Differences in the Distribution of Cytochrome b561 and Synaptophysin in Dog Splenic Nerve: A Biochemical and Immunocytochemical Study

Abstract: Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotrarismitter-containing vesicles, i.e., large (LDVs) and small densecore vesicles, coexist with the axonal reticulum (AR) and the well-characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b₅₆₁, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b₅₆₁ coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, Synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b₅₆₁ immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b₅₆₁, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No Synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data Clearly illustrate that Synaptophysin is absent from LDVs and AR elements in adrenergic axons.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

Binding of γ-Aminobutyric AcidA Receptors to Tubulin

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [³⁵S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.     

Depression of fast axonal transport in axons demyelinated by intraneural injection of a neurotoxin fromK. humboldtiana

Tullidinol, a neurotoxin extracted from the Karwinskia humboldtiana fruit, dissolved in peanut oil was injected into the right sciatic nerve of adult cats. The contralateral sciatic nerve received an equivalent volume of peanut oil alone. The fast axonal transport of labeled ([³H]Leucine) protein was studied in sensory and motor axons of both sciatic nerves. The radioactive label was pressure injected either into the L7 dorsal root ganglion or the ventral region of the same spinal cord segment. Several days after the toxin injection, the cat limped and the Achilles tendon reflex was nearly absent in the right hind limb. The amount of transported label was decreased distal to the site of toxin injection. Proximal to this site, the transported material was dammed. Sensory and motor axons showed similar changes. In addition, the toxin produced demyelination and axonal degeneration. Axonal transport and the structure of the axons were normal in the contralateral nerve. Both, Schwann cells and axons of the right sciatic nerve showed globular inclusions, presumably oil droplets containing the toxin. We conclude that Schwann cells and axons as well are tullidinol targets.Departamento de Química. Centro de Investigación y de Estudios Avanzados del IPN.Special issue dedicated to Dr. Sidney Ochs.     

Acrylamide and Glycidamide Impair Neurite Outgrowth in Differentiating N1E.115 Neuroblastoma Without Disturbing Rapid Bidirectional Transport of Organelles Observed by Video Microscopy

Abstract: The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene-bisacrylamide were weakly cytotoxic in a ⁵¹Cr-release assay, but only at ≥10 m M (order of efficacy: methylene-bis-acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100-fold lower concentrations, with similar EC₅₀ values for all three toxicants, i.e., acrylamide, 70 ± 15 μ M ; methylene-bis-acrylamide, 92 ± 31 μ M ; glycidamide, 120 ± 30 μ M . Only glycidamide (1 m M ) caused degeneration of established neurites within a period of 48 h. Video-enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of ≤48 h at 1 m M , neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.     

Locally Synthesized Phosphatidylcholine, but Not Protein, Undergoes Rapid Retrograde Axonal Transport in the Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phosphatidylcholine in the sciatic nerve has been demonstrated only after injection of lipid precursors into the cell body region. We now report, however, that after microinjection (1 μl) of [methyl-³H]choline chloride into the rat sciatic nerve (35-40 mm distal to the L4 and L5 dorsal root ganglia), time-dependent accumulation of ³H-labeled material occurred in dorsal root ganglia ipsilateral, but not contralateral, to the injection site. The level of radioactivity in the ipsilateral dorsal root ganglia was minimal at 2 h after isotope injection but was significantly increased at 7, 24, 48, and 72 h after intraneural isotope injection (n = 3–8 per time point); at these time points, all of the radiolabel in the chloroform/methanol extract of the ipsilateral dorsal root ganglia was present in phosphatidylcholine. The radioactivity in the water-soluble fraction did not show a time-dependent accumulation in the ipsilateral dorsal root ganglia as compared with the contralateral DRGs, ruling out transport or diffusion of precursor molecules. In addition, colchicine injection into the sciatic nerve proximal to the isotope injection site prevented the accumulation of radiolabel in the ipsilateral dorsal root ganglia. Therefore, this time-dependent accumulation of radiolabeled phosphatidylcholine in the ipsilateral dorsal root ganglia is most likely due to retrograde axonal transport of locally synthesized phospholipid material. Moreover, 24 h after injection of both [³H]choline and [³⁵S]-methionine into the sciatic nerve, the ipsilateral/contralateral ratio of radiolabel was 11.7 for ³H but only 1.1 for ³⁵S. indicating that only locally synthesized choline phospholipids, but not protein, were retrogradely transported.     

