SHORT COMMUNICATIONKnockdown of corticotropin-releasing factor 1 receptors in the ventral tegmental area enhances conditioned fear
Introduction
Corticotropin-releasing factor (CRF) is localised in numerous extrahypothalamic brain regions including the ventral tegmental area (VTA), a major structure responsible for motivated behaviour and associative learning (Koob and Heinrichs, 1999, Wise and Morales, 2010). Specifically, CRF signalling within the VTA can influence behaviours related to reward learning, including stress-induced reinstatement of cocaine-seeking (Wang et al., 2005, Blacktop et al., 2011, Chen et al., 2014). The VTA also appears important for aversive learning. For example, VTA dopamine signalling is necessary for the expression of conditioned fear (de Oliveira et al., 2009). However, little is known of the role of CRF in the VTA for conditioned fear. Therefore, we used a lenti-viral vector to deliver shRNA targeting CRF receptor 1 (CRFR1) mRNA and examined the effect of CRFR1 knockdown on conditioned fear in mice. This method enables visualisation of the viral transduction in the targeted area, as well as a high level of specificity for CRFR1 over other CRF binding partners. We chose CRFR1 because it is more highly expressed in the VTA and binds CRF with a 10-fold higher affinity than CRF receptor 2 (CRFR2) (Chen et al., 2014, Van Pett et al., 2000).
Stress has diverse effects on learning and memory, and VTA activity can be up- or down-regulated depending on the nature of the stressor (Valenti et al., 2012). If VTA CRFR1 promotes an anxiogenic state that increases the strength of the unconditioned stimulus (US) to promote fear learning, then knockdown of CRFR1 signalling in the VTA may dampen conditioned fear. However, CRFR1 signalling is necessary for stress-induced impairment in memory, and antagonism of CRFR1 reverses memory deficits caused by stress (Urani et al., 2011). Therefore, it is also possible that knockdown of CRFR1 in the VTA may enhance fear learning by alleviating stress. With these two opposing hypotheses in mind, we examined the effects of a viral knockdown of CRFR1 in the VTA on conditioned fear.
Section snippets
Animals
All experiments were performed in accordance with the Prevention of Cruelty to Animals Act 1986 under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia and approved by the Animal Ethics Committee at the Florey Institute of Neuroscience and Mental Health. In each experiment, naïve adult male C57BL/6J mice (Animal Resource Center, Perth, Australia) were used, and maintained on a reversed 12 h
Quantification of CRF receptor transcript levels in the VTA
qPCR analysis of naïve mouse VTA tissue showed significantly higher levels of CRFR1 mRNA compared to CRFR2 indicated by an average ΔΔCt value of 4.68 relative to CRFR1 (t(14)=10.21, p<0.0001). There were also greater levels of CRFR1 than CRFBP mRNA, which showed an average ΔΔCt of 1.25 (t(14)=3.94, p<0.01) (Figure 1A). Further, shCRFR1 virus treated mice showed decreased expression of CRFR1 mRNA compared to the shControl treated mice (t(11)=2.42, p<0.05). No differences were seen in CRFR2 or
Discussion
We investigated the role of VTA CRFR1 in conditioned fear. qPCR analysis of naïve mouse VTA showed that there is approximately 23 times more CRFR1 mRNA compared to CRFR2 (Figure 1A). This finding is consistent with in situ hybridisation studies showing abundant expression of CRFR1 in the VTA, but little CRFR2 (Van Pett et al., 2000). We also detected CRF-BP mRNA in the VTA at much higher levels than CRFR2, and approximately 50% of CRFR1 levels. We then demonstrated the ability of a
Funding and disclosure
This research was supported by a project Grant (APP1063140) from the National Health and Medical Research Council (NHMRC) of Australia awarded to JHK and AJL, Australian Postgraduate Award awarded to NAC, Baker Foundation Fellowship awarded to DEG, NHMRC Principal Research Fellowship (1020737) awarded to AJL, NHMRC Senior Research Fellowship (1042650) awarded to RADB, Career Development Fellowship (APP1083309) awarded to JHK. We acknowledge the Victorian Government׳s Operational Infrastructure
Contribution
NAC, AJL, and JHK designed the study. NAC and DEG conducted the study. All authors contributed to writing of the manuscript.
