A Comparative Study Of The Function Of Direct And Indirect Pathway Neurons In The Striatum

The striatum is the main input structure of the basal ganglia loop.It integrates glutamate inputs from the cortex,thalamic nuclei,hippocampal,and dopamine inputs from the substantia nigra compactus.Thus,the striatum could integrate the motor information from the cortex and the environmental information represented by the activity of midbrain dopamine neurons,transmitting the consolidated information to the basal ganglia circuits through the striatal direct and indirect pathway neurons.In this study,we investigated the mechanisms of glutamatergic synaptic transmission between direct and indirect pathway neurons in the striatum under different physiological states,including the negative reinforcement learning behavior,acute cocaine effect and valproic acid-induced autistic mice model.The striatum-basal ganglia-thalamo-cortical loops are critical for the instrumental learning,selection and organization of actions,as well as reward-based motivation behavior.Previous studies have suggested that the two striatal pathways from striatum,direct and indirect pathway,had opposing roles in movement and reinforcement learning,which proposed that the direct pathway neurons(D1-MSNs)mediated positive reinforcement learning,and promoted movement and reward-based behavior,on the contrary,the indirect pathway neurons(D2-MSNs)mediated negative reinforcement learning and punishment,and inhibited movement.Soon after publication of this theoretical model,it was discovered that,in a positive reinforcement learning paradigm using food as the positive reinforcer,potentiation in excitatory synaptic transmission on D1-MSNs and depression in the excitatory synaptic transmission on D2-MSNs occurred simultaneously and both mediated the positive reinforcement learning behavior.And other studies about examining the activity of these two striatum neurons during the initiation of movement or learned action sequences have also reported that both of direct and indirect pathway neurons were concurrently active during the processes of sequency initiation.By analogy,employing aversive and setting removal of the aversion as the reinforcer,negative reinforcement learning must recruit both the direct and the indirect pathways neurons and might rely on more complex plasticity mechanisms.However,the neural plastic mechanisms mediating negative reinforcement learning are still largely unknown.Physiological functions of the striatum are not only important for initiation and performance of actions,reinforcement learning and behavior remodeling,but are also the key target region for the effects of addictive drugs and a variety of psychiatric disorders.On the one hand,more and more young adults as the recreation users,who didn't meet criteria for abuse or dependence but took cocaine occasionally,exhibited poor adaptive behavior such as reinforcement learning.Thus,there were many studies focused on the cognitive deficits and the dysfunction of excitatory synapse caused by acute cocaine exposure.Previous studies reported that peak cocaine values were observed approximately 5-15 min after drug administration,and cocaine half-life ranges between 16 to 72 min depending on different species,drugs dosage,and experimental conditions.Although cocaine was metabolized rapidly,its metabolites,including benzoylecgonine and ecgonine methyl ester were still detected at 24 h after the first cocaine does in human beings.This persistent neuroadaptive effects left by such an acute action mediated the emotional and behavioral changes after a single cocaine injection.Previous studies have found that acute exposure to cocaine exerts significant effects on the glutamatergic transmission onto the Dl-MSNs and/or the D2-MSNs in the nucleus accumbens.However,the effect of a single injection of cocaine on the glutamatergic transmission of striatal MSNs is still largely unknown.Considering the importance of negative reinforcement learning for exploring animals to avoid aversive stimuli,the neural plastic changes induced by acute exposure to cocaine may disturb the formation of the neural plasticity required for reinforcement learning,causing impairments of the learning behavior.Unfortunately,whether a single exposure to cocaine will disturb the glutamatergic transmission of striatal MSNs required by negative reinforcement learning are still unclear.On the other hand,normal physiological activity of the striatum is essential for the control of the whole basal ganglia loop and is thought to play an important role in the mediating motor activity and other behavioral output.Abnormal activity of the striatum results in the onset of pathological conditions characterized by various motor and behavioral abnormalities,such as Obsessive-compulsive disorder,Parkinson's disease and Huntington's disease.And numerous genetic autism models displayed alterations in the structure and function of striatal circuits,dysfunction of excitatory/inhibitory synaptic transmission,as well as abnormal behaviors including repetitive grooming,stereotypic motor routines,deficits in social interaction and decision making.Meanwhile,a large number of studies on autism based on brain-imaging suggested that striatal dysfunction was postulated to underlie the repetitive motor behaviors in autism.Furthermore,because the incidence of ASD involves multiple brain regions and genotypes,the ASD-like animal model caused by Valproic acid(VPA)is a good experimental model combining the interaction between environmental factors and genetic genes.So far,it has not been reported whether there are also changes in the glutamatergic synaptic transmission of the two types of neurons in the striatum in these mice.Based on these background studies of physiological activity of the striatum neurons,the present study combined pharmacology,whole-cell patch clamp with single-cell PCR,optical fiber recording and DREADD methods to compare the normal physiological functions and the abnormal pathological conditions of striatal direct pathway neurons and indirect pathway neurons glutamatergic synaptic transmissions.We hope these results could provide a theoretical basis for