Islet-1 expression in thoracic spinal motor neurons in prenatal mouse

The LIM homeodomain protein Islet-1, an embryonic marker for motoneurons in the spinal cord, has been reported to be heterogeneously expressed among motoneuron groups in mouse. In the present study, we examined Islet-1 expression in the thoracic and rostral lumbar spinal cord in prenatal mice. In the thoracic spinal cord at embryonic day 12.5 (E12.5) and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, whereas weaker immunoreactivity was observed in the ventral group. Strong Islet-1 immunoreactivity was also observed in the intermediolateral area and more medial part of the intermediate zone. In the rostral lumbar spinal cord at E12.5 and E13.5, strong Islet-1 immunoreactivity was observed in the lateral group of the ventral horn, and in the intermediolateral nucleus, whereas weaker immunoreactivity was observed in the ventral, and dorsolateral groups. At E14.5, the number of Islet-1 immunoreactive neurons was reduced in the spinal cord, but the distribution pattern was similar to that at E12.5 and E13.5. At E15.5, Islet-1 immunoreactivity was almost completely confined to the intermediolateral area. Some weakly immunoreactive neurons were observed in the ventral horn. The findings of the present study indicated that Islet-1 expression at embryonic stages differs among the motoneuron groups in the thoracic and rostral lumbar spinal cord.     

Inducible nitric oxide synthase (iNOS) and nitrotyrosine immunoreactivity in the spinal cords of transgenic mice with a G93A mutant SOD1 gene

We performed a prospective, longitudinal immunohistochemical study of the spinal cords of transgenic mice with a G93A mutant SOD1 gene at 4 fixed points in time, using antibodies to inducible nitric oxide synthase (iNOS) and nitrotyrosine. The purpose of this study was to characterize the temporal and topographic distribution of iNOS and nitrotyrosine immunoreactivity in the spinal cord over a certain period, thus illuminating the possible role of increased oxidative damage to the motor system in the neurodegenerative process in this animal model. Specimens from age-matched non-transgenic wild-type mice served as controls. The control mice showed no positive iNOS or nitrotyrosine immuunoreactivity in the somata of anterior horn neurons or their neuronal processes at any age. On the other hand, the transgenic mice demonstrated a common immunostaining pattern of iNOS and nitrotyrosine in the anterior horn neurons. When the mice reached the age of 24 wk (early presymptomatic stage), the anterior horn neurons and their neuronal processes were occasionally immunostained for iNOS and nitrotyrosine; at 28 wk (late presymptomatic stage), the anterior horn neurons were not uncommonly immunostained; at 32 wk (early symptomatic stage) and 35 wk (end-stage), positive iNOS and nitrotyrosine immunoreactivity was frequently observed in proliferated reactive astrocytes as well as in the somata of the anterior horn cells. The selective localization of positive iNOS and nitrotyrosine immunoreactivity in the anterior horn neurons suggests that oxidative stress may be involved in the pathomechanism of degeneration of motor neurons in this transgenic animal model.     

Protein‐bound crotonaldehyde accumulates in the spinal cord of superoxide dismutase‐1 mutation‐associated familial amyotrophic lateral sclerosis and its transgenic mouse model

Growing evidence documents oxidative stress involvement in ALS. We previously demonstrated accumulation of a protein‐bound form of the highly toxic lipid peroxidation product crotonaldehyde (CRA) in the spinal cord of sporadic ALS patients. In the present study, to the determine the role for CRA in the disease processes of superoxide dismutase‐1 (SOD1) mutation‐associated familial ALS (FALS), we performed immunohistochemical and semiquantitative cell count analyses of protein‐bound CRA (P‐CRA) in the spinal cord of SOD1‐mutated FALS and its transgenic mouse model. Immunohistochemical analysis revealed increased P‐CRA immunoreactivity in the spinal cord of the FALS patients and the transgenic mice compared to their respective controls. In the FALS patients, P‐CRA immunoreactivity was localized in almost all of the chromatolytic motor neurons, neurofilamentous conglomerates, spheroids, cordlike swollen axons, reactive astrocytes and microglia, and the surrounding neuropil in the affected areas represented by the anterior horns. In the transgenic mice, P‐CRA immunoreactivity was localized in only a few ventral horn glia in the presymptomatic stage, in almost all of the vacuolated motor neurons and cordlike swollen axons and some of the ventral horn reactive astrocytes and microglia in the onset stage, and in many of the ventral horn reactive astrocytes and microglia in the advanced stage. Cell count analysis on mouse spinal cord sections disclosed a statistically significant increase in the density of P‐CRA‐immunoreactive glia in the ventral horns of the young to old G93A mice compared to the age‐matched control mice. The present results indicate that enhanced CRA formation occurs in motor neurons and reactive glia in the spinal cord of SOD1‐mutated FALS and its transgenic mouse model as well as sporadic ALS, suggesting implications for CRA in the pathomechanism common to these forms of ALS.     

