3-NP诱导大鼠纹状体损伤的组织病理学证实

目的探察证实3-NP对实验大鼠纹状体的损害作用。方法实验借助行为学探测、经典组化和免疫组化染色技术对大鼠运动行为学和纹状体组织病理学变化进行形态学探察,实验数据采用SPSS软件统计学处理。结果①运动行为学探测显示大鼠过平衡木的启动延搁时间和完成全过程时间明显延长(P0.05,P0.05),以及前爪滑落次数与3-NP处理之前比较具有统计学差异(P0.05),其抓握时间也明显比3-NP处理之前延长(P0.05)。②组织学探察显示,多种组化染色均在实验大鼠纹状体的背外侧区恒定出现一块明显的坏死灶,发生率为80%。坏死区及其周边区绝大部分神经元发生丢失,仅有少数体积较大的细胞幸存。Golgi染色显示在周边区幸存神经元的树突发生明显断裂和弯曲。酶组化显示病灶区SDH明显淡染,周边区可见一些强反应的细胞。③神经元特异性抗体探察显示坏死灶及其周边区神经元丢失严重,纹状体投射神经元的丢失更显著。对神经元死亡和损伤性质的进一步鉴定结果显示在坏死灶及其周边区均可见大量凋亡细胞。结论 3-NP特异性地诱导纹状体背外侧区神经元损伤,纹状体不同类型神经元对3-NP损伤显示不同的敏感性,凋亡和能量代谢障碍因素牵涉到此病理过程。     

大鼠纹状体parvalbumin阳性中间神经元的超微结构

目的:研究纹状体parvalbumin (Parv)阳性中间神经元在神经通路上的突触连接.方法:利用免疫组织化学和神经示踪方法标记SD大鼠纹状体Parv及其相关神经元,光镜和电镜观察阳性神经元的结构和位置关系.结果:Parv阳性中间神经元中等大小,散在分布于纹状体,以背外侧居多.光镜免疫双标记显示Parv阳性中间神经元与皮质、丘脑和中脑黑质的传入轴突终末形成明显的形态位置上的邻近关系,其轴突终末则与纹状体不同类型投射神经元在光镜下也形成邻近关系.Parv阳性中间神经元的免疫电镜观察显示阳性产物主要游离于胞体、树突和轴突的胞质内.Parv阳性中间神经元的胞体和树突均接受大量的非对称型突触传入.Parv阳性轴突终末平均大小为(0.62±0.28)μm,可见其与纹状体神经元的树突、胞体和树突棘形成对称型突触,其中与树突形成的突触占69.64%,与胞体和树突棘形成的突触分别为26.78%和3.58%.结论:纹状体Parv阳性中间神经元形态学上与皮质、丘脑、黑质以及纹状体投射神经元形成突触连接,提示其可能在调节纹状体信息传入和输出过程中具有重要作用.     

谷氨酸对大鼠海马兴奋毒性作用的形态学研究

目的：为明确谷氨酸对神经细胞有兴奋性和毒性作用。方法：选用具有较多N－甲基D－天门冬氨酸（NMDA）受体的海马作为研究对象，选用SD大鼠，给予L－谷氨酸钠盐（MSG）皮下注射，在不同的时间内观察大鼠的表现和其海马神经细胞受损的形态学的改变。结果：发现大鼠的早期行为表现较为兴奋，而后随时间的延长表现为迟钝。在光镜和电镜下，神经细胞早期为水肿，后期为水肿明显和细胞器的破坏直至细胞的因缩，损伤主要表现在CA1区。结论：提示边缘系统的海马结构易受谷氨酸的影响，同时也表明了谷氨酸对神经细胞的兴奋毒性作用，可为临床药物和食物添加剂的运用提供一定的依据。     

