Retrograde Tracing And Electrophysiological Properties Analyzing Of Dopaminergic Neuron Subgroups

Midbrain dopamine(DA)neurons system,an crucial neuroanatomical hub,participate in the regulation of important physiological functions,such as movement,working memory,and reward processing.And their dysfunction will lead to lots of neurological and psychotic disorders,for example,Parkinson's disease,schizophrenia,depression,drug addiction etc..Although these diseases are all relate to the DA neurons system,the vulnerable subgroup in each disease is different.Schizophrenia is mainly related to the DA neuron subgroup participate in meso-cortical projection pathway;drug addiction is associated with meso-limbic neural circuit;while nigro-striatal pathway is closely related to Parkinson's disease.Thus,exploring the classification and characteristics of midbrain DA subgroups will help us understand the pathogenesis of these diseases and provide the basis for targeted treatment.Midbrain DA neurons system was divided into ventral tegmental area(VTA)and substantia nigra(SN)based on anatomy location.This classification has been widely used.While recently,lots of studies have shown that the DA neurons in VTA and SN are not homogeneous.And their diverse functions might be related with their heterogeneity.Therefore,further determine the DA neuron subgroups and their characteristic could help us have a deeper understanding of midbrain DA neurons system.In distinguishing neuron subgroups,the electrophysiological characteristics were considered as one of the important indicators.The analysis of the electrophysiological characteristics of the neuron subgroups also provides a basis for understanding the relevant neural circuits and the changes of neural functions.Currently,cluster analysis based on electrophysiological characteristics reveals that there are two to four subgroups in the SN and VTA regions.Nowadays,the description of DA neuron subgroups all dependent on the morphology,biochemistry,molecular expression spectrum or electrophysiological characteristics.Thus,the comprehensive description of midbrain DA neuron subgroups' characteristics will deeper the understanding of the related diseases.In order to classify the subgroups of midbrain DA neuron and analyze their morphological and electrophysiological characteristics,we carried on the following experiment:1.The 8-weeks C57B16 mice were used in this study,and injection points were selected according to the neural circuit.Under the guidance of stereotaxic apparatus,the retrobeads were injected into the prefrontal cortex,prelimbic cortex;nucleus accumbens(NAc)core,medial and lateral part of NAc shell;anterior corpus striatum(CPu),ventral and dorsal part of posterior CPu.48h after the operation,conducting frozen section and tyrosine hydroxylase(TH)stained.Observing the prepared brain slices by fluorescence microscope,to determine whether the labeled neurons are DA neurons.2.Quantitative analysis the distribution of DA neuron subgroups in the midbrain.The distribution of different DA neuron subgroups was quantitatively analyzed by calculating the proportion of labeled neurons in VTA and SN subsets(mVTA,dlVTA,SMCM,dSNc,lSNc).The morphological differences of different DA neuron subgroups were recognized by measuring the area of labeled neurons.Determined the anatomy characteristics of midbrain DA subsets based on neural projection pathways.3.Perform the operation as mentioned above.Then,preparing the brain slices(300?m)using oscillating microtome for patch-clamp.Recording spontaneous action potential of the labeled neuron subtypes in 'Gap-free' mode.4.Then,using step current stimulation to detect the evoked electric activity:-100pA current was used to detect whether the neuron could generate "sag" in the potential diagram.+50pA and +100pA current were used to record the evoked action potential feature in those subgroups.5.Conduct the first temporal derivative and phase plane plots anlysis of AP,Through the above experiments,we found that1.Midbrain DA neurons involved in the meso-cortical system are mainly locate in mVTA,and the projection to prelimbic cortex is more intensive than other regions.Detailed tracing of NAc revealed that NAc core receives the innervation from DA neurons locate in mVTA and dIVTA evenly,DA neurons project to the medial shell of NAc are mainly located in mVTA,while neurons innervating the lateral shell of NAc are mainly locating in dlVTA.When it comes to nigro-striatal system,DA neurons of mSNc are densely projecting to aCPu.DA neurons innervate pdCPu are mainly locating in dSNc and mSNc,while DA neurons project to pvCPu not only from dSNc but also from dlVTA.2.In midbrain,DA neurons locate in mVTA mainly project to the NAc core and medial shell,dlVTA DA neurons mainly project to the NAc lateral shell.60.32%of DA neurons locate in mSNc projected to the aCPu,DA neurons from dSNc mainly innervate pdCPu and pvCPu;DA neurons distribute in ISNc mainly participate in the projection to the pvCPu3.Spontaneous discharge detection shows that the second type of meso-limbic DA neuron subgroups' excitatory is stronger than the other subgroup,followed by the second type of nigro-striatal system DA neurons subgroup.While,the first type of meso-limbic system projection and the first type of nigro-striatal system DA neurons subgroup's spontaneous discharge frequency is low.The meso-cortical projection pathways' discharge frequency is the lowest.It can be seen that DA neurons locate in the ventral area of the midbrain have lower basal excitability than those locate in the dorsal area4.,"sag" can only be observed in DA neurons with GIRK2,which participate in the form of meso-limbic and nigro-striatal system.+50-+100pA step current stimulations' results show that there is no difference in the discharge frequency of the DA subgroups of the meso-cortical and the meso-limbic pathway,but there is significant difference in the amplitude of action potential,which could be used to distinguish these subgroups.While the discharge frequency and amplitude of DA neuron subgroups in the nigro-striatal pathway are significantly different from the above three subgroups.Among them,the second type of DA neuron subgroup in the nigro-striatal pathway is the most unique one.Under the stimulation of 100pA current,its action potential is directly blocked.5.First temporal derivative and phase plane plots anlysis of AP shows that in the process of the AP,the first kind of meso-limbic pathway DA neurons' membrane permeability is the highest.While,the second type of nigro-striatal pathway DA neurons' membrane permeability is the lowest.In conclusion,midbrain DA neuron subgroups,projecting to PFC,NAc and CPu,break the anatomical boundary of VTA and SN.There are obvious differences in their spontaneous,evoked and the dynamic changes of AP discharges are also different.According to the neuroanatomical and electrophysiological characteristics,midbrain DA neurons can be divided into the following subgroups:meso-cortical system,meso-limbic system ?,meso-limbic system ?,nigro-striatal system ?,nigro-striatal system ?.