A Prelimbic Cortex NRG1-ErbB4 Pathway Regulates Expression Of Tone-cued Fear

The term "Fear" is often used when describing a feeling induced by perceived threat that occurs.Fear can cause a change in metabolic and organ functions,such as sweating and accelerating heartbeats,both in humans and in animals.Fear can also cause a change in behavior,such as fleeing,hiding or freezing from perceived danger.The fear response usually leads to confrontation with the threat,or escape from it,which is referred to as the fight-or-flight response.In extreme cases,the fear response may become a freeze response or paralysis.Fear can be modulated by learning,so fear is often judged as appropriate or inappropriate.Phobia,an irrational fear,can be harmful.The study of fear may help people overcome fear.Among scientists,there is a consensus that studing the threat responses neural circuits in experimental animals,is promising for understanding fear.Researching into mechanisms of the defensive responses in animals has a long history.One distinction can be made between 'innate fear',which is activated by intrinsically threatening stimuli;and 'learned fear',which is activated by stimuli associated with innate threats.Scientists have believed that amygdale were the center of the fear neuronal circuit for a long time.Amygdale receives information of conditioned(learned)and unconditioned(innate)stimuli and stores these stimuli as associative memories.Rats with lesions of the amygdala are not able to learn fear associations.Lesions made after conditioning abolish previously learned fear responses.The mPFC,despite negative lesion findings,is also considered important for fear acquisition or expression in an animal.The prelimbic(PL)subregion of mPFC sends projections to the basal nucleus of the amygdale.Lesions of PL do not prevent acquisition or expression of conditioned fear,but it is not known whether PL activity in the intact animal is essential for either process.One potential candidate molecule may be NRG1 which is known to play an important role in synaptic plasticity,neuronal transmission and survival12.The receptors of NRG1,ErbBs are transmembrane receptor tyrosine kinases acting in a dimerization paradigm with multiple functions related to cell proliferation and differentiation.As a ligand for ErbBs,NRG1 contains an epidermal growth factor(EGF)-like domain which can stimulate ErbB receptor.But all of the members of ErbB receptors,ErbB4 is the only autonomous NRG1-specific activated tyrosine kinase.Thus we focus our research on NRGl-ErbB4 signaling.In the past research,there are some evidences showing that NRG1 and ErbB4 are susceptibility genes for contextual fear memory expression defect which mainly acting through parvalbumin-positive interneurons13.But the role of NRG1-ErbB4 signaling in the brain region mPFC which is intimately correlated with fear expression has yet to be investigated.In the present study,we addressed this issue by applying pharmacological methods,virus-induced methods and conditional knock out mice and observing the resulting effects on fear expression.1.Neutralizing the endogenous NRG1 in PL impaired fear expression.Former studies indicated that PL affected fear expression in opposite manners.We first investigated the role of NRG 1 in tone cued fear expression in PL.We utilized ecto-ErbB4,a polypeptide which preserved the binding capacity of ErbB4 to NRG1 without inducing downstream reactivities,to competitively abolish the effect of endogenous NRG1.We found that fear expression was impaired when ecto-ErbB4 was injected in PL,suggesting that NRG1 in PL was important in fear expression.2.Administration of exogenous NRG1 in PL enhanced fear expression Next we injected synthesized NRG1 into PL,and found that fear expression was significantly increased,proved the regulatory role of NRG 1 in PL for fear expression.3.blocking ErbB4 receptor in PL down-regulated fear expression ErbB4 is the functional receptor of NRG1.We injected AG1478,a selective inhibitor for ErbB4,into the PL region.The AG1478 injected mice showed impaired fear expression,suggesting that ErbB4 receptor in PL was also involved in the regulation of fear expression.4.Fear expression was impaired upon deletion of ErbB4 receptor in PV positive neurons,but not in CaMKIIa positive neuronsIn the cortex,ErbB4 receptor is mainly expressed in inhibitory interneurons,among which the PV positive neurons are the majority.We cross-bred a PV-cre mouse line with ErbB4f/f mouse line to obtain the PV-Cre;ErbB4f/f mouse line,in which ErbB4 receptor was deleted only in PV positive neurons,but not in other cell types.We also obtained the CamKII-Cre;ErbB4f/f mouse line,in which ErbB4 receptor was deleted only in pyramidal neurons.We found that fear expression was impaired on PV-Cre;ErbB4f/f mouse line,but not on CamKII-Cre;ErbB4f/f mouse line,proved that ErbB4 receptor on PV positive neurons played a role in the regulation of fear expression.5.Knocking down ErbB4 in PL down regulated fear expression We injected AAV-CAG-Cre-GFP,a viral vector which expressed Cre recombinase,in PL subregion of ErbB4f/f mice,to knock out the ErbB4 gene selectively in PL subregion.We also used AAV-CAG-GFP,a viral vector which did not expresse Cre recombinase,as the control.Mice received AAV-CAG-Cre-GFP injection showed decreased fear expression.6.Injecting LV-ErbB4 in the PL subregion rescued the impaired fear expression of PV-Cre;ErbB 4f/f mice.To further validate of the role of ErbB4 receptor in fear expression,we injected LV-ErbB4-GFP,a viral vector which expressed exogenous ErbB4 receptor,in the PL subregion of PV-Cre;ErbB 4f/f.We found that the over expressed ErbB4 rescued the impairment of fear expression.This result proved the important role of ErbB4 receptor in the PL subregion.Our data suggest that in the PL subregion,the NRG1-ErbB4 signaling participated in the modulation of fear expression through its effects on PV positive interneurons.We found that exogenous NRG 1 increased fear expression while neutralizing endogenous NRG1,inhibition,or viral ablation ErbB4 in PL cortex inhibited fear expression Despite that previous studies indicated that activities in the PL subregion was positively correlated with fear expression,which was suggested by injection of TTX into the PL subregion or optogenetic activation of the PV positive neurons in the PL subregion,our studies showed opposite effects of the PV positive neurons in PL via modulating NRG1-ErbB4 signaling in these cells.The conflicting results reminded us that the regulatory mechanisms of fear expression by PV positive interneurons in PL might be more complex than simply up-regulation or down-regulation.The activity level of neurons,the chemical substances released from PV interneurons,in the PL subregion,may have different roles in modulation of fear expression,which might be fully clarified by future studies.