Neuroprotective Effects of Diazoxide and Its Antagonism by Glibenclamide in Pyramidal Neurons of Rat Hippocampus Subjected to Ischemia-Reperfusion-Induced Injury

Mitochondrial ATP-sensitive potassium channel opener, diazoxide, is shown to have protective effect on the heart and brain following ischemia-reperfusion-induced injury (IR/II). However, the detailed effect of diazoxide and its antagonist on neuronal death, mitochondrial changes, and apoptosis in cerebral IR/II has not fully studied. IR/II was induced in rats by the 4-vessel occlusion model. Neuronal cell death and mitochondrial changes in CA1–CA4 pyramidal cells of the hippocampus were studied by light and electron microscopy, respectively. Apoptosis was assessed by measuring the amount of protein expressed by Bax and Bcl-2 genes. In light microscopy studies, the number of total and normal cells were increased only following 18 mg/kg of diazoxide. Lower doses (2 and 6 mg/kg) failed to change the cell numbers. All three doses of glibenclamide (1, 5, and 25 mg/kg) decreased the number of total and normal cell populations. In electron microscopy studies, different doses of diazoxide and glibenclamide prevented and aggravated the IR-induced morphological changes, respectively. Western blot analysis showed that diazoxide and glibenclamide inhibited and enhanced Bax protein expression respectively. Regarding Bcl-2 expression, only diazoxide showed a significant enhancement of gene expression. In conclusion, the results show that diazoxide can exhibit neuroprotective effects against IR/II in hippocampal regions, possibly through the opening of mitochondrial ATP-sensitive K⁺ channels.     

Comparison of Stress-induced Changes in Noradrenergic and Serotonergic Neurons in the Rat Hippocampus Using Microdialysis

The effects of stress on the serotonergic and noradrenergic projection to the hippocampus were compared in freely moving rats using microdialysis. Stress-induced changes in 5-hydroxytryptamine (5-HT), noradrenaline and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in the presence of their respective uptake blockers. Local infusion of tetrodotoxin and replacement of Ca²⁺ with Cd²⁺ were used to test dependence on impulse traffic. A 5 min tail pinch or 10 min restraint stress increased 5-HT, 5-HIAA, noradrenaline and DOPAC levels. A subcutaneous saline injection produced an increase in 5-HT and DOPAC but not noradrenaline or 5-HIAA. Although α₂ adrenoceptor agonists and antagonists produced changes in the baseline values of noradrenaline and DOPAC, they had little or no effect on stress-induced changes. Both the abolition of impulse traffic and its enhancement by stress had a greater effect on transmitter than on metabolite levels. Although the responses to stress of the noradrenergic and serotonergic pathway showed many similarities, there was evidence for their activation by separate pathways.     

托吡酯对戊四氮点燃慢性癫痫大鼠海马神经细胞黏附分子的影响

目的探讨神经元活化在癫(癎)发生、发展中的作用及托吡酯对其的影响.方法采用戊四氮制备慢性癫(癎)模型,利用托吡酯干扰,选取不同时间点,观察大鼠行为学变化及神经细胞黏附分子在海马回的表达(免疫组织化学染色).结果托吡酯组点燃率在27 d明显低于模型组;模型组及托吡酯组神经细胞黏附分子表达增加,并随时间延长明显;而不同时间点该表达的增加程度,托吡酯组均低于模型组.结论神经细胞黏附分子表达的增加,说明出现了神经元活化及脑可塑性变化,而托吡酯明显地抑制了该表达的增加.     

托吡酯对戊四氮点燃慢性癫(癎)大鼠海马神经细胞黏附分子的影响

目的:探讨神经元活化在癫发生、发展中的作用及托吡酯对其的影响。方法:采用戊四氮制备慢性癫模型,利用托吡酯干扰,选取不同时间点,观察大鼠行为学变化及神经细胞黏附分子在海马回的表达(免疫组织化学染色)。结果:托吡酯组点燃率在27d明显低于模型组;模型组及托吡酯组神经细胞黏附分子表达增加,并随时间延长明显;而不同时间点该表达的增加程度,托吡酯组均低于模型组。结论:神经细胞黏附分子表达的增加,说明出现了神经元活化及脑可塑性变化,而托吡酯明显地抑制了该表达的增加。     

