Cerebellar injury due to phenytoin. Identification and evolution of Purkinje cell axonal swellings in deep cerebellar nuclei of mice

Summary The present study describes the identification and the ultrastructural and numerical evolution of Purkinje cell axonal swellings induced by phenytion. Thirty male C57BI/6J mice received phenytion orally in doses up to 100 mg/kg daily and were killed after 3, 6, 10, 14, and 48 days of treatment. Light and electron microscopic investigations as well as morphometric analysis of cut surface area and numerical density of axonal swellings were performed. The swellings appeared as early as 6 days after initiation of treatment and gradually increased in size and frequency. Use of an anti-lymphocyte monoclonal antibody (CD 3), specifically cross-reacting with Purkinje cells, identified the swellings as dystrophic Purkinje cell axons. On grounds of their ultrastructural appearance they were classified into three distinct types occurring at different time intervals after phenytoin exposure. At 6 days, most axonal swellings contained loosely aggregated membranous vesicles and tubules in a finely granulated matrix (type 1). At 14 days, larger axonal swellings appeared characterized by the presence of three-dimensional networks of branched and anastomosing membranous tubules (type 2). At 48 days, even larger axons contained bodies of highly condensed membranous material of sometimes paracrystalline appearance (type 3). It is suggested that phenytoin-induced axonal pathology of Purkinje cells is a dynamic process characterized by the progressive accumulation of proliferating membranous material arranged in an increasingly complex fashion.Supported by the Deutsche Forschungsgemeinschaft, grant VO 272, 5-1     

The wobbler mouse mutant: An animal model of hereditary motor system disease

Summary The homozygous wobbler (wr) mouse mutant manifests shortly after birth a form of progressive neural atrophy characterized pathologically by vacuolization and degeneration of neurons of the brainstem and ventral horn of the spinal cord. Ultrastructural features include the presence of autophagic vacuoles containing tubular structures indistinguishable in appearance from neurotubules. The present study correlates certain of these histological features with the characteristics of axoplasmic flow in the homozygous 5–8 week old (wr) mouse.Slow phase axoplasmic flow was found to be identical in both (wr) mice and normal littermate groups and was estimated to be 1–2 mm per day. However, the homozygous (wr) mouse consistently demonstrated more label in the proximal segments at 7 and 10 days suggesting the presence of an additional population of neurons with impaired flow but with a relatively intact protein synthetic mechanism. This is accompanied by histologic evidence for the elaboration of abnormal neurotubular protein under genetic control. It is hypothesized that the neurotubular transport system underlying the slow phase of axoplasmic flow is thus rendered defective resulting in impairment in axonal continuity and the progressive histologic picture so described.Presented in part at the American Academy of Neurology Meeting, Miami, May 2, 1970.Supported in part by grants: CA 08543 from the National Cancer Institute, General Research Support Grant Number 5409, National Institutes of Health. The Jack N. Meeks Research Fund. CA 11224 and GM 1052, National Institutes of Health.     

Differential regenerative response of purkinje cell and inferior olivary axons confronted with embryonic grafts: Environmental cues versus intrinsic neuronal determinants

Regeneration of severed central axons is supposed to depend on two factors: a permissive local environment and the particular intrinsic properties of axotomized neurones. To assess the role of each of these factors in axonal regeneration, the capability of two particular axon populations of the adult mouse cerebellum to grow into target-specific (cerebellum) and target-unspecific (neocortex) embryonic grafts was determined. Purkinje cell and inferior olivary axons were transected by passing a microscalpel through the axial white matter of the cerebellar folia, particularly, those of the anterior lobe. Immediately after the injury, solid transplants were placed in the lesion cavity. Purkinje cell axons were labelled by using anticalbindin immunocytochemistry, and olivocerebellar fibres were visualized by biotinylated dextran amine anterograde axonal tracing. Following axotomy, Purkinje cell axons appeared as thickened processes ending with large terminal clubs. Their morphology and number did not change up to the longest survival time considered (2 months), thereby confirming previous demonstrations that Purkinje cells survive axon injury (I. Dusart and C. Sotelo, 1994, J. Comp. Neurol. 347:211–232). Inferior olivary axons were thinner and bore smaller terminal bulbs. When embryonic cerebellar grafts, containing cortical and deep nuclear, precursors, were placed close to the injured axons, olivocerebellar fibres vigorously regenerated into the transplants and ended in new climbing fibies along the dendrites of grafted Purkinje cells. By contrast, host Purkinje cell axons never showed any outgrowth towards the graft. Similarly, these axons failed to regenerate into grafts containing solely the rostromedial portion of the cerebellar anlage, mostly consisting of deep nuclear neurones, their main targets. Comparable results were obtained by transplanting embryonic neocortical tissue: inferior olivary axons also regenerated into the grafts, although with distinct terminal arbours without the climbing fibre phenotype, whereas Purkinje cell axons always failed to grow. These results provide the first direct demonstration that severed inferior olivary axons are able to regenerate. In addition, they show that the growth-permissive/-promoting conditions created by embryonic nervous tissue are not sufficient to induce the regeneration of every axonal type and allow us to hypothesise that successful regenamtion depends on the mterplay between environmental cues and intrinsic properties of the axotornized neurones. © 1995 Wiley-Liss, Inc.     

