The Role Of Potassium Channel In Regulation Of Neuronal Excitability Of VTA DA Neurons And Its Role In The Depression-like Behavior Of A Social Defeat Model Of Depression

Depression is a chronic mood disorder, in which patients suffer from repeated continuous low mood, negative sentiment and are prone to suicide. World Health Organization predicts that depression will become the second most prevalent human disease by 2020. The current available treatments for major depression,such as selective serotonin/norepinephrine re-uptake inhibitors and monoamine oxidase inhibitors, are unsatisfactory due to low efficacy. As such to further understand the molecular mechanism of the depression and to find new therapeutic target for antidepressants have become one of the focus of global research.Growing evidence implicates the mesolimbic dopamine(DA)system originating in the ventral tegmental area(VTA) in the pathogenesis and present a target for treatment of depression. A recent study finds that chronic social defeat stress induces an increased activity in VTA DA neurons which plays a key role in depression-like behavior of this mice model of depression.Therefore, a key to understand the molecular and cellular mechanisms underlying depression lies in deciphering the firing activity of VTA DA neurons.VTA DA neurons fire either more regular tonic or phasic bursts of action potentials. The phasic burst firing of VTA DA neurons elicits dopamine transients with magnitudes not achieved by the longer, lower-frequency spiking, therefore more efficiently regulates the VTA and the projection neuron activity. It has beenobserved that repeated social defeat stress increases bursting activity of VTA DA neurons which contribute directly to the depression-like behavior of the model mice. It is also importantly noted in this case that an elevated expression of K+channels renders some mice not having increased activity in VTA DA neurons and not having the depression-ilke behavior, indicating a crucial role for K+channels in regulation of the VTA DA neuronal excitability and the depression behaviors. It is further demonstrated that an increased K+function could completely eliminate the depression-like behavior in social defeat mice model of depression. Other evidence also suggests a role for K+channels in depression: genetic deletion of the TREK1 K+channels results in a resistance to several inducible depression-like behaviors;selective blockade of TREK1 exerts a fast anti-depressant effect.Therefore, one emerging concept for treatment of depression is based on modulation of the neuronal K+channels.Kv7/KCNQ K+channel is expressed widely in the nervous system. Kv7 channels make up the M-type currents which plays an important role in regulation of neuronal excitability. Activation of Kv7/M channels limits repeated discharge, thus potentially limits sustained bursting firing. Studies suggest that Kv7 channels can be therapeutic targets of central nervous system disease associated with abnormal excitability, such as pain, anxiety and drug addiction and epilepsy. There are five members of this familyKv7.1 to Kv7.5. In ventral midbrain, while Kv7.2-Kv7.5 widely expressed, Kv7.4 channels prominently expressed in dopaminergic neurons of substantia nigra pars compacta(SNc) and VTA. In the first part and the second part of this study we focus on the effect of Kv7.4 channel in regulation of VTA DA neuronal excitability, and the role of this modulation in depression-like behavior of social defeat mice model of depression.Apart from Kv7 channels, past studies also implicate other K+channels in the regulation of neuronal excitability of VTA DA neurons, these include G protein-coupled inwardly rectifying K+channel(Kir3/GIRK), A-type K+channels, Ca2+-activated K+(SK)channels and two-pore-domain potassium channel. Yet, it is not understood how these K+channels modulate the excitability of VTA DA