电点燃癫痫大鼠海马白细胞介素-6的表达包

目的 通过测定杏仁核电点燃癫痫大鼠海马白细胞介素-6(IL-6)表达情况,探讨IL-6变化规律及其作用.方法 把双极电极插入大鼠左侧杏仁基底外侧核,给予慢性电刺激使大鼠达到点燃状态,观察其发作过程并记录脑电图.采用半定量RT-PCR法检测电点燃大鼠海马IL-6 mRNA表达并设药物治疗组对照观察.结果 大鼠平均经(13.50±3.99)次刺激达到点燃状态,记录到的后发放时程在21 450～119 720 ms之间.电点燃大鼠海马IL-6 mRNA表达升高.结论 癫痫大鼠海马IL-6 mRNA表达升高,可能参与了点燃过程.     

Subcellular Distribution of Persistent Sodium Conductance in Cortical Pyramidal Neurons

Cortical pyramidal neurons possess a persistent Na⁺ current (I_NaP), which, in contrast to the larger transient current, does not undergo rapid inactivation. Although relatively quite small, I_NaP is active at subthreshold voltages and therefore plays an important role in neuronal input–output processing. The subcellular distribution of channels responsible for I_NaP and the mechanisms that render them persistent are not known. Using high-speed fluorescence Na⁺ imaging and whole-cell recordings in brain slices obtained from mice of either sex, we reconstructed the I_NaP elicited by slow voltage ramps in soma and processes of cortical pyramidal neurons. We found that in all neuronal compartments, the relationship between persistent Na⁺ conductance and membrane voltage has the shape of a Boltzmann function. Although the density of channels underlying I_NaP was about twofold lower in the axon initial segment (AIS) than in the soma, the axonal channels were activated by ∼10 mV less depolarization than were somatic channels. This difference in voltage dependence explains why, at functionally critical subthreshold voltages, most I_NaP originates in the AIS. Finally, we show that endogenous polyamines constrain I_NaP availability in both somatodendritic and axonal compartments of nondialyzed cortical neurons.SIGNIFICANCE STATEMENT The most salient characteristic of neuronal sodium channels is fast inactivation. However, a fraction of the sodium current does not inactivate. In cortical neurons, persistent current (I_NaP) plays a prominent role in many important functions. Its subcellular distribution and generation mechanisms are, however, elusive. Using high-speed fluorescence Na⁺ imaging and electrical recordings, we reconstructed the I_NaP in soma and processes of cortical pyramidal neurons. We found that at near-threshold voltages I_NaP originates predominately from the axon, because of the distinctive voltage dependence of the underlying channels and not because of their high density. Finally, we show that the presence of endogenous polyamines significantly constrains I_NaP availability in all compartments of nondialyzed cortical neurons.     

Abnormal Excitability of Hippocampal CA3 Pyramidal Neurons of Spontaneously Epileptic Rats (SER), a Double Mutant

The spontaneously epileptic rat (SER:zi/zi, tm/tm), a double mutant, shows both tonic convulsions and absence-like seizures characterized by low-voltage fast waves and by 5-7 Hz spike and wave-like complexes in the cerebral cortical and hippocampal EEG, respectively. Characteristics of hippocampal CA3 pyramidal neurons were examined to determine whether these neurons are abnormally excitable. When a single stimulus was given to the mossy fiber, there was repetitive firing and a depolarization shift in neurons of mature SER (over 12 weeks old), in which epileptic seizures had fully developed. However, in young SER (7-8 weeks old) and littermates (zi/zi, tm/+), which did not show any seizures, only a single spike was elicited with each single stimulation of the mossy fiber. Intracellular recording showed that the resting membrane potential was not significantly different among young and mature SER and littermates, but a long-lasting (100-200 ms) depolarizing shift accompanied by repetitive firing was observed following a single stimulation of the mossy fiber in half of the CA3 neurons of mature SER. Furthermore, the input impedance of the CA3 neurons in mature SER was lower than that in young SER and in littermates. These results indicate that SER hippocampal CA3 neurons become abnormally excitable in conjunction with the development of epileptic seizures.     

