Animals have evolved specialized neural circuits to defend themselves from pain- and injury-causing stimuli. Using a combination of optical, behavioral and genetic approaches in the larval zebrafish, we describe a novel role for hypothalamic oxytocin (OXT) neurons in the processing of noxious stimuli. In vivo imaging revealed that a large and distributed fraction of zebrafish OXT neurons respond strongly to noxious inputs, including the activation of damage-sensing TRPA1 receptors. OXT population activity reflects the sensorimotor transformation of the noxious stimulus, with some neurons encoding sensory information and others correlating more strongly with large-angle swims. Notably, OXT neuron activation is sufficient to generate this defensive behavior via the recruitment of brainstem premotor targets, whereas ablation of OXT neurons or loss of the peptide attenuates behavioral responses to TRPA1 activation. These data highlight a crucial role for OXT neurons in the generation of appropriate defensive responses to noxious input.
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All data, code (hardware control and analysis) and resources (transgenic lines/mutants generated) will be made available by the corresponding authors upon request.
Live versions of the analysis code are maintained at https://www.github.com/carolinewee.
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The authors thank E. Glasgow (Georgetown University) for providing the Tg(oxt:GFP) transgenic line and oxt:GFP plasmid, which we used to generate the oxt:Gal4 line, L. O’Connell and H. Gainer (NINDS) for providing an OXT antibody and M. Manning (University of Toledo) for providing the pharmacological tools for preliminary experiments. The authors are especially grateful to D. Schoppik, M. Haesemeyer, D. Guggiana-Nilo and J. Yang for helping with preliminary experiments and to R. Portugues for sharing software. They also thank C.-T. Yang and M. Ahrens for sharing transgenic lines used in preliminary experiments and the Zebrafish International Resource Center (ZIRC) for providing the Tg(UAS:nfsb-mCherry) transgenic line. Support from S. Turney and the CBS imaging facility, as well as A. Viel and the NorthWest Undergraduate Teaching Laboratories at Harvard, were essential for the successful completion of many experiments. J. Miller, S. Zimmerman, K. Hurley and B. Hughes at Harvard, as well as the University of Utah Centralized Zebrafish Animal Resource (CZAR), provided invaluable fish care. Finally, the authors thank N. Uchida, W. Carlezon, R. Dorsky and C. Gregg for helpful discussions and comments. This work was supported by the Sloan Foundation (to A.D.D.), NIH grants R01-NS111067 (to A.D.D.), U01-NS090449 (to F.E.), R24-NS086601 (to F.E.), U19-NS104653 (to F.E., A.D.D. and S.K.), T32-HL007901 (to A.M.B.L.), T32-NS076067 (to S.L.-M.), F31-NS100412 (to J.P.B.), DP1-HD094764 (to A.F.S.), Simons Foundation grant SCGB 325207 (to F.E.), and NSF CAREER IOS-10043082 (to A.D.D.). C.L.W. was supported by the National Science Scholarship from the Agency for Science, Technology and Research (A*STAR), Singapore.
The authors declare no competing interests.
Peer review information: Nature Neuroscience thanks Alexandre Charlet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
(a)We developed an automated analysis pipeline (Fiji/ImageJ) to extract single-cell MAP-mapping data from high-resolution brain images. Acquired images are first cropped and reduced to a region of interest (ROI) spanning the OXT population. The tERK channels are inverted, allowing individual particles to be extracted based on size/shape criteria. The GFP (that is OXT-labeling) channel is likewise processed to isolate individual GFP-positive cells. pERK, tERK and GFP intensities, as well as cell position are extracted from all identified cells. Data are normalized to control means and pooled across multiple experiments. One of two independent criteria was used to define OXT-positive cells: 1) All particles identified in the GFP channel; 2) Particles identified in the tERK channel that also exceeded a threshold for GFP fluorescence. Neurons with a mean GFP intensity below 400 are automatically classified as non-OXT neurons. Since GFP-dependent cell segmentation is more accurate than the tERK-dependent process, we utilized the GFP-dependent segmentation method for the data shown in Figures 1 and 2; however, tERK-based segmentation generally produced similar conclusions. For example, for electric shock (6V/cm) treatment, an average increase in normalized pERK/tERK ratios of 0.35 and 0.33 was quantified for tERK versus GFP-extracted OXT neurons respectively.
