Purkinje Cell Degeneration Mutant Mice Research Articles

Expression of the yes proto-oncogene in cerebellar Purkinje cells.

To identify the kinds of cells in the brain that express the yes proto-oncogene, we examined chicken brains by using immunofluorescent staining and in situ hybridization. Both approaches showed that the highest level of the yes gene product was in cerebellar Purkinje cells. In addition, we analyzed Purkinje cell degeneration (pcd) mutant mice. The level of yes mRNA in cerebella of pcd mutants was four times lower than that found in cerebella of normal littermates. Our studies point to Purkinje cells as an attractive model for functional studies of the yes protein.

Expression of the yes proto-oncogene in cerebellar Purkinje cells.

To identify the kinds of cells in the brain that express the yes proto-oncogene, we examined chicken brains by using immunofluorescent staining and in situ hybridization. Both approaches showed that the highest level of the yes gene product was in cerebellar Purkinje cells. In addition, we analyzed Purkinje cell degeneration (pcd) mutant mice. The level of yes mRNA in cerebella of pcd mutants was four times lower than that found in cerebella of normal littermates. Our studies point to Purkinje cells as an attractive model for functional studies of the yes protein.

Exploration and habituation in Purkinje cell degeneration mutant mice

Purkinje cell degeneration ( pcd) mutant mice lose virtually all Purkinje cells. It was found that the pcd mutants did not show within-sessions habituation in terms of hole-poking responses. This type of perseverative responding has been found in rats with limbic system damage and implies a role for the cerebellum in behavioral inhibition.

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.

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.

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.

Electrophysiological demonstration of a synaptic integration of transplanted purkinje cells into the cerebellum of the adult purkinje cell degeneration mutant mouse

After implantation of solid pieces of cerebellar primordia from 12-day-old C57BL embryos into the cerebellar parenchyma of 3- to 4-month-old “Purkinje cell degeneration” mutant mice, Purkinje cells from the donor leave the implant and differentiate while migrating into the host molecular layer. Electrophysiological studies were performed using in vitro cerebellar slice preparations from “Purkinje cell degeneration” mutants 1–2 months after grafting, when grafted Purkinje cells have reached their final location in the host molecular layer and have completed their morphological differentiation. Intracellular recordings obtained from 45 Purkinje cells in mutant mice demonstrated that such grafted neurons have normal bioelectrical properties including sodium and calcium conductances and inward rectification. Moreover, all grafted Purkinje cells responded to electrical white matter stimulation by a typical all-or-none climbing fiber response. Responses mediated through the activation of mossy and parallel fibers, as well as inhibitory postsynaptic potentials, were also recorded in a significant number of grafted Purkinje cells. On the whole, all these excitatory and inhibitory responses in grafted “Purkinje cell degeneration” mutant mice have characteristics comparable to those in control mice. After electrophysiological studies, Purkinje cells were further characterized by their positive staining by calbindin antibody. Neurons of this class were dispersed throughout the molecular layer of the host folia in which the electrophysiological recordings had been performed. The ectopic location of their perikarya, the presence of dendritic trees spanning most of the molecular layer (without entering the granular layer), and the occasional presence of axons emerging from the ectopic neurons and forming loose bundles at the white matter axis of the folia, corroborate the grafted nature of the Purkinje cells studied. Therefore, these experiments demonstrate that embryonic Purkinje cells from the graft can complete differentiation in the adult host cerebellum, and establish specific synaptic contacts with the presynaptic elements previously impinging on the missing neurons of “Purkinje cell degeneration” mutants. This process leads to a qualitative functional synaptic restoration of the cortical cerebellar network.

Stability of inferior olivary neurons in rodents. I. Moderate cell loss in adult Purkinje cell degeneration mutant mouse

A light microscopic study and cell counts of the inferior olivary nucleus (ION) were performed in the Purkinje cell degeneration mutant mouse ( pcd/pcd). Six groups of animals aged from 24 to 210 days were studied and compared to age matched (+/+) C57BI.6J mice. A cell deficit of about 30% was found in the youngest animals studied; it did not change significantly with age. The deficit affects the 4 subnuclei of the ION, but predominates clearly in the medial accessory olive. This cell loss is already established at the moment when the massive loss of Purkinje cells that has been reported in this mutant occurs. Thus it is not yet known if the ION represents a primary site of gene action or if the deficit is secondary to another cellular event, presumably the loss of Purkinje cells.

