Hydrogen peroxide mediates damage by xanthine and xanthine oxidase in cerebellar granule neuronal cultures

The free radical-generating system of xanthine and xanthine oxidase is commonly used experimentally as a source of superoxide anion, which can produce oxidative stress, leading to cellular damage and death. Models of oxidative stress are important in elucidating pathologies associated with increased levels of reactive oxygen species, including stroke and neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We therefore, examined the effect of the xanthine/xanthine oxidase system on the viability of postnatal cerebellar granule neurones obtained from 8-day old Sprague–Dawley rat pups. Xanthine (100 μM) and xanthine oxidase (0.02 U/ml) applied for 1 or 6 h reduced the viability of cells at 8 div assessed using the alamar blue assay, and induced morphological changes, such as shrinkage of the cell bodies and neurites. Heat-inactivation of xanthine oxidase resulted in complete loss of its activity. Superoxide dismutase (250 U/ml) failed to modify the damage by xanthine and xanthine oxidase, while catalase (250 U/ml) completely prevented it. When applied alone, xanthine oxidase significantly lowered cell viability, an effect that was blocked by allopurinol and catalase, but not by superoxide dismutase. The results indicate that xanthine and xanthine oxidase can produce predominantly hydrogen peroxide instead of the superoxide anion. Cerebellar granule cells in culture may also possess significant levels of endogenous xanthine.     

On the identification of the afferent axon terminals in the nucleus lateralis of the cerebellum an electron microscope study

Summary Axon terminals in the neuropil of the lateral nucleus can be divided into six classes, each with a specific constellation of characteristics that consistently occur together. Two of these classes have synaptic varicosities with elliptical synaptic vesicles, one in a dense, the other in a sparse matrix, and both make axosomatic and axodendritic synapses. The remaining four classes all have round synaptic vesicles and do not make axosomatic synapses. In the first of these four, the vesicles are tightly packed in a dense matrix, in another they are loosely dispersed, and in the third they are clustered. In the fourth, large granular vesicles predominate. Of these six classes, the most numerous belong to the axons of the Purkinje cell terminal arborization. These boutons resemble their counterparts in the cerebellar cortex, the recurrent collaterals of the Purkinje axon. They have elliptical and flat synaptic vesicles in a dark matrix. The varicosities terminate on somata and dendrites of large and small neurons and constitute the majority of their input. Purkinje axons constitute 86% of the total population of terminals on large neuronal perikarya and 50% of those on their dendrites, but only 78% on the somata of small neurons and 31% on their dendrites. The terminals of climbing fiber collaterals are recognized by their resemblance in electron micrographs to the terminals of the climbing fiber arborization in the cerebellar cortex. They bear round synaptic vesicles packed into a dense axoplasmic matrix and make Gray's type 1 axodendritic synapses with large and small neurons. These axons are restricted to the lateral and ventral aspects of the nucleus and constitute 5% of the terminals on large cell dendrites and 6% of those on small neurons. The axons tentatively identified as collaterals of mossy fibers are myelinated fibers with a light axoplasm containing round synaptic vesicles, dispersed throughout their varicosities. They make Gray's type 1 synapses and constitute a fair percentage of the total axodendritic contacts in the neuropil, 22% on large neurons and 28% on small neurons. The bases for these tentative identifications are discussed in detail, as are the various synaptic relationships undertaken by each class of axon. The remaining 4 classes of axons of the neuropil will be described in subsequent papers.Supported in part by U.S. Public Health Service grants NS 10536 and NS 03659, Training grant NS 05591 from the National Institute of Neurological Diseases and Stroke, and a William F. Milton Fund Award from Harvard University.     

An HRP and autoradiographic study of cerebellar corticonuclear-nucleocortical reciprocity in the monkey

Summary Combined injections of ³H-leucine and HRP were made into the monkey cerebellar cortex in order to identify any reciprocal connections between the corticonuclear and the nucleocortical pathways. These combined intraaxonal labeling experiments have demonstrated a considerable overlap of orthogradely labeled Purkinje cell axons and terminals with retrogradely labeled HRP-positive neurons in the ventrolateral region of the dentate nucleus following combined injections into the lateral hemisphere, and in the dorsal area of the dentate following combined injections into medial cortical areas of the anterior lobe. There were also areas within the deep cerebellar nuclei where orthogradely labeled corticonuclear terminals did not overlap with retrogradely labeled nucleocortical neurons.     

