During infection, animals exhibit adaptive changes in physiology and behaviour aimed at increasing survival. Although many causes of infection exist, they trigger similar stereotyped symptoms such as fever, warmth-seeking, loss of appetite and fatigue1,2. Yet exactly how the nervous system alters body temperature and triggers sickness behaviours to coordinate responses to infection remains unknown. Here we identify a previously uncharacterized population of neurons in the ventral medial preoptic area (VMPO) of the hypothalamus that are activated after sickness induced by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid. These neurons are crucial for generating a fever response and other sickness symptoms such as warmth-seeking and loss of appetite. Single-nucleus RNA-sequencing and multiplexed error-robust fluorescence in situ hybridization uncovered the identity and distribution of LPS-activated VMPO (VMPOLPS) neurons and non-neuronal cells. Gene expression and electrophysiological measurements implicate a paracrine mechanism in which the release of immune signals by non-neuronal cells during infection activates nearby VMPOLPS neurons. Finally, we show that VMPOLPS neurons exert a broad influence on the activity of brain areas associated with behavioural and homeostatic functions and are synaptically and functionally connected to circuit nodes controlling body temperature and appetite. Together, these results uncover VMPOLPS neurons as a control hub that integrates immune signals to orchestrate multiple sickness symptoms in response to infection.
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Raw and processed data have been deposited in the Gene Expression Omnibus and are available under accession number GSE197547.
The pipeline used to perform MERFISH analyses is freely available at https://github.com/ZhuangLab/MERlin. Other custom code is available upon request.
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We thank K. Prichett-Corning and other veterinary staff at the Harvard Office of Animal Resources for advice on working with sick mice; R. Hellmiss and staff at MCB Graphics for help with illustrations; A. Emery and J. Emery for assistance with histology and RNAscope experiments; members of the Dulac laboratory for helpful advice on experiments, analysis and the manuscript; and F. Engert and R. Losick for comments on the manuscript. This work was supported by NIH awards K99NS114107 to J.A.O. and F31MH120911 to E.V., NIH grant R01NS050835 to L.L, and NIH grant R01NS112399 and Simons Foundation Award 572189 to C.D. C.D., X.Z. and L.L. are investigators at the Howard Hughes Medical Institute.
X.Z. is an inventor on patents applied for by Harvard University related to MERFISH.
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Extended data figures and tables
(a) Mean body temperature 2 h following injection of saline (n = 13), LPS (n = 16) or Poly(I:C) (n = 10). (b-c) Representative images of Fos expression in brain areas displaying significant increases in number of Fos+ cells following LPS administration (b) or saline (c), scale bar for all panels = 200μm. (d) mRNA expression of inhibitory neuronal marker Vgat (green) and Fos (magenta) in the VMPO after LPS injection. (e) Fraction of Fos+ cells that express Vgat or Vglut2 (n = 4). (f) Quantification of the fraction of Fos+ cells within individual snRNA-seq clusters in LPS-injected sample. (g) Dotplot of average expression of marker genes and genes with immunological significance in each cluster: dot size indicates percent of cells in cluster with measurable expression and color indicates average expression levels; ependymal cluster cluster #5 and neuronal cluster #19, found significantly activated after LPS injection are highlighted in red. (h) Quantification of the fraction of Fos+ cells in individual MERFISH neuronal clusters, with statically significant enrichment for Fos+ cells indicated in red, n = 3. (i) mRNA expression of markers for VMPOLPS neurons (calcR, gal and amigo2) in LPS-injected mice. (j) Mean of overlap of markers for VMPOLPS neurons and for warm-sensitive neurons (adcyap1) with LPS-mediated Fos expression in the VMPO, n = 3 mice/experiment. All scale bars = 200μm. All error bars = SEM.
Extended Data Fig. 2 Spatial distribution of activated neuronal and non-neuronal cell type classes in the preoptic area during inflammation.
