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Cited by 246Cannabis and Cannabinoid ResearchStress Responses and CortisolNeuropeptides and Animal Physiology

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Corticotropin-Releasing Hormone Drives Anandamide Hydrolysis in the Amygdala to Promote Anxiety

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Gray, J. Megan, Vecchiarelli, Haley A., Morena, Maria, Lee, Tiffany T.Y., Hermanson, Daniel J., Kim, Alexander B., McLaughlin, Ryan J., Hassan, Kowther I., Kühne, Claudia, Wotjak, Carsten T., Deussing, Jan M., Patel, Sachin, Hill, Matthew N.·2015·The Journal of Neuroscience
DOI 10.1523/jneurosci.2737-14.2015PMC PMC4348185
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Abstract

Corticotropin-releasing hormone (CRH) is a central integrator in the brain of endocrine and behavioral stress responses, whereas activation of the endocannabinoid CB1 receptor suppresses these responses. Although these systems regulate overlapping functions, few studies have investigated whether these systems interact. Here we demonstrate a novel mechanism of CRH-induced anxiety that relies on modulation of endocannabinoids. Specifically, we found that CRH, through activation of the CRH receptor type 1 (CRHR1), evokes a rapid induction of the enzyme fatty acid amide hydrolase (FAAH), which causes a reduction in the endocannabinoid anandamide (AEA), within the amygdala. Similarly, the ability of acute stress to modulate amygdala FAAH and AEA in both rats and mice is also mediated through CRHR1 activation. This interaction occurs specifically in amygdala pyramidal neurons and represents a novel mechanism of endocannabinoid-CRH interactions in regulating amygdala output. Functionally, we found that CRH signaling in the amygdala promotes an anxious phenotype that is prevented by FAAH inhibition. Together, this work suggests that rapid reductions in amygdala AEA signaling following stress may prime the amygdala and facilitate the generation of downstream stress-linked behaviors. Given that endocannabinoid signaling is thought to exert "tonic" regulation on stress and anxiety responses, these data suggest that CRH signaling coordinates a disruption of tonic AEA activity to promote a state of anxiety, which in turn may represent an endogenous mechanism by which stress enhances anxiety. These data suggest that FAAH inhibitors may represent a novel class of anxiolytics that specifically target stress-induced anxiety.

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Figures

Figure 1 Response of amygdala endocannabinoid levels, FAAH activity, and circulating CORT following intracerebroventricular CRH. A-D , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH infusions 10 min after administration on ( A ) AEA, ( B ) FAAH hydrolytic activity, ( C ) 2-AG, and ( D ) circulating plasma CORT levels. E-H , Effects of intracerebroventricular vehicle or 1 𝛍 gCRHinfusions 30 min after administration on ( E ) AEA, ( F ) FAAH hydrolytic activity, ( G ) 2-AG, and ( H ) CORT. I-L , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH infusions 120 min after administration on ( I ) AEA, ( J ) FAAH hydrolytic activity, ( K ) 2-AG, and ( L ) CORT. Data are mean + SEM; n = 6-8/groupforAEAand2-AGlevels; n = 4/group for FAAH hydrolytic activity; n = 10-12/groupfor CORT analysis. * p < 0.05.
