Biomedical subjects

E A Neale

Publications and source records attributed to E A Neale.

At least 19 recordsLinked to original sources

PubMed1994

Bafilomycin A1 inhibits the action of tetanus toxin in spinal cord neurons in cell culture.

Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release. However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively. In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT. To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium-evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA). baf A1, which inhibits the vacuolar-type H(+)-ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose-dependent manner. TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 nM baf A1, respectively, compared with cultures treated with the inhibitor alone. The effects of baf A1 are fully reversible. In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action. These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1-sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.

Animals↗

PubMed1992

Differential effects of tetanus toxin on inhibitory and excitatory neurotransmitter release from mammalian spinal cord cells in culture.

The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.

Animals↗

PubMed1990

Cholinergic function in cultures of mouse spinal cord neurons.

Cholinergic synapses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetyltransferase (ChAT). Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recordings showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparations indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co-cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non-neuronal cells from cerebral cortex, than in co-cultures with VH alone or with SC and dorsal root ganglion cells. Consistency between cholinergic physiology and staining for ChAT-positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG. Application of acetylcholine, muscarine, and/or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synaptic interactions between SC neurons in only one-third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures. The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.

Acetylcholine↗

PubMed1990

Mechanisms involved in activity-dependent synapse formation in mammalian central nervous system cell cultures.

Differences in neuronal activity produced by electrical stimulation lead to competition between synapses from sensory afferents converging on a common spinal cord neuron. Studies were performed on neurons dissociated from the mouse spinal cord and grown in culture dishes with three compartments. Synaptic efficacy from stimulated afferents was increased compared with unstimulated convergents, and the number of functional connections was increased by stimulation compared with control cultures. Blocking NMDA channel activation with 100 microM APV in medium containing 1.8 mM calcium inhibited this synaptic plasticity, but plasticity was not blocked by APV in medium in which the calcium concentration was elevated to 3 mM. These experiments support the view that electrical activity differentially influences processes that cause a persistent decrease in synaptic efficacy or lead to synapse elimination and those that increase synaptic strength or lead to synapse augmentation. We interpret our results in terms of a model in which these antagonistic mechanisms are both regulated via changes in calcium levels and second messengers that are modulated by electrical activity. A significant portion of the activity-related calcium influx relevant to synaptic plasticity passes through the NMDA channel, but other sources of calcium are involved. In particular, competitive elimination of synapses appears to occur during blockade of NMDA channels if the extracellular concentration of calcium is elevated moderately. The outcome of competition between the two calcium-dependent but antagonistic processes may depend either on their differential sensitivity to intracellular calcium concentration or separate specificities to NMDA and non-NMDA receptor-linked mechanisms.

Action Potentials↗

PubMed1990

Cloning and expression of functional fragment C of tetanus toxin.

A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the vector fused to the C-terminal 460 amino acids of tetanus toxin. This protein (rFragment C) was recognized by an antipeptide antibody specific for fragment C in an enzyme-linked immunosorbent assay and on immunoblots. rFragment C could be purified significantly in one step by immunoaffinity chromatography. Immunization of mice with rFragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. rFragment C bound to ganglioside GT1b and to neuronal cells in a manner indistinguishable from that of fragment C obtained by papain cleavage of tetanus toxin. For many applications, rFragment C appears to be a suitable alternative to tetanus toxin or toxin-derived fragment C.

Animals↗

PubMed1990

Effects of patterned electrical activity on neurite outgrowth from mouse sensory neurons.

A noninvasive method of electric stimulation was used in cell culture preparations to determine the effects of patterned electrical activity on the morphology and motility of mammalian central nervous system growth cones. Neurites from dorsal root ganglion (DRG) neurons of fetal mice were allowed to grow under the barrier of an insert placed in culture dishes. The insert confined the cell bodies within separate experimental and control compartments, and provided a means of exciting action potentials in the growing neurites by extracellular current pulses delivered across the barrier. A phasic pattern of stimulation caused immediate retraction of the filopodia and lamellipodium. Further outgrowth was halted and in many cases retraction of the neurite ensued. No changes in morphology or growth cone motility were evoked by electric stimulation when action potentials were blocked with 1 microM tetrodotoxin (TTX). These effects depended on the rate, pattern, and duration of stimulation. Phasic stimulation was more effective than stimulation with the same number of impulses delivered at a constant frequency. An important new observation was that cultures exposed to phasic stimulation for several hours contained actively growing neurites with normal growth cones which were insensitive to the stimulus. This apparent accommodation in neurites exposed to chronic stimulation may involve processes that regulate calcium conductance or buffering. Cessation of neurite outgrowth by action potentials could represent one mechanism linking morphological and functional characteristics in the developing CNS of mammals, by stabilizing the outgrowth of neurites forming appropriate synaptic contacts and leading to the retraction of growth cones from collaterals that have not formed appropriate contacts at the time the neuron enters into a functionally active circuit.

