Motor Neurons In Anterior Horn Research Articles

Evaluation of neuronal loss, astrocytosis and abnormalities of cytoskeletal components of large motor neurons in the human anterior horn in aging.

In order to identify possible morphological changes which occur in the anterior horn of normal individuals during aging, 40 controls with no neurological disease were studied. Brain and spinal cord tissue was processed according to a brain banking protocol. Controls were grouped according to age in 10 year intervals. Serial sections (20 microm) of formalin fixed, paraffin-embedded tissue were obtained, from each cervical, thoracic and lumbar spinal cord segment. Every 5th section (until 2 mm) was stained with haematoxylin and eosin and the numbers of motor neurons in the anterior horn counted at x400 magnification. Descriptive statistical analysis was performed using the SPSS program. Parallel sections (5 microm) of the same spinal segments were immunostained with a panel of antibodies including glial fibrillary acidic protein (GFAP), tau, ubiquitin and two phosphorylated neurofilaments subunits. Significant neuronal loss with aging was found by regression line analysis where three equations were used to calculate the number of motor neurons by age in each spinal segment. In 24/40 cases spheroids were observed and they were more numerous in the lumbar segment. GFAP staining revealed a distinctive cellular pattern in the anterior horn of oldest cases. Large and intensely stained astrocytes were seen in the anterior horn of cases aged over 75 years. The number of astrocytes increased progressively with age up to 70 years. Some of the changes observed in the present study may be the result of a selective vulnerability of large motor neurons to aging which could play an important role in the progression of MND. Most of these changes may also have similar pathophysiological mechanisms.

Distribution of B-50(GAP-43) mRNA and protein in the normal adult human spinal cord.

B-50(GAP-43) is a phosphoprotein mainly found in the nervous system which plays a major role in neurite growth during development and regeneration as well as in synaptic remodelling. In the mature intact central nervous system, intense B-50 immunoreactivity (B-50-IR) can still be detected in regions which maintain residual capacity for structural re-organization. B-50 expression has been studied extensively in laboratory animals; however, its distribution and regulation in the human spinal cord is largely unknown. As a first step to analyze lesion-induced structural alterations, we investigated the distribution of B-50 protein and mRNA in the normal adult human spinal cord and dorsal root ganglia. Intense B-50-IR was localized to the superficial laminae of the dorsal horn at all segmental levels, the intermediolateral nucleus at thoracic levels and Onuf's nucleus at sacral levels. Scattered neurons, particularly in the ventral horn of lumbar and sacral segmental levels (and occasionally also in Clarke's nucleus) displayed intense B-50-IR in close apposition to the perikaryal and proximal dendritic surfaces. Nonradioactive in situ hybridization indicated that B-50 mRNA could also be detected in neurons of the ventral horn and also in the intermediolateral nucleus. The distribution of B-50 mRNA and protein in the normal human spinal cord shows a marked similarity to that reported in experimental animals, including the selective labelling of Onuf's nucleus. However, the strong B-50-IR on the surface of some large anterior horn motor neurons has not been observed in other mammals. This finding might reflect a particular state of readiness for synaptic plasticity.

Selective involvement of large motor neurons in the spinal cord of rats treated with methylmercury

Mercury is thought to be a possible epidemiological factor for the pathogenesis of motor neuron disease, since it has been reported that metallic, inorganic and organic mercury causes a syndrome clinically resembling amyotrophic lateral sclerosis. We administered 10 mg/kg/day methylmercury chloride to adult rats for 10 consecutive days. The hind-limbs became flaccid and atrophic, and 14 out of the 34 rats had died by the 18th day after methylmercury treatment began. Light microscopical examination of the large motor neurons in the spinal anterior horn revealed cytoplasmic vacuolation and loss of Nissl substance on the 14th day, and neuronophagia appeared on the 16th day. On the 18th day, the loss of large motor neurons was almost complete, whereas small to medium-sized neurons were preserved. Silver acetate autometallography to detect mercury revealed the selective accumulation of mercury in the large motor neurons. These findings suggest that although a high dose is required, organic mercury can cause the definite loss of large spinal motor neurons in rats.

