Brain Polyamine Stress Response: Recurrence After Repetitive Stressor and Inhibition by Lithium

Abstract: We recently demonstrated that, unlike in peripheral tissues, the increase in activity of polyamine synthesizing enzymes observed in the brain after acute stress can be prevented by long-term, but not by short-term, treatment with lithium. In the present study we sought to examine the effects of chronic intermittent stress on two key polyamine synthesizing enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and their modulation by lithium treatment. Adult male rats were subjected to 2 h of restraint stress once daily for 5 days and to an additional delayed stress episode 7 days later. Enzyme activities were assayed 6 h after the beginning of each stress episode. In contrast to the liver, where ornithine decarboxylase activity was increased (300% of the control) only after the first stress episode, the enzyme activity in the brain was increased after each stress episode (to ～170% of the control). Unlike ornithine decarboxylase activity, S-adenosylmethionine decarboxylase activity was slightly reduced after the first episode (86% of the control) but remained unchanged thereafter. After cessation of the intermittent stress period, an additional stress episode 7 days later led again to an increase in ornithine decarboxylase activity in the brain (225% of the control) but not in the liver, whereas S-adenosylmethionine decarboxylase activity remained unchanged. The latter increase in ornithine decarboxylase activity was blocked by lithium treatment during the intervening 7-day interval between stressors. The results warrant the following conclusions: (a) Repetitive application of stressors results in a recurrent increase in ornithine decarboxylase activity in the brain but to habituation of this response in the liver. (b) This brain polyamine stress response can be blocked by long-term (days) lithium treatment. (c) The study implicates an overreactive polyamine response as a component of the adaptive, or maladaptive, brain response to stressful events and as a novel molecular target for lithium action.     

Modeling the electrical behavior of anatomically complex neurons using a network analysis program: Excitable membrane

We present methods for using the generalpurpose network analysis program, SPICE, to construct computer models of excitable membrane displaying Hodgkin-Huxley-like kinetics. The four non-linear partial differential equations of Hodgkin and Huxley (H-H; 1952) are implemented using electrical circuit elements. The H-H rate constants, and , are approximated by polynomial functions rather than exponential functions, since the former are handled more efficiently by SPICE. The process of developing code to implement the H-H sodium conductance is described in detail. The Appendix contains a complete listing of the code required to simulate an H-H action potential. The behavior of models so constructed is validated by comparison with the space-clamped and propagating action potentials of Hodgkin and Huxley. SPICE models of multiply branched axons were tested and found to behave as predicted by previous numerical solutions for propagation in inhomogeneous axons. New results are presented for two cases. First, a detailed, anatomically based model is constructed of group Ia input to an -motoneuron with an excitable soma, a myelinated axon and passive dendrites. Second, we simulate interactions among clusters of mixed excitable and passive dendritic spines on an idealized neuron. The methods presented in this paper and its companion (Segev et al. 1985) should permit neurobiologists to construct and explore models which simulate much more closely the real morphological and physiological characteristics of nerve cells.     

Histochemical localization of ornithine decarboxylase with a labelled suicidal enzyme inhibitor

We describe a new technique for cytochemical localization of ornithine decarboxylase by the use of a synthesized conjugate of rhodamine bound to α-difluoromethylornithine a suicidal inhibitor of the enzyme. The labelled inhibitor retained its specificity and irreversibility towards ornithine decarboxylase inhibition. Using this technique we have localized the enzyme in specific regions of the developing rat cerebellum. This novel technique may be generally applicable to other enzymes.     

Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.     

