.2015 Aug 5;87(3):605-20.

doi: 10.1016/j.neuron.2015.07.002. Epub 2015 Jul 23.

CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety

Jordan G McCall¹, Ream Al-Hasani², Edward R Siuda¹, Daniel Y Hong³, Aaron J Norris³, Christopher P Ford⁴, Michael R Bruchas⁵

Affiliations

¹ Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Washington University Pain Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
² Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Washington University Pain Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
³ Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
⁴ Department of Physiology and Biophysics, Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
⁵ Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Washington University Pain Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: bruchasm@wustl.edu.

PMID:26212712
PMCID: PMC4529361
DOI: 10.1016/j.neuron.2015.07.002

CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety

Jordan G McCall et al. Neuron.2015.

.2015 Aug 5;87(3):605-20.

doi: 10.1016/j.neuron.2015.07.002. Epub 2015 Jul 23.

Authors

Jordan G McCall¹, Ream Al-Hasani², Edward R Siuda¹, Daniel Y Hong³, Aaron J Norris³, Christopher P Ford⁴, Michael R Bruchas⁵

Affiliations

¹ Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Washington University Pain Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
² Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Washington University Pain Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
³ Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
⁴ Department of Physiology and Biophysics, Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
⁵ Division of Basic Research, Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Washington University Pain Center, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: bruchasm@wustl.edu.

PMID:26212712
PMCID: PMC4529361
DOI: 10.1016/j.neuron.2015.07.002

Abstract

The locus coeruleus noradrenergic (LC-NE) system is one of the first systems engaged following a stressful event. While numerous groups have demonstrated that LC-NE neurons are activated by many different stressors, the underlying neural circuitry and the role of this activity in generating stress-induced anxiety has not been elucidated. Using a combination of in vivo chemogenetics, optogenetics, and retrograde tracing, we determine that increased tonic activity of the LC-NE system is necessary and sufficient for stress-induced anxiety and aversion. Selective inhibition of LC-NE neurons during stress prevents subsequent anxiety-like behavior. Exogenously increasing tonic, but not phasic, activity of LC-NE neurons is alone sufficient for anxiety-like and aversive behavior. Furthermore, endogenous corticotropin-releasing hormone(+) (CRH(+)) LC inputs from the amygdala increase tonic LC activity, inducing anxiety-like behaviors. These studies position the LC-NE system as a critical mediator of acute stress-induced anxiety and offer a potential intervention for preventing stress-related affective disorders.

PubMed Disclaimer

Figures

**Figure 1. Chemogenetic inhibition of LC-NE neurons prevents stress-induced anxiety**
**(A)** Cartoon of restraint stress-induced anxiety paradigm.(B and C) Stressed animals spend significantly less time exploring the center of the OFT than non-stressed controls with no change in locomotor activity (Data represented as mean ± SEM, n=7–8/group: Student’s t-test, p<0.001).(D and E) Representative immunohistochemistry (IHC) and quantification show restraint stress increases c-fos immunoreactivity (IR) in LC neurons (Red=c-fos, green=tyrosine hydroxylase, arrows indicate example co-localization, scale bars=100 μm; data represented as mean ± SEM, n=3 slices from 3 animals/group: Student’s t-test, p<0.0001). See locomotor activity data in Fig. S1.(E) Cartoon of viral strategy with high power confocal image of post-CNO internalized mCherry expression in LC-NE neurons. Scale bar=10 μm.(G and H) Representative IHC and quantification show hM4Di inhibition of LC neurons decreases c-fos IR in LC neurons (Red=c-fos, green=tyrosine hydroxylase, Scale bars=100 μm; data represented as mean ± SEM, n=3 slices from 3 animals/group: One-Way ANOVA, Bonferroni post-hoc, No stress/Cre⁻ vs. No stress/Cre⁺ **p<0.01, No stress/Cre⁻ vs. Stress/Cre⁻ ****p<0.0001, No stress/Cre⁻ vs. Stress/Cre⁺ ****p<0.0001, No stress/Cre⁺ vs. Stress/Cre⁻ ****p<0.0001, No stress/Cre⁺ vs. Stress/Cre⁺, not significant). See also Fig. S1.(I) Cartoon of LC-NE inhibition paradigm.(J) Inhibition of LC-NE neurons during stress blocks stress-induced anxiety. Data represented as mean ± SEM, n=8–13/group: One-Way ANOVA, Newman-Keuls post-hoc, No stress/Cre⁻ vs. No stress/Cre⁺, not significant, No stress/Cre⁻ vs. Stress/Cre⁻ **p<0.01, No stress/Cre⁻ vs. Stress/Cre⁺, not significant, No stress/Cre⁺ vs. Stress/Cre⁻ **p<0.01, No stress/Cre⁺ vs. Stress/Cre⁺, not significant).(K) Inhibition of LC-NE neurons has no effect on stress-induced bowel motility. Data represented as mean ± SEM, n= n=8–13/group: One-Way ANOVA, Newman-Keuls post-hoc, No stress/Cre⁻ vs. No stress/Cre⁺, not significant, No stress/Cre⁻ vs. Stress/Cre⁻ *p<0.05, No stress/Cre⁻ vs. Stress/Cre⁺ *p<0.05, No stress/Cre⁺ vs. Stress/Cre⁻, not significant, No stress/Cre⁺ vs. Stress/Cre⁺, not significant).(L) Inhibition of LC-NE neurons has no effect on locomotor activity. Data represented as mean ± SEM.(M) Representative heat maps of activity during OFT.