Action Potentials↗

PubMed1989

Synaptic connections in vitro: modulation of number and efficacy by electrical activity.

The functional architecture of synaptic circuits is determined to a crucial degree by the patterns of electrical activity that occur during development. Studies with an in vitro preparation of mammalian sensory neurons projecting to ventral spinal cord neurons slow that electrical activity induces competitive processes that regulate synaptic efficacy so as to favor activated pathways over inactive convergent pathways. At the same time, electrical activity initiates noncompetitive processes that increase the number of axonal connections between these sensory and spinal cord neurons.

Action Potentials↗

PubMed1989

A fractal analysis of cell images.

Methods of digital image analysis have been adapted to measure the fractal dimension of cellular profiles. The fractal dimension is suggested as a useful measure of the complexity of a contour. Three methods produce similar results when applied to constructed, near-ideal fractal figures. Comparison of the measurements for a variety of image types indicates the measurement accuracy in each case and may help in interpreting the results when applied to real, non-ideal cell images of unknown fractal dimension. Two of the methods are currently adopted as appropriate for use on neuronal contours. A correlation exists between the complexity of these contours and the magnitude of the estimated fractal dimension.

Animals↗

PubMed1988

Edge detection in images using Marr-Hildreth filtering techniques.

Details of the morphology of light microscope images of horseradish peroxidase labeled mammalian neurons in cell culture were investigated. A modified Marr-Hildreth edge-detecting algorithm was used in an image processor to obtain a continuous border of the labeled neurons. The interior of the border was filled to obtain isolated binary silhouettes of the neurons. These silhouettes can be used for further quantitative studies.

Animals↗

PubMed1987

Morphological and biochemical differences expressed in separate dissociated cell cultures of dorsal and ventral halves of the mouse spinal cord.

The neuronal properties of separate dissociated cell cultures of dorsal and ventral halves of the embryonic mouse spinal cord (E 13.5) were investigated. Ventral-half cultures grew on a variety of substrates and in a variety of media; dorsal-half cultures required a non-neuronal feeder layer and supplemented medium for survival. The two types of cultures differed in their morphological and biochemical properties. Ventral-half neurons remained well separated on the culture plate, whereas dorsal-half neurons tended to aggregate. Lucifer yellow fills showed that ventral-half neurons were substantially larger and had more processes than dorsal-half neurons. Because of the large size and good separation of the neurons, ventral-half cultures provide an especially attractive system for electrophysiologic and morphologic studies. Ventral-half cultures were highly enriched for choline acetyltransferase (ChAT) activity and had more neurons that stained for intracellular acetylcholinesterase (AChE); dorsal-half cultures were enriched for glutamic acid decarboxylase (GAD) activity, and high-affinity gamma-aminobutyric acid (GABA) uptake. The clear differences between the two cultures indicate that many morphological and biochemical properties are already specified on embryonic day 13.5.

Animals↗

PubMed1987

Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide.

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.

Animals↗

PubMed1986

Tetanus toxin in dissociated spinal cord cultures: long-term characterization of form and action.

The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin with the degradation of cell-associated toxin in primary cultures of mouse spinal cord neurons. From these studies we can conclude that the toxin has a half-life of 5-6 days. Both the heavy and the light chains of tetanus toxin degrade at similar rates. Labeled toxin, visualized by radioautography, is associated with neuronal cell bodies and neurites, and its distribution is not altered during a 1-week period following toxin exposure. Blockade of synaptic activity persists for weeks at the concentration of radiolabeled toxin used in these studies. This blockade of transmission is reversed as the toxin is degraded, suggesting that degradation of toxin may be a sufficient mechanism for recovery from tetanus.

Action Potentials↗

PubMed1986

Autoradiographic localization of benzodiazepine receptor binding in dissociated cultures of fetal mouse cerebral cortex.

Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.

Affinity Labels↗

PubMed1986

Active and inactive central synapses in cell culture.