弥漫性脑损伤诱发的前脑胆碱能神经元变化

目的 本实验通过实验性研究,以了解弥漫性脑损伤（ＤＢＩ）是否引起胆碱能神经元的显性减少。方法 采用Ｍａｒｍａｒｏｕ打击装置建立ＤＢＩ动物模型,行胆碱酯酶组织化学染色以显示基底前胆碱能神经元。结果 ①基底前脑胆碱能神经元在重伤组、轻伤组、对照组有显差异（Ｐ〈０．０１）;②损伤后二周组神经元减少与一周组比较有显差异（Ｐ〈０．０１或Ｐ〈０．０５）。结论 ①在本实验所观察到的ＤＢＩ后基底前脑胆碱能神     

红藻氨酸诱导的大鼠癫痫模型中海马神经元caspase-9活性的变化

目的 :观察红藻氨酸 (kainicacid ,KA)诱导的癫大鼠海马神经元caspase 9活性的变化。方法 :应用KA腹腔注射诱导大鼠癫模型 ,检测癫大鼠海马神经元caspase 9的活性。caspase 9活性的测定采用荧光底物分析法。结果 :注射KA后 1h大鼠海马神经元caspase 9活性开始升高 ,但与对照组相比 ,无统计学差异 ;注射KA后 3h大鼠海马神经元caspase 9活性达到高峰 ,与对照组相比P <0 0 5 ;然后开始下降 ,于注射KA后 12h接近于正常对照组水平。结论 :在KA腹腔注射诱导的大鼠癫模型中 ,海马神经元caspase 9活性仅在早期表达升高 ,提示caspase 9活性升高可能启动了癫大鼠海马神经元的损伤     

阿尔茨海默病大鼠海马额叶Bcl-2蛋白表达的改变

目的研究珂尔茨海默病(AD)大鼠学习记忆及Bcl-2蛋白表达的改变。方法β淀粉样蛋白(βAP)注入大鼠迈内特基底核(NBM)建立AD型．Y迷宫测定学习记忆能力,流式细胞仪检测Bcl-2蛋白含量。治疗组腹腔注射尼莫的平2周。结果模型组大鼠学习记忆能力下降,海马和额叶Bcl-2蛋白含量增高;治疗组Bcl-2水平下调,学习记忆有所改善,但与对照组比仍有差异。结论βAP注入NBM引起大鼠学习记忆损害,Bcl-2蛋白的表达增加。     

BCL—2蛋白在癫痫过程中海马细胞内的变化

癫痫全面性强直阵挛发作（ＧＴＣＳ）可诱导脑细胞的损伤与修复过程。ＢＣＬ—２蛋白是具有介导多种细胞外信号保护细胞存活的重要的细胞浆蛋白之一。应用流式细胞免疫荧光技术对马桑内酯（ＣＬ）致痫大鼠海马细胞中ＢＣＬ—２蛋白进行检测。结果显示：ＣＬ诱导癫痫发作海马细胞中ＢＣＬ—２蛋白表达增高，发作６小时以内荧光指数（ＦＩ）（１．１２９±０．０４７，ｎ＝７）明显高于对照组（０．９９９±０．０９６，ｎ＝９），Ｐ＜０．００５，６～２４小时组（１．０２６±０．０６５，ｎ＝３）及２４小时以后组（０．９８２±０．０４３，ｎ＝６）ＦＩ回落，Ｐ＞０．０５。提示这种ＢＣＬ—２变化可能对损伤和凋亡细胞具有保护作用。     

Functional Properties of ES Cell–Derived Neurons Engrafted into the Hippocampus of Adult Normal and Chronically Epileptic Rats