**Figure 2. Optogenetic targeting to selectively drive tonic LC-NE activity**
**(A)** Cartoon of viral strategy for slice experiments.(B and C) Representative IHC shows selective targeting of ChR2-eYFP to TH⁺ LC neurons (Red= tyrosine hydroxylase, green=ChR2-eYFP, 4V = 4^th ventricle, Scale bars= 100 and 50 μm, respectively).(D and E) Whole cell current- and voltage-clamp recordings of an LC-NE neuron expressing ChR2.D, scale bar is 200 pA x 100 ms andE, scale is 20 ms.(F) Slice recording of a single LC-NE neuron demonstrating action potentials over 30 min in response to 5 Hz 470 nm light.(G) Cartoon of viral and multielectrode delivery for anesthetized,*in vivo* recordings.(H) Peristimulus time histogram (PSTH) showing increased LC neuron firing during a 20 s optical stimulation at 5 Hz.(I) Firing rate of n=16 cells before and during 5 Hz photostimulation (Paired Student’s t-test, p<0.01).(J) Correlation of baseline activity to photostimulated activity (r=0.8938, p<0.0001).

**Figure 3. High tonic LC-NE neuronal activity is sufficient to induce anxiety-like behavior**
**(A)** Cartoon of viral and fiber optic delivery.(B) Calendar of pre-stimulation OFT studies.(C) 5 Hz photostimulation prior to OFT causes an anxiety-like phenotype of ChR2-expressingTh-Cre⁺ animals compared toTh-Cre⁻ controls (Data represented as mean ± SEM, n=14–15/group: Mann-Whitney t-test, p<0.01) with(D) a significant decrease in locomotor activity (Data represented as mean ± SEM, n=14–15/group: Student’s t-test, p<0.05).(E) Calendar of concurrent stimulation OFT studies.(F) 5 Hz photostimulation drives anxiety-like behavior in OFT ofTh-Cre^LC:ChR2 animals compared toTh-Cre^LC:eYFP controls (Data represented as mean ± SEM, n=10/group: Student’s t-test, p<0.01) with(E) no change in locomotor activity (Data represented as mean ± SEM, n=10/group). (H) Representative heat maps of activity in the OFT. See also Fig. S2.(I) Calendar of systemic antagonism in EZM studies.(J) Representative heat maps of activity in the EZM.(K) 5 Hz photostimulation drives anxiety-like behavior in EZM ofTh-Cre^LC:ChR2 animals compared toTh-Cre^LC:eYFP controls, which is blocked by β-adrenergic antagonism (Prop), but not α1 antagonism (Praz) (Data represented as mean ± SEM, n=6–9/group; Kruskal-Wallis One-way ANOVA, *p<0.05, **p<0.01).

