Biomedical subjects

E A Neale

Publications and source records attributed to E A Neale.

At least 37 records · Page 2Linked to original sources

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.

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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.

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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.

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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.

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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.

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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.

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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.

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PubMed1983

Synaptic interactions between mammalian central neurons in cell culture. II. Quantal Analysis of EPSPs.

The presynaptic release mechanism involved in excitatory synaptic connections between neurons in cell cultures of fetal mouse spinal cord were studied by statistical analysis of intracellularly recorded postsynaptic responses. Quantal parameters were determined for the EPSPs evoked in spinal cord (SC) neurons by stimulation of either other SC or dorsal root ganglion (DRG) neurons. Transmitter release was manipulated by varying the Ca2+ and Mg2+ content of the culture medium. The release process was represented better by binomial than by Poisson statistics. A method was derived for obtaining the probability of release and the number of release elements. The quantal content and the number of release elements were substantially higher for the SC-SC connection than for the DRG-SC connection. This was partially compensated for by a larger quantal amplitude for the DRG-SC connection. There was some indication that the probability of release was higher for the SC-SC connection. The relationship between transmitter output and effective external Ca2+ ion concentration was approximately linear.

Animals↗

PubMed1983

Synaptic interactions between mammalian central neurons in cell culture. III. Morphophysiological correlates of quantal synaptic transmission.

The statistical properties of excitatory synaptic transmission between neurons in cell cultures of fetal mouse spinal cord were studied and the anatomical extent of these connections demonstrated by horseradish peroxidase (HRP) injection of the presynaptic cell. In conjunction with previous experiments (7), we have correlated the number of boutons involved in a given synaptic connection with the physiologically determined number of release elements. The number of boutons is somewhat greater than the number of release elements, and our results are supportive of the conclusion of others (1, 2) that the physiologically defined release element corresponds to the bouton. We interpret this to suggest that a rate-limiting mechanism may operate at the level of the bouton to allow release of no more than one quantal unit of transmitter with each presynaptic action potential.

Animals↗

PubMed1983

Glutamate decarboxylase immunoreactivity and gamma-[3H] aminobutyric acid accumulation within the same neurons in dissociated cell cultures of cerebral cortex.

In order to evaluate the reliability of high affinity [3H]GABA accumulation as a marker for GABAergic neurons, murine cerebral cortical neurons were studied in dissociated cell culture. Cultures which had been incubated in [3H]GABA were stained immunohistochemically for the GABA-synthesizing enzyme, glutamate decarboxylase, fixed with paraformaldehyde, and subsequently processed for radioautography. In mature cultures, there was an 84 to 94% correlation between the presence of the enzyme and [3H]GABA uptake within the same cortical neurons. These data provide direct evidence that those neurons which synthesize GABA are the same neurons which are labeled by high affinity [3H]GABA uptake.

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PubMed1982

A method for large-scale production of mouse brain cortical cultures.

Fetal cerebral cortex can be grown for several weeks in dissociated cell culture using a relatively simple protocol for culture preparation. It is possible to establish large numbers of very similar cultures which serve as an effective test system for studies of toxicity and mechanism of action of neuroactive compounds. Light microscopic and ultrastructural studies document the neuronal component of the cultures as well as the developmental sequence. Cell counts, protein concentration and choline acetyltransferase activity demonstrate the reproducibility of the cultures from dish to dish.

Animals↗

PubMed1982

Kainic acid sensitivity of mammalian Purkinje cells in monolayer cultures.

Purkinje cells from fetal mouse cerebellum were studied in a monolayer culture system. These neurons, labeled by intracellular iontophoresis of HRP, showed one or more thick, spine-covered dendrites and an axon whose swellings contacted other neuronal processes and Purkinje cell somata. In short-term experiments, kainic acid was cytotoxic to Purkinje neurons and not to granule cells in monolayer cultures of fetal rat cerebellum.

Animals↗

PubMed1982

Cerebellar macroneurons in microexplant cell culture. Methodology, basic electrophysiology, and morphology after horseradish peroxidase injection.

