Search

The expression of AMPA receptor subunit mRNAs and the binding of [(3)H]AMPA were studied in the cerebellum of normal and "Purkinje cell degeneration" ( pcd) mutant mouse. In the pcd cerebellum, [(3)H]AMPA binding was decreased significantly in both the molecular and granule cell layers by 63% and 36%, respectively. In those mutants, GluRA, GluRB and GluRC mRNAs were not detected in the Purkinje cell layer, and the levels of GluRB and GluRD mRNAs were significantly decreased in the granule cell layer by 16% and 57%, respectively. Cerebellar grafts transplanted into the pcd cerebellum expressed only GluRB and GluRC mRNAs, suggesting that donor cells express the appropriate subunits normally expressed by Purkinje neurons. Our results, firstly, support the idea that the expression of the GluRA subunit in Golgi epithelial cells may depend upon the sustained interaction with adjacent Purkinje cells, and secondly, suggest that granule cells which are more resistant to transsynaptic death may express higher levels of GluRB mRNA.

Quantitative analyses of transsynaptic granule cell death subsequent to the genetically determined degeneration of Purkinje cells in the cerebellum of pcd/pcd mutant mice show that granule neuron fallout follows a typical mathematical pattern of elemental decay. Biological and theoretical connotations are discussed in light of the empirical observations and a simulation model.

MAP2 is a major microtubule-associated brain protein, selectively localized in dendrites; growth-associated phosphoprotein GAP-43 is a neuron-specific protein associated with axonal outgrowth. In adult cerebellum, both of these proteins and their corresponding RNA transcripts are most strongly expressed by granule cells. Using immunocytochemistry with antibodies and in situ hybridization histochemistry with [32P] labeled oligonucleotide probes, we examined the cellular localization of MAP2, GAP-43 and their mRNAs in the cerebellum of control and weaver (wv/wv) mutant mice, which exhibit massive granule cell death. In wild-type (+/+) mice, MAP2 immunoreactivity was seen in neuronal somata and dendrites of the granule cell layer; GAP-43 immunoreactivity was present in molecular layer, corresponding to the distribution of parallel fibres. Transcripts encoding MAP2 and GAP-43 were localized in the layer of the granule cell somata. In heterozygous weaver mice (wv/+), which feature an intermediate degree of granule cell loss, MAP2 immunoreactivity was localized in the granular layer, and the pattern of GAP-43 immunostaining was also similar to +/+, the only difference being a thinner molecular layer. Heterozygotes had an anatomical pattern of MAP2 and GAP-43 mRNA hybridization qualitatively similar to that of the wild-type with some deviations in signal intensity. In homozygous weaver mutants (wv/wv), MAP2 immunoreactivity was extremely weak in the area beneath Purkinje cells and a certain GAP-43 immunoreactivity was seen in the upper part of cerebellar cortex. Hybridization signals for MAP2 and GAP-43 mRNAs were minimal. The reported alterations in regional pattern of MAP2 and GAP-43 expression in mutant mice offer a molecular correlate of dendritic and axonal protein gene transcription pertinent to the neuropathological manifestations of certain forms of heredodegenerative ataxia.

Fetal grafts of normal cerebellar tissue were implanted into the cerebellum of Purkinje cell degeneration mutant mice (pcd/pcd), a model of adult-onset recessively inherited cerebello-olivary atrophy, in an attempt at correcting their cellular and motor impairment. Donor cerebellar cells engrafted in the appropriate sites, as evidenced by the pattern of expression of insulin-like growth factor-I (IGF-I) system genes. Bilateral cerebellar grafts led to an improvement of motor behaviors in balance rod tests and in the open field, providing evidence for functional integration into the atrophic mouse cerebellum and underscoring the potential of neural transplantation for counteracting the human cerebellar ataxias.