(a) Neurons labeled by Tg(oxt:GFP) expression are oxytocinergic. A = Anterior, L = Left. Images (left to right) show 1) Tg(oxt:GFP) fluorescence; 2) Anti-OXT staining (Altstein and Gainer, 1988); 3) merged image. Scale bar = 50 μm. Right-most panel shows a higher magnification image of OXT clusters on the top (scale bar = 5 μm) and the pituitary gland (more ventrally-located, scale bar = 50 μm) on the bottom. Cytoplasmic GFP labeling is detectable but weak in the pituitary gland (Pit.), as compared to anti-OXT staining. aPO/pPO = anterior/posterior Preoptic area, PT = posterior tuberculum (where PT cluster resides). This experiment was repeated on 13 fish with similar results. (b) Maximum intensity projection image showing anti-OXT staining in another fish. Images (left to right) show 1) anti-OXT staining; 2) merged image. Scale bar = 20 μm. White asterisks denote OXT stained cells that were not GFP-positive. Only a few (<5) such cells, usually anteriorly-situated, were observed per fish, suggesting that Tg(oxt:GFP) labels almost the entire OXT population; 3) OXT neurons color-coded according to OXT antibody staining intensity. Colorbar indicates fluorescence intensity in grayscale units: Red, fluorescence intensity >3000; orange, 2000–3000; yellow, 1500–2000, cyan, 1000–1500; blue, <1000. Neurons expressing higher levels of OXT, likely magnocellular neurons, are located more anteriorly. The PT OXT cluster comprises exclusively parvocellular cell types. Magno./M = putative magnocellular, Parvo./P = putative parvocellular. This experiment was repeated on 13 fish with similar results. (c) Histograms of OXT peptide expression levels (that is OXT antibody staining intensities) in individual GFP-positive neurons for 6 different fish. (d) Histogram (top) showing the distribution of normalized OXT levels (i.e. OXT antibody staining intensities) in 13 larvae (606 neurons). OXT expression level for each neuron was normalized to the median intensity of all neurons of each fish. A two-component Gaussian mixture model (GMM, bottom) gave the lowest Bayesian Information Criterion (BIC) values across various model types, confirming bimodality of the distribution. For k=2 (covariance type = diagonal – unshared), the GMM generated two Gaussians with the following means and variance: μ1 = 0.89, σ1 = 0.034, μ2 = 1.96, σ2 = 0.21. We subsequently used K-means clustering (k=2) to divide the neurons into two categories, likely representing magnocellular and parvocellular cell types. The first group (putative parvocellular, gray) comprised 455/606 (75%) of the population (mean normalized intensity = 0.92 ± 0.010) and the second group (putative magnocellular, red) comprised 151/606 (25%) of the population (mean normalized intensity = 2.12 ± 0.028). (e) Anti-OXT staining intensity as a function of position. Left: A-P position (μm) is computed relative to the most-anterior neuron for each fish. Neurons classified as magnocellular by higher OXT staining intensity are located more anteriorly (red, median = 9.65 μm) than putative parvocellular cells of the PO (blue, median = 38.38 μm) and PT clusters (cyan, median = 125.98 μm). Magnocellular vs parvocellular (PO + PT): ***p = 1.03x10−59, magnocellular vs parvocellular (PO): ***p = 3.08x10−53, n = 151 (magno.), 369 (parvo. PO), 86 (parvo. PT), from 13 larvae, two-sided Wilcoxon rank-sum test. Right: D-V position (μm) is computed relative to the most-dorsal neuron for each fish. Neurons classified as magnocellular are distributed more dorsally (median = 8 μm) than parvocellular cells of the PO (median = 16 μm) and PT clusters (median = 34 μm). Magnocellular vs parvocellular (PO + PT): ***p = 1.48x10−24, Magnocellular