Tardive decrease of astrocytic glutamate transporter protein in transgenic mice with ALS-linked mutant SOD1

The expressions of glutamate transporter proteins were immunocytochemically examined in the spinal cord of transgenic mice harboring a Gly93 --> Ala (G93A) mutant human SOD1 gene. Astroglial EAAT2 protein level was preserved in the ventral horn even after the beginning of paralysis, and finally decreased at terminal stage of the disease (35 weeks of age), when neuronal EAAT3 protein level was also decreased. In contrast, glial fibrillary acidic protein (GFAP) immunoreactivity progressively increased from 25 weeks of age in the ventral horn. The present results show interesting dissociative expressions of astroglial proteins EAAT2 and GFAP in the same ventral horn, but suggest not an early and primary role of EAAT2 in the motoneuronal death of this model.     

Neuronal nitric oxide synthase (nNOS) immunoreactivity in the spinal cord of transgenic mice with G93A mutant SOD1 gene

Immunohistochemical and quantitative analyses were used to examine the evolution of neuronal nitric oxide synthase (nNOS) with time in spinal motor neurons of transgenic mice with a G93A mutant Cu/Zn superoxide dismutase (SOD1) gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the controls, the anterior horn including the anterior horn neurons was not immunostained for nNOS. In the transgenic mice, at the age of 24 weeks (early presymptomatic), when no pathological change was observed in the spinal cord, anterior horn neurons were only occasionally immunostained for nNOS (0.3%). At the age of 28 weeks (late presymptomatic), nNOS-positive anterior horn neurons and their neuronal processes were occasionally observed (7.6%), and at the age of 32 weeks (early symptomatic), nNOS-positive anterior horn cells, including degenerated ones showing central chromatolysis, were frequently demonstrated (27.6%) and nNOS-positive cord-like swollen proximal axons were also observed in the anterior horns. nNOS expression in the anterior horn neurons was almost always observed in the somata. At the age of 35 weeks (end stage), neuronal loss of the anterior horn cells was severe, and nNOS-positive anterior horn neurons and cord-like swollen axons in the anterior horns were less prominent compared to those at the age of 32 weeks (33.8%), but many reactive astrocytes were immunostained for nNOS. Thus, nNOS immunoreactivity in the anterior horn neurons is observed as early as the presymptomatic stage and varies with the progression of the disease. The selective localization of positive nNOS immunoreactivity in the anterior horn neurons and degenerated ones in particular, and swollen proximal axons suggests that nNOS immunoreactivity may be involved in the degeneration of anterior horn neurons in this SOD1 transgenic mouse model.     

Changing pattern of expression of parvalbumin immunoreactivity during human fetal spinal cord development

Expression of the calcium binding protein parvalbumin (PV) by different classes of spinal neuron has been shown to be developmentally regulated in both rat and monkey. From postmortem studies of eight human cervical spinal cords ranging in age from 11 to 35 weeks postconceptional age, we report that parvalbumin immunoreactivity is similarly plastic in human lower cervical spinal cord development, with many changes occurring prenatally. At 11-14 weeks postconceptional age, there was prominent immunostaining of primary sensory afferents that could be seen coursing through the dorsal horn and extensively innervating the motoneuron pools. Motoneurons were also found to be clearly immunoreactive for choline acetyltransferase by this age. A few ventral horn neurons that were not motoneurons were also parvalbumin immunoreactive. By 24-27 weeks postconceptional age, sensory afferents were still immunoreactive, as were many other axons throughout the white matter. In addition, many ventral horn neurons were now immunoreactive as well as a few dorsal horn neurons. By 31-35 weeks postconceptional age, there was extensive immunostaining of neurons throughout the spinal cord, including a few moderately immunoreactive motoneurons. There were many immunopositive axons in all the white matter tracts except the corticospinal tracts; however, staining of sensory axons traversing the grey matter was less prominent by this age. In the rat, expression of PV by primary sensory neurons coincides with the onset of fetal limb movement. The onset of expression of PV in ventral horn neurons coincides with later developmental events after the arrival of corticospinal inputs, whereas widespread PV immunoreactivity in dorsal horn neurons marks the attainment of a mature pattern of PV expression. The extent to which expression of PV immunoreactivity can be taken to indicate landmarks in human development will be discussed.     

