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.

Effects of sodium and chloride on neuronal survival after neurite transection.

An in vitro investigation was undertaken to study the roles of Na+ and Cl- in mammalian spinal cord (SC) neuron deterioration and death after injury involving physical disruption of the plasma membrane. Individual SC neurons in monolayer cultures were subjected to UV laser microbeam transection of a primary dendrite. Neurons lesioned in modified ionic environments (MIEs) where 50%-75% of the NaCl was replaced with sucrose had higher survival (65%-75%) than neurons lesioned in medium with normal (125 mM) NaCl (28%; p < 0.001). Subsequent experiments found a comparable increase in lesioned neuron survival in MIEs in which only Na+ was replaced with specific ionic substitutes; however, replacement of Cl- was not protective. Electron microscope examinations of neurons fixed <16 min after lesioning showed a dramatic decrease in vesiculation of the smooth endoplasmic reticulum and Golgi apparatus in the low NaCl or low Na+ MIEs. It is hypothesized that Na+ entry after membrane disruption may stimulate elevation of [Ca+2]i leading to ultrastructural disruption and death of injured neurons. The results of these studies suggest that a low NaCl MIE may be useful as an irrigant to limit damage spread and cell death within CNS tissues during surgery or after trauma.

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

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 microm 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 gamma-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 gamma-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.

Unmyelinated axons in thoracic ventral roots of the cat.

This study shows that approximately 30% of the axons in the T11 and T12 ventral roots of the cat are unmyelinated. The unmyelinated axons fall into two categories. Slightly less than half are efferents from the spinal cord and slightly more than half arise from dorsal root ganglion cells. The efferent fibers are regarded as unmyelinated preganglionic sympathetics, the fibers of dorsal root ganglion origin are regarded as sensory. This organization of the T11-T12 ventral roots, which are part of the sympathetic outflow, is similar to that of cat ventral roots S3 and Cal, which are part of the parasympathetic outflow, but different from cat ventral roots L7 and S1, which are between the visceral outflows.

Categories of axons in the inferior cardiac nerve of the cat.

Myelinated and unmyelinated axons in the inferior cardiac nerve of the cat were examined to determine how many axons were (1) sensory, (2) preganglionic sympathetic, and (3) postganglionic sympathetic. In one group of cats, a segment was removed from the middle of the inferior cardiac nerve as a control, and the proximal and distal stumps of the nerve were examined one week later. In another group of cats, the control segment of nerve was removed and the first thoracic white ramus communicans and sympathetic trunk were cut proximal to the stellate ganglion, followed in one week by examination of the proximal and distal stumps of the inferior cardiac nerve. In still another group of cats, the first five thoracic spinal nerves were cut just distal to the dorsal root ganglion. The counts of myelinated and unmyelinated axons after these surgical procedures indicated that, in the cat inferior cardiac nerve, all or almost all of the approximately 30,000 unmyelinated axons and 10 percent of the myelinated axons are postganglionic sympathetic fibers, and that approximately 90 percent of the myelinated axons are sensory.

Unmyelinated and small myelinated axons in rat ventral roots.

The present study is an analysis of the ventral roots in the rat. The smallest myelinated fibers are found in segments T1-L2 and L6-S1. These fibers are regarded as preganglionic efferents and the L6-S1 outflow is more cranial than the sacral parasympathetic outflow in other animals. Large numbers of unmyelinated axons are seen in segments T1-L2 and L6-S1, the same segments that contain the smallest myelinated fibers. This data will be used as a baseline for determining the functional categories of the unmyelinated axons.

Fiber analysis of the feline inferior cardiac sympathetic nerve.

The left inferior cardiac nerves of nine adult cats were examined with the electron microscope. Electrical recordings were also made from four of these nerves. Most of the nerves contained between 25,000 and 40,000 unmyelinated fibers and 30 to 150 myelinated fibers. The diameters of the unmyelinated fibers ranged from 0.1 mu to 1.8 mu with mean diameters ranging from 0.47 to 0.78 mu in the various nerves. The myelinated fibers ranged in diameter from 1 mu to 9 mu with a mean difference of approximately 3 mu. Electrical recordings showed two peaks in both the C fiber and the Adelta fiber compound action potentials. Conduction velocities of the two C fiber peaks were approximately 0.7 and 0.9 m/sec. The two groups of A delta fibers had conduction velocities of approximately 7 and 16 m/sec.

Effects of methylprednisolone on lesioned and uninjured mammalian spinal neurons: viability, ultrastructure, and network electrophysiology.

