Axonal maturaion in development—I. Characterization of monoclonal antibodies reacting with axon-specific neurofilament epitopes

Monoclonal antibodies reacting with the high molecular weight neurofilament polypeptides (NF 150K and NF 200K) were obtained upon immunization with NF 150K and NF 200K isolated from bovine spinal cord by anion exchange chromatography. The five monoclonal antibodies obtained with NF 200K stained only axons. With three monoclonals the reactivity was abolished by digestion with phosphatase and by dilution of the supernatants in sodium potassium phosphate. The nine monoclonal antibodies obtained upon immunization with NF 150K stained both high molecular weight neurofilament polypeptides on immunoblots of bovine and rat spinal cord extracts with the exception of one monoclonal only reacting with the homologous antigen. The antibodies could be divided into two groups, axon-specific and conventional. Conventional antibodies decorated neurofilaments regardless of their location, i.e. axons, perikarya and dendrites. With all these antibodies the immunostaining was not affected by phosphatase digestion of neurofilament protein nor by dilution of the supernatants in sodium potassium phosphate. Axon-specific antibodies reacting with both NF 150K and NF 200K in rat spinal cord only stained the heterologous antigen (NF 200K) in rat optic nerve and sciatic nerve extracts. We suggest that some axon-specific neurofilament antibodies recognize neurofilament modifications other than phosphorylation; or, alternatively that they react with phosphorylated epitopes not accessible to phosphate or to exogenous phosphatases. Furthermore, we suggest that some neurofilament modifications do not occur uniformly throughout the nervous system.     

Autologous sciatic nerve grafts to the rat spinal cord: Immunofluorescence studies with neurofilament and gliofilament (GFA) antisera

Autologous sciatic nerve grafts to the spinal cord contained many regenerating nerve fibers intensely immunofluorescent with neurofilament antisera. Axonal growth was not confined to the graft but also occurred in the surrounding spinal cord. In this location regenerating nerve fibers were invariably associated with Schwann-like cells. The reverse situation, that is, invasion of the graft by GFA-positive astroglia, also occurred but was a more limited phenomenon involving only a few cells.     

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.     

Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions.

Three neuroanatomical tracers have been employed to map the axonal projections formed between transplants of fetal spinal cord tissue and the surrounding host spinal cord in adult rats. Solid pieces of embryonic day 14 (E14) rat spinal cord were placed into hemisection aspiration cavities in the lumbar spinal cord. Injections of either (1) a mixture of horseradish peroxidase and wheat germ agglutinin- conjugated horseradish peroxidase, (2) Fluoro-Gold, or (3) Phaseolus vulgaris leucoagglutinin (PHA-L) were made into the transplants or the neighboring segments of the host spinal cord at 6 weeks to 14 months post-transplantation. Injections of anterograde and retrograde tracers into the transplants revealed extensive intrinsic projections that often spanned the length of the grafts. Axons arising from the transplants extended into the host spinal cord as far as 5 mm from the host-graft interface, as best revealed by retrograde labeling with Fluoro-Gold. Consistent with these observations, iontophoretic injections of PHA-L into the transplants also produced labeled axonal profiles at comparable distances in the host spinal cord, and in some instances elaborate terminals fields were observed surrounding host neurons. The majority of these efferent fibers labeled with PHA-L, however, were confined to the immediate vicinity of the host-graft boundary, and no fibers were seen traversing cellular partitions between host and transplant tissues. Host afferents to the transplants were also revealed by these tracing methods. For example, the injection of Fluoro-Gold into the grafts resulted in labeling of host neurons within the spinal cord and nearby dorsal root ganglia. In most cases, retrogradely labeled neurons in spinal gray matter were located within 0.5 mm of the graft site, although some were seen as far as 4-6 mm away. The distance and relative density of ingrowth exhibited by host axons into the grafts, however, appeared modest based upon the results of HRP and Fluoro-Gold retrograde labeling. This was further confirmed with the PHA-L anterograde method. Whereas some host fibers were seen extending into the transplants, the majority of PHA-L containing axons formed terminal-like profiles at or within 0.5 mm of the host-graft interface. The comprehensive view of intrinsic connectivity and host-graft projections obtained in these studies indicates that intraspinal grafts of fetal spinal cord tissue can establish a short-range intersegmental circuitry in the injured, adult spinal cord. These observations are consistent with the view that such grafts may contribute to the formation of a functional relay between separated segments of the spinal cord after injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparisons of capillary maturation in control and hypothyroid rat spinal cord: an ultrastructural study.

