Electrochemical degradation of trichloroacetic acid in aqueous media: influence of the electrode material.

The electrochemical degradation of trichloroacetic acid (TCAA) in water has been analysed through voltammetric studies with a rotating disc electrode and controlled-potential bulk electrolyses. The influence of the mass-transport conditions and initial concentration of TCAA for titanium, stainless steel and carbon electrodes has been studied. It is shown that the electrochemical reduction of TCAA takes place prior to the massive hydrogen evolution in the potential window for all electrode materials studied. The current efficiency is high (> 18%) compared with those normally reported in the literature, and the fractional conversion is above 50% for all the electrodes studied. Only dichloroacetic acid (DCAA) and chloride anions were routinely detected as reduction products for any of the electrodes, and reasonable values of mass balance error were obtained. Of the three materials studied, the titanium cathode gave the best results.

Lead dioxide film sonoelectrodeposition in acidic media: Preparation and performance of stable practical anodes.

Practical lead dioxide anodes have been obtained by electrodeposition on glassy carbon and titanium substrates in the presence and in the absence of an ultrasound field. The films obtained by mechanical agitation on glassy carbon are strongly improved when the electrodeposition process is carried out with the ultrasound field, providing adherent deposits free from nodules and stress, but with pores appearing occasionally. These enhanced properties were not achieved by mechanical conditions, even when optimization of temperature, current density, additives and geometrical aspects was attempted. The best practical anodes were obtained by sonoelectrodeposition using specially treated titanium as substrate, providing comparable behavior to commercial electrodes.

Sonochemical degradation of perchloroethylene: the influence of ultrasonic variables, and the identification of products.

Sonochemistry is a technique that offers promise for pollutant degradation, but earlier studies on various chlorinated substrates do not give a definitive view of the effectiveness of this methodology. We now report a thorough study of ultrasonic operational variables upon perchloroethylene (PCE) degradation in water (variables include ultrasonic frequency, power and system geometry as well as substrate concentration) and we attempt to close the mass balance where feasible. We obtained fractional conversions of >97% showing very effective loss of pollutant starting material, and give mechanistic proposals for the reaction pathway based on cavitational phenomena inducing pyrolytic and free radical processes. We note major products of Cl(-) and CO(2)/CO, and also trichloroethylene (TCE) and dichloroethylene (DCE) at ppm concentrations as reported earlier. The formation at very low (ppb) concentration of small halocompounds (CHCl(3), CCl(4)) and also of higher-mass species, such as pentachloropropene, hexachloroethane, is noteworthy. But of particular importance in our work is the discovery of significant quantities of chloroacetate derivatives at ppm concentrations. Although these compounds have been described as by-products with other techniques such as radiolysis or photochemistry, this is the first time that these products have been identified in the sonochemical treatment of PCE; this allows a much more effective account of the mass balance and may explain earlier inconsistencies. This reaction system is now better identified, but a corollary is that, because these haloacetates are themselves species of some toxicity, the use of ultrasound here may not sufficiently diminish wastewater toxicity.

Sonoelectrochemical treatment of water polluted with trichloroacetic acid: from sonovoltammetry to pre-pilot plant scale.

The sonoelectrochemical treatment of aqueous solutions of trichloroacetic acid (TCAA) has been scaled-up from the voltammetric analysis to pre-pilot stage. The degradation in absence of ultrasound field has yield to a poor performance which has been improved in presence of ultrasound. The sonovoltametry study has provided the range of potentials and/or current densities to be used with the lowest current efficiency penalty. Sonoelectrolyses at batch scale (carried out with a horn-transducer 24 kHz positioned at about 3 cm from the surface of the electrode) achieved little improvement in the degradation. However, when a specifically designed sonoelectrochemical reactor (not optimized) was used during the scale-up, the presence of ultrasound field provided better results (fractional conversion 97%, degradation efficiency 26%, selectivity 0.92 and current efficiency 8%) at lower ultrasonic intensities and volumetric flow.

Comparative study of matrix-assisted laser desorption/ionization and gas chromatography for quantitative determination of cocoa butter and cocoa butter equivalent triacylglycerol composition.

The triacylglycerol (TAG) composition study of cocoa butter (CB) and cocoa butter equivalents (CBEs) has been performed by gas chromatography (GC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). These two techniques provided comparable results. The advantage of the MALDI technique was the detection of each compound comprising the triacylglycerol classes (Cn). Moreover, comparison of the data obtained by these two techniques indicated that TAG relative percentages could be obtained quantitatively with the MALDI technique. These techniques have been applied for the composition determination of CB + CBE mixtures. Encouraging results showed that it is possible to quantify an admixture containing as little as 4% of CBE.

Early development of neuronal activity in the primate hippocampus in utero.

