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J N Barrett

Publications and source records attributed to J N Barrett.

At least 37 records · Page 2Linked to original sources
PubMed1991

Membrane resealing in cultured rat septal neurons after neurite transection: evidence for enhancement by Ca(2+)-triggered protease activity and cytoskeletal disassembly.

Neurites of cultured septal neurons were transected with a laser under sterile conditions, and the subsequent membrane resealing was assayed using a dye exclusion method. In agreement with findings in other preparations, Ca2+ enhanced resealing: in normal culture medium the percentage of lesioned neurons that resealed within 20-30 min after transection increased with increasing bath [Ca2+] over the range 10(-7) to 2 x 10(-3) M; about 75% of cells resealed in 2 mM Ca2+. Mn2+ and Sr2+ also enhanced resealing, but Mg2+ inhibited it. The percentage of resealing neurons was sensitive to agents known to alter the stability of cytoskeletal components. Agents that tend to disassemble microtubules and/or neurofilaments (e.g., colchicine, low-ionic-strength media) strongly promoted resealing, whereas treatments that tend to stabilize microtubules (taxol, Mg2+) inhibited resealing. Addition of exogenous proteases (papain, trypsin, or dispase) enhanced resealing, whereas inhibitors of cysteine proteases (including a specific inhibitor of calpain, a Ca-activated neutral protease) strongly inhibited resealing. Calmodulin inhibitors inhibited resealing, consistent with reports that calmodulin facilitates calpain-mediated proteolysis of fodrin, a component of the cortical cytoskeleton. Based on these results, we hypothesize that one of the major mechanisms involved in resealing is activation of endogenous proteases by Ca2+ entry into the injured neurite. The resulting changes in the cellular cytoskeleton might promote fusion and resealing of the cut ends of the plasma membrane by enhancing membrane mobility and/or by removing structures that normally prevent membrane-membrane contact.

Animals↗
PubMed1990

Transplantation of human fetal dopamine cells for Parkinson's disease. Results at 1 year.

In an effort to improve the clinical signs of Parkinson's disease, we have implanted mesencephalic dopamine cells from a 7-week human embryo into the caudate and putamen of a 52-year-old man with Parkinson's disease. Fetal tissue was obtained from elective abortion. The woman and the patient with Parkinson's disease were unknown to each other. The woman gave specific consent and was not paid. The patient had a 20-year history of parkinsonism treated with multiple drug therapies including levodopa/carbidopa (Sinemet) every 2 1/2 hours. His symptoms were worse on the left side. For 5 months prior to transplantation, the patient underwent clinical evaluations by both a neurologist and a computer system installed in his home for daily measurement of walking and hand movements. Preoperative positron emission tomographic scanning with 6-L[18F]fluorodopa (fluorodopa) demonstrated severe dopamine depletion bilaterally. Fetal tissue was matched to the patient for ABO blood antigens, and maternal serum was screened for hepatitis B and human immunodeficiency virus type 1 prior to surgery. Fetal tissue was implanted stereotactically throughout the caudate and putamen on the right side of the brain via 10 needle tracks. The patient was not immunosuppressed. Results 12 months after surgery showed 42% improvement in left-hand speed before the first morning dose of drug and 40% greater response to drug therapy. Right-hand speed increased 15% before drug therapy and 23% after drug therapy. Reaction time was unaffected. Walking speed increased 33% after drug administration, although walking speed before the first morning dose of drugs declined 40%. Walking speed on an all-day basis improved 17%.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗
PubMed1987

Characterization of the altered form of the c-src gene product in neuronal cells.

