| Serotonin, as an essential monoamine neurotransmitter in the central nervous system, is involved in wide physiological processes, including emotional regulation, cognitive control of learning and memory, appetite and sleep. The serotonergic pathway originates from the raphe nuclei, which is formed from a collection of cell groups close to the midline throughout the brainstem, including rostral group, intermediate group and caudal group. The rostral section, which projects mainly to extensive forebrain areas, including the amygdala, hippocampus, hypothalamus and almost all neocortical regions, consists of the dorsal raphe nucleus and the media raphe nucleus is involved in cognition, emotional states, food intake, circadian rhythms and reproduction as a main area of emotional regulation. The caudal portion, which projects mainly to the cerebellum and the spinal cord, is in charge of motor activity, pain control, and regulation of the autonomic nervous system. Previous studies suggest that hyperactivity is a physical characteristic feature of DRN neurons in stress and anxiety states. Exposure to hostile conditions producing stress, anxiety and fear will increase the excitability of serotonergic neurons and serotonin level in the vicinity of DRN neurons. Therefore, inhibition of DRN neuron excitability and negative modulation of serotonergic activity in the DRN projection pathways may produce anxiolytic responses.Kv7 channels are voltage-gated K+ channels originally termed the ‘M-channel’ activated near at the threshold potential, with the feature of slow-activation, non-deactivation, slow-inactivation and voltage-dependence. Because their activation is relatively slow they do not contribute materially to the depolarization(and repolarization) of action potential. However, they can exert an intense inhibition on the repetitive or continuous burst-firing, the frequency of firing and the excitability of neurons. Five members Kv7.1-Kv7.5 which are coded by KCNQ1-KCNQ5 have been found for the family of Kv7. So far, no Kv7 channels except Kv7.4 channels are found to be expressed in the DRN. The DRN contains both serotonergic and non-serotonergic neurons with most of the latter neurons presumably being GABAergic. Notably, Kv7.4 is only expressed on a subpopulation but not all of serotonergic neurons in the DRN. Previous study reports that retigabine, an non selective opener of Kv7 channels, produce a robust inhibition on the excitability of neurons throughout the DRN. These results suggest that DRN has functional Kv7 channels which are most likely mediated by Kv7.4 channels. A very important mechanism of serotonergic neurotransmission is mediated by self-feedback regulation of 5-HT1 A autoreceptors. The presynaptic 5-HT1 A autoreceptors are mainly located in the 5-HT neuron dendrites of raphe nuclei in the brainstem, whose activation inhibits the electrical activity of 5-HT neurons, reduces the release of neurotransmitter and the activation of protein kinase. Based on these studies, we attempt in this project to study the role of Kv7.4 channels in modulation of neuronal excitability of DRN neurons and in depression model. The results are reported in three separate parts. The first part studied the regulation and the underlying mechanism of 5-HT modulation of Kv7.4 channel in DRN, using the brain slice patch clamp technique. In the second part, a social defeat(SD) model of depression was established and the depression-like behavior was evaluated. The third part studied the effect of 5-HT on the excitability of DRN neurons in both SD model and in Kv7.4 knockout mice. Part 1 Regulation and the underlying mechanism of 5-HT modulation of Kv7.4 channel in DRNObjective: To study the effect of 5-HT on the currents of Kv7.4 channel and the excitability of DRN neurons, and to explore the underlying mechanism.Methods: Whole cell patch clamp technique was used to observe the effect of 5-HT on Kv7.4 channel currents in acutely isolated DRN slices of C57BL/6J mice(short for C57 mice). Current clamp recording was used to record the effect of 5-HT on the membrane potential and excitability of serotonergic neurons in DRN slices. 5-HT1 A receptor antagonist WAY-100635, reducing substance DTT and Gi/o-coupled-receptor blocker PTX were used to explore the mechanism of the effect of 5-HT on Kv7.4 channels in DRN.Results:(1) 5-HT increased the currents of Kv7.4 channel in DRN slices.(2) 5-HT induced hyperpolarization of the resting potential and inhibition of excitability of DRN neurons.(3) The regulatory effect of 5-HT on Kv7.4 channel in DRN was completely abolished by the Kv7 channel blocker XE991.(4) The regulatory effect of 5-HT on Kv7.4 channel in DRN was completely abolished by the 5-HT1 A receptor blocker WAY-100635.(5) The regulatory effect of 5-HT on Kv7.4 channel in DRN was completely reversed and abolished by the reducing agent DTT.(6) The regulatory effect of 5-HT on Kv7.4 channel in DRN was completely abolished after 3 h incubation of the DRN slice with Gi/o protein inhibitor PTX.Conclusion: DRN in mice brain contains functional Kv7.4 channels which can be regulated by 5-HT, which through activation of the 5-HT1 A receptor, the Gi/o protein, and the oxidative modification of Kv7.4 channels. Part 2 The establishment of the social defeat model of depression and behavioral assessmentObjective: To establish a social defeat model of depression with depression-like behavior.Methods: Thirty C57 mice in model group were subjected to continuous social defeat stress stimulation with an aggressive CD-1 mouse for 10 days(10 min/ day). Another thirty C57 mice were used as control group with no stimulation. Behavior of depression was evaluated through following three indexes:(1) the change of sucrose preference rate 12 h and 24 h after stress stimulation;(2) body weight change;(3) Social interaction time in the interaction zone in the presence or absence of the CD-1 mouse.Results: Compared with the control group, SD model group showed significant lower body weight, reduced preference rate of sucrose intake and reduced social interaction time.Conclusion: The social defeat model of depression is successfully established. Part 3 The effect of 5-HT on the excitability of DRN neurons in social defeat depression model and in Kv7.4 knockout miceObjective: To observe the effect of 5-HT on the excitability of DRN neurons in social defeat(SD) model and in Kv7.4 knockout mice.Methods: Patch clamp technique of brain slice was used to record the effect of 5-HT on the membrane potential of serotonergic neurons in DRN slices of SD model and in Kv7.4 knockout mice.Results: 5-HT induced a significant hyperpolarizition in DRN neurons of SD model, but had no significant effect on DRN neurons of Kv7.4 knockout mice.Conclusion: 5-HT reduced the excitability of DRN neurons in SD model, which was most likely mediated by activation of Kv7.4 channels, suggesting that Kv7.4 channel could be a target for the treatment of depression.Summary: 5-HT significantly activates Kv7.4 channel in DRN neurons, which result in hyperpolarization of the membrane potential and decreased excitability of neurons. The regulatory effect of 5-HT is realized through the 5-HT1 A receptor, the Gi/o protein, and the oxidative modification of Kv7.4 channels.5-HT reduces the excitability of DRN neurons in SD model, which is most likely mediated by activation of Kv7.4 channels, suggesting that Kv7.4 channel could be a target for the treatment of depression. |
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