**Figure 4. LC-NE photostimulation drives both real-time and learned aversions**
**(A)** Current-clamp whole cell recording at 2, 3, 5 and 8 Hz.(B) Cartoon of viral and fiber optic delivery and calendar of behavioral studies.(C) Frequency response of RTPT and(D) locomotor activity at 0, 1, 2, 5, and 10 Hz. Data represented as mean ± SEM, n=6–7/group: Two-Way ANOVA, Bonferroni post-hoc, 5 Hz ChR2 vs. 5 Hz eYFP *p<0.05, 10 Hz ChR2 vs. 10 Hz eYFP ***p<0.001.(E) Representative traces of behavior at different frequencies.(F) Phasic stimulation does not drive aversion, n=6/group.(G) 5 Hz photostimulation causes a real-time place aversion in RTPT ofTh-Cre⁺ animals expressing ChR2 in the LC compared toTh-Cre⁻ control animals that do not express ChR2. This effect is not reversed by Propranolol (Prop) pre-treatment, but is by Prazosin (Praz) (Data represented as mean ± SEM, n=6–10/group; One-Way ANOVA, Bonferonni post-hoc, ****p<0.0001, **p<0.01, ns=no significance).(H) Timeline and cartoon of 5 Hz CPA experiment.(H) Mean preference (s) ± SEM, post-test minus pre-test (n=7–9; Student’s t-test, p<0.05).(I) No locomotor effect in either pre- or post-test.(J) Representative traces of behavior in the pre- and post-test. See also Fig. S3.

**Figure 5. Galanin containing LC-NE neurons are sufficient to drive place aversion**
**(A and B)** Galanin labeling inGal-Cre x Ai9-tdTomato compared toin situ images from the Allen Institute for Brain Science in a sagittal section highlighting presence of galanin in the LC. All images show tdTomato (red) and Nissl (blue) staining. See also Fig. S4.(C) Cartoon of viral and fiber optic delivery.(D) IHC of ChR2-eYFP targeting to DBH⁺ LC neurons. Scale bar=100 μm.(E) Quantification of Cre-dependent eYFP viral expression in the LC ofTh-Cre andGal-Cre mouse lines. Data represented as mean ± SEM, n=3 slices from 3 animals/group: Student’s t-test, p<0.0001. See also Fig. S5.(F) 5 Hz stimulation of LC-Gal neurons drives aversion (n=6, Paired Student’s t-test, p<0.01).(G) Representative traces of behavior at different frequencies.

**Figure 6. Identifying a CRH⁺ CeA input to the LC**
**(A)** Calendar of pharmacological experiment.(B) CRF-R1 antagonism blocks stress-induced anxiety-like behavior(C) with no significant effect on locomotor activity (n=6–8/group, Student’s t-test, *p<0.05).(D) Representative heat maps show behavior in the OFT.(E) Cartoon depicting fluorgold tracing.(F) Representative image shows robust retrograde labeling of the CeA (Fluorgold pseudocolored red, Nissl=grey). See also Fig. S6.(G) Cartoon depicting dual injection tracing for CTB-594 and DIO-ChR2-eYFP.(H) Representative IHC shows retrograde labeling in CeA of CTB-594 (red) and anterograde labeling of CRH⁺ cells (green). Arrow indicates example co-localization.(I) ~25% of each label co-labels with the other.(J) Cartoon depicting anterograde tracing.(K) 71° off of sagittal slice ofCrh-Cre mouse expressing DIO-eYFP in the CeA. Image shows intact projections from CeA to LC. Arrow indicates fiber optic placement.(L and M) Coronal image depict robust eYFP labeling in the CeA and LC of the same mouse. All scale bars=100 μm. + is 4^th ventricle.

