【作者】 孔岩；

【导师】 刘春风；

【作者基本信息】 苏州大学 ， 外科学， 2009， 博士

【摘要】 目的:研究急性脑片中内源性多巴胺(DA)在纹状体多棘神经元(MSN)电生理活动中的作用,探讨黑质-纹状体环路联系对MSN功能状态的影响。方法:建立急性皮层-纹状体-黑质脑片,采用可视脑片膜片钳全细胞记录技术,观察脑片中黑质细胞、纹状体细胞基本电生理活动。采用纹状体与皮层或黑质各种不同组合方式的矢状位脑片,观察纹状体MSN膜电位双相波动出现的条件。在皮层-纹状体-黑质脑片中,分别阻断DA受体和谷氨酸(Glu)受体,研究来自皮层的Glu和来自黑质的DA对MSN膜电位双相波动影响。对皮层进行配对刺激和短阵刺激,在背侧纹状体MSN记录兴奋性突触后电流(EPSC),观察皮层-纹状体突触短时程可塑性的变化。通过阻断皮层-纹状体-黑质脑片中的DA受体,观察内源性DA对短时程可塑性的作用。结果:1.皮层-纹状体-黑质脑片中,黑质细胞呈现出特征性自发动作电位,符合在体DA能细胞或γ-氨基丁酸能细胞的特征。部分纹状体细胞vf记录到与在体类似的膜电位双相波动,其细胞膜特性符合在体纹状体MSN的电生理特征。2.皮层-纹状体-黑质脑片中,65.2%的MSN(30/46)记录到自发的膜电位双相波动,在接近皮层的背侧纹状体有92.3%的MSN(24/26)表现为膜电位双相波动;而腹侧纹状体只有30%的MSN(6/20)出现膜电位的波动。阻断DA受体使去极化平台电位(up state)的幅度明显降低(P <0.05),其中阻断DA D1受体降低up state的水平(P <0.05),阻断动作电位的发放;而阻断D2受体使膜电位的双相状态均抬高(P <0.05)。在皮层-纹状体脑片、纹状体脑片、纹状体-黑质脑片中,MSN膜电位呈现为稳定的静息状态(down state),各组MSN静息电位值:皮层-纹状体-黑质脑片(-67.3±2.7mV,n=10);皮层-纹状体脑片(-69.7±2.6 mV,n=10);纹状体脑片(-73.2±3.3mV,n=10);纹状体黑质脑片(-71.4±2.5mV,n=10),差异无统计学意义(P>0.05)。3.皮层-纹状体-黑质脑片与皮层-纹状体脑片相比,配对刺激比率(PPR) :50ms刺激间隔时间(ISI)为139.6±18.2% / 113.3±13.7%(P<0.01);500ms ISI为67.4±10.6% /83.1±5.9% (P <0.01)。在皮层-纹状体-黑质脑片中阻断D2受体前后PPR:50ms ISI:136.5±12.6% / 113.6±10.5%,P<0.01;500ms ISI:66.2±6.4% / 79.3±7.1%,P<0.01。阻断D2受体皮层-纹状体-黑质脑片中PPR与皮层--纹状体脑片中PPR相比,差异无统计学意义。10Hz、20个刺激的短阵刺激引起EPSC幅度的依次减少,表现为短时程抑制(STD)。在皮层-纹状体-黑质脑片中,STD比率(0.20±0.03),D1受体阻断导致STD比率增大(0.58±0.03,P <0.001),抑制程度降低;而D2受体阻断后STD比率无明显差异(0.22±0.02,P >0.05)。皮层-纹状体脑片中,10Hz短阵刺激同样也引起EPSC幅度的依次减少,STD比率(0.59±0.05)与皮层-纹状体-黑质脑片(0.20±0.03)比较显著增加(P <0.001),与D1受体阻断的皮层-纹状体-黑质脑片比较STD比率相近(P >0.05)。结论:1.矢状位脑片中黑质和纹状体细胞保持与在体一致的基本电生理活动。2. MSN膜电位的双相波动是在DA的调节下对皮层Glu能兴奋性输入的反应,是纹状体与皮层、黑质间神经环路功能完整的表现。3.来自黑质的内源性DA通过对皮层-纹状体突触配对刺激和短阵刺激可塑性紧张性调控作用,可能参与调节纹状体MSN对皮层输入信息的瞬时整合和编码。更多还原