The pp60c-src protein that is expressed at high levels in cultures of neurons from rat embryos displays an altered mobility on SDS-polyacrylamide gels due to a structural difference in the amino-terminal region of the molecule. In this report we show that the expression of this unique form of pp60c-src, designated pp60c-src(+), is not restricted to cultured neuronal cells since the pp60c-src molecules expressed in tissues from avian and rat neural tissues also display a retarded electrophoretic mobility. The amino-terminal region from pp60c-src(+) was found to contain a novel phosphorylated tryptic peptide that contains phosphoserine. However, this phosphorylation does not appear to be responsible for the retarded electrophoretic mobility of pp60c-src(+), since the mobility of this protein is not altered by phosphatase treatment under conditions that remove greater than 95% of the radiolabeled phosphate on pp60c-src(+). The altered electrophoretic form of pp60c-src was also shown to be radiolabeled with [3H]myristate, indicating that pp60c-src is fatty-acylated in neurons, as is pp60c-src in fibroblasts. The pp60c-src molecules synthesized in vitro using rabbit reticulocyte lysates programmed with mRNA from embryonic brain migrated more slowly on SDS-polyacrylamide gels than the pp60c-src protein that was translated in vitro using RNA from embryonic limb tissue. These results suggest the possibility that the c-src mRNA expressed in neurons may undergo a unique form of processing to generate the structurally distinct form of neuronal pp60c-src(+).

Animals↗
PubMed1986

1-Methyl-4-phenylpyridinium (MPP+) but not 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively destroys dopaminergic neurons in cultures of dissociated rat mesencephalic neurons.

Dopaminergic neurons were studied in cultures of dissociated cells from the ventral mesencephalon of fetal rat embryos (gestational day E15-16). After a week of growth, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+) was added to the growth medium for 24 h. Dopaminergic neurons were then visualized with tyrosine hydroxylase (TH) immunocytochemistry or catecholamine (CA) cytofluorescence. Concentrations of MPTP in the range of 10 to 100 microM obliterated CA fluorescence without affecting the number of TH-positive neurons. At concentrations greater than 100 microM, MPTP decreased the number of TH-positive neurons as well as the number of all other cell types. MPP+ (0.1-10.0 microM) produced a decrease in the number of TH-positive neurons without decreasing the total number of all cell types. The findings indicate that MPP+ but not MPTP is able to selectively destroy rat dopaminergic neurons in our cultures. The selective toxicity of MPP+ for dopaminergic neurons was partially prevented by pretreatment and co-incubation with mazindol (a selective inhibitor of dopamine uptake) but not by desipramine or deprenil, in confirmation of the notion that MPP+ enters dopaminergic neurons by the specific uptake mechanism for dopamine.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1986

Cryopreservation of primary neurons for tissue culture.

We have developed new cryopreservation methods which allow storage of fetal rat central nervous system tissues for more than 1 week at 3-8 degrees C or for several months at -70 or -90 degrees C prior to tissue culture. For refrigeration, small brain regions (less than 2 mm thick) were placed intact into 35 mm petri dishes of 'hibernation medium' inside a humidified chamber. Optimal preservation was obtained with hibernation media of pH 6.8-7.4, containing 30-70 mM K+, 10-30 mM Na+, 5-50 mM PO4(2-), 20 mM lactic acid, 5 mM glucose, and less than 0.1 mM Ca2+. The media were made approximately isotonic by addition of sorbitol. For freezing, brain tissues were dissociated by gentle trituration (without enzymes) in the above medium supplemented with 5-10% dimethylsulfoxide. After refrigeration or freezing, neurons were very sensitive to damage from mechanical stress (e.g. centrifugation, harsh rinsing or trituration). Rapid changes in osmotic pressure or excessive polylysine on the tissue culture substratum also reduced neuronal survival after cryopreservation. Pretreatment of tissue culture substrata with media from lung cell cultures, or plating of neurons at higher density (2000 cells/mm2) improved neuronal survival after cryopreservation.

Animals↗
PubMed1986

Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells.

Using primary culture methods, we show that purified astrocytes from embryonic mouse or rat central nervous system (CNS) can be induced to produce interferon (IFN) activity when pretreated with a standard IFN-superinducing regimen of polyribonucleotide, cycloheximide, and actinomycin D, whereas IFN activity was not inducible in neuronal cultures derived from mouse CNS. Astrocyte IFN displays inductive, kinetic, physicochemical, and antigenic properties similar to those of IFN-alpha/beta, but is dissimilar to lymphocyte IFN (IFN-gamma). Treatment of pure astrocytic cultures or astrocytes cultured with neurons with astrocyte IFN or IFN-alpha/beta induced a dramatic increase in the expression of H-2 antigens on a subpopulation of astrocytes. Neither neurons nor oligodendroglia expressed detectable levels of H-2 antigens when exposed to astrocyte IFN, IFN-alpha/beta, or to IFN-beta. Injection of astrocyte IFN or IFN-alpha/beta directly into brains of newborn mice indicated that H-2 antigens were also induced in vivo. None of the IFNs (astrocyte, alpha/beta, or beta) tested induced Ia antigens on CNS cells in vitro or in vivo. Since H-2 antigens have a critical role in immune responses, astrocyte IFN may initiate and participate in immune reactions that contribute to immunoprotective and immunopathological responses in the CNS.

