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A combined analytical and experimental investigation was conducted to develop a flat, phase change heat spreader to enable focal thermoelectric cooling as a treatment for intractable neocortical epilepsy. The design parameters required minimum transport capacity of 5 W with an associated temperature drop of less than 0.5°C. A one-dimensional steady-state model was developed and the predicted performance characteristics compared with the results obtained from the experimental evaluation of three conceptual designs of varying complexity. These designs varied in terms of the materials used, but all three used water as the working fluid. The experimental results indicate that the minimum transport limit of 5 W can be achieved by one of the three concepts evaluated with a maximum overall temperature drop of 0.36°C at 5 W, well below the 0.5°C limit and within the experimental uncertainty of the temperature measurement technique used. A simple model was used to aid in the selection of an appropriate heat pipe. ...

The idea of establishing a market for organs is now the subject of unusual controversy. Proponents emphasize the concept of autonomy; opponents invoke fairness and justice. The controversy, however, has given sparse attention to what it would mean to society and medicine to establish a market in organs and to the intended and unintended consequences of such a practice. This article addresses these issues by exploring the tensions between 'extrinsic' and 'intrinsic' incentives, suggesting that donation might well decline were financial incentives introduced. It also contends that social relationship and social welfare policy would be transformed in negative ways and that a regulated market in organs would be extraordinarily difficult to achieve. Finally, it argues that organ sale would have a highly detrimental affect on medicine as a profession.

3-Benzyl-3-ethyl-2-piperidinone (3-BEP) belongs to a family of compounds that includes alpha- substituted gamma-butyrolactones, gamma-thiobutyrolactones, 2-pyrrolidinones and hexahydro-2H-azepin-2-ones. Many of these drugs exhibit potent in vivo anticonvulsant activity in mice. Previous electrophysiological studies demonstrated that they potentiate gamma-aminobutyric acid- (GABA) mediated chloride currents. This GABAA receptor modulation was thought to be the main mechanism of anticonvulsant activity. We report that 3-BEP also modulates sodium channels. It decreased sodium currents in cultured rat hippocampal neurons in a voltage- and concentration-dependent manner. The drug's apparent affinity increased as neurons were depolarized. At a holding potential of -60 mV, the apparent IC50 was 487 microM. This concentration is comparable to its EC50 for GABAA modulation (575 microM). Current blockade occurred over all activation voltages tested. The steady state inactivation curve was shifted by 600 microM 3-BEP from V50 = -65.3 mV to -72.0 mV, and recovery from inactivation was slowed from tau = 4.9 to 12.8 msec. Sodium current inhibition was not observed for three related compounds, suggesting a degree of chemical specificity for this activity. We conclude that in addition to its known effects on GABAA receptors, 3-BEP modulates sodium channels. Therefore this compound may prevent seizures by both enhancing inhibition and diminishing neuronal excitability.

Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones exhibit convulsant or anticonvulsant activity, depending on the alkyl substituents. alpha-Substituted lactones with small alkyl substituents are anticonvulsant and potentiate gamma-aminobutyric acid (GABA)-mediated chloride currents, whereas beta-substituted compounds are usually convulsant and block GABAA currents. We have now found that this distinction is not so clear-cut, in that some compounds can both block and augment GABAA currents, but with different time courses. For example, alpha,alpha-diisopropyl-GBL (alpha-DIGBL) potentiates exogenous GABA currents in cultured rat hippocampal neurons but diminishes GABA-mediated inhibitory postsynaptic currents. A more detailed analysis demonstrates a triphasic effect of alpha-DIGBL on GABA currents, with a rapid inhibitory phase, a slower potentiating phase, and then an "off response" when the GABA/alpha-DIGBL perfusion is stopped. Thus, alpha-DIGBL can inhibit and potentiate GABA currents with kinetically different time courses. Inhibition is more rapid, but at steady state potentiation dominates. Using a simplified model of the GABAA receptor/ionophore, we have simulated our experimental observations with alpha-DIGBL. Another lactone, beta-ethyl-beta-methyl-gamma-thiobutyrolactone, also has dual actions, with inhibition predominating at low concentrations and potentiation predominating at high concentrations. We propose two distinct GBL modulatory sites on the GABAA receptor, i.e., an inhibitory "picrotoxin" site and an enhancing "lactone site." New information on the structure of the GABAA receptor/ionophore may allow the molecular dissection of these two sites.

In 1982, while exploring methods of prolonging the survival of cultured hippocampal neurons, we found that our older, but not the newly plated, neurons were extremely vulnerable to inhibition of oxidative metabolism. When cultures at least two weeks in vitro were exposed to sodium cyanide or an atmosphere of 95% nitrogen/5% CO2, neurons swelled and eventually died (Rothman 1983). However, cultures exposed to similar insults one or two days after plating showed little damage over a comparable time period. Of special interest, we found that increasing the extracellular magnesium to 10 mM could dramatically diminish the damage induced by either cyanide or hypoxia. Our initial interpretation of these experimental results was that synaptic activity was in some way producing the neuronal damage and that, by blocking transmitter release, magnesium was protective. The minimal damage seen in young cultures reflected a lack of synaptic connections early on in culture development.

