Ion Channel Mechanism Of Phenylephrine-induced Spontaneous Firing In DRN 5-HT Neurons

The serotonergic（5-HT）system originating in the dorsal raphe nucleus（DRN）is involved in regulating various physiological and behavioral functions,including mood-and emotion-related behaviors.Many observations suggest that abnormal activity of DRN neurons is linked to psychiatric disorders,such as major depression disorder（MDD）and anxiety.Aversive-stimuli-produced stress,anxiety and fear increase neuronal activity in subpopulations of serotonergic neurons and increase serotonin levels in the vicinity of DRN neurons.Thus,activity of the 5-HT neurons needs to be well controlled to maintain the homeostasis of the nervous system.The activity of DRN 5-HT neurons is regulated by various ion channels and membrane receptors,such as GIRK channels,SK channels,TREK1channels,5-HT_1A receptor,GABA_B receptor andα1 receptor.These channels and receptors have all been reported to play an important roles in controlling the intrinsic DRN 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 potassium channels,including SK channels,TREK1 channels and GIRK channels have been reported to play important roles in controlling the intrinsic neuronal activity,and modulating these channels may alleviate emotional disorders.For example,up-regulation of SK3 channels in 5-HT neurons greatly reduced activity in these neurons from a model of isolated mice;blocking these channels restores normal activity and alleviates the depressive symptoms of the isolated mice.Similarly,blocking TREK1 channels substantially increases the firing rate of 5-HT neurons in the DRN and induces a significant antidepressant-like response in a rat model of depression.Deletion of the GIRK2 or blocking the activity of GIRK2 in DRN promotes a depression-resistant phenotype.Early electrophysiological experiments have demonstrated that when the firing of 5-HT neurons in the DRN is recorded in vivo from a awake animal,the neurons exhibit two discharge modes:tonic discharge and cluster discharge,while in anesthetized animals the 5-HT neurons in DRN fire with a slow（0.5-3 Hz）,regular（clock-like）pattern,which is described as pacemaker firing.However,5-HT neurons,in spite of their spontaneous activity seen in vivo,are not intrinsic pacemakers but rather depend on extrinsic inputs to drive their firing,mainly the noradrenergic input trigger from the locus coeruleus（LC）.Therefore,in ex vivo brain slices,5-HT neurons in the DRN are silent,and the reconstruction of spontaneous discharge depends on theα1-adrenergic agonist such as phenylephrine（PE）.For this PE-induced spontaneous activities,A-type potassium channel have been suggested to be the undelying mechanism,however it is unclear whether other mechanisms are also involved.In this study,the patch clamp and single-cell PCR techniques were used to determine whether the calcium-activated small-conductance potassium channels（SK channels）,besides the A-type potassium channels,are involved in the PE-induced spontaneous activity of DRN 5-HT neurons,through a mechanism ofα1 receptor-mediated inhibition of these potassium channles.Part one Role of A-type potassium channel in phenylepherin-induced spontaneous firing of DRN 5-HT neuronsObjective:To investigate the role of A-type potassium channel in phenylephrin（PE）-induced spontaneous firing of dorsal raphe nucleus（DRN）5-HT neurons.Methods:1.The whole-cell patch clamp and loose-cell-attached patch clamp recordings were used to observe the effect of PE on the A-type potassium currents of 5-HT neurons in DRN,and the effect of A-type potassium channel blocker on the spontaneous firing of DRN 5-HT neurons.2.Single-cell PCR were used to determine the expression of the A-type potassium channel subtypes in DRN 5-HT neurons.Results:1.Spnotaneous firing of DRN 5-HT neurons was recoded using