Responses of the superficial entorhinal cortex in vitro in slices from naive and chronically epileptic rats
Responses of the superficial entorhinal cortex in vitro in slices from naive and chronically epileptic rats
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DOI：
10.1152/jn.1996.76.5.2928
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发表时间：
1996-11-01
影响因子：
2.5
通讯作者：
Lothman, EW
中科院分区：
文献类型：
--
作者：
Bear, J;Fountain, NB;Lothman, EW
1.本研究的主要目的：1.研究慢性癫痫动物内嗅皮层(EC)浅层细胞的细胞内外反应，确定其生理改变是否依赖于与海马区的连接，并探讨是否存在兴奋增强和神经元间连接抑制的证据。在大鼠海马区-海马旁联合切片制备中，通过与脑底成30度角的振动刀切片，在体外保持了海马区和EC之间的功能连接。对三组动物进行了研究：幼稚的动物，经历过(非抽搐的)自持性边缘系统状态elilepticus(SSLSE)的动物，这些动物是由电刺激导致慢性癫痫板岩形成的，以及电极对照组的动物。TN慢性癫痫大鼠和电极对照组，对电极植入侧的对侧组织进行研究。细胞外和细胞内记录来自EC的浅层。电刺激EC深层或与EC相邻的角束可激活EC浅层神经元，角束内含有EC神经元的轴突。刺激另一部位可通过逆行和突触机制引起反应。此外，在离子亲性谷氨酸阻滞剂D(-)-2-氨基-5-磷酸-丙戊酸(APV)和6，7-二硝基-2-3-二酮(DNQX)存在下，使用单突触方案直接激活中间神经元，在记录电极附近放置刺激电极。在从极低强度到高强度的一系列刺激强度上收集响应，以构建输入/输出函数(I/O)曲线。在最低的刺激强度下测量幅度和持续时间，以获得最大的反应。5.来自电极对照的胞外场电位响应在场电位响应的形态上或在响应持续时间和幅度上与朴素的没有什么不同。SSLSE后大鼠组织中的场电位反应在形态上明显不同于未经SSLSE和电极对照的大鼠。更复杂，持续时间明显更长，幅度更小。用APV阻断N-甲基-D-天冬氨酸(NMDA)受体可显著缩短这些癫痫样反应，但这种操作不能逆转正常形态的反应。用DNQX.6阻断非NMDA介导的离子型谷氨酸受体可取消这些反应。在对对照组和癫痫动物脑片中的神经元进行细胞内记录时，在没有电刺激的情况下，神经元在静息条件下处于静止状态。电极对照组的细胞内反应与NAIVE相同，一起被认为是“对照组”。在对照组织中，诱发的细胞内反应与先前描述的相似，最常见的包括NMDA受体拮抗剂APV部分阻断的兴奋性突触后电位(EPSP)，然后是超极化电位，从电生理学和药理学上被鉴定为伽马氨基丁酸-A(GABA(A))和GABA(B)受体介导的抑制性突触后电位(IPSP)。DNQX.8可完全阻断EPSP。在慢性癫痫组织中，诱发的脱细胞反应与对照组动物的反应明显不同？表现为对单一电击反应的具有多个叠加动作电位的延长的阵发性去极化事件。在APV中，这些去极化事件的持续时间和幅度都有所减少，但并未被消除。IPSP在所有刺激强度下均未见或明显减少。这些细胞内的反应从来不像对照反应。细胞内反应在形态和持续时间上与细胞外场能精确相关。在单突触直接激活抑制性中间神经元的慢性癫痫组织中，诱发的超极化电位被电生理学和药理学鉴定为GABA(A)和GABA(B)受体介导的IPSP，与对照组动物的单突触IPSP没有区别。这些发现与假设一致，即浅层EC细胞是过度兴奋的，就像在该模型中的CA1一样，这是由于增强的兴奋和受损的抑制，可能是因为GABA能抑制中间神经元处于休眠状态(与兴奋性输入断开连接)。这些结果构成了对慢性颞叶癫痫动物模型中EC的第一次研究，并为海马区外的电生理变化提供了证据。
1. The main purposes of this study are to characterize the intracellular and extracellular responses of cells in superficial layers of entorhinal cortex (EC) in chronically epileptic animals, determine whether their altered physiology is dependent on being connected to hippocampus, and investigate whether there is evidence of augmented excitation and inhibitory interneuron disconnection.2. Functional connectivity was maintained between the hippocampal area and the EC in vitro in a combined rat hippocampal-parahippocampal slice preparation by slicing with a vibratome at a 30-deg angle to the base of the brain. Three groups of animals were studied: naive animals, animals that had experienced a previous episode of (nonconvulsive) self-sustaining limbic system status elilepticus (SSLSE) induced by electrical stimulation resulting in a chronically epileptic slate, and animals in an electrode control group. Tn chronically epileptic rats and the electrode control group, studies were done on tissue contralateral to the side of electrode implantation.3. Extracellular and intracellular recordings were made from the superficial layers of EC. Neurons in the superficial layers of the EC were activated by stimulation of the deep layers within the EC or the angular bundle adjacent to the EC, which contains axons from EC neurons. Responses could be elicited by antidromic and synaptic mechanisms by stimulation at tither site. Tn addition, a monosynaptic protocol was used that involved direct activation of interneurons with a stimulating electrode placed near the recording electrode in the presence of the ionotropic glutamate blockers D(-)-2-amino-5-phosphonovaleric acid (APV) and 6,7-dinitroquinoxaline-2-3-dione (DNQX).4. Responses were collected over a range of stimulus intensities, from very low to high intensities, to construct input/output function (I/O) curves. Amplitudes and durations were measured at the lowest stimulus intensity that elicited a maximum response. 