The Anxiolytic Effects Of Motilin In Basolateral Amygdala

BackgroundStatistical research reported that, in2010, there were273million more people sufferingfrom anxiety disorders all over the world, which accounted for4.5%of the totalpopulation. And the incidence of women was higher than that of men, respectively5.2%and2.8%. In Europe, Africa and Asia, the lifetime prevalence rate was about9-16%, andthe annual prevalence rate was between4-7%, while the incidence was significantlyhigher in the United States. It was reported that the lifetime prevalence rate was about29%, and11-18%adults in his life had experienced this state.A body of evidence has suggested that anxiety disorders are mainly caused by thefollowing factors: drugs, psychological factors, stress, persistent anxiety, genetic andfamilial factors, cultural background, and abnormal neurotransmission in the neuralcircuits. The interaction of one or more factors of them may cause the different types ofanxiety disorders.A focused effort to study the neurobiological basis of anxiety disorders has led togreater understanding of important neurophysiological and neurochemical abnormalitiesrelated to these disorders, but the underlying mechanisms of molecular biology remain unclear. In addition to a variety of neurotransmitters involved, researchers also found theneural networks related to anxiety, and hypothesized that the abnormal neurotransmissionin the neural circuits was the root cause of anxiety disorders. In view of this, somescholars have proposed a cell model of anxiety disorders. They believed that pathologicalanxiety is due to the imbalance of excitation and inhibition in neural circuits, with theamygdala playing a central role. They said that" If all molecules that play a role in anxietyare integrated into the same picture, we believe that every neurotransmitter andneuromodulator system or intracellular signaling protein could contribute to the balance ofexcitation and inhibition, thereby exerting their effects on anxiety responses. The locationof these targets could be in any nuclei in the neural circuits underlying anxiety."Clinical treatments of anxiety disorders include lifestyle change, psychotherapy anddrug therapy. Drug therapy is recommended only if other treatments are ineffective.Cognitive behavioral therapy (CBT) is the most effective psychological treatment, whichhas two major components: cognition and behaviour. The drug of current clinicaltreatment of anxiety disorders are as follows: selective5-HT reuptake inhibitors (SSRIs),serotonin-norepinephrine reuptake inhibitor (SNRIs), benzodiazepines (BDZs),monoamine oxidase inhibitors and-calcium channel anticonvulsants. Because of theserious side effects, such as work slowly, poor compliance, dependence, and possiblewithdrawal reactions, researchers are trying to find new treatments and molecular targets,one of which are neuropeptides. The types of neuropeptides involved in regulating anxietyare increasing. So far, the more extensive studies have focused on corticotropin-releasinghormone (CRF), P substance (SP), vasopressin (VP), neuropeptide Y (NPY), other typessuch as galanin, oxytocin, motilin, also received more attention in recent years.Motilin is a hormone released by the endocrine cells of the duodenal mucosa duringfasting to stimulate gastrointestinal motility, and distributes in the gastrointestinal tract andcentral nervous system (CNS). Motilin receptor (MTLR) is one of G protein-coupledreceptors, and erythromycin (EM) is its first discovered non-peptide agonists. It has beenreported that in addition to parenteral, MTLR also was expressed in fear andanxiety-related brain regions, such as amygdala, hippocampus, hypothalamus and so on. In CNS, amygdala is the core area in emotional control, especially in anxiety and fearregulation.Some evidence has suggested that microinjection of motilin into basomedial amygdaladose-dependently increases stomach contractions in conscious rats, and intraventricularinjection of motilin has significantly anxiolytic effects. Moreover, it was reported that alarge number of MTLR expressed in the basolateral amygdala (BLA). Based on the above,we hypothesized that motilin and its receptor not only controls gastrointestinal motility inamygdala, but also may play an important role in regulating anxiety behaviour.AimsTo investigate the effects of basolateral amygdala MTLR activation onanxiety-likebehaviours caused by different factors in mice, as well as exammine theexpression differences of MTLR in different neurons of amygdala, and then to reveal thepossible molecular mechanism by molecular biology and electrophysiology, and all ofthese will provide a new target for the development of anxiolytic drugs and theoreticalbasis for the treatment of anxiety disorders.Methods1. Two kinds of stresses were adopted in our experiments.1)Forced-swimming stress:mice were placed in individual bottles for forced swimming for30min one or twotimes, and allowed to swim once a day, then anxiety-likebehaviours were tested atdifferent time points to choose the best parameters for the forced swimming-inducedanxiety-like behaviours;2) Restraint stress: mice were placed in a restraint device for2h one or2times, and only allowed to restrain once a day, thenanxiety-likebehaviours were tested at different time points to choose the bestparameters for restraint-induced anxiety-like behaviours.2.10μl CFA was injected into plantar of mice and then anxiety-likebehaviours weretested at different time points to choose the best parameters for chronic inflammatorypain-induced anxiety-like behaviours.3. Anxiety-related behavioural tests 1) Elevated plus maze (EPM): Mice were allowed to habituate to the testing room for2d before the test, and pretreated with gentle handling two times per day to eliminatetheir nervousness. For each test, individual animals were placed in the center square,facing an open arm, and allowed to move freely for5min. Mice were videotapedusing a camera fixed above the maze and analyzed with a videotracking system. Thenumber of open and closed arm entries (all four