Dynamics of epileptiform activity in mouse hippocampal slices

Increase of the extracellular K⁺ concentration mediates seizure-like synchronized activities in vitro and was proposed to be one of the main factors underlying epileptogenesis in some types of seizures in vivo. While underlying biophysical mechanisms clearly involve cell depolarization and overall increase in excitability, it remains unknown what qualitative changes of the spatio-temporal network dynamics occur after extracellular K⁺ increase. In this study, we used multi-electrode recordings from mouse hippocampal slices to explore changes of the network activity during progressive increase of the extracellular K⁺ concentration. Our analysis revealed complex spatio-temporal evolution of epileptiform activity and demonstrated a sequence of state transitions from relatively simple network bursts into complex bursting, with multiple synchronized events within each burst. We describe these transitions as qualitative changes of the state attractors, constructed from experimental data, mediated by elevation of extracellular K⁺ concentration.     

Computational models of epileptiform activity in single neurons

A series of original computational models written in NEURON of increasing physiological and morphological complexity were developed to determine the dominant causes of epileptiform behavior. Current injections to a model hippocampal pyramidal neuron consisting of three compartments produced the sustained depolarizations (SD) and simple paroxysmal depolarizing shifts (PDS) characteristic of ictal and interictal behavior in a cell, respectively. Our results indicate that SDs are the result of the semi-saturation of Na+, Ca2+ and K+ active channels, particularly the CaN, with regular Na+/K+ spikes riding atop a saturated depolarization; PDS rides on a similar semi-saturated depolarization whose shape depends more heavily on interactions between low-threshold voltage-gated Ca2+ channels (CaT) and Ca(2+)-dependent K+ channels. Our results reflect and predict recent physiological data, and we report here a cellular basis of epilepsy whose mechanisms reside mainly in the membrane channels, and not in specific morphology or network interactions, advancing a possible resolution to the cellular/network debate over the etiology of epileptiform activity.     

羟基马桑毒素所致的大鼠海马锥体细胞痫样放电活动

本研究采用离体海马脑片电生理研究技术,细胞外记录海马锥体细胞群体锋电位(population spike,PS),观察羟基马桑毒素(tutin)对大鼠海马脑片CA1区锥体细胞电活动的影响,探讨tutin是否具有致痛作用及其致痫机制。结果如下:(1)用40、30和20μg/ml浓度的tutin灌流海马脑片,可显著增高由顺向刺激Schaffer侧支所诱发的PS的幅度,灌流tutin 30min时,PS第一个波的幅度分别为对照的(388.7±20.1)%、(317.2±19.1)%和(180.9±11.6)%(各组n=5,P<0.05)。(2)伴随PS波幅的增高,可出现成串痫样放电波,波数4～11个不等。(3)灌流tutin后的部分脑片(n=9/34),在未刺激Schaffer侧支时也出现自发的成串、高幅痫样放电。(4)灌流CNQX阻断非NMDA受体后,再灌流tutin,PS幅度和放电波数均无显著性变化,即CNQX可完全抑制tutin所致的痫样放电;灌流AP-5阻断NMDA受体后,tutin仍可使PS幅度增高但放电波数无显著性增加,即AP-5可部分抑制tutin所致的痫样放电。上述结果表明,tutin可使海马脑片锥体细胞兴奋活动增强,具有致痫作用;兴奋性谷氨酸受体尤其是非NMDA受体可能介导tutin的致痫作用。     

Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons

To investigate the activity of senescence-associated beta-galactosidase (SA-beta-GAL) in the hippocampus of aging rats. Hippocampi of 6-, 18-, and 24-month-old rats were observed by histochemical staining for SA-beta-GAL and cytochemical staining for SA-beta-GAL in cultured hippocampal neurons. The activity of SA-beta-GAL doubled in hippocampal pyramidal cells of the CA3 region in rats between 6 and 18 months (14.57 ± 2.74% vs. 31.66 ± 14.12% SA-beta-GAL-positive, respectively), and reached 50.76 ± 14.41% positive at 24 months. The activity of SA-beta-GAL also increased as a function of time upon prolonged culture of cultured hippocampal neurons with 95% of cells being SA-beta-GAL-positive at 20 days in vitro. Interestingly, no SA-beta-GAL-positive cells were found in neurons of the hippocampal dentate gyrus, a neurogenic region of the brain, at any age examined. SA-beta-GAL can be used as a senescence biomarker in determining senescent neurons in hippocampal pyramidal cells of the CA3 region in advanced aging.     

