Quantitative Estimation of HRP-Labeled Sensory and Motor Neurons during Nerve-Dependent and Nerve-Independent Periods of Urodele Limb Regeneration

The relationship between urodele regeneration and the possibility of regeneration in mammals is unclear, but the idea of possible regeneration of neural elements in man is being studied because of its potential clinical importance. One of the great challenges is to gain sufficient knowledge about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is known that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibers connected to their cell bodies. The nerve fibers severed at the level of limb amputation regrow and penetrate the blastema, providing blastema cells with indispensable factors. These factors are produced in the perikarya of neurons and transported via their axons to the blastema. Numerous studies have been performed to elucidate the quantitative relationships between nerve fibers and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of this investigation was to analyze the quantitative participation and qualitative distinctions of different nerve cells innervating the regenerating parts of the urodele limb and their possible roles in the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral part of the spinal cord gray matter (motor neurons). The direct involvement of these neurons in different regeneration periods was visualized by means of horseradish peroxidase (HRP) labeling. A total of 34 animals (21 experimental and 13 control) were used to study fluctuations in the numbers of labeled nerve cells. The results are summarized as follows: (a) the first nerve cells incorporating HRP within 5 days after amputation are found in the dorsal ganglia, whereas motor neurons in the gray matter are labeled within 7 days; (b) the number of labeled perikarya increases during the nerve-dependent regeneration period (0–21 days after amputation), with the percentage of implicated sensory neurons exceeding that found in the control series; and (c) during the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration on the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.     

Gas7在成年大鼠脊髓和脊神经节的表达

目的 研究生长休止蛋白7（Gas7）在成年大鼠脊髓和脊神经节的表达.方法 成年SD大鼠12只,采用逆转录聚合酶链反应（RT-PCR）方法、焦油紫染色以及免疫组织化学方法来观察Gas7基因核酸和蛋白在成年SD大鼠脊髓和脊神经节的表达.结果 RT-PCR结果显示,脊髓和脊神经节有较丰富的Gas7 mRNA表达.免疫组化结果显示：与焦油紫染色相对照,脊髓灰质各板层神经元均表达Gas7蛋白,与其它版层相比较,后角Ⅱ版层胶状质的小细胞和前角Ⅸ版层的运动神经元显色较深且数量较多.脊髓白质Gas7免疫阳性反应较弱且分布均匀.脊神经节内大型感觉神经元呈Gas7免疫强阳性反应,中、小型感觉神经元为弱阳性反应.结论 本文首次描述了Gas7在成年大鼠脊髓和脊神经节的表达,为进一步研究Gas7在成年神经系统再生和修复过程中的功能提供形态学基础.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated, nerve-dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD-NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response.     

Selective sparing of NADPH-d positive spinal cord neurons affected by ischemia

Histochemical characterization of NADPH diaphorase positive neuronal pools in the rabbit lumbosacral segments was performed during and after transient spinal cord ischemia. Strongly enhanced staining of NADPH diaphorase positive structures appeared in the superficial dorsal horn, the pericentral region and in the neurons of the sacral parasympathetic nucleus at the end of 40 min of abdominal aorta ligation or after 1 day reperfusion. Four days after ischemia, NADPH-d positive neurons and vessels were detected in the central gray matter despite well developed necrosis in this location. Regional nitric oxide synthesis and its vasodilatatory effect during the period of aortic occlusion may account for the observed selective resistance of these spinal cord neurons to transient ischemia.     

The Effect of Long-Term Reduction of Aortic Blood Flow on Spinal Cord Gray Matter in the Rabbit. Histochemical Study of NADPH-Diaphorase

1. The aim of this work was to study the influence of reduced aortic blood flow on NADPH-diaphorase (NADPH-d) staining in the gray matter of L4–S3 spinal cord segments.2. Surgery was performed on the abdominal aorta of the rabbit. Spinal cord ischemia was introduced by infrarenal aortic constriction to 30% from the normal blood flow. Animals were allowed to survive 1 week, 1 month and 3 months after surgery. Neurological outcome was studied in relation to the duration of aortic occlusion. The NADPH-d histochemistry was used for the visualisation of spinal cord sections.3. The most affected area of the spinal cord was pericentral region, and slight changes were seen in the NADPH-d activities of both dorsal and ventral horns. One week after surgery, NADPH-d positive pericentral neurons were almost unchanged in their shape and intensity of staining, the only difference was seen in slightly increased staining of the background around the central canal. One month following surgery neurons exhibited shrinkage or were swollen, NADPH-d staining was less intensive in the pericentral zone and positively stained vessels were present.4. Three months of ischemia influenced the NADPH-d activity: (a) In the pericentral region were seen intensively NADPH-d stained neurons almost normal in shape of their bodies but with shortened processes or without them; (b) NADPH-d staining of neuropil was greatly enhanced mostly around the central canal and in the dorsal commissure; (c) Numerous vessels were present in the pericentral zone and in the location of the ventral horn.5. It can be concluded that the reduction of blood flow in the abdominal aorta makes most changes in the pericentral region of the rabbit spinal cord. Increased NADPH-d staining of neuropil and the presence of positively stained vessels reflect increased NADPH-d/NOS production in the spinal cord gray matter after long-term incomplete aortic occlusion.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated,nerve‐dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF‐like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine‐rich domain (CRD‐NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD‐NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve‐dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve‐dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 150–158, 2000     

