Heidrun Straub

Spreading depression in human neocortical slices

Cortical spreading depression (CSD) occurrence has been suggested to be associated with seizures, migraine aura, head injury and brain ischemia-infarction. Only few studies identified CSD in human neocortical slices and no comprehensive study so far evaluated this phenomenon in human. Using the neocortical tissue excised for treatment of intractable epilepsy, we aimed to investigate CSD in human. CSD was induced by KCl injection and by modulating T-type Ca(2+) currents in incubated human neocortical tissues in an interphase mode. The DC-fluctuations were recorded by inserting microelectrodes into different cortical layers. Local injection of KCl triggered single CSD that propagated at 3.1+/-0.1 mm/min. Repetitive CSD also occurred spontaneously during long lasting application (5 h) of the T-type Ca(2+) channel blockers amiloride (50 microM) or NiCl(2) (10 microM) which was concomitant with a reversible extracellular potassium increase up to 50 mM. CSD could be blocked by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid in all cases. The results demonstrate that modulation of the Ca(2+) dynamics conditioned human neocortical slices and increased their susceptibility to generate CSD. Furthermore, these data indicate that glutamatergic pathway plays a role in CSD phenomenon in human.

Paroxysmal depolarization shifts induced by bicuculline in CA3 neurons of hippocampal slices : suppression by the organic calcium antagonist verapamil

Organic calcium antagonists have been reported to abolish epileptic neuronal discharges elicited by pentylenetetrazol and penicillin. It was tested whether the organic calcium antagonist verapamil is able to suppress also paroxysmal depolarization shifts (PDS) induced by bicuculline. This is of special interest, since bicuculline is assumed to produce PDS by blocking GABAergic synaptic inhibition. The experiments were performed on CA3 neurons of hippocampal slices (guinea pig). Verapamil (40, 60, and 80 microM) reduced amplitude, duration and frequency of appearance of PDS until the generation of PDS failed. The results indicate that calcium currents are also involved in bicuculline PDS, and that bicuculline exerts its epileptogenic action, at least in part, on extrasynaptic sites.

A portable chamber for long-distance transport of surviving human brain slice preparations

Incubation chambers for surviving brain slice preparations are in most cases designed to be stationary. For investigations on human brain tissue resected for the treatment of brain tumor or epilepsy, portable incubation chambers are needed in addition to stationary ones to allow transport of the slices between laboratories and hospitals located far from each other. For such purposes, interface chambers have been in use. In view of the ongoing discussion of the merits of interface versus submerged baths, here we describe an alternative chamber as a lightweight, easy to assemble portable bath of the submersion type for transport of surviving brain slice preparations over considerable distances. The chamber has been used in a variety of investigations on human brain slices. These slice preparation showed bioelectric properties comparable to those reported in investigations by other laboratories using stationary incubation chambers in cases where portable ones were not needed.

Effect of levetiracetam on epileptiform discharges in human neocortical slices.

Summary:  Purpose: The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy.

Identification and quantitative measurements of biogenic monoamines in the central nervous tissue of some gastropods

Investigations were carried out to determine levels of biogenic monoamines in the central nervous tissue of seven species of terrestrial gastropods (taxonomic family: Helicidae) using high-performance liquid chromatography (HPLC) with electrochemical detection (ELCD). The central nervous tissue of all species examined contained assayable amounts of dopamine (DA) and serotonin (5-HT). Noradrenaline (NA) was occasionally present; adrenaline (A) could not be detected. The central ganglia of two Mediterranean species generally contain 3-4 times more 5-HT relative to DA than the ganglia of the other species which contain amounts of 5-HT only slightly higher than DA.

Lowering of the potassium concentration induces epileptiform activity in guinea-pig hippocampal slices

Extra- and intracellular recording techniques were used to study the epileptiform activity generated by guinea-pig hippocampal slices perfused with low potassium containing artificial cerebrospinal fluid. Extracellular field potentials were recorded in CA1 and CA3 regions along with intracellular recordings in CA3 subfield. Reduction of the extracellular potassium concentration [K(+)](o) from 4 to 2 mM caused a transient neuronal hyperpolarisation which was followed by a repolarisation and subsequent depolarisation period. Paroxysmal depolarisation shifts occurred during the transient hyperpolarisation period while epileptic field potentials (EFP) appeared in the late repolarisation or early depolarisation phase. EFP elicited by reduction of [K(+)](o) were neither affected by blockade of N-methyl-D-aspartate (NMDA) glutamate-subreceptor or gamma aminobutyric acid receptor, nor by application of the organic calcium channel blocker nifedipine or the anticonvulsant drugs carbamazepine and valproic acid. Upon application of non-NMDA glutamate-subreceptor blocker the EFP were abolished in all trials, while application of the organic calcium channel blocker verapamil only suppressed the EFP in some cases. The results point to a novel mechanism of epileptogenesis and may provide an in vitro model for the development of new drugs against difficult-to-treat epilepsy.

Lowering the extracellular potassium concentration elicits epileptic activity in neocortical tissue of epileptic patients.

The increase in the extracellular potassium concentration ([K+]o) is a well‐established model of epilepsy (the so‐called high potassium model). Therefore, it is generally accepted that for the prevention of abnormal excitability and seizure generation, increases of [K+]o must be avoided. In this paper, however, we show that on the contrary, a reduction of [K+]o also elicits epileptic activity in brain slices of man.

Low magnesium induced epileptiform discharges in neocortical slices (guinea pig): Increased antiepileptic efficacy of organic calcium antagonist verapamil with elevation of extracellular K+ concentration

1. The antiepileptic effect of the organic calcium antagonist verapamil on low Mg2+ induced epileptiform discharges in the neocortex was tested. 2. The experiments were carried out on slices of the frontal neocortex (guinea pigs). Verapamil was tested at normal (4 mmol/l) and elevated (8 mmol/l) KCl levels. 3. Verapamil (40, 60 mumol/l) suppressed epileptiform activity in any case. 4. With elevated K+ concentration the suppressive effect of verapamil was significantly accelerated. 5. Epileptic activity reappeared when verapamil was omitted from the Mg(2+)-free superfusate.

Augmentation ofN-methyl-d-aspartate induced depolarizations by GABA in neocortical and archicortical neurons

The influence of the inhibitory transmitter gamma-aminobutyric acid (GABA) on depolarizations elicited by the excitatory amino acid N-methyl-D-aspartate (NMDA) was tested in neurons of organotypic neocortical tissue cultures (newborn rat) and in CA3 neurons of the hippocampal slice (guinea pig). Drugs were applied through a 3-barrelled micropipette by pressure ejection. Applications of GABA before the ejection of NMDA increased the amplitude of the depolarizations induced by the excitatory amino acid. It is suggested that the enhancement of NMDA responses by GABA may be mainly mediated by an intracellular common pathway.

Cutting of living hippocampal slices by a highly pressurised water jet (macromingotome).

Living brain slices are usually cut with razor blades, which compress a ca. 50-microm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100-1800 bar was ejected through nozzles of 35-100 microm to cut 300-500-microm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400-600 bar and with nozzle diameters of 60-80 microm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 microm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg(2+)-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.