Increased reactive oxygen species in familial amyotrophic lateral sclerosis with mutations in SOD1.

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder characterized by degeneration of large motor neurons of the brain and spinal cord. A subset of ALS is inherited (familial ALS, FALS) and is associated with more than 70 different mutations in the SOD1 gene. Here we report that lymphoblast cell lines derived from FALS patients with 16 different mutations in SOD1 gene exhibit significant increase of intracellular reactive oxygen species (ROS) compared with sporadic ALS (SALS) and normal controls (spouses of ALS patients). The ROS generation did not correlate with SOD1 activity. Further, cells incubated with vitamin C, catalase or the flavinoid quercetin significantly reduced ROS in all groups. The catalase inhibitor 3-amino-1,2,4-triazole resulted in a ten-fold increase of ROS in all groups. Neither L-nitroarginine, a nitric oxide synthase inhibitor or vitamin E altered the ROS levels. Thus, these studies suggest that hydrogen peroxide (H(2)O(2)) is a major ROS elevated in FALS lymphoblasts and it may contribute to the degeneration of susceptible cells. Further, we postulate a mechanism by which increased H(2)O(2) could be generated by mutant SOD1.

PGE2 suppresses mitogen-induced Ca2+ mobilization in T cells.

PGE2-mediated suppression of T cell proliferation during sepsis could result from altered Ca2+ signaling. The present study evaluated the effects of PGE2 on Ca2+ release from intracellular stores and its influx through the plasma membrane in splenic T cells from Sprague-Dawley rats. Intracellular Ca2+ concentration ([Ca2+]i) responses in individual T cells were assessed using the Ca2+ imaging technique, and the release of Ca2+ from intracellular stores and Ca2+ influx were spectrofluorometrically quantified in T cell suspensions. Under unstimulated conditions, nearly 85% of T cells exhibited [Ca2+]i

Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells.

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.

Signaling mechanisms of elevated neutrophil O2- generation after burn injury.

A full skin thickness burn injury was produced in anesthetized rats by exposing 25% of total body surface area to 98 degrees C water for 10 s. Sham (exposed to 37 degrees C water) and burn rats were killed 1, 3, 7, or 10 days later. The role of Ca2+ signaling and Ca(2+)-related protein kinase C (PKC) activation in neutrophil O2- generation was ascertained by evaluating the effect of treatment of the rats with the Ca2+ entry blocker, diltiazem. There was an overt enhancement of O2- generation by polymorphonuclear leukocytes from burn rats on days 1, 3, and 7 postburn, with the peak release occurring on day 3 postburn. O2- generation comparable to the sham was noted on day 10 after the burn. O2- releases on days 1, 3, and 7 postburn were accompanied by marked elevation of Cai2+ and PKC responses. Like the O2- release, intracellular Ca2+ concentration ([Ca2+]i) response on day 10 after burn was suppressed to levels found in the sham group. The treatment of burn rats with diltiazem prevented the upregulation of both [Ca2+]i and PKC responses as well as O2- generation in neutrophils in rats on days 1, 3, and 7 after the burn. Because previous studies have shown that increases in [Ca2+]i precede O2- generation and degranulation, our results suggest that neutrophil O2- release enhancement in the early stages after burn injury (e.g., days 1-7 postburn) results from an overactivation of the Cai2+ and PKC signaling pathways. The heightened O2- generation during the early burn injury phase might play a role in tissue damage in one or more of host organs.

Analysis of spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats.

