长时程增强诱导和维持过程中海马CA1区神经细胞粘附分子蛋白水平与mRNA表达的变化

既往研究发现,神经细胞粘附分子(neural cell adhesion molecules,NCAM)对海马CA1区突触传递长时程增强(longterm potentiation,LTP)的诱导和维持极为关键。本文采用原位杂交法和Western blot法,观察了大鼠海马腑片LTP诱导和维持过程中NCAM mRNA和蛋白水平的动态变化过程。结果显示,强直刺激诱发fEPSP斜率升高10 min时,海马CA1区NCAM mRNA染色阳性神经元数量显著增加(76.6±11.5个),NCAM蛋白水平亦明显升高(7.190±0.64任意单位/50μg蛋白)。强直刺激诱发fEPSP斜率升高1 h时,NCAM mRNA染色阳性神经元数量为73.3±14.0个,NCAM蛋白量为9.031±0.71任意单位/50 μg蛋白;与强直刺激后10 min比较,NCAM mRNA表达无显著变化,而NCAM蛋白水平变化明显。NMDA受体特异阻断剂AP-5在损害LTP的同时,显著抑制NCAM mRNA和蛋白的增加。实验结果表明,在大鼠海马LTP诱导和维持过程中,NCAM mRNA增强的表达相对稳定,而NCAM蛋白水平呈现先低后高的变化。     

双电极绑定技术记录在体大鼠海马CA1区长时程增强

目的:探讨双电极绑定条件下记录大鼠在体海马CA1区长时程增强的可行性。方法:雄性Wistar大鼠乌拉坦麻醉;脑立体定位仪上埋置脑室导管;安装自制的刺激/记录绑定电极;引导基础性场兴奋性突触后电位(fEP-SP);强直刺激诱导长时程增强(LTP)。结果:绑定后的刺激和记录电极能可靠地引起海马CA1区fEPSP,fEPSP的出现率几乎100%;基础性fEPSP记录可保持长时间稳定;高频刺激成功诱导出LTP并维持达3h以上,诱导率约67%;双脉冲易化记录稳定、可靠;脑室注射β淀粉样蛋白(Aβ)对LTP显示出明显的压抑作用。结论:采用双电极绑定技术进行在体海马LTP记录简便易行、节省资源、引导fEPSP和诱导LTP的成功率较高,有望成为一项重要的研究学习和记忆机制的电生理辅助手段。     

褪黑素对大鼠海马 CA3区长时程增强诱导的影响

目的：研究褪黑素对海马CA3区长时程增强（LTP）诱导阶段的影响及可能的机制。方法：用细胞外电生理记录方法。通过向大鼠海马CA3区分别微量注射褪黑素、Tacrine（胆碱酯酶抑制剂）和DNQX（非NMDA受体的竞争性拮抗剂）,以海马CA3区群体兴奋性突触后电位（fEPSP）斜率的改变为指标,观察PP-CA3通路的LTP变化。结果：①0．2μg/μl、1μg/μl和5μg/μl的褪黑素对CA3区的诱发电位和LTP均有抑制作用,随着剂量加大,抑制作用加强。②褪黑素能抑制Tacrine对LTP诱导的易化作用。③DNQx能部分阻断褪黑素对LTP诱导的抑制作用。结论：褪黑素对LTP诱导的抑制作用可能与胆碱能系统和非NMDA受体有关。     

