Dennis G. Emery

Direct Modulation of the Secretory Machinery Underlies PKA-Dependent Synaptic Facilitation in Hippocampal Neurons

Activation of protein kinase A (PKA) is known to facilitate synaptic transmission. Using synapses established by hippocampal neurons in culture, we show that dialysis of PKA inhibitors in the presynaptic neuron blocks synaptic facilitation produced by the adenylyl cyclase activator forskolin, demonstrating a presynaptic locus of action. Using ruthenium red, a tool that is known to stimulate exocytosis independently of Ca2+ influx, but in a manner sensitive to tetanus toxin, we find that the secretory process is directly up-regulated under conditions where the number of functional terminals remains unchanged, as revealed by imaging of FM1-43, a vital indicator of synaptic vesicle endocytosis. Taken together with our ultrastructural analysis that suggests no enhancement of docking, our data indicate that PKA causes synaptic facilitation by directly elevating the probability of exocytosis of individual vesicles in response to an invariant Ca2+ signal.

The sequence of ultrastructural changes in cultured neurons after dendrite transection

SummaryCultured mouse spinal neurons were fixed at three different intervals after dendrite amputation: within the first 15 min, at 2 h and at 24 h. Dendrites were amputated at lesion distance of either 50 μm (31% probability of cell survival) or 100 μm (53% probability of cell survival) from the edge of their perikarya. When fixed within 15 min, operated neurons showed a two-phase gradient of ultrastructural damage which spread from the transection site towards the perikaryon. At 2 h after dendrite amputation all neurons operated close to their perikarya were categorized as either viable, moribund or dead, based on their appearance with phase contrast microscopy. These categories of response to physical trauma corresponded to distinctly different ultrastructural changes. Moribund neurons were filled with membrane-bound vesicles which were derived from swollen mitochondria and grossly dilated cisternae of the smooth endoplasmic reticulum. The cytoplasm of dead neurons contained large clear areas and many condensed, dark mitochondria. Both moribund and dead neurons lacked cytoskeletal elements. All of these ultrastructural changes are hypothesized to be the result of an increase in the intracellular concentrations of free calcium. Although evidence of residual mitochondrial swelling was present in some surviving neurons at 24 h, the ultrastructure of others was comparable to that of control cells. Some surviving neurons had terminal swellings at the ends of the severed neurites which were very similar to retraction balls of transected axons after CNS trauma.

Ultrastructural damage and neuritic beading in cold-stressed spinal neurons with comparisons to NMDA and A23187 toxicity

While exposure of cultured spinal neurons to mild hypothermia provides some protection from physical trauma (dendrotomy), profound cooling (< 17 degrees C) causes unrelated neuronal injury and death, which can be prevented by treatment with NMDA receptor antagonists. To investigate the mechanism of hypothermic neuronal injury we examined the ultrastructure of cultured spinal neurons after 2 h of cooling to 17 degrees C or 10 degrees C, with or without the presence of the NMDA receptor antagonist D-2-amino-5-phosphonovalerate, and with or without rewarming to 37 degrees C. These groups were compared to cultures exposed to NMDA or to the calcium ionophore A23187. Patterns of ultrastructural change, involving cytoskeletal disruption, mitochondrial abnormalities and vacuolization of the cytoplasm, suggest a common mechanism of injury in all treatment groups, involving an elevation of intracellular calcium. Some neurons exposed to hypothermia, NMDA or ionophore developed beaded dendrites. Microtubules were fragmented in varicosities but not in the intervening constrictions; other organelles were largely excluded from the constrictions. Varicosities may form when organelles and cytoplasm accumulate as the result of disruption of transport and membrane stabilizing proteins by proteases activated by calcium influx via NMDA mediated channels. The periodic nature of the swellings may reflect inherently discontinuous distribution of molecular subunits of the cytoskeleton.