Summary:  Purpose: Embryonic stem (ES) cell–based therapy strategies are thought to bear considerable promise in chronic neurologic disorders. Nonetheless, studies addressing the functional properties of ES cell–derived progeny after transplantation into the adult, pathologically modified CNS are scarce.
Methods: We therefore transplanted ES cell–derived neural precursors expressing enhanced green fluorescent protein only in neuronal progeny bilaterally into the hippocampi of pilocarpine-treated chronically epileptic and sham-control rats. Whole-cell patch-clamp recordings of identified ES cell–derived neurons (ESNs) in hippocampal slices were performed 13 to 34 days after transplantation.
Results: Most ESNs were found in clusters at the transplant site and did not migrate into host tissue. However, they gave rise to a dense network of processes extending over large distances into the host tissue. All ESNs possessed the ability to generate action potentials and expressed voltage-gated Na⁺ and K⁺ currents, as well as hyperpolarization-activated currents. Likewise, most ESNs received non– N -methyl- d -aspartate (NMDA) and γ-aminobutyric acid (GABA)_A receptor-mediated synaptic input. Both types of synapses displayed intact short-term plasticity. An unusual feature of the majority of ESNs was the occurrence of spontaneous pacemaking activity at frequencies ∼3 Hertz. No obvious differences were found between the functional properties of ESNs in sham-control and in pilocarpine-treated rats.
Conclusions: After transplantation into adult control and epileptic rats, ESNs displayed intrinsic and synaptic properties characteristic of neurons. Even though ESNs remained close to the transplant site, the formation of extensive networks of graft-derived processes may be useful for ES cell–based substance delivery.     

Neuroligins Differentially Mediate Subtype-Specific Synapse Formation in Pyramidal Neurons and Interneurons

Neuroligins(NLs) are postsynaptic cell-adhesion proteins that play important roles in synapse formation and the excitatory-inhibitory balance. They have been associated with autism in both human genetic and animal model studies, and affect synaptic connections and synaptic plasticity in several brain regions. Yet current research mainly focuses on pyramidal neurons, while the function of NLs in interneurons remains to be understood. To explore the functional difference among NLs in the subtypespecific synapse formation of both pyramidal neurons and interneurons, we performed viral-mediated shRNA knockdown of NLs in cultured rat cortical neurons and examined the synapses in the two major types of neurons. Our results showed that in both types of neurons, NL1 and NL3 were involved in excitatory synapse formation, and NL2 in GABAergic synapse formation. Interestingly, NL1 affectedGABAergic synapse formation more specifically than NL3,and NL2 affected excitatory synapse density preferentially in pyramidal neurons. In summary, our results demonstrated that different NLs play distinct roles in regulating the development and balance of excitatory and inhibitory synapses in pyramidal neurons and interneurons.     

Ultrastructural Changes in the Deep Cortical Pyramidal Cells of Infant Rats with Inherited Hydrocephalus and the Effect of Shunt Treatment

Pathological changes in the cortical gray matter in infantile hydrocephalus vary with the age at onset and may not be reversible with shunt treatment. We have used electron microscopy to investigate the sequence of pathological change and the effect of shunt treatment on layer VI pyramidal cells from infant H-Tx rats with inherited early-onset hydrocephalus. Tissue was prepared from the frontal and visual cortex of control and hydrocephalic rats at 4, 11, and 21 days after birth, together with 21-day rats previously treated with ventriculosubcutaneous shunts at 4–5 or 10–11 days after birth. Both cortical regions gave similar results but the effects were more severe in the visual cortex. In the early stages of hydrocephalus, the pyramidal cells were in clusters with fewer mature dendrites and less cytoplasmic organization than those in control rats, and some neuronal processes were vacuolated. In intermediate hydrocephalus the changes were more severe, with vacuolated cytoplasm, fewer cytoplasmic organelles, frequent swollen processes, and infrequent synapses. In advanced hydrocephalus at 21 days, many neurons showed degenerative changes, with edematous Golgi and dilated endoplasmic reticulum, distorted mitochondria, and single ribosomes. The neuropil contained many spongy areas with distended profiles. Shunt treatment prevented most of the changes if carried out at 4 days. Shunt treatment at 11 days also gave a dramatic recovery at the cellular level, but there were more immature pyramidal cells and edematous processes in the neuropil than in the 4-day-treated rats. The changes in hydrocephalus are consistent with progressive neuronal damage, which is largely prevented by early shunt treatment.     