褪黑素对大鼠空间学习记忆的影响及其机制研究

本研究运用Morris水迷宫和电生理学方法 ,以逃避潜伏期、穿环系数和海马CA1区突触长时程增强(long termpotentiation ,LTP)为指标 ,研究褪黑素对大鼠空间学习记忆能力的影响。实验结果显示 :( 1)在Morris水迷宫 6d训练中 ,对照组大鼠后 4d平均逃避潜伏期为 18 4 4± 2 7s,褪黑素组为 3 0 0 2± 3 6s,两者有显著差异 (P <0 0 1) ;训练 6d后 ,褪黑素组穿环系数为 2 5 68± 2 3 2 % ,明显小于对照组的 4 3 3 3± 2 85 % (P <0 0 1)。( 2 )采用微量注射法给予海马CA1区褪黑素 ,强直后 60min ,fEPSP斜率为基准值的 114 2 8± 1 80 % ,显著低于对照组的 169 71±6 4 8% (P <0 0 1)。( 3 )预先给予东莨菪碱 ,不影响褪黑素对海马CA1区LTP的抑制 ,强直后 60minfEPSP斜率为基准值的 113 70± 5 5 5 %。( 4 )提前给予荷包牡丹碱后给予褪黑素 ,强直后 60minfEPSP斜率为基准值的 162 2 9±10 5 2 % ,明显大于褪黑素组 (P <0 0 1) ,而与对照组无显著差异 (P >0 0 5 )。上述结果表明 ,褪黑素对大鼠的空间学习记忆能力及海马CA1区LTP均有明显的抑制作用 ,两者相关 ;东莨菪碱不能阻断褪黑素对海马CA1区LTP的抑制作用 ,而荷包牡丹碱可以阻断褪黑素对LTP的抑制 ,提示褪黑素的作用可能不是由胆碱能系统所介     

大鼠在体海马CA1区场电位引导新技术——刺激/记录/给药一体化装置的开发和应用

大鼠海马场电位记录是研究学习记忆的一个重要手段,尤以在体记录更具生理学意义.为克服目前在体海马场电位记录的弊端和诸多不便,提高实验效率,设计并完善了一套简便易行,融刺激、记录、给药于一体的大鼠海马在体CA1区场电位实验技术.将雄性SD大鼠麻醉后固定于脑立体定位仪上,利用自制的刺激/记录/给药联合装置,引导海马CA1区场兴奋性突触后电位(fEPSP).结果表明,使用刺激/记录/给药联合装置能长时间稳定记录由测试刺激诱发的海马CA1区fEPSP.高频刺激条件下,能成功诱导早期时相长时程增强(E-LTP)和晚期时相型长时程增强(L-LTP).海马内注射AMPA受体阻断剂CNQX(100μmol/L,1μl)可迅速抑制fEPSP,注射NMDA受体拮抗剂AP-5(100μmol/L,1μl)可明显压抑LTP,药物发挥作用的时间较侧脑室给药明显缩短,剂量减少.采用此联合装置还成功实现了PPF的稳定记录.总之,采用刺激/记录/给药一体化技术进行在体海马CA1区场电位记录的特点是简单、可靠、高效,可以为开展脑认知功能活动的电生理学研究奠定良好的基础.     

丹参酮ⅡA 预防慢性缺氧大鼠认知功能障碍的电生理机制

目的:探讨丹参酮ⅡA(TⅡA)预防慢性缺氧大鼠认知功能障碍的电生理机制。方法:将18只雄性SD大鼠(200-250 g)随机分为对照组、模型组(Model组)、TⅡA(10mg/kg.d)治疗组(TⅡA组)。复制慢性缺氧大鼠认知功能障碍模型,并给予相应治疗,在脑片水平运用膜片钳技术检测海马CA1区的LTP变化,并检测海马CA1区锥体细胞的兴奋性变化。结果:(1)给予高频强直刺激(HFS)后各组兴奋性突出后电位(fEPSP)斜率均显著增加,即均可诱发LTP并持续1h以上,但模型组LTP较对照组显著减弱(P<0.05),TⅡA治疗组LTP较模型组明显增强(P<0.05);(2)慢性缺氧使海马CA1锥体细胞放电所需的刺激电流幅度显著增加、阈电位升高、兴奋性降低,同样刺激强度条件下动作电位数量减少,TⅡA干预可明显减轻慢性缺氧对海马CA1锥体细胞的上述抑制。结论:TⅡA可能是通过维持海马CA1锥体细胞的兴奋性、维持海马的突出可塑性减轻慢性缺氧对认知功能的损害。     