The Paralysé Mouse Mutant: A New Animal Model of Anterior Horn Motor Neuron Degeneration

The survival and morphometric characteristics of lumbar spinal motoneurons were examined in the paralysé mouse mutant. Affected (par/par) mice can be first recognized at approximately postnatal day (PN) 7 to 8 and are characterized by their smaller-than-normal body size, a progressive generalized muscle weakness, and lack of coordination. Mutant mice die by PN16-18, when they have become almost completely paralyzed. Previously, we have shown that this mutation involves alteration of several developmental aspects of the neuromuscular system. However, whether ventral (or anterior) horn motoneurons degenerate and die during the course of the disease was unknown. We report here that at the time the mutant phenotype can be first identified (i.e. approximately PN8), lumbar motoneuron numbers in the lateral motor column of the spinal cord of paralysé mice were not significantly different from those of control littermates. In contrast, by PN14, there was a significant (30 to 35%) decrease in motoneuron numbers in mutant compared to control mice. Furthermore, motoneuron (nuclear and soma) sizes were significantly decreased in the mutants at both stages examined, i.e. PN8 and PN14. These results show that the paralysé mutation involves atrophy and subsequent death of anterior horn motoneurons. Together with the rapid progression and the severity of the disease, these results suggest that the paralysé mouse may represent a good animal model for studying early-onset human motor neuron diseases such as spinal muscular atrophy.

Lethal arthrogryposis with anterior horn cell disease

Fifteen infants (11 families) with lethal arthrogryposis and anterior horn motor neuron loss are described. The clinical presentation was the fetal akinesia deformation sequence (FADS) with multiple contractures and facial anomalies. At autopsy neurogenic muscular atrophy was present in all infants. The spinal cord showed a paucity of anterior horn motor neurons in the 12 infants studied. Both male and female infants were affected. Nine cases were sporadic, whereas in two families there were three affected cases. Consanguinity between the parents was reported in one family with one affected child. This and the recurrence of the condition speak for autosomal recessive inheritance. Detailed neuropathological examination and documentation of the clinical features are needed for a better delineation of and genetic counseling for perinatally lethal arthrogryposis.

Assignment of the human 8.5 H gene to chromosome 5, region 5q35

The human 8.5 H probe was isolated from a human cerebellum cDNA library with a probe corresponding to the coding region of the murine 8.5 M cDNA. This cDNA isolated from a murine cDNA library constructed from newborn cerebral hemispheres was selected because of its strong expression in embryonic neurons. Consequently the corresponding human gene could be a candidate for hereditary neurodegenerative diseases. The human 8.5 H gene was assigned by somatic hybrid analysis to chromosome 5; this chromosome contains the gene(s) for spinal muscular atrophy (SMA), a group of heritable degenerative diseases that selectively affect the anterior horn motor neuron of the spinal cord. The localization by in situ hybridization of 8.5 H on 5q35 excluded the possibility that this gene is identical to SMA. The SMA gene(s) was (were) known, from linkage analysis, to be in a region (5q11.2-q13.3) very distant from 5q35.

Some observations on the localization of hyaluronic acid in adult, newborn and embryonal rat brain

Hyaluronic acid was localized in acetone-fixed cryostat sections of brain and spinal cord obtained from adult, newborn and embryonal rat. The sections were incubated with glial hyaluronatebinding protein (GHAP) of human origin and the protein was visualized by indirect immunofluorescence with monoclonal antibodies raised to human GHAP and not staining rat brain by immunofluorescence. GHAP is a brain extracellular matrix (ECM) glycoprotein, approximately 60,000 molecular weight, which is structurally related to the HA-binding region of cartilage ECM proteins. The distribution of hyaluronate in adult brain white matter and cerebellar cortex was similar to that previously reported for GHAP. In both cases, the reaction product formed a mesh surrounding myelinated axons and granule cells. Hyaluronate was also found in parts of the brain that did not contain GHAP. A finely reticulated mesh was observed in the neuropil between cell bodies in cerebral cortex and basal ganglia. Scattered cortical neurons were surrounded by a rim of reactive material. Perineural staining was the rule rather than the exception in spinal cord anterior horn motoneurons, inferior olivary nucleus, large bulbar reticular neurons and dentate nucleus of cerebellum. The only part of the brain which appeared relatively free of hyaluronate was the molecular layer of the cerebellum. In newborn and embryonal rat, the densely packed cell bodies in cerebral gray matter, periventricular germinal layer and external granular layer of cerebellum were surrounded by hyaluronate. Small droplets of hyaluronate were observed inbetween the cylindrical epithelial cells lining the neural tube in 11 day embryos. Non-myelinated fiber tracts and the molecular layer of the developing cerebellum were relatively unstained. No hyaluronate was detected in the ependyma lining the cerebral ventricles and the central canal of the spinal cord.

Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.

Localization of Mercury in CNS of the Rat III. Oral Administration of Methylmercuric Chloride (CH3HgCl)

The distribution and cellular localization of mercury in the in situ brain and upper cervical spinal cord of adult Wistar rats were studied at various time intervals after oral administration of methylmercuric chloride (CH3HgCl; 20 mg×liter−). Coronal sections of the brain and transverse sections of the cervical spinal cord were prepared for visualization of the mercury by the autometallographic silver-enhancement method. Following mercury administration there was a latent period before the menal appeared in the tissue. Mercury staining was first detected after 10 days in cell bodies of five specific areas of the brain stem: the mesencephalic nucleus of the trigeminal nerve, the red nuclei, the ventral cochlear nucleus, the superior vestibular nucleus, and the nucleus reticularis pontis caudalis. After 28 days of treatment, a fairly even distribution of mercury was seen in the brain and spinal cord. Longer periods of treatment caused no further increase in the density of mercury within the stained cell bodies. In cerebral cortex, staining commenced in piriform and entorhinal cortices. This was followed by staining in neurons of lamina III in the isocortex and ultimately all layers were stained after 28 days of treatment. After 20 days of treatment, mercury deposits in the cerebellar cortex were restricted to Purkinje cells, Golgi epithelial cells, and Golgi cells, while in the spinal cord the majority of mercury was located in the anterior horn motoneurons. Scattered ependymal cells and epithelial cells of the choroid plexus also exhibited mercury staining. The principal target cells were neurons followed by the glial and ependymal cells. Ultrastructurally, the bulk of detectable mercury was localized in lysosomes.

Localization of mercury in CNS of the rat: III. Oral administration of methylmercuric chloride (CH 3HgCl)

The distribution and cellular localization of mercury in the in situ brain and upper cervical spinal cord of adult Wistar rats were studied at various time intervals after oral administration of methylmercuric chloride (CH 3HgCl; 20 mg × liter −1). Coronal sections of the brain and transverse sections of the cervical spinal cord were prepared for visualization of the mercury by the autometallographic silver-enhancement method. Following mercury administration there was a latent period before the metal appeared in the tissue. Mercury staining was first detected after 10 days in cell bodies of five specific areas of the brain stem: the mesencephalic nucleus of the trigeminal nerve, the red nuclei, the ventral cochlear nucleus, the superior vestibular nucleus, and the nucleus reticularis pontis caudalis. After 28 days of treatment, a fairly even distribution of mercury was seen in the brain and spinal cord. Longer periods of treatment caused no further increase in the density of mercury within the stained cell bodies. In cerebral cortex, staining commenced in piriform and entorhinal cortices. This was followed by staining in neurons of lamina III in the isocortex and ultimately all layers were stained after 28 days of treatment. After 20 days of treatment, mercury deposits in the cerebellar cortex were restricted to Purkinje cells, Golgi epithelial cells, and Golgi cells, while in the spinal cord the majority of mercury was located in the anterior horn motoneurons. Scattered ependymal cells and epithelial cells of the choroid plexus also exhibited mercury staining. The principal target cells were neurons followed by the glial and ependymal cells. Ultrastructurally, the bulk of detectable mercury was localized in lysosomes.

