D₂R And GABA_BR Modulation Of Midbrain VTA Dopaminergic Neurons In A Depression Mice Model

Depression is a chronic affective disorder caused by multiple factors,and is characterized by significant and long-lasting changes in mood and emotion.Depression severely affects the daily lives of the patients,and places a heavy burden on families and society.About 15%of depression patients die of suicide.A joint study by the World Health Organization,the World Bank,and Harvard University shows that depression has become China’s second-most threat to human health.However,the current anti-depression drugs are still not satisfactorily effective in treating depression patients.Therefore,it is important to understand the pathophysiology of depression and find new therapeutic targets and strategies.Recent studies have shown that changes in excitability of dopamine（DA）neurons in the Ventral tegmental area（VTA）play a key role in the pathogenesis of depression.VTA DA neurons fires actionpotential spontaneously,which is further regulated:by excitatory input of glutamate and inhibitory input of GABA systems.The inhibitory GABAergic regulation is imposed through the transmitter GABA acting on GABA_A and GABA_Breceptors（GABA_BR）on DA neurons.In addition,firing of DA neurons is also self-regulated,through a feed-back regulation by activation of D₂receptor（D₂R）.Both D₂R and GABA_BR are G protein-coupled receptors coupled to Gi protein,which when activated,inhibit firing of DA neurons through Gi-mediated activation of potassium channels（GRIK and K_V7.4）.Our previous study found that:DA could activate the K_V7.4 channel in VTA DA neurons.Also we found that the excitability of VTA DA neurons from a mouse depression model of social defeat was increased in comparison with the control mice,and in parallel with this a decreased K_V7.4 current density.This leads us to postulate here that a diminished D₂R inhibitory auto-regulation of VTA DA excitability may underlie the elevated firing of the VTA DA neurons in social defeat.Futhermore,other previous studies have shown that in animal model of drug addicition,GABA_BR-mediated inhibitory regulation of VTA DA neurons was also reduced,which is accompanied with a reduced activation of GRIK current;it will be interesting to know if this will also be the case in the social defeat depression.Therefore,in this study,by using patch clamp and single-cell PCR rechniques,we aim to study the inhibitory regulation of VTA DA neurons by D₂R and GABA_BR in the in the social defeat model of depression,to clealy if the increased excitability of VTA DA neurons from these mice is caused by a reduced inhibitory regulation through these receptors.The results show that DA and D₂ receptor agonist quinpirole inhibited similarly the firing of VTA DA neurons from the depression model and the control mice.However,the inhibitory effect of GABA on excitability of VTA DA neurons in depression model mice was greatly weakened,but the effect of GABA_Breceptor agonist baclofen was not changed.Part one Effects of dopamine and D₂ receptor agonist quinpirole on model of depressionObjective:To observe the difference of dopamine-and quinpirole-induced inhibition on excitability of VTA DA neurons among the control,the depression-susceptible,and the depression-resilent mice,respectively.Methods:1. A depression model of social defeat was established using C57 mice, and the firing of action potential from DA neurons was recorded in the VTA brain slices using loose-cell-attached patch-clamp technique.2.Single-cell PCR technology was used to identify DA neurons.Results:1. In the social defaet depression model,the sugar preference and social interaction rate of the depression-susceptible mice were significantly lower than those of control mice（P<0.05）;the sugar preference and social rate of depression-resilent mice were not significantly different from those of control mice（P>0.1）.2.DA（20μM）,when applied in the bathing solution of VTA brain clices,reduced the firing frequency of DA neurons in the VTA region of control mice,and the depression-susceptible mice from 1.4±0.1 Hz to 0.2±0.1 Hz（n=15）,and from 1.7±0.2 Hz to 0.2±0.2 Hz（n=13）,respectively,which is not significantly different（P>0.1）.3.Quinpirole（10μM）when applied in the bathing solution of VTA brain clices,reduced the firing frequency of DA neurons in the VTA region of control mice,and the depression-susceptible mice from 1.0±0.2 Hz to 0.3±0.3 Hz（n=9）,and from 1.6±0.2 Hz to 0.3±0.2 Hz（n=18）,respectively,which is not significantly different（P>0.1）.Conclusions:1. The depression-model of social defeat was successfully established with the reduced sugar preference and social interaction rate.2. VTA DA neurons from depression-susceptible mice have a tendency of increased excitability.3.Dopamine as well as D₂eceptor agonist quinpirole inhibits the firing of action potential of VTA DA neurons from control and depression-susceptible mice with similar efficacy,indicating that the increased excitability of VTA DA neurons is not caused by a reduced D₂ receptor-mediated inhibition of DA neurons activity.Part two Effects of GABA and GABA_B receptor agonist baclofen on model of depressionObjective:To observe the difference of GABA-and baclofen-induced inhibition on excitability of VTA DA neurons among the control,the depression-susceptible,and the depression-resilent mice,respectively.Methods:1.A depression model of social defeat was established using C57 mice,and the firing of action potential from DA neurons was recorded in the VTA brain slices using loose-cell-attached patch-clamp technique.2.Single-cell PCR technology was used to identify DA neurons.Results:1.In the social defaet depression model,the sugar preference and social rate of the depression-susceptible mice were significantly lower than those of control mice（P<0.05）;the sugar preference and social interaction rate of depression-resilent mice were not significantly different from those of control mice（P>0.1）.2. GABA（100μM）,when applied in the bathing solution of VTA brain clices,reduced the firing frequency of DA neurons in the VTA region of control mice,and the depression-susceptible mice from 1.7±0.3 Hz to 0.2±0.2 Hz（n=12）,and from 1.8±0.2 Hz to 0.7±0.2 Hz（n=15）,respectively,which is significantly different（P<0.05）.3. Baclofen（10μM）when applied in the bathing solution of VTA brain clices,reduced the firing frequency of DA neurons in the VTA region of control mice,and the depression-susceptible mice from 1.6±0.2 Hz to 0 Hz（n=9）,and from 1.9±0.2 Hz to 0.1±0.1 Hz（n=9）,respectively,which is not significantly different（P>0.1）.Conclusions:1.The depression-model of social defeat was successfully established with the reduced sugar preference and social interaction rate.2.VTA DA neurons from depression-susceptible mice have a extremely small tendency of increased excitability.3. GABA inhibit the firing of action potential of VTA DA neurons from control and depression-susceptible mice with significantly different efficacy;hower GABA_BR receptor agonists baclofen inhibits the firing of action potential of VTA DA neurons from control and depression-susceptible mice with similar efficacy,indicating that the increased excitability of VTA DA neurons is not caused by a reduced GABA_BR receptor-mediated ihibition of DA neurons activity,but may be mediated by a alterd GABA_AR activity.