Inhibition of spontaneous synchronous activity of hippocampal neurons by excitation of GABAergic neurons

The molecular mechanisms of the neuronal spontaneous synchronous activity (SSA) regulation by population of GABAergic neurons have been investigated in rat hippocampal culture. The neurons from this population contain Ca²⁺-permeable KA receptors on the presynaptic membrane. Using image analysis, confocal microscopy and immunocytochemistry, we identified by the shape of Ca²⁺ signal the population of GABAergic neurons with unique charachteristics allowing these neurons to control SSA. The SSA in a neuronal network was suppressed by the KA-receptor mediated [Ca²⁺]i increase in neurons of this population. Agonists of GluR5/GluK1-containing KA receptors (domoic acid (DA), SYM2081, and ATPA) evoked a fast high-amplitude Ca²⁺ signal without desensitization only in this population of neurons. This fact points to Ca²⁺ permeability of KA receptors in these neurons. The GABA(A) receptor antagonist bicuculline increased the activity of AMPA but not KA receptors of these neurons, indicating presynaptical localization of KA receptors. Depolarization of cells induced by KCl (unlike bicuculline-induced depolarization) increased the activity of AMPA and KA receptors twofold, which points to the dependence of the activity on depolarization. A tenfold increase of the SSA frequency in neurons of this population caused an increase in the basal [Ca²⁺]i level, which was accompanied by inhibition of SSA in another numerous population of neurons, suggesting that an increased GABAergic inhibition takes place. Prolonged high-frequency oscillations causes a global [Ca²⁺]i increase in the neurons of this population and their subsequent death. Thus, KA receptors in the population of fast GABAergic neurons may implement a negative feedback under hyperexcitation by glutamate enhancing GABA release due to the fast and prolonged [Ca²⁺]i increase. It has been shown that this mechanism can be used to suppress hyperactivation of a certain population of neurons under high-frequency SSA and ischemia. It is obvious that selective death of inhibitory neurons from this population may lead to hyperexcitability of certain brain regions.     

Negative occurrence between hippocampal rhythmic slow activity and epileptiform spikes: influence of the striatum

The effects of caudate and septal stimulation on hippocampal activity were studied. Sodium penicillin was intravenously injected in order to induce a steady rate of interictal epileptic activity. Penicillin injection caused significant reduction of the rate of occurrence and duration of hippocampal rhythmic slow activity (RSA). Spontaneous RSA as well as RSA-eliciting caudate and septal stimulation induced a marked inhibition on frequency of epileptiform complexes. Lesions of the medial septal nucleus made it impossible to record RSA or to observe any sort of influence on hippocampal epileptiform activity by caudate stimulation. This result suggests that the septum is important for RSA genesis in the striato-hippocampal pathway or in the reciprocal septo-hippocampal connections.     

Nicotine modifies the activity of ventral tegmental area dopaminergic neurons and hippocampal GABAergic neurons

While trying to mimic the dose and time course of nicotine as it is obtained by a smoker, we found the following results. The initial arrival of even a low concentration of nicotine increased the firing rate of dopaminergic neurons from the ventral tegmental area (VTA) and increased the spontaneous vesicular release of GABA from hippocampal neurons. Longer exposure to nicotine caused variable, but dramatic, desensitization of nicotine receptors and diminished the effects of nicotine. The addictive properties of nicotine as well as its diverse effects on cognitive function could be mediated through differences in activation and desensitization of nicotinic receptors in various areas of the brain.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

The activity of hippocampal neurons during several types of behavior]

The activity of 87 hippocampal units and the EEG (field CAI) were studied in unrestrained rabbits during calm and active alertness and at different stages of sleep. Correlation has been established between the characteristics (mean frequency and the pattern) of unit firing, EEG and the animal's activity. For most of the neurones, fixed values of mean frequency and the discharge pattern corresponded to a definite functional state. With transition from sleep to alertness, 63.6% of the units became active, 29.1% were inhibited, and the rest of the units changed the firing pattern only. The cells which became activated during awakening, showed a reduced firing frequency during a more profound sleep and higher discharge frequency during the paradoxal phase of sleep, while the inhibitory cells revealed reverse dynamics of discharge frequency. In a state of alertness, the most pronounced shifts in firing activity were observed in 33.3% of the nerve cells at orienting investigating behaviour, in 27.1%, during attention reaction, in 22.9% at some kinds of movement, and in 16.7%, in the course of feeding and drinking. A conclusion has been drawn that the role of the hippocampus in achieving different behavioral reactions is probably to a great extent determined by its participation in setting up a level of the brain central tone, specific for each state.     

Dose-dependent effects of phencyclidine on hippocampal CA1 neurons

The dose-dependent effects of phencyclidine were examined in guinea pig hippocampal slices using intracellular and extracellular recordings. Orthodromically evoked population potentials from the CA1 cell body layer were enhanced by low doses (0.2-0.4 microM) and depressed by high doses (0.01-10 mM). Medium doses of the drug (2.0-10.0 microM) showed little effect. Intracellular recordings from CA1 pyramidal neurons gave similar dose-dependent results. Low doses increased spontaneous firing rates and caused silent cells to fire. Medium doses both increased and decreased firing rates, whereas high doses depressed firing rates. Large transient depolarizing shifts were seen in some phencyclidine-treated cells at medium and high doses. Phencyclidine effects took 15-30 min to develop and were only partially reversible after a washout of up to 1 h.     