Quantitative and qualitative features of capillary maturation were examined in the ventral horn of the lumbar spinal cord of control and neonatally induced hypothyroid rats from birth to 6 weeks, using light (LM) and electron microscopy (EM). Quantification of the capillary densities by LM in the control animals and their hypothyroid litter mates have shown three- and twofold increases, respectively, from birth to Postnatal (Pn) Day 21. The following features were observed in the control animals at the EM level: (a) The newborn animals showed varying degrees of capillary maturation; (b) The majority of capillaries possessed mature characteristics by Pn Day 21; (c) By Pn Day 42, mature characteristics were found in nearly all capillary profiles. The hypothyroid animals demonstrated: (1) reactive perivascular cells and astrocytes; (2) delayed appearance of glycogen in the early Pn period and its persistence in extensive amounts during the latter part (3-6 weeks) of development; (3) cytoplasmic extensions of endothelial cells and perivascular cells, and (4) the presence of mitotic endothelial cells and perivascular cells even during the latter period of development. The observations suggest that the peak period of vasculogenesis in the lumbar spinal cord of the normal rat occurs during the second and third Pn weeks. The results from the hypothyroid rats point toward a delay in development and maturation of capillaries resulting in a hypoplastic vascular bed of the ventral horn. The reactive cells and the accumulation of glycogen particles could be morphological expressions of biochemical changes in hypothyroidism during the critical period of CNS development.     

Axonal regeneration in lamprey spinal cord

Spinal cords of sea lamprey larvae were transected at one of two levels: (a) rostral, at the last gill, or (b) caudal, at the cloaca. Following various recovery times, regeneration of the posteriorly projecting giant reticulospinal axons (RAs) was demonstrated by intra-axonal injection of horseradish peroxidase (HRP). Regeneration of axons of anteriorly projecting dorsal cells (DCs) and giant interneurons (GIs) was demonstrated by intrasomatic HRP injection into cells located just below the transection scar. After 40 days of recovery, 55% of proximally transected RAs (rostral cut) regenerated at least as far as the center of the scar, whereas only 15% of distally transected RAs (caudal cut) did so. Maximum distance of regeneration was 5.3 mm beyond the scar for proximally transected RAs but only 38 u for distally transected RAs. Proximally transected RAs also branched more profusely than distally transected ones. These data (when combined with others in the literature) suggest that the regenerative capacity of RAs may decrease with distance of axotomy from the cell body. Distance of regeneration and degree of branching of proximally transected RAs peaked between 40 and 100 days. Thereafter, there appeared to be a tendency toward neurite retraction. Of axotomized GIs, 76% regenerated anteriorly at least as far as the center of a caudal transection scar (GIs are located only in the caudal part of the cord). The maximum distance of regeneration was 1.3 mm beyond the scar. Of DC axons, 56% regenerated anteriorly at least as far as the transection site. The maximum distance was 1.1 mm beyond the scar. DCs located just below a caudal transection regenerated at least as well as those located below a rostral transection. Axonal regeneration was also demonstrated for a few lateral cells, edge cells, and crossed caudally projecting interneurons.     

Axonal regeneration in the adult lamprey spinal cord

Larval sea lampreys recover from complete spinal transection by a process involving directionally specific axonal regeneration. In order to determine whether this is also true of adults, 14 adult lampreys were transected at the level of the 5th gill and allowed to recover for 10 weeks. Müller and Mauthner cells and their giant reticulospinal axons (GRAs) were impaled with microelectrodes and injected with horseradish peroxidase (HRP). The tissue was processed for HRP histochemistry and wholemounts of brain and spinal cord were prepared. All animals recovered coordinated swimming; 61 of 121 (50%) neurites emanating from 30 axons regenerated caudal to the scar into the distal stump. Of the neurites which had grown beyond the scar, 92% were correctly oriented, i.e., caudalward and ipsilateral to the parent axon. Retransection in two additional animals eliminated the recovered swimming. Thus, behavioral recovery in adult sea lampreys is accompanied by directionally specific axonal regeneration.     

Phosphorylation-dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord.

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.     

Axonal and dendritic development of substantia gelatinosa neurons in the lumbosacral spinal cord of the rat