Whole-cell patch recording techniques were used to analyze spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats. Spontaneous activity was present in 65% of the cells examined, and it included simple and complex firing patterns which persisted under conditions that eliminated residual or reformed synaptic contacts. Under voltage clamp, both spontaneous and quiescent cells displayed similar voltage-dependent conductances. Inward current was carried by Na+ through tetrodotoxin (TTX)-sensitive channels and by Ca2+ through P-type and T-type Ca channels. P-type current was present in all cells examined. T-type current was found in <50%, and it did not correlate with spontaneous activity. We found no evidence of a transient (A-type) potassium current or hyperpolarization-activated cationic current in either spontaneous or quiescent cells. Spontaneous activity did correlate with a lower activation threshold of the Na current, resulting in substantial overlap of the activation and inactivation curves. TTX reduced the holding current of spontaneous cells clamped between -50 and -30 mV, consistent with the presence of a Na "window" current. We were unable, however, to measure a persistent component of the Na current using voltage steps, a result which may reflect the complex gating properties of Na channels. An Na window current could provide the driving force underlying spontaneous activity, as well as plateau potentials, in Purkinje cells.

Is laminin-1 a guidance cue for cerebellar granule cell migration?

Laminin-1 is a glycoprotein found in the basement membrane of many tissues. In the cerebellum of rodents, it has also been localized along Bergmann glial fibers, where it is thought to be involved in promoting granule cell migration by enhancing adhesion and neurite outgrowth along these fibers. Recent reports, however, indicate that laminin-1 is not present on Bergmann fibers, but instead is associated with blood vessels and meninges. Furthermore, attempts to block granule cell migration using antibodies against laminin-1 have yielded conflicting results. In this report, we provide further evidence that laminin-1 is associated exclusively with blood vessels and meninges in the cerebellum of postnatal rats. In addition, we show that adhesion and neurite outgrowth of granule cells was impeded on laminin-coated surfaces. In fact, cerebellar cells dramatically and consistently avoided laminin-1 regions of patterned surfaces. Cells did adhere to laminin regions if it was coadsorbed with polylysine or tested in serum-containing medium. Avoidance of laminin-1 regions in culture was not, however, blocked by pretreatment with laminin-1 antibodies. By comparison, mouse neuroblastoma cells adhered preferentially to laminin-1 regions in serum-free medium, a response which was blocked by laminin-1 antibodies. These results indicate that laminin-1 is not involved in granule cell migration along Bergmann glial fibers. Instead, they suggest that laminin-1 may function as a repulsive guidance cue preventing granule cells from following inappropriate pathways during development.

Kinetics of bone cell organization and mineralization on materials with patterned surface chemistry.

Materials with spatially resolved chemistries (i.e. patterned surfaces) have been used to guide and organize the position of mammalian cells in vitro. A common theme in guiding the spatial distribution of cells has been the use of patterned alkylsiloxanes, where one region contains an aminosilane and the other an alkylsilane. The regions of the aminosilane served as preferential sites for cell attachment and spreading, presumably dependent on the association between cell surface proteoglycans the positively charged amine. In this study, experiments were conducted with patterns of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS) to determine the kinetics of spatial organization of bone-derived cells, and whether initial attachment and spreading affected the rate of matrix mineralization (i.e. bone formation) in extended cultures. The bone cells required the presence of serum or preadsorption of serum proteins to the patterned EDS/DMS surface to organize according to the lithographically defined surface chemistry. Time-lapse video microscopy indicated that cells were randomly distributed over the EDS/DMS surface at the time of plating, but organized on the EDS regions within 30 min. When cultures were extended for 15 and 25 days, the matrix synthesized by the cells was preferentially mineralized on the EDS chemistry. These results demonstrate the ability of surface chemistry modifications to organize cells and form mineralized tissue in vitro. The methods employed should have general value to the engineering of tissues in vitro.

Bistability in cerebellar Purkinje cell dendrites modelled with high-threshold calcium and delayed-rectifier potassium channels.

Phase-plane analysis of the ionic currents underlying dendritic plateau potentials was carried out to study the nonlinear dynamics and steady-state transfer properties of the dendritic tree in cerebellar Purkinje cells. The results of an analysis of the P-type calcium and delayed rectifier potassium channel system are presented in this study. These channels constitute a simple system that can support bistability and plateau potentials. By requiring both the steady-state current-voltage curve and nullclines to mimic basic plateau potential properties, we obtained well-defined ranges of specific conductance that can support bistability. Hysteresis was found to be surprisingly prevalent in this simple ion-channel system. Using the steady-state current voltage relationship, we derive concise, algebraic expressions for the voltage and current thresholds of state transitions as functions of specific conductance. The significance of bistability in this ion-channel system is discussed with respect to the generation of plateau potentials in Purkinje cells dendrites and the role of the cerebellum in motor control.