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.

Chapter 18 Integration of grafted Purkinje cell into the host cerebellar circuitry in Purkinje cell degeneration mutant mouse

Publisher Summary This chapter aims at analyzing the progressive fate of grafted Purkinje cells (PCs), and their interactions with the host cerebellar cells. Despite time mismatch, embryonic PCs grafted into adult Purkinje cell degeneration (pcd) cerebellum migrate along stereotyped pathways to their final position in the deficient molecular layer, where they develop monoplanar dendritic trees composed of proximal thick branches and distal spiny branchlets, and receive appropriate synaptic contacts from adult host neurons. Despite the heterogeneous composition of the solid grafts, only neurons of the same category as those missing are able to leave the graft and to invade the host. The fact that the embryonic PCs can migrate, differentiate dendritic trees, and integrate synaptically into the deficient cerebellar circuitry, strongly suggests that signals for neurotropism, migration, neuronal differentiation and synaptogenesis are not limited to embryos, but can be expressed in the adult as well, at least in the pcd mouse cerebellum. The chapter concludes that embryonic neurons can induce a new type of plasticity in adult neural cells by generating a permissive microenvironment that could regulate gene expression of the adult neurons and glial cells.

Delayed spontaneous alternation in Purkinje cell degeneration mutant mice

Purkinje cell degeneration ( pcd) mutant mice lose cerebellar Purkinje cells. It was found that pcd mice, contrary to normal mice, did not alternate spontaneously at any of 3 inter-trial intervals (15 s, 3 or 6 min). Deficits in spontaneous alternation are typical of animals with brain damage in areas important in spatial learning. Results are discussed in terms of a role for the cerebellum in spatially mediated behavior and behavioral inhibition.

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.

Reconstruction of the defective cerebellar circuitry in adult purkinje cell degeneration mutant mice by Purkinje cell replacement through transplantation of solid embryonic implants

Solid pieces of cerebellar primordia taken from 12-day-old C57BL embryos were implanted into the cerebellar parenchyma of 3- to 4-month-old “Purkinje cell degeneration” mutant mice and analysed 2–3 months later. Purkinje cell replacement was followed by means of immunocytochemistry with antisera against either cyclic guanosine monophosphate-dependent protein kinase or vitamin D-dependent calcium-binding protein, which allows the complete staining of these neurons. Although all solid graft implants survived, their fate within the mutant cerebellum varied in three ways: (1) Often, a more or less large fragment of the solid graft remained in the white matter, close to the cortex or even partially replacing it. These remnants contained a few distorted Purkinje cells and a region corresponding to the transplanted deep nuclei, composed of numerous immunostained axons and axon terminals surrounding immunonegative neurons. (2) Less frequently remnants of the graft were extruded to an extracerebellar location, between two adjacent folia. They contained a few Purkinje cells intermixed with granule cells and other neurons. (3) In a few cases corresponding to superficial deposition, the implants developed lobulated and trilaminated minicerebella which were located outside the mutant cerebellum but integrated into it. In all three situations, a large number of grafted Purkinje cells succeeded in moving out of the implants and in invading the host molecular layer. These Purkinje cells develop flattened dendritic trees perpendicular to host bundles of parallel fibres. Ultrastructural examination of the synaptic investment of Purkinje cells which have reached the host molecular layer revealed that they acquire normal synaptic inputs although complex pericellular baskets and pinceau formation do not develop. Axons from molecular layer interneurons synapse on perikaryal and smooth dendritic membranes, climbing fibres synapse on stubby spines emerging from thick dendritic branches and parallel fibres contact almost exclusively the long-necked spines of the distal spiny branchlets. Finally, Purkinje cells which succeed in migrating to molecular layer regions no further than 0.6 mm from the host deep nuclei are able to grow axons which reach appropriate target areas and establish synaptic connections on nuclear neurons. The results obtained from this series of long-term survival cerebellar transplantations point to the possibility of fulfilling most of the conditions necessary for functional restoration of neural grafts in systems in which neurons are connected in a point-to-point manner. Transplanted Purkinje cells leave the graft and migrate to the location of the missing neurons. Once there, they provoke the sprouting of host axon terminals, leading to the formation of specific synaptic inputs. The grafted neurons are also able to grow axons which reach the host deep nuclei and, by providing appropriate outputs, reconstruct to a certain extent the defective adult cerebellar circuitry of mice with heredo-degenerative ataxia.

