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Parallel visual circuitry in a basal chordate

  1. Matthew J Kourakis
  2. Cezar Borba
  3. Angela Zhang
  4. Erin Newman-Smith
  5. Priscilla Salas
  6. B Manjunath
  7. William C Smith  Is a corresponding author
  1. University of California, Santa Barbara, United States
Research Article
Cite this article as: eLife 2019;8:e44753 doi: 10.7554/eLife.44753
9 figures, 7 videos, 1 table and 9 additional files

Figures

Figure 1 with 2 supplements
Cartoon of a Ciona tadpole larva with outline of the central nervous system.

The minimal visuomotor circuit is shown with circles representing classes of neurons with the number of cells of each class indicated in the parentheses of the key. Abbreviations: dor., dorsal; vent., ventral; ant., anterior; post., posterior; PR-II, photoreceptor group II; PR-I, photoreceptor group I; pr-AMG RN, photoreceptor ascending motor ganglion relay neuron; prRN, photoreceptor relay neuron; MGIN, motor ganglion interneuron; MN, motor neuron. L, left; R, right. Cell types are color coded according to Ryan et al. (2016).

https://doi.org/10.7554/eLife.44753.002
Figure 1—figure supplement 1
Chemical synapse connectivity of minimal visuomotor system of Ciona.

Electrical synapse connectivity of minimal visuomotor system of Ciona. Both panels derived from data in Satoh (1994). Thickness of lines is proportional to synapse strength. Abbreviations: PR-II, photoreceptor group II; PR-I, photoreceptor group I; pr-AMG RN, photoreceptor ascending motor ganglion relay neuron; prRN, photoreceptor relay neuron; MGIN, motor ganglion interneuron. L, left; R, right.

https://doi.org/10.7554/eLife.44753.003
Figure 1—figure supplement 2
Electrical synapse connectivity of minimal visuomotor system of Ciona.
https://doi.org/10.7554/eLife.44753.004
Figure 2 with 1 supplement
Neurotransmitter use in the ocellus.

(a) Coexpression of opsin and VGAT reporter constructs in the ocellus (white and orange arrowheads). Insets show expression of Opsin-1 and VGAT individually. (b) Expression of VGLUT and VGAT in the brain vesicle and epidermis by in situ hybridization. VGAT was observed in an anterior (white arrowhead) and posterior (orange arrowhead) domain of the ocellus. Blue arrowhead indicates VGLUT expression in the ocellus, and red arrowheads indicate VGLUT-expressing epidermal sensory neurons. (c) Posterior VGAT-expression in the ocellus consists of two cells (orange arrowheads), one exclusively expressing VGAT, and one coexpressing VGAT and VGLUT. Two cells in the anterior exclusively express VGAT (white arrowheads). Nuclei are shown as red spheres. Asterixis indicate overlap of VGAT and VGLUT. (d) Neurotransmitter predictions color-coded on a schematic diagram of the ocellus photoreceptors. Lines between photoreceptors indicate chemical synaptic connections taken from Satoh (1994), with red lines indicating projections to the relay neurons. (e) Heat map of neurotransmitter predictions from registration for photoreceptor group I (cells 01–23). Scale assigns color to proportion of iterations predicting VGAT or VGLUT within a particular cell. (f) Confusion matrix of registration of photoreceptor group I cells (cells 01–23). High values (light colors) in the diagonal indicate higher confidence. Abbreviations: dor., dorsal; vent., ventral; ant., anterior; post., posterior; em., eminens cell; RN, relay neuron; AC, antenna cells; pr-AMG RN, photoreceptor ascending motor ganglion relay neuron; prRN, photoreceptor relay neuron; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter; PR-I, photoreceptor group I (01–23).

https://doi.org/10.7554/eLife.44753.005
Figure 2—figure supplement 1
Neurons in the visuomotor circuit postsynaptic to the Group-I Photoreceptors (PR1-PR23).

Lines indicate chemical synapses and their thickness indicates synaptic strength. Data from Ryan et al. (2016). Abbreviations: PR-I, photoreceptor group I; PR-II, photoreceptor group II; pr-AMG RN, photoreceptor ascending motor ganglion relay neuron; prRN, photoreceptor relay neuron.

https://doi.org/10.7554/eLife.44753.006
Figure 3 with 1 supplement
Neurotransmitter use in the relay neurons.