Identification of acutely isolated cells from developing rat cerebellum.

Immunocytochemical staining was used to identify nerve and glial cells from postnatal rat cerebelli in situ and following tissue dissociation. Purkinje cells were identified using antibodies for the calcium-binding proteins calbindin and PEP19. Purkinje cells isolated during the second postnatal week were 15-20 microns in diameter and relatively abundant and displayed thin perisomatic processes. These features were used to identify Purkinje cells with scanning electron microscopy, which revealed extensive membrane infoldings. Golgi and nuclear cells were identified using antibodies against rat-303 antigen. Pale, nuclear, and Purkinje cells were identified using antibodies for rat-302 antigen. Although staining for rat-302 and rat-303 was weak during the second postnatal week, we were able to identify Golgi and pale cells even after tissue dissociation. Isolated Golgi cells were 8-10 microns in diameter and fewer in number than Purkinje cells and did not counterstain with calbindin antibodies. Isolated pale cells were 8-10 microns in diameter, rare, and resistant to calbindin antibodies. Isolated neurons from cerebellar nuclei were not located with either 302 or 303 staining, suggesting that they remained in the tissue. Golgi-Bergmann cells and astrocytes were identified using antibodies for glial fibrillary acidic protein. Isolated glial cells were 12-15 microns in diameter, more numerous than Purkinje cells, and unstained with calbindin antibodies. With phase-contrast optics, glial cells appeared flatter than neuronal cell types and had acentric nuclei. These results demonstrate that specific cell types in developing rat cerebellum can be identified after acute isolation, which should facilitate analysis of their endogenous properties.

Spatial distribution of mammalian cells dictated by material surface chemistry.

Anisotropic cell culture surfaces patterned with amino and alkylsilanes can guide cell distribution and provide an approach to study important processes involved in tissue engineering, such as cell attachment and locomotion. By combining photolithographic and silane coupling techniques, glass coverslips were patterned with either n-octadecyldimethylchlorosilane (ODDMS) or dimethyldichlorosilane (DMS), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). The alkylsilanes, theoretically, have similar methyl and methylene groups exposed at the surface but different structures, with DMS being amorphous and ODDMS ordered. Neuroblastoma cells, osteosarcoma cells, and fibroblasts plated on surfaces patterned with EDS/ODDMS and EDS/DMS specifically localized on the EDS regions, but distributed randomly on ODDMS/DMS patterned surfaces. The preferential assembly of cells onto EDS regions did not depend on the structure of the adjacent alkylsilane regions and was a time-dependent process. Angle dependent x-ray photoelectron spectroscopy (XPS) and contact angle measurements indicated that EDS was immobilized on glass as a fractional hydrophilic monolayer, and ODDMS and DMS were bound as patchy amorphous hydrophobic multilayers. Neither surface coverage nor thickness of the overlayer seemed to be as important as surface chemistry, or charge, in guiding mammalian cell distribution. These results are consistent with the concept that mammalian cells attach to and are guided by positively charged surfaces.

High-voltage-activated calcium current in developing neurons is insensitive to nifedipine.