In the present study, developing neurons of the substantia gelatinosa (SG) are examined at short interval sequential stages from 15 days of gestation through 20 days postpartum. Rapid Golgi preparations are utilized to examine axonal and dendritic development and toluidine blue preparations are employed to study the overall growth pattern of SG cells by measuring changes in mean cell body area. Results show that there are two maturation periods, which involve two separate groups of SG neurons. The sequence and pattern of development for each group is different. The first period occurs prenatally and involves the axonal and dendritic development of presumptive projection and propriospinal neurons. In classical terminology, these cells can be classified as limiting, large find small central, and transverse cells. These neurons have axons that enter the white matter and their dendritic arbors develop through a relatively simple process of elongation and branching. The second maturation period occurs postnatally and involves the development of presumptive nonprojection intrinsic neurons that have axons which remain within the gray matter. These neurons are identified as islet, stalk, inverted stalklike, and vertical cells. Unlike projection or propriopinal neurons, the intrinsic nonprojection neurons sprout numerous short, beaded dendrites that radiate from the cell body in a starlike fashion. Star-shaped cells undergo a metamorphosis involving a rearrangement of dendrites along adult dendritic patterns. Measurements taken from toluidine blue preparations indicate that the nonprojection intrinsic population makes up the greatest percentage of SG neurons, as evidenced by a marked increase in the size of the average SG nerve cell during the second maturation period.     

Axonal growth cones in the developing amphibian spinal cord

Axonal growth cones in the spinal cord of embryonic and larval Xenopus (stages 24-48) were filled with the anatomical tracer horseradish peroxidase (HRP). Growth cones of lateral and ventral marginal zones, including those of descending spinal and supraspinal pathways, were labeled by application of tracer to the caudal medulla or to one of several levels of the spinal cord. Central axons of sensory neurons were filled via their peripheral processes. Growth cone configuration varied widely but fell into five general categories: complex with both filopodia and veils, filopodia only, lamellipodia only, clavate, and fusiform. Several general patterns emerged from the distribution of these various configurations. Growth cones of younger animals generally were more complex than those of older ones. Growth cones closer to the leading edge of descending fiber bundles were more elaborate than those that followed. Growth cones of the dorsolateral fascicle, which carries ascending central processes of Rohon-Beard and sensory ganglion neurons, were very simple and followed a straight course rostrally, whereas those of descending axons of the lateral fiber areas were more complex and sometimes spread over almost the entire lateral marginal zone. Growth cones of Rohon-Beard central ascending axons were fusiform or clavate, while those of sensory ganglion axons showed several fine filopodia at their tips. Growth cones of both types of sensory axons change configuration as they approached the hindbrain, becoming more complex. This study demonstrates that the configurations of growth cones belonging to the same axonal pathway vary with age and with position along their routes, and that growth cones of different neuron classes exhibit characteristic ranges of morphological variation.     

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia.

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.     

Postnatal maturation of primary afferent terminations in the substantia gelatinosa of the rat spinal cord. An electron microscopic study

Axodendritic dark sinuous endings occurred on the day of birth (PO) in the synaptic areas of lamina II. These terminals (TI) turned very electron dense and shrunken after capsaicin administration. From day P2, TI-terminals exhibited fluoride-resistant acid phosphatase (FRAP) reactivity. Such findings revealed their origin from primary afferent fine fibers. Dark scalloped, FRAP-reactive, central terminals of type I glomeruli (CI) were first observed on P5. During the ensuing survival times up to P20 an increasing number of CI-terminals seemed to evolve from the less mature TI-endings that gradually disappeared. A few large clear boutons, similar to the central terminals of type II glomeruli (CII) of the adult which arise from thick afferents, were also present in deep lamina II on the day of birth and became more numerous thereafter. From P5, both CI- and CII-endings developed pre- and postsynaptic contacts with vesicle-containing profiles though it was sometimes difficult to distinguish the axonic (V2) from the dendritic (V1-presynaptic dendritic) profiles. CI-boutons established as many presynaptic as postsynaptic contacts with vesicle-containing profiles. In contrast, CII-terminals were mostly postsynaptic to vesicle-containing profiles. Thus, the boutons generated by thin (CI-boutons) and thick (CII-boutons) primary axons gradually develop synapses with vesicle-containing profiles probably arising from local interneurons. The resulting pre- and/or postsynaptic interactions may contribute to the physiological maturation of somatosensory integration that occurs postnatally.     

免疫介导的脊髓前角运动神经元损伤过程中神经丝异常的观察

目的 研究免疫介导的脊髓前角运动神经元损伤过程中神经丝(NF)磷酸化及超微结构的特征,探讨免疫与肌萎缩侧索硬化发病之间的关系.方法 通过透射电镜技术及免疫组化方法,对免疫介导的脊髓前角运动神经元损伤过程中NF异常聚集的超微结构特征及异常磷酸化状态进行研究.结果 电镜观察发现免疫后动物脊髓前角运动神经元胞质及轴索近端有神经丝异常聚集;免疫组化证实抗非磷酸化神经丝(SMI-32)抗体阳性的脊髓前角运动神经元(个/张脊髓切片)数量(12.00±1.05)与对照组(18.00±1.83)相比,明显减少(P＜0.05),而抗磷酸化NF抗体(SMI-31)阳性的脊髓前角运动神经元数量(13.00±1.60)与对照相比(3.23±1.33)明显增加(P＜0.01).结论 在免疫介导的运动神经元损伤过程中存在类似于肌萎缩侧索硬化的神经丝结构及代谢异常特征,两者之间可能存在共同的发病机制.     

