rank Test. C. Swim directions following lights-off in wild-type and homozygous pristine mutants. The analysis was performed as in B (*: p<0.05; *** p<0.005). D. Frames from time-lapse recordings showing representative reaction times to lights-off at 21- and 25-hpf. Times in milliseconds (msec) are indicated.

Figure 6.

Figure 6.. Model of Gravitaxis Circuitry.

Each panel shows both a cartoon larva with the relative activation of the muscle cells shown from low (yellow), to intermediate (orange) and high (red), and a diagram of the gravitaxis and photoreceptor circuits. In the circuit diagrams, proposed inhibitory synaptic projections are in red, excitatory are in green, and electrical in black. A. Behavior and circuitry of a down-facing larva before (left), and after (right) visible light being turned off (lights-off). Asymmetric inhibition from the antRNs leads to asymmetric L/R muscle activation and turning (green arrow). B. Behavior and circuitry of downward swimming larva. Note that inhibition of the antRNs by the pr-AMG RNs allows for symmetrical activation of muscles (orange) before lights-off. C. Behavior and circuitry of up-facing larva. Note that the antenna cells are inactive when the larva is in this position. At lights-off the larvae can go directly to symmetric swim. D. Behavior and circuitry of upward swimming larva. In the model release of inhibition by the pr-AMG RNs on the MGINs at lights-out leads to acceleration. Abbreviations: PR-II: photoreceptor group II; pr-AMG RN: photoreceptor-ascending motor ganglion relay neurons; Ant: antenna cells; antRN: antenna relay neurons; MGIN: motor ganglion interneurons; R: right: L: left.

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