Persistent changes of corticostriatal plasticity in dt(sz) mutant hamsters after age-dependent remission of dystonia.

Abnormal plasticity in the cortico-basal ganglia-thalamocortical loop has been suggested to represent a key factor in the pathophysiology of dystonia. In a model of primary paroxysmal dystonia, the dt(sz) mutant hamster, previous experiments have shown a strongly increased long-term potentiation (LTP) in comparison to non-dystonic control hamsters. These basal changes, i.e. in the absence of dystonia, were found in young animals at an age of 5 weeks, when the age-dependent dystonia in dt(sz) mutant reaches highest severity. In the present study we examined in corticostriatal slices (1) whether the increases in synaptic plasticity can be modulated by stressful stimuli which induce dystonic episodes in young mutant hamsters, and (2) whether increases of LTP persist after spontaneous remission of dystonia in animals older than 10 weeks. The present data show that in slices of young mutant hamsters the extent of LTP was not influenced by the presence of dystonia: In comparison to age-matched control hamsters, LTP was increased in mutant hamsters independent of preceding stressful stimulation. After remission of dystonia, i.e., in older dt(sz) mutant hamsters >10 weeks, only LTP could be elicited, while in preparations from age-matched control hamsters, either LTP or long-term depression developed, depending on previous behavioral challenge. We conclude that in mature brain, corticostriatal connections have the potential for changes in metaplasticity, while in dt(sz) mutant hamsters this metaplasticity is persistently infringed even though stress-inducible dystonic symptoms are lost.

The afterhyperpolarizing potential following a train of action potentials is suppressed in an acute epilepsy model in the rat Cornu Ammonis 1 area.

In hippocampal Cornu Ammonis 1 (CA1) neurons, a prolonged depolarization evokes a train of action potentials followed by a prominent afterhyperpolarizing potential (AHP), which critically dampens neuronal excitability. Because it is not known whether epileptiform activity alters the AHP and whether any alteration of the AHP is independent of inhibition, we acutely induced epileptiform activity by bath application of the GABA(A) receptor blocker gabazine (5 µM) in the rat hippocampal slice preparation and studied its impact on the AHP using intracellular recordings. Following 10 min of gabazine wash-in, slices started to develop spontaneous epileptiform discharges. This disinhibition was accompanied by a significant shift of the resting membrane potential of CA1 neurons to more depolarized values. Prolonged depolarizations (600 ms) elicited a train of action potentials, the number of which was not different between baseline and gabazine treatment. However, the AHP following the train of action potentials was significantly reduced after 20 min of gabazine treatment. When the induction of epileptiform activity was prevented by co-application of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 10 µM) and D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 µM) to block α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and N-methyl-d-aspartate (NMDA) receptors, respectively, the AHP was preserved despite of GABA(A) receptor inhibition suggesting that the epileptiform activity was required to suppress the AHP. Moreover, the AHP was also preserved when the slices were treated with the protein kinase blockers H-9 (100 µM) and H-89 (1 µM). These results demonstrate that the AHP following a train of action potentials is rapidly suppressed by acutely induced epileptiform activity due to a phosphorylation process-presumably involving protein kinase A.

Isolation and purification of medium chain length poly(3-hydroxyalkanoates) (mcl-PHA) for medical applications using nonchlorinated solvents.

A novel process was developed to isolate poly([R]-3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHO) and poly([R]-3-hydroxy-ω-undecenoate-co-3-hydroxy-ω-nonenoate-co-3-hydroxy-ω-heptenoate) (PHUE) from Pseudomonas putida species. Methyl tert-butyl ether (MTBE), ethyl acetate, acetone, and methylene chloride efficiently extracted PHO from freeze-dried biomass. The ratio of solvent to biomass was 15:1 (vol/wt). The nonchlorinated solvents required 18 h of extraction to achieve methylene chloride's yield of 15 wt % within 60 min. In the case of PHUE, the yield was 15-17 wt % after 60 min of extraction at room temperature, independently of the solvent used. MTBE performed best in life cycle assessment (LCA) if contamination of the environment is avoided. Filtration of the extract containing 8 wt % of raw polyhydroxyalkanoate (PHA) through activated charcoal revealed colorless polymers with less than one endotoxin unit/g. The ratio (v/v) of the solution to activated charcoal was 2:1. The loss (impurities and polymers) amounted up to 50 wt %.

