Hyperexcitability associated with localizable lesions in epileptic patients

Intracellular recordings from neurons were carried out in cortical slices obtained from tissue removed from patients suffering from intractable seizures. The patients were divided into two groups based on the presence or absence of an anatomical abnormality that could be imaged preoperatively. The lesion or its surround was the presumptive epileptogenic area. The tissue removed from the patients without lesions was removed either for biopsy purposes or for access to epileptic tissue and was not considered epileptogenic. All neurons from patients without an imageable lesion, and some (19%) from patients with an imageable lesion, responded to orthodromic stimuli with a sequence of synaptic excitation followed by inhibition; these properties resembled those of normal rodent cortical slices. Different responses, classified as abnormal, were observed in 81% of the neurons in tissue specimens obtained near lesions. The most common was prolonged synaptic excitation with no noticeable inhibition, even at high stimulus strengths. In three resections, long latency all-or-none depolarization shifts were observed that resemble the classic paradoxical depolarization shift seen in in vivo extracellular recordings. Loss of specific inhibitory systems within the cortex may contribute in part to these abnormal responses.     

Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamtergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar of hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 μM, the maximum increase was at about 1 μM, but levels higher than 10 μM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC₅₀, about 0.5 μM), but not by the metabotropic QA receptor agonist trans-(±)-1-aino-cyclopentyl-1,3,dicarboxyalte (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, -2-amino-5-phosphonovalerate, γ- -glutamyl aminomethyl sulphonic acid and γ- -glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca²⁺-dependent. The increase in Ca²⁺ influx essentially through the activation of L-type voltage-operated Ca²⁺ channels, and not the mobilization of internal Ca²⁺ stores, was responsible for these QA receptor-mediated long-term plastic changes in hippocampal and cerebellar neurons.     

Substance P augments a persistent slow inward calcium-sensitive current in voltage-clamped spinal dorsal horn neurons of the rat

Rat spinal dorsal horn neurons in slice preparations perfused with Ringer solution containing 0.5-1 microM TTX and/or 10-20 mM tetraethylammonium at 29 degrees C, were studied by using a single microelectrode voltage-clamp technique. Slow persistent inward currents were recorded during depolarizing voltage commands to membrane potentials positive to about -40 mV. The inward current was depressed by removing external Ca, or by adding 0.1-0.2 mM Cd, 5 mM Co or 0.1 mM verapamil, and was increased by adding Ba or Bay-K 8644. Substance P (SP) augmented a persistent slow inward Ca-sensitive current in a dose-dependent manner. It is suggested that this effect may be instrumental in generating the SP-evoked slow depolarization, increase in membrane excitability, and the 'bursting' behavior in the immature rat dorsal horn neurons. In addition, in some neurons SP reduced the M-like current, which effect may contribute to, but not explain, generation of the SP-induced slow depolarization.     

Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre-and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.     

Estimating neuron dendritic length in 3D from total vertical projections and from vertical slices

The recently developed method of total vertical projections is illustrated to estimate the total dendritic length of a human Substantia Nigra neuron. Next, the length of the different orders of dendritic branches, and the mean segment length for each order - commonly regarded as important parameters in neuron physiology - are also estimated. Finally, it is shown how to estimate the mean dendritic length in a population of neurons from vertical slices of arbitrary and unknown thickness. Being unbiased and highly efficient, the proposed methods offer interesting alternatives to current procedures used for the metric analysis of neuron arborizations.     

Effects of Glucocorticoid on Electrical Activities of Hippocampal Neurons in vitro

Intracellular recording of the rat hippocampal slices was established and usedto analyse the effects of glucocorticoid on resting membrane potential as well as inputresistance of the cells in CA1 and CA2 of hippocampal slices.The results indicate thatwithin 1-2 min the steroid hyperpolarized some of the hippocampal neurons with atendency of increasing the cells' input resistance,suggesting that glucocorticoid caninfluence the function of central neurons in a non-genomic pattern,and the underlyingmechanism involves the change of resting membrane potential.     