Pathfinding of peripheral neurons in the central nervous system of an embryonic grasshopper (Chorthippus biguttulus)

The peripheral leg nerves of grasshoppers are initially formed by a set of pioneer neurons and guidepost cells. These cells are used as guiding structures for later-arising axons of sensory neurons. The development of the central projections of the pioneer cells, the guidepost cells and some sensory cells is shown with Lucifer Yellow injection or with DiI application. The axons of the pioneer cells Ti1 enter the central nervous system at 38% of embryonic development. They turn anteriorly close to the midline and ascend with no major branching to the brain. The axons of the guidepost cells Fe1 and Tr1 follow the same path but do not ascend to the brain. Sensory axons of the subgenual organ and the femoral organ probably do not follow the central path pioneered by the former neurons. They end ipsilaterally in the respective thoracic neuromere, as is found in the adult.     

“Axonal spheroids” in the spinal cord of normal rabbits

Summary Within the gray matter and the white matter of the spinal cord of apparently healthy rabbits, myelinated and unmyelinated axonal swellings, so called axonal spheroids, occur. Most of the spheroids contain mitochondria, dense bodies, vesicles and fragments of the tubular or smooth endoplasmic reticulum. In myelinated spheroids the process of swelling is effected by slippage of the myelin leaflets. At the periphery of the unmyelinated parts of the spheroids, synapses are regularly found. The presynaptic terminal bouton is formed by the spheroid. A few myelinated and unmyelinated spheroids are packed with fine granular material while mitochondria are lacking. The axonal spheroids may represent a physiological, perhaps age dependent phenomenon.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70^th birthdayThe author wishes to thank Mrs. Helga Zuther-Witzsch, Mrs. Elisabeth Schöngarth and Miss Hildegard Schöning for excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft, Projekt Le 69/7-13     

The ventral lateral geniculate nucleus,pars lateralis of the rat

Summary The synaptic organization of the pars lateralis portion of the ventral lateral geniculate nucleus is similar to that of other thalamic nuclei. There are four types of synaptic knobs (RL, RS, F1, F2). RL knobs are large and irregularly shaped, contain round synaptic vesicles and make multiple asymmetrical junctions. They are found primarily in synaptic islands making contact with gemmules, spines, small dendrites, and other synaptic profiles containing pleiomorphic synaptic vesicles (F2). Smaller RS knobs contain round vesicles and make asymmetrical junctions with the same type of elements as RL knobs, with the exception of the F2 profiles, but are seldom found in synaptic islands. F1 knobs contain flattened synaptic vesicles and form symmetrical junctions with F2 knobs, gemmules, spines, and small-medium dendrites in synaptic islands, throughout the neuropil, and on the proximal dendrites and soma of the largest type of neuron. F2 knobs are irregularly shaped, contain pleiomorphic synaptic vesicles and make symmetrical junctions primarily with gemmules and spines in synaptic islands. They are postsynaptic to RL and F1 knobs. Occipital decortication indicates that cortical terminals are of the RS type. Bilateral enucleation indicates that retinal terminals are of both the RL and RS type. The large amount of geographic overlap of retinal and cortical terminals on gemmules, spines, and small dendrites found in the neuropil outside of synaptic islands logically would maximize axonal sprouting between these two sources.We would like to thank Mr. Peter Rossetti for his excellent technical assistance on a major portion of this project, Ms. Judith Strauss for photographic assistance, and Ms. Nancy Wood for typing. Supported by grants NS 10579, NS 08724, 5 S01 RR 05402, and 2 T01 GM 00326