GABAA receptor-dependent synchronization leads to ictogenesis in the human dysplastic cortex.

Patients with Taylor's type focal cortical dysplasia (FCD) present with seizures that are often medically intractable. Here, we attempted to identify the cellular and pharmacological mechanisms responsible for this epileptogenic state by using field potential and K+-selective recordings in neocortical slices obtained from epileptic patients with FCD and, for purposes of comparison, with mesial temporal lobe epilepsy (MTLE), an epileptic disorder that, at least in the neocortex, is not characterized by any obvious structural aberration of neuronal networks. Spontaneous epileptiform activity was induced in vitro by applying 4-aminopyridine (4AP)-containing medium. Under these conditions, we could identify in FCD slices a close temporal relationship between ictal activity onset and the occurrence of slow interictal-like events that were mainly contributed by GABAA receptor activation. We also found that in FCD slices, pharmacological procedures capable of decreasing or increasing GABAA receptor function abolished or potentiated ictal discharges, respectively. In addition, the initiation of ictal events in FCD tissue coincided with the occurrence of GABAA receptor-dependent interictal events leading to [K+]o elevations that were larger than those seen during the interictal period. Finally, by testing the effects induced by baclofen on epileptiform events generated by FCD and MTLE slices, we discovered that the function of GABAB receptors (presumably located at presynaptic inhibitory terminals) was markedly decreased in FCD tissue. Thus, epileptiform synchronization leading to in vitro ictal activity in the human FCD tissue is initiated by a synchronizing mechanism that paradoxically relies on GABAA receptor activation causing sizeable increases in [K+]o. This mechanism may be facilitated by the decreased ability of GABAB receptors to control GABA release from interneuron terminals.

Characterization of a fast transient outward current in neocortical neurons from epilepsy patients.

A-type currents powerfully modulate discharge behavior and have been described in a large number of different species and cell types. However, data on A-type currents in human brain tissue are scarce. Here we have examined the properties of a fast transient outward current in acutely dissociated human neocortical neurons from the temporal lobe of epilepsy patients by using the whole-cell voltage-clamp technique. The A-type current was isolated with a subtraction protocol. In addition, delayed potassium currents were reduced pharmacologically with 10 mM tetraethylammonium chloride. The current displayed an activation threshold of about -70 mV. The voltage-dependent activation was fitted with a Boltzmann function, with a half-maximal conductance at -14.8 +/- 1.8 mV (n = 5) and a slope factor of 17.0 +/- 0.5 mV (n = 5). The voltage of half-maximal steady-state inactivation was -98.9 +/- 8.3 mV (n = 5), with a slope factor of -6.6 +/- 1.9 mV (n = 5). Recovery from inactivation could be fitted monoexponentially with a time constant of 18.2 +/- 7.5 msec (n = 5). At a command potential of +30 mV, application of 5 mM 4-aminopyridine or 100 microM flecainide resulted in a reduction of A-type current amplitude by 35% or 22%, respectively. In addition, flecainide markedly accelerated inactivation. Current amplitude was reduced by 31% with application of 500 microM cadmium. All drug effects were reversible. In conclusion, neocortical neurons from epilepsy patients express an A-type current with properties similar to those described for animal tissues.

RNA editing (R/G site) and flip-flop splicing of the AMPA receptor subunit GluR2 in nervous tissue of epilepsy patients.

Editing and alternative splicing of mRNA are posttranscriptional steps probably involved in pathophysiological aspects of epilepsy. The present study analyses the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR2 with respect to the expression of (i) editing at the R/G site and (ii) flip-flop cassettes. Nervous tissue from patients with temporal lobe epilepsy was analysed by RT-PCR followed by restriction enzyme assays. Human autoptic tissue served as control. R/G editing status: the relative amount of edited RNA was significantly increased in the hippocampal tissue, whereas no changes were found in neocortical tissues. Flip-flop expression: no significant alterations were found in relative abundance of spliced variants containing the flip exon. The increased editing at the R/G site in the hippocampal tissue of epilepsy patients may enhance responses to glutamate, resulting in a synapse operating at an increased gain.

Stimulus-induced patterns of bioelectric activity in human neocortical tissue recorded by a voltage sensitive dye.