vs parvocellular (PO): ***p =7.56x10−17, two-sided Wilcoxon rank-sum test. (f) OXT cell size (area) as a function of position. Sample sizes are same as in (e). Left: OXT cell area as a function of A-P position. Cells classified as magnocellular based on anti-OXT staining intensities are significantly larger and located more anteriorly (normalized area = 1.18 ± 0.02) than putative parvocellular cells in the PO (normalized area = 0.97 ± 0.01) or PT cluster (normalized area= 0.97 ± 0.03). Cell areas are normalized to the median cell area per fish. Magnocellular vs. parvocellular: ***p = 5.59x10−18, Magnocellular vs. parvocellular (PO) ***p = 2.07x10−17, two-sided Wilcoxon rank-sum test). Right: OXT cell area as a function of D-V position. Larger cells are typically located more dorsally. (g) In situ hybridization shows overlap of OXT mRNA with Tg(oxt:GFP) expression in the PO cluster. Stronger mRNA expression is observed more anteriorly, likely corresponding to magnocellular cell types. No staining of the PT cluster was observed, consistent with results from Wircer et al., 2017, which reported extremely low mRNA levels that were only observable using a highly-sensitive fluorescent in-situ method. This experiment was repeated on 15 fish with similar results. Scale bar = 100 μm. (h) Left: Maximum intensity projection image showing overlap of Tg(oxt:Gal4) (magenta) and Tg(oxt:GFP) (green) expression. Tg(oxt:Gal4) labels fewer neurons in the PT cluster. Scale bar = 20 μm. Right: Lower magnification maximum intensity projection of Tg(oxt:Gal4) and Tg(oxt:GFP) expressing fish showing extensive OXT neuron projections to many parts of the brain, including the pituitary gland and hindbrain/spinal cord. This experiment was repeated on 9 fish with similar results. Scale bar = 100 μm. Altstein, M., and Gainer, H. (1988). Differential biosynthesis and posttranslational processing of vasopressin and oxytocin in rat brain during embryonic and postnatal development. J. Neurosci. 8, 3967–3977. Wircer, E., Blechman, J., Borodovsky, N., Tsoory, M., Nunes, A.R., Oliveira, R.F., and Levkowitz, G. (2017). Homeodomain protein Otp affects developmental neuropeptide switching in oxytocin neurons associated with a long-term effect on social behavior. Elife 6.
(a) Comparison of OXT neuron response to shock, mustard oil, heat and taps (n = 231 neurons from 7 fish), from the same experiments shown in Figure 2d. Here, the integrated calcium response per unit time was summarized by averaging the Δf/f signal over a 40 s window either beginning at stimulus onset (for tap and shock signals) or spanning the peak of the response (for heat and mustard oil signals), and dividing by window size. The neurons were further sorted according to their positions along the A-P axis. Responsive OXT neurons were distributed across the entire A-P extent, and multiple stimuli typically activated single neurons. A = anterior, P = posterior. (b) Examples of calcium responses from individual fish. Only a single z-plane was imaged for each fish, covering either dorsal (Fish 1) or ventral (Fish 2) populations. Shock, mustard oil and heat tended to activate the same neurons, whereas tap responses were weaker overall. Scale bar = 20 μM. (c) Example tail angle traces showing behavioral responses to aversive stimuli. Red dots indicate stimulus onset for taps and shock, red box indicates stimulus period for heat and mustard oil. Mustard oil and heat induce large angle tail bends, whereas shock, an unnatural stimulus, drives a mixture of forward swims and tail bends. Taps induce C-bends, as further described in Figure S7. This experiment was repeated on more than 5 fish per treatment similar results.