paws in one arm) and time spent ineach arm were recorded.2) Open field test: Mice were placed in the center of the box and were videotapedusing a camera fixed above the floor and analyzed with a video-tracking system. The“center” field is defined as the central15×15cm2area of the open field, one fourth ofthe total area. Each subject was placed in the center of the open field, and its activitywas measured for15min. Times in the center areas and total distance travelled wererecorded.4. Motilin concentration in amygdala was tested by ELISA, and the samples were fromthe anxious mice induced by forced swimming stress or chronic inflammatory pain.5. MTLR expression in amygdala was detected by Western blot, and the samples werefrom the anxious mice induced by the stresses or chronic inflammatory pain. Theeffects of EM and motilin on MTLR expression were also tested.6. Amygdala Cannulation and Microinjection1) Surgery: Two stainless steel cannulas were bilaterally aimed at0.5mm above theintended sites of injection, namely the BLA (1.45mm anterior to the bregma,±3.5mm lateral from the midline, and4.5mm beneath the surface of the skull). The guidecannulae were affixed to the skull with machine screws and dental cement, and styletswere inserted into the cannulae to keep them patent. After1week recovery, the micereceived microinjection.2) Microinjection into BLA: The animals firstly were placed individually in aninduction chamber, and were anesthetized with2.5%isoflurane in100%oxygen witha delivery rate of0.5l/min until loss of righting reflex. Anesthesia was thenmaintained with1.5%isoflurane in100%oxygen with a flow of0.5l/min delivered by face mask. Drugs or saline were bilaterally delivered at0.5μl/min using a syringedriven by an infusion pump. The drugs and saline were injected over a1min periodwith the injector left in place for an additional1min to permit the diffusion.30minlater, mice suffered from forced swimming and injected with CFA receivedbehavioural tests.7. Dual immunohistochemical analyses of MTLR and the GABAergic inhibitory neuronmarker GAD67in the amygdala were preformed to detect the cell-specificdistribution.8. The effects of MTLR activation on neuronal excitability and excitatory and inhibitoryneurotransmission were tested by whole-cell patch clamp recording.Results1. Behavoural tests suggested that24h after one restraint stress, mice had increasedanxiety-likebehaviours were increased in the mice, which were more significant24hafter the two restraint stresses; mice with one or two forced swimming stresses spentless time in the open arms and the center area in the EPM and open field tests, and theanxiety-likebehaviors were more significant in the mice tested24h after the two stresses.;14dafter CFA injection mice expressed increased anxiety-likebehaviours, which at leastlasted two weeks.2. Western blot analysises showed that MTLR expression in amygdala significantlydecreased with the times of restraint stress and forced swimming stress, and its levelwas also significantly reduced14d after CFA injection. ELISA assay showedsignificantly reduction of motilin concentration of amygdala at the corresponding timepoint. Because the restraint devices likely cause the cannulas to fall off, so thefollowing experiment used forced swimming stress and chronic inflammatorypain-induced models.3. Behavior tests after local injection into BLA showed that microinjection of motilinand EM significantly reduced the time mice spending in the open arms and in the central area respectively in EPM and open-field tests, which experienced the forcedswim stress or chronic inflammatory pain, while the number of entries into the closedarm or total distance travelled were not affected. Meantimem, MTLR selectiveantagonist MA-2029reversed the effects of EM.4. Immunofluorescence showed that the amygdala MTLR+neurons were colocalizedwith GAD67+interneuron in amygdala, and the number of MTLR+neurons in theBLA was significantly more than that in lateral amygdala.5. MTLR activation in pyramidal neurons of BLA did not affect the firing rate of actionpotentials and resting membrane potential (RMP). But MTLR activation ininterneurons significantly increased firing rate of action potentials, and reduced itsRMP. In addition, MA-2029and G protein inhibitor GDP-β-S both blocked the effectsof MTLR receptor activation on neuronal excitability.6. Forced swimming stress and chronic inflammatory pain statistically decreased theamplitude and frequency of GABAergic sIPSCs in BLA pyramidal neurons. Thenbath application of EM increased the amplitude and frequency of sIPSCs both incontrol and model mice, and this effect was inhibited by MA-2029. However, MTLRactivation did not affect GABAergic mIPSCs. In addition, motilin significantlyreduced paired-pulse ratio (PPR) in BLA pyramidal neurons, and this effect also wasblocked by MA-2029.7. MTLR activation did not affect the amplitude and the frequency of sEPSCs ormEPSCs in BLA pyramidal neurons.8.14d after CFA injection, the thresholds of thermalgesia and tactile pain in theipsilateral hind paws of injection were significantly lower, compared with that in thesaline injection group. However, local injection of EM or motilin had no effect on thepain threshold.ConclusionsThis study has demonstrated that MTLR receptor selectively expresses in interneuronsof amygdala; both motilin concentration and MTLR expression decrease significantly in mice suffering from restraint stress, forced swim stress and chronic inflammatory pain;MTLR agonists, motilin and EM, have significant anxiolytic effects after microinjectioninto BLA of amygdala; the possible mechanisms are that MTLR activation selectiveincreases neuronal excitability of interneurons in BLA and enhances GABAergicneurotransmission, which is dependent on action potential.Our results reveal the electrophysiological basis of anxiolytic effects of motilin for thefirst time. These findings suggest that motilin receptor is a new target for development ofanxiolytic drugs, and provide the theoretical basis of the anxiolytic effects based onmotilin receptor agonists.