Tail regeneration and ependymal outgrowth in the adult newt, Notophthalmus viridescens, are adversely affected by experimentally produced ischemia

Spinal axons of the adult newt will regenerate when the spinal cord is severed or when the tail is amputated. Ischemia and associated hypoxia have been correlated with poor central nervous system regeneration in mammals. To test the effects of ischemia on newt spinal cord regeneration, the spinal cord and major blood vessels of the newt tail were severed 2 cm caudal to the cloaca as a primary injury. This primary injury severely reduced circulation in the caudal direction for 7 days; by day 8, circulation was largely restored. After various periods of time after primary injury, tails were amputated 1 cm caudal to the primary injury (in the area of ischemia) and tested for regeneration. If the tail was amputated within 5 days of the primary injury, regeneration did not occur. If amputation was 7 days or longer after the primary injury, a regenerative response occurred. Histology showed that in the non-regenerating tails the spinal cord and associated ependyma, known to be important to tail regeneration, had degenerated in the rostral direction. Such degeneration was prevented when tails were first amputated and allowed to form blastemas before the primary injury. The data indicate that the first 5-7 days of blastema formation are particularly sensitive to compromised blood flow (ischemia/hypoxia). It follows that mechanisms must be present in the adult newt to reduce ischemia to a minimum and thus allow ependymal outgrowth and tail regeneration.     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents.

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early- and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revealed no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Decreased Release of D-Aspartate in the Guinea Pig Spinal Cord After Lesions of the Red Nucleus

This study attempts to determine if fibers that project from the guinea pig red nucleus to the spinal cord use L-glutamate and/or L-aspartate as transmitters. Unilateral injections of kainic acid were placed stereotaxically in the red nucleus to destroy the cells of origin of the rubrospinal tract. Six days after the injection, Nissl-stained sections through the lesion site showed that the majority of neurons in the red nucleus ipsilateral to the kainic acid injection were destroyed. In addition, the lesioned area included parts of the surrounding midbrain reticular formation. Silver-impregnated, transverse sections of the cervical spinal cord revealed the presence of degenerating fibers contralaterally in laminae IV-VII of the gray matter. Ipsilaterally, very sparse degeneration was evident in laminae VII and VIII of the gray matter. Two to six days after surgery, the electrically evoked, Ca2(+)-dependent release of both D-[3H]aspartate, a marker for glutamatergic/aspartatergic neurons, and gamma-amino[14C]-butyric acid ([14C]GABA) was measured in dissected quadrants of the spinal cervical enlargement. Lesions centered on the red nucleus depressed the release of D-[3H]aspartate by 25-45% in dorsal and ventral quadrants of the cervical enlargement contralaterally. The release of [14C]GABA was depressed by 27% in contralateral ventral quadrants. To assess the contribution of rubro- versus reticulospinal fibers to the deficits in amino acid release, unilateral injections of kainic acid were placed stereotaxically in the midbrain reticular formation lateral to the red nucleus. Nissl-stained sections through the midbrain revealed the presence of extensive neuronal loss in the midbrain and rostral pontine reticular formation, whereas neurons in the red nucleus remained undamaged. In the spinal cord, degenerating axons were present ipsilaterally in laminae VII and VIII of the gray matter. Some fiber degeneration was also evident contralaterally in laminae V and VI of the gray matter. This lesion did not affect the release of either D-[3H]aspartate or [14C]GABA in the spinal cord. The substantial decrements in D-[3H]aspartate release following red nucleus lesions suggests that the synaptic endings of rubrospinal fibers mediate the release of D-[3H]aspartate in the spinal cord. Therefore, these fibers may be glutamatergic and/or aspartatergic. Because other evidence suggests that rubrospinal neurons are probably not GABAergic, the depression of [14C]GABA release probably reflects changes in the activity of spinal interneurons following the loss of rubrospinal input.     

