Pcd Mutant Research Articles

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

Cerebellar cell suspensions were prepared from normal mouse embryos and implanted into the cerebellum of Purkinje cell degeneration (pcd) mutant mice, which are characterized by a virtually complete degeneration of Purkinje cells between postnatal day (P) 17 and P45. The expression of immunoreactivity for PEP-19, a developmentally-regulated brain-specific polypeptide, was analyzed in normal mouse cerebellum, as well as in pcd mutants with or without grafts. In the normal cerebellum, PEP-19 immunoreactivity was present in Purkinje cells. In unoperated mutants, 45 days of age or older, Purkinje cells were absent. In grafted pcd mice, numerous PEP-19 immunoreactive, neuroblast-like cells were seen in the graft at 5 days after transplantation. By 9 days, large PEP-19 immunoreactive neurons were found in the host molecular layer; by 17 days after transplantation, such neurons displayed an extensive dendritic tree and resembled differentiated Purkinje cells. The vast majority of PEP-19 immunoreactive cells was located in the molecular layer of the host at 9 days after transplantation and beyond; nonetheless, the same cells extended axonal processes toward the graft, indicating an affinity for co-grafted (possibly deep nuclei) neurons. These results point to the ability of donor Purkinje cells for survival, migration into the host brain and morphological and chemical differentiation following transplantation to the degenerated cerebellar cortex of the recipient mutants.

Serotonin concentration and turnover in cerebelum and other brain regions of pcd mutant mice

The Purkinje cell degeneration ( pcd) mutant mouse is characterized by loss of Purkinje cells in the cerebellum. Loss of granule cells occurs and is severe in pcd mutants after 9 months of age. Since Purkinje cells and granule cells represent two groups of target cells for serotonin neurons projecting from raphe nuclei and other brain areas, the content and turnover of serotonin in the cerebellum were determined in pcd mice aged 3–15 months. The content of serotonin was not decreased in pcd mouse cerebellum but tended to be slightly increased after 7 months. The ratio of 5-hydroxyindoleacetic acid (5-HIAA) to serotonin was significantly decreased in cerebellum at 7–15 months but not at 3 or 6 months. The decrease in this ratio is indicative of decreased serotonin turnover. Similar changes were not seen in brainstem or hypothalamus in mice up to 14 months old, but slight decreases were observed at 15 months. Another index of turnover, the accumulation of 5-HIAA after administration of probenecid to block its efflux from brain, was decreased by 46% in 7-month-old pcd mice in the cerebellum but not in the brainstem or hypothalamus. The decrease in serotonin turnover in pcd mouse cerebellum occurs subsequent to and perhaps due to the loss of the target Purkinje and granule cells.

Autoradiographic localization of insulin-like growth factor II receptors in cerebellar cortex of weaver and Purkinje cell degeneration mutant mice

Autoradiography was used to visualize insulin-like growth factor II (IGF-II) receptors in the cerebellar cortex of weaver and Purkinje cell degeneration ( pcd) mice. These mutants were selected for their respective absence of granule or Purkinje cells. Histological preparations confirmed a severe loss of granule cells in the cerebella of weaver mutants and an absence of Purkinje cells in those of pcd mutants. Autoradiographs showed specific IGF-II binding to the granule cell layer of the cerebellar cortex in control mice, and in pcd specific IGF-II receptor sites are located on the granule cells of the cerebellum.

Changes in the content of glutamate and GABA in the cerebellar vermis and hemispheres of the Purkinje cell degeneration (pcd) mutant.