丘脑束旁核-纹状体神经元轴突终末的超微结构证实

目的研究丘脑束旁核(PFn)-纹状体神经元轴突终末的分布和超微结构特征,以及与纹状体神经元之间的形态学联系。方法借助于神经示踪剂BDA10K注射方法顺行标记PFn神经元,光镜和电镜下观察和计数阳性纤维在纹状体内的数量和分布形式以及超微结构特征,实验资料借助于SPSS软件统计处理分析和体视学观察。结果①PFn神经元轴突终末在纹状体内呈散在不均匀分布;背侧区显著多于腹侧区(P<0.001),而外侧区与内侧区之间差异无统计学意义(P=0.387)。②光镜高倍镜观察显示大量的BDA阳性纤维与纹状体不同类型神经元(包括NeuN阳性、Darpp-32阳性以及Parv阳性神经元)的胞体和突起在形态学上存在明显的相邻关系。③电镜观察和测量结果显示BDA阳性终末平均直径为(0.648±0.288)μm,同时发现它们与纹状体神经元之间主要形成兴奋性(非对称型)突触连接;阳性轴突终末的65.18%与树突形成兴奋性突触,其直径为(0.651±0.304)μm;剩余的34.82%[平均直径为(0.642±0.257)μm]与树突棘形成兴奋性突触连接。结论丘脑PFn-纹状体神经元轴突终末主要分布于纹状体的背侧区,并与纹状体不同类型的神经元显示相邻关系,电镜超微结构下证实其突触形式主要为轴-树兴奋性突触连接,结果提示丘脑PFn神经元对纹状体不同类型的神经元的影响可能具有选择性和特异性。     

纹状体对体外培养中脑多巴胺神经元形态发育的影响

靶细胞在神经细胞的生长发育过程中起着重要的调节作用。本文通过胚胎腹侧中脑和纹状体联合培养研究纹状体对中脑多巴胺(DA)神经元形态发育的影响。纹状体(Str)和腹侧中脑(YMA)细胞悬液取自胚胎14天SD大鼠,联合培养3到14天后取出,用酪氨酸羟化酶(TH)免疫组化ABC法观察不同时期DA神经元的生长。与VMA单独培养相比,联合培养3天,发现细胞聚集程度较低,开始形成单层分布,培养7天TH阳性细胞数增加,多突起阳性细胞较易发现,细胞突起粗短,呈树枝状分枝。培养14天,TH阳性细胞数进一步增加,平均可达49个/25mm~2。本文对纹状体调节DA神经元生长发育的机制进行了讨论。     

纹状体神经元调控SVZ区域神经再生的作用机制

目的:纹状体神经元可以投射突触到室管膜下区(SVZ),并影响SVZ区域前体细胞的活性,但是纹状体神经元活性如何影响SVZ区域神经再生未知,本文旨在探讨纹状体神经元与SVZ区域神经再生的关系。方法:利用光遗传学的方法,特异调控纹状体神经元活性后,利用免疫组织化学方法观察其活性对SVZ区域神经再生的影响,并且探讨其可能机制。结果:当以CaM KⅡ作为启动子时,光敏感蛋白可以在纹状体神经元中表达,而且纹状体神经元的发放频率能够被激光控制。纹状体神经元活性的抑制促进NT-1的分泌(P0.05),免疫组织化学结果显示纹状体神经元活性的降低促进SVZ区域BrdU与DCX双阳性细胞数目的增多(P0.05)。结论:纹状体神经元活性是影响SVZ区域神经再生的重要因素,抑制纹状体神经元活性可以促进神经再生。     

右美沙芬对培养大鼠皮层神经元谷氨酸兴奋毒性和缺氧损伤的保护作用

本文采用16～18d胎龄的大鼠皮层细胞进行分离培养，以培养上清液中乳酸脱氢酶（LDH）活性作为细胞损伤的指标，分别观察了缺氧和过量谷氨酸对皮层神经元的影响以及右美沙芬的保护作用。实验结果表明，外源性谷氨酸（10和50μmol／L）和缺氧（5h）均引起LDH释放增加，而这种作用皆被右美沙芬所抑制。提示右美沙芬对此有明显的保护作用。以上结果说明谷氨酸兴奋毒性与NMDA受体在缺血性脑损伤过程中起着重要的作用。     

