Multiple convergent hypothalamus-brainstem circuits drive defensive behavior

Abstract

The hypothalamus is composed of many neuropeptidergic cell populations and directs multiple survival behaviors, including defensive responses to threats. However, the relationship between the peptidergic identity of neurons and their roles in behavior remains unclear. Here, we address this issue by studying the function of multiple neuronal populations in the zebrafish hypothalamus during defensive responses to a variety of homeostatic threats. Cellular registration of large-scale neural activity imaging to multiplexed in situ gene expression revealed that neuronal populations encoding behavioral features encompass multiple overlapping sets of neuropeptidergic cell classes. Manipulations of different cell populations showed that multiple sets of peptidergic neurons play similar behavioral roles in this fast-timescale behavior through glutamate co-release and convergent output to spinal-projecting premotor neurons in the brainstem. Our findings demonstrate that homeostatic threats recruit neurons across multiple hypothalamic cell populations, which cooperatively drive robust defensive behaviors.

Extended Data Fig. 1 ∣. Responses to rapid environmental changes do not influence forward swimming, do not habituate over trials, and are not influenced by ablation of pomc+ cells in the pituitary.

a, Schematic of behavior (left), and measurement of threat onset/offset kinetics using a thermocouple (heat) or two-photon imaging of fluorescence in flow solution (flow - salinity, acidity) (right). b, Threat onset does not influence forward swimming (mean ± s.e.m., 1 s bins, N = 57 fish (5 trials each), same fish as shown in Fig. 1c). c, Fraction of trials with turning movements during threat presentation, across all 5 trials (mean ± s.e.m., N = 57 fish). Comparison of first two trials vs last two trials. All p > 0.1, Two-sided Wilcoxon signed-rank test. d, Ventral sections of Tg(pomc:Gal4;UAS:NTR-mCherry) fish, showing ablation of pomc+ cells in the pituitary (red) upon treatment with metronidazole (MTZ; right). Similar ablation observed in N = 3 fish examined. e, Summary data from control and ablated fish. N = 9 fish per group, mean ± s.e.m., individual fish are points. One-tailed Mann-Whitney U tests, Benjamini-Hochberg false discovery rate correction for multiple comparisons. NS = ‘not significant’, all p > 0.4. f, Maximum tail angle of responsive movements - summary data from control and ablated fish. Note that control fish have lower response magnitude on salinity trials. N = 9 fish per group, mean ± s.e.m., individual fish are points. One-tailed Mann-Whitney U tests, Benjamini-Hochberg false discovery rate correction for multiple comparisons. *p < 0.05. NS = ‘not significant’, all p > 0.4. See Supplementary Table 2 for test statistics.

Extended Data Fig. 2 ∣. Coverage, registration, and anatomical segregation of brain-wide imaging data.

a, All z-planes functionally imaged in a single Tg(elavl3:H2B-GCaMP6s) fish - 14 z-planes, 15 μm separation, 2 volumes/second. All N = 8 fish were imaged with the same x/y/z-sampling. b, Example z-planes from N = 6 fish, after registration to a common volume. Fish were registered to a common ‘bridge’ brain; the standard Tg(elavl3:H2B-RFP) brain from the Z-Brain atlas (along with atlas masks) were registered to this bridge brain, to allow for anatomical region analysis. c, Example z-planes from a single fish, with a subset of overlaid anatomical regions from the registered Z-Brain atlas. The same regions were aligned to each of the N = 8 fish. d, Step-by-step protocol for registering each recorded brain to a common volume, and assigning recorded neurons to brain regions in the Z-brain atlas.

Extended Data Fig. 3 ∣. Brain-wide imaging and linear models of single neurons reveal feature-selective neurons across brain regions.

a, Schematic of experiment, and example tail angle over threat presentation. b, Schematic of single-neuron analyses, and data from an example neuron. A linear model of each cell’s denoised fluorescence time series is composed from behavioral events (convolved with calcium indicator decay), and the importance of each event is determined by the change in model performance (ΔR²) upon shuffling of each event time-series (unique model contributions, UMC). UMC plot for the example neuron (right). This example neuron preferentially encodes salinity. c, Spatial distribution of UMC for each variable, shown in side projection of the medial 40 μm of whole-brain volume. Values are displayed as the percentile within the distribution of UMC for that task component across all cells. Brain regions are outlined in brown. d, Violin plots showing the distribution of unique model contributions (UMC, ΔR²) for brain regions denoted by the Z-brain atlas. Density does not extend beyond the minima and maxima of the data (0-100^th percentile). Quartiles are not shown. The number of neurons in each group are noted at bottom, along with the region name.

