The Role Of Kv7.4 In The Modulation Of The Excitability Of DRN 5-HT Neurons And The Depression-like Behavior In A Mice Model Of Social Defeat

Serotonin（5-HT）,as an essential monoamine neurotransmitter in the central nervous system,is implicated in various physiological processes and emotion-related disorders.The serotonergic system originates from the raphe nucleus,which is formed from a collection of cell groups close to the midline throughout the brainstem,and is divided into rostral group,intermediate group and caudal group.The rostral part includes the dorsal raphe nucleus（DRN）and the media raphe nucleus（MRN）,which projects extensively to the forebrain areas,including the amygdala,hippocampus,hypothalamus and almost all neocortical regions,and is the major region involved in emotional regulation.Accumulating evidence suggest that abnormal activity of DRN neurons is linked to psychiatric disorders,such as major depression disorder and anxiety.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.Thus,activity of the 5-HT neurons has to be tightly controlled to maintain emotional homeostasis.The activity of DRN 5-HT neurons is controlled by various membrane receptors as well as by the intrinsic activity of ion channels,such as GABA_B receptors,5-HT_1A receptors,α1 receptors,SK channels,TREK1 channels and GIRK channels.These receptors and channels have all been reported to play an important role in controlling the intrinsic 5-HT neuronal activity.However,complete understanding of the firing activities in the DRN and what controls these activities are currently lacking.For 5-HT neurons,some K⁺channels have been suggested to play important roles in controlling the intrinsic neuronal activity,and modulating these channels may alleviate emotional disorders.For example,blocking TREK1 channels substantially increases the firing rate of5-HT-ergic neurons in the DRN and induces a significant antidepressant-like response in a depression model of rats.Deletion of the GIRK2 or blocking the activity of GIRK2 in DRN promotes a depression-resistant phenotype.Up-regulation of SK3 channels in serotonin-producing neurons are responsible for greatly reduced activity in these neurons from a model of isolated mice;blocking these inhibitory SK3 channels restores normal activity in the serotonin-producing cells and alleviates the depressive symptoms of the isolated mice.Kv7/KCNQ channels are voltage-dependent potassium channels widely expressed in the central nervous system,with the feature of slow-activation,non-inactivation,slow-deactivation.Opening of Kv7 channels leads to neuronal hyperpolarization,thereby stabilizing the membrane potential and decreasing excitability.Five members（Kv7.1-Kv7.5）have been found for the family of Kv7.As far as we know,no Kv7 channels except Kv7.4 channels are found to be expressed in the DRN.Some studies have indicated Kv7 channels being as targets in various nerve system disorders including anxiety,pain,epilepsy and drug addiction.However,the functional role of Kv7.4 in regulating the excitability of DRN 5-HT neurons has not been elucidated.The 5-HT neurons release 5-HT,which acts on their receptors to exert its cellular and physiological functions.Among 14 different reported 5-HT receptors,the 5-HT_1AA receptor has been shown to be involved in mood-related behavior including anxiety and depression.The presynaptic 5-HT_1AA auto-receptors are believed to mediate the serotonin-induced auto-inhibition,which inhibits the electrical