Projection Of The Doral Raphe Nucleus To Separate Amygdala Nucleus Is Critical For The Regulation Of Anxiety-like Behavior

Anxiety is a complex stress related disorder,and includes generalized anxiety,social phobia,post-traumatic stress disorders,obsessive-compulsive disorder and so on.Generalized anxiety is the most common type of psychiatric disorder with a life incidence of 4.1%-6.6%;however,the pathogenesis remains to be further explored.In the past 50 years,anxiety is mainly treated with benzodiazepine drugs.Although these drugs are generally well tolerated,there is a risk of drug dependence or unnecessary sedative effect with the long-term use.For these reasons,the current medical practice guide lines recommended antidepressant drugs,especially the selective serotonin reuptake inhibitors(SSRIs)or serotonin and norepinephrine reuptake inhibitors,as a first-line choice for the treatment of anxiety disorders,with benzodiazepines as second-line agents or in the immediate management of anxiety before SSRIs treatment.For non-drug treatment therapy,cognitive behavioral therapy is considered more effective.Based on behavior and imaging studies,previous studies have proposed basic neuron circuits about anxiety and fear.Hippocampus is involved in information processing during learning and memory.Hippocampus dysfunction may cause an overestimation of the potential threat,which lead to pathological anxiety.Animal experiments have shown that damage of the ventral frontal lobe will affect fear extinction.As a result,the ventral prefrontal dysfunction may also result in pathological anxiety.The amygdala is considered to be the key structure to regulate alert and fear.Most of the studies in the amygdala focus on the basolateral amygdala(BLA)and central nucleus(CeA).Studies on fear conditioning showed that BLA function as an integration point of diverse environmental information and decodes behaviors.In fact,some cells of the BLA response to the threat environment,while others are activated by situations that are no longer dangerous.Most of the BLA terminals project to the CeA,which receives excitatory inputs mainly from the BLA.CeA is divided by the lateral and central medial parts.The central lateral amygdala inhibit central medial nucleus,which is the main output of the amygdala.CeA plays an important role in immediate behavior induced by threatening stimuli..Studies have shown that central nucleus deactivation can reduce fear responses induced by voice,partially due to the fact that CeA projects strongly to the hypothalamus and brainstem,which mediate various anxiety-relevant physiological and behavioral responses.Interaction of different brain regions regulates the anxiety behavior.Studies have showed that vHPC injection of gap junction blocker reduces vHPC-mPFC synchronicity and decreases the anxiety level on the high plus maze(EPM)and open field(OFD)test.It was reported that light activation of BLA-vHPC projection induces anxiogenic effects on on the EPM and OFD test,and inhibition of this projection induces anxiolytic effects.Just as BLA,BNST also play an important role in anxiety.Light activation of adBNST to lateral hypothalamus projection induces anxiolytic effects on the EPM and OFD test,and light activation of adBNST to parabrachial nuclei projection selectively decreases respiratory rate(a physical sign of anxiety),with no significant effects on anxiety-relevant behavioral responses.Finally,optical activation of adBNST to VTA projection induced location preferences.Neurotransmitter systems involved in anxiety mainly include noradrenaline,dopamine,GABA and serotonin.Norepinephrine,especially in the locus coeruleus,can improve alert responses to danger.The cortical dopaminergic system is related to emotional behavior,including expression and suppression of different emotions.GABA is the major inhibitory neurotransmitter.5-HT of the dorsal raphe nucleus(DRN)plays an important role in modulation of anxiety.These neurotransmitters may interact with each other and lead to anxiety related clinical manifestations.This study mainly focused on the serotonin system,which plays an important role in generalized anxiety.Serotonin is thought to regulate development and the plasticity of adult-born neurons,which can improve the flexibility and adaptability of neural circuits in response to environmental challenges.And 5-HT in amydala participates in the processing of stressful information and regulation of stress sensitivity.Pharmacological regulation of serotonin level leads to changes of the amygdala activity patterns,and genetic mutations of serotonin related molecule can lead to increased risk of mental illness.These results suggest that serotonin in the amygdala have an important role in anxiety.In the amygdala neurotransmitter systems,serotonin is gaining more attention.Pharmacological behavior studies have shown that serotonin is involved in the modulation of anxiety-like behavior,fear conditioning,stress,and the regulation of reward.From rodents to primates,the amygdala receives intensive serotonin inputs,and levels of serotonin in the amygdala are closely correlated with anxiety level.Similarly,pharmacological manipulation of the amygdala serotonin regulates stress response.In healthy volunteers,pharmacological regulation of serotonin system leads to changes of the amygdala activity.This functional imaging study suggest that the emotion regulation of amygdala serotonin also exists in humans beings.DRN is rich in serotonin neurons.Although the DRN contains heterogeneous types of neurons,most of previous studies focus on the effects of serotonin levels on behavior.DRN projects to several brain regions which are involved in the regulation of anxiety,and DRN serotonin neurons are activated by stress and anxiety-related stimuli.Previous tracing studies have shown that in rats,the amygdala receives serotoninergic efferents mainly from the DRN,with a minority from the medial raphe nucleus(MRN).Mouse and primate amygdala receive far higher rate of serotonin afferents from DRN;therefore,mice might be a better model to study stress and anxiety disorders than rats.Compared with other brain