Extended Data Fig. 4 ∣. Details of MultiMAP experimental protocol for registration of live functional imaging to multi-color fluorescent in situ hybridization.

a, Step-by-step protocol for performing MultiMAP procedure, and assigning recorded neurons to molecularly-defined populations. b, Overlay of single live-imaging z-plane (mean of all time-points in motion-corrected plane) with the associated z-plane from registered volumes. Similar results were obtained from all N = 7 fish. c, Validation of registration in the hypothalamus, using the red fluorescent protein (RFP, mCherry in this case) as a “held out” marker in both live and fixed volumes to assess accuracy of fixed-to-live registration of H2B-GCaMP (in Tg(oxt:Gal4; UAS:NTR-mCherry; elavl3:H2B-GCaMP6s) fish (N = 4). Note that after registration, the held-out RFP signals precisely overlap. Scale bar = 10 μm.

Extended Data Fig. 5 ∣. Cellular-resolution registration of live functional imaging with up to three rounds of triple fluorescent in situ hybridization.

a, All z-planes functionally imaged in a single Tg(elavl3:H2B-GCaMP6s) fish - 5 z-planes, 15 μm separation, 4 volumes/second. Showing live GCaMP z-plane (grey), and fixed GCaMP z-planes from round 1 and round 2 of in situ hybridization (green and red, respectively), before (left) and after (middle) volume registration. Overlap of live GCaMP and the six registered in situ hybridization labels from two rounds of multicolor fluorescent in situ hybridization (right). b, Example z-planes from all N = 7 fish used in Figs. 2 and 3. c, Overlap of molecular labels in cell types identified (N = 7 fish, Pearson’s R). Note the co-expression of avp with the medial crf population. d, Example z-planes functionally imaged in a single Tg(elavl3:H2B-GCaMP6s) fish, showing live GCaMP z-plane (grey), and fixed GCaMP z-planes from round 1, 2, and 3 of fluorescent in situ hybridization (green, red, and cyan, respectively), before and after volume registration. Overlap of live GCaMP and the nine registered in situ hybridization labels from three rounds of triple fluorescent in situ hybridization (right). Similar expression patterns are obtained from each of the N =3 fish tested.

Extended Data Fig. 6 ∣. Functional clusters of hypothalamic neurons.

Stimulus-aligned activity averaged across all recorded neurons and trials, and unique model contribution (UMC) values, for all functional clusters identified in Fig. 3c. These plots include all recorded neurons in Tg(elavl3:H2B-GCaMP6s) fish, rather than just the identified peptidergic neurons.

Extended Data Fig. 7 ∣. Characterization of Gal4 lines, optical excitation, and electrophysiological validation of ChR2 expression.

a, Overlap of GFP expression in the preoptic area of Tg(oxt:Gal4; UAS:GFP) and Tg(sst3:Gal4; UAS:GFP) with in situ hybridization labels, and overlap of GFP expression in Tg(otpba:Gal4;UAS:GFP) line with fluorescent in situ hybridization for oxt, crf, and avp. Similar expression for N = 3 fish per genotype. Crf and avp are co-localized in the same cells. b, Co-localization of GFP and crf in Tg(crf:Gal4; UAS:GFP) fish. Note that GFP expression is specific to crf+ neurons, but that only 29% of crf+ neurons are GFP+ (n = 2 fish). c, Imaging oxt+ and crf+ neurons in transgenic lines. Image of GCaMP expressing neurons and event-averaged responses of neurons recorded from Tg(oxt:Gal4; UAS:GCaMP6s) and Tg(crf:Gal4; UAS:GCaMP6s) fish (top and bottom, respectively). Neurons pooled from N = 2 fish per genotype. d, Localization of optical stimulation for optogenetics experiments. 405 nm light was delivered through the same optic fiber used for ChR2 or GtACR1 stimulation, and converts kaede from green to red. The increased green-to-red (pseudocolored magenta) change in the preoptic area demonstrates limited spatial extent of optogenetic stimulation. Similar results obtained for N =3 fish tested. e, in vivo cell-attached recordings from single ChR2-mCherry+ and neighboring ChR2-mCherry-negative neurons in the preoptic hypothalamus of a Tg(oxt:Gal4; UAS:ChR2-mCherry) fish, during stimulation with the same parameters used for behavioral experiments. 5 Hz blue light trains evoke spiking in the opsin-expressing neuron. Similar results were obtained from 2 other neurons.