understanding the working rules of different types of neurons in the striatum under the different outside environmental conditions and selfstates,including the footshocks mediated negative reinforcement learning behavior,a single in vivo exposure to cocaine as well as VPA induced autism.The main investigations and results are as follows:1.Striatal direct pathway neurons and indirect pathway neurons were indispensable during the negative reinforcement learningSince dopamine neurons play an important role in regulating the glutamatergic synaptic transmission of the striatum neurons in the context of rewarding and aversive stimuli,we first investigated the substantia nigra compacta(SNc)dopaminergic neuronal activity throughout the learning phase and habituation phase of the negative reinforcement learning process.The results indicate that the learning phase,in the trials before successful completion of learning to escape the footshocks,enhancement of dopamine neuron activity followed the termination of the footshocks,but after the completion,defining the habituation phase,follows the presence of it.And during the learning phase,Ca2+ signals decreased along the action of the footshocks,the hypoacti vity of dopamine neurons represented negative value of the aversive stimuli.The striatum is the target region of dopamine release from the SNc.These increased or decreased responses of dopamine neurons could enhance or suppress dopamine release in DMS.Microinjection of dopamine D1 receptor(DlR)or D2 receptor(D2R)antagonist in dorsomedial striatum(DMS)significantly impaired the learning behavior,suggesting that the modulation of dopamine on both the direct and indirect pathways is required.Meanwhile,during the learning phase,excitatory synaptic transmission to DMS D2R-expression medium spiny neurons(D2-MSNs)was potentiated.But,with completion of the learning and the habituation phases,the synapses onto D1R-expression medium spiny neurons(D1-MSNs)were potentiated and those onto D2-MSNs were restored to the normal level.The bidirectional changes in both SNc dopamine neuron activity and DMS synapse plasticity might be the critical neural correlates for negative reinforcement learning.2.A single exposure to cocaine 24 h before reinforcement learning significantly impairs the reinforcement learning through enhancing the glutamatergic synapses on striatal direct pathway neurons.Based on the discussion of the normal physiological function of striatum D1-MSNs and D2-MSNs during the negative reinforcement learning,we further explored the effect of a single exposure cocaine to the negative reinforcement learning behavior in mice.The present work found that the negative reinforcement learning,escaping mild footshocks by correctly nosepoking,was impaired by a single in vivo exposure to 20 mg/kg cocaine 24 h before the learning in mice.Either the single exposure to cocaine or the reinforcement learning was able to potentiate the glutamatergic synaptic transmission on striatal D1-MSNs.The potentiation of synaptic transmission was demonstrated by increased miniature excitatory postsynaptic(mEPSC)amplitude and AMPAR/NMDAR ratio.However,24 h after the exposure to cocaine,experience of cocaine impaired the potentiation in striatal direct pathway neurons induced by the negative reinforcement learning,confirmed by which HFS induced LTP in D1-MSNs was impaired after a single exposure to cocaine 24 h.However,this process of cocaine exposure did not affect the glutamatergic synaptic transmission in D2-MSNs.Then in this paper,we selectively manipulation of the activity of DMS D1-MSNs,using DREADD methods,to testify the sufficiency and necessity relationship between the potentiation of glutamatergic synaptic transmission in D1-MSNs and the impairment of the negative reinforcement learning behavior caused by cocaine.The results indicated that activation of striatal D1-MSNs impaired the reinforcement learning in normal D1cre mice,but inhibition of these neurons reversed the reinforcement learning impairment induced by cocaine.The results suggested that cocaine could potentiate activity of striatal direct pathway neurons in dorsomedial striatum and this potentiation might disrupt the potentiation produced during and required for the reinforcement learning.3.Dysfunction of striatum neurons in ASD mice model induced by VPATo explore the pathological changes of striatum neurons in VPA induced ASD model,the mice model of autism was obtained in offspring of the female mice who received a 500 mg/kg VPA during the pregnancy at E 12.5 day.Compared to the normal mice,VPA mice showed increased repetitive stereotyped behavior and abnormal social communication ability.These results indicated that a single injection of VPA to the pregnant female mice could successfully induce the animal model of autism.Then,using the patch clamp with single-cell PCR techniques,we observed the excitatory glutamatergic synaptic transmission and the intrinsic excitability of neurons in the striatum MSNs between the control and VPA groups.Compared with the control mice,the glutamatergic synaptic strength in the striatum D1-MSNs was remarkably increased in the VPA group.The potentiation of synaptic strength was characterized by an enhanced frequency and amplitude of mEPSC,and an increased the AMPAR/NMDAR ratio,as well as a decreased the paired pulse ratio(PPR)in DMS D1-MSNs.Meanwhile,the intrinsic excitability of D1-MSNs in the VPA group was also significantly improved,presented as decreased rheobase current,increased spike number and the average instantaneous frequency.On the contrary,compared with the control mice,the glutamatergic presynaptic strength in striatum D2-MSNs was significantly decreased in the VPA group,which proved by a decreased frequency of mEPSC,as well as an increased the paired pulse ratio(PPR)in DMS D2-MSNs.Activation of mGluR5 by bathing its agonist DHPG could induce long-term depression(LTD)on glutamatergic synaptic on D2-MSNs.But this mGluR-LTD was absent in the group of VPA mice.Meanwhile,the intrinsic excitability of D2-MSNs in the VPA group was significantly suppressed,presented as increased rheobase current,decreased spike number and the average instantaneous frequency.