The contents of glutamate and GABA, as well as aspartate, glycine, and alanine, were examined in the cerebellar vermis and hemispheres of normal and Purkinje cell degeneration (pcd) mutant mice at 6, 9, and 12 months of age. Relative to normal values, the content of glutamate was approximately 50% lower in the vermis for the 3 age groups. In the hemispheres, the content of glutamate was also lower than control values and showed a progressive loss from 30 to 47% with age. On the other hand, in the case of GABA in the vermis, the level was 39% lower in the pcd mutant at 6 months of age but no different from control values at 12 months. However, relative to data for normal mice, the content of GABA in the hemispheres was consistently lower (20%) for all age groups. The level of aspartate was approximately 60% lower in the cerebellar vermis and 45 to 55% lower in the hemispheres of the mutant with respect to control data for all three age groups. Likewise, alanine showed a reduced content in the hemispheres (36-46%) and vermis (24%) in the mutant relative to normal values at 6, 9, and 12 months of age. On the other hand, the level of glycine was 43-64% higher in the vermis and 77-100% greater in the hemispheres of the mutant than in the control group. The higher values for glycine were observed at the two oldest ages. In conclusions, the data are consistent with the idea that glutamate and GABA are present in high concentrations in granule and Purkinje cells, respectively, and provide additional support for a transmitter function for both amino acids in the cerebellum.

Cerebellar mutations affecting the postnatal survival of Purkinje cells in the mouse disclose a longitudinal pattern of differentially sensitive cells

The pattern of surviving Purkinje cells (PCs) was investigated in three cerebellar mutant mice with severe postnatal PC death. Two of these mutations, nervous ( nr) and Purkinje cell degeneration ( pcd) mutations are already well characterized. The third mutation is a new one, which appeared spontaneously in DW/J-Pas mice and was called tambaleante ( tbl). PCs were identified by immunocytochemistry using an antibody against vitamin D-dependent calcium-binding protein which labels all the PCs in adult control mice. In each of the three mutations, surviving PCs are arranged according to a different and reproducible pattern which is symmetric relative to the midline. In NR and young PCD mutants, PCs are closely packed in broad sagittal bands. In TBL, they are more loosely arranged in a rather patchy pattern. In PCD and in TBL mutants the death of resistant PCs is only shortly delayed but in NR there is little change in the number of surviving PCs after 3 months. The differential sensitivity of subsets of PCs to the effect of nr, pcd, and tbl mutations is topographically determined. These results provide a new evidence of the PC heterogeneity which has been previously demonstrated by histochemical and immunohistochemical techniques. Moreover, in the anterior vermis of control mice, three thin sagittal bands of PCs are labeled by the Q113 monoclonal antibody. Similarly, in the anterior lobe of the NR cerebellum, the thin longitudinal strips of missing PCs coincide with the absence of Q113 immunoreactivity: in this region the nr mutation affects specifically the survival of Q113 positive cells. However, other clusters of Q113 immunoreactive PCs do survive in NR mice suggesting that susceptibility to the nr mutation and Q113 positivity are two independent markers of the underlying PC compartmentalization.

Spontaneous alternation and habituation in Purkinje cell degeneration mutant mice

Purkinje cell degeneration (pcd) mutant mice were found to be as active as normal mice in a T-maze. The pcd mutants, contrary to normal mice, did not alternate spontaneously in either a 2-trial or a 4-trial test. Results are discussed in terms of a role for the cerebellum in behavioral inhibition and visuo-spatial organization.

Norepinephrine metabolism in the cerebellum of the purkinje cell degeneration (pcd) mutant mouse

Norepinephrine (NE) metabolism in brain regions was studied in the Purkinje cell degeneration (pcd) mutant mouse. The Purkinje cells, which are target cells for noradrenergic (NA) neurons, degenerate completely in the pcd mutant mouse between 17 and 45 days of age, but the NA terminals remain intact. The purpose of this study was to determine if cerebellar NE turnover is altered after Purkinje cell loss. The concentrations of NE and of its major metabolite in mouse brain, 3-methoxy-4-hydroxy-phenylglycol (MHPG) were measured by liquid chromatography with electrochemical detection. The concentration of MHPG and the concentration ratio MHPG/NE were taken as indices of NE turnover. Although the cerebellar content of NE in the pcd mice was not different from control mice, MHPG and the ratio MHPG/NE were decreased slightly in 3-month old, and more in 6 and 9-month old, mutants compared to controls. No decrease in MHPG content or in the MHPG/NE ratio was detectable in younger mutants (22 or 45-day old). The accumulation of dopa (3,4-dihydroxyphenylalanine) after administration of NSD 1015 to inhibit aromatic l-amino acid decarboxylase was determined as an index of NE synthesis. Dopa accumulation was decreased in cerebellum to 69%, 53% and 37% of control values, respectively, at 3, 6 and 12 months in pcd mice, but was not changed in brain stem and was only slightly decreased in hypothalamus. No decrease in MHPG (content or concentration) or in the ratio MHPG/NE was found in brain stem. In hypothalamus, NE concentration and content were slightly increased in pcd mice at all ages, and the ratio MHPG/NE was decreased in 6 and 9-month old mice. We conclude that although NA axons in the cerebellum are maintained in pcd mice after Purkinje cell degeneration, NE turnover is decreased, suggesting that the synthesis and release of neurotransmitter does not continue at normal rates when the target cells have degenerated.