(a) Correlation between stimulus and motorstim regressors in DMSO and Optovin. Mean Pearson’s correlation coefficient (r value) is 0.36±0.03 in DMSO, and 0.67±0.04 in Optovin. N = 14 of each regressor type (corresponding to 14 fish from 14 independent experiments). Bar plots show mean ± SEM. (b) Pearson’s correlation coefficients for mCherry signals in OXT-positive neurons, which estimate the probability of spurious correlations that may occur due to motion artifacts, laser power fluctuations or other factors. At a threshold of r = 0.35/−0.35, no false positives for either positively or negatively-correlated neurons would be identified, with the exception of motorspon neurons in Optovin, which had some spurious negative correlations (highlighted with asterisks). We thus used the thresholds of r= 0.35/−0.35 to cluster neurons in in our analysis of GCaMP signals, and since we did not identify any negatively-correlated neurons at these thresholds we conclude that the low residual false-positive rate for the motorspon group was not a confounding factor in our analyses. (c) Examples of “multimodal” cells that are correlated with more than one regressor. Each panel corresponds to a single OXT neuron. Calcium traces (gray) show the neuron’s activity (Δf/f) in both DMSO and Optovin (highlighted in pink). The corresponding six regressors and Pearson’s correlation coefficients (r-value) are also displayed below in their respective color codes. Bolded r-values are those that exceed the threshold of r = 0.35. Panels (i) and (ii) show neurons that are correlated to stim/motorstim regressors in both DMSO and Optovin. Panels (iii) and (iv) show neurons that show strong correlation specifically to spontaneous movements. Panels (v) and (vi) show neurons whose activity correlates both with spontaneous movements and the TRPA1 stimulus/behavior in Optovin.
(a) Mean intensity projection image showing OXT neurons (magenta) overlaid on a pan-neuronal GCaMP6s background (grayscale). One ventral and one dorsal plane are shown. Non-OXT neurons were selected from the area shown in the grey boxes. This experiment was repeated on 13 fish with similar results. (b) Bout-triggered calcium average of OXT (top) and non-OXT (bottom) neurons in the PO, for the first post-stimulus bout and all large-angle (>100°) spontaneous bouts. Post-stimulus bouts (OXT): n = 2001 (DMSO; 1mW), 2686 (Optovin; 1mW), 1438 (DMSO; 7mW), and 2165 (Optovin; 7mW) calcium traces from 13 fish. Post-stimulus bouts (non-OXT): n = 4753 (DMSO; 1mW), 6527 (Optovin; 1mW), 3480 (DMSO; 7mW), and 5179 (Optovin; 7mW) calcium traces from 13 fish. Large angle non-stimulus bouts (OXT): n = 3527 (DMSO) and 1999 (Optovin) calcium traces from 13 fish. Large angle non-stimulus bouts (non-OXT): n = 9510 (DMSO) and 5257 (Optovin) calcium traces from 13 fish. (c) Histograms showing Pearson’s correlation coefficients (r-values) of OXT (top) and non-OXT (bottom) neurons to all 6 regressors. OXT neurons have a much stronger right-shift in stim and motorstim r values in Optovin (mean increasestim = 0.12, p = 1.96x10−31; mean increasemotorstim = 0.13, p = 4.78x10−44) than non-OXT neurons (mean increasestim = 0.045, p = 2.52x10−12; mean increasemotorstim =0.053, p =4.24x10-18, one-sided Wilcoxon rank-sum test). (d) Percentage of cells that would be classified into each cluster as a function of the cutoff r-value used. At all thresholds, the largest clusters would correspond to neurons showing high correlation to the stimulus and motorstim regressors in Optovin (i.e. TRPA1 responsive neurons). Color code as in S5c. (e) Classification of neurons into 6 different response types. Each row corresponds to a single neuron, and the Pearson’s correlation coefficients to the corresponding regressors are represented by color intensity. At r >0.35 or r <-0.35, a smaller fraction of non-OXT neurons