The microtubular apparatus of cerebellar purkinje cell dendrites during postnatal development of the rat: The density and cold-stability of microtubules increase with age and are sensitive to thyroid hormone deficiency

A quantitative ultrastructural study of microtubules in Purkinje cell dendrites of normal and hypothyroid developing rats was performed after fixation either at room or at low temperature (4°C). In normal animals, the density of microtubules and their fold-stability increased with age, more especially during the period of intense dendritic growth. Thyroid deficiency delayed the appearance of microtubules and still more the acquisition of their fold-stability. These effects might explain the defects in Purkinje cell dendritic growth and branching observed in hypothyroid animals.     

On the cellular localization of cerebellar muscarinic receptors: An autoradiographic analysis of weaver, reeler, purkinje cell degeneration and staggerer mice

Light microscopic autoradiography of [3H]quinuclidinyl benzilate binding sites was used to study the distribution of muscarinic cholinergic receptors in mouse mutants which have abnormalities affecting specific cerebellar cell types. In the normal C57BL/6J mouse, binding sites were distributed throughout the cerebellar cortex, with the highest levels in the granule cell layer and deep cerebellar nuclei. Normal binding site density was observed in the cerebellum of the weaver mutant in which the majority of granule cells had degenerated. The density of [3H]quinuclidinyl benzilate binding sites was elevated in the cortex of the reeler, despite a reduction in the number of granule cells. The concentration of binding sites was also high over the Purkinje cell masses where granule cells were largely absent. No significant reduction in cortical [3H]quinuclidinyl benzilate binding site density was detected in the Purkinje cell degeneration mutant, in which essentially all Purkinje cells had degenerated. In contrast, receptor binding in the deep cerebellar nuclei of this mutant was significantly increased. A substantial increase in labeling was observed in the cortex and deep nuclei of the staggerer cerebellum in which a large fraction of Golgi II cells, Purkinje cells, granule cells and mossy fibers have degenerated. We discuss the possibility that the persistence of [3H]quinuclidinyl benzilate binding sites in all four mutants may imply a non-neuronal localization for a large proportion of muscarinic receptors in the mouse cerebellar cortex.     

Knockdown of amyloid precursor protein normalizes cholinergic function in a cell line derived from the cerebral cortex of a trisomy 16 mouse: An animal model of down syndrome

We have generated immortal neuronal cell lines from normal and trisomy 16 (Ts16) mice, a model for Down syndrome (DS). Ts16 lines overexpress DS-related genes (App, amyloid precursor protein; Sod1, Cu/Zn superoxide dismutase) and show altered cholinergic function (reduced choline uptake, ChAT expression and fractional choline release after stimulation). As previous evidence has related amyloid to cholinergic dysfunction, we reduced APP expression using specific mRNA antisense sequences in our neuronal cell line named CTb, derived from Ts16 cerebral cortex, compared to a cell line derived from a normal animal, named CNh. After transfection, Western blot studies showed APP expression knockdown in CTb cells of 36% (24 hr), 40.4% (48 hr), and 50.2% (72 hr) compared to CNh. Under these reduced APP levels, we studied 3H-choline uptake in CTb and CNh cells. CTb, as reported previously, expressed reduced choline uptake compared to CNh cells (75%, 90%, and 69% reduction at 1, 2, and 5 min incubation, respectively). At 72 hr of APP knockdown, choline uptake levels were essentially similar in both cell types. Further, fractional release of 3H-choline in response to glutamate, nicotine, and depolarization with KCl showed a progressive increase after APP knockdown, reaching values similar to those of CNh after 72 hr of transfection. The results suggest that APP overexpression in CTb cells contributes to impaired cholinergic function, and that gene knockdown in CTb cells is a relevant tool to study DS-related dysfunction.     