2 Hz与100 Hz刺激在诱导大鼠视皮层长持续长时程增强中的不同作用

在哺乳动物的视皮层,多种不同参数的刺激可诱导出长时程增强(long-term potentiation,LTP)现象。但关于刺激参数与持续时间长于3h的长持续LTP(long lasting LTP,L-LTP)之间关系的研究较少。本研究用3周龄的大鼠视皮层脑片标本,在Ⅳ层刺激而在Ⅱ／Ⅲ层记录场电位,待场电位稳定后施加强直刺激诱导LTP,探讨2Hz与100Hz的强直刺激在诱发持续时间长于3h的L-LTP中的作用。结果表明,多于300个脉冲不同频率的刺激可稳定地诱导出L-LTP;2Hz与100Hz的刺激诱发的L-LTP有明显不同的表达形式,100Hz刺激可诱导出较大的L-LTP;频率相同而脉冲数不同的强直刺激诱发的L-LTP有相同的表达形式。以上结果提示,不同频率的强直刺激诱发的L-LTP机制可能不同;相同频率的刺激(脉冲数不同)诱发的L-LTP可能有相同的机制。     

小鼠海马CAl区高频刺激诱发的突触可塑性分析

通过细胞外记录方法记录场兴奋性突触后电位（field excitatory postsynaptic potential,fEPSP）的变化是研究突触可塑性,诸如长时程增强（long-term potentiation,LTP）和双脉冲可塑性（paired-pulse plasticity,PPP）的最常见方法之一。fEPSP波形的起始斜率、起始面积、峰值及总面积等的变化常用作判断突触可塑性增强或减弱的标准。在相同记录结果中测量fEPSP波形不同部位通常会有不同的结果,因此可能得出不同的结论,这些往往会被研究者忽略。本文通过测量小鼠海马CA1区细胞fEPSP波形的起始斜率、起始面积、峰值、总面积及时间参数等,分析比较高频刺激（high-frequency stimulation,HFS）诱发的突触可塑性,包括LTP和PPP的变化。结果显示,LTP过程中AMPA受体动力学变化加快,且在同一记录中,fEPSP波形不同部位的测量分析可以产生较大幅度的LTP和PPP差异。给予HFS后,双脉冲诱发fEPSP的比率在测量起始面积时略有下降,但在测量起始斜率时则显著增加,这些结果可能导致相反的结论。因此,全面仔细地分析fEPSP波形在整个实验中的变化对正确了解突触可塑性至关重要。     

急性神经损伤引起脊髓背角C-纤维诱发电位长时程增强

神经损伤引起神经病性疼痛,表现为持续性痛超敏和痛觉过敏。目前对神经病性疼痛的机制尚缺乏了解。我们以往的工作表明强直电刺激坐骨神经可引起脊髓背角C-纤维诱发电位的长时程增强(long-term potentiation,LTP),该LTP被认为是病理性疼痛的突触模型。本研究的目的在于探讨急性神经损伤是否能在完整动物的脊髓背角诱发出C-纤维诱发电位LTP。在以测试刺激(10～20V,0．5ms)电刺激坐骨神经的同时在脊髓背角用微电极记录C一纤维诱发电位。分别用强直刺激、剪断或夹捏坐骨神经诱导LTP。结果发现：(1)剪断或夹捏坐骨神经都可以诱导脊髓背角C-纤维诱发电位的LTP,该LTP可持续到实验结束(3～9h),在剪断神经前10min用利多卡因局部阻滞坐骨神经则可完全阻断LTP的产生;(2)神经损伤诱导的LTP可被NMDA受体阻断剂AP5所阻断;(3)用单次强直刺激引起LTP后,切断坐骨神经可使LTP的幅度进一步增大,而用多次强直电刺激使LTP饱和后,损伤神经则不能使LTP进一步增大。切断神经引起LTP后,强直电刺激也不能使LTP进一步增大。这些结果表明,急性神经损伤可以诱导脊髓背角C纤维诱发电位LTP,且切断神经能更有效地诱导LTP。该试验进一步支持我们的设想,即脊髓背角C-纤维诱发电位LTP可能在病理性疼痛的形成中起重要作用。     