(a) In situ hybridization of VGAT and VACHT to the relay neurons in the brain vesicle. Also visible is the anterior tip of the motor ganglion. Nuclei are shown as spheres. (b) Confusion matrix for relay neuron registration. (c) Confusion matrix for relay neurons grouped by type. (d) Heat map of neurotransmitter predictions from cell registration of relay neurons, with scale showing color by proportion of iterations predicting either VGAT or VACHT. Abbreviations: ant., anterior; post., posterior; dor., dorsal; vent., ventral; MG, motor ganglion; pr-AMG RN, photoreceptor ascending motor ganglion relay neuron; prRN, photoreceptor relay neuron; AntRN, antenna cell relay neuron; PBRN, photoreceptor-bipolar tail neuron relay neuron; PCRN, photoreceptor-coronet relay neuron; PNRN, peripheral relay neuron; VGAT, vesicular GABA transporter; VACHT, vesicular acetylcholine transporter.

https://doi.org/10.7554/eLife.44753.007
Figure 3—figure supplement 1
Relay neuron centroids projected in two dimensions.

The top left panel is from the connectome, and the remaining panels show centroids from seven larvae. Anterior is to the left and dorsal is at the top.

https://doi.org/10.7554/eLife.44753.008
Figure 4 with 1 supplement
Neurotransmitter use in the motor ganglion.

(a and b) Expression of VGAT and VACHT by in situ hybridization in the motor ganglion, lateral (a) and dorsal (b) views. Asterisks indicate predicted ependymal cells. (c) Lateral view of VGAT expression in the AMGs. (d) shows same view as c, but with VACHT expression. (e) Diagram of neurons in the motor ganglion (derived from Figure 1 of Ryan et al., 2017). Box indicates approximate positions of panels c and d. Lateral view; anterior is to the left. (f) Dorsal view of VGAT expression in the AMGs. Asterisk indicates central non-VGAT expressing cell. (g) Three dimensional surface rendering of VGAT expressing cells in the AMGs. (h) Diagram of a dorsal view of the motor ganglion. AMG cells are numbered. Abbreviations: dor., dorsal; vent., ventral; ant., anterior; post., posterior; AMG, ascending motor ganglion neuron; MGIN, motor ganglion interneuron; ddN, descending decussating neurons; ACIN, ascending contralateral inhibitory neurons; MN, motor neuron; VGAT, vesicular GABA transporter; VACHT, vesicular acetylcholine transporter.

https://doi.org/10.7554/eLife.44753.009
Figure 4—figure supplement 1
Representative larvae showing expression pattern for VGAT (green) and VACHT (red) by HCR in situ.

Top four larvae show the most common expression pattern (also observed in larva shown in Figure 4). Larvae 5 and 6 have offset ACINs (arrows). Larva seven is missing both a motor neuron (red asterisk) and an ACIN (green asterisk). Anterior is to the left in all samples.

https://doi.org/10.7554/eLife.44753.010
Figure 5 with 1 supplement
AMPA receptors in negative phototaxis.

(a) Coexpression of an AMPA-receptor and VACHT expression constructs in the relay neurons (white asterisks). The main panel shows the merge while smaller panels at right show single channels. (b) Negative phototaxis assay in control larvae. Yellow arrow indicates direction of 505 nm light. By 60 min (m) the majority of the larvae have swum to the side of the dish away from the light (red arrow). (c) Perampanel-treated larvae do not show negative phototaxis. (d) Quantification of negative phototaxis in control and perampanel-treated larvae. Points indicate the averages from three independent assays, ±standard deviation. Y-axis represents the percentage of larvae found on the side away from the light source (distal third). Abbreviations: VGAT, vesicular GABA transporter; VACHT, vesicular acetylcholine transporter.

https://doi.org/10.7554/eLife.44753.011
Figure 5—figure supplement 1
AMPA-receptor neurons in the Ciona brain vesicle identified with an AMPA-receptor promoter construct driving GFP.
https://doi.org/10.7554/eLife.44753.012
Perampanel does not disrupt the light dimming response.

(a) Light dimming response in control larvae. Shown are 5 s (s) projections from time-lapse videos in which swims appear as lines. Left panel shows a projection 5 s before dimming, and right panel 5 s after dimming. (b) same as a, but larvae were perampanel-treated. (c) Quantification of light dimming response in control and perampanel treated larvae. Larvae were exposed to dimming of 505 nm light from 2- to 60-fold. Dimming response was scored as percent of larvae responding. Bars show averages of three independent assays ± standard deviation.

https://doi.org/10.7554/eLife.44753.015
Effects of the GABA receptor antagonist picrotoxin on swimming behavior.

