β-adrenergic receptors mediate stress-induced transcriptomic changes in the hippocampus and are independent of subregion and sex.

a, Experimental design for assessing stress-induced cortical noradrenaline (NA) turnover at various time points following stress exposure. b, Stress-dependent changes in cortical NA turnover, as measured by the ratio of 3-Methoxy-4-hydroxyphenylglycol (MHPG) and NA levels. NA turnover significantly increased within 45 min and returned to baseline within 90 min of stress onset (one-way ANOVA with Tukey’s post hoc tests; F(5, 24) = 10.55, p < 0.0001). c, Experimental design for assessing the effect of prazosin (Pra, 1 mg/kg, i.p.) and propranolol (Pro, 10 mg/kg, i.p.) on stress-dependent transcriptomic changes in the dorsal (dHC) and ventral (vHC) hippocampus 45 min after stress exposure. d, Heatmap showing the expression of all differentially expressed genes across dHC and vHC and pharmacological treatments 45 min after acute swim stress exposure. n = 6 per group. e, Heatmap selectively showing those stress-responsive genes that are affected by the β-adrenergic receptor antagonist propranolol 45 min after acute swim stress exposure in the dHC and vHC (FDR-adjusted p < 0.05). f, Strength of the propranolol effect on the transcriptomic stress response in the dHC and vHC for genes with a significant propranolol effect in either region (same genes as in panel e). Data are sorted by interaction strength in the vHC (orange) and the corresponding interaction strength in the dHC are shown in black for the same gene. g, Experimental design for assessing propranolol-dependent changes in the vHC of female and male mice. h, Heatmap showing expression of all stress-dependent genes that are affected by propranolol treatment between male and female mice in the vHC 45 min after acute swim stress exposure (FDR-adjusted p < 0.05). n = 4 per group. Individual data points are shown with bars representing mean ± s.e.m. n = 5 per group. ***p < 0.001.

Locus Coeruleus-mediated transcriptomic changes in the hippocampus.

a, Experimental design for assessing LC activation and cortical NA release induced by injection of yohimbine (3 mg/kg, i.p.). b, Representative images and quantification of LC activation in mice 90 min after injection of vehicle or yohimbine as measured by cFos (red) and tyrosine hydroxylase (TH, green) coexpression within LC neurons. Yohimbine injection increased cFos expression within LC neurons compared to vehicle-injected animals (unpaired t test; t(8.9) = −8.814, p = 1.083e-05). Vehicle, n = 4; Yohimbine n = 7. Scale bar: 100μm. c, Experimental design for comparing molecular changes in the hippocampus after acute swim stress exposure or yohimbine administration. d, Volcano plots showing differentially expressed RNA transcripts in the dorsal (dHC) and ventral (vHC) hippocampus between control (Veh) and yohimbine (Yoh) injected animals 45 min after injection. Red and blue values represent changes with FDR-adjusted p<0.05 (Veh n = 6, Yoh n = 6). e, Strength of the yohimbine effect in comparison to the transcriptomic stress response. Data are sorted by interaction strength in the stress group (orange) and the corresponding interaction strength of the yohimbine group are shown in dark red for the same gene. f, Experimental design for assessing LC activation and hippocampal transcriptomic changes induced by chemogenetic LC activation. g, Representative images and quantification of LC activation in mice 45 min after injection of clozapine (0.03 mg/kg) in hM3Dq- and hM3Dq+ animals as measured by cFos (green) and tyrosine hydroxylase (TH, red) coexpression within LC neurons. Neurons are stained with Nissl (blue). Clozapine injection increased cFos expression within LC neurons in hM3Dq+ animals compared to hM3Dq- animals (one way anova; F(3, 23) = 135.4, p = 9.34e-15). hM3Dq- n = 6, hM3Dq+ n = 7. Scale bar: 100μm. h, Volcano plots showing differentially expressed RNA transcripts between hM3Dq- and hM3Dq+ animals 45 min after injection of clozapine (0.03 mg/kg) in the dHC and vHC. Red and blue values represent changes withwith FDR-adjusted p<0.05 (hM3Dq- n = 6, hM3Dq+ n = 7). i, Experimental design for assessing LC activation, cortical NA release, pupillometry and hippocampal transcriptomic changes induced by optogenetic 5 Hz LC activation. j, Representative images and quantification of LC activation in mice after 5 Hz optogenetic LC activation as measured by cFos (red) and tyrosine hydroxylase (TH, green) coexpression within LC neurons in stimulated and non-stimulated LC hemispheres of ChR2- and ChR2+ animals. 5 Hz stimulation increased cFos expression within LC neurons in stimulated LC hemispheres of ChR2+, but not in ChR2-animals 45 min (one-way ANOVA with Tukey’s post hoc tests; F(3, 14) = 12.91, p = 0.000256) and 90 min after stimulation onset (one way ANOVA with Tukey’s post hoc tests; F(3, 14) = 5.866, p = 0.00824). ChR2-(45 min), n = 5; ChR2-(90 min), n = 5; ChR2+ (45 min), n = 4; ChR2+ (90 min), n = 4. Scale bar: 100μm. k, Quantification of cortical MHPG/NA ratio, as measured by uHPLC, after 5 Hz optogenetic LC activation in ChR2- and ChR2+ animals. 5 Hz stimulation increased cortical NA turnover in ChR2+ animals (unpaired t test; 45 min: t(3.6) = 8.444, p = 0.001681; 90 min: t(4.0854) =3.4127, p = 0.02608). ChR2-45min, n = 5; ChR2-90min, n = 5; ChR2+ 45min, n = 6; ChR2+ 90min, n = 5. l, Average pupil size changes in response to 5 Hz optogenetic LC activation in ChR2- and ChR2+ animals. 5 Hz stimulation increased pupil size in ChR2+, but not ChR2-animals. m, Volcano plots showing differentially-expressed RNA transcripts between ChR2- and ChR2+ animals 45 and 90 min after 5 Hz optogenetic LC activation in the ventral (vHC) hippocampus. Red and blue values represent changes with FDR-adjusted p<0.05 (ChR2-n = 10, ChR2+ n = 11). n, Heatmap showing genes that are commonly differentially expressed by yohimbine, chemogenetic and optogenetic induced LC activation. o, Logarithmic cumulative rank of genes across all experiments from figure 1 and 2 in terms of their NA responsiveness. A lower cumulative rank indicates that a gene is among the more significant hits across all analyses (for full list of included analyses see methods). Labels indicate the 10 genes identified to be most responsive to LC-NA stimulation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