Morphological studies suggest that the primate hippocampus develops extensively before birth, but little is known about its functional development. Patch-clamp recordings of hippocampal neurons and reconstruction of biocytin-filled pyramidal cells were performed in slices of macaque cynomolgus fetuses delivered by cesarean section. We found that during the second half of gestation, axons and dendrites of pyramidal cells grow intensively by hundreds of micrometers per day to attain a high level of maturity near term. Synaptic currents appear around midgestation and are correlated with the level of morphological differentiation of pyramidal cells: the first synapses are GABAergic, and their emergence correlates with the growth of apical dendrite into stratum radiatum. A later occurrence of glutamatergic synaptic currents correlates with a further differentiation of the axodendritic tree and the appearance of spines. Relying on the number of dendritic spines, we estimated that hundreds of new glutamatergic synapses are established every day on a pyramidal neuron during the last third of gestation. Most of the synaptic activity is synchronized in spontaneous slow ( approximately 0.1 Hz) network oscillations reminiscent of the giant depolarizing potentials in neonatal rodents. Epileptiform discharges can be evoked by the GABA(A) receptor antagonist bicuculline by the last third of gestation, and postsynaptic GABA(B) receptors contribute to the termination of epileptiform discharges. Comparing the results obtained in primates and rodents, we conclude that the template of early hippocampal network development is conserved across the mammalian evolution but that it is shifted toward fetal life in primate.

Presynaptic kainate receptors that enhance the release of GABA on CA1 hippocampal interneurons.

We report that kainate receptors are present on presynaptic GABAergic terminals contacting interneurons and that their activation increases GABA release. Application of kainate increased the frequency of miniature inhibitory postsynaptic currents recorded in CA1 interneurons. Local applications of glutamate but not of AMPA or NMDA also increased GABA quantal release. Application of kainate as well as synaptically released glutamate reduced the number of failures of GABAergic neurotransmission between interneurons. Thus, activation of presynaptic kainate receptors increases the probability of GABA release at interneuron-interneuron synapses. Glutamate may selectively control the communication between interneurons by increasing their mutual inhibition.

Human and monkey fetal brain development of the supramammillary-hippocampal projections: a system involved in the regulation of theta activity.

The supramammillary (SUM)-hippocampal pathway plays a central role in the regulation of theta rhythm frequency. We followed its prenatal development in eight Cynomolgus monkeys (Macaca fascicularis) from embryonic day E88 to postnatal day 12 (term 165 days) and in eight human fetuses from 17.5 to 40 gestational weeks, relying on neurochemical criteria established in the adult (Nitsch and Leranth [1993] Neuroscience 55:797-812). We found that 1) SUM afferents reached the dentate juxtagranular and CA2 pyramidal cell layers at midgestation in human fetuses, earlier than in monkeys (two-thirds of gestation [E109]). They co-expressed calretinin, substance P, and acetylcholinesterase but not gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD); 2) the presumed parent neurons in the monkey SUM expressed calretinin or both calretinin and substance P; 3) most of them were surrounded by GAD-containing terminals that might correspond to the septo-SUM feedback pathway (Leranth et al. [1999] Neuroscience 88:701); and 4) in addition, a large band of calretinin-labeled terminals that did not co-express substance P, GAD, or acetylcholinesterase was present in the deepest one-third of the dentate molecular layer in both the Cynomolgus monkey and human fetuses. It persisted in the adult monkey but not in adult human hippocampus; it remains questionable whether it originates in the SUM. In conclusion, the early ingrowth of the excitatory SUM-hippocampal system in human and non-human primates may contribute to the prenatal activity-dependent development of the hippocampal formation. The possibility and the functional importance of an in utero generation of hippocampal theta-like activity should also be considered.

Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy.

Impaired inhibition is thought to be important in temporal lobe epilepsy (TLE), the most common form of epilepsy in adult patients. We report that, in experimental TLE, spontaneous GABAergic inhibition was increased in the soma but reduced in the dendrites of pyramidal neurons. The former resulted from the hyperactivity of somatic projecting interneurons, whereas the latter was probably due to the degeneration of a subpopulation of dendritic projecting interneurons. A deficit in dendritic inhibition could reduce seizure threshold, whereas enhanced somatic inhibition would prevent the continuous occurrence of epileptiform activity.

What is GABAergic inhibition? How is it modified in epilepsy?

