Mechanism Of Eph-ephrin Receptors Mediated Neural Circuit Development For Early Emotion

Emotion and memory related neural circuits are formed through long-time developing period.Innate emotional response to environmental stimuli is a fundamental brain function that is controlled by these specific neural circuits.Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia.Amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to threat stimuli.During the postnatal development,EphB(Erythropoietin-producing human hepatocellular receptors subtype B)tyrosine receptor kinases and their ligand ephrin-B mediated initiates nerve wiring and synapse formation upon axon-dendrite/cell contact which allows neural circuit formation to support brain function.Moreover,Ephs and ephrin-Bs continue to express highly at synapses and modulate emotion,learning and memory through interacting with NMDA receptor.The presented studies are mainly focused on development and function of neural circuit by using a series of Eph/ephrin knockout/knockin mice combined with morphology,electrophysiology and behavioral test to clarify the precise roles of EphB and ephrin in developing and adult brain.The study should lead to an increased understanding of new mechanisms for neural circuit formation.Firstly,we set up an early behavioral paradigm of innate fear to identify the timing of innate fear onset during development period.Using either a testing trial with an elevated plus maze(EPM)or an exposing trial to a predator odour TMT during postnatal development(postnatal days 9,12,16,20),innate defensive responses can be triggered in juvenile and c-Fos positive cells are observed in several brain regions.The result showed that c-Fos activation in basolateral amygdala(BLA)was dramatically increased starting from postnatal day(P16).To determine the neuronal properties of BLA area responsible for the defensive behavioral elicitation,we examined the density of mushroom spines along the dendrites of BLA neurons using the Thyl-GFP-M transgenic mouse reporter line and investigated for potential correlation between active response and morphogenesis.We found that two factors became significantly correlated in either EPM or TMT paradigm at P16 solely but not at P12 or P20 after the innate response was elicited.The study clarified the intrinsic properties of amygdala neurons that were critical for generating of innate fear behavior,and strongly support the presumption that neuronal development circuits assembly modulate innate emotional behavior specially which is distinct from cognitive modulation.Secondly,Efnb3-/-mutant mice were subjected to EPM,and significant longer exploring time and more entries in open-arm of the maze,reflecting a defective defensive response,were clearly observed from P16 till P42,compared to the WT littermates.We used confocal and two-photon images to identify long projecting axons and observed a bundle of downward projecting fibers starting from the ipsilateral edge of the hippocampus and extending into the amygdala.We found that in Efnb3-/-mutant the nerve fibers in the more distal region of the bundle as the axon terminals enter the amygdala were loosely packed and not well-wired with BLA neurons in contrast to the tightly and neatly fasciculated bundles observed in wild-type mice.Meanwhile,Efnb3-/-mice showed significantly reduced correlation of spine density and c-Fos activation in BLA.Therefore,to examine if the cytoplasmic domain of ephrin-B3 is required for innate defensive responses,Efnb3lacZ/lacZ mice were subjected to EPM.These ephrin-B3 mutant mice express a truncated ephrin-B-?-gal fusion protein that retains the extracellular and transmembrane domains to provide ligand like activity but are unable to transducer reverse signals.Unlike Efnb3-/-knockout,no difference was observed compared to the WT littermates.This genetic data indicates that ephrin-B3 is required as a ligand to promote nerve wiring and the behavior elicitation.To further verify the temporal and spatial specificity of Ephrin-B3 for defensive responses,we utilized tamoxifen injection and an injected AAV2-NLS-Cre induced rescue strategy in the Efnb3-/-mice,which could improve the defect of behavior and morphology in development period.Taken together,we find ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala.The morphological and behavioral abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated.These results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period.Finally,we found that glutamatergic neurons were activated in amygdala when the innate fear behavior are evoked in juvenile mice.Loss of EphB2,a tyrosine receptor kinase expressed in amygdala neurons,suppressed the reactions and led to defects in spine morphogenesis and fear behaviors.We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2.To determine brain nucleus specificity of EphB2 effects in BLA for fear behavior and spinogenesis,we over-expressed EphB2 protein through injecting an adeno-associated virus(AAV2)encoding EphB2(AAV2-HA-EphB2)into the amygdala of EphB2-/-mice at P0-P1 and found that the deficit was rescued.The mice exhibited normal innate defensive behavior and neuronal morphology.Furthermore,constitutively active form of EphB2(F620D mutant)was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3.These data suggested that kinase-dependent EphB2 intracellular signaling play a major role for innate fear responses in the critical developing period,in which functional spinogenesis in amygdala was involved.In summary,in the study we firstly set up an early behavioral paradigm of innate fear and identify the timing of innate fear onset is postnatal day 16.Meanwhile,a specific population of spine morphogenesis in amygdala that was coupled to neuronal reaction to aversive cue.Furthermore,we identify a class of molecules specifically expressed in the synapse,EphB2/ephrin-B3,that play a critical role for early innate fear behavior.This study reveals for the first time that ephrin-B3 functions as a ligand in hippocampus to integrate pre-and post-synaptic structures and thereby coordinates axon targeting,spinogenesis,and neuronal reactions in the amygdala.Moreover,we identify that EphB2 as the receptor expressed specifically in the glutamatergic neurons of amygdala during postnatal developing period and is required for fear initiation in juvenile mice.