LC-NA mediated molecular responses in the hippocampus are independent of LC firing pattern and frequency and are directly stimulated via hippocampus-projecting LC neurons.

a, Experimental design for assessing molecular changes in the hippocampus induced by optogenetic LC activation with tonic (3 Hz and 5 Hz) and phasic (15 Hz) firing patterns. b, Radial plots showing expression changes (based on the logFC) of the most LC-NA-responsive genes after optogenetic LC activation in ChR2+ animals compared to controls (Sham n = 6, 3 Hz n = 6, 5 Hz n = 7, 15 Hz n = 6). Black borders indicate that the gene is upregulated, blue border downregulated. c, Experimental design for assessing molecular changes in the hippocampus induced by retrograde optogenetic 5 Hz activation of hippocampus projecting LC neurons (LCHC). d, Representative images of retrograde mCherry (mCh, red) and ChR2-EYFP (EYFP, green) expression in tyrosine hydroxylase (TH, blue) positive LC neurons across hemispheres. e, Cortical and right dorsal hippocampal (RH-dHC) NA turnover as measured by ultra-high performance liquid chromatography 45 min after 5 Hz optogenetic activation of LCHC neurons in ChR2- and ChR2+ animals. 5 Hz stimulation of LCHC neurons increased dorsal hippocampal but not cortical NA turnover in ChR2+ animals (unpaired t test; t(17.43) = −5.5997, p = 2.911e-05). ChR2-, n = 12; ChR2+, n = 12. ****p < 0.0001. f, Average pupil size changes in response to 5 Hz optogenetic activation of LCHC projecting neurons in ChR2- and ChR2+ animals. g, Radial plots showing expression changes (based on the logFC) of the top ten LC-NA responsive genes in response to optogenetic LCHC activation with tonic 5 Hz stimulation in ChR2+ animals compared to ChR2-45 min after stimulation onset (ChR2-n = 12, ChR2+ n = 12). h, Selective boxplots of NA-responsive genes Dio2, Ppp1r3c, Ppp1r3g, Sik1 and Nr4a1 in response to 5 Hz optogenetic activation of LCHC projecting neurons in ChR2- and ChR2+ animals 45 min after stimulation onset. (ChR2-n = 12, ChR2+ n = 12). 5 Hz optogenetic activation of LCHC projecting neurons increased hippocampal expression of Dio2, Ppp1r3c, Ppp1r3g and Nr4a1. *p < 0.05, **p < 0.01, ***p < 0.001,, ****p < 0.0001.

Screening of publicly available datasets shows that the noradrenaline-regulated genes Dio2, Ppp1r3c, Ppp1r3g, Sik1 and Nr4a1 are induced by various stressors predominantly in astrocytes.

a, Experimental design for assessing transcriptomic changes in the hippocampus induced by different stressors as performed by Floriou-Servou et. al (Floriou-Servou et al. 2018). These stressors included a 10 min exposure to the open field test (Novelty), a 6 min cold swim stress (Swim), a 30 min immobilization stress (Restraint) and exposure to a 1 mA footshock (Footshock). b, Selective boxplots of top NA-responsive genes Dio2, Ppp1r3c, Ppp1r3g, Sik1 and Nr4a1 in response to different stressors. Control n = 10, Novelty n = 5, Swim n =5, Restraint n = 10, Footshock n = 5. c, Experimental design for assessing transcriptomic changes in the dorsal and ventral hippocampus across 4 hours following acute swim stress exposure as performed by von Ziegler et. al. d, Selective boxplots showing expression changes of top NA-responsive genes Dio2, Ppp1r3c, Ppp1r3g, Sik1 and Nr4a1 across 4 hours following acute swim stress exposure. Control n = 8, 45 min n = 8, 90 min n = 7, 120 min n = 7, 180 min n = 7, 240 min n = 7. e, Experimental design for assessing single cell transcriptomic changes in the hippocampus 45 min following acute swim stress exposure by single-nucleus RNA sequencing as performed by von Ziegler et. al (von Ziegler et al. 2022). f, Selective boxplots showing expression changes of top NA-responsive genes Dio2, Ppp1r3c, Ppp1r3g, Sik1 and Nr4a1 across cell types of the hippocampus 45 min following acute swim stress exposure. Control n = 2, Stress n = 2. g, Experimental design for assessing actively translated RNA in the somatosensory cortex 90 min following a 20 min acute swim stress exposure by TRAP sequencing as performed by Murphy-Royal et. al (Murphy-Royal et al. 2020). h, Selective boxplots of top NA-responsive genes Dio2, Ppp1r3c, Ppp1r3g, Sik1 and Nr4a1 in the somatosensory cortex 90 min following a 20 min acute swim stress exposure. Acute stress increases the binding of Dio2, Ppp1r3c, Ppp1r3g and Nr4a1 mRNA to the ribosome. Control n = 4, Stress n = 4. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.