(a) Cumulative distribution of Fos+ (purple) and Fos- (blue) VMPOGal/Amigo2 neurons as a function of the distance to the bottom of the section (top) or the midline (bottom), n = 3 replicates. (b) MERFISH analysis indicating the fraction of Fos+ cells in major cell type classes in samples from mice injected with LPS versus mice displaying other behaviors: aggression, mating or parenting. Significantly activated populations are indicated in red. (c) Spatial distribution of major cell type classes in MERFISH analysis, cell type (green), Fos+ cell (red), Fos+ cells in the indicated cell type (purple). (d) Cumulative distribution of Fos+ (purple) and Fos- (green) cells in major cell type classes as a function of the distance to the bottom of the section (top) or the midline (bottom), n = 3 mice. All scale bars = 200μm. All error bars = SEM.
(a-i) mRNA expression in the VMPO at 2hrs post LPS administration, genes of interest in green, LPS-induced Fos is in magenta. Scale bar for a-i = 200μm (a) Prostaglandin E synthase 2 (ptgs2) and its receptors ep2 (d), ep1 (g), ep3 (h), ep4 (i). (b) interleukin-1β (il1β) and its receptor il1rap (e). (c) chemokine ligand 2 (ccl2) and it’s receptor chemkine receptor 2 (ccr2) (f). (j) Expression of ptgs2 in absence of LPS stimulation and at 60min and 120min post LPS. No or weak expression is found without LPS stimulation, and expression after LPS challenge shows overlap with markers for endothelial cells and microglia. (k) Expression of il1β with no stimulation and at 60min and 120min post LPS, overlap with markers for ependymal cells and microglia. Scale bar for j-l = 50μm (l) Expression of ccl2 with no stimulation and 120 min post LPS inejction, overlap with gfap+ astrocytes. (m) Expression of il1r1 in the VMPO after LPS injection, Scale bar = 200μm.
Extended Data Fig. 4 Effects of PGE-2 and cytokines on the intrinsic properties and synaptic activity of VMPOLPS neurons.
(a) Wide field microscope images depicting tdTomato expression in the VMPO of TRAP2;Ai9 during whole cell patch clamp recording with pipette containing Alexa 488. (b) Changes in rheobase current for VMPOLPS neurons in presence of ACSF (black), CCL2 (pink), PGE2 (green), addition of EP2 antagonist in presence of PGE2 (blue), IL-1β (orange) and further addition of PGE2 (yellow), and (c) IL-1β (orange), addition of COX-2 inhibitor in the presence of IL-1β (light orange), further addition of PGE2 (light green) and further addition of EP2 antagonist (light blue). During whole cell voltage clamp recordings, changes in (d) amplitude of miniature EPSCs, (e) inter event interval for miniature EPSCs, (f) amplitude of miniature IPSCs and (g) inter event interval for miniature IPSCs for VMPOLPS neurons in presence of ACSF (black), CCL2 (pink), PGE2 (green), addition of EP2 antagonist in presence of PGE2 (blue), IL-1β (orange) and further addition of PGE2 (yellow). Changes in (h) amplitude of miniature EPSCs, (i) inter event interval for miniature EPSCs, (j) amplitude of miniature IPSCs and (k) inter event interval for miniature IPSCs for VMPOLPS neurons in the presence of IL-1β and COX-2 inhibitor (light orange), further addition of PGE2 (light green) and further addition of EP2 antagonist (light blue). (l) Cumulative plots showing total charge transferred over 1s for inhibitory events and excitatory events for control (black) and CCL2 (pink). (m, top) Cumulative charge transferred by IPSCs for control (mean value of 24.81 ± 0.14 μC/s) and CCL2 (mean value of 19.06 ± 0.09 μC/s p = 2.0 * 10−03 for). (m, bottom) Cumulative charge transferred by miniature EPSCs for control (mean value of 18.81 ± 0.09 μC/s) and CCL2 (mean value of 23.37 ± 0.11 μC/s p = 9.0 * 10−03). All other values and statistical tests are catalogued in Extended Data Table 2. For violin plots, central white circle depicts the mean value and thick black line depicts the interquartile range. All p values shown are for two-sided Wilcoxon rank sum test with * = p< 0.05, ** = p< 0, *** = p< 0.005 and ns = p > 0.05. All error bars = SEM. See Statistics and Reproducibility section for exact n values.