Stimulus-induced pattern of bioelectric activity in human neocortical tissue was investigated by use of the voltage sensitive dye RH795 and a fast optical recording system. During control conditions stimulation of layer I evoked activity predominantly in supragranular layers showing a spatial extent of up to 3000 microm along layer III. Stimulation in white matter evoked distinct activity in infragranular layers with a spatial extent of up to 3000 microm measured along layer V. The mean amplitude of optical signals close to the stimulated sites in layer I and white matter determined 25 ms following the stimulus, decreased by 50% at a lateral distance of approximately 900 microm and 1200 microm, respectively. Velocity of spread along the vertical stimulation axis reached 0.24 m/s in the supragranular layers (layers I to III) and then decreased to 0.09 m/s following layer I activation; stimulation of white matter induced a velocity of spread in layer V of 0.38 m/s, which slowed down to 0.12 m/s when passing the lower border of lamina IV. The horizontal velocities of spread determined from the stimulation site to a lateral distance of 500 microm reached 0.26-0.28 m/s and 0.28-0.35 m/s for layer I and white matter stimulation, respectively. At larger distances velocity of spread decreased. Increased excitability (Mg(2+)-free solution) had no significant effect on the spatio-temporal distribution of evoked activity as compared with control conditions. There were also no obvious differences between the results obtained in slices, which generated spontaneously sharp waves and those which were not spontaneously active. About 30% of the slices (n=7) displayed a greatly different response pattern, which seemed not to be related in a simple way to the stimulation as was the case in the majority of the investigated slices. The activity pattern of those slices appeared atypical in regard to their deviations of the vertical and horizontal extent of activity, to their reduced spatial extent of activity during increased excitability, to their layer-related distribution of activity, and to the appearance of afterdischarges.Concluding, in 30% of the human temporal lobe slices atypical activity pattern occurred which obviously reflect intrinsic epileptiform properties of the resected tissue. The majority of slices showed stereotyped activity pattern without evidence for increased excitability.

Differential sensitivity to induction of spreading depression by partial disinhibition in chronically epileptic human and rat as compared to native rat neocortical tissue.

Spreading depression (SD) is characterized by a transient breakdown of neuronal function concomitant with a massive failure of ion homeostasis. It is a phenomenon that can be induced in neocortical tissue by raising excitability, e.g. injection of K(+), application of glutamatergic agonists, or blocking Na(+)/K(+) ATPase. Here we report a novel method of SD induction using minimal disinhibition with application of low concentrations (5 microM) of the GABA(A) receptor blocker bicuculline. This procedure-while subthreshold for epileptiform activity-readily induced spontaneous SDs in native rat neocortical slices, accompanied by typical depolarizations of neurons and glial cells. In contrast, in human neocortical preparations obtained from epilepsy surgery, in approximately 20% of the slices spontaneous epileptiform activity appeared with this bicuculline dosage without SDs. Raising the concentration of bicuculline to an epileptogenic dose (10 microM) in human tissue also resulted in the generation of epileptiform activity only. Likewise, in slices from pilocarpine-treated, chronically epileptic rats, bicuculline also only induced epileptiform activity without eliciting SDs. The experiments indicate that chronic epilepsy causes a differential sensitivity to partial GABA(A) receptor blockade with regard to induction of SD.

Quinine suppresses extracellular potassium transients and ictal epileptiform activity without decreasing neuronal excitability in vitro.

The effect of quinine on pyramidal cell intrinsic properties, extracellular potassium transients, and epileptiform activity was studied in vitro using the rat hippocampal slice preparation. Quinine enhanced excitatory post-synaptic potentials and decreased fast- and slow-inhibitory post-synaptic potentials. Quinine reduced the peak potassium rise following tetanic stimulation but did not affect the potassium clearance rate. Epileptiform activity induced by either low-Ca(2+) or high-K(+) artificial cerebrospinal fluid (ACSF) was suppressed by quinine. The frequency of spontaneous inter-ictal bursting induced by picrotoxin, high-K(+), or 4-aminopyridine was significantly increased. In normal ACSF, quinine did not affect CA1 pyramidal cell resting membrane potential, input resistance, threshold for action potentials triggered by intracellular or extracellular stimulation, or the orthodromic and antidromic evoked population spike amplitude. The main effects of quinine on intrinsic cell properties were to increase action potential duration and to reduce firing frequency during sustained membrane depolarizations, but not at normal resting membrane potentials. This attenuation was enhanced at increasingly depolarized membrane potentials. These results suggest that quinine suppresses extracellular potassium transients and ictal activity and modulates inter-ictal activity by limiting the firing rate of cells in a voltage-dependent manner. Because quinine does not affect 'normal' neuronal function, it may merit consideration as an anticonvulsant.

