Involvement Of Locus Coeruleus-hippocampus TH⁺ Projection In The Surgical Incision Pain-induced Memory Consolidation Enhancement In Mice

Background:Clinical evidence shows that 20%of patients have post-traumatic stress syndrome（PTSD）after surgery,but the exact mechanism of PTSD after surgery is still unclear.The brain is the center of public to deal with pain and memory.Studies have shown that there are some common neural circuits or molecular mechanisms between pain and cognition,and they may interact with each other,causing changes in cognition after pain.In addition,previous studies show that acute postoperative pain is an independent risk factor for PTSD.Therefore,does acute pain after surgery enhance preoperative malignant memory and contribute to the development of postoperative PTSD?If the above conjectures is correct,then what is the neural circuits or molecular mechanisms of memory enhancement caused by acute pain?Clarifying these issues has a great significance to explain or preventing the occurrence of PTSD after surgery.It is well known that the occurrence of PTSD is closely related to the enhancement of memory consolidation.Memory consolidation is the process of stabilizing short-term memory to long-term memory which depends on changes in neural activity and synaptic plasticity.Surgical incision pain,as a common acute postoperative pain,its effect on memory consolidation of preoperative adverse events is largely unknown.Moreover,preoperative patients often experience traumatic events or have some unpleasant treatment experiences,while surgery-induced pain often occurs during the memory consolidation phase of these events or unpleasant treatment experiences,so exploring the effect of postoperative acute pain on memory consolidation may help explain why acute postoperative pain induces PTSD.The locus coeruleus（LC）,located in the dorsal pons,is involved in regulating various physiological functions,including arousal,memory,cognition and pain.LC is the main source of norepinephrine（NE）and dopamine（DA）in the central nervous system.Recent studies have shown that norepinephrine and dopamine released by TH⁺（lysine hydroxylase）neurons projected from locus coeruleus to hippocampus are key transmitters that depend on the formation of hippocampal memory.Behavioral studies have confirmed that the activation of dopamine receptors 1 and 5（D1/D5）and norepinephrine receptors in hippocampus not only contributes to memory coding,but also is necessary to transform short-term memory into long-term memory dependent on protein synthesis.In addition,the activation ofβ-adrenoceptors and D1/D5 receptor is indispensable for maintaining long-term potentiation（LTP）of hippocampal CA1.Studies have demonstrated that acute stress increased the activity of neurons in the locus coeruleus.Therefore,surgical incision pain,as a common stress stimulus after operation,may activate the locus coeruleus-hippocampal TH⁺projection,release norepinephrine and dopamine in the hippocampus,and enhance memory consolidation.Based on the above background,the purpose of this study is to observe the effect of surgical incision pain on emotional memory and non-emotional memory consolidation,and to explore the possible role of the LC-Hip pathway in the effect of surgical incision pain on memory consolidation.Objective:1.To explore the influence of surgical incision pain on emotional memory and non-emotional memory consolidation.2.To explore the role of locus coeruleus-hippocampus TH⁺projection in the influence of surgical incision pain on the consolidation of emotional memory and non-emotional memory.3.To explore the role of norepinephrine and dopamine released after activation of locus coeruleus-hippocampus TH⁺projection in the consolidation of emotional memory and non-emotional memory induced by surgical incision pain.Methods:1.The effect of surgical incision pain on emotional memory and non-emotional memory consolidation1.1 After C57BL/6 mice acquired certain emotional and non-emotional memory through inhibitory avoidance and novel object recognition training,we observed the influence of surgical incision pain on the consolidation of emotional and non-emotional memory in mice.1.2 Using the open field test,the exercise ability and anxiety of the mice were detected 24 hours after the surgical incision pain,and the possible influence of the surgical incision pain on the movement and anxiety of the mice was excluded.1.3 After 6 hours（memory consolidation window period）of inhibitory avoidance and new object recognition training,the pain model of the surgical incision was made,and the memory test was performed 18 hours after the model was established to exclude the possible