Animals↗
PubMed1986

Effects of ischemia-like conditions on cultured neurons: protection by low Na+, low Ca2+ solutions.

An in vitro system was used to mimic several aspects of ischemia, including low oxygen pressure, low nutrient levels, and the accumulation of cellular products thought to contribute to damage during ischemia. We replaced normal culture medium from 3-week-old basal ganglia cultures with oxygen-depleted, nutrient-deficient medium. After incubation in an atmosphere of 94% N2, 6% CO2 for 5 hr at 37 degrees C, the cultures were returned to normal medium. After a 24 hr recovery period, cell viability was assessed in terms of cell number, electrophysiological properties, and immunohistochemical markers. When the medium used during the ischemic period was a normal balanced salt solution, more than 70% of the cells were damaged by the low-oxygen, low-glucose stress. Loss of cell processes and cell swelling were the most evident signs of damage. The majority of the cells remaining viable were astrocytes. Neuronal damage was observed only when both glucose and oxygen were deficient. Some damage was evident even at oxygen tensions of 60 mm Hg when glucose was absent from the medium; much more extensive damage was observed at tensions below 1.0 mm Hg. Lowering both extracellular sodium and calcium resulted in more than a 2-fold increase in survival (70 vs 28%). These results indicate that damage to neurons during conditions of extreme energy deprivation such as ischemia may be mediated by the influx of calcium and/or sodium.

Animals↗
PubMed1985

Differential effects of calcium antagonists on viability of adult rat ventricular myocytes.

This study was designed to determine whether the various classes of Ca2+ channel blockers have differential protective effects on isolated adult rat ventricular myocytes exposed to high K+ under anoxic (100% N2) conditions. Calcium-tolerant myocytes were incubated under control (4mM K+) aerobic conditions and then subjected to high K+ (75 mM) and N2. The cells were assessed by morphological criteria (i.e. absence of blebbing, granulation etc.), maintenance of ATP levels, exclusion of trypan blue, and the presence or absence of spontaneous contractile activity. Under control conditions, the cells were quiescent and declined at a rate of approximately 10%/h. In the absence of O2, the rate of cell decline was significantly faster. Verapamil, diltiazem and the dihydropyridines had no significant effects on cell decline under these conditions. Cells exposed to 75 mM K0+ exhibited contractile activity and accelerated rate of decline under anoxic conditions; these effects were independent of lowering Na0+ to 75mM. Cells in high K0+ and N2 were significantly protected (i.e. contractile activity and rate of decline were decreased) by verapamil, less so by diltiazem, and not at all by the dihydropyridines. The uptake of 45Ca2+ into cells in high K0+ was not significantly altered by verapamil or diltiazem. Caffeine induced the immediate cessation of contractile activity of cells incubated in high K0+, but did not affect the accelerated rate of cell declined under anoxic conditions. Verapamil and diltiazem still conferred significant protection in this non-beating cell preparation. Neither verapamil nor diltiazem had any effect on the oscillation frequency of skinned heart cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1985

Characterization of tissue-derived macromolecules affecting transmitter synthesis in rat spinal cord neurons.

Rat spinal cord cells maintained in neuron-rich cultures were exposed to extracts of skeletal muscle or to medium conditioned by non-neuronal cells. The conditioned media enhanced neuronal acetylcholine (ACh) synthesis, choline acetyltransferase activity, and protein synthesis, and decreased gamma-aminobutyric acid (GABA) synthesis. Muscle extract prepared from newborn rats produced similar enhancements but did not depress GABA synthesis. Muscle extracts prepared from normal and denervated adult rat limbs contained relatively little activity. These results suggest that different molecular factors might mediate the effects on GABA and ACh synthesis. Gel filtration of conditioned media and muscle extracts revealed that all of these activities were confined to a macromolecular fraction with an apparent Mr of 40,000. These tissue-derived factors affecting neuronal protein and transmitter synthesis are in turn distinct from a neuronal survival-promoting factor obtained from serum (Kaufman, L. M., and J. N. Barrett (1983) Science 220: 1394-1396).