Sodium currents in striatal neurons from dystonic dt(sz) hamsters: altered response to lamotrigine.

Dystonic mutant dt(sz) hamsters are a model for paroxysmal dystonia. Handling/stress provoke the dystonic attacks. This phenomenon subsedes with maturation, but can be reinvoked when these animals receive sodium channel blockers such as lamotrigine, suggesting a dysfunction of striatal sodium channels. Voltage-gated fast sodium currents (I(Na(+))) were studied in acutely isolated striatal neurons from healthy and dt(sz) hamsters in whole-cell voltage clamp recordings. The action of lamotrigine was tested on (a) current/voltage relationship, (b) kinetics, and (c) steady-state inactivation and activation. Under control conditions, properties of I(Na(+)) were not different between healthy and dt(sz) neurons. With lamotrigine, however, (a) peak currents were significantly less depressed by the drug in neurons from dt(sz) hamsters as compared to healthy cells, and (b) the steady-state inactivation curve shift of I(Na(+)) was less pronounced in dt(sz) neurons. The results suggest that in dt(sz) hamsters, fast sodium currents in striatal neurons are more resistant to blockade. This sodium channel alteration might be causal for a functional imbalance between input and output structures of the basal ganglia under conditions of compromised I(+)(Na).

Spatio-temporal patterns of neuronal activity: analysis of optical imaging data using geometric shape matching.

Optical imaging of neuronal network activity yields information of spatial dynamics which generally is analyzed visually. The transient appearance of spatial activity patterns is difficult to gauge in a quantifiable manner, or may even altogether escape detection. Here, we employ geometric shape matching using Fréchet distances or straight skeletons to search for pre-selected patterns in optical imaging data with adjustable degrees of tolerance. Data were sampled from fluorescence changes of a voltage-sensitive dye recorded with a 464-photodiode array. Fluorescence was monitored in a neuronal network in vitro. Neuronal activity prompting fluorescence fluctuations consisted of spontaneous epileptiform discharges in neocortical slices from patients undergoing epilepsy surgery. The experiments show that: (a) spatial activity patterns can be detected in optical imaging data; (b) shapes such as "mini-foci" appear in close correlation to bioelectric discharges monitored with field potential electrodes in a reproducible manner; (c) Fréchet distances yield more conservative matches regarding rectangular, and less conservative hits with respect to radially symmetric shapes than the straight skeleton approach; and (d) tolerances of 0.03-0.1 are suited to detect faithful images of pre-selected shapes, whereas values >0.8 will report matches with any polygonal pattern. In conclusion, the methods reported here are suited to detect and analyze spatial, geometric dynamics in optical imaging data.

Prolonged epileptiform bursting induced by 0-Mg(2+) in rat hippocampal slices depends on gap junctional coupling.

The transition from brief interictal to prolonged seizure, or 'ictal', activity is a crucial event in epilepsy. In vitro slice models can mimic many phenomena observed in the electroencephalogram of patients, including transition from interictal to ictaform or seizure-like activity. In field potential recordings, three discharge types can be distinguished: (1) primary discharges making up the typical interictal burst, (2) secondary bursts, lasting several hundred milliseconds, and (3) tertiary discharges lasting for seconds, constituting the ictal series of bursts. The roles of chemical synapses in these classes of burst have been explored in detail. Here we test the hypothesis that gap junctions are necessary for the generation of secondary bursts. In rat hippocampal slices, epileptiform activity was induced by exposure to 0-Mg(2+). Epileptiform discharges started in the CA3 subfield, and generally consisted of primary discharges followed by 4-13 secondary bursts. Three drugs that block gap junctions, halothane (5-10 mM), carbenoxolone (100 microM) and octanol (0.2-1.0 mM), abolished the secondary discharges, but left the primary bursts intact. The gap junction opener trimethylamine (10 mM) reversibly induced secondary and tertiary discharges. None of these agents altered intrinsic or synaptic properties of CA3 pyramidal cells at the doses used. Surgically isolating the CA3 subfield made secondary discharges disappear, and trimethylamine under these conditions was able to restore them.We conclude that gap junctions can contribute to the prolongation of epileptiform discharges.

