RAPID AXOPLASMIC TRANSPORT IN DYSTROPHIC MICE

Abstract— Rapid axoplasmic transport was studied in dystrophic mice of the 129/ReJ-dy strain. Proteins transported in vivo through α-motoneurons of the sciatic nerve were labeled by injections of [³H] or [³⁵S] amino acids into the ventral horn of the lumbar spinal cord. Following an 18 h incubation, axoplasmic transport was quantitated by summing the radioactivity in the 10 mm length of sciatic nerve proximal to a ligation. Although the amount of transported radioactivity was small, transport appeared depressed when adult dystrophic mice were compared to controls. Transport was also studied in the sensory fibers of the sciatic nerve under in vitro conditions, resulting in high levels of transported radioactivity. In this system transport was strongly depressed. The severity of the deficiency varied with age, being small in animals with early clinical signs and becoming maximal (80–90%) in animals over 60 days of age. Proteins transported by adult dy/dy and +/+ animals were compared by gel electrophoresis using double-label techniques. Transport of nearly all proteins was depressed in dy/dy mice, although the possibility exists that small differences occur. The data suggest that the dystrophic state produces a significant deficiency in rapid axoplasmic transport in both motor and sensory fibers, and may interfere with transport processes in all neurons. Since rapid axoplasmic transport has been associated with membranes, the data are consistent with a general alteration of cellular membranes in dystrophic animals.     

Changes in the rate of axonal flow of proteins through the branches of motor and sensory neurocytes during the period of intense growth of the sciatic nerve in rats

14C-glycin was microinjected into the ventral horns of the spinal cord or spinal ganglions. The rate of fast and slow axoplasmic transport of proteins in the axons of motor and sensory neurons was studied by liquid scintillation. Motor fibers of the sciatic nerve manifested a marked decrease (P less than 0.05) in the rate of slow axoplasmatic transport of the labeled protein from 5.25 +/- 0,31 in 2-week-old rats to 3.45 +/- +/- 0.23 mm/day in 4-week-old animals and a significant increase in the rate of fast axoplasmic transport (P less than 0.05) from 99 +/- 13.2 (2-week-old rats) up to 198 +/- 18.9 mm/day (in 4-week-old rats). The two-week-old rats had higher rates (4.5 +/- 0.3 mm/day) of slow axoplasmic transport of the labeled protein in the central and peripheral axons of sensory neurocytes and lower rates of fast axoplasmic transport (126 +/- 14.7 mm/day) as compared with 4-week-old animals (3.75--4.1 +/- 0.25 -- slow transport; 144 +/- 23.34 mm/day -- fast transport). However, the differences described are not significant.     

Anatomical specificity of regenerated muscle sensory afferents in the spinal cord of the bullfrog

The specificity of central projections made by regenerated muscle sensory fibers in the brachial spinal cord was studied with anatomical tracing methods. Sensory fibers were interrupted by freezing dorsal roots in postmetamorphic bullfrogs. After several months, regenerated sensory fibers were labeled with horseradish peroxidase applied to the triceps brachii muscle nerve, and their arborizations within the spinal cord were reconstructed from serial cross sections. Most of the regenerated projections from triceps muscle sensory afferents ended in or near their normal terminal field. A few branched and appeared to terminate more dorsally than normal, however, sometimes within the region where cutaneous afferents normally terminate. In contrast to the normal pathway followed by muscle afferents within the spinal cord, many regenerated afferents grew along the circumference of the spinal cord, just under the pial surface, and then turned abruptly toward the midline and into their appropriate terminal region. This suggests that regenerating afferents may actively seek out their appropriate targets and are not simply passively guided to them.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion.

After injection of the L7 dorsal root ganglion with 3H-leucine, fast axoplasmic transport carries some 3--5 x more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Rapid axoplasmic transport of insulin-like growth factor I in the sciatic nerve of adult rats