Acetylcholine↗
PubMed1984

Neurite growth cone-substratum adherence increases in vitro.

During experiments characterizing the turning response of dorsal root ganglion neurites toward NGF, it was observed that growth cone-substratum adherance increased with time in culture. The experiments reported here indicate that the observed increase in growth cone-substratum adherance is significant and can be detected with both collagen and poly-L-lysine substrates. The increased adherance is apparently due to a substance(s) produced and released by the ganglia which binds to the substrate, increasing adherance. Flow chamber studies indicate that the substrate-bound substance(s) may be necessary for neurite growth onto artificial tissue culture substrata.

Animals↗
PubMed1983

Serum factor supporting long-term survival of rat central neurons in culture.

Gel filtration of serum at pH 3.6 yielded a fraction that supported long-term (months) survival of dissociated rat central neurons in monolayer culture more reliably than the traditionally used unfractionated serum. The cultures remained neuron-rich, because this fraction did not support the proliferation of glia and fibroblasts that occurs in whole serum. With an apparent molecular weight of 55,000 and an isoelectric point of 5.6, the active factor (or factors) in this fraction is distinct from any well-defined growth factor.

Animals↗
PubMed1983

An investigation of the growth characteristics of oil-palm (Elaeis guineensis) suspension cultures using 31P NMR.

High-resolution 31P nuclear-magnetic-resonance (NMR) spectra are reported for oil-palm (Elaeis guineensis) cells in suspension culture. The spectra are a significant improvement on the results that have appeared for other cultures and they are comparable with the spectra of the meristematic tissue in seedling roots. The NMR technique was used in parallel with other analytical methods to investigate the growth characteristics of the suspension culture, including the effect of 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1982

Two components of conditioned medium increase neuritic outgrowth from rat spinal cord explants.

Conditioned medium (CM) from muscle or fibroblast cultures dramatically increases the outgrowth of neurites from fetal rat spinal cord slices in vitro. We report here that there are two separable fractions in conditioned medium that cause this increase in neuritic outgrowth. One fraction, with a molecular weight of approximately 50,000 daltons, enhances neuritic outgrowth only when it is present in the culture medium so that the slices are directly exposed to it. The second component has a much larger molecular weight (above 300,000 daltons), and can enhance neuritic outgrowth by directly binding to the culture substrate on pretreatment. Only when these two fractions are combined by pretreating the substrate with the higher molecular weight fraction and then growing the slices in the lower molecular weight fraction is the full outgrowth promoting activity of whole conditioned medium reconstituted. These two components act synergistically to reproduce nearly the full outgrowth promoting activity of non-fractionated, whole conditioned medium.

Animals↗
PubMed1982

Characterization of neuritic outgrowth-promoting activity of conditioned medium on spinal cord explants.

Conditioned medium (CM) from muscle or fibroblast cultures dramatically increases the outgrowth of neurites from fetal rat spinal cord slices in vitro. The factor(s) in CM responsible for this enhanced outgrowth is chymotrypsin-sensitive, but neuraminidase-insensitive. At neutral pH, the factor(s) binds to a concanavalin A-agarose affinity column, a zinc metal chelate affinity column, a DE-52 anion exchange column, but not to a carboxymethyl-52 cation exchange column. These results suggest that the active CM factor(s) is a glycoprotein that is negatively charged at neutral pH. Following gel chromatography the major peak of outgrowth-promoting activity elutes at a molecular weight of approximately 50,000 daltons.

Animals↗
PubMed1982

Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotential.