neurons. In the third part of this study we will examine the expression of these K+channels in the VTA DA neurons, assess their contribution to the regulation of neuronal excitability, and we will focus our study mainly on A-type K+channels.Part one The role of Kv7.4 channel in regulation of VTA DA neurons excitabilityObjective: To investigate the role of Kv7.4 channel in regulation of VTA DA neuronal excitability of miceMethods:(1) Immunohistochemistry, whole-cell and cell-attached patch clamp technique were used to identify VTA DA neurons and to record the neuronal excitability in the brain slice of mice.(2) Immunofluroscence technique was used to observe the expression of Kv7.4 channel on VTA DA neurons.Results:(1) VTA DA neurons were identified by the following electrophysiological characteristics: i) A sag hyperpolarization potential during injection of a hyperpolarizing current pulse(mean amplitude of 60 ± 3.7 m V with a-300 p A current injection);ii) An action potential width from start to negative trough > 1.1 ms(2.6 ± 0.1 ms); iii) An action potential with a negative deflection,and an action potential width from start to rest > 2.5 ms(5.5 ± 0.2ms); iii) Firing rate less than 5 Hz(1.3 ± 0.1 Hz);(2) Kv7.4immunopositive staining was found on the TH immunopositive neurons in lateral VTA and part of the SNc, and no Kv7.4 staining was observed in Kv7.4-/-mice. Non-selective Kv7 channel openers RTG increased Kv7/M-like currents in VTA DA neurons in aconcentration-dependent manner, with an EC50 of 3.4 ± 0.3 μM. 10μM RTG increases Kv7/M-like currents from 45.6 ± 9.1 p A to 80.2 ±14.1 p A(P < 0.01); 3 μM XE911 decreased Kv7/M-like currents from 59.1 ± 6.4 p A to 26.0 ± 3.1 p A(P < 0.01)(inhibition rate is45.6 ± 6.0%). Deletion of Kv7.4(Kv7.4-/-) greatly reduced the XE991 sensitive Kv7/M-like K+current(inhibition rate is 11.8 ±4.3%) and completely abolished the RTG-induced enhancement of Kv7/M-like K+current in VTA DA neurons.(3) In CHO cells exogenously expressed Kv7.4, fasudil(0.1, 1, 10, 30, 100 μM)enhanced Kv7.4 current in a concentration-dependent manner,with an EC50 of 14.2 ± 0.6 μM; fasudil had no effect on Kv7.2 and Kv7.2/Kv7.3. Similarly in the DA neurons of VTA brain slice, fasudil also concentration-dependently increased the Kv7/M current, with an EC50 of 16.8 ± 1.1 μM, which was reversed by 3 μM XE991;fasudil did not induce current in VTA DA neuron of Kv7.4-/-.(4)XE991(3 μM) significantly depolarized the resting membrane potential(RMP) of VTA DA neurons from-65.3 ± 3.1 m V to-62.9 ±3.0 m V(P < 0.01). Fasudil(10 μM) significantly hyperpolarized the RMP from-66.6 ± 3.6 m V to-72.4 ± 4.0 m V(P < 0.001). In stark contrast, in the Kv7.4-/-mice these effects of XE991 and fasudil disappeared.(5) Fasudil(10 μM) dampened the spike number with the increased magnitude of the injected currents in wild-type mice but not in Kv7.4-/-mice. Injection of 200 p A depolarizing current induced 13.0 ± 1.7 spikes in wild-type mice whereas a same magnitude of current induced 21.6 ± 2.9 spikes in Kv7.4-/-mice(P< 0.05).(6) Fasudil inhibited the spontaneous firing, recorded using the whole-cell patch clamp technique, from 1.5 ± 0.3 Hz to0.6 ± 0.2 Hz in VTA DA neurons from wild-type mice(P < 0.05), the effect of fasudil disappeared in Kv7.4-/-mice. Similarly in a loosely cell-attached patch recording, fasudil reduced the firing of VTA DA neurons in WT mice(from 1.7 ± 0.3 Hz to 0.8 ± 0.3 Hz, P < 0.05)but no effect of fasudil was found in Kv7.4-/-mice. Average firing frequencies of VTA DA neurons from wild-type and Kv7.4 mice was1.7 ± 0.3 Hz and 3.6 ± 0.6 Hz, respectively(P < 0.05).Conclusions:(1) Kv7.4 is mainly expressed in the TH immunopositive neurons of the lateral VTA and part of the SNc.(2)Fasudil selectively activates Kv7.4 and did not affect Kv7.2 and Kv7.2/Kv7.3 expressed in CHO cells; Fasudil increases Kv7/M currents in VTA DA neurons in wild type mice but not in Kv7.4-/-mice; Kv7.4 is the dominant Kv7 channel subunit in VTA DA neurons.