D-Aspartate Uptake and Release in the Guinea Pig Spinal Cord After Partial Ablation of the Cerebral Cortex

This study attempts to determine if L-glutamate and L-aspartate may be transmitters of the guinea pig corticospinal tract. Unilateral ablations were made of the frontal and parietal neocortex which destroyed most of the motor and somatosensory areas in the right cerebral hemisphere. In lesioned animals, transverse sections of the cervical enlargement of the spinal cord (segments C6--T1) were stained to reveal degenerating fibers. Degeneration of axons first appeared 4 days after surgery, reached a maximum on the seventh day, and began to wane by the ninth day. The most prominent loss of axons appeared deep in the dorsal funiculus and in laminae IV-IX of the gray matter contralateral to the cortical lesion. Ipsilaterally, there was very sparse degeneration of fibers in the dorsal and ventral funiculi and in the spinal gray matter. The uptake and release of D-[3H]aspartate, a putative nonmetabolizable marker for L-glutamate and L-aspartate, were measured in dissected quadrants of the cervical enlargement taken from intact and lesioned animals. The uptake and the electrically evoked, Ca2+-dependent release of D-[3H]aspartate were depressed by 29-35% at 4 and 7 days after surgery, but only in tissue that was contralateral to the cortical ablation. The lesion had no effect on the uptake and release of exogenous gamma-[14C]aminobutyric acid, which were measured as indices of the postlesion integrity of neurons in the spinal gray matter. These findings suggest that the synaptic endings of corticospinal fibers probably mediate the uptake and release of D-[3H]aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.     

The organization of serotonin-immunoreactive neuronal systems in the brain of the crested newt,Triturus cristatus carnifex Laur.

Summary The distribution of serotonin (5-HT) immunoreactive structures has been investigated in the brain of the crested newt by means of indirect immunofluorescence, and unlabeled antibody peroxidase-antiperoxidase-complex (PAP) or biotin-avidin-system (BAS) techniques. In the newt, the bulk of the serotoninergic system extends from the raphe region of the medulla oblongata, through the isthmus, toward the mesencephalic tegmentum, and is characterized by pyriform neurons mainly located in a subependymal position, close to the midline. Also in the caudal hypothalamus, in addition to some 5-HT-positive adenohypophysial cells, many immunoreactive CSF-contacting neurons are found lining the paraventricular organ and the nucleus infundibularis dorsalis. A rich serotoninergic innervation was observed in the preoptic area and in the habenular complex. Concerning the telencephalon, immunopositive nerve fibers are encountered in the dorsal pallium, primordium hippocampi, striatum and olfactory bulbs. The general organization of serotoninergic systems in the newt brain exhibit close similarities to that described in higher vertebrates.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

Blastema cell proliferation in vitro: effects of limb amputation on the mitogenic activity of spinal cord extracts

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts of non-amputated animals in a dose-dependent manner up to 60 micrograms protein/ml of culture medium; at this concentration the mitotic index is increased 4-fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.     

Comparative analysis of NADPH-diaphorase positive neurons in the rat, rabbit and pheasant thoracic spinal cord. A histochemical study.

The distribution of NADPH-diaphorase (NADPH-d) activity was investigated and compared in the rat, rabbit and pheasant thoracic spinal cord. The investigation of all spinal cord regions (laminae) in three experimental species revealed marked differences in the distribution of NADPH-d activity. Cross sectional analysis of the spinal cord of the rat, rabbit and pheasant confirmed differences in the shape of the gray matter in all examined species. More detailed investigation of Rexed's laminas showed similar distribution of NADPH-d activity in the spinal cord of the rat and rabbit, which were different when compared with the spinal cord of the pheasant. Ventral horn of the rat and rabbit showed no labelling whereas in pheasant this area possessed a number of scattered, intensively stained neurons. In the location of autonomic preganglionic neurons, differences were found as well. In the rat there was seen a number of densely packed, clearly dark blue coloured neurons. Similarly, these neurons were present in the rabbit spinal cord but they were less numerous. No staining was found in this region of pheasant. Pericentral area (lamina X) and intermediate zone (laminaVII) revealed the presence of NADPH-d positive neurons in all examined species although they differed in number and shape of their bodies. The dorsal horn showed the presence of NADPH-d staining in all three animals but its distribution was different in medio-lateral direction. It can be suggested that observed differencies in the presence and distribution of NADPH-d activity across the examined species may reflect different fylogenetic development.     

Increased protein kinase C activity in the central nervous system of the newt during limb regeneration.