KA1表达再分布对神经元兴奋毒性的影响****

背景：特异的兴奋性神经递质受体过度活化时便发生细胞的兴奋性中毒,有关NMDA受体和AMPA受体活化致细胞兴奋性中毒死亡通路较为明确,但关于红藻氨酸(Kainate,KA)受体的功能到目前为止仍未明了。 目的：探究小鼠海马内注射KA之后KA1受体的再分布状况对中枢神经元兴奋性中毒的影响。 方法：成年雄性C57BL6小鼠海马内注射KA或PBS,2 h后进行Bederson体征评分、全脑切片免疫组化分析和死亡神经元检测。 结果与结论：海马内注射KA 2 h后,Bederson体征评分表明中枢神经功能出现明显损伤;KA1受体表达的变化主要出现在海马CA1和CA3区,在CA1区的表达上调明显,并开始出现神经元死亡,4~6 h后可见大量神经元死亡。结果表明 KA促使KA1受体亚单位在海马神经元CA区重新分布,增加神经元对兴奋性氨基酸毒性的敏感性,导致神经细胞死亡,中枢神经功能缺失。     

大鼠纹状体一氧化氮合酶阳性神经元的生后分布及发育

目的：探讨一氧化氮合酶(NOS)阳性神经元在生后不同日龄(1、5、10、15、30、120d)大鼠纹状体中的分布及形态变化特征。方法：NADPH-d组织化学方法。结果：NOS阳性神经元首先出现于尾壳核前上部，随生长发育逐渐扩展至外侧部、内侧部和苍白球；1～5d时胞体直径10μm左右，无突起，或仅1～2个短小突起，10～15d胞体增大，数量增多，突起明显增多伸长，并有许多膨体，30d时胞体直径达20～35μm，突起200μm以上，并反复分支交织成网状，120d与30d比较无明显变化。结论：纹状体内NOS阳性神经元的发育变化在生后1～30d之间，NO可能与运动机能的建立及调控有关。     

羊膜细胞对帕金森病鼠纹状体中残存多巴胺神经元的营养作用

目的：探讨非多巴胺能组织移植治疗帕金森病（PD）的作用机理。方法：通过6-羟基多巴胺（6-OHDA）立体定向流向破坏-侧黑质,制成PD大鼠模型,然后将鼠的成活羊膜细胞悬液植入受损侧纹状体,与鼠的死羊膜细胞移植及生理盐水假移植对照。结果：发现活羊膜细胞移植可减少大鼠的异常旋转行为,酪氨酸羟化酶（TH）免疫组化结果显示在活羊膜细胞移植靶点周围出现了（TH）疫阳性物质。结论：说明活羊膜细胞诱导了宿主纹状体残存多巴胺（DA）神经元的再生,提示羊膜细胞能够分泌对在体DA神经元具营养作用的物质。     

Parvalbumin-containing interneurons in rat hippocampus have an AMPA receptor profile suggestive of vulnerability to excitotoxicity

-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate excitatory neurotransmission in the central nervous system, and contain combinations of four subunits (GluR1-4). We developed a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and most interneurons. In addition, we found a subset of parvalbumin (PV)-containing interneurons in the hippocampus and neocortex that was notable for its intense GluR3 immunoreactivity and lack of GluR2 immunoreactivity. Such an expression pattern of AMPA receptor subunits is likely to make these interneurons selectively vulnerable to excitotoxicity.     

Quinolinic acid stimulates luteinizing hormone secretion in female rats: evidence for involvement of N-methyl-D-aspartate-preferring receptors

Summary Pharmacological evidence suggests that endogenous excitatory amino acid neurotransmitters stimulate luteinizing hormone (LH) secretion in neonatal and adult rats. Recent studies have identified quinolinic acid (QUIN), an endogenous brain and peripheral metabolite of tryptophan, as a potent agonist at N-methyl-D-aspartate (NMDA)-preferring excitatory amino acid receptors. The present studies examined whether QUIN alters LH secretion in ovariectomized, estradiol-primed rats and whether such effects are mediated by specific amino acid receptor subtypes. In one experiment, animals received intracisternal injections of either quinolinic acid, N-methyl-DL-aspartate (NMA), aspartate (ASP), quisqualic acid (QA), or monosodium glutamate (GLU) five minutes prior to decapitation. In a second study, animals receiving central QUIN or NMA were treated simultaneously with either 2-amino-7-phosphonoheptanoic acid (APH) or kynurenic acid (KYA), both antagonists of NMDA-preferring receptors, or the quisqualate antagonist, glutamate diethyl ester (GDEE). Serum LH concentrations were measured by radioimmunoassay. Intracisternal administration of either QUIN or NMA resulted in an acute, dose-dependent increase of serum LH concentrations. Coadministration of APH blocked the effects of QUIN and NMA. QUIN stimulation of LH was also blocked by KYA, but not GDEE. Neither GLU nor ASP increased LH release, but QA did produce a small, significant elevation of LH. Light microscopic evaluation of brains showed no morphologic disturbance resulting from administration of these agents. The present results suggest that QUIN, or other endogenous ligands of NMDA-preferring receptors, may participate in the regulation of LH secretion in the adult female rat.This research was supported by National Research Service Award Postdoctoral Fellowship HD-06443 (MDJ); by NIH grant HD-13703 and NIH Research Career Development Award HD-00366 (WRC); by Biomedical Research Support grant GR RR-05423, and by USPHS grant NS-20509 (WOW)     