An in vitro investigation was undertaken to provide information regarding the effectiveness of methylprednisolone sodium succinate (MPSS) as a treatment for the primary mechanical injury of spinal cord (SC) trauma. Exposure of uninjured mouse SC cells to MPSS for 24 h caused neuronal stress when the concentration exceeded 150 micrograms/mL; neuronal death occurred at concentrations above 600 micrograms/mL. The concentration range for MPSS protection of SC neurons subjected to a defined physical injury (laser microbeam transection of a primary dendrite 100 microns from the perikaryon) was very narrow: survival in the 30 micrograms/mL group differed significantly from the untreated control group (68.5% +/- 14.1 vs. 47.1% +/- 14.1), treatment with 20 or 60 micrograms/mL MPSS did not increase survival, and treatment with 100 micrograms/mL MPSS accelerated ultrastructural deterioration and increased the likelihood of death. Enhanced survival of lesioned neurons was observed when 30 micrograms/mL MPSS was applied within 15 min of dendrotomy but not when MPSS was administered 2 h after lesioning. Multimicroelectrode plate (MMEP) studies of SC network electrical activity indicated that MPSS associated readily with neuronal membranes. This finding was consistent with the hypothesis that MPSS may protect lesioned neurons by stabilizing damaged membranes, enhancing lesion resealing, and limiting the spread of ion-mediated damage. However, comparisons of neurite die-back 24 h after dendrotomy found no significant difference between MPSS-treated and control neurons. Application of 30 or 100 micrograms/mL MPSS increased the spontaneous burst activity of SC networks grown on MMEPs, however, there was no evidence that the increased excitability at these concentrations was the result of specific actions of MPSS on GABA or NMDA synapses.

Neuronal survival and dynamics of ultrastructural damage after dendrotomy in low calcium.

To determine the contributions of calcium to development of ultrastructural damage and neuronal death after mechanical injury, we amputated primary dendrites from over 300 cultured mammalian spinal neurons under normal (1.8 mM) or low (less than or equal to 30 microM) calcium conditions. Two general categories of early ultrastructural change were seen in both normal and low calcium: (1) a lesion-dependent gradient of damage that moved centripetally through the proximal segment and penetrated the soma within 15 min and (2) dilation of the somal Golgi/smooth endoplasmic reticulum (SER), which preceded the wave of deterioration from the lesion. Although the somal Golgi/SER changes were similar in both normal and low calcium, the damage gradient in low calcium differed from the damage gradient in normal calcium. (1) Microtubules and neurofilaments were preserved, (2) mitochondria became more electron dense but did not develop electronlucent foci or high amplitude swelling, and (3) an extensive vesicular gradient formed consisting of rows of swollen SER vesicles. Sodium ionophores have been reported to cause similar changes. Survival studies showed that calcium reduction significantly delayed neuronal death. Survival was 63 +/- 16% vs 35 +/- 8% (p less than 0.003) at 2 h and 30 +/- 7% vs 23 +/- 8% at 6 h in low and normal calcium, respectively. Dead neurons that had been lesioned in low calcium also showed greater ultrastructural preservation than neurons that died after dendrotomy in normal calcium. We hypothesize that under low calcium conditions, the large sodium injury current plays an important role in neuronal deterioration and death after mechanical trauma.

In vitro investigations of the effects of nonfreezing low temperatures on lesioned and uninjured mammalian spinal neurons.

This two-part investigation explored the parameters and mechanisms of: (1) injury to spinal cord (SC) neurons by nonfreezing low temperatures, and (2) hypothermic protection of SC neurons subjected to a defined, physical injury (dendrite transection). Conclusions from the studies of hypothermic injury were: (1) morphologic and ultrastructural signs of stress developed in SC neurons as the temperature was decreased below 17 degrees C; (2) most neurons showing stress during cooling died upon rewarming to 37 degrees C; (3) spontaneous SC network activity was not significantly changed by cooling to 17 degrees C for 2 hours and rewarming, but cooling to 10 degrees C for 1 hour caused a reduction of burst frequency after rewarming, and cooling to 10 degrees C for 2 hours resulted in electrical silence after rewarming; and (4) application of N-methyl-D-aspartate (NMDA) antagonists before cooling prevented neuronal death, ultrastructural damage, and loss of activity upon rewarming, but application after cooling (before rewarming) was not protective. Conclusions from the studies of hypothermic protection were: (1) cooling at 17 degrees C for 2 hours followed by rewarming to 37 degrees C significantly increased lesioned neuron survival, but protection was lost when the period at 17 degrees C was increased to 6 hours; (2) NMDA blockade under normothermic (37 degrees C) or hypothermic (17 degrees C or 10 degrees C for 2 hours) conditions was not more protective of lesioned neurons than cooling to 17 degrees C (no NMDA antagonist); and (3) 200 microM thiopental or 100 microM pentobarbital increased lesioned neuron survival to a degree comparable to cooling for 2 hours at 17 degrees C.

Fine structure of olfactory epithelia of gastropod molluscs.