1. Electrophysiological techniques are described which allow intracellular recording from peripheral myelinated axons of lizards and frogs for up to several hours. The sciatic and intramuscular axons studied here have resting potentials of -60 to -80 mV and action potentials (evoked by stimulation of the proximal nerve trunk) of 50-90 mV. They show a prominent depolarizing afterpotential (d.a.p.), which is present both in isolated axons and in axons still attached to their peripheral terminals. This d.a.p. has a peak amplitude of 5-20 mV at the resting potential, and decays with a half-time of 20-100 msec.2. The peak amplitude of the d.a.p. is voltage-sensitive, increasing to up to 26 mV with membrane hyperpolarization. The d.a.p. disappears as the axon is depolarized to -60 to -45 mV, and does not appear to reverse with further depolarization.3. The d.a.p. is not reduced when bath Ca is replaced by 2-10 mm divalent Mn or Ni. The d.a.p. is not reversed when axons depleted of Cl (by prolonged exposure to Cl-deficient, SO(4)-enriched solutions) are bathed in Cl-rich solutions. These results suggest that the d.a.p. is not mediated by a conductance change specific for Ca or Cl ions. Partial substitution of tetramethylammonium for bath Na, or addition of 10(-5)m-tetrodotoxin to the normal bathing solution, reduces the amplitude of both the action potential and the d.a.p.4. The amplitude of the d.a.p. is not sensitive to bath [K] over the range 1-7.5 mm, provided that all measurements are made at the same holding potential. This result argues that the d.a.p. is not mediated by accumulation of K outside the active axon.5. Treatments expected to inhibit the Na-K exchange pump (cooling from 25 to 10 degrees C, or 0.15 mm-ouabain) do not enlarge or prolong the d.a.p., although they do abolish a slower hyperpolarizing afterpotential seen following repetitive stimulation.6. The passive voltage response of the axon to small injected pulses of depolarizing or hyperpolarizing current shows a prominent, slowly decaying component with a time course similar to that of the d.a.p. Depolarizing current reduces the input resistance of the axon, and increases the rate of decay of both the passive voltage response and the d.a.p. There is a slight conductance increase during the peak of the d.a.p., but the same conductance increase can be produced by a comparable passive depolarization.7. We conclude that the d.a.p. is due mainly to a passive capacitative current, probably resulting from discharge of the internodal axonal membrane capacitance through a resistive current pathway beneath or through the myelin sheath. We suggest that this slow capacitative discharge becomes evident as soon as most of the nodal ionic channels activated during the action potential have closed. An electrical model of the myelinated axon that incorporates the postulated internodal leakage pathway can account both for the prolonged d.a.p. recorded inside the axon, and for the potential profile recorded extra-axonally in or near the internodal periaxonal space.

Action Potentials↗
PubMed1982

Properties of single calcium-activated potassium channels in cultured rat muscle.

1. Properties of the Ca-activated K channel were studied in excised patches of surface membrane from cultured rat muscle cells using single channel recording techniques.2. Increasing the concentration of calcium at the intracellular membrane surface [Ca](i), increased both the frequency and effective duration of channel openings. Ca at the extracellular membrane surface was not sufficient to activate the channels.3. An approximate third power relationship (slope = 2.7) was observed between [Ca](i) and the percentage of time the channels spent in the open state.4. Both the frequency and effective duration of channel openings increased as the intracellular membrane surface was made more positive; the percentage of time spent in the open state increased e-fold for a 15 mV depolarization for low levels of activity.5. The percentage of time spent with 1, 2,...n channels open in membrane patches with n channels was described by the binomial distribution, suggesting that the channels opened and shut independently of one another.6. Single channel conductance (144 mM-K on both sides of the membrane) was essentially independent of membrane potential (-50 to +50 mV) and [Ca](i) (0.1 muM -1 mM), but did increase with temperature, from 100 pS at 1 degrees C to 300 pS at 37 degrees C.7. Channel activity occurred in apparent bursts, with the duration of the apparent bursts increasing with increasing [Ca](i).8. Two exponentials were required to describe the distribution of observed channel open times, suggesting two different open channel states of apparently normal conductance. The observed mean channel open time of these states at +30 mV was 0.34 and 2.2 msec with 0.1 muM-Ca(i) and was 0.47 and 6.9 msec with 0.5 muM-Ca(i).9. The channel occasionally entered an apparent third open channel state with a single channel current amplitude about 40% the amplitude of the normally observed single channel currents. The reduced conductance state was immediately preceded and followed by a normal conducting state.10. While the kinetics of the Ca-activated K channel appear complex, its large conductance and high Ca and voltage sensitivity suggest that it is uniquely suited to resist depolarizations of the cell membrane potential that are accompanied by increases in intracellular Ca.

Animals↗