Analgesic Effect And Mechanism Of Ventrolateral Midbrain Aqueductal Gray Matter-bed Nucleus Of Stria Terminalis Circuit

Object:To clarify the anatomical basis of the new neural circuit of the ventrolateral mesencephalic aqueductal gray matter-bed nucleus of stria terminalis,plumb the mechanism of the neural circuit involved in analgesia,further analyze the types and mechanisms of neurons involved in the regulation of food-related pain threshold,and clarify the neurobiological mechanism of the new neural circuit of the ventrolateral mesencephalic aqueductal gray matter-bed nucleus of stria terminalis specifically involved in the regulation of food-related pain threshold.Methods:1.Determine the anatomical connection of the ventrolateral periaqueductal gray（vl PAG）-bed nucleus of stria terminalis（BNST）circuit:through virus loop tracing:inject r AAV-EF1α-DIO-EYFP into the vl PAG nucleus using vglut2-cre transgenic mice through brain stereotactic injection technology,after 21days of complete expression of the virus,take the whole brain section of the mouse brain,and observe the projection of the vl PAG glutamate neuron(vl PAG^{v Glu2+})-BNST neural circuit.2.Using photogenetics to manipulate vl PAG ^{v Glu2+}-BNST neural circuit to clarify its analgesic effect:C57BL/6 mice were randomly divided into control group（mcherry）,experimental excitation group（chr2）,and experimental inhibition group（nphr）.r AAV-VGLUT2-CRE-WPRE-h GHPA（Retro）was injected into BNST,and r AAV-EF1α-DIO-mcherry/chr2/nphr-WPRE-h GHPA（AAV9）was injected into vl PAG in turn,after 21 days of virus expression,embed optical fiber in BNST.Under normal physiological conditions and 24 hours of food deprivation in mice,photogenetics was used to control the vl PAG ^{v Glu2+}-BNST neural circuit,and the pain behavior was tested by von Frey wire,hot plate,and controlled electric shock test box,and the unprompted locomotion and anxiety of mice were tested by open-field experiment.3.To identify the type of neurons in the downstream nucleus of the vl PAG ^{v Glu2+}-BNST nerve circuit:activate the upstream nucleus vl PAG ^{v Glu2+}using the photogenetic technology,and write down the activity of the downstream BNST（excitatory glutamate and inhibitory GABA）using the calcium imaging technology;The mice were electrocuted by different intensities in a controlled shock test box,and the activity of BNST glutamate neurons and GABA neurons in the downstream nuclei were noted by optical fiber calcium signal recording system;Further verify the neuronal type of this loop by using the anterograde transmonosynaptic virus strategy.Results:1.The results of virus tracing showed that the fluorescence labeling of the upstream vl PAG glutamate neurons was clear,and the fluorescence labeling of the downstream BNST fibers was clear.2.Photogenetic activation of vl PAG ^{v Glu2+}-BNST neural circuit increased the pain threshold of mice:von Frey wire and hot plate detection of mechanical pain and thermal pain threshold displayed that after photogenetic manipulation,compared with the control group（mcherry）,experimental excitation（chr2）after this circuit,the mechanical pain threshold and thermal pain threshold of thermal pain increased（p<0.05）;In the experimental excitation group（chr2）,the threshold of mechanical pain and thermal pain after the photogenetic activation（chr2-light-on）increased compared with that before the photogenetic activation（chr2-light-off）（p<0.05）.The results of controlled electric shock test box showed that the number of times,time and food intake of mice in the chr2 group to explore the electric shock and food areas were increased compared with those in the mcherry group when they were given electric shock and food at the same time after photogenetic manipulation（p<0.05）;After photogenetic manipulation,only food was given,After 24hours of food deprivation,compared with mcherry group,the number,time and amount of food intake in chr2 group decreased（p<0.05）.The field experiment showed that the spontaneous activity path of the experimental inhibition group（nphr）increased after the photogenetic activation（nphr-light-on）compared with that before the photogenetic activation（nphr-light-off）（p<0.05）.3.Photogenetics activates vl PAG glutamate neurons,downstream BNST can record the signals of glutamate and GABAergic neurons,indicating that glutamate nerve cells andγ-Aminobutyric acid nerve cells of BNST all receive the signal input of glutamate nerve cells in vl PAG nucleus.but only BNST glutamate neurons are activated under electric shock pain stimulation;the anterograde transmonosynaptic virus tracing shows that the vl PAG glutamate neurons project to the BNST glutamate neurons.Conclusion:Activation of vl PAG ^{v Glu2+}-BNST ^{v Glu2+}circuit,which can specifically raise the pain threshold associated with food intake.