Fast axonal transport in extruded axoplasm from squid giant axon

Development of video-enhanced contrast-differential interference contrast for light microscopy has permitted study of both orthograde and retrograde fast axonal transport of membranous organelles in the squid giant axon. This process was found to continue normally for hours after the axoplasm was extruded from the giant axon and removed from the confines of the axonal plasma membrane. It is now possible to follow the movements of the full range of membranous organelles (30-nanometer vesicles to 5000-nanometer mitochondria) in a preparation that lacks a plasma membrane or other permeability barrier. This observation demonstrates that the plasma membrane is not required for fast axonal transport and suggests that action potentials are not involved in the regulation of fast transport. Furthermore, the absence of a permeability barrier surrounding the axoplasm makes this an important model for biochemical pharmacological, and physical manipulations of membranous organelle transport.     

Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease

We identified axonal defects in mouse models of Alzheimer's disease that preceded known disease-related pathology by more than a year; we observed similar axonal defects in the early stages of Alzheimer's disease in humans. Axonal defects consisted of swellings that accumulated abnormal amounts of microtubule-associated and molecular motor proteins, organelles, and vesicles. Impairing axonal transport by reducing the dosage of a kinesin molecular motor protein enhanced the frequency of axonal defects and increased amyloid-beta peptide levels and amyloid deposition. Reductions in microtubule-dependent transport may stimulate proteolytic processing of beta-amyloid precursor protein, resulting in the development of senile plaques and Alzheimer's disease.     

Slow transport of tubulin in the neurites of differentiated PC12 cells

In order to study the rate and form of tubulin transport in cultured neuronal cells, the fluorescence recovery after the photobleaching of a fluorescent tubulin analog has been followed within the neuritic processes of differentiated PC12 cells. In these cells, as in peripheral axons, tubulin is transported in coherent, nondiffusing waves at two different slow rates that are within the range of the slow components a and b of axonal transport measured in vivo. Finally, it appears that most, if not all, of the tubulin analog is moving out these processes. Thus, slow neuroplasmic transport in cultured neuron-like cells is a good model of axonal transport, in which experimental manipulations of the system can be performed that would be difficult in the whole animal.     

Fast axonal transport in squid giant axon

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.     

Preferred microtubules for vesicle transport in lobster axons

The hypothesis that transported vesicles are preferentially associated with a subclass of microtubules has been tested in lobster axons. A cold block was used to collect moving vesicles in these axons; this treatment caused the vesicles to accumulate in files along some of the microtubules. Quantitative analysis of the number of vesicles associated with microtubule segments indicated that lobster axons have two distinct populations of microtubules--transport microtubules that are the preferred substrates for vesicle transport and architectural microtubules that contribute to axonal structure.     

Axonal transport of tritium-labeled putrescinein the embryonic visual system of zebrafish

The transport of [(3)H]putrescine is demonstrated by autoradiography in the retino-tectal tract of Brachydanio rerio embryos. Transport of [(3)H]putrescine appears to be more rapid than that of tritium-labeled protein and is not inhibited by a colchicine effect on axonal neurotubules as is protein transport. The radioactivity transported to the brain is found, on electrophoresis, in the putrescine fraction.     

Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes

The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.     

Biochemistry of information storage in the nervous system

The use of molecular biological approaches has defined new mechanisms that store information in the mammalian nervous system. Environmental stimuli alter steady-state levels of messenger RNA species encoding neurotransmitters, thereby altering synaptic, neuronal, and network function over time. External or internal stimuli alter impulse activity, which alters membrane depolarization and selectively changes the expression of specific transmitter genes. These processes occur in diverse peripheral and central neurons, suggesting that information storage is widespread in the neuraxis. The temporal profile of any particular molecular mnemonic process is determined by specific kinetics of turnover and by the geometry of the neuron resulting in axonal transport of molecules to different synaptic arrays at different times. Generally, transmitters, the agents of millisecond-to-millisecond communication, are subject to relatively long-lasting changes in expression, ensuring that ongoing physiological function is translated into information storage.     

Differential regulation of dynein and kinesin motor proteins by tau

Dynein and kinesin motor proteins transport cellular cargoes toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single-molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction, whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at about a tenth of the tau concentration that inhibited dynein, and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.     

Axonal proteins of presynaptic neurons during synaptogenesis

Changes occur in the synthesis and axonal transport of neuronal proteins in dorsal-root ganglia axons as a result of contact with cells from the spinal cord during synapse formation. Dorsal-root ganglia cells were cultured in a compartmental cel culture system that allows separate access to neuronal cell bodies and their axons. When cells from the ventral spinal cord were cultured with the dorsal-root ganglia axons, synapses were established within a few days. Metabolic labeling and two-dimensional electrophoresis revealed that four of more than 300 axonal proteins had changed in their expression by the time synapses were established. The highly selective nature of these changes suggests that the proteins involved may be important in the processes of axon growth and synapse formation and their regulation by the regional environment.     

AMP-activated protein kinase regulates neuronal polarization by interfering with PI 3-kinase localization

Axon-dendrite polarization is crucial for neural network wiring and information processing in the brain. Polarization begins with the transformation of a single neurite into an axon and its subsequent rapid extension, which requires coordination of cellular energy status to allow for transport of building materials to support axon growth. We found that activation of the energy-sensing adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway suppressed axon initiation and neuronal polarization. Phosphorylation of the kinesin light chain of the Kif5 motor protein by AMPK disrupted the association of the motor with phosphatidylinositol 3-kinase (PI3K), preventing PI3K targeting to the axonal tip and inhibiting polarization and axon growth.     

