Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

  1. Marcus M Weera  Is a corresponding author
  2. Abigail E Agoglia
  3. Eliza Douglass
  4. Zhiying Jiang
  5. Shivakumar Rajamanickam
  6. Rosetta S Shackett
  7. Melissa Herman
  8. Nicholas J Justice
  9. Nicholas W Gilpin
  1. Louisiana State University Health Sciences Center New Orleans, United States
  2. University of North Carolina at Chapel Hill, United States
  3. The University of Texas Health Science Center, United States

Abstract

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the precise role of CRF1-expressing neuronal populations in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat, which expresses a bicistronic iCre-2A-tdTomato transgene directed by 200kb of promoter and enhancer sequence surrounding the Crhr1 cDNA present within a BAC clone, that has been transgenically inserted into the rat genome. We report that Crhr1 and Cre mRNA expression are highly colocalized in CRF1-Cre-tdTomato rats within both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be selectively targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like behavior and nocifensive responses. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the rostrocaudal axis of the CRF1-Cre-tdTomato rat brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons and circuits can now be performed in assays that require the use of rats as the model organism.

Data availability

All data generated during this study are included in the manuscript.

Article and author information

Author details

  1. Marcus M Weera

    Department of Physiology, Louisiana State University Health Sciences Center New Orleans, New Orleans, United States
    For correspondence
    mweera@lsuhsc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2451-0350
  2. Abigail E Agoglia

    Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Eliza Douglass

    Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Zhiying Jiang

    Institute Of Molecular Medicine, The University of Texas Health Science Center, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Shivakumar Rajamanickam

    Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Rosetta S Shackett

    Department of Physiology, Louisiana State University Health Sciences Center New Orleans, New Orleans, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Melissa Herman

    Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Nicholas J Justice

    Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Nicholas W Gilpin

    Department of Physiology, Louisiana State University Health Sciences Center New Orleans, New Orleans, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8901-8917

Funding

National Institute on Alcohol Abuse and Alcoholism (R01,AA023305)

  • Nicholas W Gilpin

National Institute on Alcohol Abuse and Alcoholism (R21,AA026022)

  • Melissa Herman
  • Nicholas W Gilpin

National Institute on Alcohol Abuse and Alcoholism (R00,AA023002)

  • Melissa Herman

National Institute on Alcohol Abuse and Alcoholism (National Research Service Award,AA027145)

  • Marcus M Weera

National Institute on Alcohol Abuse and Alcoholism (Institutional Training Grant,AA007577)

  • Marcus M Weera

United States Department of Veterans Affairs (Merit Award,#I01 BX003451)

  • Nicholas W Gilpin

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal procedures were conducted in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and were approved by the Institutional Animal Care and Use Committee of the respective institutions at which procedures occurred (Louisiana State University Health Sciences Center, University of North Carolina - Chapel Hill, University of Texas Health Sciences Center). (LSUHSC IACUC Protocol #3749; UNC IACUC Protocol #19-190; UTHSC IACUC Protocol #21-075)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 2,089
    views
  • 293
    downloads
  • 15
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Marcus M Weera
  2. Abigail E Agoglia
  3. Eliza Douglass
  4. Zhiying Jiang
  5. Shivakumar Rajamanickam
  6. Rosetta S Shackett
  7. Melissa Herman
  8. Nicholas J Justice
  9. Nicholas W Gilpin
(2022)
Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior
eLife 11:e67822.
https://doi.org/10.7554/eLife.67822

Share this article

https://doi.org/10.7554/eLife.67822

Further reading

    1. Neuroscience
    Mathias Guayasamin, Lewis R Depaauw-Holt ... Ciaran Murphy-Royal
    Research Article

    Early-life stress can have lifelong consequences, enhancing stress susceptibility and resulting in behavioural and cognitive deficits. While the effects of early-life stress on neuronal function have been well-described, we still know very little about the contribution of non-neuronal brain cells. Investigating the complex interactions between distinct brain cell types is critical to fully understand how cellular changes manifest as behavioural deficits following early-life stress. Here, using male and female mice we report that early-life stress induces anxiety-like behaviour and fear generalisation in an amygdala-dependent learning and memory task. These behavioural changes were associated with impaired synaptic plasticity, increased neural excitability, and astrocyte hypofunction. Genetic perturbation of amygdala astrocyte function by either reducing astrocyte calcium activity or reducing astrocyte network function was sufficient to replicate cellular, synaptic, and fear memory generalisation associated with early-life stress. Our data reveal a role of astrocytes in tuning emotionally salient memory and provide mechanistic links between early-life stress, astrocyte hypofunction, and behavioural deficits.

    1. Neuroscience
    Li Shen, Shuo Li ... Yi Jiang
    Research Article

    When observing others’ behaviors, we continuously integrate their movements with the corresponding sounds to enhance perception and develop adaptive responses. However, how the human brain integrates these complex audiovisual cues based on their natural temporal correspondence remains unclear. Using electroencephalogram (EEG), we demonstrated that rhythmic cortical activity tracked the hierarchical rhythmic structures in audiovisually congruent human walking movements and footstep sounds. Remarkably, the cortical tracking effects exhibit distinct multisensory integration modes at two temporal scales: an additive mode in a lower-order, narrower temporal integration window (step cycle) and a super-additive enhancement in a higher-order, broader temporal window (gait cycle). Furthermore, while neural responses at the lower-order timescale reflect a domain-general audiovisual integration process, cortical tracking at the higher-order timescale is exclusively engaged in the integration of biological motion cues. In addition, only this higher-order, domain-specific cortical tracking effect correlates with individuals’ autistic traits, highlighting its potential as a neural marker for autism spectrum disorder. These findings unveil the multifaceted mechanism whereby rhythmic cortical activity supports the multisensory integration of human motion, shedding light on how neural coding of hierarchical temporal structures orchestrates the processing of complex, natural stimuli across multiple timescales.