Kv7.4 And Kir3.2 K~+ Channels Underlie Projection-specific Auto-inhibition Of Dopamine Neurons In Ventral Tegmental Area

The brain's reward circuitry is composed of the ventral tegmental area?VTA?and their projection targets including the nucleus accumbens?NAc?,medial prefrontal cortex?mPFC?,and the amygdala.Dopamine?DA?in this system has been shown to be involved in reward-related behaviors,particularly the depression and drug addiction,and also in non-reward-related behaviors,such as pain and aversion.Different projection targets of VTA DA neurons are correlated with their different anatomic distributions,electrophysiological characteristics and different aspects of behaviors.Electrophysiological characteristics of VTA DA neurons are represented by different firing patterns,presence/absence of hyperpolarization-activated cyclic nucleotide-gated currents?HCN;Ih?,presence/absence of D₂R and associated Kir3/GIRK mediated auto-inhibition.These findings highlight the importance of differentiating and defining DA neuron subpopulations.VTA DA neurons fire either more regular tonic or phasic bursts of action potentials which encode different types of behavior.It has been observed that repeated social defeat stress increases bursting activity of VTA DA neurons which elicits dopamine transients with magnitudes,directly contributing to the depression-like behavior of the model mice.Ionotropic NMDA and GABA receptors as well as Ca²⁺-activated K⁺?SK?channels,Kv7 and Kir3/GIRK channels have all been suggested to play important roles in controlling the intrinsic DA neuronal activity.K⁺channels play a crucial role in regulation of the VTA DA neuronal excitability,and is also the basis for the generation and transmission of neuronal excitability.Dopamine-mediated auto-inhibition of DA activity is an important mechanism for the modulation of DA firing/activity.Somatodendritic DA release in the VTA decreases the excitability of dopamine neurons through activating D2-autoreceptors on DA neurons.This auto modulation involves D2 receptor and K⁺channels of Kir family?GIRK?,which are linked by G?_i/o/o type of G protein,leading to hyperpolarization of the cells and inhibition of neuronal excitability.Recent work suggests that there is further variability in VTA DA neurons in the context of D₂R-Kir3/GIRK modulatory pathway.Another K⁺channel family that appears to be an important regulator of VTADA firing/activity is the Kv7/KCNQ channels.Four members of Kv7/KCNQ channels?Kv7.2-7.5?are expressed in the CNS.Stimulation of Kv7 channel leads to hyperpolarization of the cells and inhibition of neuronal activity.Kv7.4 is the dominant isoform of Kv7 family expressed in VTA DA neurons.Kv7 channels could serve as targets in neurological disorders linked to hyperexcitability including pain,epilepsy,anxiety as well as addiction to psychostimulants.Outside the known D₂R-Kir3/GIRK pathway,the Kv7channel is another mechanism that influences the excitability of VTA DA neurons,especially in the low expression on the Kir3/GIRK channel of neurons.The purpose of this study is to investigate the role of Kv7.4 and Kir3.2 in the regulation of the excitatory activity of NAc-and BLA-projecting dopamine neurons.Part 1 Heterogeneity of dopaminergic neurons in VTA region projectingto different brain targetsObjective:To investigate heterogeneity of dopaminergic neurons in VTA region projecting to different brain targetsMethods:Identification of VTA DA neurons with different projection targets was confirmed by retrogradely labeled DA neurons using retrograde beads and immunohistochemistry;the electrophysiological characteristics of dopaminergic neurons projecting to target area of VTA were studied by cell-attached patch clamp technique;single cell PCR technology was used to observe the expression difference of VTA dopaminergic markers and Kv7.4and Kir3.2 ion channels.Results:?1?The distribution of projection-specific dopaminergic neurons in the VTA:Immunohistochemical staining showed DA neurons projecting to NAc core clustered in the VTA subregions of medial paranigral?PN?nucleus and the adjacent medioventral aspects of the parabrachial?PBP?nucleus,the DA-mPFC projection DA neurons mainly located in the interfascicular?IF?nucleus close to midline,while DA neurons projecting to BLA were scattered throughout the PBP.?2?The electrophysiological characteristics of dopaminergic neurons projecting to different targets:The firing frequency of VTA DA neurons projecting to NAc and BLA was lower than that projecting to mPFC?P<0.05?.Also,the action potential duration of firing of VTA DA neurons projecting to the NAc was significantly shorter than that of VTA DA neurons projecting to mPFC?P<0.05?.?3?The effect of DA and baclofen on the excitatory of dopaminergic neurons projecting to each target area could be summarized as follows:in a loosely cell-attached patch recording DA inhibited the spontaneous firing of dopaminergic neurons projecting to NAc and BLA?P<0.05?,but no such effect was found in DA-mPFC neurons?P>0.05?.In contrast,after application of baclofen,the firing frequency of dopaminergic