Moduation Kv7 Channel Is A Key Regulator Of Dopaminergic Neuronal Excitability And Depression-like Behaviour

Depression is one of the most serious mood disorders which has devastating consequences for the affected individuals, their family, and friends and affects up to 20% of the global population. The World Health Organization predicts that major depression will be the second leading cause of global disability burden by 2020. Current treatments available for major depression are unsatisfactory due to multiple side effects and low efficacy,leaving more than one-third of depressed individuals resistant to drug treatments. In addition, antidepressants usually take several weeks to months to produce a therapeutic response. Thus, new strategies are needed to understand the pathophysiology of major depression and develop new therapeutic treatments. Growing evidence implicates the mesolimbic dopamine(DA) system originating in the ventral tegmental area(VTA) in the pathogenesis and treatment of depression. VTA DA neurons fire either more regular tonic or phasic bursts of action potentials which encode different types of behavior. The phasic burst firing of VTA DA neurons is sufficient to drive behavioral conditioning and elicit dopamine transients with magnitudes not achieved by longer, lower-frequency spiking. More importantly, it has been demonstrated recently that the phasic burst firing pattern of VTA DA neurons directly controls the depression-like behavior in rodent animal models.A key to understand the molecular and cellular mechanisms underlying depression lies in deciphering the firing patterns of VTA DA neurons as well as in the identification of the main molecular players controlling such patterns.Ionotropic NMDA and GABA receptors as well as Ca2+-activated K+(SK)channels, HCN and Kir3/GIRK channels have all been suggested to play important roles in controlling either the intrinsic DA neuronal activity or the abusive drug-induced modulation of such activity. Yet, despite of progress,complete understanding of firing patterns in VTA and what controls these is currently lacking. One emerging concept for treatment of depression is based on modulation of the VTA DA neuron K+ channels; it is based on the several experimental findings. Thus, i) genetic deletion of the TREK1 K+ channels results in a resistance to several inducible depression-like behaviors and to SSRI antidepressants. ii) Selective blockade of TREK1 with spadin or its analogues exerts a fast anti-depressant effect. iii) Alteration of K+ channel expression/activity in VTA DA neurons correlates with the susceptibility of mice to social depression stress.Kv7/KCNQ channels are voltage-dependent potassium channels with four members(Kv7.2–5) being expressed in the CNS. Opening of Kv7 channels leads to neuronal hyperpolarization, thereby stabilizing the membrane potential and decreasing excitability. This makes Kv7 channels particularly interesting as targets in neurological disorders linked to hyperexcitability, including epilepsy, anxiety, pain, migraine as well as addiction to psychostimulants. Interestingly Kv7 channels are expressed in DA neurons and have been suggested to modulate DA neuron excitability and dopaminergic transmission. On a transcriptional level Kv7.4 was found to be a predominant KCNQ gene expressed in VTA DA, yet, functional expression and the potential role in depression of KCNQ channels have not been elucidated. In this study we provide strong evidence that Kv7.4 is a dominant and potent modulator of VTA DA neuron excitability. Due to the much more restricted expression of Kv7.4 in CNS, targeting this channel may offer a new way for treating depression and such a strategy could be less prone to on-target side effects as compared to current treatments.Section 1 Kv7.4 activity controls the excitability of VTA DA neurons Objective: To investigate the effects of fasudil on the excitability of VTA DA neuronMethods:(1) The firing of VTA DA neuron from mice was recorded using in vivo single unit recording technique, and the electrophysiological properties of the firing action potential(AP) were characterized;(2) Effects ofKv7 openers fasudil and retigabine(RTG) injected intraperitoneally on VTA DA firing were investigated.Results:In a separate study, we investigated the effect of Kv7.4 modulators on the spontaneous firing of VTA DA neurons using the in vitro whole-cell patch clamp and cell-attach techniques. In that study, we observed the inhibition of spontaneous firing of VTA DA neurons by fasudil in wild-type mice but not in Kv7.4-/- mice, indicating a selective activation of Kv7.4 by fasudil. However,as stated in the introduction, the burst firing of VTA DA neurons, which is crucial for the release of DA and the related behavior, could not be recorded in these in vitro recordings. As such in this study, we recorded spontaneous firing of VTA DA neurons using the in vivo single unit recordings and studied the effect of fasudil and RTG. In this study using the single unit recording,two patterns of firing were often recorded in the same neurons, the tonic firing and the burst firing, and both total firing frequency and the burst firing properties were analyzed. The electrophysiological characteristics of action potential in VTA DA neuron include:(1) Typical triphasic action potential with a negative deflection;(2) The width of action potential from start to negative trough ≥ 1.1 ms;(3) A characteristic long duration(> 2 ms) and(4)A slow firing rate(< 10 Hz); the burst firing were identified by(1) start of the