Among gastropod molluscs the chemical senses are most important for location of distant objects. They are used in food finding, locating mates, avoiding predators, trail following, and homing. Chemoreceptors are commonly associated with the oral area, the tentacles, and the osphradium, which lies in the mantle cavity. Most chemosensory neurons are primary sensory neurons, although secondary sensory cells have been reported in the osphradium of some prosobranch gastropods. Most chemosensory organs contain sensory cells with ciliated sensory endings that are in contact with the external environment. Some sensory endings have only microvilli or have no surface elaborations. Cilia on sensory endings are commonly of the conventional type, but some species have modified cilia; some lack rootlets, some have an abnormal microtubular content, and some have paddle-shaped endings. The perikarya of sensory neurons may be within the sensory epithelium, below it, or in ganglia near the sensory surface. In some groups of gastropods there are peripheral ganglia in the olfactory pathway; in others chemosensory axons appear to pass directly to the CNS. Olfactory epithelia of terrestrial pulmonates have modified brush borders with long branching plasmatic processes and a spongy layer of cytoplasmic tubules which extend from the epithelial cells. Sensory endings of the olfactory receptors are entirely within this spongy layer. Aquatic pulmonates may have a similar spongy layer in their olfactory epithelia, but the cilia of sensory endings, as well as motile cilia of epithelial cells, extend well beyond the spongy layer.

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 histology and fine structure of the olfactory organ of the squid Lolliguncula brevis Blainville.

The olfactory organ of the squid has a thick, pseudostratified epithelium containing five morphological types of ciliated receptors. In the simplest receptors the cilia originate separately in the distal pole of the cell. All other receptors have some type of cilia filled cavity, varying from a simple pocket of cilia at the surface to a completely closed vesicle filled with cilia in cells deep in the epithelium. The receptors are compared to cells in the rhinophore of Nautilus and the olfactory organs of coleoid cephalopods. Possible functions of the olfactory organ, based on its morphology, are discussed.

Observations on the olfactory organ of adult and juvenile Octopus joubini.

The olfactory organ has an epithelium containing many sense cells and a large subepithelial mass of receptor cells. The epithelium includes cells with cup-shaped, ciliated endings, and hollow, flask-shaped sense cells with ciliated cavities that open to the surface, through a small pore. Below the epithelium are large hollow cells with ciliated cavities and distal processes that either form patent connections between the ciliated cavity and the surface or have a ciliated ending at the surface. There are many synapses between processes in the olfactory nerve. The possible chemosensory function of the olfactory organ is discussed.

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells of Helisoma 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 of Helisoma. 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 of L-homocysteic acid, but not by NMDA, L-aspartate, D-glutamate or kainate. The response is blocked by the presence of L- or D-glutamate in the bath, but not by CNQX, DNQX, DGG, D-AP5, or L-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When either L- or D-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 that L-glutamate is a strong candidate for the fast excitatory neurotransmitter at the Helisoma neuroglandular synapse.

Ciliated sensory neurons in the lip of the squid Lolliguncula brevis Blainville.

The lip of Lolliguncula brevis is a muscular fold covered by a simple columnar epithelium and overlayed medially and distally by a non-cellular cuticle. Bipolar sensory cells in the epithelium have a shallow pocket with cilia and microvilli at the free end of their dendrite. The cilia project out onto the surface through pores in the cuticle. Cells with intracellular cilia were observed below the epithelium. These cells send a process toward the surface of the lip. These two receptors are compared to the receptors previously described from the lip of Sepia.

Ciliated sensory cells and associated neurons in the lip of Octopus joubini Robson.

The lip of Octopus joubini is a fleshy fold around the beak that is subdivided distally into finger-like papillae and overlayed by an uninterrupted noncellular cuticle. The muscular core of the lip has a high proportion of nervous tissue. The simple epithelium contains numerous ciliated sensory cells, especially in the papillae. In many of these cells the cilia lie deep within the cytoplasm and usually appear to extend toward the surface. Receptors with intracellular cilia also lie below the epithelium and send dendrites bearing cilia to the surface. Large unipolar interneurons that may receive synapses from the ciliated receptors lie in the musculature near the papillae. The sensory system of the octopus lip is more advanced than that of the squid, and it is very similar to that of Sepia. The relationship of these findings to the phylogeny and ecology of cephalopods is discussed.

Contributions of sodium and chloride to ultrastructural damage after dendrotomy.

To 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.

Intracellular acidification reversibly reduces endocytosis at the neuromuscular junction.

The close spatial and temporal coupling of endocytosis and exocytosis in nerve terminals has made it difficult to elucidate the mechanisms and the regulation of endocytosis per se. Despite significant advances in our knowledge of the molecules involved in endocytosis, it has not yet been possible to selectively manipulate endocytosis in nerve terminals. We report that the substitution of propionate for chloride in the saline bathing a lizard neuromuscular junction reduces internal pH and reversibly blocks activity-dependent endocytosis. When intraterminal pH is reduced by approximately 0.7 pH units, the uptake of FM1-43 in nerve terminals, but not activity-dependent destaining, is reduced. Normalization of intracellular pH by removing the propionate, raising extracellular pH, or adding ammonium chloride immediately restores FM1-43 uptake. Electron microscopy indicates that intracellular acidification reversibly reduces activity-dependent endocytosis in nerve terminals, because depolarization in propionate saline leads to a depletion of vesicles and the appearance of large intramembraneous infoldings.