Synaptic interactions between pairs of spinal cord (SC) neurons and between dorsal root ganglion neurons and SC neurons were studied in dissociated cell cultures prepared from fetal mouse. Combined injection of horseradish peroxidase into presynaptic neurons and Lucifer yellow into postsynaptic neurons allowed detailed correlation of morphological-physiological analyses of synaptically linked cells. Statistical analysis of trains of evoked EPSPs under conditions of high and of low transmitter output was used to determine the number of physiological release elements, n, involved in a given synaptic connection. When n was compared with the number of boutons subserving a synaptic connection, it was found that in 80% of cases the number of boutons was equal to or greater than the number of release elements. In some cases, the bouton count was more than fivefold greater than n. The simplest explanation is that, in general, one bouton can release no more than one quantum of transmitter and, in a significant proportion of synaptic connections, a large fraction of boutons do not participate in the release process. Theoretical consideration and analysis of the electrotonic structure of some of the neurons studied indicate that the dendritic location of synaptic inputs does not affect our results. Variations in the probability of release, p, may contribute to the apparent disparity between n and bouton number. If so, this variation must be large with many boutons having a very low p, difficult to distinguish experimentally from zero.

Animals↗

PubMed1984

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons.

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.

Animals↗

PubMed1983

Toxic effect of phenytoin on developing cortical neurons in culture.

Studies were undertaken to determine the effect of chronic phenytoin exposure on developing neurons. Cerebral cortex from 16-day fetal mice was utilized to prepare primary dissociated cell cultures. Phenytoin was added to the cultures 10 days after plating and the cultures were harvested on day 17. Cortical cultures were assayed for neuronal cell number by phase microscopy and for high-affinity uptake of 3H-labeled gamma-aminobutyric acid (GABA) by both radioautography and scintillation spectrometry. Neuronal cell counts demonstrated a highly significant decrement in the number of neurons in cultures exposed to phenytoin at 15, 25, and 50 micrograms/ml. 3H-GABA-labeled neurons constituted 13% of the neurons present in both control and phenytoin-exposed cultures. These data indicate that phenytoin is toxic to cortical neurons in culture and that GABAergic neurons are affected to the same extent as the total neuronal population.

Animals↗

PubMed1983

Developmental and neurochemical specificity of neuronal deficits produced by electrical impulse blockade in dissociated spinal cord cultures.

Blockade of spontaneous electrical activity in dissociated fetal spinal cord cultures produced neuronal deficits as measured by biochemical and morphological techniques. Spinal cord cultures exhibited an age-dependent vulnerability to impulse blockade with tetrodotoxin (TTX) or xylocaine. Neuronal cell counts, [125I]tetanus toxin fixation and [125I]scorpion toxin binding indicated that TTX application produced neuronal deficits during the second or third week in culture. Application of TTX during the first or fourth week did not produce a difference in tetanus toxin fixation from controls. Radioautography of [125I]tetanus toxin revealed no obvious change in the label distribution after TTX treatment. Suppression of electrical activity during the first 6 days in culture had no effect on choline acetyltransferase (CAT) activity and no apparent effect on the appearance of the cultures. Application of TTX during the seventh day in culture decreased CAT activity to 68% of control. Chronic electrical blockade produced a progressively greater loss of CAT activity through 21 days in culture. GABAergic neurons, as indicated by high-affinity GABA uptake, glutamic acid decarboxylase activity and [3H]GABA radioautography, were not affected by electrical blockade. These data indicate that there is developmental and neurochemical specificity in the neuronal death produced by blocking spontaneous electrical activity in dissociated spinal cord cultures.

Animals↗

PubMed1983

Synaptic interactions between mammalian central neurons in cell culture. I. Reversal potential for excitatory postsynaptic potentials.

Intracellular recording and stimulation techniques were used to study the electrical properties of neurons in cell cultures from fetal mouse spinal cord (SC). The morphology of SC neurons and the distribution on SC neurons of boutons formed by synaptically connected SC or dorsal root ganglion (DRG) neurons were demonstrated with horseradish peroxidase (HRP) injection. Postsynaptic polarization in conjunction with synaptic activation of SC neurons was used to determine the reversal potential for excitatory postsynaptic potentials (EPSPs). Tetraethylammonium ions were injected postsynaptically in order to obtain reversal of the EPSPs. Both SC-SC and DRG-SC excitatory connections could be reversed by postsynaptic depolarization. The average reversal potential for the SC-SC EPSP was -4 +/- 12.2 (SD) mV and that for the DRG-SC EPSP was +8 +/- 7.9 (SD) mV, a statistically significant difference (Wilcoxon two-sample rank; P less than 0.05). Scatter was quite large, particularly for the SC-SC connection. While some neurons gave clear electrophysiological evidence of significant dendritic effects, the average total electrotonic length was small (0.58 +/- 0.65 (SD) of a length constant). The morphological extent of the dendrites of SC neurons was substantially less than that of mature motoneurons in vivo. We concluded that both SC-SC and DRG-SC EPSPs were mediated by a conventional conductance increase and that most synaptic input was not far removed electrically from the recording site in the neuron cell body.

Animals↗