a loose-cell-attached patch clamp method in brain slices,and the results showed that theα1 receptor agonist PE（10μM）induced a slow（<5Hz）,clock-like discharge.2.Bath-application ofα1 receptor agonist PE（10μM）significantly inhibited a typical transient outward current of 5-HT neurons from DRN brain slices recorded using whole-cell patch clamp at a voltage of-20 mV（from1409.1±134.3 pA to 1087.6±117.8 pA,n=6,P<0.001）;the use ofα1 receptor blocker prazosin（5μM）reversed the above effects of PE（n=6,P<0.05）.3.Spnotaneous firing of DRN 5-HT neurons was recoded using a loose-cell-attached patch clamp method in brain slices,and A-type potassium channel blocker 4-AP（500μM）induced spontaneous firing in these neurons.With continued presence of 4-AP,further additation of PE（10μM）,increased the firing frequency significantly from 0.6±0.2 Hz to 1.4±0.3 Hz（n=5,P<0.01）.4.Single-cell PCR results showed that Kv4.2 and Kv4.3 were highly expressed in 5-HT neurons in the dorsal ventral part（DRV）of DRN.Conclusions:1.PE induces spontaneous firing of 5-HT neurons in the DRN via theα1receptor.2.A-type potassium channel subtypes Kv4.2 and Kv4.3 channels are highly expressed in 5-HT neurons in the ventral region of the dorsal raphe nucleus.3.PE can inhibit the currents of A-type potassium channels,which are likely composed mainly by Kv4.2 and Kv4.3 in DRN 5-HT neurons.4.PE induced significantly higher frequence of spontaneous firing than A-type potassium channel blocker 4-AP did,indicating that other mechanisms,besides the A-type potassium channels are also involved in PE-induced spontaneous firing in DRN 5-HT neurons.Part two Role of SK channel in phenylepherin-induced spontaneous firing of DRN 5-HT neuronsObjective:To investigate the role of SK channel in phenylepherin（PE）-induced spontaneous firing of DRN 5-HT neurons.Methods:1.The whole-cell patch clamp and loose-cell-attached patch clamp recordings were used to observe the effect of PE on the SK potassium currents of 5-HT neurons in DRN,and the effect of SK potassium channel blcoker on the spontaneous firing of DRN 5-HT neurons.2.Single-cell PCR were used to determine the expression of the SK channel subtypes in DRN 5-HT neurons.Results:1.Single cell PCR results showed that SK channel subtypes SK2 and SK3 channels were highly expressed in DRN 5-HT neurons.2.In whole-cell patch clamp recording,PE（10μM）significantly inhibited SK currents in DRN 5-HT neurons,from initial current amplitudes of 44.7±1.3 pA to 25.6±7.8 pA（n=6,P<0.05）.3.After spontaneous firing of DRN 5-HT neurons was first induced by SK channel blocker apamin（100 nM）,4-AP（500μM）induced further increase in spontaneous firing rate from 0.1±0.04 Hz to 0.9±0.3 Hz（n=8,P<0.05）,and furthermore the discharge mode was converted from tonic firing to burst firing.Under the blocking of both SK channel and A-type potassium channel,application of PE had no further effect on the firing frequency of 5-HT neurons in the DRN（from 0.9±0.3 Hz to 1.1±0.3 Hz,n=8,P>0.05）.4.When extracellular Ca²⁺was omitted from the bath solution（zero calcium）,spontaneous firing was induced in DRN 5-HT neurons.In addition,the HCN channel blocker ZD7288 had no effect on the spontaneous firing of zero-calcium-induced DRN 5-HT neurons.Extracellular zero calcium also induced a membrane depolarization of DRN 5-HT neurons（from-66.2±1.7mV to-49.7±0.9 mV,n=6,P<0.001）.Conclusions:1.SK channel subtypes SK2 and SK3 channels are highly expressed in DRN 5-HT neurons.2.Extracellular zero calcium significantly depolarized the membrane potential and induces spontaneous discharge in DRN 5-HT neurons.3.In addition to the A-type potassium channel,the SK channel also plays an important role in PE-induced spontaneous firing in 5-HT neurons of DRN.4.Interplay between SK channel and A-type potassium channel contributes to burst firing property of the 5-HT neuron in DRN.