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.

Non-Purkinje cell GABAergic innervation of the deep cerebellar nuclei: A quantitative immunocytochemical study in C57BL and in Purkinje cell degeneration mutant mice

Purkinje cell degeneration (pcd) mutant mice, 3–4 months old, were used to identify and quantify the non-Purkinje cell GABAergic innervation of deep cerebellar nuclei. Glutamic acid decar☐ylase (GAD) immunoreactive structures appeared as dark dots throughout the 4 nuclei. Ultrastructural examination confirmed that each dot corresponded to an axon terminal. GAD-labeled boutons were large, contained tightly packed flattened vesicles and established Gray type II synapses with all nuclear neuronal populations. Thus, cytological criteria did not distinguish between Purkinje cell and non-Purkinje cell GAD-positive nerve terminals, since they shared many common features. The number of GAD-immunoreactive axon terminals in the deep nuclei of pcd cerebella was compared to that of normal C57BL mice. Despite an almost complete disappearance of Purkinje cells in the pcd mouse (less than 0.05% of these neurons remained in the mutants), the surface density of GAD-positive nerve terminals in the deep nuclear region was 37% of control value. Taking into account a volumetric decrease of 58% for the deep nuclei of the mutant cerebellum, we estimated the percentage of GAD-positive boutons innervating these nuclei to be 15% of normal values. This important residual innervation of the deep nuclei might arise from local GABAergic neurons, which were identified in the normal and mutant cerebella by immunostaining with an antiGABA antibody.

Cerebellar benzodiazepine receptor distribution: An autoradiographic study of the normal C57BL/6J and Purkinje cell degeneration mutant mouse

The distribution of [ 3H]flunitrazepam binding sites in the cerebella of normal mice and Purkinje cell degeneration mutant mice was studied by light microscopic autoradiography. In the cerebellar cortex of normal mice, a high density of [ 3H]flunitrazepam binding was observed over the molecular layer, an intermediate density over the Purkinje cell layer and a low density over the granule cell layer; the white matter was devoid of labeling. The deep cerebellar nuclei were labeled to an intermediate density. In the 54-day-old Purkinje cell degeneration mutant cerebellum, which is depleted of Purkinje cells, a 36% reduction in labeling density of the cerebellar cortex was observed. The density was reduced by approximately equal amounts in both the molecular and granule cell layers; labeling in the deep cerebellar nuclei was, however, substantially increased.

Monoaminergic nerve terminals in the cerebellar cortex of purkinje cell degeneration mutant mice: Fine structural integrity and modification of cellular environs following loss of Purkinje and granule cells

The cerebellar cortex of normal and Purkinje cell degeneration mutant mice was examined by electron microscopy after fixation with potassium permanganate for the demonstration of small granular vesicles in monoaminergic nerve terminals. In control mice, monoaminergic terminals were found mainly in apposition to Purkinje cell dendrites. After the degeneration of Purkinje cells, which constitute the major target for monoaminergic fibres in the cerebellum, monoaminergic terminals persisted in the cerebellar cortex of Purkinje cell degeneration mutant mice. They were ensheathed by astroglial processes in most of the instances. They were also apposed to boutons that contained agranular vesicles, and to stellate cells in the molecular layer. Clear synaptic specializations in the form of thickening of the synaptic membranes were not observed in either control or mutant mice. It is hypothesized that the survival of monoaminergic axons following loss of their target cells may be attributed to the lack of intimate adhesion to their target elements, to a possible functional interaction with the glia, or to the integrity of the extracerebellar terminal fields of the monoamine axon collaterals.