were correlated with all the stimulus regressors in Optovin (12% (non-OXT) vs 32% (OXT)) or motorstim (10% (non-OXT) vs 28% (OXT)). They were also less correlated to DMSO regressors (stim: 4% (non-OXT) vs 7% (OXT), 0.5% (non-OXT) vs 3% (OXT)). Since on average only ~70% of the OXT population was labeled, it is possible that some of these may be non-labeled OXT neurons. The proportion of identified stim and motorstim neurons were comparable to Tg(oxt:Gal4;UAS:GCaMP6s), suggesting that the activation we observe is consistent across multiple genotypes and datasets. Similarly, a smaller subset of fish showed activity in DMSO as compared to in Optovin. In contrast, the proportion of motor-correlated neurons during spontaneous movements in DMSO was much lower than in the Tg(oxt:Gal4;UAS:GCaMP6s) dataset. This might be a consequence of incomplete or selective GCaMP labeling of the OXT population by the HuC promoter. (f) Clustered neurons, color-coded to identify cells imaged in specific fish, highlight similarities between OXT and non-OXT neurons within and between fish. The numbers at the top of each column denote the number of animals in which each neuronal subtype was observed out of the 13 total fish that were imaged. (g) 2D spatial distributions (left) of OXT and non-OXT neurons that are either more stimulus or motor-tuned, and histograms of their positions along the anterior-posterior axis (right). For visualization purposes, the scale for Optovin stimulus-correlated neurons is different from other groups. Non-OXT neurons that were TRPA1-responsive occupied the full extent of the AP axis, from the aPO to the PT. Similar to the Tg(oxt:Gal4;UAS:GCaMP6s) dataset, motor-tuned OXT neurons appeared to be more concentrated at the posterior end, but non-OXT neurons did not show the same trend. Interestingly, there appeared to be a left-right asymmetry (i.e. left bias) in non-OXT neuron activity, particularly in response to TRPA1 stimulation. Whether this is related to the slight asymmetry observed in behavioral output remains to be explored (mean behavioral symmetry (from 0-1, where 0.5 is perfectly symmetrical): DMSO = 0.57 ± 0.03 (right bias), Optovin = 0.40 ± 0.01 (left bias) (HuC:GCaMP6s fish); DMSO = 0.56 ± 0.03 (right bias), Optovin = 0.41 ± 0.02 (left bias) (oxt:Gal4;UAS:GCaMP6s fish)).
(a) Volumetric imaging of an example Tg(HuC:GCaMP6s; oxt:Gal4; UAS:nsfb-mCherry) fish. Raster plots show Δf/f activity traces for all visible OXT and non-OXT neurons (n = 30 OXT neurons, n = 53 non-OXT neurons, 3 non-overlapping z-planes) for the entire experiment (10 UV stimuli of 100 ms duration alternating between 2 intensities (1 mW and 7 mW), 120 s inter-stimulus interval (ISI)). In this plot, neurons are sorted along the A-P axis. The same neurons were visually identified in both the DMSO and Optovin conditions. Bottom: Average Δf/f for all neurons and simultaneously recorded tail angle traces. Red dots represent UV stimuli, with larger dots representing the higher intensity stimulus. Non-OXT neurons had similar responses to UV in DMSO, but weaker responses to UV in Optovin, than OXT neurons. (b) Regressors for 6 neuron classes (corresponding to the stimulus, stimulus-locked behavior (motorstim) and spontaneous (motorspon) behavior in both DMSO and Optovin), and example OXT and non-OXT neurons from Tg(HuC:GCaMP6s) experiments that show strong correlation (Pearson’s) with each regressor. The neuron’s response in DMSO and Optovin (shaded pink) are shown. Note that each neuron may correlate with more than one regressor.
