The Molecular And Cellular Mechanism Of5-HT1AR In The Regulation Of Anxiety

Anxiety is thought to be a way of controlling an animal’s response to threateningor potentially threatening stimuli, whereas excessive levels of anxiety, or pathologicalanxiety, are accompanied by distress and suffering. As a common human emotionalexperience, anxiety can cause people to feel tired, tense and worried, and can stripaway the ability to concentrate, sleep, experience pleasure or carry on a peacefulexistence. Anxiety-related disorders are becoming a leading cause for decreasedquality of life worldwide. As many as25%of adults will, at one point in their lives,suffer from anxiety disorders, and the collective economic cost of these disorders isestimated to be over$40billion per year.The treatment options currently available for anxiety, however, are limited. Themost widely prescribed classes of anxiolytic drugs are selective serotonin reuptakeinhibitors (SSRIs), which target the serotonergic system, and the benzodiazepines,which modulate the GABAergic system.Serotonergic system is intensively implicated in anxiety. Among the various5-HT receptor subtypes, the5-HT1A receptor (5-HT1AR) is well studied for its rolein anxiety. Some researches found that knockout of5-HT1AR led to an anxiogenicphenotype. Using a tissue-specific expression of5-HT1AR, primarily in thehippocampus and cortex, is sufficient to rescue the behavioral phenotype of the5-HT1AR knockout mice. The mechanism of the effects is still unknown.The hippocampus is one of the brain structures making up the limbic system,which are structurally and functionally affected in mood disorders. The involvementof the hippocampus in mood disorders is suggested by magnetic resonance imagingstudies demonstrating a small reduction in hippocampal volume in depressed patients.It has been proposed that reduced neurogenesis contributes to these volumetricchanges, as well as the symptoms associated with depression and anxiety, and that theefficacy of antidepressant treatments may depend on their ability to restore neurogenesis. The importance of5-HT1AR in neurogenesis has been proved, whichis required to SSRIs-induced neurogenesis.5-HT1AR agonists promote neurogenesisand5-HT1AR antagonists inhibit it. However, the anxiolytic effects of somemanipulations, such as drugs that directly target the HPA axis, can be achieved evenafter suppression of hippocampal neurogenesis is at least one indication thatneurogenesis independent mechanisms exist. These may include other types ofstructural change, a possibility that is supported by the demonstration thathippocampal spine synapse loss occurs in animal models of depression.Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO), a free radicalwith signaling functions in the central nervous system, may be an importantdownstream signaling molecule of5-HT1AR in regulating anxiety-related behaviors.Recent studies have shown that nNOS inhibitors have antidepressant-like effectsunder physiological conditions. More recently, our study has shown thatnNOS-derived NO contributes to chronic stress-induced depression by suppressinghippocampal neurogenesis. These findings suggest that endogenous NO is involved inboth the pathogenesis and treatment of depression.cAMP-responsive element-binding protein (CREB) is well known for its role inactivity-dependent gene regulation and is involved in many neuronal processes,including synaptic plasticity and memory. CREB phosphorylation (pCREB) plays animportant role in regulating neurogenesis in hippocampus. Recently, it was found thatCREB is also engaged in emotional behaviors, such as anxiety. CREB knockout miceexhibited increase in anxiety-like behaviors in several paradigms including theelevated plus maze, light/dark box and open field. The mechanism by which CREB isinvolved in anxiety is not known.Collectively, these findings raise the possibility that the5-HT1AR-mediatedhippocampal structural plasticity via nNOS-CREB pathway may, at least partially,account for the role of5-HT1AR in anxiety-related behaviors. This study includesthree parts. In part I, we focus on the importance of nNOS-CREB pathway in themodulation of5-HT1AR in anxiety. In part II, we worked on whetherCREB-mediated hippocampal structural plasticity involve in the regulation of 5-HT1AR