Multiple receptors modulate the ion channel gated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). gamma-Butyrolactones and gamma-thiobutyrolactones are compounds that act at the picrotoxin recognition site on the GABA receptor complex as either agonists or inverse agonists, depending on the nature of the alkyl substituents. Here we have compared the effects of two gamma-butyrolactones, alpha-ethyl-alpha-methyl-gamma-butyrolactone (alpha EMGBL) and alpha-isopropyl-alpha-methyl-gamma-butryolactone (alpha IMGBL), on GABA currents and inhibitory postsynaptic currents (IPSCs) in cultured, voltage-clamped, rat hippocampal neurons. alpha EMGBL also decreased the rate of IPSC decay without altering IPSC peak amplitude. At higher GABA concentrations (30 microM), alpha EMGBL has already been shown to block picrotoxin receptor agonists and inverse agonists. Thus, alpha EMGBL is a mixed antagonist/inverse agonist. In contrast to alpha EMGBL, alpha IMGBL had no effect on responses to either 0.5 or 30 microM GABA or on IPSCs, but it was able to block the effects of picrotoxin receptor agonists and inverse agonists. Therefore, alpha IMGBL is the first pure antagonist to be described for the picrotoxin receptor. The main conductance state of the GABA-gated channel probably has two or more open states, brief openings associated with binding of a single GABA molecule and longer openings due to the binding of two GABA molecules. We were able to simulate the results obtained with alpha EMGBL, using a computer model, by assuming that alpha EMGBL altered only the opening and closing rate constants for the monoliganded open channel of the GABA receptor. In addition to having site-selective actions, these results suggest that drugs modulating the GABA-linked chloride ionophore may be specific for the kinetic state of the GABA-gated channel.

A number of investigators have shown that gamma-butyrolactones bind to the picrotoxin site on the gamma-aminobutyric acid (GABA) receptor. We examined the effects of three fluorinated gamma-butyrolactone derivatives on GABA currents and synaptic currents in cultured hippocampal neurons. alpha-(2,2,2-trifluoroethyl)-alpha-methyl-gamma-butyrolactone was a partial picrotoxin agonist, meaning that like picrotoxin, it blocked GABA currents, but not completely. This action is in marked contrast to that of its parent compound, alpha-ethyl-alpha-methyl-gamma-butyrolactone, which is a mixed inverse agonist/antagonist, enhancing GABA currents at low GABA concentration (less than or equal to 1 microM) and antagonizing picrotoxin block of GABA currents at higher GABA concentrations (approximately 30 microM). The properties of two difluorinated derivatives were found to be very different from the trifluorinated compound. Both alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone and alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-thiobutyrolactone+ ++ dramatically increased the current produced by low concentrations of GABA. The former had less marked inverse agonist effects at higher GABA concentrations but behaved instead like a picrotoxin antagonist. In addition, alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone reduced both excitatory and inhibitory synaptic currents suggesting a separate effect on presynaptic transmitter release through an unknown mechanism. These experiments support the hypothesis that gamma-butyrolactones can have a variety of effects at the picrotoxin receptor and indicate that specific properties of these compounds can be altered drastically by fluorination.

Identifying the factors responsible for the exquisite sensitivity of the brain to anoxia and ischemia has been very difficult with currently available preparations. During the past three years, our laboratory has utilized two fairly well characterized in vitro preparations to study the effects of anoxia on central mammalian neurons. The experiments described below, which employed cultures of dissociated hippocampal neurons and slices of rat hippocampus, implicate the synaptic release of glutamate (Glu) in the pathophysiology of anoxic neuronal death.

For the eventual purpose of isolating and studying a single animal cell replicon, we have developed a methotrexate-resistant Chinese hamster ovary cell line that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. We estimate the total length of the unit repeated sequence to be 135 +/- 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR cDNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. These data suggest that an early replicon can be isolated from this region, and that this entire, normally unique, genomic segment can be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.

Cultures of dissociated rat hippocampal neurons were used to study the physiology and pharmacology of excitatory synaptic transmission. Rat hippocampal neurons depolarized when they were exposed to the excitatory transmitter candidates, glutamate (Glu) and aspartate (Asp), as well as to the pure excitatory amino acid agonists, N-methyl-D-aspartate (NMDA) and kainate (KA). Quisqualate (QUIS) produced responses in about two-thirds of these cells. Glu responses were much more effectively blocked by the excitatory amino acid antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and gamma-D-glutamylglycine (DGG) than by D-2-amino-5-phosphonovaleric acid (APV) or D-alpha-aminoadipic acid (DAA). Asp depolarizations were depressed by all four antagonists. Monosynaptic excitatory postsynaptic potentials (EPSPs) were only decreased by PDA and DGG. Postsynaptic responses to both Glu and Asp were very voltage dependent, decreasing as the membrane potential was hyperpolarized up to 70 mV below resting levels. The EPSP, however, increased linearly in the hyperpolarized range. NMDA responses were also voltage dependent, while KA and QUIS responses behaved like EPSPs. DGG very effectively blocked KA, but not QUIS, depolarizations. APV, which only partially depressed Glu responses, markedly diminished their voltage sensitivity. These results all suggest that EPSPs in this preparation are produced by Glu acting at KA-type synaptic receptors. Exogenous Glu probably acts at both synaptic KA receptors and extrasynaptic NMDA receptors, which explains why it produces a voltage-dependent response different from the EPSP.