The endogenous antioxidant glutathione as a factor in the survival of physically injured mammalian spinal cord neurons

Abstract. Glutathione is part of the system of cellular defenses against lipid peroxidation and other free radical-mediated damage. An established in vitro trauma model was utilized to evaluate whether glutathione is a factor in the survival of mammalian spinal cord neurons following physical injury. Cultured murine spinal neurons were subjected to a standard lesion: transection of a primary dendrite 100 u,m from the perikaryon. Prior reduction of glutathione with ethacrynic acid or buthionine sulfoximine caused a dose-dependent decrease in neuronal survival 24 hours after dendrotomy. Prior glutathione augmentation with -γ-glutamylcysteine or L-2-oxo-4-thiazolidine carboxylic acid significantly increased survival, but N-acetyl-cysteine was not protective. Gamma glutamylcysteine effected the most rapid increase in glutathione (peak at 10 min), and survival was 72% ± 10 when 0.2 mM γ-glutamylcysteine was added immediately after dendrotomy compared with 38% ± 4 in the control group (p < 0.0001). These results indicate that the level of glutathione is a factor in spinal cord neuron survival after physical trauma, and that glutathione augmentation may be an effective acute phase spinal cord injury (SCI) intervention strategy.

Contributions of sodium and chloride to ultrastructural damage after dendrotomy

SummaryTo determine the contributions of sodium and chloride to ultrastructural changes after mechanical injury, we amputated primary dendrites of cultured mouse spinal neurons in low calcium medium in which sodium chloride had been replaced with either choline chloride or sodium isethionate or sodium propionate. Uninjured cultured neurons were also exposed to the sodium ionophore, monensin. A third set of neurons was injured in medium in which all sodium and calcium chloride had been replaced with sucrose. Neurons injured in low-calcium, low-sodium medium exhibited few ultrastructural changes, except very near the lesion, where there was some dilation of mitochondria and cisternae of the smooth endoplasmic reticulum (SER). Mitochondria in other regions of the neurons developed an electron opaque matrix, and those nearer to the lesion converted to the condensed configuration, characterized by expanded intracristal spaces as well as a dense matrix. If sodium but not chloride was present in the medium, there was some dilation of the Golgi cisternae after injury, as well as some increased electron opacity of the mitochondria. Monensin treated neurons also exhibited dilation of the Golgi cisternae. Neurons injured in sucrose-substituted medium showed none of the changes associated with injury in normal culture medium. These results indicate that sodium influx through the lesion is involved in the dilation of the SER, which is seen even in low-calcium medium, and that a permeant anion, such as chloride, is also involved. This dilation of the SER may result from uptake of calcium released from mitochondria in response to elevated cytosolic sodium. Dilation of the Golgi cisternae appears to be a response only to elevated intracellular sodium. Condensation of the mitochondria after injury is thought to be due to increased demands for ATP synthesis and may involve a “futile cycling” of calcium across the mitochondrial membrane, involving sodium-mediated calcium release in response to elevated intracellular calcium.

REVIEW ■ : Physical Injury of Neurons: Important Roles for Sodium and Chloride Ions:

There is growing evidence that ions other than Ca2+ play important roles in the deterioration of neuronal elements in both gray and white matter after physical injury. This review features information gathered with a tissue culture model of dendrite transection regarding the contributions of Na+ and CI- to ultrastructural damage and neuronal death. This information and the results of other in vitro investigations of physical and ischemic/excitotoxic injuries indicate that elevation of internal Na+ is an early event that may contribute significantly to neuronal injury through effects on Na+-driven transport mechanisms. Proposed deleterious consequences include cytoplasmic acidification, reduced mitochondrial energy production, and elevation of intracellular Ca2+ and extracellular excitatory amino acids to toxic levels. Prevention of Na+ entry into neurons after injury has been found to limit ultrastructural damage, prevent death, and preserve electrophysiological function. Although the role of CI- in neuronal injury is less well defined, there is also evidence that elevation of intracellular CI- contributes to structural damage, particularly to the smooth endoplasmic reticulum. In terventions that limit Na+- and CI--mediated damage to injured neurons may have utility in neurosurgery and as acute phase treatments for nervous system trauma and other pathological states. NEURO SCIENTIST 3:89-101, 1997