A synopsis is presented of the recent history of cerebellar tissue transplantation over the past 25 years. The properties of growth and differentiation of cerebellar grafts placed intraocularly or intracranially are reviewed, as well as the interaction of heterotopic and orthotopic grafts with the host brain. Particular emphasis is placed on the use of ataxic mouse mutants as recipients of donor cerebellar tissue for the correction of their structural deficits and the functional recovery of behavioural responses.

Fetal cerebellar cell suspensions, prepared from wild-type (+/+) mice, were implanted bilaterally into the cerebellum of Purkinje cell degeneration' (pcd) mutant mice, a model of adult-onset recessively inherited cerebellar ataxia, to study the functional effects of the grafts on motor coordination and fatigue resistance in a rotating rod treadmill paradigm. The viability of transplanted Purkinje cells was verified with immunocytochemistry for calbindin-D28k and for glutamate receptor 2/3 subunits and with in situ hybridisation histochemistry for insulin-like growth factor I mRNA, biochemical markers normally expressed by Purkinje cells in the cerebellum. Sham injections of vehicle did not appreciably modify the performance of pcd mutants in the rota-rod tests. On the other hand, bilateral cerebellar grafts led to a 3.5-fold increase in the time period that recipient pcd mice were able to stay on the rotating drum based on the comparison of mean scores (of three trials) or a 5.5-fold increase based on the comparison of maximum scores (of the three trials). These findings provide evidence for a motor enhancement in the pcd mouse model of hereditary cerebellar ataxia following intracerebellar transplantation of primordial Purkinje cells.

The IGF system components play important roles in cerebellar development as demonstrated by their specific spatial-temporal expression. IGF-I, type I IGF receptor (IGFR-I), IGFBP2 and IGFBP5 mRNA are localized in distinct cell populations, and all are expressed at the highest levels at the peak of Purkinje cell growth, active synaptogenesis and dendritic formation. To understand IGF-I's action at the cellular level, in situ hybridization was employed to investigate the distribution of IGF system gene transcripts in the cerebellum of weaver mutant mice (wv/wv). Although located ectopically, the surviving Purkinje cells express IGF-I mRNA at the same level in wv/wv as in +/+. No alteration in the cellular distribution or mRNA levels was observed with IGFBP2, or IGFR-I mRNAs. However, the pattern of IGFBP5 expression is altered in the external germinal layer of wv/wv mice. Not only is IGFBP5 expressed by more granule cell precursors of wv/wv cerebellum, but its mRNA level is 2.3 fold that of +/+. The altered IGFBP5 gene expression in granule cell precursors may modulate the interaction of IGF-I with IGFR-I in ways that contribute to their massive death occurring in the development of wv/wv cerebellum.

In order to obtain cDNAs of genes that are expressed in cerebellar granule cells (GC), an antiserum was raised against GC isolated from mouse cerebella. Western blot analysis demonstrated that antibodies against multiple proteins were present and immunohistochemical analysis showed that at least some of these proteins were localized to cerebellar GC. The antiserum was used to screen an expression library derived from mouse cerebellar cDNA. Twenty-two granule cell antibody-positive (GCAP) clones were obtained. Of these, eight represented genes previously described and 14 were novel clones (not found in the GenBank database). In situ hybridization histochemistry showed that eight of the novel clones had moderate to strong expression in cerebellar GC and some of these clones were expressed also in the hippocampal formation. One such clone, GCAP-7, appears to represent a single-copy gene and the entire cDNA insert (2,688 bp) has been sequenced. The clone appears to consist primarily of the 3' untranslated portion, including a poly(A) tail and polyadenylation signals, of a 5 kb transcript. The GCAP clones should be useful for future studies of molecular biology of GC in normal individuals and in inherited neurologic disease with GC degeneration.