Amelioration of hypoxia-induced striatal 5-HT(2A) receptor, 5-HT transporter and HIF1 alterations by glucose, oxygen and epinephrine in neonatal rats

Alterations in neurotransmitters and its receptors expression induce brain injury during neonatal hypoxic insult. Molecular processes regulating the serotonergic receptors play an important role in the control of respiration under hypoxic insult. The present study focused on the serotonergic regulation of neonatal hypoxia and its resuscitation methods. Receptor binding assays and gene expression studies were done to evaluate the changes in 5HT_2A receptors and its transporter in the corpus striatum of hypoxic neonatal rats and hypoxic rats resuscitated with glucose, oxygen and epinephrine. Total 5HT and 5HT_2A receptor number was increased in hypoxic neonates along with an up regulation of 5HT_2A receptor and 5HT transporter gene. The enhanced striatal 5HT_2A receptors modulate the ventilatory response to hypoxia. Immediate glucose resuscitation was found to ameliorate the receptor and transporter alterations. Hypoxia induced ATP depletion mediated reduction in blood glucose levels can be encountered by glucose administration and oxygenation helps in overcoming the anaerobic condition. The adverse effect of immediate oxygenation and epinephrine supplementation was also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.     

Poly(ADP-ribose) polymerase-1 is involved in the neuronal death induced by quinolinic acid in rats

Reactive oxygen and nitrogen species formation leads to DNA damage in animals treated with quinolinic acid. Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein involved in the DNA base excision repair system. Its overactivation promotes cellular energy deficit and necrosis. Here, we evaluated the effect of PJ-34, a potent inhibitor of PARP-1, on the neuronal damage induced by quinolinic acid. Animals were administered with PJ-34 (10 mg/kg, i.p.), 1 h before and 1 h after a striatal infusion of 1 μl of quinolinic acid (240 nmol). PJ-34 clearly attenuated the circling behavior produced by quinolinic acid and completely prevented the histological damage induced by the toxin. The protective effect of PJ-34 suggests that PARP-1 activation is playing an active role in the neuronal death induced by quinolinic acid.     

Persistence of classical conditioned heart rate after extensive lesions of the striatum in rats

The participation of the Corpus Striatum in the acquisition and maintenance of conditioned heart rate was determined. After a 25 min period of adaptation in a sound attenuated chamber 8 rats were presented with a single association of a sound (CS) and a nociceptive stimulus (US) applied to the paws, while electrocardiographic recordings were made. Twenty-four hours later the sole application of CS produced a significant increment in the heart rate. The same results were obtained in 8 rats that were previously electrolytically lesioned in the corpus striatum. In another 8 rats the electrolytic lesions were performed after the acquisition session. These rats also showed the increment in heart rate produced by the CS during the testing session. Finally, in 8 more rats in which the association of CS-US was inversed no conditioned response was obtained. The electrolytic lesions comprised several milimeters of the corpus striatum involving the anterior medial and posterior part and even extended to surrounding areas. Nevertheless the animals were able to acquire and retain the vegetative conditioned response.     

The effects of A1 and A2A adenosine receptor agonists on kainic acid excitotoxicity in the guinea pig cochlea

The present study aimed to clarify the protective effect of adenosine receptors against the excitotoxicity of cochlear afferent dendrites. The effects of 2-chloro-N6-cyclopentyladenosine (CCPA), an A1 adenosine receptor agonist, and 5′-N-cyclopropyl-carboxamidoadenosine (CPCA), an A2A adenosine receptor agonist, on cochlear excitotoxicity induced by kainic acid (KA) were examined using guinea pigs. KA was applied to the round window membrane at a concentration of 10 mM for 30 min. CCPA or CPCA was given at the onset of KA application. KA morphologically induced the swelling of cochlear afferent dendrites and significantly elevated the threshold of the compound action potential (CAP) of the cochlea. CCPA inhibited the KA-induced CAP threshold shift and swelling of the cochlear afferent dendrites. However, CPCA did not affect cochlear excitotoxicity induced by KA. The results suggest that adenosine A1 receptor activation could prevent the excitotoxicity of cochlear afferent dendrites.     