Buthionine sulfoximine induced growth inhibition in human lung carcinoma cells does not correlate with glutathione depletion

Treatment of A549 human lung carcinoma cells with L-buthionine-[S,R]-sulfoximine (BSO) results concomitantly in cellular glutathione (GSH) depletion and growth inhibition. The nature of BSO effects on cell growth and the relationships between BSO inhibition of cell growth and BSO effects on cellular GSH levels were determined in this study. A dose dependent effect of BSO on cell growth was observed, but this effect was found not to correlate with BSO effects on cellular GSH levels. Treatment with BSO for 60 h at concentrations of 5 and 10 mM was found to deplete cellular GSH at similar rates and to an undetectable level (below 0.5 nmol/mg protein). However, cessation of growth occured in 10 mM BSO whereas growth continued at better than one half the control rate in 5 mM BSO. The results suggest there may be a distinct threshold level of intracellular G GSH (on the order of or less than 0.5 nmol/mg protein) required for cell growth and for cells to protect themselves from the antiproliferative effects of BSO. At a concentration of 10 mM, BSO inhibited both DNA and protein synthesis and arrested growth of A549 cells throughout rather than at a specific phase of the cell cycle. BSO inhibition of growth was not, as indicated by colony-forming efficiency (CFE) and electron microscopy studies, accompanied by indications of cytotoxic effects. A stimulatory effect of 0.1 mM BSO on the growth of A549 cells was found also.

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells ofHelisoma trivolvis

In this study, we have further characterized the morphology and physiology of the neuroglandular synapse between the identified buccal neuron, B4, and the salivary gland ofHelisoma. We demonstrate that the coupling coefficient between salivary cells within an individual acinus is approximately 1.0. We also demonstrate that synapses within the salivary gland are located near a superficial muscle layer. We examine the effects of glutamate on the salivary gland and on the B4-salivary gland EPSP.l-glutamate produces a transient, rapid onset depolarization of salivary gland cells. The response is mimicked by high concentrations ofl-homocysteic acid, but not by NMDA,l-aspartate,d-glutamate or kainate. The response is blocked by the presence ofl- ord-glutamate in the bath, but not by CNQX, DNQX, DGG,d-AP5, orl-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When eitherl- ord-glutamate is present in the bathing solution, the amplitude of the B4-salivary gland EPSP is reversibly reduced. The similar pharmacological properties of the response of the salivary gland to glutamate and the B4 epsp indicate thatl-glutamate is a strong candidate for the fast excitatory neurotransmitter at theHelisoma neuroglandular synapse.

Histology and ultrastructure of the olfactory organ of the freshwater pulmonate Helisoma trivolvis

SummaryThe olfactory organ of Helisoma trivolvis is located on the surface of the body at the base of the cephalic tentacles. An evagination of skin, the olfactory plica, at the base of the tentacle extends over the olfactory organ dorsally. The epithelium of the olfactory organs contains unspecialized epithelial cells, ciliated epithelial cells, basal cells, mucous secretory cells, and sensory dendrites. The surface of the epithelium has a complex brush border of thick plasmatic processes, which branch to form several terminal microvillar twigs. Long slender cytoplasmic processes form a dense spongy layer among the plasmatic processes beneath the level of the terminal twigs. Bipolar primary sensory neurons clustered beneath the epithelium of the olfactory organ send dendrites through the epithelium to the free surface. Some sensory endings have a few short cilia, but most bear only microvilli. Cilia of sensory endings and epithelial cells extend beyond the brush border of the epithelium. Small axons arise from the perikarya of the sensory neurons and enter a branch of the olfactory nerve. HRP tracing indicates that the axons pass to the cerebral ganglion without interruption. Histochemical tests indicate that the sensory neurons are neither aminergic nor cholinergic.