Figure 1 Response of amygdala endocannabinoid levels, FAAH activity, and circulating CORT following intracerebroventricular CRH. A-D , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH infusions 10 min after administration on ( A ) AEA, ( B ) FAAH hydrolytic activity, ( C ) 2-AG, and ( D ) circulating plasma CORT levels. E-H , Effects of intracerebroventricular vehicle or 1 𝛍 gCRHinfusions 30 min after administration on ( E ) AEA, ( F ) FAAH hydrolytic activity, ( G ) 2-AG, and ( H ) CORT. I-L , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH infusions 120 min after administration on ( I ) AEA, ( J ) FAAH hydrolytic activity, ( K ) 2-AG, and ( L ) CORT. Data are mean + SEM; n = 6-8/groupforAEAand2-AGlevels; n = 4/group for FAAH hydrolytic activity; n = 10-12/groupfor CORT analysis. * p < 0.05.CC-BY-NC-SA · From the PDF
Image 2
CC-BY-NC-SA · From the PDF
Figure 3 FAAH protein and mRNA levels in the amygdala and PFC following intracerebroventricular CRH. A , B , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH on amygdala FAAH, ( A ) protein and ( B ) mRNA levels, 30 min after intracerebroventricular infusion. C , D , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH on PFC FAAH, ( C ) protein and ( D ) mRNA levels, 30 min after intracerebroventricular infusion. E , Representative Western blot images of FAAH (63 kDa) and GAPDH (40 kDa) bands in the amygdala and PFC. A , C , For protein analysis, the optical density for FAAH was divided by the optical density for GAPDH and was normalized to the vehicle control group and represented using arbitrary units. B , D , For FAAH mRNAanalysis, fold change of FAAH mRNAlevels is shown normalized to vehicle controls (relative to RRLP2 ) and represented as arbitrary units. Data are mean + SEM; n = 5 or 6/group for FAAH protein; n = 6-8/group for FAAH mRNAlevels.
Figure 3 FAAH protein and mRNA levels in the amygdala and PFC following intracerebroventricular CRH. A , B , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH on amygdala FAAH, ( A ) protein and ( B ) mRNA levels, 30 min after intracerebroventricular infusion. C , D , Effects of intracerebroventricular vehicle or 1 𝛍 g CRH on PFC FAAH, ( C ) protein and ( D ) mRNA levels, 30 min after intracerebroventricular infusion. E , Representative Western blot images of FAAH (63 kDa) and GAPDH (40 kDa) bands in the amygdala and PFC. A , C , For protein analysis, the optical density for FAAH was divided by the optical density for GAPDH and was normalized to the vehicle control group and represented using arbitrary units. B , D , For FAAH mRNAanalysis, fold change of FAAH mRNAlevels is shown normalized to vehicle controls (relative to RRLP2 ) and represented as arbitrary units. Data are mean + SEM; n = 5 or 6/group for FAAH protein; n = 6-8/group for FAAH mRNAlevels.CC-BY-NC-SA · From the PDF
Figure 4 Analysis of amygdala and PFC levels of endocannabinoids and FAAH activity after CRH receptor agonism. A-C , Effects of intracerebroventricular vehicle, cortagine (CRHR1 agonist; R1, 300ng)orurocortin 2 (CRHR2 agonist; R2, 1 𝛍 g) on amygdala 30 min after infusion, ( A ) AEA, ( B ) FAAH hydrolytic activity, ( C ) 2-AG. D-F , Effects of intracerebroventricular vehicle, cortagine (CRHR1 agonist; R1, 300 ng) or urocortin 2 (CRHR2 agonist; R2, 1 𝛍 g) on PFC 30 min after infusion, ( D ) AEA, ( E ) FAAH hydrolytic activity, ( F ) 2-AG. Data are mean + SEM; n = 6-8/groupfor AEA and 2-AG levels; n = 4/group for FAAH hydrolytic activity. * p < 0.05.