Coordination of reinnervated muscle and reorganization of spinal cord motoneurons after nerve transection in mice

Combining physiological and anatomical methods, the dependence of muscle coordination on the alteration in connections between anterior horn motoneurons and muscle following peripheral nerve repair has been examined in adult mice. Fourteen weeks after transection and readaptation of the right common peroneal nerve electromyographic activity patterns of the tibialis anterior (TA) and medial gastrocnemius (MG) muscles were recorded simultaneously during free running. The number and locomotion of motoneurons supplying the TA were determined bilaterally with horseradish peroxidase. In control animals with normal muscle coordination the number of TA motoneurons varied considerably, but was bilaterally symmetrical. The average distribution of cells in both anterior horns was nearly identical. After reinnervation muscle coordination was significantly impaired. Prolonged TA activity phases overlapped antagonistic MG activity with large inter-individual variability. The number of TA motoneurons was reduced to 70.2 ± 24.0% compared to the contralateral control side. The distribution of the cells was significantly shifted to the caudal direction. This indicated inappropriate innervation of the TA by motoneurons which originally served the peroneal muscles, as suggested by the results in another control group. There was no correlation between the number of motoneurons reinnervating the TA and the restoration of motoneuron somatotopy. These two factors promoted the reestablishment of muscle coordination significantly and independently of one another.

Localization of mercury in CNS of the rat: II. Intraperitoneal injection of methylmercuric chloride (CH 3HgCl) and mercuric chloride (HgCl 2)

The autometallographic method has been used to determine the precise localization of mercury in the brain and spinal cord of adult Wistar rats which had been treated with repeated ip injections of methylmercuric chloride (CH 3HgCl; 0.2 to 10.0 mg) or mercuric chloride (HgCl 2; 0.2 to 10.0 mg). The distribution of mercury was uneven following administration of HgCl 2, while it was fairly homogeneous following CH 3HgCl. With both compounds, however, heavy deposits of mercury were present in the motor nuclei of rhombencephalon. In contrast, cerebellar Purkinje cells, Golgi cells, and Golgi epithelial cells only contained mercury in sections from rats exposed to CH 3HgCl. In cerebral sections from rats exposed to CH 3HgCl, staining intensity in cortical cells varied among the layers, being greatest in laminae III, V, and VI. On the other hand, sections from rats exposed to HgCl 2 showed only staining in scattered cells of lamina VI. Following administration of either compound, mercury was detected in the gray matter of the spinal cord mercury. Particularly large deposits were present in the anterior horn motoneurons. At the cellular level, the heaviest staining intensity was seen in neurons, although the cytoplasm of glia and ependymal cells also showed significant deposits in sections from rats exposed to CH 3HgCl. In HgCl 2-treated rats, the largest accumulations of mercury were seen in the neurons. The ependymal cells were stained to a lesser extent, while glia were devoid of mercury. Ultrastructurally, mercury deposits were located exclusively in lysosomes. The present results demonstrate that the pattern of mercury distribution and its staining intensity in individual cells in the rat CNS are dependent upon the chemical structure of the compound and the duration of its administration.

Dendritic Development of Motor Neurons in the Cervical Anterior Horn and Hypoglossal Nucleus of Normal Infants and Victims of Sudden Infant Death Syndrome

Morphometric Golgi methods were used to study dendritic development of neurons in the cervical ventral and dorsal horns and the hypoglossal nucleus of 8 victims of sudden infant death syndrome and 20 age-matched control infants. The dendrites and spines of these neurons proliferated rapidly until 28 weeks gestation, then increased slowly until shortly after birth. Dendritic spine density of motor neurons in the cervical anterior horn and hypoglossal nucleus of victims of sudden infant death syndrome was similar to that of the controls. Thus, the delayed maturation of dendritic spines seen in the reticular formation and vagal nuclei is not present in motor neurons of the cervical anterior horn and hypoglossal nucleus.