Action of beta-carboline derivatives on the evoked activity of hippocampal neurons

The effects of tetrahydro-beta-carboline-3-carbonic acid methyl ester (I), 1-phenyl-tetrahydro-beta-carboline-3-carbonic acid methyl ester (II), tetrahydro-beta-carboline-3-carbonic acid methylamide, and beta-carboline-3-carbonic acid methylamide on evoked potentials (EP) of neurons were investigated in experiments with hippocampal slices. Each compound was tested in 5 experiments. These derivatives applied in a concentration 4 M had the following features in common: 1) a 10-20% augmentation of the amplitude of the population spike (PS) evoked in the CAI area by Schaffer collaterals stimulation; 2) appearance of additional PSs; 3) potentiation of the substance effect after beginning of washing; 4) poor washing (incomplete recovery of EP after 40-60 min of washing). In one of five experiments, I administration led to a decrease in the PS amplitude (by 15%). A similar effect was observed in one of five experiments during perfusion of II. The authors assume that there are hippocampal mechanisms responsible for realization of opposite effects associated with occupation of benzodiazepine receptors by ligands.     

一氧化氮的释放对海马脑片CA1区痫样放电的影响

用自制的一氧化氮（ＮＯ）敏感电极－Ｎａｆｉｏｎ－壳聚糖合镍修饰铂电极（Ｎａｆｉｏｎ－ＣＴＳ（Ｎｉ）－Ｐｔ）连续测定了青霉素致痫海马脑片ＣＡ１区锥体层神经元ＮＯ的释放,并同时观察了ＮＯ合酶抑制剂７－ｎｉｔｒｏ－ｉｎｄａｚｏｌｅ（７－ＮＩ）及Ｎ＾ω－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ（Ｌ－ＮＮＡ）对诱发痫波及ＮＯ释放量的影响。研究观察到：（１）在青霉素致痫脑片模型上,诱发的痫波随青霉素浓度的增加而增多,     

Culturing hippocampal neurons

We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2-3 h.     

Time relations between the evoked potential and activity of hippocampal neurons

The time relations between the evoked potential (EP) and neuronal activity of the dorsal hippocampus in response to sciatic nerve stimulation were investigated in experiments conducted on rabbits paralyzed by tubocurarine. Two groups of neurons were distinguished on the basis of the type of their reaction to sciatic stimulation. Inhibition of background spike activity was found in the neurons of the first group (70.9%); in 37% of them inhibition was preceded by excitation in the form of a spike discharge or excitatory postsynaptic potential (EPSP) which coincided in time with the positive phase of the EP. During inhibition of spike activity the hyperpolarization potential was recorded intracellularly in a number of neurons, the latent period of which coincided with the latent period of the negative phase of the EP. Neurons of the second group (20%) were characterized by protracted excitation of spike activity, and the start of their excitation coincided with the start of the negative phase of the EP and hyperpolarization potential of the neurons of the first group. Different sensitivity of the two groups of neurons was noted. It is concluded that the EPSP of the pyramidal neurons of the hippocampus participates in generation of the positive phase of the EP, and the hyperpolarization potentials of these neurons participate in the generation of its negative phase. The possibility is not precluded that hippocampal neurons closer to the surface participate in the development of the negative phase of the EP.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 285–292, November–December, 1969.     

Phencyclidine actions measured intracellularly in hippocampal CA1 neurons

The electrophysiological effects of phencyclidine (PCP) were measured intracellularly in guinea pig hippocampal CA1 neurons in vitro. At all doses tested (0.2 microM - 10 mM), PCP increased the width of action potentials (APs). Doses of 10 microM and higher were associated with decreased action potential amplitude. PCP decreased inhibitory postsynaptic potentials and excitatory postsynaptic potentials but did not alter responses to focally applied GABA. At the lowest dose (0.2 microM), PCP decreased the input resistance (Rin), while at all other doses Rin was increased. PCP decreased post-spike train afterhyperpolarizations at low and medium doses. PCP effects persisted in low calcium medium and also in medium containing 10(-6) M tetrodotoxin. It is concluded that in these central neurons, PCP primarily blocks potassium conductances at all doses and, at anesthetic doses, depresses sodium-dependent spikes.     

Leptin protects hippocampal CA1 neurons against ischemic injury

Leptin is an adipose hormone with well characterized roles in regulating food intake and energy balance. A novel neuroprotective role for leptin has recently been discovered; however, the underlying mechanisms are not clearly defined. The purpose of this study was to determine whether leptin protects against delayed neuronal cell death in hippocampal CA1 following transient global cerebral ischemia in rats and to study the signaling mechanism responsible for the neuroprotective effects of leptin. Leptin receptor antagonist, protein kinase inhibitors and western blots were used to assess the molecular signaling events that were altered by leptin after ischemia. The results revealed that intracerebral ventricle infusion of leptin markedly increased the numbers of survival CA1 neurons in a dose-dependent manner. Infusion of a specific leptin antagonist 10 min prior to transient global ischemia abolished the pro-survival effects of leptin, indicating the essential role of leptin receptors in mediating this neuroprotection. Both the Akt and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathways appear to play a critical role in leptin neuroprotection, as leptin infusion increased the phosphorylation of Akt and ERK1/2 in CA1. Furthermore, pharmacological inhibition of either pathway compromised the neuroprotective effects of leptin. Taken together, the results suggest that leptin protects against delayed ischemic neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt and ERK1/2 MAPK signaling pathways.