(3) Fasudil hyperpolarizes the membrane potential,inhibits the evoked firing and the spontaneous firing of VTA DA neurons in wild type mice but not in Kv7.4-/-mice. Fasudil inhibits neuronal excitability of VTA DA neurons through activation of Kv7.4.Part 2 The role of Kv7.4 in dopamine-mediated auto-modulation of neuronal excitabilty of VTA DA neurons and its role in depression-like behavior of mice model of depressionObjective:(1) To test whether Kv7.4 plays a role in the dopamine-mediated auto-modulaton of VTA DA neuronal excitability.(2) To study whether Kv7.4 plays a role in the development of depression-like behavior in the social defeat mice model of depression.Methods:(1) The whole-cell patch clamp, cell-attached patch clamp technology and Kv7.4-/-mice were used to observe the role of Kv7.4 channels in dopamine-mediated auto-regulation.(2) A social defeat mice model of depression was established.(3)Cell-attached patch clamp technique was used to observe the excitability of VTA DA neurons from social defeat model.Results:(1) In wild-type mice, 20 μM and 100 μM dopamine(DA) increases Kv7/M current of VTA DA neuron by 42% and 53%,respectively, which was significantly reduced in Kv7.4-/-mice(100μM dopamine, 14%), and this effect of DA in WT mice was blocked or prevented by XE991.(2) 20 μM DA hyperpolarized the RMP from-65.3 ± 2.9 m V to-75.9 ± 2.1 m V in WT mice(P < 0.01), and this effect of DA was reduced in Kv7.4-/-mice(from-65.0 ± 3.3 m V to-67.9 ± 3.2 m V, P < 0.05) and blocked by XE991. Dopamine changed the RMP of VTA DA neurons from WT mice(pretreated by XE991) and Kv7.4-/-mice by 2.5 ± 2.0 m V å'Œ 2.9 ± 1.2 m V,respectively(P > 0.05).(3) In WT and Kv7.4-/-mice, DA reduced average firing rate of VTA DA neurons from 1.9 ± 0.3 Hz and 2.5 ±0.3 Hz to 0.6 ± 0.2 Hz and 1.7± 0.4 Hz, respectively(P < 0.01,P <0.05); the inhibition rate was 62.1 ± 11.3% and 39.1 ± 10.3%,respectively(P < 0.001).(4) Kv7-dependent neurons(73%) and Kv7-independent neurons(27%) were identified based on their sensitivity to XE991. In Kv7.4-dependent neurons, DA induced an inhibition of firing which was prevented by XE991, and in Kv7.4-independent neurons DA induced an inhibition of firing which was not blocked by XE911.(5) D2 receptor blocker sulpiride(300 n M) but not a D1 receptor blocker SCH23390(1 μM),inhibited the DA-induced activation of Kv7/M-like currents. PTX(Gi/o blocker, 500 ng/ml) and DTT(dithiothreitol, a reducing agent,1 m M) has similar effect. D2 receptor antagonist sulpiride also abolished the effect of DA on membrane potential hyperpolarization and firing inhibition.(6) The amplitude of Kv7/M currents were reduced in the mice susceptible to social defeat stimulation(SUS mice) compared with these in the control and in the mice unsusceptible to social defeat stimulation(UNSUS mice).(7) DA failed to affect significantly the firing rate of VTA DA neurons from the SUS mice. Fasudil reduced the firing rate of VTA DA neurons in SUS mice although it was less efficacious than in the control mice(1.46 ± 0.25 Hz and 42.2 ± 13.5% reduction in thecontrol and 2.53 ± 0.39 Hz and 31.6 ± 12.2%, P > 0.05) compaired with reduction in the SUS mice in the presence of fasudil).Conclusions:(1) The DA-induced enhancement of Kv7.4currents in VTA DA neurons is mediated by the D2(but D1)receptor, with a down stream Gi/o and redox mechanism. The DA-induced hyperpolarization of the RMP and inhibition of spontaneous firing is mediated by the D2 but not by the D1 receptor.(2) ~27% DA neurons in VTA use mechanisms other than Kv7.4 to mediate DA-induced inhibition of firing activity, but the majority of the neurons(~73%) rely on Kv7.4.(3) The Kv7/M currents and expression are reduced in the mice susceptible to social defeat stress compared with these in the control and in the mice unsusceptible to social defeat stress.