Extended Data Fig. 8 ∣. Controls related to cellular ablation.

a, Expression of UAS:NTR-mCherry in the preoptic hypothalamus of each Gal4 line, and neuronal ablation upon treatment with MTZ (related to Fig. 4e, f). A similar extent of ablation was observed in all fish examined. b, Maximum tail angle of responsive movements - summary data from control and ablated fish. Same fish as Fig. 4f. Mean ± s.e.m., individual fish are points. One-tailed Mann-Whitney U tests, Benjamini-Hochberg false discovery rate correction for multiple comparisons within genotype. NS = “not significant”, all p > 0.1. c, Inactivation of preoptic neurons in Tg(otpba:Gal4; UAS:GtACR1-eYFP) fish. Schematic of experiment (left), image of GtACR1-eYFP expression (middle; similar expression observed in all N = 8 fish.), and behavioral results (right). N = 8 fish per group, mean ± s.e.m., individual fish are points. One-tailed Mann-Whitney U tests, Benjamini-Hochberg false discovery rate correction for multiple comparisons. NS = “not significant”, all p >0.1. *p < 0.05. While light ON trials increases total movement in control and opsin fish, GtACR1-expressing fish move less under these conditions. d, Ablation of neurons in Tg(th:Gal4) line, including the dopaminergic posterior tuberculum neurons also labeled in the Tg(otpba:Gal4) line, does not influence behavior in this assay. A similar extent of ablation was observed in all fish examined. N = 8 fish per group, mean ± s.e.m., individual fish are points. One-tailed Mann-Whitney U test, Benjamini-Hochberg false discovery rate correction for multiple comparisons. NS = “not significant”, all p > 0.4. See Supplementary Table 2 for test statistics.

Extended Data Fig. 9 ∣. Details of RoL1 location, two-photon single-cell ablation of spinal projection neurons, and local injection of NBQX.

a, Location of spinal projection neurons (SPNs), with cell names, and axonal projections in Tg(oxt:Gal4; UAS:GFP) fish. b, Location of SPNs relative to monoaminergic cells, labeled with spinal backfill in a Tg(vmat2:eGFP) line. RoL1 neurons reside slightly anterior and medial to the noradrenergic neurons of the locus coeruleus. c, Example images showing sequential two-photon single-cell ablation of three RoL1 neurons in a Tg(elavl3:H2B-GCaMP6s) fish backfilled with Texas red dextran. This specificity and extent of ablation was confirmed across all N = 10 fish. d, Images of an example fish, backfilled with Texas red dextran, before and after bilateral ablation of the Mauthner neurons and segmental homologues (“M-array” ablation). This specificity and extent of ablation was confirmed across all N = 11 fish. e, Maximum tail angle of responsive movements - summary data from control and ablated fish. Mean ± s.e.m., individual fish are points. N = (11,11,10) - (sham, M-array, RoL1), left to right. Note that RoL1 neuron ablation reduced the maximum tail angle in response to some stimuli. One-tailed Mann-Whitney U tests, Benjamini-Hochberg false discovery rate correction for multiple comparisons. Same fish as Fig. 5h. NS = “not significant”, all p > 0.2. *p < 0.05. f, DIC image of embedded fish (agar around nose removed), with location of injection pipette entering from the fissure between the optic tectum and lateral cerebellum (left). Close-up image of injection area in DIC and fluorescence imaging to visualize drug/dye flow (right). Localized dye spread was confirmed across all injected fish. g, Maximum tail angle of responsive movements - summary data from in RoL1-injected fish (NBQX or vehicle). Mean ± s.e.m., individual fish are points. N = (6,6) - (vehicle, NBQX). One-tailed Mann-Whitney U tests, Benjamini-Hochberg false discovery rate correction for multiple comparisons. Same fish as Fig. 5k. NS = “not significant”, all p > 0.1. See Supplementary Table 2 for test statistics.