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

Immunohistochemical localization of choline acetyltransferase in rabbit spinal cord and cerebellum

Guinea pig antiserum specific for purified bovine choline acetyltransferase has been shown to cross-react with rabbit enzyme. We used the peroxidase-antiperoxidase immunohistochemical method to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded sections of rabbit spinal cord and cerebellum. In the spinal cord, in agreement with our and others' previous results using immunofluorescent techniques, choline acetyltransferase was found in the cell bodies of the ventral horn motor neurons. In the cerebellum, choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. The immunohistochemical findings in the cerebellum reveal the morphological detail of cholinergic axons and their terminals. The results are consistent with published biochemical data on the cerebellar distribution of choline acetyltransferase.     

Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury

Spinal cord injury was induced in rat by weight drop. The extent of degradation of neurofilament proteins in the lesion following trauma was examined and served as a measure of calpain activity. Calpain was identified in the samples by myelin mealpain antibody and the content was estimated from the immunoblot. There was progressive degradation of both 68 kDa and 200 kDa neurofilament proteins in the cord lesion at intervals after injury. At 30 min after injury there was 20% degradation of both neurofilament proteins while the breakdown of 68 kDa and 200 kDa NFRs amounted to more than 60% at 24 h and beyond. Calpain content progressively increased in the lesion by 22% at 30 min to 91% at 4 h after trauma compared to control and then decreased but remained elevated for up to 72 h following injury. These results suggest that calpain is a primary responder synthesized early in injury and involved initially in the breakdown of cytoskeletal proteins in spinal cord trauma. Later in the injury cascade, increased calpain activity is derived from inflammatory as well as endogenous cells supporting a pivotal role for calpain throughout the process of secondary and evolving tissue damage in spinal cord trauma.     

Axonal regeneration in spinal cord injury: a perspective and new technique

A set of techniques is described for determining the response of mammalian spinal axons to transection. The logical selection and the advantages of these techniques are discussed. The dorsal column of guinea pig thoracic spinal cord was transected with a tungsten needle and the position of the lesion was marked by a staple-shaped wire device (Foerster: J. Comp. Neurol. 210:335-356, '82). The morphology of dorsal column axons projecting rostrally toward the lesion was examined between 1 and 50 days postlesion by anterograde staining with horseradish peroxidase, applied to a second lesion of the dorsal column two to three vertebral segments caudal to the first. Axons damaged by the original lesion were found to die back 1-2 mm from the plane of transection and at 18-20 hours were characterized by terminal club-shaped swellings attached to the proximal axon by a thin connection. At 50 days postlesion there was some evidence of limited regenerative responses in terms of growth-cone-like axon terminals, and the presence of aberrant axonal branching, but no evidence of regenerating axons approaching close to the plane of transection. These findings are in agreement with previous studies indicating little or no effective regrowth of myelinated axons in the mammalian spinal cord. These same techniques were used in a succeeding study to examine the effects of applied electric fields on the axonal response to transection.     

Suppression of neurofilament degradation by protease inhibitors in experimental spinal cord injury

Intraperitoneal administration of the neutral protease inhibitors leupeptin and E-64c substantially suppressed the degradation of neurofilament proteins (NFP) at the site of mechanical insult and secondary axonal degeneration, and facilitated the recovery of motor functions in acute spinal cord injury in rats. The drug effects were assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of NFP fractions from the injured tissue and by morphometry of degenerating axons revealed by the Fink-Heimer method in distal spinal cord segments with the aid of an automated image analyzer. The role of calcium-activated neutral proteases in acute central nervous tissue damage and potential use of protease inhibitors as therapeutic modalities are discussed.     

Distribution of somatostatin receptor subtypes in rat lumbar spinal cord examined with gold-labelled somatostatin and anti-receptor antibodies

Using gold-labelled somatostatin, somatostatin binding sites were predominantly found in laminae I–III, X and on motorneurones of the rat lumbar spinal cord. A comparison with immunohistochemical staining using antisera against somatostatin receptor sequences revealed that the marked binding in laminae I–III coincided with the presence of somatostatin receptor-like immunoreactivity for the receptor subtypes 1, 2 and 3. Binding sites on motorneurones were only paralleled by an immunoreaction for subtype 3. In lamina X, however, the lack of a positive immunoreaction indicates that in this part other subtypes may be present.