A deficit of gamma-aminobutyric acid-ergic (GABAergic) inhibition is hypothesized to underlie most forms of epilepsy. Although apparently a straightforward and logical hypothesis to test, the search for a deficit of GABAergic inhibition in epileptic tissue has revealed itself to be as difficult as the quest for the Holy Grail. The investigator faces many obstacles, including the multiplicity of GABAergic inhibitory pathways and the multiplicity of variables that characterize the potency of inhibition within each inhibitory pathway. Perhaps more importantly, there seems to be no consensual definition of GABAergic inhibition. The first goal of this review is to try to clarify the notion of GABAergic inhibition. The second goal is to summarize our current knowledge of the various alterations that occur in the GABAergic pathways in temporal lobe epilepsy. Two important features will emerge: (a) according to the variable used to measure GABAergic inhibition, it may appear increased, decreased, or unchanged; and (b) these modifications are brain area- and inhibitory pathway-specific. The possible functional consequences of these alterations are discussed.

Distribution of spontaneous currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons.

Excitatory and inhibitory pathways have specific patterns of innervation along the somato-dendritic axis of neurons. We have investigated whether this morphological diversity was associated with variations in the frequencies of spontaneous and miniature GABAergic and glutamatergic synaptic currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons. Using in vitro whole cell recordings from somata, apical dendrites and basal dendrites (for which we provide the first recordings) of CA1 pyramidal neurons, we report that over 90% of the spontaneous currents were GABAergic, <10% being glutamatergic. The frequency of spontaneous GABAergic currents was comparable in the soma and in the dendrites. In both somata and dendrites, the Na(+) channel blocker tetrodotoxin abolished more than 80% of the spontaneous glutamatergic currents. In contrast, tetrodotoxin abolished most dendritic (>90%) but not somatic (<40%) spontaneous GABAergic currents. Computer simulations suggest that in our experimental conditions, events below 40pA are electrotonically filtered to such a degree that they are lost in the recording noise. We conclude that, in vitro, inhibition is massively predominant over excitation and quantitatively evenly distributed throughout the cell. However, inhibition appears to be mainly activity-dependent in the dendrites whereas it can occur in the absence of interneuron firing in the soma. These results can be used as a benchmark to compare values obtained in pathological tissue, such as epilepsies, where changes in the balance between excitation and inhibition would dramatically alter cell behaviour.

Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy.

In the pilocarpine model of chronic limbic seizures, subpopulations of glutamic acid decarboxylase (GAD)-containing neurons within the hilus of the dentate gyrus and stratum oriens of the CA1 hippocampal region are vulnerable to seizure-induced damage. However, many gamma-aminobutyric acid (GABA) neurons remain in these and other regions of the hippocampal formation. To determine whether long-term changes occur in the main metabolic pathway responsible for GABA synthesis in remaining GABA neurons, the levels of mRNA and protein labeling for the two forms of GAD (GAD65 and GAD67) were studied in pilocarpine-treated animals that had developed spontaneous seizures. Qualitative and semiquantitative analyses of nonradioactive in situ hybridization experiments demonstrated marked increases in the relative amounts of GAD65 and GAD67 mRNAs in remaining hippocampal GABA neurons. In addition, immunohistochemical studies demonstrated parallel increases in the intensity of terminal labeling for both GAD65 and GAD67 isoforms throughout the hippocampal formation. These increases were most striking for GAD65, the isoform of GAD that is particularly abundant in axon terminals. These findings demonstrate that, in a neuronal network that is capable of generating seizures, both GAD65 and GAD67 are up-regulated at the gene and protein levels in the remaining GABA neurons of the hippocampal formation. This study provides further evidence for the complexity of changes in the GABA system in this model of temporal lobe epilepsy.

Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) in humans and animals is associated with axonal sprouting of glutamatergic neurons and neosynaptogenesis in the hippocampal formation. We examined whether this plasticity of excitatory pathways contributes to an increased level of glutamatergic excitation in the CA1 region of rats experiencing chronic spontaneous limbic seizures following kainic acid or pilocarpine treatment. In chronic cases, we report an extensive axonal sprouting of CA1 pyramidal neurons, with many axonal branches entering the pyramidal cell layer and stratum radiatum, regions that are not innervated by axonal collaterals of CA1 pyramidal neurons in control animals. Concurrently with this anatomical reorganization, a large increase of the spontaneous glutamatergic drive is observed in the dendrites and somata of CA1 pyramidal cells. Furthermore, electrical activation of the reorganized CA1 associational pathway evokes epileptiform bursts in CA1 pyramidal cells. These findings suggest that reactive plasticity could contribute to the hyperexcitability of CA1 pyramidal neurons and to the propagation of seizures in these two models of TLE.

Interneurones are not so dormant in temporal lobe epilepsy: a critical reappraisal of the dormant basket cell hypothesis.

