| Addiction has been defined as a chronic, relapsing disorder characterized by ①a compulsion to seek and take drugs, ② loss of control over drugs taking, and ③ emergence of a negative emotional state (e.g., dysphoria, anxiety, and irritability) that defines a motivational withdrawal syndrome when access to the drug is prevented. The occasional, limited, recreational use of a drug is clinically distinct from escalated drug use, the loss of control over drug intake, and the emergence of compulsive drug-seeking behavior that characterize addiction.All drugs of abuse exert their primary rewarding effects on the mesolimbic dopamine system, which consists of dopamine neurons originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc) and the prefrontal cortex (PFC). Psychomotor stimulant, such as opiates through binding with μ opioid receptor (MOR), which located in the tail of ventral tegmental area (tVTA) GABAergic neurons, exert their effects on dopamine neuron, and increase dopamine release in the nucleus accumbens, and finally produce euphoria. Addiction is a complex disorder, because many factors contribute to the development and maintenance of this neurological disorder.Stress plays an important role in the initiation, maintenance and relapse to drug addiction. It has been suggested that the rate of acquisition to drug intake would be after exposure to stressful events. Repeated exposure to stress can positively drive the rewarding effect of cocaine. Researches have also illustrated a positive correlation of stress and drug craving in human, and stress has the ability to activate the mesolimbic dopamine system following exposure to some kind of stress events. Controversially, it has been hypothesized that simultaneous exposure to abused drugs and stress might have a reverse effect. Since there are interactions between the neural pathways involving stress and addiction. It has been demonstrated that chronic stress causes a special interruption of rewarding behaviors, which are caused by drugs of addiction. And stress can also decrease the score which are used for evaluating the performance of the animals’addictive behaviors, and even block the establishment of some addictive behaviors, such as the self-administration, etc.Recently, findings in the depression research have indicated that uncontrollable stress can increase the metabolic activity of habenula neurons. Foot-shock stress can increase the excitatory input compared to controls on neurons, which are from lateral habenula, projecting to the VTA, and the degree of synaptic potentiation positively correlated with the animal’s helplessness behavior. When the basal ganglia (a major source of input to the habenula) is specifically activated, animals display avoidance behavior which is consistent with the notion that habenula activation is aversive. And, taking advantage of optogenetic, Stamatakis and Stuber have shown that direct activation of lateral habenula (LHb) efferents onto the dopaminergic midbrain to can be aversive specifically through neurons that synapse onto GABAergic neurons in the tVTA. As the relay station between forebrain (limbic and basal ganglia) and midbrain structures, Habenula plays an important role depression. Habenula activation has been illustrated to be associated with disappointment and anticipation of an aversive outcome. And this is just opposite to the results of the dopaminergic neurons activation. At the same time, clinical data suggest that there are a lot of comorbidity between depression and drug addiction.Therefore we speculate that forced swimming stress may exert its negative effect on mesolimbic dopamine system through activating LHb neurons, and finally interfere the addictive behaviors of animals.In the present study, we used the morphine-induced conditioned place preference model, the impact of forced swimming stress (FSS) were observed on morphine-induced conditioned place preference (CPP) in SD rats and ICR mice. Retrograde and anterograde trace tracing method were employed to demonstrate the neural pathways among nucleus (e.g., LHb, VTA and tVTA, etc.) which were associated with addiction and stress. Immunohistochemistry detection method and other methods were applied to search for possible internal molecular biological mechanisms. Our results are as follows:1. Forced swimming stress influenced morphine-induced conditioned place preferenceAccording to the protocol morphine (10mg/kg, i.p.) was administrated on day 1, day 3, day 5, and day 7; Saline (10mg/kg, i.p.) was administered on day 2, day 4, day 6, and day 8. Intraperitoneal injection of morphine could induce conditioned place preference on SD rats and ICR mice. Before intraperitoneal injection of morphine (10mg/kg) on day 1, day 