activity of 5-HT neurons,reduces the release of neurotransmitter.Up to now,GIRK channel has been the sole proposed mechanism for 5-HT_1A receptor-mediated auto-inhibition,and it is not known if other mechanisms are also involved in this regulation process.In this study we provide evidence that activation of Kv7.4 significantly reduce the excitability of DRN 5-HT neurons and alleviate the depression-like behavior of a social defeat model.Moreover,we characterized the Kv7.4channels as the regulation target of 5-HT_1A receptors which play a role in the5-HT-mediated auto-inhibition.Part 1 Kv7.4 activity modulates the excitability of the DRN 5-HT neuronsObjective:To investigate the role of Kv7.4 channel in regulation of DRN5-HT neuronal excitability.Methods:（1）Immunohistochemistry and single-cell PCR were used to determine the Kv7.4 channel distribution pattern and the proportion of Kv7.4 positive neurons in TPH positive neurons that are regarded as 5-HT neurons.（2）Whole-cell and cell-attached patch clamp recording were used to investigate the effect of Kv7.4 opener on the Kv7/M current,membrane potential,induced firing and spontaneous firing of DRN 5-HT neurons.Results:（1）Immunohistochemistry results showed a strong expression of the Kv7.4 channel in DRN 5-HT neurons,especially in the ventral part（DRV）.Single-cell PCR results showed that Kv7.4 positive neurons account for approximately 67%of the 5-HT neurons in the DRN.（2）Bath-application of Kv7.4 opener fasudil（30M）significantly augmented the outward potassium currents recorded from a holding potential of-20 mV（from 90.7±14.9 p A to 109.9±18.0 pA,n=8,P<0.05）in DRN5-HT neurons and the augmentation was completely reversed by XE991（3M）;deletion of Kv7.4(Kv7.4^-/-mice)had a trend to reduce the basal Kv7/M currents（from 90.7±14.9 pA to 54.9±8.9 pA,n=10,P=0.056）and completely abolished the augmentation of fasudil on Kv7 currents in DRN5-HT neurons（from 54.9±8.9 pA to 51.3±9.1 pA,n=10,P>0.05）.（3）Fasudil（30M）significantly hyperpolarized the resting membrane potential（RMP）（from-61.4±3.8 mV to-64.0±3.4 mV,n=9,P<0.001）of DRN 5-HT neurons in WT mice and had no significant effect on Kv7.4^-/-mice（from-62.6±2.0 mV to-61.5±1.7 mV,n=10,P>0.05）.（4）Fasudil（30 M）also reduced neuronal discharge induced by 250 pA depolarizing current.In WT neurons,the mean spike number during 500 ms was 8.0±1.4,fasudil reduced this to 6.7±2.3（n=9,P<0.05）;fasudil did not affect neuronal discharge in Kv7.4^-/-mice（from 8.0±1.2 to 8.0±1.0,n=10,P>0.05）.（5）In cell-attach patch clamp mode,an agonist ofα1 adrenergic receptor phenylephrine（PE）was used to induce the spontaneous firing of 5-HT neurons in DRN brain slices.Fasudil significantly reduced the average firing frequency of DRN 5-HT neurons from 1.52±0.15 Hz to 1.06±0.18 Hz（n=11,P<0.001）in WT mice and had no effect on the firing frequency in Kv7.4^-/-mice（from 1.22±0.12 Hz to 1.14±0.10 Hz,n=14,P>0.05）.Conclusions:（1）Kv7.4 is strongly expressed in DRN 5-HT neurons,especially in the ventral part（DRV）.（2）Kv7.4 is the functionally dominant Kv7 channel subunit in DRN 5-HT neurons.（3）Activation of Kv7.4 by Fasudil significantly hyperpolarizes the membrane potential and inhibits neuronal excitability of DRN 5-HT neurons,in WT mice but not in Kv7.4^-/-mice,which demonstrated a crucial role of Kv7.4in modulation of excitability in DRN 5-HT neurons.Part 2 The modulation of Kv7.4 activity by 5-HT in DRN 5-HT neurons and the role in the depression-like behaviorObjective:To study the effect of 5-HT on the Kv7.4 activity and explore the underlying mechanism;To test the role of Kv7.4 channel in the development of depression-like behavior in the social defeat model of depression.Methods:（1）Whole-cell patch clamp recording was used to study the effect of 5-HT on the Kv7.4 activity in DRN slices.