Cell Type-Specific Decrease of the Intrinsic Excitability of Motor Cortical Pyramidal Neurons in Parkinsonism

The hypokinetic motor symptoms of Parkinson''s disease (PD) are closely linked with a decreased motor cortical output as a consequence of elevated basal ganglia inhibition. However, whether and how the loss of dopamine (DA) alters the cellular properties of motor cortical neurons in PD remains undefined. We induced parkinsonism in adult C57BL/6 mice of both sexes by injecting neurotoxin, 6-hydroxydopamine (6-OHDA), into the medial forebrain bundle. By using ex vivo patch-clamp recording and retrograde tracing approach, we found that the intrinsic excitability of pyramidal tract neurons (PTNs) in the primary motor cortical (M1) layer (L)5b was greatly decreased in parkinsonism; but the intratelencephalic neurons (ITNs) were not affected. The cell type-specific intrinsic adaptations were associated with a depolarized threshold and broadened width of action potentials (APs) in PTNs. Moreover, the loss of midbrain dopaminergic neurons impaired the capability of M1 PTNs to sustain high-frequency firing, which could underlie their abnormal pattern of activity in the parkinsonian state. We also showed that the decreased excitability in parkinsonism was caused by an impaired function of both persistent sodium channels and the large conductance, Ca²⁺-activated K⁺ channels. Acute activation of dopaminergic receptors failed to rescue the impaired intrinsic excitability of M1 PTNs in parkinsonian mice. Altogether, our data demonstrated a cell type-specific decrease of the excitability of M1 pyramidal neurons in parkinsonism. Thus, intrinsic adaptations in the motor cortex provide novel insight in our understanding of the pathophysiology of motor deficits in PD.SIGNIFICANCE STATEMENT The degeneration of midbrain dopaminergic neurons in Parkinson''s disease (PD) remodels the connectivity and function of cortico–basal ganglia–thalamocortical network. However, whether and how dopaminergic degeneration and the associated basal ganglia dysfunction alter motor cortical circuitry remain undefined. We found that pyramidal neurons in the layer (L)5b of the primary motor cortex (M1) exhibit distinct adaptations in response to the loss of midbrain dopaminergic neurons, depending on their long-range projections. Besides the decreased thalamocortical synaptic excitation as proposed by the classical model of Parkinson''s pathophysiology, these results, for the first time, show novel cellular and molecular mechanisms underlying the abnormal motor cortical output in parkinsonism.     

The Number of IgG-Positive Neurons in the Rat Hippocampus Increases after Dosed Traumatic Brain Injury

To evaluate the consequences of traumatic brain injury (TBI), we used a model of lateral fluid percussion brain injury in freely moving male Wistar rats. The immediate response to TBI included development of motor excitation and tonic–clonic seizures. Morphological analysis was performed 7 day after TBI. To localize IgG in the brain, rat brain slices were double stained with antibodies against IgG and NeuN (neuronal marker). To evaluate the state of microglia, we performed staining with Isolectin B4 (a microglial marker). The number of neurons was measured in sections stained using the Nissl method. The results show the IgG accumulation in neurons adjacent to cortical focus of trauma. In the hippocampus, IgG was accumulated in the neurons of the ipsilateral hippocampal CA1 and CA2 fields and the dentate gyrus, while in the contralateral hemisphere IgG was accumulated in the neurons of the CA1 field. These changes were accompanied by activation of microglia in the hippocampus, as well as by a decrease in neuronal density in the dentate gyrus of the ipsilateral hippocampus. The results show that TBI leads to bilateral damage to the hippocampus.     

Host Brain Regulation of Fetal Locus Coeruleus Neurons Grafted to the Hippocampus in 6-Hydroxydopamine-Treated Rats. An Intracerebral Microdialysis Study

