Circuits Mechanism Of Dopaminergic Neurons In Rewarding Memory Consolidation

Objective:Dopaminergic neurons(DANs)play an important role in cognitive function and the regulation of sleep and wakefulness,and the abnormality of dopamine(DA)system is considered to be an important factor causing cognitive and sleep disorders.However,the neural mechanism of how the DA system integrates cognition and sleep has not been fully decoded.Therefore,the purpose of this study is to analyze the specific types of DANs that bridge memory and sleep,carry out research on the relevant neural circuit mechanisms under physiological conditions,and use the hypoxic-ischemic brain damage(HIBD)model to carry out research on the molecular mechanisms related to cognition under pathological conditions.Methods:In this study,we take flies and mice as the research objects,and use neuroscience research methods such as genetics,immunohistochemistry,behavior and functional imaging to analyze the above problems.The specific method is as follows:1.Taken flies as the research object:1.1 At the cellular level,immunohistochemical methods were used to identify the projections and their interrelationships of specific DANs labelled by trans-Tango,Green Fluorescence Protein Reconstitution Across Synaptic Partners(GRASP)and Activity dependent GRASP(Active-GRASP)genetic tools in the brain;1.2 At the behavioral level,the classical olfactory reward conditioning paradigm and the sleep behavior research paradigm combined with the genetic tools of thermal activation/inhibition were used to detect the memory and sleep changes of specific DANs;1.3 In terms of function,the activity changes of DANs after training and memory consolidation were detected by in vitro functional calcium imaging;1.4 Using the new split-GAL4 genetic technology combined with immunohistochemistry and the classical olfactory reward conditioning paradigm to identify specific fine subtypes of DANs;1.5 Establishing the hypoxia model of flies during development to carry out relevant research under pathological conditions;1.6 The sleep behavior research paradigm and the classical olfactory reward conditioning paradigm were used to detect the changes of sleep and cognitive function of hypoxic flies in development stage.2.Taken mice as research objects:2.1 Using the improved Rice-Vannucci method to establish a mouse model of hypoxic-ischemic brain injury;2.2 Assessing the degree of damage after HIBD by gross observation of brain tissue,TTC staining and laser speckle cerebral blood flow imaging;2.3 Immunohistochemical method was used to identify the morphological changes and different brain regions after brain damage.Results:1.Under physiological conditions,protocerebral anterior medial(PAM)DANs related to the memory acquisition stage and DPM neurons related to the memory consolidation stage project to the horizontal lobe of the mushroom body,and the downstream of PAM-DANs is MB-α’β’ lobe,which is related to long-term memory.2.Regardless of the internal physiological state of flies(starvation/non starvation),activation of PAM-DANs can induce the generation of transient reward memory.However,starvation as a reward motive,under the condition of starvation,blocking PAM-DANs during the process of memory acquisition leads to 3-hour(h)short-term memory and 24 h long-term memory(LTM)impairment.Activation of PAM-DANs s or inhibition of DPM neurons in the memory consolidation stage both inhibit the formation of 24 h LTM.3.Under the condition of starvation,the activation of PAM-DANs reduces the total sleep during the day,resulting in short-term sleep fragmentation,and at this stage,flies are more likely to be awakened,inhibiting DPM neurons.The total sleep at night is significantly reduced,resulting in sleep fragmentation and easier to be awakened.4.After the classical olfactory reward conditioning paradigm training and 1 h after memory consolidation,the activity of PAM-DANs did not change significantly compared with the untrained group.5.By analyzing the projection and memory of PAM-DANs subtypes,it was found that activation of long-term memory PAM(LTM-PAM)DANs impaired 24 h memory formation.After further analysis,it was found that activation/blockade of PAM-α1 neurons,24 h memory formation was impaired,total sleep was reduced,sleep was fragmented,and it was easier to be awakened.6.The results of immunohistochemical staining after hypoxia in the development of flies showed that the inflammatory factor TNF-α increased.7.Under pathological conditions,flies after hypoxia show impairment of sleep and LTM formation.8.After HIBD,the cortex and hippocampus at right of mouse brain showed obvious infarction liquefaction necrosis.9.After HIBD,a large number of inflammatory cells infiltrated in the marginal area of the striatum(the projection area of the dopamine system),and a large number of neurons in the cortex and hippocampus died.Conclusion:1.Under physiological conditions,PAM-DANs not only participate in the process of memory acquisition,but also play a role in the process of memory consolidation,and affect the formation of LTM by interfering with sleep.In the first hour of the memory consolidation phase,PAM-DANs or their more specific subset are transiently activated,PAM-α1 neurons,which will damage 24 h LTM.Interestingly,transient activation of PAM-DANs or PAM-α1 neurons also lead to reduced and fragmented sleep.Structurally,we found that PAM-DANs form functional synapses with DPM neurons.Transient inhibition of DPM neurons results in a phenotype similar to that of PAM-DANs activation.2.Under pathological conditions,it was found that flies after hypoxia and mice after HIBD showed inflammatory response.Flies exposed to hypoxia during development exhibited sleep and memory disorders similar to activating/inhibiting specific DANs in adulthood,suggesting that DANs play a role in brain damage caused by hypoxia.After HIBD in mice,the areas related to the projection of DANs were damaged,including the striatum,cortex and hippocampus.A large number of neurons were necrotic,nuclear pyknosis,dissolution and fragmentation,and inflammatory cells increased.This suggests that the cognitive dysfunction and long-term behavioral abnormality of mice after brain injury may be related to the abnormality of DA system.