Horizontal optokinetic ocular nystagmus in wildtype (B6CBA+/+) and weaver mutant mice

Summary Horizontal optokinetic nystagmus (OKN) evoked by a random dot pattern moving at a constant speed around the animal was investigated in wild-type mice and Weaver mutants (cerebellar impairment) by means of chronically implanted EOG-electrodes. The shape of OKN in the homozygotic Weaver mouse was clearly different from that in normal mice. The OKN in the mutant showed inconstant velocity during the slow phase. Nystagmus frequency of the mutant was significantly below that of normal controls for velocities of 1.4 to 25 degrees · s^-1. In one group of normals the mean slow-phase gain was relatively constant for stimulus angular velocities between 1.4 and 15 degrees · s^-1 and declined thereafter. In a second group the mean slow-phase gam decreased gradually between stimulus angular velocities from 1.4 to 15 degrees · s^-1 and thereafter with a steeper slope. In mutants gain decreases with increasing stimulus velocity over the entire range tested (1.4 to 42 degrees · s^-1). Normals and mutants with one eye occluded exhibited strong OKN when the pattern was moved in a temporonasal direction; little response was obtained by stimuli moving in a naso-temporal direction.     

Spinal projections to the cerebellar nuclei in the cat

Summary Projections from the spinal gray matter to the cerebellar nuclei in the cat have been studied using Nauta's silver technique. Following unilateral section of the ventrolateral cord at the cervical level, heavy degeneration is seen in the nucleus medialis on both sides. Scanty degeneration is present bilaterally in the nucleus interpositus. The degeneration is most intense on the contralateral side. Scanty degeneration is also present bilaterally in subnucleus medialis parvicellularis (SMP) (Flood and Jansen, 1961). No degeneration is seen in nucleus lateralis. Following unilateral section of the dorsolateral cord at the cervical level, scanty degeneration is present bilaterally in nucleus medialis and nucleus interpositus anterior. The degeneration is more pronounced ipsilaterally and is also seen in SMP on both sides. No degeneration is present in nucleus lateralis. Fibers from the ventral and dorsal spinocerebellar tracts (VSCT and DSCT) terminate bilaterally in nuclei medialis and interpositus, with the VSCT as the most important connection.     

Lateral and medial sub-divisions within the olivocerebellar zones of the paravermal cortex in lobule Vb/c of the cat anterior lobe

Summary The olivocerebellar projection to the c₁,c₂ and c₃ zones in the paravermal cortex of lobule Vb/c has been investigated in the cat using a combined electrophysiological/neuroanatomical tracing technique. The zonal boundaries in the paravermal cortex were located by recording, on the cerebellar surface, climbing fibre field potentials evoked in response to percutaneous stimulation of one or more paws. A small (10–30 nl) injection of WGA-HRP was then made either into the centre or into the medial or lateral geographical half of a chosen zone and the resultant distribution of retrogradely labelled cells within the contralateral inferior olive was plotted. The c₁ and c₃ zones were each found to consist of two mediolaterally oriented sub-zones which could be distinguished by their olivocerebellar input. The medial part of the c₁ zone received climbing fibre input from the rostromedial part of the dorsal accessory olive (DAO) while the lateral part of the c₁ zone received climbing fibre input from middle/rostral regions of the medial accessory olive (MAO). Both medial and lateral sub-zones within the c₃ zone were found to receive climbing fibre input from the rostral pole of DAO but, whereas there was heavy overlap between the olivary territories projecting to the medial c₁ and medial c₃ subzones, olivary cells projecting to the lateral part of c₃ were located more rostrally within DAO. The c₂ zone was found not to be divisible into mediolaterally oriented subzones and to receive climbing fibre input from a region of MAO located rostral and somewhat lateral to the region projecting to the lateral part of the c₁ zone. The sub-zonal organisation of the olivocerebellar projection to the c₁, c₂ and c₃ zones is discussed in relation to the functional properties of the different zones.     