We have analyzed the effect of nifedipine on the macroscopic high-threshold, voltage-activated (HVA) calcium current in four cell types: postnatal rat Purkinje and dorsal root ganglion (DRG) neurons, embryonic chick DRG neurons, and adult cat ventricular myocytes. As is consistent with previous reports, nifedipine reduced HVA current in myocytes in a voltage-sensitive manner. Analysis of nifedipine actions on neurons, however, was compromised by slow inactivation of the current at holding potentials between -80 mV and -40 mV. The slow inactivation was voltage-dependent, irreversible after 5 min, and contributed to "rundown" of the current. At -40 mV, slow inactivation displayed two time constants: 12 +/- 8 s and 7 +/- 4 min. When slow inactivation was taken into account, we found no evidence for a nifedipine-sensitive component of the HVA current in these neurons. Consistent with previous studies, DRG neurons were reduced irreversibly by omega-conotoxin, whereas cardiac and Purkinje cells were unaffected. Our biophysical and pharmacological results are consistent with two types of neuronal HVA currents (N type and P type) in developing neurons that are distinct from cardiac HVA currents (L type).

A versatile technique for patterning biomolecules onto glass coverslips.

A fast, inexpensive, and versatile technique for patterning the surface of glass coverslips with molecules of biological interest is described. The technique combines photolithographic, silane-coupling, and protein adsorption procedures to pattern coverslips with amines, alkanes, and proteins with micrometer spatial resolution. The attachment of amines and alkanes was verified using contact angle and X-ray photoelectron spectroscopic (XPS) measurements. XPS results showed that amines and alkanes were attached in 1-4 nm thickness covering approximately 20% and 45%, respectively, of the surface. Patterns of amines were visualized using fluorescent staining, and patterns of proteins were detected immunochemically. Patterned coverslips were used to investigate adhesion and neurite outgrowth of mouse neuroblastoma (N1E-115) cells. Cells were examined on the following patterns: alkane-glass, protein-glass, amine-alkane, and amine-protein. Cell attachment and neurite outgrowth on patterned coverslips displayed the following preferences: laminin, fibronectin, or collagen IV > amine or glass > alkane or bovine serum albumin. This patterning method should be useful for studies of cell-surface interactions, cell migration, nerve regeneration, and the formation of neural networks in vitro.

Pathfinding by neuroblastoma cells in culture is directed by preferential adhesion to positively charged surfaces.

Pathfinding is a fundamental behavior of migrating neuroblasts and advancing growth cones. We have analyzed this behavior in culture using mouse neuroblastoma (N1E-115) cells grown on a chemically patterned surface. The patterned surface was defined photolithographically and consisted of intersection 10-micron-wide pathways. The pathways were coated with positively charged amines and separated by regions bound with uncharged alkanes. Cells and growth cones were guided along the pathways and made choices at intersections. Whereas migrating cells made random choices at intersections, growth cones displayed a preference for advancing straight ahead. Interference reflection microscopy (IRM) revealed that pathfinding by cells and growth cones was correlated with greater overall attachment to aminated regions, although cell bodies and appendages also attached to adjacent alkanated regions. Thus guidance was not simply due to contact inhibition by alkanes; rather, it was due to "preferential" adhesion to aminated surfaces. Gray level analysis of IRM images demonstrated that focal and close contacts were made on both surfaces, indicating that preferential adhesion was not the result of tighter attachment to aminated surfaces. Fluorescent labeling of F-actin and microtubules indicated that preferential adhesion was not due to compartmentalization of these cytoskeletal structures on aminated regions. We propose that preferential adhesion involved a signal transduction mechanism that discriminated between positively charged and uncharged molecules. Such a mechanism could contribute to pathfinding by neuroblasts and growth cones along extracellular matrix proteins in vivo.

Divalent ions released from stainless steel hypodermic needles reduce neuronal calcium currents.

