The Mechanism Of Dopaminergic Neurons In Midbrain Ventral Tegmental Area Involved In Depression

Depression is a chronic neurobiological disorder, and has become a major mental health problem all over the world, affecting over 1/50 people worldwide.Up to now, the underlying electrophysiological and molecular mechanism remains unclear though various depressive models are used in the pathogenesis of depression study and some breakthrough research progress of the neurological mechanism have been reached. VTA dopaminergic neurons play a role in reward, motivation and drug addiction. According to some research, the variety of dopaminergic neurons’ plasticity in midbrain ventral tegmental area(VTA) are closely related with depression. In the different kinds of depressive animal models, specifically active or inhibit VTA dopaminergic neuron to regulate its firing rate have different effect on the depressive behaviors, suggesting that the functional change of dopaminergic neurons in midbrain VTA probably involved in the formation of depression, however, the specific mechanisms need to be further explored.Objection: This research was built on chronic unpredictable mild stress depressive rats’ models and chronic neuropathic depressive rats’ models to simulate the clinical depressive and chronic pain patients, we used the multichannel acquisition processor neurophysiology system to record and analysis the firing activity of dopaminergic neurons in midbrain tegmental area when the depression is occurred. After the treatment of drugs, the depressive behaviors was reversed, we analyzed the characterizes of dopaminergic neurons firing activity and used the pathological morphology method to study a key ion channels protein which have the effect on the regulation of the excitability of VTA dopaminergic neurons to explore the depression mechanism.Methods: 1. The establishment of depression models:(1) Chronic neuropathic depression We used a chronic neuropathic model of spare nerve injury(SNI) and choose some behavioral test such as paw withdrawal mechanical threshold, sucrose preference test, focus swimming test and open field test. Rats divided into two groups including Sham group and SNI group were adapted to the experimental environment before test, and then we measured the baseline data of paw withdrawal mechanical threshold, sucrose preference test, the immobility of focus swimming test and the moving distance of open field test. For the surgery procedure, the Sham group rats were exposed the nerve without injury, while the SNI group rats ligated and cut the peroneal nerve and tibia nerve, reserved the sural nerve. We measured the paw withdrawal mechanical threshold on the day of 3, 14, 28, 56 after SNI, and on the day of 14, 28, 56 we did the open field test, sucrose preference test, forced swimming test after SNI to estimate the depressive disorder. On the day 56 after surgery, we divided the SNI rats in two group, one was injected the gabapentin and another was injected saline for 14 days continually, then did the behavioral test mentioned above.(2)For the CUMS depressive rats model, sucrose preference test, moving distance in open field test and Body weight change were operated as the indicator of the depressive behavior test. Rats were divided into two groups including control group and CUMS group randomly after adapted to the CUMS experimental surrounding before test. The control rats were feeding normally as usual, but the CUMS rats were give these experimental treatment as followed. After six or eight-weeks stimulation with nine different stimulations, the rats were induced depression-like behaviors which were confirmed with the behavioral test mentioned before. Then, the CUMS rats were divided into two groups randomly. For one group, they were injected with saline as control while another group were injected by fluoxetine for 21 days. After injection, we did the behavioral tests to see whether the rats had been recovered from the depression. 