Neural Circuits And Mechanisms Underlying The Medial Septum Regulating Negative Emotions

Anxiety and fear are two common negative emotional states in our daily life.During evolution,these negative emotions emerged appropriately to facilitate animal avoiding danger or diminishing threat,which increase the survival probability for individuals.However,abnormal negative emotions(persistent or overwhelming)always accompany with abnormal behavioral and physical responses,including improper defense strategies,disrupted hormone secretion,and unbalanced body homeostasis.Thus,these emotion disorders impact negatively on individual’s life and survival,and cause significant financial burden on the family and whole society.In this situation,there is an urgent need of comprehensive and systematic research to explore the mechanisms underlying these emotional abnormality.Many efforts had been made by researchers around the world to figure out the neural substrates of negative emotion occurring and maintaining.Previous results indicated that the medial septum(MS)involved in the regulation of these negative emotions,but the certain neuronal circuits and involved mechanisms remain unclear.In this study,we developed a paradigm to induce a fear state by direct predator information inputs,and then we combined optogenetics,in vivo photometry recording,molecular methods and electrophysiological techniques,to identify the specific subtypes of MS neurons and related neural circuit responding to fear information inputs of predator stressor,and explore the electrophysiological mechanism underlying the regulation of negative emotions like fear by MS.First of all,we developed a new polymer optical fiber and applied it in the optogenetic manipulation of animals in vivo.Due to its low mechanical and biological compatibility with brain tissue,long-term implantation of traditional silica optical fiber in brain caused tissue damage and inflammation,thus impaired the effectiveness of optogenetic manipulation.To address inflammation and mechanical incompatibility in long-term optogenetic stimulation via traditional fiber,a flexible polymer optical fiber(POF)with low optical loss and mechanically compatible with biological tissues has been developed and test.A series of studies have been made to confirm the photoconductivity of POF and its efficacy in the optogenetic manipulation of central nervous system.Compared with silica optical fiber,results showed that neuronal damage and astrocyte activation evoked by POF implantation is fairly milder,which verifies the excellent histocompatibility of POF.These findings imply that the POF fibers with large deformation,low optical loss,and mechanical compatibility is suitable for long-term optogenetics experiments on neural tissues in vivo,and may provide new tool for the study of mental disorders treatment.Secondly,we have identified that the medial septal nucleus and its neural circuitry involved in the regulation of negative emotions induced by predator information inputs.By exposing mice to predator,and then collecting and analyzing behavioral and physiological readouts,we have proved that predator stressor can induce fear emotional states in mice,decrease travelling distance and increase immobility,which accompanying with higher heart rate and corticosterone level,and anxiety-like behavior.Furthermore,these predator information inputs increased the c-fos signal in MS neurons,and Cholinergic neurons(ChAT)in MS were activated,but Vesicular Glutamate Transporter 2-expressing neurons(VGlut2)were inhibited,GABAergic neurons(Parvalbumin,PV)showed no obvious difference.Optogenetics experiemnts on MS suggested that both Cholinergic and glutamatergic neurons can bi-directionally regulate behavioral changes in response to the predator information,but these two subtypes showed mutually exclusive modulatory effects.Viral tracing data verified the structural connections specifically from MS to RN(Red nucleus,RN).Light stimulation of projection of MS ChAT or VGlut2 neurons to RN elicited same effects with manipulating their soma.in vivo photometry recording results demonstrated that calcium signal of cholinergic neurons increased(amplitude and frequency)when the mice contact with a predator but reduced when they switched from locomotion to immobility,indicating that MS ChAT neurons involved in response to the predator information input.In contrast,calcium signal of glutamatergic neurons displayed opposite changed compared with ChAT neurons during predator contacting.Combined with behavioral tests of optogenetic modulation,our results suggested that VGlut2 neurons were inhibited by the input of predator information.Finally,we have also explored the neural circuit underlying the regulation of predator fear by MS.To mimic the predator fearful scene in environment,a predator threat test(PTT)experimental paradigm was adopted.c-fos staining indicated that MS were activated after predator threat,co-stained with other markers demonstrated that cholinergic neurons are activated more significantly after threat.Result of optogenetic manipulation and behavior tests suggested that mice displayed anxiety-like behavior when activating MS ChAT neurons,and entered the danger zone for shorter periods of time and more frequently;while activation of MS ChAT neurons caused opposite effects.Activating PV neurons caused no obvious change in anxiety-like behavior.These behavior tests suggested the different function of these neuronal subtypes in MS in regulating predator threat response.Antero-and retrograde viral tracing studies revealed the projection of the MS-MHb(Medial habenula,MHb),optogenetic manipulation of MS-MHb circuit indicated this circuit is crucial in regulating the behavioral responses to predator-induced fear.In conclusion,this study has mainly shed light on the neural circuits and mechanisms underlying MS regulating negative emotions like fear and anxiety,which provide new perspective for understanding the mechanisms underlying fear or anxiety disorder,as well as potential targets and methods for clinical interventions of negative emotion disorders.