Fig. 1 ∣. Hypothalamic population dynamics distinguish between avoidance-promoting threats.

a, Schematic of the experiment. Head-restrained larval zebrafish were presented with 40 s of threats (30 s of no-stimulus interval), while tail movements were recorded. b, Tail angle of an example fish during threat presentation, showing turn-like or escape-like movements to each threat type. c, Rate of turning movements evoked by each threat and by a blank stimulus (no change in environment). Data shown as the mean ± s.e.m., 1-s bins, N = 57 fish (average of 5 trials each). d, Top and side projections show location of analyzed neurons from all fish (N = 48,331 cells from 8 fish), registered to a common brain volume. a, anterior; d, dorsal; p, posterior; v, ventral. e, All active neurons recorded by two-photon Ca²⁺ imaging in an example Tg(elavl3:H2B-GCaMP6s) fish, with the times of threats (above) and tail angle (below) for an example trial (five such trials during imaging experiment). Broad anatomical regions are noted on the left. f, Trajectory of population activity in two anatomical regions of a single example fish. The z-scored and baseline-subtracted time series of the first three principal components (PC 1–3) is shown from the threat onset (black dot) to 3 s later (gray dots) (heat, red; salinity, blue; acidity, green). g, Accuracy of a threat classifier on held-out data, derived from the top ten principal components in two anatomical regions (N = 8 fish, 2-s bins, mean ± s.e.m.). h, Differences in the mean classifier accuracy between the time after threat onset compared with before threat onset across anatomical subregions (N = 8 fish). Arrows and beige text denote regions that are significantly different from zero (two-tailed one-sample t-tests, Benjamini–Hochberg false discovery rate correction for multiple comparisons across all brain regions). *P < 0.05. See Supplementary Table 2 for test statistics.

Fig. 2∣. Large-scale cellular registration of neural dynamics and neuropeptide gene expression across the hypothalamus.

a, Schematic of the process for registering multiple rounds of multicolor fluorescent in situ hybridization to cellular-resolution live-brain imaging during behavior. b, Location of volumetric two-photon functional imaging encompassing the preoptic hypothalamus (top and side views), and functional imaging z-planes from an example Tg(elavl3:H2B-GCaMP6s) fish. c, Select z-plane images from volumes in an example fish showing live functional imaging (gray GCaMP signal) overlapping with the GCaMP signal after fixation and both rounds of in situ hybridization (left, pre-registration; middle, post-registration) or with in situ hybridization markers of neuropeptides from both rounds (right). Similar results were obtained for N = 7 fish (Extended Data Fig. 5b). d, Same volume as shown in c, but higher magnification of a small area. Arrowheads indicate labeled cells. e, Location of analyzed neurons and fluorescence activity traces of simultaneously recorded labeled neurons in an example fish. Similar activity traces were observed for all N = 7 fish. Crf is split into medial (m.Crf) and lateral (l.Crf) populations. Npy is split into dorsal (d.Npy) and ventral (v.Npy) populations. Vip is split into anterior (a.Vip) and posterior (p.Vip) populations.

Fig. 3 ∣. Behavior-specific neural ensembles span multiple neuropeptidergic hypothalamic populations.

a, All identified peptidergic neurons recorded (N = 452 peptidergic neurons from 28,211 total recorded cells in 7 fish), with the cell population denoted by color, event onset-averaged responses indicated by gray intensity and UMC values from the linear model fit displayed as a heatmap (percentage of full model). Left: neurons were sorted by molecular and anatomical identity. Right: after clustering UMC values, the same neurons were sorted by functional identity (cluster identity written on the right). b, Cluster identities for each molecular and anatomical cell population examined (N = 7 fish, number of neurons in each cell population (from left to right) = 33, 53, 37, 44, 75, 48, 63, 28 and 71). ‘Movement’ encompasses ‘tail angle’ and ‘move onset’ clusters. The ‘not tuned’ cluster is not displayed. c, The cell populations within each functional cluster, plotted as percentage of all neurons in the cluster (including unlabeled neurons). Details of each functional cluster is presented in Extended Data Fig. 6. d, Differences in the mean threat classifier accuracy between the time after threat onset compared with before threat onset across cell populations (N = 7 fish). Data shown for all peptidergic neurons together and with neurons from individual cell classes removed. Data are shown as the mean ± s.e.m., with points representing individual fish. All comparisons are two-tailed t-tests with the ‘All combined’ group (all recorded peptidergic neurons), Benjamini–Hochberg false discovery rate correction for multiple comparisons. NS, not significant, all P > 0.3. See Supplementary Table 2 for test statistics.