We have investigated the effects of saline solutions exposed to stainless steel hypodermic needles on their ability to reduce voltage-dependent calcium currents in chick dorsal root ganglion neurons and rat cerebellar Purkinje cells. Salines exposed to needles with brass hubs, but not those with plastic hubs, for as little as 2-3 sec reduced calcium currents in both cell types. The amplitude of the response was exposure-dependent and reversible. Elemental analysis of the exposed salines using inductively-coupled-plasma atomic emission spectroscopy revealed that Cu and Zn (but not Cd, Cr, Co, Fe, Mn, Ni or Pb) were released from the brazed needles. The amount of Cu plus Zn released in 30 sec was estimated to be 12-26 microM, depending upon the specific needle examined. Longer exposures produced proportionately higher concentrations of the metals. Dose-response curves for Cu or Zn ions applied directly to cells confirmed that similar concentrations of these ions reduced neuronal calcium currents. Our results indicate that divalent ions released from stainless steel hypodermic needles with brass hubs can interfere with measurements of calcium currents. In addition the results contribute new information regarding potential physiological and pathological actions of copper and zinc ions in biological tissues.

Transferrin can alter physiological properties of retinal neurons.

The role of transferrin as a possible neurotransmitter was examined in cultured chick retinal cells. Brief exposure to transferrin caused a dramatic and transient increase in intracellular calcium levels in approximately 20% of the total population of cultured retinal neurons. The increase in intracellular calcium was observed in cell bodies and neuronal processes. Electrophysiological analysis of a subset of the population, bipolar-like neurons, demonstrated that more than half of these cells responded to the application of transferrin with a transient membrane depolarization. Under voltage clamp conditions, the currents evoked by transferrin were similar to glutamate in that they both displayed non-linear voltage dependence. Furthermore, acute transferrin exposure resulted in a 200% increase in the amount of Na+ independent [3H]glutamate binding observed in these cultures. These results suggest that transferrin may function as a neurotransmitter or neuromodulator in the developing vertebrate nervous system.

Fura-2 measurements of cultured rat Purkinje neurons show dendritic localization of Ca2+ influx.

The specific objectives of this study were the following: (1) to characterize the types of calcium currents in cultured PCs using whole-cell voltage-clamp techniques; (2) using fura-2 imaging techniques, to monitor intracellular Ca2+ levels during application of high potassium, glutamate, or glutamate analogs; and (3) to evaluate the types of calcium channels contributing to the calcium fluxes using pharmacological blocking agents. Voltage-clamp analysis of calcium currents proved to be difficult due to space-clamping problems. The latter was presumably due to the unfavorable geometry of cultured PCs. Nevertheless, we found no evidence for inward currents in cells bathed in TTX-TEA-BaCl2 saline. On the other hand, fura-2 measurements demonstrated that free Ca2+ levels were elevated in PCs following local application of either high-potassium saline or glutamate. When individual cells were injected with fura-2 and analyzed in TTX-containing saline, the Ca2+ elevation was usually greater in the dendrites. Since Ca2+ levels were not elevated in all dendrites of the same cell, the smaller responses in the soma wre not simply due to volumetric differences. Together with the voltage-clamp results, the fura-2 data indicate that calcium channels were localized to certain dendrites. Using selective calcium channel blockers, we found evidence for 2 types of calcium conductances in the dendrites of cultured PCs. The Ca conductance induced by high potassium was reduced in a dose-dependent manner by nifedipine (ED50 = 5 X 10(-7) M), indicating that a high-threshold voltage-dependent calcium channel was present. The Ca response to glutamate (or NMDA) was reduced by 2-amino-5-phosphonovaleric acid (ED50 = 10(-4) M), as well as by nifedipine or 10(-4) M LaCl3, indicating that both voltage-dependent and glutamate-coupled channels were opened by glutamate application.

Development of rat cerebellar Purkinje cells: electrophysiological properties following acute isolation and in long-term culture.