2.Electrophysiological techniques to analyze the VTA dopaminergic neurons’ firing characteristic: Use the multichannel acquisition processor neurophysiology system to record and analyze the VTA dopaminergic neurons’ firing activity. Recent evidences have shown that dopaminergic neurons in VTA have important roles in reward, motivation, drug behaviors as well as in depression. We use the Plexon MAP neurophysiology system to recorded and analyzed the VTA dopaminergic neurons’ firing characterize in vivo. After eight weeks stimulation during the CUMS experiment, we chose the depression-like rats, and then gave continually injection of fluoxetine, we analyzed VTA dopaminergic neurons’ firing characteristic such as frequency and burst firing activity in CUMS-fluoxetine treated rats, CUMS-saline rats and control rats.In the experiment of chronic neuropathic depression, we chose depressive rats and gave continually injection of gabapentin for 14 days, then we analyzed the dopaminergic neurons’ frequency and burst firing activity in SNI-gabapentin treated rats, SNI rats and Sham rats 3. Immunofluorescence stain in VTA dopaminergic neurons: We used the technology of immunofluorescence to double stain both the tyrosine hydroxylase and hyperpolarization-activated cyclic nucleotide-gated channel(HCN) in VTA area in rats to measure the expression of HCN protein in VTA dopaminergic neurons.Result: 1. In the neuropathic depression, SNI rats’ paw withdrawal mechanical threshold was decreased and showed obvious statistical difference compare to Sham rats(n = 6, P<0.01). The sucrose preference was decreased(n = 6, P < 0.01) and the moving distance in open field was decreased as well(n = 8, P < 0.01), the immobility time in focus swimming test was increased(n = 10, P < 0.01). After 14 days injection of gabapentin, the depressive behaviors in SNI rats were reversed. Compared with saline-treated SNI rats, gabapentin-treated SNI rats’ showed higher paw withdrawal mechanical threshold(n = 10, P<0.01) with the moving distance in open field test increased(n=6, P<0.001). And the immobility time in focus swimming test was decreased(n=8,P<0.01) while the sucrose preference was increased(n = 8, P<0.01). The firing frequency and burst activity of VTA dopaminergic neurons in SNI rats were increased compare to Sham rats(P<0.01). After 14 days gabapentin injection, the gabapentin-treated SNI rats’ dopaminergic neurons’ firing rate was decreased compared to the saline-treated SNI rats(P<0.05), while the burst activity was increased(P<0.01). Compared to the Sham rats, VTA dopaminergic neurons’ firing frequency for gabapentin-treated SNI rats were increased(P<0.01), and the burst activity was increased as well(P<0.001, n = 132 in SNI+Saline, n = 81 in SNI+GBP; n = 71 for Sham). At the same time, HCN1 protein expression had no difference in gabapentin-treated SNI rats, saline-treated rats and Sham rats(P>0.05); HCN2 protein expression was increased in saline-treated SNI rats(P<0.01) and gabapentin-treated SNI rats(P<0.05) compare to Sham rats, but there was no statistically difference in saline and gabapentin-treated SNI rats. We did not find the HCN3 protein in VTA, we did not find the cell both stained HCN4 and TH in VTA as well. 2. In CUMS depression model: after 8 weeks CUMS, CUMS rats’ sucrose preference was significantly decreased compare to control rats(n=12, P<0.001), the body weight increased slowly(n = 12, P < 0.05), there was no difference in moving distance in open field test(n = 12, P > 0.05). After 21 days injection of fluoxetine, fluoxetine-treated CUMS rats’ sucrose preference was increased(n = 12, P < 0.01) and there were no difference in both body weight change and moving distance in open field test(n = 12, P > 0.05). CUMS rats’ VTA dopaminergic neurons’ firing rates was increased compare to control rats(P < 0.01), burst activity was increased as well(P < 0.001); after 21 days fluoxetine treatment, fluoxetine-treated rats VTA firing rats was decreased but no difference compare to the saline-treated CUMS rats(P > 0.05), and it was increased compare to Sham rats(P < 0.05); the percent spikes in burst of gabapentin-treated rats was decreased compare to saline-treated CUMS rats(P < 0.05) and is was increased compare to Sham rats(P < 0.05); there was no difference in burst activity between gabapentin and saline-treated CUMS rats(P > 0.05), but it was significantly increased compare to control rats(P < 0.001, n = 61 in control, n = 58 for CUMS+saline, n = 83 in CUMS+fluoxetine).Conclusion: 1. In both chronic pain – induced depression rat model and in the CUMS rat model, we found that the firing rate for VTA dopaminergic neurons which closely related to the neural excitability were significantly changed, which have a major correlation for the underlying electrophysiological mechanism for depression 2. In the chronic neuropathic pain – induced depression rat model, we found that the expression of HCN2 protein that could influence the neural excitability in VTA dopaminergic neurons were significantly increased, which indicated the underlying molecular mechanism of depression.