姜黄素对癫痫大鼠认知功能障碍的预防作用及其可能机制

目的:探讨姜黄素(curcumin)对癫痫大鼠认知功能障碍的预防作用及其可能机制。方法:将30只成年雄性SD大鼠分为正常对照组、单纯致痫组(SE组)、姜黄素[60mg/(kg.d)]干预组(curcumin组)。采用Morris水迷宫方法检测大鼠学习记忆功能变化,并检测脑片水平的长时程增强(LTP)变化,处死大鼠后取脑组织并匀浆,测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、谷胱甘肽(GSH)、丙二醛(MDA)的水平。结果:(1)SE组大鼠寻找平台的潜伏期明显长于对照组,具有统计学意义(P<0.05),姜黄素组寻找平台的潜伏期相对于SE组显著缩短(P<0.05)。撤离平台后,SE组大鼠在平台所在象限的停留时间明显短于对照组(P<0.05),姜黄素治疗后大鼠在平台所在象限的停留时间较SE组显著延长(P<0.05)。(2)给予HFS刺激后各组兴奋性突出后电位(fEPSP)斜率较前明显增加,均可持续1h以上,与对照组比较SE组HFS刺激后fEPSP斜率明显减小(P<0.05),姜黄素可减轻SE所致的fEPSP斜率减小(P<0.05)。(3)SE组SOD、GSH-PX、GSH显著下降,MDA明显增高,姜黄素可逆转上述现象,有统计学意义(P<0.05)。结论:姜黄素可显著减轻癫痫持续状态所致的大鼠认知功能障碍,减轻海马区的氧化应激反应从而保护海马海马是其可能机制之一。     

Unique Induction of CA1 LTP Components After Intake of Theanine, an Amino Acid in Tea Leaves and its Effect on Stress Response

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. This study was undertaken to evaluate the effect of theanine intake on long-term potentiation (LTP) induction at hippocampal CA1 synapses and exposure to acute stress. Young rats were fed water containing 0.3% theanine after birth. Key findings: Serum corticosterone level was markedly decreased by theanine intake. Because this decrease can modify synaptic plasticity, the effect of theanine intake was examined focused on CA1 LTP induction. CA1 LTP induced by a 100-Hz tetanus for 1 s was almost the same extent in hippocampal slices from theanine-administered rats, whereas that induced by a 200-Hz tetanus for 1 s was significantly attenuated. 2-Amino-5-phosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly attenuated CA1 LTP induced by a 200-Hz tetanus in the control rats, but not in theanine-administered rats. Interestingly, APV completely blocked CA1 LTP induced by a 100-Hz tetanus in the control rats, while scarcely blocking it in theanine-administered rats. These results indicate that theanine intake reduces NMDA receptor-dependent CA1 LTP, while increasing NMDA receptor-independent CA1 LTP. Furthermore, neither 100-Hz tetanus-induced LTP nor 200-Hz tetanus-induced LTP was attenuated in theanine-administered rats after exposure to tail suspension stress, suggesting that the lack of NMDA receptor-dependent CA1 LTP by theanine intake is involved in ameliorating the attenuation of CA1 LTP after tail suspension. This study is the first to indicate that theanine intake modifies the mechanism of CA1 LTP induction.     

Sequential activation of soluble guanylate cyclase, protein kinase G and cGMP-degrading phosphodiesterase is necessary for proper induction of long-term potentiation in CA1 of hippocampus. Alterations in hyperammonemia

Long-term potentiation (LTP) is a long-lasting enhancement of synaptic transmission efficacy and is considered the base for some forms of learning and memory. Nitric oxide (NO)-induced formation of cGMP is involved in hippocampal LTP. We have studied in hippocampal slices the effects of application of a tetanus to induce LTP on cGMP metabolism and the mechanisms by which cGMP modulates LTP. Tetanus application induced a transient rise in cGMP, reaching a maximum at 10s and decreasing below basal levels 5 min after the tetanus, remaining below basal levels after 60 min. Soluble guanylate cyclase (sGC) activity increased 5 min after tetanus and returned to basal levels at 60 min. The decrease in cGMP was due to sustained tetanus-induced increase in cGMP-degrading phosphodiesterase activity, which remained activated 60 min after tetanus. Tetanus-induced activation of PDE and decrease of cGMP were prevented by inhibiting protein kinase G (PKG). This indicates that the initial increase in cGMP activates PKG that phosphorylates (and activates) cGMP-degrading PDE, which, in turn, degrades cGMP. Inhibition of sGC, of PKG or of cGMP-degrading phosphodiesterase impairs LTP, indicating that proper induction of LTP involves transient activation of sGC and increase in cGMP, followed by activation of cGMP-dependent protein kinase, which, in turn, activates cGMP-degrading phosphodiesterase, resulting in long-lasting reduction of cGMP content. Hyperammonemia is the main responsible for the neurological alterations found in liver disease and hepatic encephalopathy, including impaired intellectual function. Hyperammonemia impairs LTP in hippocampus by altering the modulation of this sGC-PKG-cGMP-degrading PDE pathway. Exposure of hippocampal slices to 1 mM ammonia completely prevents tetanus-induced decrease of cGMP by impairing PKG-mediated activation of cGMP-degrading phosphodiesterase. This impairment is responsible for the loss of the maintenance of LTP in hyperammonemia, and may be also involved in the cognitive impairment in patients with hyperammonemia and hepatic encephalopathy.     