Figure 4 Analysis of amygdala and PFC levels of endocannabinoids and FAAH activity after CRH receptor agonism. A-C , Effects of intracerebroventricular vehicle, cortagine (CRHR1 agonist; R1, 300ng)orurocortin 2 (CRHR2 agonist; R2, 1 𝛍 g) on amygdala 30 min after infusion, ( A ) AEA, ( B ) FAAH hydrolytic activity, ( C ) 2-AG. D-F , Effects of intracerebroventricular vehicle, cortagine (CRHR1 agonist; R1, 300 ng) or urocortin 2 (CRHR2 agonist; R2, 1 𝛍 g) on PFC 30 min after infusion, ( D ) AEA, ( E ) FAAH hydrolytic activity, ( F ) 2-AG. Data are mean + SEM; n = 6-8/groupfor AEA and 2-AG levels; n = 4/group for FAAH hydrolytic activity. * p < 0.05.CC-BY-NC-SA · From the PDF
Figure5 RoleofCRHR1receptorsintheeffectsofstressonendocannabinoidlevelsandFAAHactivityinamygdalaandPFC. A-F , Effects of intraperitoneal CRHR1 antagonism (R1-ANT, 20 mg/kg) on ( A-C ) amygdala, ( A ) AEA, ( B ) FAAH hydrolytic activity, ( C ) 2-AG and ( D-F ) PFC, ( D ) AEA, ( E ) FAAH hydrolytic activity, ( F ) 2-AG, either under basal conditions or immediately following the end of 30 min of restraint stress. G-L , Effects of CRHR1-deletion off of glutamatergic dorsal telencephalon neurons (R1-Glu KO) on ( G-I ) amygdala, ( G ) AEA, ( H ) FAAH hydrolytic activity, ( I ) 2-AG and ( J-L ) PFC, ( J ) AEA, ( K ) FAAH hydrolytic activity, ( L ) 2-AG, similarly, under basal conditions or immediately after the end of 30 min of restraint stress. Data are mean + SEM; n = 6-8/groupforAEA and 2-AG levels; n = 4/group for FAAH hydrolytic activity. * p < 0.05.
Figure5 RoleofCRHR1receptorsintheeffectsofstressonendocannabinoidlevelsandFAAHactivityinamygdalaandPFC. A-F , Effects of intraperitoneal CRHR1 antagonism (R1-ANT, 20 mg/kg) on ( A-C ) amygdala, ( A ) AEA, ( B ) FAAH hydrolytic activity, ( C ) 2-AG and ( D-F ) PFC, ( D ) AEA, ( E ) FAAH hydrolytic activity, ( F ) 2-AG, either under basal conditions or immediately following the end of 30 min of restraint stress. G-L , Effects of CRHR1-deletion off of glutamatergic dorsal telencephalon neurons (R1-Glu KO) on ( G-I ) amygdala, ( G ) AEA, ( H ) FAAH hydrolytic activity, ( I ) 2-AG and ( J-L ) PFC, ( J ) AEA, ( K ) FAAH hydrolytic activity, ( L ) 2-AG, similarly, under basal conditions or immediately after the end of 30 min of restraint stress. Data are mean + SEM; n = 6-8/groupforAEA and 2-AG levels; n = 4/group for FAAH hydrolytic activity. * p < 0.05.CC-BY-NC-SA · From the PDF
Figure 6 FAAHprotein and mRNA levels in the amygdala and PFC in response to acute restraint stress. A , B , Effects of 30 min of restraint stress on amygdala FAAH ( A ) protein and ( B ) mRNAlevels. C , D , Effects of 30 min of restraint stress on PFC FAAH ( C ) protein and( D ) mRNA levels. E , Representative Western blot images of FAAH (63 kDa) and GAPDH (40 kDa) protein bands in the amygdala and PFC. A , C , For protein analysis, the optical density for FAAH was divided by the optical density for GAPDH and was normalized to the basal control group and represented using arbitrary units. B , D , For FAAH mRNAanalysis, fold change of FAAH mRNAlevels is shown normalized to basal controls (relative to RRLP2 ) and represented as arbitrary units. Data are mean + SEM; n = 5 or 6/group for FAAH protein; n = 6-8/group for FAAH mRNAlevels.