Conflict of interest statement
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgement
We would like to thank the Florey Behaviour Core Facility.
References (28)
- et al.
Fear extinction learning can be impaired or enhanced by modulation of the CRF system in the basolateral nucleus of the amygdala
Behav. Brain Res.
(2014) - et al.
Circuit architecture of VTA dopamine neurons revealed by systematic input–output mapping
Cell
(2015) - et al.
Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats
Neuropharmacology
(2016) - et al.
The corticotropin-releasing factor receptor 1 antagonist CP-154,526 reverses stress-induced learning deficits in mice
Behav. Brain Res.
(2003) - et al.
A role for corticotropin releasing factor and urocortin in behavioral responses to stressors
Brain Res.
(1999) - et al.
GABA neurons of the VTA drive conditioned place aversion
Neuron
(2012) - et al.
The corticotropin-releasing factor 1 receptor antagonist, SSR125543, and the vasopressin 1b receptor antagonist, SSR149415, prevent stress-induced cognitive impairment in mice
Pharmacol. Biochem. Behav.
(2011) - et al.
A ventral tegmental CRF-glutamate-dopamine interaction in addiction
Brain Res.
(2010) - et al.
Augmented cocaine seeking in response to stress or CRF delivered into the ventral tegmental area following long-access self-administration is mediated by CRF receptor type 1 but not CRF receptor type 2
J. Neurosci.
(2011) - et al.
Ventral tegmental area GABA projections pause accumbal cholinergic interneurons to enhance associative learning
Nature
(2012)
Knockdown of CRF1 receptors in the ventral tegmental area attenuates cue- and acute food deprivation stress-induced cocaine seeking in mice
J. Neurosci.
Role of dopamine receptors in the ventral tegmental area in conditioned fear
Behav. Brain Res.
Dopamine is necessary for cue-dependent fear conditioning
J. Neurosci.
Cell-type specific deletion of GABA(A)α1 in corticotropin-releasing factor-containing neurons enhances anxiety and disrupts fear extinction
Proc. Natl. Acad. Sci. USA
Cited by (10)
Tailored behavioural tests reveal early and progressive cognitive deficits in M1000 prion disease
2023, Neurobiology of DiseaseDissociated roles of dorsal and ventral hippocampus in recall and extinction of conditioned fear in male and female juvenile rats
2020, Experimental NeurologyCitation Excerpt :Additionally, corticotropin-releasing factor receptor 1 (CRFR1) expression is significantly lower at P18 relative to adulthood (Avishai-Eliner et al., 1996). CRFR1 knockdown transiently delays extinction acquisition (Chen et al., 2016). Thus, naturally low HPC CRFR1 expression at P18 may delay extinction acquisition, consistent with the present findings on accelerated extinction following dHPC inactivation.
Postnatal development of neurotransmitter systems and their relevance to extinction of conditioned fear
2017, Neurobiology of Learning and MemoryCitation Excerpt :Both CRFR1 and CRFR2 undergo interesting changes in binding profiles and mRNA expression during the first two weeks of postnatal life in rodents, which is the time period when most early life adversity treatments are aimed at in rodent studies (Callaghan & Richardson, 2011; Roth & Sullivan, 2005). Using chronic ways to suppress CRF systems during this period (Chen et al., 2016), future studies should examine whether the dynamic development of CRF signaling plays a role in the behavioral and neural consequences of early life adversity. Acetylcholine is an endogenous agonist to both nicotinic ion channel receptors and G protein-coupled muscarinic receptors.
Type 1 Corticotropin-Releasing Factor Receptor Differentially Modulates Neurotransmitter Levels in the Nucleus Accumbens of Juvenile versus Adult Rats
2022, International Journal of Molecular SciencesSex differences in the neurochemistry of frontal cortex: Impact of early life stress
2021, Journal of Neurochemistry