Mechanistic Study Of Mu Opioid Receptors In Glutamatergic, γ-aminobutyric Acid Neurons And Astrocytes In Stress Analgesi

Stress can induce changes in the body’s pain response.The downregulation of pain response mediated by acute stress is called stress induced analgesia（SIA）,whereas the upregulation of pain response mediated by chronic stress is called stress-induced hyperalgesia（SIH）.In SIA,the pathway mediated by endogenousβ-endorphin（β-EP）binding to mu opioid receptors（MORs）is considered to be one of the important endogenous mechanisms of analgesia.Endogenousβ-EP mediated experimental SIA through MORs can be triggered by a variety of stress stimuli including bondage stress,intermittent plantar shock,social defeat,and forced swimming,etc.However,the exact cellular mechanisms of opioid-dependent SIA still remain unclear.Firstly,the occurrence of SIA requires the stress with a certain intensity and duration,but the stress intensity and duration required to trigger the release ofβ-EP are much lower than that to induce SIA,for example,some weak stress enhanceβ-EP level without accompany SIA.Secondly,in the central nervous system,MORs are expressed widely on glutamatergic neurons,gamma-aminobutyric acid（GABA）-ergic neurons and glias including astrocytes,but the regulation of MORs in these different types of cells on the pain response and their contributions to the whole effect of pain regulation under different stress intensity has not been clearly addressed.Therefore,in the present study,Oprm1 flox mice,total MORs knockout mice（MOR-/-）,and mice selectively deletion of MORs expressed on glutaminergic neurons(MOR_Glu-/-),GABAergic neurons(MOR_GABA-/-)and astrocytes(MOR_Astro-/-)were prepared,and various recombinant adeno-associated virus（r AAV）techniques containing Calcium signal monitoring,projection marking,chemical genetics and Cre-target gene knockout were used to study and discuss the above questions at morphological,behavioral,cellular and electrophysiological levels.The research results of this paper are as follows:1.In 32℃ warm water forced swim stress induced SIA model,the moderate（3min）swim exposure induced a MOR-dependent SIA,whereas transitory（1.5 min）swim exposure produced no analgesic effect and prolonged（6 min）swim exposure elicited a MOR-independent SIA.However,all of these three stresses enhancedβ-endorphin release.MOR_GABA-/-obviously attenuated analgesia and blocked the enhancement of activity of PAG-RVM neurons induced by moderate swim exposure.In contrast,under transitory swim exposure,MOR_Glu-/-oppositely elicited an analgesia behavior which was accompanied with strengthened activity of PAG-RVM neurons.These results indicated that MOR was involved in both transitory and moderate swim stress induced regulation of pain response.In addition,MORs expressed on glutaminergic neurons and GABAergic neurons had opposite regulating effects on SIA,and these antagonistic effects of these two neurons also changed in response to different stress intensity,that is,from balance between excitation and inhibition under transitory stress to biasing towards disinhibition under moderate stress.2.In intermittent foot shock stress induced SIA,both MORs expressed on GABAergic neurons and astrocytes had contributions to SIA.In addition,PAG was one of target brain area of abovementioned mode of action for MOR expressed on astrocytes.This were supported by 1)MORs were expressed on astrocytes of PAG to a certain degree and had the ability to activate astrocytes;2)c-fos protein could be expressed on PAG astrocytes after foot shock stress and SIA could be attenuated by inhibition of astrocytes’energy metabolism,which were mediated by MOR_Astro;3)The functional activity and the frequency of miniature excitatory postsynaptic currents（m EPSCs）of PAG-RVM projection neurons were enhanced in foot shock stress,which could be antagonized by the downregulation of the activity of astrocytes and the directional knock down MOR_Astroin PAG.These results suggested that PAG astrocytes were involved in the regulation of SIA mediated by MOR_Astro,and one of the mechanisms of this regulation was to enhance the excitatory synaptic inputs on PAG-RVM projection.In conclusion,MORs expressed on glutaminergic neurons,GABAergic neurons and astrocytes in the central nervous system are all involved in regulating the acute pain response.MORs expressed on GABAergic neurons and astrocytes mainly elicit analgesia,whereas MORs expressed on glutamate neurons play an anti-analgesic like effects.The contribution of MORs expressed on glutamatergic and GABAergic neurons to the regulation of pain response changes with stress intensity.In addition,the effects of MORs expressed on all of three types of neurons on pain response are processed through regulating activities of PAG-RVM projecting neurons.