Summary Somatomedin C (Sm-C; insulin-like growth factor I; IGF-I) is a polypeptide (M_r 7649), often dependent on growth hormone (GH), with trophic effects on several different tissues. Monospecific IGF-I antisera were used to investigate its localization in the sciatic nerve and corresponding nerve cells, as well as its possible axoplasmic transport in the adult rat. IGF-I-like immunoreactivity was demonstrated in anterior horn motor nerve cells in the spinal cord and in spinal- and autonomic ganglion nerve cells. Faint IGF-I immunoreactivity was under normal conditions observed in axons of the sciatic nerve and in the Schwann cells. Using crush technique, accumulation of IGF-I immunoreactivity was seen in dilated axons within 2 h, both proximal and distal to the crush. However, only a small fraction of the anterogradely transported IGF-I immunoreactive material could be demonstrated to be transported in retrograde direction. Colchicine injected proximal to a crush prevented accumulation of IGF-I immunoreactivity proximal to the crush, but not distal to it.IGF-I-immunoreactive material is synthesized in the cell bodies of peripheral sensory and motor nerve cells. It is transported at rapid rates in the axoplasm of the sciatic nerve of adult rats both in anterograde and retrograde directions. We propose that axonally transported IGF-I may be released and exert trophic influence on innervated cells, tissues and organs.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion

After injection of the L7 dorsal root ganglion with ³H-leucine, fast axoplasmic transport carries some 3–5 × more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Primary projections of the trigeminal nerve in two species of sturgeon: Acipenser oxyrhynchus and Scaphirhynchus platorynchus

Horseradish peroxidase histochemical studies of afferent and efferent projections of the trigeminal nerve in two species of chondrostean fishes revealed medial, descending and ascending projections. Entering fibers of the trigeminal sensory root project medially to terminate in the medial trigeminal nucleus, located along the medial wall of the rostral medulla. Other entering sensory fibers turn caudally within the medulla, forming the trigeminal spinal tract, and terminate within the descending trigeminal nucleus. The descending trigeminal nucleus consists of dorsal (DTNd) and ventral (DTNv) components. Fibers of the trigeminal spinal tract descend through the lateral alar medulla and into the dorsolateral cervical spinal cord. Fibers exit the spinal tract throughout its length, projecting to the ventral descending trigeminal nucleus (DTNv) in the medulla and to the funicular nucleus at the obex. Retrograde transport of HRP through sensory root fibers also revealed an ascending bundle of fibers that constitutes the neurites of the mesencephalic trigeminal nucleus, cell bodies of which are located in the rostral optic tectum. Retrograde transport of HRP through motor root fibers labeled ipsilateral cells of the trigeminal motor nucleus, located in the rostral branchiomeric motor column.     

Quantitative Changes in Myelin Proteins in a Peripheral Neuropathy Caused by Tullidora (Karwinskia humboldtiana)

Peripheral nerve demyelination was induced in cats by oral administration of ether extracts of Tullidora (Karwinskia humboldtiana). Proteins from several hindlimb nerves, spinal roots, and dorsal columns of the spinal cord were subjected to slab gel electrophoresis and quantified by densitometry. In Tullidora-treated cats with severe motor disturbances, specific myelin proteins were reduced by at least 50% in motor nerves and less than 25% in cutaneous axons. There was a greater decrease of these proteins in the distal than in the cephalad segments of the sciatic nerve; no changes were detected either in the spinal roots or in the white matter of the spinal cord. Electron microscopy revealed intense demyelination in the motor nerves only. Both the density of the 100 A-thick neurofilaments and the relative proportion of a polypeptide with a molecular weight of 68,000 were considerably increased in the affected nerves. It is tentatively concluded that the active principles of Tullidora may enter the axons through the motor nerve terminals. The distal segments of the motor nerves would then be preferentially affected and demyelination could result from axonal damage.     

大鼠脊髓损伤后感觉、运动及电生理变化

在应用磁控机械夹断法复制的大鼠脊髓损伤模型上,动态地观察了脊髓损伤后的感觉及运动机能变化,并进行了电生理学研究。结果表明,0.3A电流未能导致永久性瘫痪。术后2周,后肢的感觉及运动功能逐渐恢复;可记录到体感诱发电位(SEP)。0.4,0.5和0.8A电流均能导致大鼠永久性瘫痪;倾斜板及开阔场地行走分数均显著低于0.3A组;术后4周这些大鼠可产生行走样动作,于损伤部位再次切断脊髓后仍能出现这些动作;0.4A组可记录到早期SEP,再次切断脊髓后SEP消失。结果提示:(1)脊髓不全横断后,由于残留纤维活动,可在相当程度上导致大鼠感觉和运动机能的恢复;(2)脊髓完全横断后,后肢的上行冲动可能经再生的神经纤维向中枢端传导至脑;(3)大鼠脊髓内可能存在行走中枢模式发生器(CPG),适当刺激可激发其活动,并产生行走样运动。     

Blockade of retrograde axoplasmic transport induces transganglionic degenerative atrophy of central terminals of primary nociceptive neurons