Cerebellar macroneurons, including Purkinje cells, survive and differentiate in long-term monolayer cultures, which are prepared by a partial dissociation procedure we refer to as a microexplant technique. Intracellular recording demonstrated that these neurons were functional, showing spontaneous spiking activity and electrical excitability, and both spontaneous and evoked synaptic activity. In order to further characterize cell types, light and electron microscopic studies were performed after intracellular iontophoresis of horseradish peroxidase. Purkinje neurons were identified by their form of dendritic arborization and numerous dendritic spines. Cortical granule cells and macroneurons derived from the deep nuclei could also be demonstrated.

Animals↗

PubMed1982

Cerebellar macroneurons in microexplant cell culture. Postsynaptic amino acid pharmacology.

Cerebellar neurons derived from 17- to 19-day-old fetal rats have been grown in a monolayer in microexplant cell culture, and intracellular recording coupled with iontophoresis of amino acid neurotransmitters has been employed to characterize their amino acid chemosensitivity. Although these cultures contain at least 3 different neuronal cell types, intracellular recordings were obtained from large neurons (diameter greater than 15 microns) with 1-5 dendritic shafts and fine dendritic arborizations and which could, on morphological grounds, be identified as Purkinje cells. All neurons with resting membrane potentials greater than 25 mV and with action potentials evoked by intracellular stimulation, responded to iontophoretically applied glutamate and GABA. There was essentially no chemosensitivity to glycine, beta-alanine or taurine. Aspartate application evoked only small responses at high iontophoretic currents. GABA reversibly increased membrane conductance and produced hyperpolarization at resting membrane potential with reversal potentials between -50 and -40 mV (5-10 mV more negative than resting membrane potential). Glutamate reversibly increased membrane conductance and produced depolarizing responses with extrapolated reversal potentials between 0 and -10 mV. Aspartate augmented glutamate responses at low iontophoretic currents which did not directly alter membrane potential or conductance. Thus Purkinje cells grown in the absence of parallel fiber and climbing fiber input develop autonomous neuropharmacologic specificity similar to that of Purkinje cells in vivo.

Amino Acids↗

PubMed1982

Cerebellar macroneurons in microexplant cell culture: ultrastructural morphology.

Microexplant cell cultures of fetal rat cerebellum contain essentially monolayer networks of Purkinje cells, occasional granule cells and neurons from the deep nuclei. The neurons and occasional filament-packed glial cells develop on top of a sheet of flattened, non-neuronal cells. In the absence of extrinsic input to the cerebellum and greatly reduced numbers of granule cells, the Purkinje cells develop a stunted and non-oriented dendritic arbor similar to that observed in agranular cerebella. The Purkinje cell dendritic branches, however, are spine-covered. Although the spines are not enveloped by glia and are only rarely contacted by a presynaptic bouton, most spines display a patch of electron-dense material resembling a postsynaptic membrane specialization. The Purkinje cells develop synaptic interactions among themselves and with granule cells. The ultrastructural morphology of boutons derived from both Purkinje cells and large neurons of the deep nuclei, identified after intracellular injection of horseradish peroxidase, is consistent with that observed in vivo. The present study indicates that cerebellar Purkinje cells survive and differentiate in a culture system in which individual neurons are accessible for electrophysiological and morphological analyses.

Animals↗

PubMed1982

Substance P-like immunoreactivity in neurons in dissociated cell cultures of mammalian spinal cord and dorsal root ganglia.

Dissociated cell cultures prepared from fetal mouse spinal cords and dorsal root ganglia were stained for endogenous substance P using the peroxidase-antiperoxidase technique. Substance P-like immunoreactivity was localized within a small percentage of rounded or multipolar neuronal somata and in varicose processes. The substance P-positive multipolar neurons were derived from spinal cord, while the small rounded neurons were possibly of spinal cord and/or sensory ganglion origin. Large dorsal root ganglion neurons were unreactive. These results are consistent with in vivo findings and indicate the feasibility of electrophysiologic studies in culture to analyze the synaptic connections between substance P neurons and their target cells.

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