neurons projecting to each of the targets?NAc,mPFC and BLA?was significantly reduced?P<0.05?.?4?Approximately 80%–95%of the NAc core-and BLA-projecting neurons were positive for TH and DAT expression,indicating that most of them were indeed DA neurons.In contrast,less than 30%of VTA neurons projecting to mPFC expressed detectable levels of TH and none of them expressed DAT.Furthermore,while D2 receptor mRNA was highly abundant in the NAc core-and BLA-projecting VTA DA neurons,it was absent in the mPFC-projecting VTA TH-positive neurons.The expression of Kv7.4 in projection-specific DA neurons is similarly high?50%-70%?in DA-NAc,DA-BLA and DA-mPFC neurons;Kir3.2 was also found to be abundantly expressed among these three population neurons?60%⁹0%?.Conclusions:?1?The firing frequency of VTA DA neurons projecting to NAc and BLA is lower than that of VTA DA neurons projecting to mPFC.?2?DA inhibited the spontaneous firing of dopaminergic neurons projecting to NAc and BLA,but no effect was found in DA-mPFC neurons.?3?The expression of Kir3.2 and Kv7.4 in projection-specific VTA DA neurons was similarly distributed.Dopaminergic markers such as TH,DAT and D₂R were expressed in neurons projecting to NAc and BLA,but their expression decreased significantly in neurons projecting to mPFC.Part 2 Kv7.4 and Kir3.2 contribute to DA-induced inhibition on firing ofNAc-projecting VTA DA neuronsObjective:To investigate the effect of Kv7.4 and Kir3.2 on the DA-mediated auto-inhibition in the VTA DA neurons projecting to NAc.Methods:mRNA in situ hybridization and immunofluorescence technique was used to detect the expression of Kv7.4 and Kir3.2 in the VTA;the role of Kv7.4 and Kir3.2 in DA-mediated inhibition on excitability of VTA DA neurons projecting to NAc in wild type?Kv7.4^-/-and Kir3.2^-/-mice was studied using patch clamp technique.Results:?1?In situ hybridization and immunofluorescence results showed that Kv7.4 and Kir3.2 channels were expressed in TH immunoreactive neurons in VTA region.?2?The effect of Kv7.4 and Kir3.2 on the DA-mediated auto-inhibition in the VTA-NAc projection pathway:DA?20?M?significantly reduced the average firing frequency of VTA DA-NAc neurons in WT mice?P<0.05??Kv7.4^-/-?P<0.05??Kir3.2^-/-?P<0.05?and Kv7.4^-/-/Kir3.2^-/-mice.In WT mice,the DA-induced inhibition was mostly reversed by XE991?P<0.05?;In Kv7.4^-/-mice,the DA effects were not significantly affected by XE991?P>0.05?.In Kir3.2^-/-mice,the DA effect was partly reversed by XE911?P<0.05?.Tertiapin-Q partially reversed the DA-induced inhibition of firing of DA neurons in WT and Kv7.4^-/-mice?P<0.05?but had no significant effect in Kir3.2^-/-mice?P>0.05?.?19/23 cells?of DA-NAc neurons in WT mice,71%?17/24 cells?of neurons from Kv7.4^-/-mice?43%of neurons?10/23 cells?from Kir3.2^-/-mice and 20%?2/10?neurons from Kv7.4^-/-/Kir3.2^-/-mice were completely inhibited by DA.?3?DA?20?M?potentiated Kv7/M-like currents from 71.0±13.6 pA to93.0±15.1 pA?P<0.05?,which were blocked by XE911 from 93.0±15.1pA to 39.0±7.1 p A?P<0.05?.This slowly-deactivating M-like current was completely absent in the neurons from Kv7.4^-/-mice.Similarly,DA also activated the Kir3.2-like currents in DA neurons projecting to NAc from WT mice?P<0.05?,but not in Kir3.2^-/-mice?P>0.05?.Conclusions:?1?Kv7.4 and Kir3.2 contribute to DA-induced inhibition on firing of NAc-projecting VTA DA neurons.?2?In addition to Kv7.4 and Kir3.2 channels,there are other ionic mechanisms involving in the DA-induced inhibition on firing of NAc-projecting VTA DA neurons.Part 3 Kv7.4 and Kir3.2 contribute to DA-induced inhibition on firing ofBLA-projecting VTA DA neuronsObjective:To investigate the effect of Kv7.4 and Kir3.2 on the DA-mediated auto-inhibition in the VTA DA neuronsprojecting to BLA.Methods:The role of Kv7.4 and Kir3.2 in DA-mediated inhibition on excitability of VTA DA neurons projecting to BLA in wild type?Kv7.4^-/-and Kir3.2^-/-mice was studied using patch clamp technique.Results:?1?The effect of Kv7.4 and Kir3.2 on the DA-mediated auto-inhibition in the BLA-NAc neurons could be summarized ad follows:The average firing frequency of VTA DA-BLA neurons in WT mice?P<0.05??Kv7.4^-/-?P<0.05?and Kir3.2^-/-?P<0.05?mice was significantly reduced by DA?20?M?.The Kv7 blocker,XE991 reversed the DA-induced inhibition in WT mice?P<0.05?;but in Kv7.4^-/-mice,XE991 did not affect the effects of DA?P>0.05?.In Kir3.2^-/-mice,the DA effect was partly reversed by XE911?P<0.05?.Tertiapin-Q partially reversed the DA-induced inhibition of firing of DA neurons in WT and Kv7.4^-/-mice?P<0.05?but had no significant effect in Kir3.2^-/-mice?P>0.05?.?2?DA?20?M?potentiated Kv7/M-like currents from 58.8±10.3 pA to85.0±9.4 pA?P<0.05?,which were blocked by XE911 from 85.0±9.4 pA to 33.8±4.3 pA?P<0.05?.This slowly-deactivating M-like current was completely absent in the neurons from Kv7.4^-/-mice.Similarly,DA also activated the Kir3.2-like currents in DA neurons projecting to BLA from WT mice?P<0.05?,but not in Kir3.2^-/-mice?P>0.05?.Conclusions:Kv7.4 and Kir3.2 contribute to DA-induced inhibition on firing of VTA DA neurons projecting to BLA.