Partial reconstruction of the adult Lurcher cerebellar circuitry by neural grafting

doi:10.1016/0306-4522(93)90450-T

Neuroscience

Volume 55, Issue 1, July 1993, Pages 1-21

https://doi.org/10.1016/0306-4522(93)90450-T Get rights and content

Abstract

Solid cerebellar grafts, taken from normal mouse embryos (gestational day 12–14), were transplanted into the cerebellum of adult Lurcher mice. The degree of Purkinje cell replacement was analysed one to three months after transplantation by means of immunoycytochemistry (antibodies against calbindin, cGMP-dependent protein kinase and neurofilament proteins) and electron microscopy. Grafted Purkinje cells succeed in moving out of the graft and migrate into the host cerebellar cortex. They are present next to the graft in the granule cell and molecular layers, and far from the graft remnant, only in the molecular layer, indicating that, although both layers subserve Purkinje cell migration, the molecular layer is the ultimate target. In the host molecular layer, axons of transplanted Purkinje cells form thick bundles running in the frontal plane over long distances. Most of them terminate in the upper granule cell layer by enlarged bulbs resembling collapsed growth cones. Axons reaching their normal targets (the neurons of the deep cerebellar nuclei) are observed only in cases where the granule cell layer is disrupted and/or grafted Purkinje cells remain in the white matter. The projection is massive only from grafts lying in the close vicinity of the target neurons. Electron-microscopic analysis of grafted Purkinje cells populating the host cerebellar cortex reveals that their synaptic investment is abnormal. In the molecular layer, where the normal inputs are reduced, the compartmentation in proximal and distal dendritic segments is severely affected, climbing fibre synapses only form on a minority of grafted cells and “pinceau” formations are absent. In the granule cell layer, the synaptic investment is similar to that of Purkinje cells in agranular cerebellum, and even heterelogous synapses with mossy fibres have been observed.

These results, compared to those previously obtained with grafting experiments in Purkinje cell degeneration mutant mouse, allow us to conclude that:

(i) the Purkinje cell-deficient molecular layer of the host, despite its severe atrophy and reactive gliosis, still exerts a positive neurotropism specific for grafted Purkinje cells;
(ii) the unlesioned host granule cell layer underlying the molecular layer containing grafted Purkinje cells, even if almost depleted of granule cells, remains an obstacle for the re-establishment of a corticonuclear projection;
(iii) the degree of synaptic integration of grafted Purkinje cells is directly related to the nearby presence of available host axon terminals. Hence, owing to the atrophy of the Lurcher cerebellum, the postgrafting restoration of the cerebellar cortical circuit is much less complete in this mutant.