（2）The social defeat mice model of depression was established and effect of fasudil injected locally in DRN on depression-like behavior was investigated.Results:（1）Bath-application of 5-HT（30μM）induced a sharp augmentation of Kv7 currents（form 107.0±13.1 pA to 146.8±20.3 p A,n=14,P<0.001）,which were completely reversed by XE991（3μM）in DRN 5-HT neurons of WT mice.5-HT did not affect the Kv7 currents in Kv7.4^-/-mice（from 93.9±14.0 pA to 86.4±14.5 pA,n=10,P>0.05）.（2）5-HT_1A agonist 8-OH-DPAT induced a similar activation of the Kv7currents as 5-HT（from 72.9±13.2 pA to 104.4±16.2 pA,n=8,P<0.01）,while 5-HT_1A receptor antagonist WAY-100635 blocked the effect of 5-HT in DRN 5-HT neurons of WT mice（from 75.4±13.5 pA to 74.8±11.3 p A,n=11,P>0.05）.（3）5-HT activation of Kv7.4 currents was reversed by a reducing agent,dithiothreitol（DTT,1 mM）,or prevented by DTT（from 118.5±28.3 pA to83.8±22.6 pA,n=11,P<0.05）.（4）In social defeat mice,the social interaction time was significantly increased by local injection of fasudil in DRN（from 18.5±5.0 s to 61.9±4.6 s,n=11,P<0.001）,whereas the move distance of the mice in the open field test was not affected by fasudil.Conclusions:DRN in mice brain contains functional Kv7 channels which can be regulated by 5-HT,which is mediated by the 5-HT_1AA receptor,with a redox mechanism.Activation of Kv7.4 in DRN can alleviate the depression-like behavior in the mice model of social defeat.Part 3 The role of Kv7.4 and GIRK2 in 5-HT-mediated auto-inhibition of neuronal excitability of DRN 5-HT neuronsObjective:To observe the role of Kv7.4 and GIRK2 in 5-HT-mediated auto-inhibition of neuronal excitability of DRN 5-HT neurons.Methods:The whole-cell patch clamp and cell-attach patch clamp technology were used to study the effect of 5-HT on the excitability of DRN5-HT neurons in WT mice,Kv7.4^-/-mice,GIRK2^-/-mice and Kv7.4^-/-&GIRK2^-/-mice.Results:（1）In the presence of 5-HT（30μM）,RMP was markedly hyperpolarized in WT mice（from-55.7±1.7 mV to-66.3±2.0 mV,n=9,P<0.001）;5-HT also significantly hyperpolarized RMP（from-55.2±1.5 mV to-61.5±0.9 mV,n=11,P<0.01）of DRN 5-HT neurons in Kv7.4^-/-mice and in GIRK2^-/-mice（from-53.5±2.2 mV to-62.2±2.0 mV,n=9,P<0.01）;5-HT did not affect the RMP in the Kv7.4^-/-&GIRK2^-/-mice（from-57.5±1.9 mV to-60.6±2.0mV,n=7,P>0.05）.（2）The current/spike relationship was more prominently suppressed by5-HT in DRN 5-HT neurons from WT mice compared to Kv7.4^-/-mice（P<0.05）and that from the GIRK2^-/-mice（P=0.385）.5-HT did not affect the evoked firing in the Kv7.4^-/-&GIRK2^-/-mice.（3）The spontaneous activity of DRN 5-HT neurons from WT mice was completely eliminated by 5-HT（15/15 neurons）.In contrast,5-HT eliminated spontaneous activity in only 15/19 neurons from the Kv7.4^-/-mice and in only13/17 neurons from the GIRK2^-/-mice;the remainder showed an incomplete suppression of activity.In the Kv7.4^-/-&GIRK2^-/-mice,5-HT eliminated spontaneous firing activity in only 3/8 5-HT neurons.Conclusions:Kv7.4,similar to GIRK2,plays a major role in5-HT-mediated auto-inhibition of DRN 5-HT neuronal activity.However,there are also other channels which are likely involved in the 5-HT induced inhibition of spontaneous firing of DRN 5-HT neurons.