Intrahippocampal Injection of Endothelin-1: A New Model of Ischemia-induced Seizures in Immature Rats

Summary:  The goal of this study was to develop a new model of ischemia-induced seizures in immature rats using injection of vasoconstrictor Endothelin-1 (ET-1) into the brain. ET-1 (10, 20, or 40 pmol) was infused into the left dorsal hippocampus of freely moving Wistar rats 12 (P12) and 25 (P25) days old. Animals were then video/EEG-monitored for 100 min and monitoring was repeated 22 h later. Parameters of electrographic seizures (frequency and mean duration) as well as pattern of their behavioral correlates were evaluated. The pattern of behavioral seizures was used to develop model-specific scoring system. Cresyl violet and Fluoro Jade-B-staining were used to evaluate brain damage. Extension of the lesion was correlated with seizure severity. After ET-1-injection, seizures occurred in 83–100% animals of all age-and-dose groups and persisted for 24 h except P12 rats with 10 pmol. There were no differences in average seizure duration (18–40 s) or seizure frequency (3–7 seizures/100 min) among individual dose-groups. Between the 1st and 2nd observation period, total seizure duration decreased in 71% of P12 and 47% of P25 rats. Electrographic seizure activity was most frequently accompanied by clonus, incidence of more severe convulsions (barrel rolling or generalized clonic seizures) increased with dose of ET-1. Morphologic examination did not reveal any dose-related difference in damage severity, hippocampal damage was however more extensive in P12 compared to P25 animals. Seizure severity correlated positively with severity of the damage in both age groups. Our study presents focal injection of ET-1 into the brain as a new and practical model of ischemia-induced seizures in immature rats.     

Adrenocorticotropic Hormone Immunoreactivity in the Hippocampal Formation of Temporal Lobe Epilepsy Patients

Summary: Purpose: We wished to identify immunocytochemically the distribution of proopiomelanocortin-related peptides in the hippocampal formation of patients with epilepsy.
Methods: Surgical hippocampal specimens from temporal lobe epilepsy (TLE) patients and autopsy control tissue were examined immunocytochemically for ACTH, α-melanocyte-stimulating hormone (α-MSH) and α-endorphin.
Results: There was a dense distribution of ACTH-immunoreactive neurons in the hippocampal formation of patients with mesial TLE syndrome (MTLE). These hippocampal specimens showed significant cell loss. ACTH-positive neurons were most prominent in the subiculum, with scattered ACTH-immunoreactive neuronal elements distributed in the cornu ammonis fields and hilus. Light ACTH immunoreactivity was detected in the tumor-related epileptic hippocampal specimens, which showed minimal cell loss. Although autopsy control tissue from the hypothalamus showed intense ACTH staining patterns in cells and fibers, there was little or no ACTH immunoreactivity in the autopsy hippocampal tissue. The expression of ACTH immunoreactive elements was correlated with patterns of cell loss. No α-MSH- or β-endorphin-immunoreactive neurons were detected in any of the hippocampal specimens.
Conclusions: ACTH has anticonvulsant properties, and its novel expression in the glutamatergic subicular neurons, which provide the main outflow of the hippocampal formation, may represent an attempt by the damaged hippocampal circuit to restore the balance of excitatory/inhibitory neurotransmission in TLE.     

辽宁产东亚钳蝎毒的镇痛作用——离体海马脑片的细胞内电位记录

应用离体脑片技术及细胞内生物电记录方法，研究辽宁产东亚钳蝎毒及某些活性物质对海马ＣＡ１区痛敏神经元的影响，以了解蝎毒的作用机制，在海马ＣＡ１区记录到细胞内放电５４个单位，其静息电位平均值为４５±５ｍＶ，当给予海马伞入口处一定强度刺激时，在海马ＣＡ１区记录到细胞内放电，向海马脑片灌注蝎毒液时，ＣＡ１区细胞内放电受抑制，说明蝎毒液有镇痛作用     

Distributive profiles of free and protein-incorporated tryptophan and valine in cerebral cortex slices from adult and 2-day-old rats

The distribution of the specific radioactivity and the incorporation into protein of [³H]-tryptophan and [³H]valine at varying layers from surface to centre were measured in incubated slices of cerebral cortices from infant and adult rats. Specific radioactivity in free amino acids was in both age groups highest in the intact surface layer. Incorporation of tryptophan into protein was even in slices from adult rats but much less than the average in the surface layers in slices from infant rats. Incorporation of valine exhibited similar heterogeneity in both age groups. The results suggest in brain slice preparations a zonal compartmentation of amino acid and protein metabolism which varies for different amino acids.