Age-dependent poliomyelitis of mice: expression of endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons

The widespread presence of endogenous retroviruses in the genomes of animals and humans has suggested that these viruses may be involved in both normal and abnormal developmental processes. Previous studies have indicated the involvement of endogenous ecotropic murine leukemia virus (MuLV) in the development of age-dependent poliomyelitis caused by infection of old C58 or AKR mice by lactate dehydrogenase-elevating virus (LDV). The only genetic components which segregate with susceptibility to LDV-induced paralytic disease are multiple proviral copies of ecotropic MuLV and the permissive allele, at the Fv-1 locus, for N-tropic, ecotropic virus replication (Fv-1n/n). Using in situ hybridization and Northern (RNA) blot hybridization, we have correlated the expression of the endogenous MuLV, both temporally and spatially, with LDV infection of anterior horn motor neurons and the development of paralysis. Our data indicate that treatment of 6- to 7-month-old C58/M mice with cyclophosphamide, which renders these mice susceptible to LDV-induced paralytic disease, results in transient increases in ecotropic MuLV RNA levels in motor neurons throughout the spinal cord. Peripheral inoculation of C58/M mice with LDV, at the time of elevated MuLV RNA levels, results in a rapid spread of LDV to some spinal cord motor neurons. LDV infections then spread slowly but progressively throughout the spinal cord, involving an increasing number of motor neurons. LDV replication is cytocidal and results in neuron destruction and paralysis of the infected animals 2 to 3 weeks postinfection. The slow replication of LDV in the spinal cord contrasts sharply with the rapid replication of LDV in macrophages, the normal host cells for LDV, during the acute phase of infection. The data indicate that the interaction between the endogenous MuLV with the generally nonpathogenic murine togavirus LDV occurs at the level of the motor neuron. We discuss potential mechanisms for the novel dual-virus etiology of age-dependent poliomyelitis of mice.

Diabetic and hypoglycemic neuropathy — a comparison in the BB rat

Functional and structural neuropathy was examined in hyperglycemic (diabetic) BB rats maintained on small maintenance doses of insulin, hyperglycemic BB rats receiving no insulin, and BB rats in whom hypoglycemia was induced by the administration of excessive insulin doses. The data were compared with those of non-diabetic age- and sex-matched BB rats. Functional deficits and structural abnormalities were comparable in diabetic rats with and without insulin supplementation, suggesting that the generally necessary insulin dosing in this model does not per se account for the neuropathy. Hypoglycemic neuropathy was characterized by slowing of nerve conduction velocity, marked loss of anterior horn motoneurons and Wallerian degeneration, as well as loss of large myelinated fibers, suggesting a neuropathy involving predominantly motoneurons. Diabetic neuropathy was not associated with nerve cell loss but showed marked axonal atrophy involving predominantly sensory fibers. Thus, diabetic and hypoglycemic neuropathies are two distinguishable entities under strict experimental conditions, but may overlap in human diabetic subjects in whom tight insulin control is desirable.

Accumulation of phosphorylated neurofilaments in anterior horn motoneurons of amyotrophic lateral sclerosis patients.

Perikaryal collections of intermediate filaments have been described in the anterior horn motoneurons of patients with amyotrophic lateral sclerosis (ALS), but these inclusions have generally been considered rare and mainly associated with the familial form of ALS. Using the monoclonal antibody NF2F11, which recognizes phosphorylated neurofilament epitopes, we showed that focal collections of neurofilaments in anterior horn motoneurons were a characteristic finding in sporadic as well as in familial ALS; they were present in seven of nine ALS patients, but in none of nine control spinal cords. These neurofilamentous collections are not cross-reactive with antibodies directed against paired helical filaments and the microtubule associated protein tau. In addition, diffuse staining for phosphorylated neurofilament epitopes in chromatolytic anterior horn perikarya was significantly more frequent in ALS patients than in controls.