(a) Histogram showing response latencies during 5 s stimulation of Tg(oxt:Gal4; UAS:ChR2-YFP) fish (same as in Fig. 5g); overlaid with responses from stimulating a subset of trigeminal neurons in Tg(isl2b:Gal4;UAS:ChR2YFP) fish, also for 5 s (mean = 0.32 ± 0.10 s, n = 16 responses from 3 fish, magenta). Stimulating trigeminal neurons produces predominantly short-latency responses, unlike stimulating OXT-expressing neurons under the same conditions. (b) Comparison of bout kinematics during optogenetic activation of Tg(oxt:Gal4) or Tg(isl2b:Gal4)-expressing neurons and vibrational-acoustic tap stimulus. Labels define parameters that are used to compare bout kinematics across different stimuli, for example traces elicited by taps and Tg(oxt:Gal4;UAS:ChR2-YFP) stimulation. C-bends are characterized by a directional bend, counter-bend and forward swim, whereas tail bends elicited by OXT neuron activation tend to be more variable in directionality and have a larger cumulative angle. (c) Behavior elicited by optogenetic stimulation of OXT neurons is kinematically distinct from that elicited by optogenetic stimulation of isl2b-positive (somatosensory) neurons, as well as from C-bends produced by strong vibrational-acoustic tap stimuli. Kinematic parameters include the integrated areas under the entire bout (bout area), under the first tail bend (1st bend area) and under the first and second tail bends (1st + 2nd bend area); bout duration, duration of the first tail bend (1st bend duration), magnitude of the first tail bend (θ), and number of undulations. For each of these parameters, asterisks below bars denote groups with kinematics that are significantly different (p<0.05) from Tg(oxt:Gal4;UAS:ChR2-YFP), n = 9/20/20/10 fish (oxt (5 s), isl2b (5 s) / isl2b (100 ms) / Taps), Kruskal-Wallis Test. Bar plots show mean ± SEM. Bout area: p = 0.51/0.025/9.37x10-4; 1st bend area: p = 0.29/0.072/0.020; 1st + 2nd bend area: p = 0.090/0.030/5.3x10-4; Bout duration: p = 0.61/0.028/2.67x10-5; 1st bend duration: p = 0.80/0.48/0.012; Magnitude: p =0.0030/0.013/0.0021; Undulations: p = 0.30/0.16/0.042 (isl2b (5 s) / isl2b (100 ms) / Taps compared to oxt (5 s) stimulation). (d) The probability (left) and frequency (right; bouts/second) of optogenetically-induced swim bouts correlates with the number of ChR2-expressing OXT neurons in the PO (OXTPO) neurons, for a 5 s stimulus (Response probability: r = 0.64, ***p = 2.4x10-6; Response frequency: r = 0.73, ***p = 6.25x10-6, n = 44 fish, Pearson’s correlation). Mosaic expression of ChR2 in OXT neurons was achieved by injecting the oxt:Gal4 construct into Tg(UAS:ChR2-YFP) embryos, whereas stronger expression was attained by outcrossing or incrossing Tg(oxt:Gal4;UAS:ChR2-YFP) transgenics. Colored points correspond to the respective fish shown in (e). (e) Swim behaviors could be induced even in fish that had weak/few pituitary projections (top row), suggesting that hindbrain/spinal projections are sufficient to drive behavior. Note that these fish tend to lack aPO labeling, consistent with the observation that more anterior OXT neurons project to the pituitary (Supplementary Fig. 2). a/pPO = anterior/posterior preoptic area, PT = posterior tuberculum, Pit. = Pituitary. Scale bar = 50 μm. These experiments were repeated in more than 25 fish with similar results.
(a) Distribution of the duration, frequency, and duty cycle of swim bursts (as measured by EMG output) within the 5 s time window after the onset of the Optovin or ChR2 stimulus. Swim burst duration: Optovin, n = 997 bursts from 4 fish; Tg(oxt:Gal4;UAS:ChR2-YFP), n = 216 bursts from 5 fish. Swim burst frequency: Optovin, n = 909 bursts from 4 fish; Tg(oxt:Gal4;UAS:ChR2-YFP), n = 161 bursts from 5 fish. Duty cycle: Optovin, n = 909 bursts from 4 fish; Tg(oxt:Gal4;UAS:ChR2-YFP), n = 161 bursts from 5 fish. Number of swim bouts and the summed duration of all swim bouts within the 5 s time window after stimulus onset: Optovin, n = 4 fish; Tg(oxt:Gal4;UAS:ChR2-YFP), n = 5 fish; *p = 0.0159 (duration); NS, p = 0.0635 (number of bouts), two-sided Wilcoxon rank-sum test. Bar plots show mean ± SEM. (b) Probability distributions