regions,all serotonin receptors in the amygdala of rodents,primates and human beings,are relatively highly expressed.The expression of these receptors in different types of neurons may explain the differential effects following infusion of different serotonin agonists or antagonists into the amygdala.And somehow some results are even controversial.Moreover,serotonin levels induced by stress are not always consistent by electrophysiology,neural chemistry and behavior tests,which might lead to different interpretations of the function of serotonin signals in the amygdala.Based on the above-mentioned backgrounds,we hypothesize that the DRN serotonin projections to the amygdala may play an important role in anxiety disorder.Thus,with a combination of electrophysiology,optogenetics,and behavior tests,we tried to dissect the DRN projections onto separate amygdale nucleui,in particular the basolateral and central nuclei,in the regulation of anxiety.In order to explore the projection of the dorsal raphe nucleus to the amygdala and its role in anxiety,we injected AAV carrying ChR2-EGFP driven by the ubiquitous CAG promoter into the DRN brain regions of C57 BL/6J mice.In vitro slices showed that EGFP was expressed strongly not only in the DRN,but also in the amygdala,mainly in the BLA and CeA subnuclei.Blue light stimulation induced photocurrents and action potentials under voltage-and current-clamp,respectively.For behavioral analysis we mainly focused on the EPM and OFD tests.Experimental results showed that the stimulation of the DRN-CeA projection in the CeA increased the time both in the open arms on the EPM test and in the center on the OFD test,with the motor function largely unaffected.These results showed that selective stimulation of the DRN-CeA fibers reduced innate anxiety levels,producing an anxiolytic effect.However,stimulation of the DRN-BLA projection had no significant effect on either the time in the open arms on EPM,or in the central area on OFD.Still,the motor function was not affected.These results showed that selective stimulation of the DRN-CeA,but not DRN-BLA projection,exerts significant effect on anxiety.To further confirm the above results,we injected AAV carrying Arch-EGFP driven by the ubiquitous CAG promoter into the DRN of C57 BL/6J mice.Similarly,in vitro slices showed that EGFP was strongly expressed in the DRN,confirming successful expression of Arch,and it was also found that in the amygdala,mainly in the BLA and CeA.589 nm yellow light induced outward photocurrents and inhibited the action potential firing induced by the injection of superthreshold currents.Inhibition of CeA-projecting fibers from DRN decreased both the time in the open arms on the EPM test and the time in the center on the OFD test,without affecting the motor function.These results showed that selective inhibition of DRN-CeA projection was anxiogenic.Howefer,inhibition of the DRN-BLA projection had no significant effects on the time spent in the open arms of EPM or in the central area of OFD tests.These results showed that selective inhibition of the DRN-CeA,but not DRN-BLA projection,exerts significant effect on anxiety.The DRN is home to many subtypes of neurons expressing serotonin,dopamine,glutamate or GABA,with serotonin neurons accounting for the majority.And coexpression of two types of neurotransmitters is common in the DRN;in particular,glutamate and serotonin have been shown to coexpress in DRN serotonin neurons.To better distinguish whether serotonin neurons play a predominant role in anxiety,we specially expressed light-activated ion channels on serotonin neurons in the DRN.We injected cre-dependent DIO ChR2-mCherry AAV virus into the DRN of Slc6a4-Cre transgenic mice,which express Cre recombinase specifically in serotonin neurons.Red fluorescence(mCherry)and 5-HT-positive neurons colocalized together,suggesting specific expression of ChR2 on serotonin neurons.To check the effect of activating DRN-CeA projection on postsynaptic neurons,we recorded postsynaptic currents mainly in CeL neurons with whole-cell recordings in in vitro slices.Illuminating the CeA induced robust excitatory postsynaptic currents,which could be blocked by CNQX.Behavior studies showed that injection of glutamate receptor blockers into the CeA abolished the increase in the time in open arms on the EPM test and in central area on the OFD test induced by blue light stimulation of the DRN-CeA terminals.These results demonstrated that anxiolytic effects induced by blue light stimulation are mainly due to direct projection of the DRN serotonin neurons to the CeA,rather than projections that pass through the CeA or others.Our results showed that the projection of the DRN to the CeA plays an important role in anxiety;we next asked whether directly manipulating the activity of CeA neurons alter anxiety levels?In order to explore the function of CeA neurons in anxiety,we injected DIO-ChR2 or DIO-Arch AAV virus into the CeA brain regions of synapsinl-Cre mice.Immunofluorescence and electrophysiological experiments showed the successful expression of light-sensitive ion channels in the CeA.Behavioral results showed that activation of CeA neurons increased the time in the open arms on the EPM test and the time in the center on the OFD test.But the locomotor ability was not affected.These results showed that the selective activation of the CeA neurons can reduce anxiety levels.Moreover,inhibition of the CeA neurons decreased the time in the open arms on the EPM test and the time in the center on the OFD test.Still,the motor function was not affected.These results showed that selective inhibition of the CeA neurons were anxiogenic.Together,these results are consistent with previous studies and further confirm the importance of CeA in the regulation of anxiety.Taken together,our results show that the projection of the DRN to the amygdala,mainly the CeA,involved in the regulation of anxiety.These results deepen our understanding of the cellular and circuit mechanisms of anxiety that may shed new light on the pathological mechanisms of emotional disorders and provide a potential target for intervention in anxiety disorders.