The objectives of this study were 2-fold: (1) to characterize the electrical properties of Purkinje cells (PCs) acutely isolated from rat cerebella at different stages of development, and (2) to compare these properties with those recorded from PCs grown in long-term culture. PCs under both conditions were identified with the aid of cell-specific immunocytochemical staining, and the electrical properties were analyzed using whole-cell-recording techniques. PCs acutely isolated during late embryonic and early postnatal periods displayed a progressive change in electrical properties. Between embryonic days 20 and 22 (stage 1), PCs were inexcitable, did not respond to glutamate, and displayed only small outward currents under voltage clamp. During postnatal days 1-4 (stage 2), current stimulation elicited nonovershooting action potentials, and small inward and outward currents were evoked under voltage clamp. Glutamate application depolarized the cells resulting in an increase in intracellular free calcium measured with fura-2. Stage 3 and 4 cells spanned postnatal days 5-9 and 10-14, respectively, and the cells showed progressively larger voltage-dependent conductances and greater sensitivity to glutamate. We found no evidence for either spontaneous or complex spikes in PCs isolated at any of these stages. In agreement with previous studies, we found that PCs dissociated from postnatal rats did not survive well in culture. On the other hand, PCs from embryonic rats cultured for 2-3 weeks in high-potassium, serum-supplemented medium developed extensive dendritic processes and excitability. Current stimulation or glutamate application elicited depolarizing waveforms reminiscent of climbing fiber-evoked responses in vivo. The results suggest that dendritic processes are important in the generation of complex spikes and that PC excitability can develop in the absence of the highly structured architecture of the intact cerebellum.

A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation.

Diacylglycerol analogues (for example 1,2-oleoylacetylglycerol, OAG) and phorbol esters are activators of protein kinase C, and have been widely used to study the function of this enzyme in both intact cells and cell-free preparations. Electrophysiological studies have shown that these activators can either depress or increase Ca2+ currents, or decrease K+ currents when applied outside the cell. It has been assumed that these effects are mediated by protein kinase C activation. Here we report that micromolar levels of OAG and phorbol esters depress Ca2+ currents in chick sensory neurons independently of their effect as activators of protein kinase C. The depression of the Ca2+ current is rapid and is unaffected by intracellular application of the protein kinase C inhibitors staurosporin, sphingosine and H-7. Furthermore, the activators were ineffective when applied intracellularly, indicating that their site of action is on the outside of the membrane.

Controlled outgrowth of dissociated neurons on patterned substrates.

The cytoarchitecture of nervous tissue is lost during the dissociation procedures used to form primary cell cultures. As a first step toward reestablishing an ordered arrangement of these cells in vitro, we developed a set of procedures for patterning the outgrowth of cells cultured on 2-dimensional substrates. These procedures used a combination of surface chemistry and photolithographic techniques. The adhesive properties of either silicon or silicon dioxide (quartz) surfaces were controlled by covalently binding small organic molecules to the surface with silane coupling agents. The attachment and growth of either embryonic mouse spinal cells or perinatal rat cerebellar cells were found to be promoted by binding certain amine derivatives to the surface. In particular, cells grown on surfaces bound with diamines and triamines, but not with monoamines, formed cultures whose morphology was similar to that of cells cultured on conventional substrates, i.e., glass coated with poly(D-lysine). The attachment of cells to a substrate was inhibited by binding alkane chains (e.g., n-tetradecane) to the surface and plating the cells in media containing 5-10% (vol/vol) serum. Patterns of selected adhesivity were formed using photochemical resist materials and lithographic masking techniques compatible with the silane chemistry. Cultures of either spinal cord cells or cerebellar cells could be confined to square regions on the scale of 50 micron. Cerebellar cells could be confined to grow on lines with widths less than 10 micron. This width is comparable to the diameter of granule cell somata. The patterned growth of cerebellar cells was maintained up to 12 d in vitro. Over this time period the granule cells were observed to develop electrical excitability and immunoreactivity for neuron-specific enolase. Purkinje neurons also developed electrical excitability when grown on the chemically modified surfaces. Immunochemical reactivity of the patterned cultures for glial fibrillary acid protein (GFAP) showed that glia are patterned along with the associated granule cells. Interestingly, the GFAP-positive glia that proliferated on surfaces bound with amine derivatives attained primarily a tile-shaped, fibroblast-like morphology, while those proliferating on glass coated with poly(D-lysine) developed primarily a spindle-shaped, process-bearing morphology. Granule cells preferentially associated with the spindle-shaped glia.