| Background and objective:Prefrontal cortex(PFC) is closely related to brain function in many advanced features, such as cognition, decision making, motivation, emotion, memory, social functions regulation and so on. In psychology study, prefrontal cortex function is typically closely related to personality. In a sense, the function of the prefrontal cortex is a key feature that human distinguishes from other non-human animals. Anatomically, the prefrontal cortex closely linked to other brain regions extensive, including many cortical, subcortical and brainstem regions, to formation of complex neural circuits, and plays the appropriate nerve function. Dysfunction of the prefrontal cortex associated with a number of neuropsychiatric disorders, such as drug addiction, schizophrenia, depression and so on.Previous studies, especially intracranial electrical self-stimulation, had found that some regions of the frontal cortex have rewarding effects, indicating that the prefrontal cortex involved in reward circuit. However, due to the technical limitations of electrical stimulation itself, it cannot be determined how the prefrontal cortex processes the rewarding signal? By exciting the neurons in the frontal cortex? Or by inhibiting? What is the detail of the reward circuit?Recent clinical studies have shown that the prefrontal cortex is closely related to emotion and motivation. Dysfunction of the prefrontal cortex is closely associated with depression, and deep brain stimulation(DBS) specific area of prefrontal cortex(Cg 25) can "corrective" PFC abnormal activity of refractory depression and has a good therapeutic effect. However, its mechanism of action is unclear.Optogenetics is one of the most cutting-edge experimental techniques in the field of neuroscience in recent years. It can be implemented selectively specific excitatory or inhibitory neurons, rather than have a direct impact on the other neurons and peripheral nerve crossing fibers, with a quite high temporal and spatial resolution. Optogenetics is a powerful tool used to resolve specific neural circuits underlies certain behavior. In this study, we performed to dissect the reward circuit involving PFC by using optogenetics, in order to deeper understand the function of the prefrontal cortex further, and contribute to the clinical treatment of prefrontal cortex dysfunction associated with neuropsychiatric diseases, especially in drugs addiction and depression. Methods:Several methods were used in this study, such as optogenetics, multi-channel extracellular in vivo recording in freely moving mice, body, in vivo detection of dopamine by FSCV in freely moving mice, and self-stimulation behavioral paradigm. By using Cre recombinase transgenic mice combined with Cre recombinase-induced virus expression strategy, we can express a light-sensitive protein in specific types of neurons and achieve selective regulation of specific types of neurons. Experiment was divided into four parts, firstly to explore the role of the prefrontal cortex in the rewarding, and then the role of different types of neurons in the rewarding process was studied, followed by exploring the details of prefrontal related reward circuit and the role of dopamine in rewarding involving the prefrontal cortex. Results: Experiment one(1) Excited neurons located in m PFC(DP, Pr L, MO), and DTT or VTT, mice can quickly learn to press the lever paired with laser to get the stimulation which excited the neurons. When the laser turned off, the mice significantly reduced their responses to the lever. The mice pressed as much as previous and even more when the laser was reloaded, suggesting that the activation of neurons in m PFC and DTT or VTT has rewarding effect.(2) Excited neurons located above m PFC(cg1 / M2) and the outer area(VO / LO), mice were unable to learn to self-stimulate, suggesting that exciting neurons in these brain regions have no reward effect.(3) Reversal the lever paired, the mice can quickly learn to press the lever that paired with laser, suggesting that the self-stimulation responses of mice is specific associated with neuron exciting.(4) The effect of various laser stimulation frequencies was different frequencies, 50 Hz and 25 Hz was significantly stronger than the 12.5 Hz and 6.25 Hz(p <0.0001) in self-stimulation.(5) Multi-channel in vivo recording displayed, laser stimulation intensity 0.1--10 m W, frequency of 6.25 Hz, 12.5 Hz, 25 Hz and 50 Hz can cause neuronal discharge in time-locked pattern, and the discharge frequency consistent with laser stimulation frequency. Experiment two(1) Excited v Glut1 positive neurons located in the m PFC supports self-stimulation, the lever press rate as high as 501.83 ± 359.27.(2) Excited v Glut2 positive neurons located in the m PFC supports self-stimulation, the lever press rate no more than 198.25 ± 117.99, significantly lower than that excited v Glut1 positive neurons.(3) The self-stimulation effect of exciting v GAT positive neurons in m PFC was weaker than that of v Glut1 and v Glut2 positive neurons and required much more laser stimulation(25 Hz, 72 pulses) to support self-stimulation.(4) CPP results showed excited v GAT positive neurons have rewarding effect. Experiment three(1) Exciting the projection from m PFC to striatum(m CP, Ac Sh) supported self-stimulation, suggesting that activation of this neural pathway has rewarding effect.(2) Exciting the projection from m PFC to internal capsule(ic) supported self-stimulation, suggesting that activation of this neural pathway has rewarding effect.(3) Exciting the projections from m PFC to thalamus(VM, MD) and hypothalamus(LHA) supported self-stimulation, suggesting that activation of these neural pathways have rewarding effect.(4) Local(m CP) pharmacological blockade can be significantly reduced the self-stimulation induced by exciting the projection from m PFC to m CP, indicated that the neurons in m CP involved in the reward circuit and it is an important node. Experiment four(1) The dose of dopamine D1 and D2 receptor antagonists which is sufficient to block the dopaminergic neurons in VTA to support self-stimulation had no significant effect on m PFC self-stimulation, suggesting that the rewarding effects of exciting m PFC is dopamine independent.(2) Successfully achieved real-time detection dopamine concentrations in striatum by using FSCV in wake freely moving mice.(3) FSCV DA concentration assay showed that electrical stimulation mfb significantly increased striatal DA concentration; while optogenetics excited m PFC neurons which expressing Ch R2 had no significant effect on the striatal DA concentrations, suggesting that activation of m PFC neurons by optogenetics did not induce striatal DA release. Conclusion:(1) We firstly found that the direct activation of the medial prefrontal cortex neurons are highly rewarding by using optogenetics, and confirmed the optimal optogenetics stimulation parameters.(2) The m PFC reward effect mediated mainly by excited v Glut1 positive neurons, partially by v Glut2 positive neurons, while the overall effect of activating inhibitory neurons v GAT positive neurons is rewarding, rather than aversion.(3) By taking the advantages of optogenetics in neural circuits, we firstly discovered two pathways(m PFC--- m CP and m PFC--- ic) play an important role in the reward.(4) For the first time, by using optogenetics combined with FSCV in freely moving mice, we studied the role of dopamine in rewarding involving m PFC, we found the rewarding effect of m PFC did not depend on the release of dopamine in the brain, and optogenetics stimulation of m PFC did not cause dopamine release in the striatum. |
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