Expressions of nitrotyrosine and TUNEL immunoreactivities in cultured rat spinal cord neurons after exposure to glutamate, nitric oxide, or peroxynitrite

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanism is not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosine (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number greatly increased at 48 hr in contrast to the vehicle. Pretreatment of cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulation of NT nitration and the apoptotic pathway in cultured rat spinal neurons.     

Transient ischemia-induced changes of excitatory amino acid carrier 1 in the ventral horn of the lumbar spinal cord in rabbits

OBJECTIVES: To examine temporal changes of EAAC1 immunoreactivity and its protein level in the spinal ventral horn after transient ischemia in the rabbit to investigate the correlation between neuronal cell death and EAAC1 in the ventral horn of spinal cord. METHODS: White rabbits weighing 2.5-3.0 kg were anesthetized with a mixture of 2.5% isoflurane in 30% oxygen and 70% nitrous oxide, and the abdominal aortic artery below the left renal artery was occluded for 15 minutes. At designated times after reperfusion, the immunohistochemical and Western blot analysis for EAAC1 was conducted using tissues of the seventh lumbar spinal segment. RESULTS: EAAC1 immunoreactivity was detected in the neurons of the normal spinal cord. EAAC1 immunoreactivity and protein level reduced significantly 30 minutes after ischemia/reperfusion, but EAAC1 immunoreactivity and protein level again increased by 80% versus sham 3 hours after ischemia. At this time point, neurological defect in hindlimb was also detected. Thereafter, EAAC1 immunoreactivity and protein levels remained to be attenuated in the ventral horn of spinal cord until 48 hours after ischemia. CONCLUSION: The significant change in EAAC1 expression and motor defects at early time after transient spinal cord ischemia relates to the acute events following ischemia/reperfusion. These results indicate that EAAC1 has an important role in the modulation of glutamate homeostasis in ischemic neurons in the spinal ventral horn.     

Development changes in the distribution of gamma-aminobutyric acid-immunoreactive neurons in the embryonic chick lumbosacral spinal cord

The development of γ-aminobutyric acid (GABA)-immunoreactive neurons was investigated in the embryonic and posthatch chick lumbosacral spinal cord by using pre- and postembedding immunostaining with an anti-GABA antiserum. The first GABA-immunoreactive cells were detected in the ventral one-half of the spinal cord dorsal to the lateral motor exception of the lateral motor column, appeared throughout the entire extent of the ventral one-half of the spinal gray matter by E6. Thereafter, GABA-immunoreactive neurons extended from ventral to dorsal regions. Stained perikarya first appeared at E8 and then progressively accumulated in the dorsal horn, while immunoreactive neurons gradually declined in the ventral horn. The general pattern of GABA immunoreactivity characteristic of mature animals had been achieved by E12 and was only slightly altered afterwards. In the dorsal horn, most of the stained neurons were observed in laminae I–III, both at the upper (LS 1–3) and at the lower (LS 5–7) segments of the lumbosacral spinal cord. In the ventral horn, the upper and lower lumbosacral segments showed marked differences in the distribution of stained perikarya. GABAergic neurons were scattered in a relatively large region dorsomedial to the lateral motor column at the level of the upper lumbosacral segments, whereas they were confined to the dorsalmost region of lamina VII at the lower segments. The early expression of GABA immunoreactivity may indicate a trophic and synaptogenetic role for GABA in early phases of spinal cord development. The localization of GABAergic neurons in the ventral horn and their distribution along the rostrocaudal axis of the lumbosacral spinal cord coincide well with previous physiological findings, suggesting that some of these GABAergic neurons may be involved in neural circuits underlying alternating rhythmic motor activity of the embryonic chick spinal cord. © 1994 Wiley-Liss, Inc.     