Protein kinase C (PKC) activity was examined in the CNS of the newt Pleurodeles waltlii undergoing regeneration after limb amputation. In the spinal cord and brain of control newts, the level of PKC activity was virtually the same for the cytosolic and the particulate fractions. At days 7 and 14 after amputation of two limbs, a twofold increase in overall PKC activity occurred in the spinal cord and accounted for increased membrane-bound activity, while cytosolic activity was not significantly impaired. In contrast, overall PKC activity was not affected in brain. However, a twofold increase in the brain particulate fraction occurred at day 14 while cytosolic activity decreased proportionately. Similar alterations were observed in newts undergoing one or multiple limb amputations. Such changes in PKC activity neither occurred in the CNS of newt after limb denervation nor in the CNS of limb amputated frog Rana temporaria, an Amphibian which is unable to regenerate. Taken together, these results provide evidence that PKC of the CNS is involved in the regeneration process of newts. Changes in activation-associated PKC distribution proceeded through different mechanisms: long-lasting increase in membrane bound activity with a net increase of overall activity in the spinal cord, and long-term redistribution of enzyme activity to the particulate fraction in brain.     

Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructural and immunohistochemical studies of the origin of the cells.

In the regenerating newt tail, epimorphic regeneration--which recapitulates morphologically normal embryonic development--proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca++ independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways.     

Lack of evidence for cell death among avian spinal cord interneurons during normal development and following removal of targets and afferents

Chick embryos and posthatched chicks were examined at several ages for the presence of pyknotic interneurons in the lumbar spinal cord. Because no pyknotic interneurons were found, direct cell counts of healthy interneurons were carried out and a comparison made between early-and late-stage embryos and hatchlings. There was no decrease in the number of interneurons in the ventral intermediate gray matter of the spinal cord between embryonic day (E) 8 and 2 weeks posthatching (PH) or in the dorsal horn between E10 and 2 weeks PH. To study whether interneuron survival is regulated by targets or afferents, a situation known to exist in other developing neural populations, early embryos were subjected to (1) removal of one limb, resulting in the loss of lateral motor column motoneurons and dorsal root ganglion sensory afferents; (2) transection of the thoracic spinal cord, thereby removing both descending afferents and rostral targets of spinal interneurons, or (3) a combination of the two operations. No reductions in interneuron numbers were found as a result of these operations. Furthermore, morphometric analysis also revaled no change in neuronal size following these experimental manipulations. By contrast, there was a slight decrease in the total area of spinal gray matter that was most prominent in the dorsal region following limb bud removal. Our results indicate (1) that spinal interneurons fail to exhibit the massive naturally occurring death of postmitotic neurons that has been observed for several other populations of spinal neurons, and (2) spinal interneurons appear to be relatively resistant to induced cell death following the removal of substantial numbers of afferent inputs and targets.     

Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system

We prepared a monoclonal antibody to microtubule-associated protein 1 (MAP 1), one of the two major high molecular weight MAP found in microtubules isolated from brain tissue. We found that MAP 1 can be resolved by SDS PAGE into three electrophoretic bands, which we have designated MAP 1A, MAP 1B, and MAP 1C in order of increasing electrophoretic mobility. Our antibody recognized exclusively MAP 1A, the most abundant and largest MAP 1 polypeptide. To determine the distribution of MAP 1A in nervous system tissues and cells, we examined tissue sections from rat brain and spinal cord, as well as primary cultures of newborn rat brain by immunofluorescence microscopy. Anti-MAP 1A stained white matter and gray matter regions, while a polyclonal anti-MAP 2 antibody previously prepared in this laboratory stained only gray matter. This confirmed our earlier biochemical results, which indicated that MAP 1 is more uniformly distributed in brain tissue than MAP 2 (Vallee, R.B., 1982, J. Cell Biol., 92:435-442). To determine the identity of cells and cellular processes immunoreactive with anti-MAP 1A, we examined a variety of brain and spinal cord regions. Fibrous staining of white matter by anti-MAP 1A was generally observed. This was due in part to immunoreactivity of axons, as judged by examination of axonal fiber tracts in the cerebral cortex and of large myelinated axons in the spinal cord and in spinal nerve roots. Cells with the morphology of oligodendrocytes were brightly labeled in white matter. Intense staining of Purkinje cell dendrites in the cerebellar cortex and of the apical dendrites of pyramidal cells in the cerebral cortex was observed. By double-labeling with antibodies to MAP 1A and MAP 2, the presence of both MAP in identical dendrites and neuronal perikarya was found. In primary brain cell cultures anti-MAP 2 stained predominantly cells of neuronal morphology. In contrast, anti-MAP 1A stained nearly all cells. Included among these were neurons, oligodendrocytes and astrocytes as determined by double-labeling with anti-MAP 1A in combination with antibody to MAP 2, myelin basic protein or glial fibrillary acidic protein, respectively. These results indicate that in contrast to MAP 2, which is specifically enriched in dendrites and perikarya of neurons, MAP 1A is widely distributed in the nervous system.