(a) Frequency of spontaneous swims for control (vehicle only) and picrotoxin-treated larvae in dark conditions (i.e., 700 nm illumination). Each open circle represents a single tracked larva with the number of swim bouts in one minute presented. Also shown are the averages (red circles)±standard deviation. (b) Duration of spontaneous swims in seconds (s). Each circle is one swim bout recorded during a one-minute capture session. (c) Picrotoxin-treated larvae retain negative phototaxis. Bars show the averages of three trails and present the percentage of larvae in the side of the petri dish opposite the illumination (distal third of dish). (d) Dimming response is diminished in picrotoxin-treated larvae, and further diminished by cotreatment with picrotoxin and perampanel. Shown are the averages of three independent trials (37–99 larvae quantified per trial). Data shows the duration in seconds (s) of swims. (e) Tortuosity of sustained swims (>10 s) for control and picrotoxin-treated larvae. Data is presented as in panel a. *, p<0.05; **, p<0.01; ***, p<0.001.

https://doi.org/10.7554/eLife.44753.019
Behavior of homozygous frimousse (frm) larvae.

(a) VGAT and VACHT reporter construct expression in wild type (wt) and frm larvae. (b) Frequency of spontaneous swims of wt and frm larvae in dark conditions (i.e., 700 nm illumination). Each open circle represents one tracked larva with the number of swim bouts in one minute presented. Also shown are the averages (red circles) ±standard deviation. (c) Duration of all spontaneous swims in one-minute recording presented in seconds (s) for wt and frm larvae. (d) Standard deviation (S.D.) of the duration of swim bouts over one minute for each individual larva recorded. (e) S.D. of the interval between swim bouts over one minute for each individual larva recorded. For d and e, larvae with <4 swim bouts were not included in the analysis. ***, p<0.001; n.s., not significant.

https://doi.org/10.7554/eLife.44753.023
Models showing parallel visuomotor pathways for negative phototaxis (top) and light dimming response (bottom).

Neurotransmitters in parentheses are thought to play a lesser role in the proposed pathway. Abbreviations: PR-II, photoreceptor group II; PR-I, photoreceptor group I; pr-AMG RN, photoreceptor ascending motor ganglion relay neuron; prRN, photoreceptor relay neuron; MGIN, motor ganglion interneuron; MN, motor neuron; Glu, glutamate; GB, GABA; ACh, acetylcholine. Cell types are color coded according to Satoh (1994).

https://doi.org/10.7554/eLife.44753.025

Videos

Video 1
Negative phototaxis of control and perampanel-treated Ciona larvae in 10 cm petri dishes.

Directional 505 nm illumination is from the left. Frames were taken at 1 per minute over five hours. In the video the 5 hr is compressed to 15 s (i.e., 1200X normal speed). Black and white tones were inverted to make the larvae more visible.

https://doi.org/10.7554/eLife.44753.014
Video 2
Swimming of control and perampanel-treated Ciona larvae in a directional light field.

Larvae in 10 cm petri dishes were recorded at nine frames/second. Black and white tones were inverted to make the larvae more visible. The video plays at 5X normal speed.

https://doi.org/10.7554/eLife.44753.017
Video 3
Dimming response of control and perampanel-treated Ciona larvae in 10 cm petri dishes.

Larvae were imaged for 70 s at five frames/second, with dimming of 505 nm ambient light at 10 s. Black and white tones were inverted, and thus the dimming appears as a brightening. The video plays at 5X normal speed.

https://doi.org/10.7554/eLife.44753.018
Video 4
Spontaneous swimming behavior in control and picrotoxin-treated Ciona larvae.

Larvae in 6 cm petri dishes were recorded for 1 min at nine frames/second with 700 nm illumination. Black and white tones were inverted to make the larvae more visible. The video plays at 5X normal speed.

https://doi.org/10.7554/eLife.44753.020
Video 5
Negative phototaxis of control and picrotoxin-treated Ciona larvae in 10 cm petri dishes.

Picrotoxin-treated larvae can be seen to stop swimming at ~1 hr. Directional 505 nm illumination is from the left. Frames were taken at one per minute over five hours. In the video the 5 hr is compressed to 15 s (i.e., 1200X normal speed). Black and white tones were inverted to make the larvae more visible.

https://doi.org/10.7554/eLife.44753.021
Video 6
Dimming response of control and picrotoxin-treated Ciona larvae in 10 cm petri dishes.