influence of pain on memory retrieval.2.The role of locus coeruleus-hippocampus TH⁺projection in the consolidation of emotional memory and non-emotional memory affected by surgical incision pain.2.1 Using virus tracer technology to verify the existence of TH⁺projection from LC to Hip in TH-Cre mice2.2 Immunofluorescence method was used to observe the effect of surgical incision pain on c-Fos of TH⁺neuron in LC nucleus.At the same time,retrograde tracer virus was injected into CA1 area of hippocampus,and the changes of discharge frequency of TH⁺neurons from locus coeruleus to the hippocampus were observed by electrophysiology.2.3 The chemogenetic viruses r AAV-EF1a-DIO-h M3D（Gq）-m Cherry and r AAV-EF1a-DIO-h M4D（Gi）-m Cherry were injected into the locus coeruleus nuclei,and the hippocampal CA1 region was injected CNO to regulates the activity of TH⁺neurons projecting from LC to Hip,and observes the memory performance of mice in inhibitory avoidance and novel object recognition experiments.Finally,theβ-adrenoceptors blocker propranolol（3.125μg/0.5μl/side）and the dopamine receptor blocker SCH23390（0.5μg/0.5μl/side）were micro-injected into the hippocampus CA1,to find out which mediators released by the TH⁺projection of LC-Hip mediated the enhancement effect of surgical incision pain on memory consolidation.Results:1.Surgical incision pain enhances memory consolidation of emotional and non-emotional memory in miceAnimals in surgery incision pain group and control group had a median retention latency of 392.5S（interquartile range:255.5-600S）and 187.0S（interquartile range:142.8-600S）,respectively.In the novel object recognition experiment,the novel object preference index of the control group was 20.5±8.1%while the object preference index of the surgical incision pain group was 36.1±12.7%.Compared with the control group,the surgical incision pain significantly increased the retention latency and the object recognition preference index（P<0.05,n=14）.In addition,surgical incision pain had no significant effect on memory retrieval,voluntary movement,and anxiety in mice（P>0.05,n=14）.2.The activation of LC-Hip TH⁺projection mediate the enhancement of emotional memory and non-emotional memory consolidation after surgical incision painThe retention latency of the chemogenetic h M4D（Gi）-m Cherry virus injection mice was 218.0S（interquartile range:177.5-345.0S）,and the novel object recognition index was 26.75±9.77%.In contrast,the median retention latency latency of the pain group was 410.0S（interquartile range:282.5-600S）,and the object preference index for novel object recognition was 40.64±13.52%.Compared with the pain group,the retention latency and novel object preference index were significantly reduced in the chemogenetic h M4D（Gi）-m Cherry virus injection group（P<0.05,n=14）.Similarly,the retention latency of h M3D（Gq）-m Cherry virus injection mice was448.5S（interquartile range:315.5-600S）,and the novel object recognition index was36.29±10.08%.The retention latency of control mice was 196.5S（interquartile range:120.8-581S）,and the novel object recognition index was 22.46±6.65%.Compared with the control group,h M3D（Gq）-m Cherry injection group significantly improved emotional and non-emotional memory（P<0.05,n=14）.In addition,the retention latency of mice in the pain group injected with h M3D（Gq）-m Cherry virus was 414.5S（interquartile range:272.5-600.0S）,and the new object recognition index was37.16±7.60%.Activation of locus coeruleus-hippocampal TH⁺projections by injection of r AAV-EF1a-DIO-h M3D（Gq）-m Cherry virus did not enhance memory consolidation after surgical incision pain compared with incision pain group mice.（P>0.05,n=14）.3.Hippocampal CA1 dopamine receptors mediate emotional and non-emotional memory consolidation enhancement after surgical incision painMicroinjection of dopamine D1/5 receptor antagonist SCH23390（0.5μg/0.5μl/side）in bilateral hippocampal CA1 area immediately after inhibitory avoidance and novel object recognition training reduced the retention latency and novel object recognition preference（P<0.05,compared with the surgical incision pain group,n=14）.However,compared with the surgical incision pain group,the injection of propranolol（3.125μg/0.5μl/side）,aβ-adrenoceptors blocker,into the hippocampal CA 1 area had no significant effect on the retention latency and the object recognition preference index.（P>0.05,n=14）Conclusion:Surgical incision pain enhances the memory consolidation of emotional memory and non-emotional memory in mice.The release of dopamine after the activation of TH+projection from locus coeruleus-hippocampus may be one of the mechanisms of memory consolidation enhancement induced by surgical incision pain.