of swim bouts evoked by TRPA1 (i.e. 50 μM Optovin, n = 147 bouts from 6 fish), Tg(oxt:Gal4;UAS:ChR2-YFP) activation (n = 158 bouts from 6 fish) and acoustic-startle stimuli (n = 58 bouts from 6 fish). Peak angles (°), maximum and mean velocities (°/s), bout durations (ms), and latencies (ms) are plotted per bout. Only the largest-angle bouts for each trial (10 trials per fish in total) were included in this analysis. Trials that failed to elicit a behavior were excluded. Peak angles induced by Optovin are more variable than for OXT stimulation, but can reach similarly large magnitudes, unlike startle responses. Acoustic startle durations are relatively long due to a prolonged forward swim following the C-bend. Note that the ChR2 response latencies are shorter than in other figures as 405 nm light (rather than 488 nm) was used for activation. Similar to 488 nm light, this wavelength does not induce behavioral responses in control animals. *p<0.05, **p<0.01, ***p<0.001, two-sided Wilcoxon rank-sum test. Peak angle: p = 4.5x10-7 / 1.3x10-8 / 4.1x10-19; Max velocity: p = 2.1x10-5 / 5.8x10-26 / 9.2x10-22; Mean velocity: p = 1.5x10-7 / 6.3x10-18 / 1.5x10-11; Duration: p = 0.27 / 0.66 / 0.38; Latency: p = 0.52 / 4.5x10-28 / 2.3x10-29 (Optovin vs ChR2 / Optovin vs Startle / ChR2 vs Startle). (c) Comparison of TRPA1, ChR2 and acoustic startle behavior per fish, including response frequencies. Bar plots show mean ± SEM. The largest angle bouts per trial (10 trials per fish in total) were used the analysis. *p<0.05, **p<0.01, ***p<0.001, two-sided Wilcoxon rank-sum test. Peak angle: p = 0.086 / 0.018 / 0.0017; Max velocity: p =0.128 / 5.3x10-4 / 4.6x10-4; Mean velocity: p = 0.031 / 7.1x10-4 / 0.0022; Duration: p = 0.45 / 0.46 / 0.85; Latency: p= 0.26 / 5.3x10-4 / 4.6x10-4; Frequency: p = 5.6x10-4 / 3.8x10-4 / 3.9x10-4 (Optovin vs ChR2 / Optovin vs Startle / ChR2 vs Startle). (d) Examples of tail angle traces for each type of stimulus from the dataset reported above.
(a) Data from a single clutch of incrossed heterozygotes, stimulated with blue light and monitored behaviorally as described in the main text. Gray = WT, Blue = het, Red = null mutant. Response probability: F(1,168) = 1.14, p=0.29 (homozygous WT vs het), F(1,102) = 3.9, p=0.051 (WT vs null), F(1,126) = 1.99, p= 0.16 (het vs null); Response frequency: F(1,168) = 0.48, p=0.49 (WT vs het), F(1,102) = 5.71, *p=0.017 (WT vs null), F(1,126) = 3.55, p = 0.062 (het vs null); Number of bouts: F(1,168) = 0.28, p=0.59 (WT vs het), F(1,102) = 6.15, *p = 0.015 (WT vs null), F(1,126) = 4.52, *p=0.036 (het vs null); n = 13 (WT), 17 (het), 6 (null), two-way ANOVA. Center black line = Mean, Box = 1 SD, range = 95% confidence interval. (b) Data from a single clutch (het crossed to null mutant). Response probability: F(1,210) = 8.29, **p=0.0044 (het vs null); Response frequency: F(1,210) = 12.46, ***p = 0.0005 (het vs null); Number of bouts: F(1,210) = 13.66, ***p= 0.0003 (het vs null); n = 21 (het), 16 (null), two-way ANOVA. Center black line = Mean, Box = 1 SD, range = 95% confidence interval. (c) Comparing ChR2-induced responses in heterozygous and WT fish (het crossed to WT, 2 clutches pooled). Response probability: F(1,185) = 0.02, p=0.90 (WT vs het); Response frequency: F(1,185) = 0.62, p=0.43 (WT vs het); Number of bouts: F(1,185) = 0.91, p=0.34 (WT vs het); n = 10 (WT), 21 (het), two-way ANOVA. Center black line = Mean, Box = 1 SD, range = 95% confidence interval. (d) While the absolute number of ChR2-evoked tail bends was smaller in null mutants than in heterozygous siblings, we found no significant differences in the mean peak turn angles between heterozygous and null mutants for a 5s stimulation duration. Mean peak tail angles: 75.54 ± 2.16° (hets) vs 68.18 ± 1.28° (null), n =704 (het), 253 (null) detected bouts, p = 0.53, two-sided Wilcoxon rank-sum test.