Release properties of intrahippocampal transplants of noradrenergic neurons were monitored by microdialysis in awake and halothane-anaesthetized rats. Fetal locus coeruleus neurons were implanted as a cell suspension into hippocampi deprived of their innate noradrenalin (NA) innervation by intraventricular 6-hydroxydopamine treatment. Dialysis probes of the loop type were implanted into the dorsal hippocampus 1 - 2 days before each experiment, i.e. 7 - 11 months after grafting. Age-matched intact and lesion-only animals served as controls. Microscopic analysis showed a graft-derived tyrosine hydroxylase immunoreactive, presumably noradrenergic, fibre network throughout the dorsal hippocampal formation, surrounding the probe site. The innervation density varied from sub- to supranormal. The grafts restored baseline NA release in the graft-reinnervated hippocampus to near-normal levels both in awake and halothane-anaesthetized animals. Potassium chloride (100 mM) in the perfusion fluid induced a dramatic increase in NA release that was similar in magnitude in the grafted and intact hippocampi. A NA uptake blocker (desipramine) added to the perfusion fluid at 5 microM induced a similar increase in NA output in the grafted and intact hippocampi, and the output was substantially reduced by tetrodotoxin, added at 1 microM in the presence of uptake blockade. Electrical stimulation of the lateral habenular nucleus (15 Hz, 0.5 mA) in halothane-anaesthetized rats induced a significant increase in NA output both in the intact and grafted hippocampi. This effect was abolished by transection of the fasciculus retroflexus, which carries the efferent projections of the habenular complex. Behavioural activation through handling induced a consistent increase in NA release only in the intact animals, but in a few grafted rats (which also responded to habenular stimulation) the NA output was clearly elevated by handling. Forced immobilization induced a significant increase in NA output both in the intact and grafted hippocampi, but in the grafted ones the response was somewhat smaller and more transient. In the same set of animals, swimming in warm water (25 - 30 degrees C) induced a sharp increase in NA output in the intact animals, whereas only one of the grafted rats responded by increased NA output. The results indicate that the locus coeruleus grafts, despite their ectopic location, can become functionally integrated with the host brain, and that the activity of the transplanted noradrenergic neurons can, under some circumstances, be modulated from the host brain in response to environmental challenges.     

Electrophysiologic Analysis of the Actions of Valproate on Pyramidal Neurons in the Rat Hippocampal Slice

Summary: Purpose: Studies in invertebrates and cultured mammalian neurons suggested that valproate (VPA) mediates its main antiepileptic effect by slowing the recovery from inactivation of voltage-dependent sodium channels. This predicts an effect on the refractory period of the action potential and, consequently, on the bursting behavior of neurons.
Methods: We investigated this prediction using intracellular and extracellular recording techniques in hippocampal slices prepared from adult rats. The refractory period (RFP) and the ratio of the slopes (SR) of a pair of action potentials were used as indices of the recovery from inactivation of sodium channels. They were measured by injecting a series of paired depolarizing current pulses into CAI pyramidal neurons.
Results: No significant changes were observed in the RFP or SR measured during a I-h recording period when VPA was bath-applied (1 mM), or when it was present in the recording electrode (10–50 mM). Lowering the temperature from 34.5°C to 26.4°C resulted in an increase of the RFP by 100% and a decrease of the SR by 40%. However, VPA did not affect any of the measured action potential parameters at this lower temperature. VPA was also without effect on the presynaptic fiber volley of axons recorded extracellularly in the stratum radiatum. The antidromic population spike was unaffected by VPA (2 mM), whereas phenytoin (50 μM) clearly affected this spike in the same slices. The absence of effect of VPA on each of the measured parameters could not be attributed to poor penetration through the slice because bath-applied VPA reduced the frequency of extracellularly recorded spontaneous interictal bursts, induced by bicuculline and elevated K⁺, within 10 min.
Conclusions: These findings suggest that at least in the hippocampal slice the drug's principal antiepileptic effect cannot be explained by its action on voltage-dependent sodium channels.     

Effects of Chronic Epileptic Foci on Control of Pyramidal Tract Neurons in Monkeys

Five Macaca mulatta monkeys were operantly conditioned to control the firing patterns of single precentral pyramidal tract neurons. The accuracy with which the monkeys could control normal PTNs from within the focus was significantly poorer than PTNs from contralateral, homotopic cortex. In comparison to nonepileptic monkeys, there was no significant difference in the accuracy with which PTNs from cortex contralateral to interictal foci were controlled. By contrast, comparison of the time necessary to gain accurate control over individual PTNs from contralateral cortex showed the epileptic monkeys to be significantly encumbered when compared to nonepileptic monkeys. These data suggest that interictal foci produce "noise" in remote regions of brain that are involved in an operant task requiring a high degree of discrimination.