Ganglioglial Differentiation in Medulloblastoma

A case of cerebellar medulloblastoma with clusters of mature ganglion cells and glial cells is described. The patient, a 15 -year -old girl, underwent three operations followed each time by radiation and chemotherapy during the four-year clinical course. Histologically, the ganglion cells were clearly identifiable by their abundant eosino-philic cytoplasm, round nuclei with prominent nucleoli, tigroid granules, and argyrophilic fibrils and axons. Im-munohistochemically, the cells were NSE- and NF positive, and ultrastructurally they contained abundant tubules and filaments, neurosecretory granules and well developed rough endoplasmic reticulum. There were many cells transitional in appearance between primitive cells and mature ganglion cells. The tumor also had many mature yet atypical astrocytes and oligodendrocytes. The exact mechanism of the extensive neuronal and glial maturation of medulloblastoma cells is unclear, but the repetitive surgical interventions, radiation and chemotherapy might have had certain cytostatic effects on rapidly dividing medulloblastoma cells, giving them a chance to mature into postmitotic cells with potential for neuronal and glial differentiation. Acta Pathol Jpn 40: 50–56, 1990.     

Evidence for reactive synaptogenesis in the ventrolateral thalamus and red nucleus of the rat: changes in high affinity glutamate uptake and numbers of corticofugal fiber terminals

Summary High affinity glutamate uptake into corticofugal fiber terminals was measured in the ventrolateral thalamus and red nucleus at varying time intervals after lesions were made by kainic acid in the contralateral interpositus nucleus of the cerebellum in rats. Under similar conditions the density of cortical fiber terminals was estimated using the Fink-Heimer impregnation technique. 1. Glutamate uptake steadily increased in the ventrolateral thalamus up to 60 days after lesions in the contralateral cerebellum. 2. Similar changes were noted in the red nucleus. 3. The changes were dependent on the integrity of corticofugal fibers to the thalamus and red nucleus. 4. No changes in uptake of gammaaminobutyric acid were noted. 5. Saturation curves for glutamate uptake suggested a change in the maximal number of transport sites. 6. Fink-Heimer degeneration studies showed an increase in cortical terminals in the ipsilateral ventrolateral thalamus and in both rostral and caudal regions of the red nucleus following lesions in the contralateral interpositus nucleus. The data are consistent with an increase in the number of cortical fiber terminals in reaction to loss of cerebellar input to the ventrolateral thalamus and red nucleus. This study correlates anatomical and biochemical evidence for collateral sprouting in a model based on electrophysiologic data in the red nucleus and extends the model to include the thalamus.     

Compensatory motor function of the somatosensory cortex for dysfunction of the motor cortex following cerebellar hemispherectomy in the monkey

Summary Electrical activities of the motor and somatosensory cortices preceding visually-initiated hand movements were recorded with electrodes chronically implanted on the surface and at 2.5–3.0 mm depth in the cortex of monkeys, and changes in field potentials in these cortices after cerebellar hemispherectomy were observed for many weeks. As previously reported, a unilateral cerebellar hemispherectomy including the lateral and interpositus nuclei eliminates the cerebellar-mediated superficial thalamo-cortical (T-C) responses recorded in the forelimb motor cortex contralateral to the hemispherectomy. These T-C responses normally precede the hand movement, and the operation results in the delay of movement initiation. The electrodes in the forelimb area of the contralateral primary somatosensory cortex showed an enhancement of superficial T-C responses of the somatosensory cortex for 30–40 days after the operation. The enhanced potentials preceded the delayed movement as do the cerebellar-mediated superficial T-C responses of the motor cortex in normal situations. Local cooling of the somatosensory cortex following the cerebellar hemispherectomy disturbed the reaction time movement for a few weeks after the operation. This effect was rarely encountered in normal monkeys. The present study suggests the compensatory motor function of the somatosensory cortex for the dysfunction of the motor cortex in early weeks after cerebellar hemispherectomy.Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

家兔小脑中央核向脊髓直接投射的研究

本实验将WGA-HRP分別注入家兔脊髓一侧的颈、胸、腰等不同节段,逆行追踪小脑中央各核向脊髓的直接投射。结果表明,小脑中央核的标记神经元恒定地见于颈段(C_1～C_4)注入例动物,而未见于下颈段(C_6～C_8)和胸腰段注入例动物。标记神经元仅出现于注入侧对侧的小脑顶核及间位核的尾侧部。研究结果提示,家兔小脑顶核及间位核的尾侧部,有向颈髓的直接越边投射。本结果为进一步探讨小脑对躯体运动的调节功能提供形态学基础。