Larvae were imaged for 20 s at five frames/second, with dimming of 505 nm ambient light at 10 s. Black and white tones were inverted, and thus the dimming appears as a brightening. The video plays at 2X normal speed.

https://doi.org/10.7554/eLife.44753.022
Video 7
Spontaneous swimming behavior in wild type and homozygous frimousse larvae.

Larvae in 6 cm petri dishes were recorded for 1 min at 9 frames/second with 700 nm illumination. Black and white tones were inverted to make the larvae more visible. The video plays at 5X normal speed.

https://doi.org/10.7554/eLife.44753.024

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Chemical
compound, drug
fluorescently-labeled
RNA probes for VGAT,
VGLUT, VACHT
Molecular instruments
Chemical
compound, drug
SlowFade GoldInvitrogen
Chemical
compound, drug
perampanelSanta Cruz Biotech;
Adooq Bioscience
Chemical
compound, drug
picrotoxinTocris
Equipment700 nm and 505 nm
led light sources
Mightex
Equipmentlight meterExtech Instruments
Recombinant
DNA reagent
Ciona robusta
pVGAT > CFP
Yasunori Sasakura,
University of Tsukuba,
Japan
Recombinant
DNA reagent
C. robusta opsin
promoter
(as pSP-Ci-opsin1)
Takeo Horie, Tsukuba University,
Japan; Takehiro
Kusakabe, Konan University, Japan
Recombinant
DNA reagent
synthesized RFPGene Block; IDT
Recombinant
DNA reagent
C. robusta
pAMPAR > GFP
Haruo Okado, Tokyo Metropolitan
Institute of Medical Science, Japan
See Hirai et al.,
(Hirai et al., 2017).
Recombinant
DNA reagent
C. robusta
pOpsin > RFP
This paper.This construct was created
using the opsin promoter
(Horie and Kusakabe) and
a synthesized RFP (Gene
Block, IDT).
Recombinant
DNA reagent
C. robusta
pVGAT > H2B::RFP
This paper.This construct was created
using the Gateway cloning
system, cloning pVGAT into
pDONR-221-P3-P5, then
recombining with an
H2B::RFP entry clone.
Software,
algorithm
Python v 2.7Python Software
Foundation
Software,
algorithm
Microsoft ExcelMicrosoft
Software,
algorithm
Imaris v 9.1Bitplane
Software,
algorithm
MATLABWolframAlpha
Software,
algorithm
ELIANE script for
MATLAB
This paper.Estimator of Locomotion
Iterations for Animal
Experiments; a MATLAB
script for tracking
swimming larvae during
behavioral assays.
Strain, strain
background
C. intestinalisMarine Biological Laboratory,
Woods Hole, Massachusetts,
USA
Formerly known as
C. intestinalis type B.
Strain, strain
background
C. robustaSanta Barbara Harbor, USA;
S. Lepage, M-REP, Carlsbad,
USA
Formerly known as
C. intestinalis type A.
Strain, strain
background
pVGAT > kaede,
C. robusta stable line
National Bioresource
Project, Japan

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 5 and 6.

Additional files

Source code 1

Calculates transformation between each dataset in a folder and the EM data based on nuclei location in 3D.

https://doi.org/10.7554/eLife.44753.026
Source code 2

Calculates cell assignments based on an affine transformation between each dataset and the EM data based on nuclei location in 3D.

https://doi.org/10.7554/eLife.44753.027
Source code 3

Plots the nucleus locations in 3D for each dataset along with the EM dataset using Plot.ly.

https://doi.org/10.7554/eLife.44753.028
Source code 4

Estimator of locomotion iterations for animal experiments: Stimulus Response Tracker v0.07 (Matlab script).

https://doi.org/10.7554/eLife.44753.029
Source data 1

Photoreceptor matrix.

https://doi.org/10.7554/eLife.44753.030
Source data 2

Relay neuron matrix.

https://doi.org/10.7554/eLife.44753.031
Source data 3

Grouped relay neuron matrix.

https://doi.org/10.7554/eLife.44753.032
Supplementary file 1

Nucleotide sequences for HCR probe selection.

https://doi.org/10.7554/eLife.44753.033
Transparent reporting form
https://doi.org/10.7554/eLife.44753.034

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