Elaine A. Neale

Persistence of botulinum neurotoxin action in cultured spinal cord cells1,2

Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2–3 days, which converted approximately half of the SNAP‐25 to an altered form lacking the final nine C‐terminal residues. The distribution of toxin‐damaged to control SNAP‐25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/A‐truncated SNAP‐25 into a single population lacking the final 26 C‐terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP‐25 was monitored by Western blot analysis. The BoNT/E‐truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A‐truncated SNAP‐25. Return of BoNT/A‐truncated SNAP‐25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for >11 weeks, while recovery from BoNT/E is complete in <3 weeks. This longer duration of enzymatic activity appears to account for the persistence of serotype A action.

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.

Intracellular horseradish peroxidase injection for correlation of light and electron microscopic anatomy with synaptic physiology of cultured mouse spinal cord neurons.

Synaptic interactions between spinal cord neurons grown in dissociated cell culture were studied electrophysiologically, and presynaptic cells were subsequently injected by intracellular iontophoresis with horseradish peroxidase (HRP). Following histochemical processing, injected cells were filled with dense reaction product which facilitated the light and electron microscopic identification of the individual physiologically typed neurons. This technique applied to neurons in monolayer culture allowed the visualization of complex intercellular relationships in essentially two dimensions. The number and distribution of morphologically defined synaptic contacts was determined for correlation with individual evoked postsynaptic potentials. HRP-filling of inhibitory and excitatory neurons revealed differences with respect to cellular geometry, axonal projection, and the number, location and ultrastructure of synaptic contacts.

Differential Effects of Tetanus Toxin on Inhibitory and Excitatory Neurotransmitter Release from Mammalian Spinal Cord Cells in Culture

Abstract: 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 × 10−10M), 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.

Bafilomycin A1 Inhibits the Action of Tetanus Toxin in Spinal Cord Neurons in Cell Culture

Abstract: 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.

Syntaxin and 25-kDa synaptosomal-associated protein: Differential effects of botulinum neurotoxins C1 and A on neuronal survival

The Clostridium botulinum neurotoxins (BoNTs) A and C1 cleave specific proteins required for neuroexocytosis. We demonstrated that, in intact neurons, BoNT A cleaves 25‐kDa synaptosomal‐associated protein (SNAP‐25), and BoNT C1 cleaves both syntaxin and SNAP‐25 (Williamson et al.: Mol Biol Cell 6:61a, 1995; J Biol Chem 271:7694–7699, 1996). Here, we compare the actions of BoNT A and BoNT C1 on mature and developing mouse spinal cord neurons in cell culture and demonstrate that BoNT C1 is severely neurotoxic. In mature cultures, synaptic terminals become enlarged shortly after BoNT C1 exposure, and, subsequently, axons, dendrites, and cell bodies degenerate. Electron microscopy confirms that early degenerative changes occur in synaptic terminals when the somatic cytoplasm appears normal. In newly plated cultures, few neurons survive exposure to BoNT C1. Whereas both BoNT A and BoNT C1 cleave SNAP‐25, BoNT A has no adverse effect on neurite outgrowth, synaptogenesis, or neuron survival. This cytotoxicity is unique to BoNT C1, is specific to neurons, and is initiated at the synaptic terminal, suggesting either a novel role for syntaxin or additional actions of BoNT C1. The neurodegeneration induced by BoNT C1 may be significant in terms of its efficacy for the clinical treatment of dystonia and spasticity. J. Neurosci. Res. 52:569–583, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Evaluation of [3H]proline for radioautographic tracing of axonal projections in the teleost visual system

The efficacy of [3-H]proline radioautography for tracing retinal ganglion cell projections to the optic tectum of the jewel fish, Hemichromis bimaculatus, has been compared with that of degeneration techniques. There was good agreement between the various methods. Retinal projections to the optic tectum of two other teleosts, the oscar, Astronotus ocellatus, and the goldfish, Carrasius auratus, were examined radioautographically. In addition to conventional methods of analysis, radioautograms were scanned in a slit microdensitometer and by an automated isodensity scanning system. Results of studies with the protein synthesis inhibitor, cycloheximide, are compatible with the suggestion that axonally transported proteins labeled with [3-H]proline may release diffusible precursors that are reincorporated into protein in adjacent regions. The possible advantages and limitations of radioautography of [3-H]proline-labeled axonally transported protein in brief or extended studies are discussed in terms of the results obtained in the teleost visual system.

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.

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.

Chapter 5 The VAChT/ChAT “cholinergic gene locus”: new aspects of genetic and vesicular regulation of cholinergic function

Publisher Summary This chapter focuses on the structural features of vesicular acetylcholine transporter (VAChT) deduced from its primary sequence, which give insight into its transporter function, describes the distribution of VAChT in the rat and primate nervous systems and its intracellular targeting to synaptic vesicles in neuronal cells, presents the ontogeny of VAChT and choline acetyltransferase (ChAT) expression in an in vitro model for developing spinal cord motor neurons, and discusses the implications of the phylogenetic conservation of the VAChT/ChAT cholinergic gene locus for the developmental and evolutionary ontogenesis of the cholinergic nervous system. The vesicular acetylcholine transporter, together with ChAT, is a part of a gene locus that is nominally sufficient to confer cholinergic function on a neuronal cell. As such, the VAChT/ChAT gene locus can be looked upon as a cholinergic operon responsible for assembling a metabolic/sequestration pathway in cholinergic cells. The extracellular signals, which is ultimately responsible for the regulation of this operon, may include trophic factors and other neurotransmitters. Recent work suggests that adrenergic neurotransmission, by stimulating production of cholinergic differentiation factor(s) from presumptive cholinergic target tissue, is a decisive stimulus for the development of the cholinergic phenotype in the autonomic nervous system, and cholinergic neurotransmission may be necessary to stabilize the peripheral cholinergic phenotype.