(a) The mean differences (post-pre values) in peak angle (°), maximum and mean velocity (°/s), bout duration (ms) and latency (ms) for sham and M-cell ablated fish reveal no specific deficits except in the latency of ChR2-induced behavior. All bouts were aggregated per fish. Bar plots show mean ± SEM. Fish with a post-ablation response probability of <0.5 were excluded from the kinematic analysis. *p < 0.05. Δ Peak angle: p = 0.13 (Optovin Sham), 0.31 (Optovin Ablated), 0.084 (ChR2 Sham), 0.22 (ChR2 Ablated); Δ Max velocity: p = 1 (Optovin Sham), 0.69 (Optovin Ablated), 0.28 (ChR2 Sham), 0.56 (ChR2 Ablated); Δ Mean velocity: p = 0.20 (Optovin Sham), 0.84 (Optovin Ablated), 0.49 (ChR2 Sham), 0.31 (ChR2 Ablated); Δ Duration: p = *0.012 (Optovin Sham), 0.69 (Optovin Ablated), 1 (ChR2 Sham), 0.84 (ChR2 Ablated); Δ Latency: p = 0.91 (Optovin Sham), 0.0625 (Optovin Ablated), 0.85 (ChR2 Sham), *0.03 (ChR2 Ablated); N = 9 (Optovin Sham), 6 (Optovin Ablated), 10 (ChR2 Sham), 6 (ChR2 Ablated) fish, two-sided Wilcoxon rank-sum test. (b) Bootstrapped mean differences confirm an increase in ChR2 response latencies (mean = +359 ms, 95%CI [+134 613], ***p= 4x10-4) in ablated (red) but not sham (blue) fish. TRPA1 (Optovin) response latencies also appeared to be significantly decreased (*p=0.02) using this permutation test. Black line indicates zero mean difference. All bouts extracted per fish, as well as the set of fish, were resampled with replacement for 10000 iterations to generate a bootstrapped distribution of mean differences (post-pre values) between pre and post ablation behavior. P<0.05 (one-tailed) if % below (or above) zero is less than 5%. * = p < 0.05, **p<0.01, ***p<0.001.
(a) Effective ablation of Tg(oxt:Gal4;UAS:nfsb-mCherry)-labeled neurons using MTZ. Representative images are shown of non-ablated (left) and ablated fish brains (right). Scale bar = 20 μm. Fish were treated with MTZ from 4-6 dpf, and their behavior was tested at 8 dpf. Note that due to variability in expression, ablation of OXT population is likely to be partial (~50-75%). This experiment was performed on more than 40 fish with similar results.
Supplementary Figs. 1–11.
Optovin-mediated photochemical activation of TRPA1 receptors in the tail using a mercury arc light source. A head-restrained fish was treated with optovin (10 μM), and a 150-μm diameter area of the tail was illuminated with ultraviolet light for 100 ms. Imaging rate, 400 f.p.s., playback rate is in real time. A ultraviolet laser rather than an arc light source was used for calcium imaging and other behavioral experiments, but the tail was similarly stimulated at a single point.
Optogenetic activation of OXT neurons. A head-restrained fish was illuminated with a 150-μm spot of blue light (6 mW mm–2) focused on the preoptic area for 3 s while imaging at 400 f.p.s. Playback rate is in real time.
OXT reduced preparation immediately after OXT. Behavior of reduced larval zebrafish preparation (brain tissue above hindbrain removed) immediately after OXT (5 μm) application to the bath. Imaging rate, 400 f.p.s., playback rate is in real time.
OXT reduced preparation 5 min after OXT. Behavior of reduced larval zebrafish preparation (brain tissue above hindbrain removed) 5 min after OXT (5 μm) application to the bath. Imaging rate, 400 f.p.s., playback rate is in real time.
Behavioral response to blue light flash in DMSO. Behavior of a freely-swimming fish in DMSO (0.1%) before and after a 100-ms blue light (470 nm) pulse. This setup was the same as used in Fig. 8.
Behavioral response to blue light flash in optovin. Behavior of a freely-swimming fish in optovin (10 μM, 0.1% DMSO) before and after a 100-ms blue light (470 nm) pulse. This setup was the same as used in Fig. 8.