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of 'Purkinje cell degeneration' (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss in pcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11-13 normal mouse embryos and were intraparenchymally grafted into the cerebellum of pcd mutants either directly or after pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca(2+)-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Using a serotonin (5-HT)-specific antibody, we examined the 5-HTergic innervation of the cerebellum in the normal mouse (+/+) and in two neurological mutants: weaver (wv/wv), which are characterized by a genetically determined loss of granule cells, and 'Purkinje cell degeneration' (pcd/pcd), which are characterized by a genetically determined loss of Purkinje cells. In normal cerebellum, serotonin-immunoreactive (5-HT-ir) fibers are discrete and ascend to all three layers of the cerebellar cortex. Serotonin-immunoreactive fibers have a much higher density in the atrophic cerebella of both weaver and pcd mutants, where they form multidirectional contours. These anatomical findings provide a profile of 5-HT axon innervation of mouse cerebellum and extend previous neurochemical observations on the metabolic state of cerebellar 5-HT in neurological mutants.

Weaver (wv/wv) mutant mice lose most granule cells of the cerebellum during the first 2 weeks of postnatal life; 'Purkinje cell degeneration' (pcd/pcd) mutants lose virtually all Purkinje cells between postnatal days 17 and 45. Both these neurological mutations are autosomal recessive. We designed a breeding protocol that, in theory, should result in the production of mice with a doubly mutant, wv/wv*pcd/pcd, genotype. Some of the offspring of such crosses had a novel cerebellar phenotype in which both granule and Purkinje cells underwent degeneration, leading to a highly atrophic cortex. This phenotype is what would be expected in wv/wv*pcd/pcd double mutants, and the proportion of such progeny obtained fits with genetic expectations. We propose that (1) wv/wv*pcd/pcd double mutant mice are viable, and (2) the anatomical phenotype of such mice is a combined expression of the component phenotypes.

Embryonic cerebellar grafts from genetically normal donors were implanted into the cerebellomedullary cistern of adult 'Purkinje cell degeneration' (pcd) and weaver mutant mice, which are respectively characterized by the selective loss of Purkinje and granule cells. Grafts placed into both mutant recipients exhibited a layered cellular organization reminiscent of the normal cerebellar cortex. Molecular, Purkinje, and granule cell layers were identifiable. Grafted Purkinje cells displayed characteristic cytological features, such as hypolemmal cisterns in association with mitochondria in the perikaryon, and lamellar structures in their axons. The cytological features of granule cell somata in the grafts appeared similar to those of mature granule cells. Electron microscopic examination of the molecular layer of the grafts revealed the presence of parallel fibers, which were not oriented in a parallel fashion; axon terminals of such fibers were often presynaptic to dendritic spines. The number of parallel fibers was markedly reduced in grafts implanted into both mutants compared to the normal cerebellar cortex; however, this phenomenon is commonly seen in cerebellum in tissue culture and in cerebellar transplants into normal hosts. It is concluded, therefore, that the environment of the mutant hosts does not affect the survival of Purkinje or granule cells and that transplantation of solid cerebellar grafts in the neurological mutants studied does not seem to pose any apparent limitations beyond those inherent to the process of cerebellar growth and differentiation outside its normal environment.

Summary. The expression of AMPA receptor subunit mRNAs and the binding of [3H]AMPA were studied in the cerebellum of normal and “Purkinje cell degeneration” (pcd) mutant mouse. In the pcd cerebellum, [3H]AMPA binding was decreased significantly in both the molecular and granule cell layers by 63% and 36%, respectively. In those mutants, GluRA, GluRB and GluRC mRNAs were not detected in the Purkinje cell layer, and the levels of GluRB and GluRD mRNAs were significantly decreased in the granule cell layer by 16% and 57%, respectively. Cerebellar grafts transplanted into the pcd cerebellum expressed only GluRB and GluRC mRNAs, suggesting that donor cells express the appropriate subunits normally expressed by Purkinje neurons. Our results, firstly, support the idea that the expression of the GluRA subunit in Golgi epithelial cells may depend upon the sustained interaction with adjacent Purkinje cells, and secondly, suggest that granule cells which are more resistant to transsynaptic death may express higher levels of GluRB mRNA. [ABSTRACT FROM AUTHOR]