Improvement Of An Animal Model Of Learned Helplessness And Its Dopamine Mechanism Study

Learned helplessness,the failure to escape shock induced by uncontrollable aversive events,was discovered by Seligman in 1967 based on animal experiments in dogs.After decades of behavioral research on the learned helplessness,Maier and Seligman theorized that individuals have acquired uncontrollability over aversive events and expected it occurred in the future to be important in the development of learned helplessness.Maier and Seligman reviewed the researches and the theory of learned helplessness in 2016,and proposed that helplessness is not learned in original experiment.In fact,there are different between the neural results of learned helplessness in rodents and the theory of learned helplessness.Environment was change from the controllable to the uncontrollable results in expectation of uncontrollability undermining trying active coping,which is key to the development of learned helplessness.Therefore,it is necessary to highlight the environment from controllable to uncontrollable for modelling the abnormal emotion and behaviors in human after prolonged experiencing loss of control over trauma and social defeat.The dopamine system mediated reinforcement learning,and its hyperactivity is closely related to the occurrence of emotional and behavioral disorders,such as depression,suggesting that learned helplessness may be related to the abnormal activity of dopamine neurons.According to the original behavioral results and original theory of learned helplessness,we modified the original triadic design.A model of Loss Of Control(LOC)was established and currently applied in C57BL/6 mice,which is an animals model of learned helplessness,highlighting that control over aversive stimulation is firstly acquired,and then lost.Utilizing in vivo and ex vivo electrophysiological techniques and fiber photometry,we investigated the neural mechanisms of dopamine neurons in the substantia nigra pars compacta(SNC)by which LOC resulted in emotional and behavioral disorders.The main investigations and results are as follows:(1)Loss of control leads to more remarkable escape deficitC57BL/6J mice were randomly divided into three groups:LOC,yoked(L-Yoked),and no shock(NS).A LOC group of mice was firstly exposed to escapable mild footshocks over 3 days to acquire control,and then to inescapable footshocks over the following 3 days to lose control.In order to observe the stressful effects of shock per se,each mouse in the LOC group was paired with one mouse over 6 days to form the L-Yoked group.Each mouse in the L-Yoked group received identical shocks in intensity,duration,and temporal pattern to its paired mouse in the LOC group but was not able to control the shocks.A third group received no shocks(NS)and was used as a baseline control.And then,all groups were measured by shuttle box test,forced swimming test and so on.The results showed that compared with the L-Yoked group,the mice of LOC group exhibited more remarkable escape deficit,indicating that shock per se leads to passivity,and loss of control over shock produces more remarkable helpless behavior.To investigate the effects of control or loss of control over shock on escape behavior,we firstly added an experiment without extinction learning that mice were exposed to negative reinforcement learning over 3 days or 6 days to acquire control over shock,with the yoked groups receiving the identical but inescapable shock.The results showed that the mice of yoked group exhibited significantly escape deficit,while the escapable groups exhibited the escape behaviors,suggesting that the protective effects of control over footshocks.Secondly,we added another experiment in which ES/IS mice were exposed to escapable shock over three days and then to inescapable shock for only one day,with EI-Yoked mice received the identical but inescapable shock.The results showed that EI-Yoked mice showed significantly escape deficit,while ES/IS mice showed the escape behaviors,suggesting that initial experience with control over shock immunized against the effect of subsequent uncontrollable shock.These results demonstrate that by prolonged exposure to inescapable shock,a complete extinction of learned control is critical for inducing loss of control over shock to produce abnormal emotion and behaviors.(2)Control and loss of control would not modulate the response to stressTo investigate the response to escapable and unescapable shocks in mice,serum corticosterone levels were determined on day 1,day 4,day 6 and day 7 after shuttle box test,respectively.The result showed that compared with NS group,serum corticosterone concentrations were significantly increased in the Learning and Learning-Yoked groups on day 1;serum corticosterone concentrations were not significantly altered in the LOC and the L-Yoked groups compared with the NS group after the exposure to footshocks on Day 6,but significantly increased in the ES/IS group compared with the EI-Yoked and the NS groups after the exposure to footshocks on Day 4,and there are no significant different for all groups after exposure to shuttle box test on Day 7,indicating that the levels of serum corticosterone were not related to controllability,and loss of control over shock did not change the response to stress in shuttle box test.Those resulted demonstrated that control and loss of control over shock do not modulate the response to stress,and their difference would modulate by the advanced neuronal structure.(3)Loss of control over footshocks increased spontaneous activity and intrinsic excitability of SNc dopamine neurons,and decreased the SNc dopamine neurons response to natural rewardTo investigate the effect of loss of control over footshocks on activity and intrinsic physiological properties,spontaneous activity of putative SNc dopamine neurons was measured by in vivo electrophysiology,and cell excitability of SNc dopamine neurons were measured in current-clamp modes with ex vivo from brain slices.The results showed that LOC mice have significantly more increase in vivo spontaneous firing rates,bursting events and action potentials elicited to current injections,suggesting that loss of control increases the baseline activity of SNc dopamine neurons.To investigate the effect of loss of control on the SNc dopamine neurons response to natural reward,fiber photometry was used to record the population activity of neurons expressing a genetically encoded Ca2+indicator,which could detect real-time activity changes in cell bodies during behavior.The result showed that a decrease in SNc GCaMP fluorescence when mice licked for sucrose after experiencing loss of control over footshocks,an effect that was absent in their yoked group and control group,indicating that loss of control over footshocks decreased the SNc dopamine neurons response to natural reward.(4)Dopamine dynamically encodes negative reinforcement learningTo investigate the SNc dopamine neurons modulated negative reinforcement learning during established model of LOC,we firstly applied fiber photometry during consumption of sucrose solution to validated expression of GCaMP in SNc neurons.The results showed that shock onset can enhance the initial dopamine activation.The activation of dopamine neurons showed decrease with shock continued.When the shock was offset,the activation of dopamine neurons showed increases.Of course,when mice of Learning group did not learn that shock offset is dependent of their response,the response of dopamine neurons to shock decreases with shock repetition.However,once mice learned that shock offset is dependent of their responses,the dopamine response increase with a shock onset.The dopamine responses decease as shock is onset,if mice have never experienced learning.Those results suggest that shock as a novel stimulus enhances the activity of SNc dopamine neurons,while it as a negative value depresses the activity of SNc dopamine neurons.However,after experiencing negative reinforcement learning,shock as a conditional cue which predicts the end of aversive stimulus,can enhance the activity of SNc dopamine neurons.In summary,shock per se produces passivity,loss of control over shock produces more remarkable helplessness and depression-like behaviors,while the experience of control over shock prevents or reduces the deleterious effect induced by shock per se or subsequent uncontrollable stressors.Therefore,we established a true "learned helplessness" animal model,in which psychological stress induced by the transition from attain control to lose control over shock is critical to develop helplessness and depression-like behaviors.Control and loss of control over shock would not modulate the response to stress in mice.Its possible mechanism is that SNc dopamine neurons dynamically encode negative reinforcement learning.Loss of control over shock which induces the increase baseline activity of SNc dopamine neurons and decrease response to reward,produces the depression-like behaviors.