Study On The Mechanism Of Morphine Conditioned Place Preference Mediated By Different Subregions Of Interstitial Nucleus Of The Posterior Limb Of The Anterior Commissure

Background and Objective:Morphine,a classic opioid drug,is one of the most potent analgesics in the clinic,yet it remains one of the most addictive drugs.Drug addiction represents a chronic recurrent encephalopathy characterized by excessive drug-seeking behavior,severe withdrawal symptoms upon cessation of drug use,and compulsive drug intake.The rewarding effects of opioid drugs are a key driving force behind addiction,as they create a sense of pleasure and motivate individuals to form specific memories associated with drug-taking environments.Once addicted,exposure to these environments can trigger the recall of these memories,leading to relapse.The conditioned place preference（CPP）model is a classic paradigm used to assess drug-related rewards.Previous research has shown that the interstitial nucleus of the posterior limb of the anterior commissure（IPAC）,a key brain region associated with opioid addiction,has neural fiber projections to the ventral tegmental area（VTA）and the shell of the nucleus accumbens（NAc Sh）,both of which are critical brain regions involved in addiction.The two subregions of IPAC may participate heterogeneously in the formation of the CPP model mediated by morphine.Therefore,exploring the neurophysiological mechanisms of IPAC’s involvement in morphine-induced CPP could further enhance our understanding of opioid addiction and offer new targets for clinical treatment and relapse prevention.The objective of our experimental study is to further examine the anatomical structure and boundaries of the IPAC brain region,determine the types of neurons that are activated or inhibited in the inner and outer subregions of the IPAC after morphine CPP,investigate the neural fiber projections from the IPAC to the VTA and NAc Sh and their intricate projection circuits,and verify whether these two neural circuits are involved in the rewarding effects of opioid addiction or the recall of drug-taking memories.Subjects and Methods:Adult male SD rats with body weight ranging from 220-320g were selected as experimental subjects.and the classic drug addiction model-morphine conditioned place preference model was selected.Through chemogenetic,optogenetics,stereotactic technique combined with viral for neural circuit tracing,whole cell patch clamp technique,Ca²⁺signal fiber photometry,immunofluorescence staining and FISH,the effects of IPACL and IPACM subregions on conditioned place preference model of morphine were studied,and two inhibitory circuit of VTA^GABA→IPACL^GABAand NAc Sh ^D1→IPACM^GABAmediated morphine conditioned place preference.Result:1.Immunofluorescence staining was used to distinguish between the medial（IPACM）and lateral（IPACL）boundaries of the IPAC as well as the border between the IPAC and the ventral pallidum（VP）,a brain region adjacent to the IPAC.2.Immunofluorescence staining of the immediate-early gene c-fos confirmed that following morphine-conditioned place preference（CPP）,GABAergic neurons in the IPACL region were activated while those in the IPACM region were inhibited.Calcium signal fiber recordings and chemogenetic results showed that activation of IPACL^GABAneurons mediated morphine CPP,while inhibition of IPACM^GABAneurons also mediated morphine CPP.3.Results from virus circuit tracing and immunofluorescence staining confirmed that IPACL receives projections from VTA^GABAneurons but not VTA^DAneurons,and that VTA^GABAneurons primarily project to IPACLGABA neurons.Meanwhile,IPACM receives projections from NAc Sh ^D1neurons but not NAc Sh ^D2neurons,and NAc Sh ^D1neurons primarily project to IPACM^GABAneurons.4.Results from virus tracing,electrophysiology patch clamp,calcium signal fiber recordings,and chemogenetic experiments confirmed that inhibition of the VTA^GABA→IPACL^GABAcircuit mediates the acquisition of morphine CPP,while activation of the NAc Sh ^D1→IPACM^GABAcircuit also mediates the acquisition of morphine CPP.5.Results from chemogenetic,optogenetic,sucrose preference,Morris water maze,and novel object recognition experiments revealed that the VTA^GABA→IPACL^GABAcircuit is involved in the rewarding effects of morphine in rats,but does not affect short-term or long-term memory.In contrast,the NAc Sh ^D1→IPACM^GABAcircuit does not affect the rewarding effects or short-term memory of morphine in rats,but is involved in the long-term environmental memory.Conclusion:Two intricate neural circuits,the VTA-IPAC and NAc Sh-IPAC,mediate opioid addiction through the interstitial nucleus of the posterior limb of the anterior commissure（IPAC）.Repeated morphine treatment induces disinhibition of the VTA ^GABA→IPACL^GABAcircuit,which mediates the rewarding effects of morphine.Additionally,repeated morphine treatment inhibits the excitability of the NAc Sh ^D1→IPACM^GABAcircuit,which promotes the formation of drug-taking memories and participates in the acquisition of morphine-conditioned place preference（CPP）.These two circuits work in tandem to mediate the formation of morphine CPP.