**Figure 7. CRH⁺ CeA-LC terminals modulate LC activity and drive anxiety through CRFR1 activation**
**(A)** Cartoon of viral and multi-electrode array delivery for anesthetized,*in vivo* recordings.(B and C) Representative PSTHs of putative LC neurons responding to 20s of 10 Hz, 10 ms pulse width photostimulation (473 nm, ~10 mW).(D) Representative principal component analysis plot showing the first two principal components with clear clustering of units.(E) Total recorded sample shows significant increase in firing rate to 10 Hz photostimulation (n=35, Wilcoxon matched- pairs signed rank test, p<0.05).(F) n=15 units increase firing rate by >10% during 10 Hz photostimulation (Wilcoxon matched- pairs signed rank test, p<0.0001).(G) Response latency following onset of photostimulation for cells that increase firing.(H) n=15 units decrease firing rate by >10% during 10 Hz photostimulation (Wilcoxon matched- pairs signed rank test, p<0.0001). See also Fig. S7A.(I) Response latency following onset of photostimulation for cells that increase firing.(J) Correlation of baseline activity to activity during photostimulation (r=0.7029, p<0.0001). Representative voltage-clamp traces following(K) 10 Hz, 3 ms photostimulation and(L) 50 Hz, 3 ms photostimulation ofCrh-Cre^CeA-LC:ChR2 terminals. Scale bars 30 pA; 50 ms.(M) Magnitude of the event after the first photostimulation. Data represented as mean ± SEM, n=35 cells from 4 brains.

**Figure 8. CRH⁺ CeA-LC terminals drive aversion and anxiety-like behavior through CRFR1 activity**
**(A)** Cartoon of viral and fiber optic delivery and (B) calendar of CPA behavior.(C and D)*Crh*-Cre^CeA-LC:ChR2 show a significant CPA compared toCrh-Cre^CeA-LC:eYFP controls. Representative traces of behavior in the pre- and post-test and mean preference (s) ± SEM, post-test minus pre-test (n=10–12/group; Student’s t-test, p<0.05).(E) Cartoon of viral, cannula, and fiber optic delivery and calendar of EZM behavior.(F) 10 Hz photostimulation drives anxiety-like behavior in EZM ofCrh-Cre^CeA-LC:ChR2 animals compared toCrh-Cre^CeA-LC:eYFP controls, which is reversed by intra-LC α-helical-CRF (αHCRF) pretreatment (Data represented as mean ± SEM, n=7/group: One-Way ANOVA, Newman-Keuls,*Crh*-Cre^CeA-LC:eYFP vs.Crh-Cre^CeA-LC:ChR2 *p<0.05, Cre^CeA-LC:eYFP vs.Crh-Cre^{CeA-LC:ChR2+AHCRF} **p<0.01, Cre^CeA-LC:ChR2 vs.Crh-Cre^{CeA-LC:ChR2+AHCRF} ***p<0.001). See also Fig. S7B&C.(G) Systemic CRHR1 antagonism reverses photostimulation-induced anxiety-like behavior (n=6–8/group, Student’s t-test, p<0.001).(H) Model and summary of results. See also Fig. S7D.

See this image and copyright information in PMC

Comment in

Norepinephrine and Corticotropin-Releasing Hormone: Partners in the Neural Circuits that Underpin Stress and Anxiety.
Sun Y, Hunt S, Sah P.Sun Y, et al.Neuron. 2015 Aug 5;87(3):468-70. doi: 10.1016/j.neuron.2015.07.022.Neuron. 2015.PMID:26247856