Noradrenergic innervation of the cerebellar cortex in normal and in Purkinje cell degeneration mutant mice: Evidence for long term survival following loss of the two major cerebellar cortical neuronal populations

Purkinje cell degeneration mutant mice were examined during the course of Purkinje cell death (26 and 35 days old) and at 3, 5, 9 and 12 months of age. Glyoxylic acid fluorescence histochemistry for catecholamines was used to investigate possible alterations or reorganization of the noradrenergic fibers from the coeruleo-cerebellar system in response to the degeneration of two major cell types in the cerebellar cortex, of which one, the Purkinje cell, is reported to be the major target neuron. In control mice, noradrenergic fibers traveled in linear and tortuous profiles through the granule cell layer, formed pericellular arrays alongside Purkinje cell somata, and branched profusely into both radially oriented and longitudinally oriented chains. The density of noradrenergic varicosities diminished in the molecular layer with age. In the mutants, concomitant with the progressive shrinkage of the molecular layer, there was a progressive increase in the density of noradrenergic varicosities. This was most conspicuous at 9 and 12 months of age, at which time the molecular layer has been depleted not only of Purkinje cell dendrites, but also of parallel fibers. Noradrenergic fibers in these zones formed dense parallel bundles of varicose profiles whose density reached 621.3 ± 122.8% (mean ± SD, n = 4) at 9–12 months of age, compared with age-matched controls. Neurochemical measurement of norepinephrine content in whole cerebellum of the Purkinje cell degeneration mutants revealed no change compared with age-matched controls. We conclude that noradrenergic innervation persists in the cerebellar cortex despite the death of Purkinje cells and most of the granule cells. Although we found an increased density of varicosities in the molecular layer of mutant mice, progressing with age, we believe that this can be explained on the basis of the resultant geometry of the altered cerebellar cortex. It appears that the health of the environment surrounding the noradrenergic fibers in cerebellar cortex has little influence on their anatomical integrity.

Growth and differentiation of cerebellar suspensions transplanted into the adult cerebellum of mice with heredodegenerative ataxia.

Cell suspensions from cerebellar primordia of 12-day mouse embryos were grafted into the cerebellum of 4-month-old Purkinje cell degeneration (pcd) mutant mice and examined 2-3 months later. In contrast to those of nontreated mutants, all of the grafted cerebella exhibited Purkinje cells that had migrated into the molecular layer, where they were clustered over its superficial two-thirds. These Purkinje cells develop flattened dendritic trees perpendicular to bundles of parallel fibers. Ultrastructural examination of their synaptic inputs and outputs disclosed that (i) as in normal cerebella, climbing fibers and axons from basket and stellate cells synapse on thick dendrites, whereas parallel fibers almost exclusively contact the distal spiny branchlets, and (ii) a substantial number of Purkinje cell axons reach their appropriate targets in the deep cerebellar nuclei, where they establish synaptic connections on large and small neurons. These results indicate that embryonic Purkinje cells grafted into the cerebellum of adult mice with heredodegenerative ataxia integrate themselves very specifically into the cerebellar circuitry of the recipient mouse, where they can replace the missing Purkinje cells. They also provide a morphological basis favoring the notion of functional restorative capabilities of neural grafts in systems in which neurons are connected in an almost point-to-point manner.

Effects of Purkinje cell degeneration on the noradrenergic projection to mouse cerebellar cortex

We have examined the effects of a genetically programmed target cell death on the noradrenergic afferent projection to mouse cerebellar cortex. We have observed that the noradrenergic axon terminals originating in locus coeruleus are maintained in the cerebellar cortex of the Purkinje cell degeneration ( pcd) mutant mouse in spite ofthe absence of Purkinje cells, the targets for the noradrenergic projection. The number of noradrenergic terminals in the atrophic mutant cerebellar cortex is approximately normal as assessed by counts of fibers exhibiting catecholamine fluorescence and by measurement of high affinity uptake of tritium-labeled norepinephrine (NE) by synaptosomes prepared from cerebellar cortex. An increased density of NE fibers is observed which appears to be a consequence of reduced cerebellar mass in the mutant. Although the number of noradrenergic terminals is unaffected, morphological and biochemical alterations are observed in this system. The fibers are more brightly fluorescent in mutant than in normal mice andd their pattern is less orderly. The content of the endogenous transmitter, NE, is increased from 150 to 170% whereas the activity of the rate-limiting enzyme tyrosine hydroxylase (TH) is reduced to about 60% of normal values. These changes appear to be permanent as they are still present in 6 month-old mutant animals, the oldest studied. No alterations in either NE content or TH activity are found in pcd/pcd hippocampus, another target for the locus coeruleus axons.