Axonal transport of protein in the optic nerve of Octopus vulgaris, Lam.
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Opiate receptors measured in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature. The time-course of accumulation was completely different in the proximal and the distal segments; the labelling was maximal 4 h after injection of [3H]lofentanil above the ligature but 16-24 h below the ligature. In unligated rats, a peak of radioactivity appeared in the nodose ganglion 16 h after injection; vagotomy, vinblastine or chronic treatment with capsaicin prevented the appearance of this delayed accumulation in the ganglion. These foregoing experiments suggest that opiate may act in the cell body of sensory neurones after being internalized at the nerve terminals and then transported retrogradely through fast axoplasmic mechanisms.
The effect of a neurotoxic dose of 5.0 mg/kg, s.c. diisopropyl phosphorofluoridate (DFP) on anterograde transport of 35S-methionine labeled proteins in cat peripheral nerve was studied. Seven days after dosing, after 24 h of flow there was 48% less radioactivity in the distal portion of the nerve. A lesser effect was also found at 4 and 14 days after dosing. After 10 or 14 h of flow, the height of the crest was unchanged, but the distance of the crest from the ganglia was greater in the DFP-treated animals. These experiments indicate that DFP treatment accelerates fast anterograde transport.
UV light fluorescent tracers (True Blue and Fast Blue) are placed in contact with sensitive receptors after dermabrasion. The abraded surfaces are limited to two hemidorsal areas and two hemi plantar areas on the animals' foot. A five days survival is allowed to enable the axonal retrograde transport of the tracers, then the animals are sacrificed and perfused with an intracardiac injection of 10% formaldehyde in phosphate buffer at 7.4 pH. The lumbar spinal ganglions are immediately dissected out, examined as a whole with a Leitz Dialux fluorescence microscope, then frozen and cut with a cryotome. The results of this analysis show that: i-only ipsilateral dorsal root ganglions are labeled by blue dye. ii-the number of fluorescent cells varies between 20 to 60 per ganglion. iii-a map of the distribution of the dermatomes on the rat hind foot can be deducted from the study of the labelled ganglions. They spread from L2 to L5 from the cranial to the caudal part, and from the medial to the lateral side of the rat foot, on both dorsal and plantar areas.
The retrograde labeling of cortical neurons with horseradish peroxidase (HRP) was used to investigate the morphological features of neurons in various cortical areas projecting to the superior colliculus in the cat. Corticotectal cells were found to be labeled in layer V of the entire cerebral cortex. The number of labeled cells and their locations varied according to the sites of injections of HRP in the colliculus. Most of the corticotectal cells identified in the present study were small (9--20 micrometer in diameter, 66%) and medium (20--40 micrometer, 30%) pyramidal neurons and only 4% of them were large (more than 40 micrometer). The labeled cells, 261 in total number, had somal diameters of 20.8 +/- 8.0 micrometer (mean and SD). The range of sizes of the labeled neurons was different in different cortical areas. For example, the labeled neurons in the Clare-Bishop area had a greater proportion of large diameter cells than in other areas. The present findings are largely in agreement with the previous data of anterograde degeneration methods with respect to the topographical correlation of the corticotectal projections. However, in some cortical areas, e.g., the sensorimotor and the first visual (area 17) cortex of the lateral surface of the hemisphere, relatively small numbers of corticotectal neurons appear to have been labeled by retrogradely transported HRP. The sparsity of the labeled neurons in certain cortical areas may reflect the existence of corticotectal neurons with axon collaterals supplying brain structures other than the superior colliculus.