(a) Example of viral injection specificity that qualified for inclusion in the study. Note viral expression in the VMPO and absence in surrounding brain areas. Injections found to be more broadly dispersed were not included in analysis. (b-d) Chemogenetic activation of Calcr+ and Gal+ neurons in the VMPO. (b) CNO injection elicited strong increase in body temperature in both genetic backgrounds (n = 6 mice/group). (c) Activation of Gal-Cre but not Calcr-Cre increased preferred temperature (n = 7mice/group). (d) Activation of Calcr-Cre (n = 6 mice) but not Gal-Cre (n = 8) decreased chow consumed (saline n = 6 mice). (e) DTA-mediated Ablation of VMPOLPS neurons had no effect on circadian temperatures, dark bar indicates dark phase, WT mice were used as controls. (f-g) Effect of DTA-mediated ablation of VMPO neurons in Saline-TRAP mice had no effect on body temperature or preferred temperature following LPS injection (n = 7 mice/group). (h) Overlap of warm-TRAP reporter expression with markers of warm-sensitive neurons adcyap1 and sncg. (i) Overlap of hunger-TRAP-mediated reporter expression (magenta) with markers of appetite-increasing neurons, agrp, or appetite-decreasing neurons, pomc, (green). Scale bars = 200μm. All error bars = SEM. For all graphs * = p< 0.05, ** = p< 0.01, *** = p< 0.001.
(a) Widefield microscope images of VMPOLPS neurons in the VMPO of TRAP2 injected with AAV8-hSyn-DIO-hM3D(Gq)-mCherry. (b) Whole cell current clamp recording of a VMPOLPS neuron showing the effects of bath application of 1mM CNO (baseline: black, CNO: pink and washout: green). (c) Effects of CNO application on the firing rate of VMPOLPS neurons; black (n = 5 cells with mean values of 0.20 ± 0.05 Hz for baseline), pink (0.91 ± 0.06 Hz for CNO application and p = 7.0 * 10−03 compared to baseline), green (0.25 ± 0.06 Hz for washout and p = 6.9 * 10−03 compared to CNO application), boxes represent the 25th and 75th percentile, whiskers extend to the minimum and maximum data points (d) Widefield microscope images of VMPOLPS neurons in the VMPO of TRAP2 injected with AAV5-EF1a-DIO-hChR2(C128S/D156A)-EYFP. (e) Whole cell current clamp recording of a VMPOLPS neuron showing activation of hChR2(C128S/D156A) by 20 ms blue light (460 ± 10 nm) light pulse and inactivation by 50 ms green light (525 ± 9 nm) pulse. (f) Firing rate of VMPOLPS neurons (n = 4 cells) in response to activation of hChR2(C128S/D156A), baseline (black, mean firing frequency of 0.21 ± 0.08 Hz), following blue light activation (blue, mean firing frequency of 1.46 ± 0.72 Hz and p = 2.1 * 10−02) and following green light mediated inactivation of hChR2(C128S/D156A) (green, mean firing frequency of 0.12 ± 0.05 Hz and p = 2.0 * 10−02), boxes represent the 25th and 75th percentile, whiskers extend to the minimum and maximum data points. All p values shown are for two-sided Wilcoxon rank sum test with * = p< 0.05, ** = p< 0, *** = p< 0.005 and ns = p > 0.05.
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Osterhout, J.A., Kapoor, V., Eichhorn, S.W. et al. A preoptic neuronal population controls fever and appetite during sickness. Nature (2022). https://doi.org/10.1038/s41586-022-04793-z
Nature Reviews Neuroscience (2022)