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.

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.

Effects of retigabine on rhythmic synchronous activity of human neocortical slices.

The antiepileptic effects of the novel antiepileptic drug retigabine (D-23129) [N-(2-amino-4-(4-flurobenzylamino)phenyl) carbamid acid ethyl ester] were tested in neocortical slice preparations (n=23) from 17 patients (age, 3-42 years) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves, as well as of epileptiform field potentials (EFP) elicited by superfusion with Mg(2+)-free solution without or with addition of 10 micromol/l bicuculline. (1) Spontaneous rhythmic sharp waves (n=6), with retigabine application, the repetition rate was decreased down to 12-47% of initial value (10 micromol/l, n=3) after 180 min or suppressed completely within 12 min (50 micromol/l, n=3). (2) Low Mg(2+) EFP (n=9), with retigabine application, the repetition rate was decreased down to 50 and 65% of initial value (10 micromol/l; n=2) after 180 min or suppressed completely after 9-55 min (10, 50 and 100 micromol/l; n=2 in each case). In one slice only a transient reduction of the repetition rate was seen with 10 micromol/l retigabine. (3) Low Mg(2+) EFP with addition of bicuculline (n=8), with retigabine application, the repetition rate was decreased down to 12-55% of initial value (10 micromol/l; n=4) after 180 min or suppressed completely after 6-30 min (50 and 100 micromol/l; n=2 in each case). The depressive effect of retigabine was reversible in all but one slice. The results show a clear antiepileptic effect of retigabine in human neocortical slices on spontaneously occurring rhythmic sharp waves and different types of induced seizure activity.

Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding.

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.

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.

Effects of nifedipine on rhythmic synchronous activity of human neocortical slices.

The antiepileptic effect of the dihydropyridine calcium channel blocker nifedipine was tested in neocortical slice preparations (n=27) from patients ranging in age from four to 46 years (mean=25) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves as well as of untriggered epileptiform field potentials induced by omission of Mg(2+) from the superfusate, or epileptiform field potentials elicited by application of bicuculline and triggered by single electrical stimuli. (1) Spontaneous rhythmic sharp waves (n=6): with nifedipine (40micromol/l), the repetition rate was decreased down to 30% of initial value, whereas the area under the field potential remained nearly unchanged. (2) Untriggered low Mg(2+) epileptiform field potentials (n=6): with nifedipine (40micromol/l) the area under the field potentials was reduced while the action on the repetition rate was ambiguous. (3) Triggered bicuculline epileptiform field potentials (n=15): with nifedipine (40micromol/l; n=4), no antiepileptic effect was found. There was, however, a marked increase in the area under the epileptiform field potentials. The area under the field potentials was reduced only at a dosage of 60micromol/l (n=11). This effect was stronger when nifedipine was applied with a K(+) concentration raised from 4 to 8mmol/l. The results show that the calcium channel blocker nifedipine is able to reduce differential epileptiform discharges in human neocortical tissue. These observations are in line with previous findings, suggesting that calcium flux into neurons is involved in epileptogenesis. The present results therefore support the idea that some organic calcium antagonists may be useful in human epilepsy therapy, although the etiology of epileptic seizures seems to be a critical factor for the efficacy of the drug.

Optical monitoring of neuronal activity during spontaneous sharp waves in chronically epileptic human neocortical tissue.

Functional changes in neuronal circuitry reflected in spontaneously occurring synchronous sharp field potentials (SSFP) have been reported to occur in human brain suffering from chronic epileptogenicity but not in primary nonepileptic tissue from peritumoral resectates. Voltage sensitive dyes and fast imaging were used to visualize spontaneously occurring rhythmic depolarizations correlated to SSFP in chronically epileptic human neocortical slices obtained during epilepsy surgery. Localized and spatially inhomogeneous neuronal depolarizations were found to underlie spontaneous SSFP, which remained unchanged and spatially restricted to foci <750 micrometer diam even under epileptogenic (low-Mg(2+)) conditions. In cases where ictaform paroxysmal activity occurred in low-Mg(2+) medium, neuronal depolarizations were wide-spread but still spatially inhomogeneous, and the events were preferentially initiated at distinct foci. The findings suggest that small neuronal networks are able to establish and maintain synchronous rhythmic and epileptiform activity.

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.