Effect of the acute and chronic administration of the selective neurokinin2 receptor antagonist SR 48968 on midbrain dopamine neurons in the rat: An in vivo extracellular single cell study

In this study, we examined the effect of the acute and chronic administration of the selective neurokinin₂ (NK₂) receptor antagonist SR 48968 on the activity of spontaneously active dopamine (DA) cells in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized, male rats. This was accomplished using the technique of in vivo, extracellular single cell recording. The intravenous (i.v.) administration of SR 48968 (10–1280 μg/kg) did not significantly alter the basal firing rate or pattern of either spontaneously active SNC or VTA DA neurons compared to control. However, the acute administration of 1 mg/kg, i.p., of SR 48968, but not its inactive enantiomer SR 48965, produced a significant increase in the number of spontaneously active DA cells in the SNC (48%) and VTA (28%) compared to vehicle controls. The i.p. administration of SR 48968 did not alter the basal firing pattern of either SNC or VTA DA neurons compared to vehicle controls. The pretreatment of animals with 1 mg/kg, i.p., of SR 48968 significantly potentiated the suppressant action of (+)-apomorphine on spontaneously active SNC and VTA DA cells. In contrast to its acute effects, the administration of 1 mg/kg, i.p., of SR 48968 once daily for 21 days produced a significant decrease in the number of spontaneously active DA cells in the SNC and VTA. The decrease in the number of spontaneously active VTA DA cells was not reversed by (+)-apomorphine administration; in fact, a further decrease in the number of VTA DA cells was observed. This suggests that the SR 48968-induced decrease in the number of spontaneously active DA neurons may not be the result of depolarization block. Overall, these results suggest that the acute and chronic administration of SR 48968 alters the number of spontaneously active midbrain DA neurons in anesthetized rats. Synapse 25:196–204, 1997. © 1997 Wiley-Liss, Inc.     

The acute and chronic administration of the 5-HT(2B/2C) receptor antagonist SB-200646A significantly alters the activity of spontaneously active midbrain dopamine neurons in the rat: An in vivo extracellular single cell study

This study examined the effect of the acute and chronic administration of the 5-HT(2B/2C) receptor antagonist N-(1-methyl-5-indolyl)-N'-(3-pyridyl) urea hydrochloride (SB-200646A) on the activity of spontaneously active DA cells in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized, male Sprague-Dawley rats. This was accomplished using in vivo extracellular single cell recording. The i.v. administration of 4-16 mg/kg of SB-200646A significantly increased the firing rate and % events as bursts in spontaneously active VTA DA neurons and significantly increased the % events as burst in SNC DA neurons. The acute i.p. administration of 20 and 40 mg/kg of SB-200646A significantly increased the number of spontaneously active VTA DA neurons when compared with vehicle-treated controls. The acute administration of 10 mg/kg of SB-200646A significantly increased the coefficient of variation in spontaneously active SNC and DA neurons when compared with vehicle-treated controls. However, the acute i.p. administration of 20 mg/kg of SB-200646A significantly decreased the degree of bursting of VTA DA neurons. Similary, chronic i.p. administration of 10 mg/kg of SB-200646 did not significantly alter firing, whereas chronic administration of 20 mg/kg of SB-200646A or 20 mg/kg of clozapine significantly decreased the number of spontaneously active VTA DA neurons when compared with vehicle-treated controls. The SB-200646A-induced decrease in the number of spontaneously active VTA DA neurons was reversed by the i.v. administration of (+)-apomorphine or (-)-baclofen. The chronic i.p. administration of either 10 or 20 mg/kg of SB-200646A did not significantly alter the firing pattern of spontaneously active SNC DA neurons. However, the chronic administration of 20 mg/kg of SB-200646A significantly increased the degree of bursting in VTA DA neurons when compared with vehicle. Overall, the acute and chronic administration of SB-200646A produces in vivo electrophysiological effects, resembling that of atypical antipsychotic drugs.