| BackgroundAs a severe neuropsychiatric disease,major depressive disorder(MDD)has become a leading cause of disability with more than 264 million sufferers around the world.The mechanisms and pathogenesis of depression are complicated.Depression is also viewed as ‘circuitopathy’ as a great deal of brain areas such as prefrontal cortex,amygdala,nucleus accumbens,hippocampus,lateral habenula and ventral tegmental area,etc.and related neural circuits are implicated in it.Even though a lot of neural circuits have been involved in modulation of depression-like behaviors,the networks were not intact and the functions of these circuits are highly heterogeneous.For example,some of these circuits can both regulating anxiety-like and depression-like phenotypes but the others can work only on part of these symptoms.Some neural circuits positively promote antidepression-like behaviors and others modulate depression-like phenotypes negatively.The heterogeneity of networks impeded us to further understand the pathogenesis of depression and to develop efficient therapies.Therefore,uncovering new neural nodes and circuits which can modulate depression can greatly benefit uncovering the mechanisms and treatments of depression.Hippocampus plays a crucial role in regulating depression.Some studies have demonstrated that there are some memory engram cells encoding emotion-related memory.The changes in activities of these engram cells could modulate depression-like behaviors.Adult neurogenesis in dentate gyrus also plays a pivotal role in modulation of depression.Furthermore,the elevated activity of pyramidal neurons in CA1 is antidepressant.The entorhinal cortex(Ent)is commonly viewed as a key node linking the hippocampus to certain neocortical and subcortical areas.However,the role of Ent in depression is not fully known.Clinical and preclinical evidences suggest that abnormal Ent is implicated in pathogenesis of depression.For example,the Ent of MDD patients or rodent models of depression has reduced volume,decreased functional connectivity with other brain region(such as medial orbitofrontal cortex),diminished neural activity and abnormal expression of molecules.However,it remains unclear how the Ent modulates core symptoms of depression.It is shown that activation of Ent II-DG(dentate gyrus)pathway,via targeted knockdown of TRIP8 b in Ent II or enhancing neural activity using chemogenetic activation,increases hippocampal neurogenesis and promotes antidepression in mice.But it is unknown whether the deep layers of Ent and related outputs are involved in modulating depression.ObjectiveIn this study,we aimed to test whether Ent Va and one of its main neocortical outputs to medial part of the secondary visual cortex(Ent→V2M pathway)are involved in depression and if so,what the underlying mechanism is.Methods(1)To understand the distribution of the axonal projections of Ent Va neurons in bilateral dorsal neocortex and the specificity of the main projection pathways,anterograde and retrograde AAV tracing,fluorescent and confocal imaging and quantitative measurement were applied.(2)Next,we applied 21-day and 10-day chronic social defeat stress(CSDS)paradigms to produce mouse model of depression.The behavioral phenotypes of these two paradigms were compared systematically.Then c-Fos immunostaining and clarity of brain tissue and light-sheet fluorescence microscopy were employed to explore the neuronal activity change in Ent Va of susceptible mice in 21-day CSDS paradigm.(3)To determine the necessity of the Ent→V2M pathway for depressionlike behavioral phenotypes,chemogenetic and optogenetic approaches were applied to inactivation of the Ent→V2M pathway at cell bodies and axon terminals respectively.(4)To further determine the sufficient role of the Ent→V2M pathway for antidepressant-like behavioral symptoms,21-day CSDS paradigm,behavioral tests combined with optogenetic activation of the Ent→V2M pathway were used.(5)During optogenetic stimulation,a small fraction of light can leak out at the interface of fiber optic cannulae and ferrules,potentially leading to the observed antidepressant effect by disturbing periphery visual pathways from retina to visual cortex.To control this,we blocked the laser at the fiber optic cannula to prevent optogenetic stimulation of the Ent→V2M pathway while leaving leaked light unchanged and the behavioral phenotypes were checked under this condition.(6)To investigate how the Ent→V2M pathway mediates depression-like behaviors,we virally expressed a newly developed genetically encoded Ca2+ indicators(j GCa MP7 b,by researchers from Janelia Research Campus)in Ent and used fiber photometry to monitor neural dynamics of the projection terminals of Ent Va neurons through optical fibers implanted in V2 M.Meanwhile,the related behavioral behaving of each mouse was recorded and analyzed with Ca2+ activity.