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In previous studies it has been shown that nerve growth factor (NGF) is taken up with high selectivity by adrenergic and sensory nerve terminals and is transported retrogradely to the corresponding cell bodies by a colchicine sensitive mechanism 10,11,23. The present study was designed to investigate whether this retrograde transport of NGF depends on properties of the nerve terminals which are common to all the neurons or restricted to those which respond to NGF either during the whole life cycle (adrenergic neurons) or during a restricted period of embryonic development (sensory neurons). In order to investigate the retrograde transport of NGF in motor neurons we injected [125I]NGF into the musculus deltoideus and measured the side differences of accumulation of radioactivity in the spinal cord (C6-C8) from 4-48 h. At no time was there a preferential accumulation of radioactivity on the injected side. In contrast, tetanus toxin was accumulated preferentially on the injected side and an approximate rate of transport of 7.5 mm/h was calculated. Astonishingly there was also a retrograde transport of tetanus toxin in sensory and adrenergic neurons. The rate of transport was identical to that of NGF and the transport could be blocked by transection of the corresponding nerve fibers and local administration of colchicine. After unilateral injection of [125I]tetanus toxin into the forepaw or the musculus deltoideus light microscopic autoradiographs revealed heavily labeled neuronal cell bodies in the dorsal root ganglia or the ventrolateral spinal cord of the injected side. It is concluded that the retrograde transport of [125I]NGF depends on properties of the neuronal membrane which are specific for adrenergic and sensory neurons, whereas that of tetanus toxin is determined by features which are common to all, or at least to all peripheral, neurons. The fact that the rate of transport for both NGF and tetanus toxin is identical in all examined neurons, supports the hypothesis that the specificity of retrograde transport is determined by specific uptake sites in the neuronal membrane whereas the retrograde transport system is non-specific.
Following stereotaxic injection of [35S]methionine into the substantia nigra of adult rats, there was rapid local incorporation of radioactivity into acid-insoluble material. Incorporation peaked by 4 h and then decreased. In contrast, acid-precipitable radioactivity in the corpus striatum (the major projection site of the substantia nigra) rose markedly between 1 and 8 h followed by a plateau period and another even more marked increase between 24 h and 6 days. Experiments involving injection of [3H]fucose gave similar results except that most of the acid-precipitable radioactivity in the striatum appeared in an early wave. In each case radioactivity in the contralateral striatum was less than 11% of that on the ipsilateral side. Stereotaxic injection of colchicine (20 microgram) into the nigrostriatal pathway (within the median forebrain bundle) blocked transport of [35S]protein and [3H]glycoprotein by 90% and 50%, respectively. In animals treated with 6-hydroxydopamine (6-OHDA; treated neonatally or as adults) the accumulation of striatal [35S]protein was reduced to 7 to 26% of control levels; striatal [3H]glycoprotein was also reduced, but not as much (29% to 73% of control). In control experiments, [3H]DOPA wa injected into the substantia nigra, and [3H]dopamine was measured in corpus striatum; 6-OHDA treatment reduced the amounts of striatal [3H]dopamine recovered to 3% of control values. The failure of colchicine or 6-OHDA to block transport of incorporated fucose as effectively as the transport of incorporated methionine is possible due to greater diffusion of fucose away from the injection site to non-dopaminergic nuclei projecting to the striatum. The molecular weight distribution of radioactive proteins at the substantia nigra and corpus striatum was analyzed by polyacrylamide gel electrophoresis. For both [35S]methionine and [3H]fucose, the gel electrophoretic pattern of radioactive proteins in the injection site (substantia nigra) was complex and did not change greatly between 2 h and 6 days. At the projection site (striatum) the electrophoretic distribution pattern was initially different from that of the substantia nigra, and changed markedly over the course of several days. In 6-OHDA-treated animals (treated neonatally or as adults), the bulk of proteins transported in nigro-striatal non-dopaminergic neurons appears to be very similar to that transported in the intact pathway in control rats. However, in striata of 6-OHDA-treated animals, a consistent reduction in striatal 35S- and 3H-radioactivitiy was observed in proteins with molecular weight from about 67,000 to 77,000. Assuming that the 6-OHDA treatment did not substantially affect the non-dopaminergic neurons, we interpret this to mean that some of the proteins in this molecular weight range are transported primarily by dopaminergic neurons.