Channel modulators affect PGE(2) binding to bovine aortic endothelial cells

PGE(2), PGF(2alpha) and the thromboxane agonist U-46619 bind to bovine aortic endothelial cells and compete on the same binding site with similar affinity. In addition, binding remains unaffected by prolonged exposure to the ligand. These characteristics differ significantly from those of any known G-coupled prostaglandin receptor. Binding of PGE(2) to the cells is reduced in the presence of the cyclic nucleotides cGMP and cAMP, and is unaffected by protein kinase inhibitors. Removal of permeable cyclic nucleotides from the cell medium results in a fast and complete restoration of PGE(2) binding to the cells, suggesting that both cyclic nucleotides reduce PGE(2) binding by a reversible interaction with the prostaglandin-binding site, without the involvement of second messenger-activated protein kinases. Our data further show that binding of prostaglandins to bovine aortic endothelial cells is sensitive to heavy metals and to activators and blockers of calcium, ATP-sensitive K(+) and chloride channels. Nickel, a specific cyclic nucleotide-gated (CNG) channel activator, decreases PGE(2) binding and so do the CNG channel activators Rp-8-Br-PET-cGMPS and Sp-8-Br-PET-cGMPS. On the other hand, the calcium channel blockers pimozide, diltiazem as well as LY-83,583, a guanylate cyclase inhibitor, which were reported to block CNG channels, enhance PGE(2) binding. The sensitivity of PGE(2) binding to selective CNG channel modifying agents, as well as the rapid and reversible interaction with cyclic nucleotides, may suggest that the common low-affinity prostanoid-binding site on bovine aortic endothelial cells is associated with a molecular entity, which possess several properties of a CNG channel.     

Spatially Distributed Dendritic Resonance Selectively Filters Synaptic Input

An important task performed by a neuron is the selection of relevant inputs from among thousands of synapses impinging on the dendritic tree. Synaptic plasticity enables this by strenghtening a subset of synapses that are, presumably, functionally relevant to the neuron. A different selection mechanism exploits the resonance of the dendritic membranes to preferentially filter synaptic inputs based on their temporal rates. A widely held view is that a neuron has one resonant frequency and thus can pass through one rate. Here we demonstrate through mathematical analyses and numerical simulations that dendritic resonance is inevitably a spatially distributed property; and therefore the resonance frequency varies along the dendrites, and thus endows neurons with a powerful spatiotemporal selection mechanism that is sensitive both to the dendritic location and the temporal structure of the incoming synaptic inputs.     

Impaired neutrophil functions in the pathogenesis of an outbreak of recurrent furunculosis caused by methicillin-resistant Staphylococcus aureus among mentally retarded adults

Staphylococcus aureus is an important cause of skin infections. We recently described an outbreak of recurrent furunculosis involving methicillin-resistant S. aureus among mentally retarded adults. We sought to determine the role of impaired neutrophil functions in its pathogenesis. Blood neutrophil functions were determined during both the outbreak (1997) and a disease-free period (2000). Chemotaxis was measured by migration toward formyl-methionyl-leucyl-phenylalanine (FMLP), specifically and randomly; phagocytosis of opsonized zymosan (OZ) was assessed by microscopy; superoxide production was determined by cytochrome c reduction in unstimulated neutrophils and after stimulation with 50 ng/ml phorbol myristate acetate, 1 mg/ml OZ or 5 x 10(-7)M FMLP. Functions were compared between recurrent furunculosis (n=10) and non-recurrent furunculosis patients (n=13). During 2000, functions were normal among the 23 subjects, except for specific/nonspecific chemotaxis (mean 68%+/-26 and 69%+/-28). During infection, recurrent furunculosis patients had a significantly increased basal superoxide production as compared to disease-free period (10.5+/-4.7 vs. 4.9+/-1.9 nmol O(-)(2)/10(6) cells/min, p=0.003). During the disease-free period, recurrent furunculosis patients had lower basal superoxide production (4.9+/-1.9 vs. 7.7+/-3.5, p=0.067) and impaired specific chemotaxis (57%+/-28 vs. 76%+/-21, p<0.05) as compared to non-recurrent furunculosis patients. Only specific chemotaxis was an independent risk factor for recurrent furunculosis. Mentally retarded adults have impaired chemotaxis, with recurrent furunculosis cases having an even greater impairment. Abnormal specific chemotaxis is an independent risk factor for recurrent furunculosis. Impaired neutrophil functions thus have a role in the pathogenesis of outbreaks of recurrent furunculosis.