3, day 5 and day 7 or saline (10mg/kg) on day 2, day 4, day 6, and day 8, animals received a 5-minute FSS. The FSS was able to reduce the animals’CPP score which were tested during the test-phase of the CPP experiment. And the result suggested that physical stress affected the formation of morphine-induced CPP, reduced animals’behavior performance associated with morphine reward effect. The influence of stress on morphine induced CPP was not a short-term intervention, it was also found 24 hours after stimulation was performed. During the 15 minutes test-phase, there is no statistically significant difference in total distance traveled in both sides of the box among experimental animals of four groups. Thus, the physical stress did not affect the conditioning scores due to changes in the locomotor activity. The results of SD rats and ICR mice were the same.2. Neuroanatomical link between addiction and stress associated with the nucleusBy retrograde tracing method, under the guidance of rat stereotaxic instrument, Dil (1 μ 1) was injected into the VTA (unilateral) and the tVTA (unilateral) of SD rats respectively. Tracking results show that there was projection from tVTA GABAergic neuron to VTA, and there was projection from LHb, DRN and amygdala to tVTA.Using anterograde tracing method, under the guidance of rat stereotaxic instrument, GFP-expressing AAV-CMV-GFP (1 μ 1) microinjection was made into the amygdala to SD rats. Tracking results show that there was projection from amygdala to tVTA.3. Stress increased CaMK Ⅱ expression in the LHb neuronscommercial available CaMK Ⅱ antibody (rabbit-derived) was chosen. Immunofluorescence was performed to display and analysis the CaMK Ⅱ protein expression in LHb of all group of rats. Immunofluorescence results showed that the specific green fluorescence for CaMK Ⅱ protein in brains of control rats and the morphine-induced CPP rats were weak. While in the other two groups which were given FSS, the specific green fluorescence were moderate intensity. Application of Image-Pro plus 6.0 software for statistical analysis of immunofluorescence images, statistical results show that FSS significantly increased the expression of CaMK Ⅱ protein in rat LHb, comparing with the control group. And this indicated that repeated FSS activated LHb neurons of rat brain.4. Stress affected the increasing effect of morphine on nucleus accumbens p-DARPP-32 protein levelsCommercial available p-DARPP-32 antibody (rabbit-derived) was chosen. Immunofluorescence was performed to display and analysis the p-DARPP-32 protein levels in NAc of all group of rats. Immunofluorescence results showed that the fluorescence intensity of NAc in control rat brain was scattered and weak. While, the specific fluorescence intensity which stand for the p-DARPP-32 protein levels war very strong in the NAc of rats from morphine-induced CPP group. The fluorescence intensity of the other two groups which were given FSS during the CPP conditioning procedure were medium. Application of Image-Pro plus 6.0 software for statistical analysis of immunofluorescence images, statistical results showed that repeated injections of morphine could increase the level of p-DARPP-32 protein in the NAc. And that represented dopamine neuron activity was significantly enhanced. Comparing with the rats from morphine-induced CPP group, FSS repeated stimulation affected the increasing effect of morphine on NAc p-DARPP-32 protein levels. And it suggested that FSS interrupted the activation effect of morphine on dopamine neurons, thereby affected the induced effects of morphine addiction, and eventually resulted in the failure of establishment of morphine-induced CPP.In conclusion, The present study demonstrates that: ①FSS influenced morphine-induced CPP, and this interference effect was not due to changes in the locomotor activity; ② the neural pathway of LHb (GLU)→ tVTA (GABA)→ VTA (DA) was confirmed; ③ FSS might increase the expression of CaMKⅡby activating LHb neurons, and that directly lead to enhancement of glutamatergic synaptic transmission efficiency of neurons within LHb, then increased the activity of GABAergic neurons located in the tVTA which received neural projection from LHb glutamatergic neurons, and then the inhibitory effect of tVTA GABAergic neurons on VTA dopaminergic neurons was enhanced. This inhibitory effect exerted by tVTA GABAergic neurons weakened the excitatory effects of morphine by binding to MOR on dopaminergic neurons. As a result, the release of dopamine in NAc was reduced, DARPP-32 phosphorylation levels declined, the rewarding effects of morphine weakened or even disappeared, eventually that lead to the failure of morphine-induced CPP. |
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