If applied locally around a peripheral sensory nerve, Formyl-Leurosin, a semi-synthetic diindol alkaloid of Vinca rosea--that, just like other mitotic spindle inhibitors, induces blockade of axoplasmic transport via inhibiting microtubular function--causes transganglionic degenerative atrophy of central terminals of primary nociceptive neurons in the substantia gelatinosa Rolandi of the spinal cord. In contrast, if applied to dorsal roots, Formyl-Leurosin fails to induce such alterations. Based upon these observations it is postulated that blockade of retrograde axoplasmic transport, rather than that of the orthograde one, is the decisive factor in the pathomechanism of transganglionic degenerative atrophy.     

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.     

Study of individual Pacinian corpuscle mechanoreceptors following application of colchicine to a nerve]

Colchicine application to the cat caudal mesenteric nerve containing sensory fibers for single mechanoreceptors (Pacinian corpuscles) causes degeneration of the axis cast of the nerve endings. Ultrastructural changes in the receptors showed no difference from the axonal degeneration after the nerve section but the rate of degeneration was considerably slower. Ultrastructural, electrophysiological, and biochemical changes occurring in the Pacinian corpuscles were not the result of direct action of colchicine, but appeared to be realized through the nerve by the axoplasmic transport block. It is suggested that the receptor's structure is under the sensory neuron neurotrophic control.     

Phrenic afferents and their role in inspiratory control

In anesthetized cats, with vagi cut and the spinal cord severed at the C8 level, phrenic motor and/or sensory discharge was recorded. Small afferent phrenic fibers were identified through their activation by lactic acid, hyperosmotic NaCl solution, or phenyl diguanide. They exhibited a spontaneous but irregular low-frequency discharge. Block of their conduction by procaine had no effect on eupneic motor phrenic activity. Large afferent phrenic fibers showed a spontaneous rhythmic discharge, and cold block (6 degrees C) of these fibers significantly prolonged the phrenic discharge time (Tphr) and total breath duration (TT) during eupnea. The stimulation of all afferent phrenic fibers lowered the impulse frequency of phrenic motoneurons (f impulses) and shortened both Tphr and TT. When the stimulation was performed during cold block all of the effects on phrenic output persisted, but changes in timing were less pronounced. Under procaine block, only the effects of phrenic nerve stimulation on Tphr persisted. These results suggest that both large and small afferent phrenic fibers control the inspiratory activity with a prominent role of small fibers on phrenic motoneuron impulse frequency.     

Morphologic and histochemical characteristics of frog skeletal muscle following application of colchicine to a motor nerve]

Experiments with application of colchicine to the muscle motor nerve carried out; this was done for the purpose of disturbance of rapid axoplasmic transport. A reduction of the areas of transverse sections of the muscle fibers, an increase in the number of fibers with a low succinic dehydrogenase (SDH) activity a greater homogeneity of the muscle fibers by the degree of optic density in staining for detection of the SDH activity was noted. Analogous changes were revealed under conditions of section of the motor nerve. However, denervation was accompanied by the block of conductivity and by degenerative changes in the nerve endings. As to the preparations treated with colchicine, transmission of excitation in the nerve and through the synapse was retained and was recorded by the end plate miniature potentials, end plate potentials and the action potentials of the muscle fibers. A conclusion was drawn that rapid axoplasmic transport brought substances maintaining differentiated state of the muscle fibers.     

Immunogenesis and axoplasmic transport in Wistar rats]

Immunization of Wistar rats with thymus dependent antigens (sheep red blood cells-SRBC) is accompanied by a reliable increase in the synthesis of RNA and proteins in thalamic cerebral cortex and spinal marrow (48 hrs after antigen injection) and also in an increase in the intensity of rapid axoplasmic transport (RAT) along motor fibers of sciatic nerve (5,48,72 hrs following the beginning of immunization). There was a consecutive augmentation in AFC number in mesenteric and partly in inguinal lymph nodes (96 hrs after SRBC injection). Thus, time dependence between immunogenesis and axoplasmic transport in experimental animals (Wistar rats) was determined for the first time. It identifies another, previously unstudied, channel in interactions of immune and nervous systems.     

