Medium Spiny Neuron Abnormalities In Parkinson's Disease Model Mice

Parkinson's disease(PD)is the second most common neurodegeneration disorder.Clinically PD is characterized by tremor,rigidity,bradykinesia and postural instability.A pathological hallmark of PD is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta(SNc).The prevalence of PD for those aged 65 years or older is 1.7%in China,similar to the incidence in the developed western countries.The estimated number of individuals over age 50 with PD in China was 1.99 million in 2005.And this number is expected to be 4.99 million in 2030.As the life expectancy increases,the economical and societal burdens of PD will likely also grow in China.As a critical movement control neuronal circuit,the basal ganglia(BG)are a group of interconnected subcortical nuclei that include the caudate nucleus and putamen(these two nuclei comprise the striatum),the globus pallidus external and internal components(GPe and GPi),the subthalamic nucleus,and the substantia nigra pars compacta and pars reticulata(SNc and SNr).The striatum is the main input station receiving and processing information from the cortex and thalamus under the regulation of rich dopamine(DA)innervation originating in SNc.The processed information is then transmitted,via GPi and SNr,to other brain areas such as the thalamus(to influence the thalamocortical motor circuit and hence movements)and brainstem motor nuclei such as the superior colliculus that control eye and head movements.In the parkinsonian state,the dopaminergic neurons in the SNc degenerate progressively and selectively and lead to dopamine denervation of the dorsal striatum.The consequence of dopamine depletion in the dorsal striatum is decreased activities of direct pathway and increased activities of indirect pathway.And the net outcome is increasing the activities of GPi and SNr,which in turn inhibit the targets of the basal ganglia and inhibit the movement initiation.Most of researchs used to focus on the STN and GP,which show the activities of neuron in these area change a lot.Recently studies have also demonstrated that after dopamine depletion in the striatum,both D1 and D2 dopamine receptors became supersensitive to dopamine modulation.Our hypothesis of this study is that after dopamine depletion:l.The density of dopamine D1 and D2 receptor in the striatum changes;2.Presynaptic D2 receptor at the corticostriatal and thalamostriatal axon terminals are supersensitized;3.D1-medium spiny neuron intrinsic excitability is abnormally high;4.D1-MSN is more sensitive to dopamine modulation.To test this hypothesis,we used a new genetic mouse model of PD,Pitx3 deficient aphakis mice(Pitx Homo mice).The dopaminergic neurons in SNC of Pitx Homo mice were consititutionally lost.And the dopamine loss in the dorsal striatum of those mice is close to 90%.Those characteristics of the nigrostriatal dopamine pathway of Pitx Homo mice are similar to late stages of PD.Moreover,Pitx Homo mice also show motor deficits that resembling the movement disorder of PD.Compared with the traditional toxin-induced mice model of PD,Pitx Homo mice as a PD model has several advantages and complementary features.First,the of lesion of nigrostriatal dopamine neuron by neurotoxin was usually partial and had variations.Furthermore,in some cases the nigrostriatal pathway could recover from a partial SNC lesion.In contrast,the loss of nigrostriatal dopamine system is more complete,consistent and irreversible in Pitx Homo mice.Second,in the traditional neurotoxin-induced PD model,the loss of dopaminergic terminal occurred selectively in the dorsal striatum with relative sparing of ventral striatum.Third,Pitx Homo mice are suitable for electrophysiological studies,since most traditional neurotoxin-induced PD models use adult animals and to perform patch clamp experiments on adult animals is very difficult.Compared with other genetic mice PD models,Pitx Homo micealso have unique advantage.Among all the genetic mice PD models,only Pitx Homo mice displayed constitutional selective and almost complete dopamine loss in SNC,whereas other genetic only display mild dopaminergic neuron loss at very late stage.Meanwhile,there are two populations of MSNs in the striatum:direct pathway MSN or D1-MSN and indirect pathway MSN or D2-MSN.These two populations of MSNs are intermingled together and morphologically indistinguishable.We took the advantage of transgenic mice in which D2 receptor-expressing MSN are labeled with the enhanced green fluorescent protein(eGFP),we called this mice D2 GFP mice.We backcrossed the Pitx Homo mice with D2 GFP mice and produced Pitx Homo/D2-GFP phenotype and Pitx WT/D2-GFP.We performed the following experiment on the these mice.The first part of the experiment is to examine mRNA expression of dopamine receptor in striatum by Semi-quantitative RT-PCR and QRT-PCR,with dopaminergic innervation and dopaminergic denervation.Compared to the Pitx WT mice,expression of D1 and D2 was minimally affected in dorsal striatum(P>0.05,P>0.05)and in ventral striatum(P>0.05,P>0.05)in the Pitx Homo mice,by means of Semi-quantitative RT-PCR.The data from the QRT-PCR also confirmed these result,which show expression of D1 and D2 in dorsal and ventral striatum from Pitx Homo mice did not change,In contrast to those in dorsal and ventral striatum from Pitx WT mice(P>0.05,P>0.05,P>0.05,P>0.05).The second part of the experiment is to study the dopamine modulation of corticostriatal and thalamostriatal EPSC and EPSP on MSN in striatum after dopamine depletion in Pitx Homo/D2 GFP mice.We selected an oblique horizontal preparation of approximately 30° off-horizontal to cut the brain slices.We