5. Extracellular field potential responses from electrode controls did not differ from naives qualitatively with respect to morphology of field potential responses or quantitatively with respect to response duration and amplitude. Field potential responses in tissue from post-SSLSE rats differed markedly in morphology from naive and electrode controls. being more complex, significantly longer in duration, and decreased in amplitude. These epileptiform responses were shortened markedly by blockade of N-methyl-D-aspartate (NMDA) receptors with APV, but this manipulation did not con vert responses to a normal morphology. These responses were abolished by blockade of non-NMDA mediated ionotropic glutamate receptors with DNQX.6. During intracellular recordings of neurons in slices from both control and epileptic animals, neurons were quiescent under resting conditions in the absence of electrical stimulation.7. Intracellular responses in electrode controls were identical to naive, and together were considered ''controls.'' In control tissue, evoked intracellular responses were similar to those previously described and most commonly consisted of an excitatory postsynaptic potential (EPSP) that was blocked partially by the NMDA-receptor antagonist APV, followed by hyperpolarizing potentials, which were identified electrophysiologically and pharmacologically as gamma-aminobuturic acid-A (GABA(A))- and GABA(B)-receptor-mediated inhibitory postsynaptic potentials (IPSPs). EPSPs were blocked completely by DNQX.8. Tn chronically epileptic tissue, evoked inh acellular responses differed markedly from responses in control animals? exhibiting all-or-none prolonged paroxysmal depolarizing events with multiple superimposed action potentials in response to a single shock. These depolarizing events were reduced in duration and amplitude, but not abolished, in APV. IPSPs were not seen or markedly reduced at all stimulus intensities. These intracellular responses never resembled control responses. Intracellular responses correlated precisely in morphology and duration with extracellular field potentials.9. In chronically epileptic tissue with the monosynaptic protocol used to directly activate inhibitory interneurons, hyperpolarizing potentials were evoked that were electrophysiologically and pharmacologically identified as GABA(A)- and GABA(B)-receptor-mediated IPSPs and did not differ from monosynaptic IPSPs in control animals.10. The findings are consistent with the hypothesis that superficial layer EC cells are hyperexcitable, as in CA1, in this model and that this is due to augmented excitation and impaired inhibition, possibly because GABAergic inhibitory interneurons are dormant (disconnected from excitatory input).11. These results constitute the first study of the EC in an animal model of chronic temporal lobe epilepsy and provide evidence of electrophysiologic changes outside of the hippocampus.