Distinct pathways of viral spread in the host determined by reovirus S1 gene segment

The genetic and molecular mechanisms that determine the capacity of a virus to utilize distinct pathways of spread in an infected host were examined by using reoviruses. Both reovirus type 1 and reovirus type 3 spread to the spinal cord following inoculation into the hindlimb or forelimb footpad of newborn mice. For type 3 this spread is through nerves and occurs via the microtubule-associated system of fast axonal transport. By contrast, type 1 spreads to the spinal cord through the bloodstream. With the use of reassortant viruses containing various combinations of double-stranded RNA segments (genes) derived from type 1 and type 3, the viral S1 double-stranded RNA segment was shown to be responsible for determining the capacity of reoviruses to spread to the central nervous system through these distinct pathways.     

Retrograde axonal transport of endogenous proteins in sciatic nerve demonstrated by covalent labeling in vivo

Extracellularly applied N-succinimidyl [2,3-3H]propionate was used in vivo to covalently label intra-axonal proteins in the rat sciatic nerve. This technique permitted a unique view of axonal transport of proteins independent of biosynthesis. The proteins detected in slow anterograde transport (1 to 2 millimeters per day) correspond to cytoskeletal proteins described in previous papers. The slowly retrogradely transported component (3 to 6 millimeters per day) was composed primarily of a single protein with a molecular weight of 68,000.     

北京鸭脑干网状脊髓束的起源及其细胞构筑:HRP逆行追踪法研究

采用HRP法,分别在北京鸭脊髓的颈中部、颈膨大部、腰膨大部单侧注射或包埋HRP,蓝色反应显色。逆行追踪了54例北京鸭,对脑干网状脊髓束的起源及其细胞构筑进行了研究。发现网状脊髓束不仅起于延髓和脑桥内侧网状结构的大细胞部,同时还起于外侧网状结构的小细胞部和中脑网状结构。标记细胞呈双侧性分布,同侧多于对侧。其结果为生理学和药理学提供了可靠的形态依据。     

Microtubule gelation-contraction: essential components and relation to slow axonal transport

Preparations of microtubule proteins isolated by assembly and disassembly undergo gelation-contraction after addition of adenosine triphosphate (ATP). A particulate fraction from these preparations that is required, along with purified tubulin, to produce ATP-dependent microtubule gelation-contraction in vitro has been isolated. The particulates exhibited microtubule-stimulated adenosine triphosphatase activity and moved slowly (about 1 micrometer per minute) along microtubule walls in the presence of ATP. The particulates contained tubulin, neurofilament, and spectrin polypeptides. The composition, solubility, and motility of the particulates are consistent with those of slow component a of axonal transport.     

Interdigitation of contralateral and ipsilateral columnar projections to frontal association cortex in primates

The combined use of two anterograde axonal transport methods reveals that in the prefrontal association cortex of macaque monkeys, associational projections from the parietal lobe of one hemisphere interdigitate with callosal projections from the opposite frontal lobe, forming adjacent columns 300 to 750 micrometers wide. The finding of separate and alternating ipsilateral and contralateral inputs in the frontal association cortex opens up new possibilities for the functional analysis of this large but unexplored area of the primate brain.     

Opiates and pain pathways: demonstration of enkephalin synapses on dorsal horn projection neurons

The participation of the opiate peptide enkephalin in the neural circuitry of the dorsal horn was examined at the light and ultrastructural level through the use of the combined techniques of immunocytochemistry and retrograde transport of horseradish peroxidase. Enkephalin immunoreactive axonal endings made direct synaptic contact with the soma and proximal dendrites of dorsal horn thalamic projection neurons. This observation demonstrates that one major synaptic site of enkephalin modulation of the transfer of nociceptive information in the dorsla horn is on the projection neurons themselves.U     

Rapid axonal transport of sulfated mucopolysaccharide proteins

When sulfur-35-labeled sodium sulfate is injected intraocularly in the goldfish, labeled sulfated mucopolysaccharides rapidly appear in the contra-lateral optic tectum of the brain, demonstrating the axonal flow of sulfated mucopolysaccharides. The transport rate is the same as that observed for proteins labeled after intraocular injection of tritiated proline. Treatment of the sulfur-35-labeled material with precipitants and enzymes reveals the presence of substances with properties similar to those of heparan sulfate (the major component) and chondroitin sulfate. Dermatan sulfate was not detected.     

Acute axonal dystrophy caused by fluorocitrate: the role of mitochondrial swelling

Fluorocitrate, a Krebs cycle inhibitor, induices neurons to rapidly expel multitudinous lysosomes, mitochondria, and other cytoplasmic constituents into their axons. After convulsive seizures commence, spectacular axonal balloons develop owing to obstruction of axonal flow by "log jams" of extruded organelles. A swelling of neuronal mitochondria is apparently responsible for the disgorgement of cytoplasmic material into axons.     

GM1 ganglioside treatment facilitates behavioral recovery from bilateral brain damage

Adult rats with bilateral lesions of the caudate nucleus were treated with GM1 ganglioside. Although animals injected with a control solution were severely impaired in their ability to learn a complex spatial task, those treated with ganglioside were able to learn spatial reversals.