Figure 6 FAAHprotein and mRNA levels in the amygdala and PFC in response to acute restraint stress. A , B , Effects of 30 min of restraint stress on amygdala FAAH ( A ) protein and ( B ) mRNAlevels. C , D , Effects of 30 min of restraint stress on PFC FAAH ( C ) protein and( D ) mRNA levels. E , Representative Western blot images of FAAH (63 kDa) and GAPDH (40 kDa) protein bands in the amygdala and PFC. A , C , For protein analysis, the optical density for FAAH was divided by the optical density for GAPDH and was normalized to the basal control group and represented using arbitrary units. B , D , For FAAH mRNAanalysis, fold change of FAAH mRNAlevels is shown normalized to basal controls (relative to RRLP2 ) and represented as arbitrary units. Data are mean + SEM; n = 5 or 6/group for FAAH protein; n = 6-8/group for FAAH mRNAlevels.CC-BY-NC-SA · From the PDF
Figure7 Coexpression of CRHR1 and FAAH protein and mRNA in the BLA. A , Illustration demonstrating the approximate area (blue box) of the amygdala where microscope images of protein and mRNAwere taken. B-E , Representative images of immunohistochemical expression of ( B ) the neuronal marker DAPI, ( C ) FAAH, and ( D ) 𝛃 -GAL, which is expressed only in CRHR1-expressing cells (Ku ¨hne et al., 2012). E , Merged image, demonstrating that FAAH and 𝛃 -GAL (indirect CRHR1 label) are coexpressed (white arrows) in cells of the BLA. Scale bar, 40 𝛍 m. F-I , Representative in situ hybridization images of ( F ) DAPI, ( G ) FAAH , and ( H ) CRHR1 in the BLA. I , Merged image demonstrating that FAAH and CRHR1 transcripts are coexpressed in the BLA (white arrows). Scale bar, 24 𝛍 m.
Figure7 Coexpression of CRHR1 and FAAH protein and mRNA in the BLA. A , Illustration demonstrating the approximate area (blue box) of the amygdala where microscope images of protein and mRNAwere taken. B-E , Representative images of immunohistochemical expression of ( B ) the neuronal marker DAPI, ( C ) FAAH, and ( D ) 𝛃 -GAL, which is expressed only in CRHR1-expressing cells (Ku ¨hne et al., 2012). E , Merged image, demonstrating that FAAH and 𝛃 -GAL (indirect CRHR1 label) are coexpressed (white arrows) in cells of the BLA. Scale bar, 40 𝛍 m. F-I , Representative in situ hybridization images of ( F ) DAPI, ( G ) FAAH , and ( H ) CRHR1 in the BLA. I , Merged image demonstrating that FAAH and CRHR1 transcripts are coexpressed in the BLA (white arrows). Scale bar, 24 𝛍 m.CC-BY-NC-SA · From the PDF
Figure 8 CRH administration and FAAH inhibition on anxiety-like behavior. A-G , Effects of vehicle, CRH (10 ng), FAAH inhibition (URB597; 10 ng), or CRH + URB597,30minbeforetesting on the EPM on ( A ) open arm time, ( B ) percentage open arm time, ( C ) closed arm time, ( D ) percentage closed arm time, ( E ) total entries, ( F ) percentage open arm entries, and ( G ) circulating plasma CORT levels 30 min following EPM testing. H , Representative photomicrograph showinginjectorplacement(arrow).Scalebar,350 𝛍 m. I , Illustration of coronal sections showing representative cannula placements. Data are mean + SEM; n = 10-14/group. * p < 0.05.
Figure 8 CRH administration and FAAH inhibition on anxiety-like behavior. A-G , Effects of vehicle, CRH (10 ng), FAAH inhibition (URB597; 10 ng), or CRH + URB597,30minbeforetesting on the EPM on ( A ) open arm time, ( B ) percentage open arm time, ( C ) closed arm time, ( D ) percentage closed arm time, ( E ) total entries, ( F ) percentage open arm entries, and ( G ) circulating plasma CORT levels 30 min following EPM testing. H , Representative photomicrograph showinginjectorplacement(arrow).Scalebar,350 𝛍 m. I , Illustration of coronal sections showing representative cannula placements. Data are mean + SEM; n = 10-14/group. * p < 0.05.CC-BY-NC-SA · From the PDF
Figure9 ModelofCRHandFAAHinteractionswithintheamygdalafollowingexposuretostress.Underrestingbasalconditions (left), AEA activation of CB1 receptors provides an inhibitory tone on presynaptic glutamate release within the BLA, constraining excessglutamatereleaseandneuronalactivationwithintheBLA.Followingexposuretostress(rightpanel),releaseofCRH,andthe subsequent activation of postsynaptic CRHR1, rapidly increases the enzymatic activity of FAAH, resulting in a depletion of the signaling pool of AEA within the BLA. The loss of this inhibitory AEA tone may in turn result in a facilitation of glutamate release and subsequent activation of excitatory pyramidal projection neurons of the BLA targeting downstream regions regulating stressrelated endocrine and behavioral outputs.