Learning deficits produced by chronic and reversible lesions of the corpus striatum in rats.

In a first experiment it was found that the reversible disruption of the normal activity of the corpus striatum (CN) of rats by microinjections of potassium chloride produced a marked impairment on the acquisition of a one-trial passive avoidance task. Two additional experiments showed the same performance deficits on the acquisition as well as on the retention of the task when the CN was electrolytically lesioned. Since two different methods of disrupting the functional integrity of the striatum were used, it can be concluded that the results are not due to the pecularities of a single method. These results further support the hypothesis of critical involvement of the CN in the integration and storing of learned information.     

Differential localization of the GluR1 and GluR2 subunits of the AMPA-type glutamate receptor among striatal neuron types in rats

Differences among the various striatal projection neuron and interneuron types in cortical input, function, and vulnerability to degenerative insults may be related to differences among them in AMPA-type glutamate receptor abundance and subunit configuration. We therefore used immunolabeling to assess the frequency and abundance of GluR1 and GluR2, the most common AMPA subunits in striatum, in the main striatal neuron types. All neurons projecting to the external pallidum (GPe), internal pallidum (GPi) or substantia nigra, as identified by retrograde labeling, possessed perikaryal GluR2, while GluR1 was more common in striato-GPe than striato-GPi perikarya. The frequency and intensity of immunostaining indicated the rank order of their perikaryal GluR1:GluR2 ratio to be striato-GPe > striatonigral > striato-GPi. Ultrastructural studies suggested a differential localization of GluR1 and GluR2 to striatal projection neuron dendritic spines as well, with GluR1 seemingly more common in striato-GPe spines and GluR2 more common in striato-GPi and/or striatonigral spines. Comparisons among projection neurons and interneurons revealed GluR1 to be most common and abundant in parvalbuminergic interneurons, and GluR2 most common and abundant in projection neurons, with the rank order for the GluR1:GluR2 ratio being parvalbuminergic interneurons > calretinergic interneurons > cholinergic interneurons > projection neurons > somatostatinergic interneurons. Striosomal projection neurons had a higher GluR1:GluR2 ratio than did matrix projection neurons. The abundance of both GluR1 and GluR2 in striatal parvalbuminergic interneurons and projection neurons is consistent with their prominent cortical input and susceptibility to excitotoxic insult, while differences in GluR1:GluR2 ratio among projection neurons are likely to yield differences in Ca²⁺ permeability, desensitization, and single channel current, which may contribute to differences among them in plasticity, synaptic integration, and excitotoxic vulnerability. The apparent association of the GluR1 subunit with synaptic plasticity, in particular, suggests striato-GPe neuron spines as a particular site of corticostriatal synaptic plasticity, presumably associated with motor learning.     

Identification of X versus Y properties for interneurons in the A-laminae of the cat's lateral geniculate nucleus

Summary Roughly 25% of the neurons in the A-laminae of the cat's lateral geniculate nucleus are local interneurons, while the remaining 75% are relay cells that project to the visual cortex. The interneurons form the focus of our study. The relay cells are either X or Y cells and are thereby integral links in the parallel and independent retino-geniculo-cortical X and Y pathways. Little is known about the response properties of interneurons, largely because it is difficult to identify them clearly during electrophysiological recording. However, they can be identified by morphological criteria. We thus studied their response properties by recording intracellularly from geniculate neurons to characterize them and then injecting them with horseradish peroxidase (HRP); the HRP labeling subsequently allowed us to distinguish relay cells from interneurons. In this manner, we studied 171 relay cells (83 X and 88 Y) and 15 interneurons. The response properties tested for each of the interneurons were indistinguishable from those of the relay X cells. We conclude that these interneurons are directly innervated by retinogeniculate X axons and are firmly embedded in the X pathway. We found no evidence for inter-neurons in the Y pathway.