SHORT COMMUNICATION
Knockdown of corticotropin-releasing factor 1 receptors in the ventral tegmental area enhances conditioned fear

https://doi.org/10.1016/j.euroneuro.2016.06.002Get rights and content

Abstract

The neuropeptide corticotropin-releasing factor (CRF) coordinates the physiological and behavioural responses to stress. CRF receptors are highly expressed in the ventral tegmental area (VTA), an important region for motivated behaviour. Therefore, we examined the role of CRF receptor type 1 (CRFR1) in the VTA in conditioned fear, using a viral-mediated RNA interference approach. Following stereotaxic injection of a lentivirus that contained either shCRF-R1 or a control sequence, mice received tone-footshock pairings. Intra-VTA shCRF-R1 did not affect tone-elicited freezing during conditioning. Once conditioned fear was acquired, however, shCRF-R1 mice consistently showed stronger freezing to the tone even after extinction and reinstatement. These results implicate a novel role of VTA CRF-R1 in conditioned fear, and suggest how stress may modulate aversive learning and memory.

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.

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