Axonal transport: a quantitative study of retained and transported protein fraction in the cat

The fast axonal transport of proteins was studied in the cat sciatic nerve after injection of [3H]leucine into the spinal ganglion or the ventral horn of the seventh lumbar segment. The amount of transported proteins after ganglion injection was linearly related to the amount of label present at the ganglion. At variable intervals after ganglion or spinal cord injection, the sciatic nerves were sectioned in some experiments. The transport of proteins continued in the peripheral nerve stump in a wavelike manner, but the advancing wave leaves a labeled trail behind. A fraction of this trail corresponds to proteins moving at slower velocities than the velocity of proteins in the wave front. Another fraction of the trail corresponds to molecules retained by the axons. Each nerve segment of 5 mm in length retains 1.5% of the transported proteins, and the profile of retained proteins along the sciatic nerves follows a single exponential function. From the proportion of retained proteins, the concentration of transported proteins at the terminals of branching axons as a function of the branching ratio was estimated. In the case of motor axons innervating the soleus muscle of the cat, the concentration of recently transported proteins at the nerve terminals would be approximately 0.83% of the proteins leaving the spinal cord. This low concentration of transported proteins at the nerve terminals may explain the lability of neuromuscular synapses when axonal transport is decreased or interrupted.     

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.     

Single Doses of Acrylamide Reduce Retrograde Transport Velocity

Abstract: Single doses of acrylamide (0–1.3 mmol/kg) produced a dose-dependent decrease in the transport of ¹²⁵I-tetanus toxin to the perikarya of sensory neurons in dorsal root ganglia and motor neurons in ventral spinal cord. Acrylamide was a more potent inhibitor of retrograde transport in sensory axons than in motor axons. Substantially greater doses of N,N '-methylene-bis-acryl-amide, a reportedly non-neurotoxic analog of acrylamide, were required to alter the axonal transport of ¹²⁵I-tetanus toxin. Velocity of retrograde transport was assessed by determining the position of the leading edge of transported¹²⁵I-tetanus toxin at times following single doses of acrylamide. Acrylamide reduced the velocity of ¹²⁵I-tetanus toxin transport in a dose-dependent manner by up to 75%. No change in neuronal uptake of ¹²⁵I-tet-anus toxin was detected. It is concluded that single doses of acrylamide produce profound alterations in retrograde transport which precede the appearance of structural changes in affected nerve fibers.     

Origin of the silent period in somato- and viscero-sympathetic responses

The origin of the period of postactivation depression of spike activity in the renal nerve during stimulation of afferent fibers of cutaneous (cutaneous branch of the peroneal nerve) and visceral (greater splanchnic nerve) nerves was studied in unanesthetized, anesthetized, decerebrate, and spinal cats. This silent period was shown to be considerably prolonged after administration of general anesthetics to unanesthetized animals or after decerebration. Analeptics (strychnine, leptazol, picrotoxin) or division of the spinal cord at the level of the lower cervical segments weaken postactivation depression. The causes of origin of the silent period are discussed and the localization of the structures responsible for its appearance is examined.I. P. Pavlov First Leningrad Medical Institute. Translated from Neirofiziologiya, Vol. 4, No. 5, pp. 501–509, September–October, 1972.     

Low temperature slowing and cold-block of fast axoplasmic transport in mammalian nerves in vitro

1) Fast axoplasmic transport in mammalian nerve in vitro was studied using an isotope labeling technique. The rate of outflow in cat sciatic nerve fibers of 410 mm/day in vitro was reduced at temperatures below 38°C with a Q₁₀ of 2.0 in the range 38–18°C and a Q₁₀ of 2.3 at 38–13°C. 2) At a temperature of 11°C a partial failure of transport occurred. At temperatures below 11°C a complete block of fast axoplasmic transport occurred, a phenomenon termed “cold-block.” No transport at all was seen over the temperature range of 10–0°C for times lasting up to 48 hr. 3) Transport was resumed after a period of cold-block lasting up to 22 hr when the nerves were brought back to a temperature of 38°C. Some deleterious effects due to cold-block were seen in the recovery phase as indicated by a reduction in crest amplitude, change in its form, and slowed rate. 4) The ∼P level (combined ATP and creatine phosphate) remained near control level in nerves kept at low or cold-block temperatures for times as long as 64 hr. The reduction in fast axoplasmic transport rate seen at low temperatures for times up to 22 hr was therefore considered due to a decrease in the utilization of ATP, a concept in accord with the “transport filament” model proposed to account for fast axoplasmic transport. 5) The sloping of the front of the crest over the temperature range of 18–13°C suggests an additonal factor at the lower temperatures. A disassembly of microtubules is discussed as a possible explanation of the cold-block phenomenon.