【Abstract】 ObjectiveTo investigate the effects of endogenous dopamine (DA) on striatum medium spiny neurons(MSNs) in acute brain slices by the means of electrophysiology, and in an attempt to clarify the impact of the nigrostriatal circuit loop on the functional status of the MSNs.MethodsWe established the acute corticostriatonigral slices to observe the basic electrophysiological activity with the visual patch clamp whole-cell recording technique on acute brain slices.With the various kinds of the structures combination which consist of striatum and cortex or/and substantia nigra, we explored the conditions with appearance of the spontaneous two-state voltage oscillations. Then, with the methods of blocking the dopaminergic or glutamic acid receptor respectively, we inverstigated how the aminoglutaminic acid, which was come from the cortex, and the dopamine, which was from substantia nigra, made affects on the two-state voltage oscillations.Recording the excitatory postsynaptic current(EPSC) of MSNs in dorsal striatum while performing paired pulses or short train pulses to the cortex, to oberserve the change in short term plasticity. Observing the effect of dopamine on the short term plasticity by blocking the dopamine receptors in the corticostritonigral slice.Results1. Substantia nigral neurons displayed characteristical spontaneous action potentials and hyperpolarization-activated inward current in corticostriatonigral slices. They were consistent with the characteristics of dopaminergic neurons orγ-aminobutyric acid neurons in vivo. Spontaneous two-state voltage oscillations were recorded in some striatal medium spiny neurons as they were in vivo.2. 65.2 %( 30/46) MSNs recorded in corticostriatonigral slices displayed spontaneous two-state voltage oscillations. 92.3% (24/26) of MSNs in the dorsal striatum near to the cortex, only 30% of MSNs in the ventral striatum. The amplitude of depolarized plateau potential(up state) were decreased by blocking dopamine receptors (P <0.05). The potential level of up state was reduced by blocking D1 receptors (P <0.05), action potentials were stopped. The results of blocking D2 receptors were the potentials level of two states raised (P<0.05). MSNs membrane potential showed a stable resting level in any slice of corticostriatal slice(-67.3±2.7mV, n=10), striatal slice (-73.2±3.3mV, n=10) and nigrostriatal slice (-71.4±2.5mV,n=10). There was no significant difference(P >0.05).3. Compared the PPR in corticostriatonigral slices with it in corticostriatal slices, there was significant difference in PPR(50ms ISI:139.6±18.2% / 113.3±13.7%, P<0.01; 500ms ISI 67.4±10.6% /83.1±5.9% P<0.01). PPR before and after blocking the D2 receptors in corticostriatonigral slices was(50ms ISI:136.5±12.6% / 113.6±10.5%,P<0.01;500ms ISI:66.2±6.4% / 79.3±7.1%,P<0.01). there was no significant difference compared PPR in corticostriatonigral slices after blocking the D2 receptors with PPR in corticostriatal slices(P>0.05). 20 stimuli delivered at 10 Hz could induce the decrease of the EPSC amplitude, which showed STD. The STD ratio in corticostriatonigral slices was(0.20±0.03), The STD ratio increased after blocking the D1 receptors (0.58±0.03,P<0.001), the extent of STD decreased. The D2 receptors have no effect on the STD ratio. 20 stimuli delivered at 10 Hz could change the EPSC in corticostriatal slices as in corticostriatonigral slices. The STD ratio(0.59±0.05) increased than it in corticostriatonigral slices(P<0.001). But comparing with the STD ratio in the in corticostriatonigral slices after blocking the D1 receptors, there was no significant difference(P>0.05). Conclusions1. Substantia nigral neurons and striatal neurons in saggital brain slice keep the basic electrophysiological activity as in vivo.2. Spontaneous two-state voltage oscillations in striatal medium spiny neurons are the results of MSN receiving the afferent of cortical glutamatergic excitatory afferent and the regulation of dopamine. Reflecting the whole circuits between the striatum and cortex, substantia nigra neurons.3. Endogenous Dopamine from the substantia nigra maybe regulate the instantaneous integration and coding of MSNs to the information from the cortex by tonic effect on the short term plasticity of paired pulses and short train pulses in the corticostriatal synapses.更多还原