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The question also is: what kind of cells would provide specific cerebellar cell phenotypes? A reliable source of Purkinje cells is embryonic or fetal cerebellar tissue (Sotelo and Alvarado-Mallart, 1987, 1991; Kohsaka et al., 1988; Dumesnil-Bousez and Sotelo, 1993; Kaemmerer and Low, 1999; Cendelin et al., 2012; Purkartova et al., 2014; Babuska et al., 2015) (Fig. 25.1), while various types of stem cells and neural progenitors mostly failed to differentiate into specific cerebellar phenotypes (Li et al., 2006; Roybon et al., 2006; Sidman et al., 2007; Chen et al., 2009; Chintawar et al., 2009; Jaderstad et al., 2010). Treatment of demyelinating diseases of the cerebellum by substitution of oligodendrocytes with transplantation of stem cell-derived progenitors seems to be simpler and more efficient, as shown in a rat model of radiation-induced demyelination (Piao et al., 2015).
Neurotransplantation may be a promising approach for therapy of cerebellar diseases characterized by a substantial loss of neurons. Neurotransplantation could rescue neurons from degeneration and maintain cerebellar reserve, facilitate cerebellar compensation, or help reconstruct damaged neural circuits by cell substitution. These mechanisms of action can be of varying importance according to the type of cerebellar disease. Neurotransplantation therapy in cerebellar ataxias is still at the stage of experimental studies. There is currently little knowledge regarding cerebellar patients. Nevertheless, data provided by experiments in animal models of cerebellar degeneration and both clinical studies and experiences in patients with other neurologic diseases enable us to suggest basic principles, expectations, limitations, and future directions of neurotransplantation therapy for cerebellar diseases.
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C3H mice were kindly provided by Dr. K. W. T. Caddy from the University College London [4,5]. B6CBA mice were kindly provided by Prof. A. Resibois from the Université Libre de Bruxelles in 1998; these mice were derived from those used by Sotelo and collaborators [15,50]. Mice of both strains were housed in the same room of the breeding facility under identical conditions.
Mutant mice are commonly used models of hereditary diseases. Nevertheless, these mice have phenotypic traits of the original strain, which could interfere with the manifestation of the mutation of interest. Lurcher mice represent a model of olivocerebellar degeneration, which is caused by the Grid2^Lc mutation. Lurchers show ataxia and various cognitive and behavioral abnormalities. The most commonly used strains of Lurcher mice are B6CBA and C3H, but there is no information about the role of genetic background on the Grid2^Lc manifestation. The aim of this work was to compare spatial navigation in the Morris water maze, spontaneous activity in the open field and motor skills on the horizontal wire, slanted ladder and rotarod in B6CBA and C3H Lurcher mutant and wild type mice. The study showed impaired motor skills and water maze performance in both strains of Lurcher mice. Both C3H Lurcher and C3H wild type mice had poorer performances in the water maze task than their B6CBA counterparts. In the open field test, C3H mice showed higher activity and lower thigmotaxis. The study showed that genetic backgrounds can modify manifestations of the Lurcher mutation. In this case, B6CBA Lurcher mice models probably have more validity when studying the behavioral aspects of cerebellar degeneration than C3H Lurcher mice, since they do not combine abnormalities related to the Grid2^Lc mutation with strain-specific problems.
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Localization of embryonic cerebellar grafts under the granular layer of the cerebellar cortex, has been shown to be essential for connecting grafted PCs to the host's deep cerebellar nuclei, which is necessary for restoration of neural circuitries and cerebellar function in Purkinje cell degeneration (pcd) mice [16,17]. The granular layer acts as a barrier preventing nerve fiber sprouting toward the deep cerebellar nuclei when grafts are placed in the molecular layer [18,19]. In SCA2 mice, grafts developed into a compact mass with few nerve fibers sprouting into the host tissue; making our results similar to those described in B6CBA mice treated with the same type of graft [20].
SCA2 transgenic mice are thought to be a useful model of human spinocerebellar ataxia type 2. There is no effective therapy for cerebellar degenerative disorders, therefore neurotransplantation could offer hope. The aim of this work was to assess the survival and morphology of embryonic cerebellar grafts transplanted into the cerebellum of adult SCA2 mice. Four month-old homozygous SCA2 and negative control mice were treated with bilateral intracerebellar injections of an enhanced green fluorescent protein-positive embryonic cerebellar cell suspension. Graft survival and morphology were examined three months later. Graft-derived Purkinje cells and the presence of astrocytes in the graft were detected immunohistochemically. Nissl and hematoxylin–eosin techniques were used to visualize the histological structure of the graft and surrounding host tissue. Grafts survived in all experimental mice; no differences in graft structure, between SCA2 homozygous and negative mice, were found. The grafts contained numerous Purkinje cells but long distance graft-to-host axonal connections to the deep cerebellar nuclei were rarely seen. Relatively few astrocytes were found in the center of the graft. No signs of inflammation or tissue destruction were seen in the area around the grafts. Despite good graft survival and the presence of graft-derived Purkinje cells, the structure of the graft did not seem to promise any significant specific functional effects. We have shown that the graft is available for long-term experiments. Nevertheless, it would be beneficial to search for ways of enhancement of connections between the graft and host.
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Nevertheless, migration of Purkinje cells did not appear in all mice, graft-derived Purkinje cells were found only in the close surrounding of the solid grafts while they were never observed in distant areas of the graft. Purkinje cell migration from the solid graft in Lurcher mice was reported earlier by Dumesnil-Bousez and Sotelo [9] and finding of aggregates of grafted cells on the surface of the host cerebellum, organotypic organization of the graft and invasion of grafted cells into the molecular layer of the host's cerebellar cortex was reported by Heckroth et al. [11]. In Lurcher mice, some advantages for their use in cerebellar transplantation investigation [11] as well as their serious limitations with regard to the reconstruction of cerebellar circuitries [31] have been described.
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Partial reconstruction of the adult Lurcher cerebellar circuitry by neural grafting

Abstract

Expl Neurol.

Brain Res.

Curr. Opin. Neurobiol.

Neuroscience

Devl Biol.

Neuroscience

Neuroscience

Trends Neurosci.

Brain Res.

Trends Neurosci.

J. biol. Chem.

Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats

J. comp. Neurol.

Experimental reorganization of the cerebellar cortex. IV. Parallel fiber reorientation following regeneration of the external germinal layer

J. comp. Neurol.

Experimental reorganization of the cerebellar cortex. VII. Effects of late X-irradiation schedules that interfere with cell acquisition after stellate cells are formed

J. comp Neurol.

Experimental reorganization of the cerebellar cortex. I. Morphological effects of elimination of all microneurons with prolonged X-irradiation started at birth

J. comp. Neurol.

Axon guidance by gradients of a target-derived component

Science

Specificity of a target cell-derived stop signal for afferent axonal growth

J. Neurobiol.

Structural and quantitative studies on the normal C3H and Lurcher mutant mouse

Phil. Trans. R. Soc. Lond., Ser. B

Studies of the dendritic tree of wild-type cerebellar Purkinje cells in Lurcher chimeric mice

J. comp. Neurol.

Developmental expression of polypeptide PEP-19 in cerebellar cell suspensions transplanted into the cerebellum of pcd mutant mice

Expl Brain Res.

Axon guidance and the patterning of neuronal projections in vertebrates

Science

Early development of the Lurcher cerebellum: Purkinje cell alterations and impairment of synaptogenesis

J. Neurocytol.

Role of the target in directing the outgrowth of retinal axons: transplants reveal surface-related and surface-independent cues

J. comp. Neurol.