first characterized the Corticostriatal and Thalamostriatal Synapses by the paired-pulse ratio(PPR).In D1 MSN,the PPR at Corticostriatal synapses was significantly>1,In contrast,the PPR at thalamostriatal synapses was significantly<1(P<0.05);In D2 MSN,the PPR of corticostriatal and thalamostriatal synapses was very similar to that found in D1 MSN(P>0.05,P>0.05).The PPR of corticostriatal synapses on striatonigral MSNs was>1,whereas as it was<1 at thalamostriatal synapses(P<0.05).The data from Pitx Homo/D2 GFP mice are the same as those from Pitx WT/D2 GFP mice.Next we compared the sensitivities to dopamine modulation of the corticostriatal and thalamostriatal EPSC and EPSP between Pitx WT mice and Pitx Homo mice.We found out that:I.When we placed the stimulating electrode in the cortex and recorded EPSC in the D1 MSN,dopamine could inhibit the amplitude of the first EPSC pulse more in Pitx Homo mice than Pitx WT mice(P<0.05);when we recorded EPSC in the D2 MSN,dopamine also could inhibit the amplitude of the first EPSC pulse more in Pitx Homo mice than Pitx WT mice(P<0.05);The inhibition of D1 MSN and D2 MSN in Pitx Homo mice were not significantly different(P>0.05).?.When we placed the stimulating electrode in the thalamus and recorded EPSC in the D1 MSN,dopamine could inhibit the amplitude of the first EPSC pulse more in Pitx Homo mice than Pitx WT mice(P<0.05);when we recorded EPSC in the D2 MSN,dopamine also could inhibit the amplitude of the first EPSC pulse more in Pitx Homo mice than Pitx WT mice(P<0.05);The inhibition of D1 MSN and D2 MSN in Pitx Homo mice were not significantly different(P>0.05),but the inhibition from thalamus stimulation was significantly larger than that from cortex stimulation(P<0.05).?.When we placed the stimulating electrode in the thalamus and recorded EPSP in the D1 MSN,dopamine could inhibit the amplitude of the first EPSP pulse more in Pitx Homo mice than Pitx WT mice(P<0.05);when we recorded EPSP in the D2 MSN,dopamine also could inhibit the amplitude of the first EPSP pulse more in Pitx Homo mice than Pitx WT mice(P<0.05);The inhibition of D1 MSN and D2 MSN in Pitx Homo mice were not significantly different(P>0.05).Then we performed paired-pulse experiments.It is established that presynaptic inhibition of neurotransmitter release may increase the paired pulse ratio(PPR)whereas postsynaptic inhibition or enhancement of GABAA receptor function generally does not alter the PPR.The results showed that after dopamine application,the PPR of the corticostriatal and thalamostriatal EPSC increased in D1 and D2 MSN(P<0.05,P<0.05,P<0.05,P<0.05).At the end we did the experiments to confirm that it was the D2 receptor that were mediating the heightened presynaptic dopamine inhibition of EPSC.We found that bath application of D2-like receptor agonist ropinirole mimicked the effect of dopamine in PitxHomo mice,reducing the peak amplitudes of the corticostriatal EPSC in D1 MSN and D2 MSN from Pitx Homo mice more than those from Pitx WT mice(P<0.05,P<0.05).The thalamostriatal datas were similar to the corticostriatal ones(P<0.05,P<0.05).The third part of the experiment is to study potential abnormalities in the intrinsic excitability of D1 MSN in the dorsal striatum in Pitx Homo mice.We injected a set of current pulses(from-100pA to 80pA)to construct I-V curves and we found D1 MSN RIn is higher in Pitx Homo mice than that in Pitx WT mice(P<0.05).We also test three different age(14-16days,17-19days,20-23days)Pitx Homo and Pitx WT mice,the difference of Ri,between Pitx Homo and Pitx WT mice is consistent(P<0.05,P<0.05,P<0.05).Then we used CsCl-based intracellular solution to distinguish that the difference of RIn is due to reduced cell surface area or reduced K channel activity.After CsCl blocked the vast majority of K channels,the difference in D1 MSN RIm between Pitx Homo and Pitx WT remained(P<0.05).However,we still need further experiment to make sure what factor contributes to the difference of RIn.We treated the Pitx Homo mice with low dose L-dopa began at P10,and started the recording from P17.We found that the L-dopa treatment could reduce the D1 MSN RIn in Pitx Homo mice(P<0.05).We also compared the threshold current for D1 MSN and D2 MSN firing between Pitx Homo and Pitx WT mice.These data showed that the threshold current to evoke spikes was smaller in D1 MSN in Pitx Homo mice than that in Pitx WT mice(P<0.05).The forth part of the experiment is to study the dopamine modulation of D1 MSN after dopamine depletion in Pitx Homo mice.After obtaining stable baseline recording,DA was bath-applied.The data showed that DA increased the evoked spikes by up to 50%in these D1 MSN in Pitx Homo mice,whereas it was only 15%increase in Pitx WT mice(P<0.05).Then we used the TTX to block the sodium channel for spikes.In this condition,the DA could aslo induce larger depolarization in D1 MSN in Pitx Homo mice than that in D1 MSN in Pitx WT mice(P<0.05).However,we still need further experiment to be clear that the larger DA depolarization attributes to which current.The major finding of this study was that:1.Compared to Pitx WT mice,D1 and D2 dopamine receptor mRNAexpression have no change in Pitx Homo mice model.2.Dopamine depletion induces the supersensitivity of D2 receptor which located on presynaptic membrane of the corticostriatal and thalamostriatal synapse.3.After Dopamine is depleted,the RIn of D1 MSN will increase.It is likely to compensate for the lost D1 receptor excitation.Higher RIn confers higher excitability and affects how MSN process input signal and produce output signal.4.Dopamine substantially increases spike firing in D1 MSN by supersensitive D1 receptor under a parkinsonian condition.