Beta-amyloid 42 accumulation in the lumbar spinal cord motor neurons of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of large motor neurons in the brain and spinal cord. Amyloid precursor protein (APP), the transmembrane precursor of beta-amyloid (A beta), accumulates in the anterior horn motor neurons of ALS patients with mild lesions. APP undergoes an alternative proteolysis mediated by caspase-3, which is activated in motor neurons in a mouse model of ALS. The ALS spinal cord motor neurons also show evidence of increased oxidative damage, which is thought to alter APP processing. We sought to determine whether A beta42, the more pathogenic A beta species, accumulates in the postmortem lumbar spinal cord of ALS patients. While there was little or no A beta42 labeling in control spinal cord tissues, elevated A beta42 immunoreactivity occurred in ALS motor neuronal perikarya and axonal swellings in the anterior horn. A few A beta42-positive neurons exhibited thioflavine S staining. No extracellular A beta42 deposits were found. A beta42 coexisted with the oxidative damage markers malondialdehyde, 8-hydroxydeoxyguanosine, heme oxygenase-1, and nitrotyrosine in abnormal neurons. The neurons with intracellular A beta42 accumulation also displayed robust cleaved caspase-3 immunoreactivity. Very little A beta40 immunoreactivity occurred in motor neurons of both control and ALS. These results suggest that aberrant accumulation of A beta42 in ALS spinal cord motor neurons is associated with oxidative stress, and may play a role in the pathogenesis of neurodegeneration in ALS.     

Oxidative damage and breakage of DNA in rat brain after transient MCA occlusion.

As thrombolytic therapy for treatment of ischemic stroke was propagated, much attention has been paid to reperfusion brain injury. Oxidative stress is one of the most important factors that exacerbate tissue damage by reperfusion. Thus, we investigated the extent of oxidative damage in rat brain after transient middle cerebral artery (MCA) occlusion by immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is one of the best markers of oxidative damage. Furthermore, in order to investigate its role in neuronal cell death, we performed terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) study, and compared the results with that of 8-OHdG immunohistochemistry. There was no immunoreactive 8-OHdG in sham-operated brain, but it became present in neurons of MCA territory at 3 h of reperfusion after 90-min ischemia. At 48 h after reperfusion, cerebral tissue of MCA territory was severely destroyed, and many cells in that area revealed TUNEL positivity. Some neurons in MCA territory showed mild immunoreactivity for 8-OHdG at that time, but it was strongest in neurons in the outer area of MCA territory. Those cells did not show TUNEL positivity, suggesting that 8-OHdG production is not necessarily followed by early cell death. Here, it was demonstrated that oxidative DNA damage occurs in more extended area than that where cell death is recognized. Although this damage does not cause early cell death, this might result in more prolonged cell dysfunction and eventual neuronal loss. Anti-oxidant therapy might be required for treatment of stroke in the future.     

Immunohistochemical detection of oxidative DNA damage induced by ischemia–reperfusion insults in gerbil hippocampus in vivo

There is much evidence to suggest that ischemic injury occurs during the reperfusion phase of ischemia–reperfusion insults, and that the injury may be due to reactive-oxygen-species (ROS)-mediated oxidative events, including lipid peroxidation and DNA damage. However, oxidative DNA damage has until now not been examined in situ. In the present study, we report for the first time observation of cell type- and region-specific oxidative DNA damages in 5 min transient ischemic model by immunohistochemical methods, using monoclonal antibody against 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative DNA product. The cell types containing 8-OHdG immunoreactivity were neurons, glia and endothelial cells in the hippocampus. The 8-OHdG immunoreactivity was present in the nucleus but not the cytoplasm of these cells. The level of 8-OHdG in CA1 increased significantly (P<0.05) at the end of 30 min after ischemia, but there was no increase within CA2 and CA3 areas. The 8-OHdG levels in the hippocampus increased significantly (about fourfold) after 3 h of reperfusion and remained significantly (P<0.01) elevated for at least 12 h. At 4 days after ischemia, 8-OHdG levels in the CA2 and CA3 areas decreased to levels of the sham without neuronal loss, while disappearance of 8-OHdG immunoreactivity in the CA1 coincided with neuronal death in this area. These findings strongly suggest that ischemia-induced DNA damage evolves temporally and spatially, and that oxidative DNA damage may be involved in delayed neuronal death in the CA1 region.     