Figure9 ModelofCRHandFAAHinteractionswithintheamygdalafollowingexposuretostress.Underrestingbasalconditions (left), AEA activation of CB1 receptors provides an inhibitory tone on presynaptic glutamate release within the BLA, constraining excessglutamatereleaseandneuronalactivationwithintheBLA.Followingexposuretostress(rightpanel),releaseofCRH,andthe subsequent activation of postsynaptic CRHR1, rapidly increases the enzymatic activity of FAAH, resulting in a depletion of the signaling pool of AEA within the BLA. The loss of this inhibitory AEA tone may in turn result in a facilitation of glutamate release and subsequent activation of excitatory pyramidal projection neurons of the BLA targeting downstream regions regulating stressrelated endocrine and behavioral outputs.CC-BY-NC-SA · From the PDF

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Authors
Gray, J. M., Vecchiarelli, H. A., Morena, M., Lee, T. T. Y., Hermanson, D. J., Kim, A. B., McLaughlin, R. J., Hassan, K. I., Kühne, C., Wotjak, C. T., Deussing, J. M., Patel, S., & Hill, M. N.
Affiliations
  • Hotchkiss Brain Institute (CA)
  • University of Calgary (CA)
  • University of British Columbia (CA)
  • Washington State University (US)
  • Max Planck Institute of Psychiatry (DE)
  • Vanderbilt University (US)
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2015-03-04
Volume
35
Issue
9
Pages
3879-3892
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.2737-14.2015
PMC
PMC4348185
OpenAlex
W2040268671
Cited by
246
References
82
Citations by year
2026: 14 · 2025: 18 · 2024: 17 · 2023: 16 · 2022: 21 · 2021: 32 · 2020: 22 · 2019: 22 · 2018: 22 · 2017: 23 · 2016: 17 · 2015: 21
FWCI
19.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • IRIS Research product catalog (Sapienza University of Rome) (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • Pfizer
  • Vanderbilt University
  • Alberta Innovates
  • Branch Out Neurological Foundation
  • National Institutes of Health (MH090412)
  • Canadian Institutes of Health Research
  • Natural Sciences and Engineering Research Council of Canada
  • National Institute of Mental Health (R21MH103515, R01MH100096, K08MH090412, MH090412)
  • National Institute on Drug Abuse (DA031572, F31DA031572)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54HD083211)
Topics
  • Health Sciences › Medicine › Pharmacology (medical) › Cannabis and Cannabinoid Research
  • Life Sciences › Neuroscience › Behavioral Neuroscience › Stress Responses and Cortisol
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuropeptides and Animal Physiology
Keywords
corticotropin-releasing factor, anandamide, fatty acid amide hydrolase, amygdala, anxiety, CRHR1, endocannabinoid system, acute stress, FAAH inhibitors, anxiolytics, stress-induced anxiety
MeSH terms
CRF Receptor, Type 1, Fatty Acid Amide Hydrolases, Adrenocorticotropic Hormone, Amidohydrolases, Amygdala, Animals, Anxiety, Arachidonic Acids, Corticotropin-Releasing Hormone, Hydrolysis, Male, Stress, Psychological, Rats, Sprague-Dawley, Receptors, Corticotropin-Releasing Hormone, Mice, Knockout, Mice, Rats, Polyunsaturated Alkamides, Endocannabinoids

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