Residual benzodiazepine (BZ) binding in the cortex of pcd mutant cerebella and qualitative BZ binding in the deep cerebellar nuclei of control and mutant mice: an autoradiographic study

In mutant mice ‘Purkinje cell degeneration’ ( pcd), there is an almost complete degeneration of Purkinje cells followed subsequently by a partial degeneration of granule cells. Recent neurochemical studies have revealed a 50% decrease in benzodiazepine (BZ) receptors in 45-day-old pcd mutants after degeneration of the Purkinje cells. At 300 days there is an 80% decrease in BZ receptors concomitant with granule cell losses. To determine the histological localization of these receptor changes this autoradiographic analysis was conducted. An in vitro autoradiographic technique was used to explore [ 3H]flunitrazepam binding. BZ receptors were found to be more concentrated in the molecular than the granular layer of mutant and control cerebellar cortices. There was, nonetheless, no statistically significant difference in grain counts between control and mutant mice in any layer. Substantial atrophy of cerebellar structures, particularly of the molecular layer, occurred in the mutant mice. It began even before 45 days of age but was extreme at 300 days. When the appropriate mathematical correction factor was introduced for the layer atrophy there was a 60% decrease in grain count in 45-day-old mutants in the molecular layer and a 84% decrease in 300-day-old mutants compared to controls. The initial decrease in total BZ receptors in the 45-day-old mutant animals is associated with a selective loss of Purkinje cells. The amount of receptor binding which persists in the 300-day-old mutants in the molecular layer would appear to reflect binding in the remaining parallel fibers from granule cells which remain. Because there is a significant decrease in the granular layer width especially in the 300-day-old mutant animals, it is proposed that other cerebellar elements present in this layer may contain BZ receptors. An unexpected finding was the substantial BZ binding in the deep cerebellar nuclei both of 45- and 300-day-old animals whether mutant or control. The density of such binding is greater in the nuclei of the mutant mice. The possibility that this receptor binding alteration may reflect a supersensitivity phenomenon is raised. Since there is complete interruption of axonal input to the deep nuclei from the Purkinje cells, particularly at 300 days, additional studies must be conducted to identify the structures accounting for the substantial residual binding in these nuclei.

Degeneration of thalamic neurons in "Purkinje cell degeneration" mutant mice. II. Cytology of neuron loss.

The cytology of thalamocortical relay neuron degeneration in the ventral medial geniculate nucleus (vMG) of mice homozygous for the autosomal recessive Purkinje cell degeneration (pcd) mutation has been studied by light and electron microscopy. More limited sampling of the submedial and mediodorsal nuclei suggested that cytological alterations in the vMG were typical of all degenerating thalamic nuclei. The number of vMG neurons in pcd mutants was comparable to controls at and prior to postnatal day 40 (P40). By P60 seventy percent, and by P90 approximately 90%, of the original complement of vMG neurons had degenerated in mutant mice. At P30, the general cytological organization of vMG neurons closely resembled that of neurons in littermate (+/+ or +/pcd) controls, but neurons in mutants were distinguished by the presence of small aggregates of fine granules (approximately 9 nm in diameter) that were commonly associated with otherwise normal cisternae of rough endoplasmic reticulum; neither the number nor the size of these granular aggregates increased in older mutants. By P50 cytoplasmic organelles were curiously distributed in more severely affected neurons: large areas of cytoplasm were occupied exclusively by polysomes, while profiles of endoplasmic reticulum and the Golgi apparatus appeared to be reduced. Before frank degenerative changes were apparent (at P50), all classes of synaptic terminals identified in normal mice were found to have made morphologically normal synaptic contacts on mutant vMG neuron dendrites. In contrast to the homologous nuclear complex in the cat, presynaptic dendrites were not apparent in synaptic glomeruli in wild-type or mutant murine vMG. Cytopathological alterations in the neuropil of P50 and older mutants were dominated by degenerating dendritic profiles; there was no evidence that the loss of thalamic neurons in pcd mutants was associated with synaptic agenesis or dysgenesis or the prior or concurrent degeneration of afferent synaptic terminals.