Expressions of caspase-3, Tunel, and Hsp72 immunoreactivities in cultured spinal cord neurons of rat after exposure to glutamate, nitric oxide, or peroxynitrite

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanisms are not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) or 72kDa heat shock protein (HSP72). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immu-noreactivity for caspase-3 became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 24h. TUNEL positive nuclei were found in spinal large motor neurons from 24 h, and the positive cell proportion greatly increased at 48 h in contrast to the vehicle. On the other hand, the immunoreactivity of HSP72 in the ventral horn was already positive at 0 h, and gradually decreased in the course of time with glutamate, NOC18 or PN than vehicle treatment. In the dorsal horn, the proportion of caspase-3 positive small neurons greatly increased at 6 to 48 h after addition of glutamate. The present results suggest that both excitotoxic and oxidative stress play important role in the apoptotic pathway in cultured rat spinal neurons.     

Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: a model for motoneuron disease

The mutant mouse, wobbler, possesses a recessively inherited degeneration of motoneurons and other ventral horn cells in the cervical spinal cord, and therefore it has been proposed as an animal model of human motoneuron disease. Affected mice have been identified by behavioral tests that also determined the extent of the motor deficit. The results from these tests were combined and used to define distinct stages of the disease process that could then be correlated histochemically with the amount of acetylcholinesterase (AChE) staining in the cervical spinal cord. AChE is used as a marker for cholinergic neurons and is known to hydrolyze the neuropeptide modulator substance P (SP). SP, a peptide neuromodulator of primary afferent transmission in the dorsal horn, excites motoneurons in the ventral horn, and may possess secondary functions in neuronal maintenance. Therefore, the levels of immunoreactive (IR) SP and AChE were examined in an attempt to determine the possible interaction between these factors in motoneuron degeneration. By enzyme histochemistry, the cervical spinal cord, taken from wobbler mice at behaviorally identified stages of the motor deficit, exhibited decreased levels of AChE throughout the ventral horn. The decrease detected in the AChe staining intensity was linear and correlated with the decrease in the number of AChE-positive cells in the ventral cervical spinal cord, as the motor deficit progressed. Presumably, the continual decrease in AChE staining represents the degeneration of cholinergic perikarya and neuronal processes in the ventral horn as the motoneuron disease proceeds. At two well-established stages of the motor deficit, the amount of immunoreactive SP increased in the ventral horn compared with the control mice. The elevated levels of immunoreactive SP suggest sprouting may have occurred preceding, or in response to, the motoneuron degeneration. Several additional hypotheses are discussed in respect to phenomena that might contribute to the increase of immunoreactive SP in the degenerating ventral horn of the wobbler mouse.     

Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene

Immunohistochemical study was performed to examine if calcium-binding proteins are involved in the degeneration of motor neurons in the brain stems and the spinal cords of transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn neurons in the spinal cord. On the other hand, calbindin D-28k immunoreactivity was much less apparent than that observed with parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k, whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn neurons were severely reduced, even at the presymptomatic stage, whereas calbindin-positive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1-transgenic mouse model.     

Development of Calbindin-D28k Immunoreactive Neurons in the Embryonic Chick Lumbosacral Spinal Cord

The development of immunoreactivity for the calcium-binding protein calbindin-D28k (CaB) was investigated in the embryonic and hatched chick lumbosacral spinal cord. CaB-immunoreactive neurons were revealed in the dorsal and ventral horns as well as in the intermediate grey matter from early stages of neuronal development. CaB immunoreactivity was first detected in large neurons in the presumptive dorsal horn at embyronic day 5, while small neurons in the lateral dorsal horn were the last to appear, at embryonic day 10. We have identified and traced the morphological maturation of six CaB-immunoreactive cell groups, three in the dorsal horn and three in the ventral horn. In the dorsal horn these groups were (1) large neurons in the lateral dorsal horn (laminae I and IV), (2) small neurons in the lateral dorsal horn (lamina II), and (3) small neurons in the medial dorsal horn (lamina III). All three groups were present throughout the entire length of the lumbosacral spinal cord and showed persistent CaB immunoreactivity. In the ventral horn, CaB-immunoreactive neurons were classified into the following three categories: (1) Neurons dorsal to the lateral motor column (lamina VII). These neurons were present exclusively in the upper lumbosacral segments (LS1 – 3), and they showed steady CaB immunoreactivity during their maturation. (2) Neurons at the dorsomedial aspect of the lateral motor column (at the border of laminae VII and IX). This population of neurons was characteristic of the lower segments of the lumbosacral cord (LS5 – 7) and presented transient CaB expression. (3) Neurons within the lateral motor column (lamina IX). These neurons were dispersed throughout the length of the lumbosacral spinal cord. They were three to four times more numerous in the upper than in the lower lumbosacral segments, and their numbers declined throughout LS1 – 7 as the animal matured. The characteristic features of the development of neurons immunoreactive for CaB are discussed and correlated with previous neuroanatomical and physiological studies concerning sensory and motor functions of the developing chick spinal cord.     

