Neuronal activation in response to IR heating and electric shock

(A) Experimental setup with an infrared (IR) laser aimed at a 7 dpf larval zebrafish to deliver a heat stimulus. (B) Heat maps of GCaMP fluorescence from a representative fish averaged over 5 presentations of IR heating. Enhanced activity is displayed in rostrolateral (lRL, rRL) and ventromedial (lVm, rVm) areas of the pallium, and habenula (lHb, rHb) in coronal and dorsal views post-laser exposure. (C) Timelapse of average neuronal activation in response to exposure to the IR laser in the forebrain of a representative zebrafish. Each square corresponds to an area outlined in the dorsal view in (B). Brain regions where activity measurements were taken are outlined with dotted lines. Background activity was subtracted from all images. (D) Region-specific ΔF/F activity traces from a representative fish, averaged across 5 trials, display pronounced increases in all measured areas following IR laser exposure. (E) Analysis of the area under the curve (AUC; 5 frames, 6.65 seconds) ΔF/F for each fish for each brain region outlined in (B, C). Mean ± SEM for n=5 fish. (F) Illustration of electric shock delivery to a 7 dpf larval zebrafish. (G) Heat maps from a representative fish averaged over 5 presentations of electric shock. Enhanced activity is displayed in rostrolateral (lRL, rRL) and ventromedial (lVm, rVm) areas of the pallium, and habenula (lHb, rHb) in coronal and dorsal views post-laser exposure. (H) Timelapse of average neuronal activation in response to exposure to electric shock in the forebrain of a representative zebrafish. Each square corresponds to an area outlined in the dorsal view in (G). Brain regions where activity measurements were taken are outlined with dotted lines. Background activity was subtracted from all images. (I) Region-specific ΔF/F activity traces from a representative fish, averaged across 5 trials, display pronounced increases in all measured areas following electric shock. (J) Analysis of the area under the curve (AUC; 4 frames, 5.32 seconds) ΔF/F for each fish for each brain region outlined in (G, H). Mean ± SEM for n=5 fish. One-way t-test was used for all statistical comparisons.

Response of neuronal components to noxious heat stimuli and electric shock.

(A) Neuronal component ΔF/F traces from an example fish as determined by CaImAn reveal a diverse response upon IR stimulation at the vertical dotted line. Red denotes activation (increase in activity ≤ 50% of background DF/F); blue, inhibition (decrease in activity ≤ -50% of background DF/F); Black, neither. Order is determined by z-score (μ/σ), which is a measure of the magnitude and consistency of the response. (B) Neuronal component ΔF/F traces as in (A) in response to electric shock at the vertical dotted line. (C) Locations of activated (small red dots) and inhibited (small blue dots) neuronal components in response to heating with an IR laser for all fish tested. Large dots are centers of mass of corresponding neuronal components. Pie graphs (inset) show the number of activated (red) inhibited (blue), and unresponsive (grey) neuronal components. (D) Same as (C), but for neurons that respond to electric shock

Neuronal activation in response to full and partial looming stimuli.

(A) Schematic of a 7 dpf zebrafish subjected to a full-looming disc stimulus. (B) Neuronal activation in a representative zebrafish immediately before and after a full looming stimulus, averaged over 5 presentations, shows an increase in average activity across all brain areas previously found to be activated by noxious stimuli (rHb, lHb, rRl, lRl, rVm, lVm). (C) Timelapse of heatmaps for the six areas outlined in (B) shows an abrupt increase in activity that commences with the full-looming stimulus. (D) Region-specific ΔF/F activity traces from a representative fish, averaged across 5 trials, display pronounced increases in all measured areas following IR laser exposure. (E) Mean increase above background ± SEM for ΔF/F A.U.C. for 5 frames, 6.65 seconds for each fish for each brain region outlined in (B) (F) Schematic of a 7 dpf zebrafish subjected to a partial-looming disc stimulus. (G) Neuronal activation in a representative zebrafish immediately before and after a partial looming stimulus, averaged over 3 presentations. (H) Time-lapse of heatmaps for the six areas outlined in (B) shows an increase in activity in the Hb and Vm regions, but no significant change in activity in the Rl region. Activity in Hb and Vm was sustained for over 10 seconds. (I) Region-specific ΔF/F and activity traces from a representative fish, averaged across 3 trials, display significant activity increases in Hb and Vm, but not in Rl, in response to the partial looming stimulus. (J) Mean increase above background ± SEM for ΔF/F after the partial-looming stimulus (12 frames, ∼16 seconds). All statistical comparisons made with one-way t-test.

Response of neuronal components to full and partial looming stimuli.

(A) Neuronal component ΔF/F traces for an exemplary fish in response to a full looming stimulus at the vertical dotted line, averaged over 5 presentations. Red denotes activation (increase in activity ≤ 50% of background ≤F/F); blue, inhibition (decrease in activity ≤ -50% of background ≤F/F); black, neither. Order is determined by z-score (μ/σ), which is a measure of the magnitude and consistency of the response. (B) Neuronal activity as in (A) in response to a partial looming stimulus averaged over 3 presentations in an exemplary fish. Activated and inhibited traces are seen in Hb and Vm, but not in Rl. (C) Locations of neurons that are activated (small red dots) and inhibited (small blue dots) in response to a full looming stimulus for all fish tested. Large dots are centers of mass of corresponding neuronal components. Pie graphs (inset) show the number of activated (red), inhibited (blue), and unresponsive (grey) neuronal components. (D) Same as (C) in response to a partial looming stimulus for all fish tested.