Cited by

Diversity of ancestral brainstem noradrenergic neurons across species and multiple biological factors.
Kelberman MA, Rodberg E, Arabzadeh E, Bair-Marshall CJ, Berridge CW, Berrocoso E, Breton-Provencher V, Chandler DJ, Che A, Davy O, Devilbiss DM, Downs AM, Drummond G, Dvorkin R, Fazlali Z, Froemke RC, Glennon E, Gold JI, Ito H, Jiang X, Johansen JP, Kaye AP, Kim JR, Kuo CC, Liu RJ, Liu Y, Llorca-Torralba M, McCall JG, McElligott ZA, McKinney AM, Miguelez C, Min MY, Nowlan AC, Omrani M, Poe GR, Pickering AE, Ranjbar-Slamloo Y, Razquin J, Rodenkirch C, Sales AC, Satyasambit R, Shea SD, Sur M, Tkaczynski JA, Torres-Sanchez S, Uematsu A, Vazquez CR, Vreven A, Wang Q, Waterhouse BD, Yang HW, Yang JH, Zhao L, Zouridis IS, Weinshenker D, Vazey E, Totah NK.Kelberman MA, et al.bioRxiv [Preprint]. 2024 Oct 16:2024.10.14.618224. doi: 10.1101/2024.10.14.618224.bioRxiv. 2024.PMID:39464004Free PMC article.Preprint.
Tripartite extended amygdala-basal ganglia CRH circuit drives locomotor activation and avoidance behavior.
Chang S, Fermani F, Lao CL, Huang L, Jakovcevski M, Di Giaimo R, Gagliardi M, Menegaz D, Hennrich AA, Ziller M, Eder M, Klein R, Cai N, Deussing JM.Chang S, et al.Sci Adv. 2022 Nov 18;8(46):eabo1023. doi: 10.1126/sciadv.abo1023. Epub 2022 Nov 16.Sci Adv. 2022.PMID:36383658Free PMC article.
A noradrenergic-hypothalamic neural substrate for stress-induced sleep disturbances.
Antila H, Kwak I, Choi A, Pisciotti A, Covarrubias I, Baik J, Eisch A, Beier K, Thomas S, Weber F, Chung S.Antila H, et al.Proc Natl Acad Sci U S A. 2022 Nov 8;119(45):e2123528119. doi: 10.1073/pnas.2123528119. Epub 2022 Nov 4.Proc Natl Acad Sci U S A. 2022.PMID:36331996Free PMC article.
Sex differences in androgen receptor, estrogen receptor alpha, and c-Fos co-expression with corticotropin releasing factor expressing neurons in restrained adult mice.
Rybka KA, Lafrican JJ, Rosinger ZJ, Ariyibi DO, Brooks MR, Jacobskind JS, Zuloaga DG.Rybka KA, et al.Horm Behav. 2023 Nov;156:105448. doi: 10.1016/j.yhbeh.2023.105448. Epub 2023 Oct 27.Horm Behav. 2023.PMID:38344954Free PMC article.
Ascending mechanisms of stress integration: Implications for brainstem regulation of neuroendocrine and behavioral stress responses.
Myers B, Scheimann JR, Franco-Villanueva A, Herman JP.Myers B, et al.Neurosci Biobehav Rev. 2017 Mar;74(Pt B):366-375. doi: 10.1016/j.neubiorev.2016.05.011. Epub 2016 May 18.Neurosci Biobehav Rev. 2017.PMID:27208411Free PMC article.Review.

See all "Cited by" articles

References

1. Alvarez VA, Chow CC, Van Bockstaele EJ, Williams JT. Frequency-dependent synchrony in locus ceruleus: role of electrotonic coupling. Proc Natl Acad Sci U S A. 2002;99:4032–4036. - PMC - PubMed
1. Anthony T, Dee N, Bernard A, Lerchner W, Heintz N, Anderson D. Control of Stress-Induced Persistent Anxiety by an Extra-Amygdala Septohypothalamic Circuit. Cell. 2014;156:522–536. - PMC - PubMed
1. Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A. 2007;104:5163–5168. - PMC - PubMed
1. Aston-Jones G, Bloom FE. Activity of norepinephrine-containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle. J Neurosci Off J Soc Neurosci. 1981a;1:876–886. - PMC - PubMed
1. Aston-Jones G, Bloom FE. Norepinephrine-containing locus coeruleus neurons in behaving rats exhibit pronounced responses to non-noxious environmental stimuli. J Neurosci Off J Soc Neurosci. 1981b;1:887–900. - PMC - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions

Related information

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- The Lens - Patent Citations Database
- scite Smart Citations
Medical
- MedlinePlus Consumer Health Information
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)

Movatterモバイル変換

Account

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Full text links

Actions

Share

CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety

Affiliations

CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety

Authors

Affiliations

Abstract

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases