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.
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We thank our colleagues for generously sharing transgenic zebrafish lines: A. Douglass, H. Baier, M. Ahrens, S. Jesuthasan, J. Bonkowsky and J.-l. Du. We thank C. Constantinople, F. Gore, T. Machado, W. Allen, R. Nath and C. Bedbrook for feedback on an earlier version of the manuscript, and the entire Deisseroth Lab for advice and discussions. This research was supported by Helen Hay Whitney Foundation Postdoctoral Fellowships (to M.L.-B. and A.S.A.), Brain and Behavior Research Foundation NARSAD Young Investigator Awards (to M.L.-B. and A.S.A.), the NIMH (to M.L.-B.: K99MH11284002), the NIDA (to S.G.: R01DA035680 and R21DA038447), and grants from the NIH, NSF, Gatsby, Fresenius, and NOMIS Foundations (to K.D.).
The authors declare no competing interests.
Peer review information Nature Neuroscience thanks Florian Engert 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.
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.
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 (0-100th 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.
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.
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.
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Lovett-Barron, M., Chen, R., Bradbury, S. et al. Multiple convergent hypothalamus–brainstem circuits drive defensive behavior. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0655-1