Localization of mercury in CNS of the rat: I. Mercuric chloride (HgCl 2) per os

Adult Wistar rats of both sexes were exposed to mercuric chloride (HgCl2) through drinking water (20 mg HgCl2 liter-1 distilled water) ad libitum during an 8-month period. Animals were subsequently sacrificed by transcardial perfusion with glutaraldehyde. Coronal sections of the brain and cervical spinal cord were examined according to a histochemical technique based on a physical development process which renders mercury deposits visible. Mercury was found unevenly distributed in the brain and spinal cord with the heaviest deposits found within the motor nuclei of the rhombencephalon. In cerebral cortex, the highest concentration of mercury was found in the striate area. Mercury was also localized within the deep nuclei of cerebellum; none was found within Purkinje cells. A proportionately high amount of mercury was additionally found in the anterior horn motoneurons of the spinal cord. In general, mercury was found primarily within neurons, but it was also observed in the cytoplasm of glia and ependymal cells.

Characteristic distribution of noradrenergic terminals on the anterior horn motoneurons innervating the perineal striated muscles in the rat. An immuno-electromicroscopic study.

Dopamine-beta-hydroxylase (DBH) immunohistochemistry was used to demonstrate the noradrenergic fibers and terminals in the anterior column of the rat lumbosacral spinal segments. PAP-positive varicose fibers were widely distributed in the gray matter with preferential accumulation in the nuclear regions containing motoneurons involved in the contraction of perineal striated muscles. Unmyelinated DBH fibers were composed of nodular enlargements (varicosities, 0.4-3.0 microns in diameter) and very fine, short intervals (intervaricose segments, 0.1-0.2 micron in diameter and 1.0-4.0 microns in length). DBH-positive dense products were electron microscopically often confined within small granular particles and less frequently within large granules. Additionally, in order to characterize the innervation pattern of noradrenergic fibers on dendritic bundles organized in the motoneuronal pools innervating the pelvic small muscles, semi-quantitative analysis was done in the area of the dorsolateral nucleus endowed with especially well-developed dendritic bundles. DBH terminals contacting with unreactive dendrites were more common (67.9%) than those with neuronal somata (15.1%), and the remainder (17%) had no contacts with surrounding neuronal elements. Furthermore, specialized synaptic formations were observed in only 20.1% of these nodules. The results suggest that bulbospinal descending noradrenergic neuron systems influence the functioning of pelvic muscles principally via the neuronal contacts with dendritic bundles in the spinal cord.

A lethal autosomal recessive syndrome of multiple congenital contractures

We describe 16 cases of a lethal syndrome with multiple congenital contractures from ten families. The main clinical findings included intrauterine growth retardation with marked fetal hydrops, multiple contractures, and facial abnormalities, especially micrognathia. At autopsy, pulmonary hypoplasia and muscular atrophy were present. There was a paucity of anterior horn motor neurons in the four studied cases. We think that the cases represent the same clinical entity, probably caused by homozygosity of an autosomal recessive gene. The syndrome resembles the Pena-Shokeir I syndrome, but seems to differ in some respects, including length of survival and presence of hydrops. Prenatal diagnosis of this syndrome is possible after the 16th week of pregnancy with ultrasound.

Effect of ionising radiation on the axon reaction of mouse anterior horn motor neurons: A histological and immunocytochemical study using a monoclonal antibody to neurofilament protein

The changes taking place in irradiated central nervous tissue prior to the onset of delayed radionecrosis are poorly understood, but functional abnormalities occurring during the latent interval after irradiation are likely to be of importance. In order to investigate functional disturbances in neurones during this period, unilateral sciatic nerve crush was performed in mice following sub-lethal X-irradiation of the lumbar spinal cord. Alterations in the axon reaction of anterior horn cells were studied using a monoclonal antibody to neurofilament protein. With irradiation immediately prior to crush, the normal, well-defined increase in perikaryal neurofilament protein was significantly diminished, although there was no concurrent radiation necrosis and no alterations were seen in contralateral neurones with intact distal axon processes. The effect was more marked in neurones irradiated one month prior to nerve crush, and in the non-irradiated nerve crush region regeneration was delayed, with diminished neurofilament protein in the regenerating axons. These observations indicate that ionising radiation can progressively impair the ability of neurones to synthesise neurofilament protein during distal axon regeneration. This may result from inadequate repair of radiation induced DNA strand-breaks, but may also follow more generalised damage to protein transcription enzymes and RNA metabolism.