(7)At last,to determine the mechanism of external visual disturb(including visual blocking and stimuli)roles in depression-like behaviors,we monitored and compared activity of the Ent→V2M pathway under both white-light and red-light conditions in EPM and restraint tests.We also delivered ten consecutive blue light flashes in front of eyes of mice and simultaneously recorded neural activity of the pathway using fiber photometry.Results1.Entorhinal-visual cortical projections(1)Results of anterograde tracing showed that neurons in Ent Va projected extensively to the caudal part of neocortex(mostly in medial part of the secondary visual cortex,V2M)in the ipsilateral hemisphere with relatively little projections in the contralateral hemisphere.(2)Notably,axons from Ent Va neurons mainly targeted the supragranularlayers(II/III)rather than infragranular layers(IV-VI)of ipsilateral V2 M.(3)Results of retrograde tracing showed that most of V2 M projecting neurons were located in sublayer Va of the ipsilateral Ent but merely in contralateral Ent.The number of labeled Ent Va neurons distributed in medial part is much more than that in lateral part.(4)Few Ent Va neurons(less than 10%)showed co-labeling of td Tomato and GFP,indicating that V2 M projecting Ent Va neurons were largely distinct from those projecting to PFC,NAc,BLA or hippocampus.2.CSDS suppresses activity in Ent Va neurons(1)After 21 days of consecutive social defeat stress,most of mice(more than 90%)are susceptibility.Compared with control mice,susceptible mice had significantly reduced time in the interaction zone and social index,increased time in the corner zone and avoidance index.Susceptible mice had significantly lower sucrose preference(indicating anhedonia),and increased immobile time in the forced-swimming test(FST,indicating despair).Susceptible mice also showed increased level of anxiety as indicated by spending much less time spent in open arms of elevated-plus maze(EPM),and by the reduced entries to open arms.The time spent in central area of open field test(OFT)and the number of entries were also significantly reduced.Susceptible mice showed no difference in body weight but lower locomotion speed relative to control mice.However,there was no significant correlation between time spent in central area of OFT and locomotion speed.(2)The resilience ratio of 21-day paradigm is significantly lower than that of 10-day paradigm(9.1% vs.30.4%).The behavioral phenotypes of susceptible mice in 21-day CSDS paradigm had greater changes,higher correlation coefficients and better prediction efficacy than those in 10-day paradigm.(3)Susceptible mice had less activated neurons compared with control mice,as characterized by reduced number of c-Fos positive neurons in Ent Va.This pattern of reduction was consistent across consecutive slices.3.Inactivation of Ent→V2M projection neurons in healthy mice recapitulates depression-like behaviors(1)h M4 Di was specially expressed in V2 M projecting Ent Va neurons.Neural activity in Ent Va was potently inactivated 40-60 min after intraperitoneal injection of CNO in h M4 Di mice,but not in m Cherry control mice.Thus,h M4 Di combined with CNO can effectively inactivate neural activity of Ent Va neurons.(2)Compared with m Cherry control mice,h M4 Di mice with Ent→V2M projection neurons inactivated was not attracted to interact with a stranger mouse at location S1,but rather spent more time with a perforated columniform cage placed at E1,resulting in a reduced social ratio in three-chamber social test.Furthermore,h M4 Di mice exhibited significant higher level of anhedonia,despair and anxiety compared with m Cherry control mice,as indicated by decreased sucrose preference,longer immobile time in FST,less central area duration and entries in OFT and less open-arm duration and entries in EPM.In contrast,h M4 Di mice injected with vehicle showed no difference in sucrose preference and anxiety compared with m Cherry control mice.In OFT,h M4 Di mice showed reduced locomotion speed.However,there was no significant correlation of duration in central area with speed,suggesting that the increased level of anxiety cannot be explained by reduced locomotion speed.The speed of locomotion in h M4 Di mice was not significantly different from control mice in TCST,and there was also no significant correlation of time spent in S1 or E1 with movement speed.(3)During behavioral test,we delivered light through optical fibers implanted over V2 M to bilaterally illuminate Ent projection terminals.Arch T mice interacted less with stranger mice in the three-chamber social test compared with control mice.Arch T mice also exhibited higher anxiety in the elevated-plus maze test with reduced exploration time in open arms compared with GFP control mice.These results further confirm that the Ent→V2M projection neurons regulate behavioral phenotypes of depression through their direct projections.4.Activation of Ent→V2M projection terminals rapidly alleviate depression-like phenotypes(1)Ch R2 was well expressed in Ent neurons and their axon terminals in V2 M.Optogenetic stimulation of Ent Va projection terminals(470 nm LED,5~6 m W)reliably induced postsynaptic Ca2+ activities in V2 M,with evoked activity having an amplitude similar to spontaneous activity.These results demonstrate that we can specifically activate the Ent→V2M pathway using blue light illumination over V2 M.