Neuronal activation in response to vibration, a loud noise, and light.

(A) Schematic of a 7 dpf zebrafish subjected to vibration. (B) Neuronal activation in a representative zebrafish immediately before and after a vibration stimulus, averaged over 5 presentations, shows an increase in average activity across Hb and Vm, but not Rl. (C) Timelapse of heatmaps for areas outlined in (B) show an increase in activity that commences with the initiation of tapping and grows until 14 s after cessation of tapping in Hb. In Vm the activity increases during the tapping stimulus and decreases abruptly after cessation. There is no increase in activity in Rl. (D) Schematic of a 7 dpf zebrafish subjected to the blast of an air horn. (E) Neuronal activation in a representative zebrafish immediately before and after an air horn blast, averaged over 5 presentations, shows an increase in average activity across Hb and Vm, but not Rl. (F) Timelapse of heatmaps for areas outlined in (E) show an increase in activity that commences with the onset of the loud noise in Hb and Vm and lasts for over 10 s. There is no increase in activity in Rl. (G) Schematic of a 7 dpf zebrafish that is first equilibrated to darkness, then exposed to light for 4 s and then returned to darkness. (H) Neuronal activation in a representative zebrafish immediately before and after transitioning from light to darkness, averaged over 5 presentations shows an increase in average activity across Hb and Vm, but not Rl. (I) Timelapse of heatmaps for areas outlined in (H) shows an increase in activity in Hb and Vm that commences ∼ 2 s after the onset of darkness and lasts for over 5 s. There is no increase in activity in Rl.

Response of total ι1F/F within different regions to vibration, a loud noise, and light

(A) Average ΔF/F activity traces within specific regions from a representative fish, averaged across 5 trials in response to a tapping stimulus. (B) Activity rates for 6 fish before and after the tapping stimulus. Significant increases in activity were seen in rHb, lVm, and rVm, but not in lHb and Rl. (C) Average ΔF/F activity traces within specific regions (AUC; 7 frames, 9.31 seconds) from a representative fish, averaged across 5 trials in response to a blast from an airhorn. (D) Responses to a blast from an airhorn from 4 fish. Significant increases in activity were seen in HB and Vm, but not in Rl. (E) Average ΔF/F activity traces within specific regions from a representative fish, averaged across 5 trials in response to a light-to-dark transition. (F) Responses to a light-to-dark transition from 6 fish. Significant increases in activity were seen in rHB and lVm, but not in lHb, rVm, and Rl.

Response of neuronal components to vibration, a loud noise, and light

(A) Neuronal component ΔF/F traces of a representative fish in response to tapping at the vertical dotted line. Red denotes activation (increase in activity ≤ 50% of background ≤F/F); blue, inhibition (decrease in activity ≤ -50% of background ≤F/F); black, neither. Order is determined by z-score (μ/σ), which measures the magnitude and consistency of the response. In Hb and Vm, the responses of neuronal components to all three stimuli tend to be better defined than those in Rl. (B, C) Neuronal component ΔF/F traces as in (A), but in response to the blast of an airhorn (B) or a transition from light to dark (C) at the vertical dotted line. (D-F) Locations of neuronal components in Hb, Rl, and Vm. Small puncta mark locations where neuronal components respond by increasing (red) or decreasing (blue) activity in response to tapping (D), a blast from an airhorn (E) or a transition from light to dark (F). Large dots represent centers of mass of corresponding neuronal components. Pie graphs (inset) show the number of activated (red), inhibited (blue), and unresponsive (grey) neuronal components. Note that in Rl, inhibited neurons dramatically outnumber activated neurons, whereas in Hb and Vm, the proportion of activated components are roughly equal to or greater than that of inhibited components.

Rl is activated by highly threatening stimuli but not by mildly threatening stimuli.

The ratio of activated to inhibited neuronal components (A/I) in Rl is greater than 1 for highly threatening stimuli but less than 1 for mildly aversive stimuli. In contrast, in Hb and Vm, the A/I ration is greater than one for all aversive stimuli.

Cells expressing tiam2a become active when exposed to IR heating.

(A) 7 dpf Tg[Tiam2a::GFP] larval zebrafish exposed 10 times to heating with an IR laser and then immunostained for pERK to label active neurons. Virtually all GFP-positive neurons were co-labeled with pERK, suggesting that Tiam2a-positive cells are activated by a painful stimulus. (B) Close-up of the area bordered by the blue rectangle in (A). (C) 7 dpf Tg[Tiam2a::GFP] larval zebrafish control that was not exposed to heating showed very little overlap between GFP labeling and labeling for pERK. (D) Closeup of the area bordered by the blue rectangle in (C). (E) Average intensity of pERK immunostaining in Tiam2a+ cells, with background (measured in the white rectangle in (A)) subtracted, for fish exposed to IR heating (n = 6) and control fish (n = 8). Significantly more labeling was found in fish exposed to heating (P = 0.0009, t-test) vs. in control fish. (F) Tg[Tiam2a::GFP] fish showing cell bodies concentrated in the rostrolateral area and local axonal connections.