burst was registered when two consecutive spikes were fired within less than80 ms and(2) end of the burst was registered when interspike interval exceeded 160 ms. In wild-type(WT) mice, fasudil, 20 mg/kg injected intraperitoneally(i.p.), reduced the total firing frequency, the proportion of the mean time spending in burst firing(10 min after injection) and increased the interspike interval(ISI). Neither the tonic firing frequency nor burst firing was affected by fasudil in Kv7.4-/- mice.Conclusion:(1) The tonic and burst pattern of spontaneous firing were recorded in VTA DA neuron in vivo using an extracellular single unit recording method;(2) Fasudil and RTG significantly decreased the spontaneous firing of VTA DA neuron.Section2 The role of Kv7.4 in the development of depression-like phenotype in social defeat model of depressionObjective: To investigate the role of Kv7.4 in the development of depression-like phenotype in social defeat model of depression.Methods:(1) The depression model of social defeat was established;(2)The firing activity of VTA DA neurons in social defeat mice was recorded using the in vivo single unit recording method;(3) The expression of Kv7.4 in VTA DA neurons was meaaured using q RCR and western blot technique and its relationship with the development of depression-like phenotype in social defeat model of depression was analyzed.Result:Since Kv7.4 plays a significant role in controlling excitability of VTA DA neurons and in the auto-regulation by DA, we investigated whether an altered Kv7.4 channel activity is a causal factor for the increased excitability of VTA DA neurons seen in the social-defeat mice model. For this, we first established the model of social defeat. Two altered behaviors: reduced sucrose preference and reduced social interaction time were used to confirm a depression-like state of mice; the animals with both parameters reduced will be referred to as a ‘susceptible mice’(SUS) henceforth. The mice without altered behaviors will be referred to as the ‘unsusceptible mice’(UNSUS).Consistent with the previous reports, the firing activity of VTA DA neurons,recorded using the in vivo single unit recording method, was higher in the SUS mice compared with the control as well as with the UNSUS mice. Importantly,crucial to the depression behaviors, the burst firing also increased in SUS mice as compared to control and UNSUS mice. As demonstrated in the study by Li Li(see Li Li’s thesis), the increased activity of VTA DA neurons is directly correlated wit a down regulation of Kv7/M currents in VTA DA neurons. We then proceeded to test if the reduced currents of Kv7/M were due to a down-regulation of Kv7.4 expression. We first measured Kv7.4 m RNA levels using the q PCR; the results demonstrated that indeed the m RNA levels of Kv7.4 were lower in the SUS mice compared with that in the control and inthe UNSUS mice. We also performed a correlation analysis and found that the firing frequency of the VTA DA neurons was strongly negatively correlated with the expression level of Kv7.4 m RNA, regardless of the SUS/UNSUS phenotype. We also measured the levels of the Kv7.4 protein in VTA and found that in good agreement with the RT-PCR data, the Kv7.4 protein abundance in VTA of SUS mice was similarly lowered as compared to these in the control and the UNSUS mice.Conclusion:(1) The firing activity of VTA DA neuron was significantly increased in social defeat susceptible mice;(2) The expression of Kv7.4 was decreased in the social defeat susceptible mice.Section 3 The effects of modulation of Kv7.4 on VTA DA neuronal excitability and depression-like behavior in social defeat susceptible mice.Objective: To investigate the effect of retigabine(RTG) and fasudil on the excitability of VTA DA neuron and depression-like behavior in social defeat susceptible mice.Methods:(1) The firing activity of VTA DA neuron was recorded using in vivo single unit recording method from the control and the social defeat stress stimulated mice.(2) The social interaction time was measured to assess the depression-like behavior of the mice.(3) The effect of fasudil and RTG injected intraperitonelly were assessed.Result:Using the in vivo single unit recordings we found that the total firing activity of DA neuron in VTA from wiild-type(WT) mice was decreased and the inter-spike interval(ISI) increased significantly following intraperitoneal injection of fasudil(20 mg/kg) or RTG(10 mg/kg) but not the vehicle(0.9%saline, 0.15 ml). Similarly, the burst firing was also decreased after treatment with fasudil and RTG. However, in Kv7.4-/- mice, fasudil failed to affect the firing of VTA DA neurons but RTG remained to be effective albeit with a reduced efficacy. Most importantly, in the wild-type SUS mice the social defeat-induced avoidance behavior was reversed by acute administration of fasudil and RTG. In Kv7.4-/- mice however, fasudil failed to affect socialinteraction time whereas RTG was still effective but with largely variable effects and reduced efficacy. Notably, under the RTG treatment mice became less active e.g. their movement in the open field test was significantly reduced,which was consistent with a previous report. Fasudil on the other hand, did not affect the general activity of WT and Kv7.4-/- mice. These effects of RTG were also observed in Kv7.4-/- mice, indicating a central, Kv7.4-independent sedative effect.Conclusion: The opening of Kv7.4(by fasudil) significantly decreased the firing of VTA DA neuron and reversed the depression-like behavior in social defeat susceptible mice.