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.     

The development of the cervical spinal cord of the mouse embryo. I. A Golgi analysis of ventral root neuron differentiation

No previous Golgi studies have described early neuron differentiation in mammals near the time of neural tube closure. By leaving embryos in utero and varying the impregnation times, the cells of the cervical spinal cord of mice of 8–11 days of gestation (E8–E11, where E0 = day vaginal plug observed) were stained and analyzed in the present study. The early stages of ventral root motoneuron differentiation described earlier (Wentworth and Hinds, '78), using serial section electron microscopy, have been confirmed in this study and include preaxonic neuroblasts (stage 1–detached from the ventrical but still attached to the basal lamina), bipolar neuroblasts (stage 2), and unipolar neuroblasts (stage 3). Later stages of differentiation described in this study include young neurons with dendrites: secondary bipolar (stage 4) and multipolar neurons (stage 5). At E8 the neural tube is just closing and consists of undifferentiated ventricular cells. On E9 a few ventral root neuroblasts are differentiating in the cervical spinal cord. The majority of these cells are in the bipolar and unipolar neuroblast stages, although the most advanced cells observed had reached the secondary bipolar neuron stage of differentiation. By E10 a large cluster of ventral root motoneurons is differentiating in the ventrolateral cord. Some cells have reached the bipolar stage, a few cells are becoming multipolar, and a subdivision into dorsolateral and vrntromedial subgroups is beginning. By E11 the medial and lateral subdivisions are more obvious, and the most advanced cells are well-differentiated multipolar neurons oriented either mediolaterally or dorsoventrally, although cells in the earlier unipolar and secondary bipolar stages are still observed.     

Age-related changes in peroxisomal membrane protein 70 and superoxide dismutase 1 in transgenic G93A mice

Abstract

Peroxisomal membrane protein 70 (PMP70) and Cu/Zn superoxide dismutase (SOD1) were examined in the spinal cords of transgenic (Tg) mice expressing a human mutant SOD1 protein (G93A) and their agematched controls at 8, 20 and 32 weeks by immunohistochemistry. At pre-symptomatic 20 weeks and symptomatic 32 weeks, PMP70 was reduced in the cytoplasm of motor neurons in Tg animals and increased in glial cells in anterior horn at late age. SOD1 showed a progressive increase of dot-like deposits in the neuropil of anterior horn of Tg mice, and a late decrease of signal intensity in the white matter and motor neurons at 32 weeks. It is conceivable that reduction of PMP70 might underlie decrease in peroxisomal functions and increase in oxidative stress that is well documented in this animal model.     

Spinal cord afferent systems containing the nerve terminal protein NT75

In the adult spinal cord, immunocytochemical staining for NT75 is concentrated in nerve terminals in the superficial laminae of the dorsal horn. Deeper laminae of the dorsal horn contain moderate immunocytochemical labeling, but the ventral horn is only sparsely stained. The origin of spinal nerve terminals containing NT75 was investigated with lesion techniques, colchicine treatment, and retrograde tracing in combination with immunocytochemical staining. Primary afferent neurons express NT75 immunoreactivity and account for most of the dense staining in the superficial dorsal horn and part of the labeling in the deeper laminae. It was found that corticospinal and virtually all brainstem neurons with descending projections to the spinal cord also express NT75 immunoreactivity, including those terminating in the ventral horn. Colchicine treatment of the spinal cord also resulted in NT75 staining in most, if not all, spinal neurons. It appears that neurons in all three major sources of spinal afferents (primary sensory, descending, and intrinsic systems) can express NT75 immunoreactivity, but that some neurons normally contain higher levels of the protein in their nerve terminals. Previous analysis of developing spinal cord has shown widespread, dense NT75 labeling throughout the spinal gray in the early postnatal period, which later becomes restricted to the adult pattern. These studies support the hypothesis that many spinal pathways express high levels of NT75 immunoreactivity during development, but that only certain pathways maintain high levels in the adult. © 1993 Wiley-Liss, Inc.