Fig. 4 ∣. Overlapping functional properties of multiple populations of hypothalamic neurons.

a, Schematic of the location of images in b, encompassing the preoptic hypothalamus. b, The location of Gal4⁺ neurons in transgenic lines used to target peptidergic cell classes displayed in example fish. Gal4 drives ‘UAS:RFP’ (UAS:NTR–mCherry or UAS:ChR2–mCherry) in a background of Tg(elavl3:H2B-GCaMP6s). This expression pattern was confirmed in at least N = 3 fish per genotype. c, Schematic of the experiment measuring the behavioral response to focal optogenetic activation of the preoptic hypothalamus in the absence of other stimuli. d, Results of optogenetic activation in ChR2⁺ and ChR2⁻ (control) fish for each Gal4 line. N ((ChR2⁺, ChR2⁻), left to right) = (8, 8), (10, 8), (7, 8), (7, 8) and (9, 8). e, Schematic and timeline of the experiment. We applied 10 mM metronidazole (MTZ) for 36 h to ablate NTR⁺ neurons before testing behavior in response to threats. f, Results of behavioral experiments in NTR⁺ and NTR⁻ (control) fish for each transgenic line (all treated with MTZ). N ((NTR⁺, NTR⁻), left to right) = (8, 8), (8, 7), (8, 8), (9, 9) and (7, 7). For d and f, data are shown as the mean ± s.e.m., with points representing individual fish. One-tailed Mann–Whitney U-tests, Benjamini–Hochberg false discovery rate correction for multiple comparisons within genotype. NS, P > 0.2 (d) or P > 0.1 (f), *P < 0.05, **P < 0.01, ***P < 0.005. See Supplementary Table 2 for test statistics.

Fig. 5 ∣. Hypothalamic outputs are glutamatergic and converge on brainstem neurons required for defensive behavior.

a, SPNs were backfilled with Texas Red dextran in fish expressing GFP in each cell population. A z-projection of an example Tg(sst3:Gal4; UAS:GFP) fish and the region of interest in RoL1 for b. b, Overlap of GFP⁺ axons with back-labeled RoL1 neurons in each Gal4 line. This expression pattern was observed in N = 3 fish per genotype. c, Two-photon imaging of axon terminals from Tg(oxt:Gal4; crf:Gal4; UAS:GCaMP6s) fish. These z-planes were imaged in N = 3 fish. d, The mean activity of axons in an example fish aligned to the onset of the first turn evoked by salinity, acidity or heat (top). Summary of peak activity in the 2 s around the stimulus onset, normalized (norm.) to the mean fluorescence 2 s before the onset (bottom). N = 3 fish. e, Two-photon imaging of SPNs in Tg(elavl3:GCaMP6f) fish. The volume was imaged in N = 4 fish. f, The mean activity of two SPN cell classes in an example fish aligned to the onset of the first turn evoked by salinity, acidity or heat (top). Summary of peak activity in the 2 s around the stimulus onset, normalized to the mean fluorescence 2 s before the onset (bottom). N = 4 fish, N = 7 and 11 cells. g, Example images from before (top) and after (bottom) bilateral two-photon ablation of backfilled RoL1 neurons (denoted by arrows). Similar results were observed in N = 11 fish (see also Extended Data Fig. 9c). h, Results of behavioral experiments in ablated (RoL1 neurons or Mauthner-array (M-array) neurons) or sham-ablated fish. N = 11 (sham), 11 (M-array) or 10 (RoL1) fish. i, Multicolor fluorescent in situ hybridization showing the overlap of neuropeptide expression (green) with Vglut2a (magenta). The bottom right image shows overlap of Vglut2a (magenta) with GFP expression in Tg(otpba:Gal4;UAS:GFP) fish. The pie charts below the images show the number of peptide⁺ neurons that are also Vglut2a⁺ (four sections from two fish each). j, Schematic of the experiment and location of NBQX injection around the RoL1 region (top). Image of drug spread in an example fish visualized by a red dye (bottom). Similar spread was observed for N = 6 injected fish. k, Results of behavioral experiments in RoL1-injected fish (NBQX or vehicle). N = 6 (vehicle) and 6 (NBQX) fish. For d, f, h and k, data are shown as the mean ± s.e.m., with points representing individual fish (d, h and k) or cells (f). Statistical tests were two-tailed paired-sample t-test (d and f) or one-tailed Mann–Whitney U-tests (h and k), all with Benjamini–Hochberg false discovery rate correction for multiple comparisons. NS, all P > 0.1. *P < 0.05, **P < 0.01. See Supplementary Table 2 for test statistics.