(4) DA fails to affect the firing of VTA DA neurons in the SUS mice; but fasudil is still able to inhibit the firing of VTA DA neurons.Part 3 The role of A-type K+channel in the regulation of excitability of VTA DA neuron and its physiological significanceObjective: To compare the expression of different K+channel including A-type K+channels, Kv7 K+channel, SK K+channel and GIRK K+channel and their effects on the excitability of mice VTA DA neurons, with a major focus on A-type K+channels; to study the relationship between A-type K+channel and glutamate receptors on the regulation of VTA DA neuronal excitability.Methods: Whole-cell and cell-attached patch clamp techniques and behavioral tests were used.Results:(1) The voltage-gated transient outward currents were increased in the mice unsusceptible to social defeat stimulation(UNSUS mice) compared with these in the control mice(P < 0.05) and in the mice susceptible to social defeat stimulation(SUS mice, P > 0.05).(2) Immunofluorescence resultsshowed that Kv7.4 was highly expressed in the lateral VTA and part of SNc; SK3 channel was highly expressed in the VTA area close to midline; Kv7.2 positive neurons distributed in SNc and the lateral VTA; Kv4.3 channel was widely distributed in VTA and SN; Kv7.5was weakly distributed in VTA and SN. All SK3 and Kv4.3 channels,as well as some of Kv7.2 and Kv7.5 channels, were expressed on TH positive neurons.(3) A-type K+channel blocker 4-AP(2 m M),Kv7/M channel blocker XE991(3 μM), GIRK channel blocker tertiapin(50 n M), Ca2+-activated K+channel SK3 blocker apamin(10 n M), hyperpolarized the resting membrane potential(RMP)from-61.3 ± 3.0,-65.3 ± 3.0 m V,-62.3 ± 6.1 m V,-60.2 ± 4.2 m V to-55.9 ± 2.6 m V(P < 0.001),-62.9 ± 3.0 m V( P < 0.01),-58.9 ± 6.1m V( P < 0.001),-56.5 ± 5.4 m V(P > 0.05), respectively; RMP was changed by 5.8 ± 1.0 m V, 2.4 ± 0.7 m V, 3.5 ± 0.3 m V, 0.93 ± 0.04 m V, respectively.(P < 0.05 for XE991 or tertiapin, P < 0.001 for apamin, compaired with 4-AP).(4) 4-AP, XE991, tertiapin, apamin changed the firing rate from 1.8 ± 0.2 Hz, 1.9 ± 0.3 Hz, 1.6 ± 0.2 Hz,1.3 ± 0.3 Hz to 2.8 ± 0.3 Hz(P < 0.01), 2.1 ± 0.3 Hz(P > 0.05), 1.8 ±0.2 Hz(P < 0.05), 1.6 ± 0.3 Hz(P > 0.05), respectively; increasing the firing rate by 65.8 ± 14.1%, 13.9 ± 5.5%, 16.9 ± 5.9%, 41.7 ±23.2%, respectively(P < 0.01 for XE991 or tertiapin, compaired with 4-AP).(5) Under condition of pretreatment and continuous presence of NMDA(20 μM), 4-AP increased the average firing rate from 1.7 ± 0.3 Hz to 2.1 ± 0.4 Hz(P < 0.05); Under condition of pretreatment and continuous presence of picrotoxin(GABAA blocker, 100 μM) and kynurenic acid(glutamate receptors blocker,100 μM), 4-AP did not affect the firing rate(from 2.0 ± 0.4 Hz to3.3 ± 1.3 Hz, P > 0.05); Under condition of pretreatment and continuous presence of picrotoxin(100 μM), CNQX(AMPA and kainate receptor blcoker, 12.5 μM) and NMDA, 4-AP increased firing rate from 4.0 ± 0.8 Hz to 5.0 ± 1.1 Hz(P < 0.05).(6) Undercondition of pretreatment and continuous presence of NMDA(20μM), 4-AP increased the coefficient variance of intern spike interval(CV-ISI) from 0.4 ± 0.08 to 0.6 ± 0.1(P < 0.05); Under condition of pretreatment and continuous presence of picrotoxin(100 μM) and kynurenic acid(100 μM), 4-AP did not affect CV-ISI(from 0.5 ± 0.1 to 0.5 ± 0.06, P > 0.05); Under condition of pretreatment and continuous presence of picrotoxin(100 μM),CNQX(12.5 μM), NMDA, 4-AP increased CV-ISI from 0.6 ± 0.1 to0.8 ± 0.3(P < 0.05).Conclusions:(1) Kv4.3 channels highly expressed in the TH-positive neurons through the midbrain.(2) Under resting condition, A-Type K+channel regulates the resting membrane potential and the spontaneous firing rate of VTA DA neurons;blockade of NMDA receptor may inhibit the effect of A-Type K+channel on spontaneous firing; A-Type K+channel alters the regularity of firing activity of VTA DA neurons, which needs an active NMDA receptor.