P400 protein characteristic to Purkinje cells and related proteins in cerebella from neuropathological mutant mice: autoradiographic study by 14C-leucine and phosphorylation.

P400 protein has been found in the cerebellum and localizes to Purkinje cells. It is a glycoprotein and reacts with plant lectins. Autoradiographic patterns of the protein profiles of mutant mice cerebella after intracranial injections of 14C-leucine were analyzed. P400 protein was one of the dominant proteins labeled in the control cerebellum. No incorporation of 14C-leucine to the position of P400 protein was found in the nervous and pcd mutant mice where no Purkinje cells and no P400 protein are present. P400 protein in weaver cerebellum was labelled more intensely than that in the control. In the staggerer mice, where dendritic arborization of Purkinje cell is poor and no spines are formed on the dendrites, no P400 protein was found and no 14C-leucine incorporation was observed to the position of P400 protein, suggesting that staggerer is a mutation where no P400 protein accumulated in the fractions being studied. P400 protein was phosphorylated independently of the presence of Ca2+. Among the proteins of the isolated Purkinje cells, P400 protein was one of the major proteins phosphorylated.

Loss of Purkinje cell-associated benzodiazepine receptors spares a high affinity subpopulation: a study with pcd mutant mice.

In order to identify the relative number of benzodiazepine (BZ) receptors in Purkinje and granule cells, the Purkinje cell degeneration (pcd) mutant mouse was used at different ages. In these mice, Purkinje cells have degenerated almost completely by 45-50 days of age. Granule cell loss occurs only later, and is most severe between 180 and 300 days. [3H]Flunitrazepam (FNZ) and [3H]ethyl-carboline-3-carboxylate (beta-CC) were used as ligands. In the 45-50-day-old pcd mice, it was found that there is approximately a 50% decrease in the number of receptors as labeled by [3H]beta-CC or [3H]FNZ, when the binding is expressed as fmol/cerebellum. The binding decreased by approximately 80% in 300-day-old pcd mice (fmol/cerebellum). [3H]FNZ was not displaced by 1 microM RO5-4864, ruling out binding to glial cells. Nonlinear regression analysis of FNZ saturation data provided evidence for two populations of receptors (high and low affinity sites). Only the low-affinity sites were reduced in number at 45 days. [3H]beta-CC saturation data showed, however, only one population of receptors. The total number of receptors (Bmax) was significantly lower for beta-CC than for FNZ in the control mice. It appears that 50% of the total BZ receptors is associated with Purkinje cells. In addition, our data on 300-day-old pcd mutants strongly suggest the existence of granule cell-associated BZ receptors.

Purkinje cell loss and the noradrenergic system in the cerebellum of pcd mutant mice

Purkinje cells in the cerebellum receive inhibitory noradrenergic input from the locus coeruleus. In pcd mutant mice all Purkinje cells degenerate by 45 days of age. The purpose of the present studies was to determine if the loss of these cerebellar neurons affects the amounts of norepinephrine in the cerebellum of mice 25–280 days of age. No significant changes in norepinephrine content were detected during or after Purkinje cell degeneration. However, since degeneration led to a reduction in cerebellar weight, the norepinephrine concentration was increased in pcd mutants. These results indicate that despite the loss of a major postsynaptic target (Purkinje cells), the cerebellar noradrenergic input remains stable.

Presence of protein I, a phosphoprotein associated with synaptic vesicles, in cerebellar granule cells.