One axiom at the basis of epilepsy research is that there exists an imbalance between excitation and inhibition. This abnormality can be achieved by an increase of excitation on principal cells, a decreased inhibition (i.e. disinhibition) or both. This review focuses on dysfunction of inhibition, and in particular on the 'dormant basket cell hypothesis'. This hypothesis states that, (1) interneurones are functionally disconnected from excitatory afferents, resulting in hyperexcitability of principal neurones and loss of paired pulse inhibition, (2) when properly activated, interneurones can still perform their task, i.e. suppress epileptiform activity and restore paired pulse inhibition. The aim of this review is to discuss the evidence in support of the 'dormant basket cell hypothesis'. We will first discuss the rationale underlying the hypothesis and the criteria needed to validate the hypothesis. We will then show that, (1) the key experimental data offered in support of the hypothesis (Bekenstein and Lothman, 1993. Dormancy of inhibitory interneurones in a model of temporal lobe epilepsy. Science 259, 97-100; Sloviter, 1991. Permanently altered hippocampal structure, excitability, and inhibition after experimental status epilepticus in the rat: the 'dormant basket cell' hypothesis and its relevance to temporal lobe epilepsy. Hippocampus 1, 41-66) are difficult to interpret, and (2) recent recordings from interneurones in epileptic tissue argue against the hypothesis. The 'dormant basket cell hypothesis' is then discussed in the broader context of disinhibition.

GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells.

We studied the modulation of GABAergic inhibition by glutamate and kainate acting on GluR5-containing kainate receptors in the CA1 hippocampal region. Glutamate, kainate or ATPA, a selective agonist of GluR5-containing receptors, generates an inward current in inhibitory interneurons and cause repetitive action potential firing. This results in a massive increase of tonic GABAergic inhibition in the somata and apical dendrites of pyramidal neurons. These effects are prevented by the GluR5 antagonist LY 293558. Electrical stimulation of excitatory afferents generates kainate receptor-mediated excitatory postsynaptic currents (EPSCs) and action potentials in identified interneurons that project to the dendrites and somata of pyramidal neurons. Therefore glutamate acting on kainate receptors containing the GluR5 subunit may provide a protective mechanism against hyperexcitability.

A novel in vitro preparation: the intact hippocampal formation.

The intact hippocampal formation (IHF) of neonatal or young rats can be kept alive for an extended period in a fully submerged chamber with excellent morphological preservation. Field or patch-clamp recordings, intracellular Ca2+ measurements, and 3-D reconstruction of biocytin-filled neurons can be performed routinely. The generation and propagation of network-driven activities can be studied within the IHF or between connected intact structures such as the septum and the hippocampus or two hippocampi, and the use of a dual chamber enables the application of drugs separately to each structure. This preparation will be useful to study intact neuronal networks in the developing hippocampus in vitro.

Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy.

Patch-clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that gamma-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.

Bicuculline induces ictal seizures in the intact hippocampus recorded in vitro.

The effects of the gamma-aminobutyric acid (GABA) receptor antagonist bicuculline on rat hippocampal neurons recorded in slices and in the intact hippocampi kept in vitro were studied using whole-cell patch-clamp recordings. Bicuculline (10 microM) evoked ictal discharges in the intact hippocampus but only interictal discharges in conventional slices. Recording from the intact hippocampus in vitro is an alternative preparation to study the organization of the hippocampal neuronal network.

Enhanced NMDAR-dependent epileptiform activity is controlled by oxidizing agents in a chronic model of temporal lobe epilepsy.

1. Graded N-methyl-D-aspartate receptor (NMDAR)-dependent epileptiform discharges were recorded from ex vivo hippocampal slices obtained from rats injected a week earlier with an intracerebroventricular dose of kainic acid. Intracellular recordings from pyramidal cells of the CA1 area showed that glutamate NMDAR actively participated in synaptic transmission, even at resting membrane potential. When NMDAR were pharmacologically isolated, graded burst discharges could still be evoked. 2. The oxidizing reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 200 microM, 15 min) suppressed the late part of the epileptiform burst that did not recover after wash but could be reinstated by the reducing agent tris (2-carboxyethyl) phosphine (TCEP, 200 microM, 15 min) and again abolished with the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV). 3. Pharmacologically isolated NMDAR-mediated responses were decreased by DTNB (56 +/- 10%, mean +/- SD, n = 6), an effect reversed by TCEP. 4. When only the fast glutamateric synaptic component was blocked, NMDA-dependent excitatory postsynaptic potentials (EPSPs) could be evoked despite the presence of underlying fast and slow inhibitory postsynaptic potentials (IPSPs). DTNB decreased EPSPs to 48 +/- 12% (n = 5) of control. 5. Since a decrease of the NMDAR-mediated response by +/- 50% is sufficient to suppress the late part of the burst, we suggest that epileptiform activity can be controlled by manipulation of the redox sites of NMDAR. Our observations raise the possibility of developing new anticonvulsant drugs that would spare alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-R (AMPAR)-mediated synaptic responses and decrease NMDAR-mediated synaptic transmission without blocking it completely.