(2)After the three-week long CSDS paradigm,susceptible mice had lower social index and higher avoidance index compared with control mice.Interestingly,the abnormality in social index and avoidance index of CSDS mice in SI test disappeared during but not before or after optogenetic activation of the Ent-V2 M pathway.The abnormality also disappeared during optogenetic activation in a variety of behavioral tests including EPM,OFT,FST and TST.Specifically,susceptible mice during optogenetic activation showed significantly increased time in social interaction zone,in open arms in EPM and in central area in OFT,and exhibited increased entries in EPM and OFT.The immobile time in FST and TST tests were significantly reduced.Consistently,the new cohort of mice exhibited a lower level of anxiety as indicated by longer exploration time in open arms during the elevated-plus maze test.The sociability of normal healthy mice was also notably improved in the three-chamber social test during optogenetic activation of the Ent→V2M pathway as indicated by significantly increased social interaction duration in S1 and decreased duration in E1 relative to GFP mice.(3)We blocked the laser at the fiber optic cannula to prevent optogenetic stimulation of the Ent→V2M pathway while leaving leaked light unchanged.Under this condition,susceptible mice showed no improvement in sociability.Susceptible mice also exhibited no change in anxiety level in EPM and OFT.(4)With eyes covered by black blinders,susceptible mice showed no significant change in sociability and anxiety related behaviors relative to the same group of mice without blinders,indicating that blocking normal vision did not alter basic behaviors of mice.However,with activation of the Ent→V2M pathway,susceptible mice with blinders still showed reduced depression-like phenotypes,as measured by increased duration in social interaction zone and significantly decreased duration in corner zone in SI.The high level of anxiety was also alleviated as measured in EPM and OFT tests.5.Activity of the Ent→V2M projection terminals promotes antidepression(1)The newly developed j GCa MP7 b has bright baseline fluorescence to enable imaging of Ent→V2M projection terminals.Fluorescent signals showed spontaneous fluctuations under both anesthesia and freely moving conditions in j GCa MP7 b mice,but not in control EYFP mice,indicating that fluorescence change reflected activity of the Ent→V2M pathway.(2)Mice freely explored in an elevated-plus maze,and neural activity of Ent→V2M pathway was significantly higher when mice were in the center and open arms relative to be in closed arms.When mice crossed from the closed arms to open arms(approach),neural activity increased typically seconds before the beginning of transition.In contrast,the activity decreased when mice moved from open to closed arms(retreat).(3)In reward test,activity of the Ent→V2M pathway responded continuously to licking of milk reward.The slope,change of amplitude during licking and after licking were opposite to each other,reflecting the upward and downward trend during and after licking respectively.The increase of amplitude during licking did not correlate with licking duration(the number of licks),suggesting that the activity of the pathway did not reflect licking movement alone.(4)During FST,the activity of the Ent→V2M projection terminals correlated with the mobile state,and the onset of activity rise was ahead of the transition from immobile to mobile states.In a restraint test,the activity of the Ent→V2M pathway also correlated with resistance state and activity increased nearly 1 s before the transition from the quiet to resistant states.This activity increase is unlikely due to movement itself since the Ent→V2M pathway did not respond to simple motion such as continuously running on a treadmill or grooming.(5)Neural activity under red-light condition is not significantly different from that under white-light condition in EPM test.The results were similar in restraint test.Consecutive visual flashes,especially late flashes,did not effectively activate the Ent→V2M pathway.Conclusion21-day CSDS paradigm is a more efficient paradigm that can robustly induce anxiety-like and depression-like behaviors in mice.The Ent→V2M pathway,a specifically projected neural projection,can bidirectionally regulate depression-like behaviors and may play a crucial role in major depression.As a shallow cortical circuit from deep brain area(Ent)to superficial cortical area(V2M,especially to superficial layer II/III),the Ent→V2M pathway is a potential target for noninvasive stimulation therapy(such as r TMS and t DCS)for major depressive disorder. |