The cerebellar levels of Protein I, a synapse-specific neuronal phosphoprotein, have been investigated in the cerebellar mouse mutants stagger (sg), weaver (wv), nervous (nr), and Purkinje cell degeneration (pcd). The Protein I concentration was reduced by about 66% in sg and wv mutants, representing a 90% loss of Protein I per cerebellum. A heterozygote effect was observed in the wv mutant. These results indicate that a great majority of Protein I in the normal cerebellum may be present in the granule cells. In nr mutants the cerebellar Protein I concentration was reduced by only 12% in 62-day-old mice, suggesting that Purkinje cells contribute little to cerebellar Protein I. However, a greater reduction was observed in pcd mutants, which may reflect on the nature of the pcd mutation.

Exaggerated cyclic AMP accumulation and glial cell reaction in the cerebellum during Purkinje cell degeneration in pcd mutant mice.

The Purkinje cell degeneration mutant (pcd) is characterized by a complete loss of cerebellar Purkinje cells. Norepinephrine causes an accumulation of cyclic AMP in the cerebellum of pcd that is far greater than in normal mice. Experiments were conducted 1) to correlated changes in the cyclic-nucleotide response with a histologic examination of the cerebellum during neuronal loss and 2) to examine the role of cyclic AMP catabolism and adenosine receptor interactions in the phenomenon. The greatest elevation in cyclic AMP occurred between 30 and 128 days of age when a severe astrocytic response was demonstrated throughout the cerebellar cortex. Purkinje cells had degenerated by 45 days of age. Norepinephrine elicited a smaller increase in cyclic AMP from 155-day-old mice than at earlier ages, and the response continued to decrease with age; at 270 days, equal accumulation, and at 365 days. lower accumulation of cyclic AMP was detected in pcd cerebella. During this time, the Purkinje cell debris had been removed, the granule cell layer was depleted of granule cells, and the molecular layer was deprived of a large number of parallel fibers. However, although phagocytosis of neuronal debris was completed, large numbers of astrocytic processes were still seen in the neuropil. Biochemical experiments in vitro established that the exaggerated accumulation of cyclic AMP in the presence of norepinephrine was not due to lower catabolism of cyclic AMP, a synergistic interaction with adenosine, or a result of lower protein in the pcd cerebellum. The correlates of heightened norepinephrine-stimulated accumulation of cyclic AMP with neuronal loss and the glial cell reaction might indicate that cyclic nucleotides play a role in controlling some glial cell functions, ie, proliferation, migration, and phagocytosis.

Exaggerated norepinephrine-stimulated accumulation of cyclic AMP in vitro in cerebellar slices from pcd mutant mice following Purkinje cell loss.

Norepinephrine caused in vitro an accumulation of cylic AMP in slices of the cerebellum from pcd mice which was 300 percent greater than in cerebella from age-matched, heterozygous, normal mice. Purkinje cells had disappeared in the pcd mice at the time when the exaggerated hormonal response was seen. The data indicate that high cyclic AMP accumulation can take place in cerebellar cells other than Purkinje cells.

The development and degeneration of Purkinje cells in pcd mutant mice

Purkinje cell degeneration (pcd), an autosomal recessive mutation in the mouse, causes the postnatal death of virtually all cerebellar Purkinje cells during the third and fourth postnatal week. We have compared the postnatal development of normal and pcd mutant Purkinje cells. The early deviations from normal development involve primarily the perikaryonal polysomes and endoplasmic reticulum. Many of the mutant Purkinje cells retain abnormally the basal accumulation of polysomes, a finding which permits the identification of affected animals at postnatal day 15, one week prior to the onset of behavioral abnormalities. In addition, the affected Purkinje cells possess unusual configurations of endoplasmic reticulum with associated electron-dense particles similar to but larger than ribosomes, mature and forming intracisternal A particles and nematosomes. Before the pcd Purkinje cells degenerate they appear to receive all their appropriate synaptic contacts. Some disruption, however, of parallel fiber: Purkinje spine synaptogenesis occurs at late stages of development. Some spines lack presynaptic elements, postsynaptic thickenings are present along the dendritic shafts and parallel fibers appear to make synaptic contacts directly onto the shafts. The spectrum of early morphological changes that has been observed in pcd mutant Purkinje cells is thus far unique to this cerebellar abnormality.