Insulin causes a transient tyrosine phosphorylation of NR2A and NR2B NMDA receptor subunits in rat hippocampus

NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 microM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 +/- 16% (NR2A) and 203 +/- 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 +/- 13% of control; NR2B: 93 +/- 10% of control). Although the consequence of insulin-stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.     

Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.     

A proteasomal stress response: pre-treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.     

Excitotoxic stimulation downregulates the ubiquitin–proteasome system through activation of NMDA receptors in cultured hippocampal neurons

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin–proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.     

磷酸化钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠脊髓背角C-纤维诱发电位长时程增强的诱导和维持中的作用

实验旨在探讨钙/钙调蛋白依赖性蛋白激酶II(calcium／calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)在脊髓背角C-纤维诱发电位长时程增强(long—term potentiation,LTP)的诱导和维持中的作用。用Western blot技术分别检测LTP形成30min和3h脊髓背角(L4-L6)CaMKⅡ的含量及其磷酸化水平。同时观察脊髓局部给予CAMKⅡ选择性抑制剂KN-93后对脊髓背角LTP和CaMKII磷酸化的影响。观察结果如下：(1)诱导LTP后30min,CaMK Ⅱ的磷酸化水平明显高于对照组,而CaMKⅡ的总量无变化;诱导LTP后3h CaMKⅡ的磷酸化水平进一步升高。而且CaMKⅡ的总量也明显增加(n=4);(2)强直刺激前30min于脊髓局部给予CaMKⅡ的特异性抑制剂KN-93(100μmol/L),可阻断LTP的诱导,同时明显抑制CaMKⅡ的磷酸化水平;(3)诱导LTP后30min给予KN-93,可显著抑制LTP的维持,同时CaMKⅡ的磷酸化水平与未用药组相比也明显降低（n=3);(4)LTP3h后给予KN-93,LTP的幅值不受影响,磷酸化的CaMKⅡ的含量与用药前相比也无差别（n=3)。根据上述实验结果可以认为,CaMKⅡ的激活参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持过程。     

The effects of fluorocitrate on renal glutamine, lactate, alanine, and oxygen metabolism in the dog

Acid-base status is considered the major factor controlling renal NH4+ production from glutamine, with maximal values found in chronic acidosis. However, metabolic inhibitors have been shown to increase NH4+ production without acid-base change; the mechanism for this increase is unclear. Fluorocitrate was administered to dogs with chronic metabolic alkalosis. Following fluorocitrate total renal NH4+ production rose from 32 +/- 5 to 104 +/- 15 mumol/(min.100 mL glomerular filtration rate (GFR] (p less than 0.01) and glutamine extraction rose from 26 +/- 8 to 65 +/- 8 mumol/(min.100 mL GFR) (p less than 0.01). These values approximate maximal values found in chronic acidosis. Lactate utilization fell from 165 +/- 19 to 99 +/- 7 mumol/(min.100 mL GFR) following fluorocitrate (p less than 0.01). Citrate extraction fell to zero and alanine production rose from 27 +/- 4 to 46 +/- 7 mumol/(min.100 mL GFR) (p less than 0.01). Oxygen consumption remained unchanged following fluorocitrate, 584 +/- 29 vs. 549 +/- 29 mumol/(min.100 mL GFR). These results demonstrate that in the presence of metabolic inhibition in the kidney, ATP production remains constant. This is achieved by increased utilization of one substrate, glutamine, when the ATP production from other substrates is reduced. Thus the necessity to maintain constant ATP production appears to modulate renal NH4+ production.     

Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in β1,4-N-acetylgalactosaminyltransferase (β1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100?Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1?Hz) at 20?min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1?Hz, 20?min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of β1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.