in anxiety-associated behaviors. In part III, we discuss the role of CREB inthe5-HT1AR-mediated building of the never circuitry.In part I, Firstly, we examined the effects5-HT1AR-selective agonist andantagonist, selective serotonin reuptake inhibitor (SSRIs) on the expression of nNOSin vivo or in vitro. The result was that5-HT1AR selective agonist and SSRIsdownregulated hippocampal nNOS expression, whereas5-HT1AR selectiveantagonist upregulated hippocampal nNOS expression. Other kinds of monaminesystem anxiolytic could not alter the expression of nNOS. At the same time, weobserved the importance of nNOS in5-HT1AR modulating anxiety-related behaviors.The results showed that the anxiolytic-like phenotype of5-HT1AR agonist or SSRIsand the anxiolygenic-like effects of5-HT1AR antagonist were disappeared in nNOSgene [knock-out (KO)] mice. We then used the stereotaxic injection to deliverselective nNOS inhibitor7-NI into bilateral hippocampi and found that7-NI rescuedthe anxiogenic-like effects of5-HT1AR antagonist.In order to clarity the downstream signal molecular of nNOS in the regulation ofanxiety-associated behaviors. We observed the expression of pCREB in presence of5-HT1AR agonist or antagonist. The result showed that5-HT1AR selective agonist8-OH-DPAT increased pCREB level, however,5-HT1AR selective antagonistNAN-190decreased it. However, in KO mice, the effects disappeared. Blockade ofhippocampal CREB acticity by microinjection of H89, a protein kinase A (PKA)inhibitor, abolished the anxiolytic-like effects of7-NI.On the whole, the part I studies indicated that both hippocampal nNOS andCREB activity involved in the anxiolytic effects of5-HT1AR agonists and SSRIs.In part I we found that hippocampal CREB activity involved in the anxiolyticeffects of5-HT1AR agonists and SSRIs. In this part of study, we focued on theinfluence of altered pCREB by5-HT1AR, major in hippocampal structural plasticity.In this part, we used H89, a PKA inhibitor, and Lentivirus packing CREB133dominant negative mutation (CREB133) to reduce CREB activity, at the same time,we used Forskolin, a PKA agonist, and Lentivirus packing CREB overexpressionfragment (VP16-CREB) to increase CREB activity. By this way, we could know whether CREB-mediated hippocampal structural plasticity involved in the mechanismof5-HT1AR regulate anxiety-associated behaviors. The results showed that8-OH-DPAT increased the expression of synaptogenesis-related peoteion, synapsinand spinophilin in vivo or in vitro. Meanwhile, the effects of8-OH-DPAT could beabolished by H89or CREB133. At the same time, NAN-190decreased the expressionof synapsin and spinophilin in vivo or in vitro. Meanwhile, Forskolin or VP16-CREBcan rescue the effects of NAN-190.We used5-bromo-2’-deoxyuridine (BrdU) to label the new-born neurons.8-OH-DPAT increased the neurogenesis in hippocampus, H89or CREB133abolishedthe effects of8-OH-DPAT. Meanwhile, NAN-190depressed the neurogenesis,Forskolin or VP16-CREB rescued the effects of NAN-190.By assessing anxiety-related behaviors using the novelty suppressed feeding,open-field, and elevated plus maze tests, we showed that intrahippocampal infusionof H89or CREB133can abolish the anxiolytic effects of8-OH-DPAT.Intrahippocampal microinjection of Forskolin or VP16-CREB rescued the anxiogeniceffects of NAN-190.In summary, the Part II studies illustrated that the pCREB-mediatedhippocampal structural plasticity involved in the regulation of5-HT1AR inanxiety-associated behaviors.In part III, we co-cultured GFP-labeled primary culture of hippocampal neuronsand differentiated neuronal stem cell (NSC), which simulated the newborn neuroninto hippocampal DG zone, to study whether5-HT1AR involved in the regulationof the synaptic contacts between the newborn neuron and original neuron and theimportance of CREB activity in it.The result showed that8-OH-DPAT up-regulated the synaptic contacts betweenthe newborn neuron and original neuron, and the effects could be abolished by H89orCREB133. Meanwhile, NAN-190down-regulated synaptic contacts between thenewborn neuron and original neuron, and Forskolin or VP16-CREB rescued theeffects of NAN-190.The result of Part III showed that CREB activity maybe involve in the building of5-HT1AR-mediated hippocampal circuitry between the newborn neuron andoriginal neuron.