Cerebellar Purkinje cells provide target support over a limited spatial range: evidence from lurcher chimeric mice.

The distribution of Purkinje cells, granule cells, and olivary neurons was quantitatively analyzed in a lurcher +/Lc in equilibrium C3H/HeJ chimera in which the surviving wild type Purkinje cells were unilaterally distributed in the left hemicerebella. The left hemisphere of this mouse contains 7600 Purkinje cells, approximately 10% of the number of Purkinje cells in inbred C3H/HeJ mice. The right hemisphere contains 300 Purkinje cells, all of which are found within 200 microns of the midline. As in other +/Lc in equilibrium wild type chimeras, the ratio of granule cells to Purkinje cells is increased in the left hemisphere, reflecting increased granule cell survival. In the right hemisphere, however, the number of granule cells is reduced to that found in +/Lc mutants. In the inferior olive, almost twice as many neurons are found in the right nucleus as opposed to the left nucleus. As the projections of olivary neurons are crossed, the number of olivary neurons is increased in the nuclei that project to the cerebellar hemisphere containing Purkinje cells compared to the olivary nuclei that project to the cerebellar hemisphere with almost no Purkinje cells. The preferential survival of granule cells and olivary neurons that either occupy or project to the hemicerebellum containing Purkinje cells suggests that the availability of trophic support from target Purkinje cell neurons is spatially restricted.     

Structure of the Purkinje cell membrane in staggerer and weaver mutant mice

The structure of the plasma membrane of Purkinje cell dendrites was examined in weaver and staggerer mutant mice. Purkinje spines in weaver mice have clusters of intramembrane particles which resemble those at normal synapses with parallel fibers, even though no parallel fibers are formed in this mutant. There are very few spines in the staggerer, and these manifest normal intramembrane structure at contacts with climbing fibers. The spines which would normally be involved in synapses with parallel fibers are never formed in the staggerer, and the intramembrane structures which would have been associated with these spine synapses are also lacking. Thus, during postnatal cerebellar development in the mutants, acquisition of intramembrane specializations requires Purkinje spine formation but can occur independently of the development of parallel fibers.     

Evidence that the loss of Purkinje cells and deep cerebellar nuclei neurons in homozygous weaver is not related to neurogenetic patterns

To determine whether the neurogenetic patterns of Purkinje cells and deep cerebellar nuclei neurons were normal in weaver homozygotes and whether the degeneration of those neuronal types was linked to their time of origin, [3H] thymidine autoradiography was applied on sections of homozygous weaver mice and normal controls on postnatal day 90. The experimental animals were the offspring of pregnant dams injected with [3H] thymidine on embryonic days 11-12, 12-13, 13-14 and 14-15. The results show that the onset of neurogenesis, its pattern of peaks and valleys, and its total span were similar between wild type and homozygous weaver in the cerebellar areas analyzed, indicating that the loss of Purkinje cells and deep cerebellar nuclei neurons is not related to neurogenetic patterns. In weaver homozygotes, the loss of Purkinje cells and deep cerebellar nuclei neurons followed a lateral to medial gradient of increasing severity. Thus, the vermis and the fastigial nucleus, which are medially located, presented the most important neuron loss, whereas in the lateral hemisphere and the dentate nucleus, neuron loss was spared.     

Numerical matching between granule and Purkinje cells in lurcher chimeric mice: a hypothesis for the trophic rescue of granule cells from target-related cell death

Previous studies of wild-type mice or mutant-wild-type mouse chimeras using the neurological mutant staggerer have supported a numerical matching hypothesis for target-related cell death. However, analyses of chimeras of a second neurological mutant, lurcher, have suggested that there may be significant flexibility in the relationship between the numbers of pre- and postsynaptic neurons. Whereas in staggerer chimeras there is a strict proportionality between the number of cerebellar granule cells and their postsynaptic target, the Purkinje cells, in lurcher chimeras, Wetts and Herrup (1983) report a relative increase in granule cell survival. We have reexamined the numerical matching between Purkinje and granule cells in an additional 5 lurcher----wild-type and 4 wild-type----wild-type chimerase. Our findings confirm and extend the results of the previous study to show that there is a close correlation between the number of granule and Purkinje cells in +/Lc chimeras, but for any given number of Purkinje cells in the +/Lc chimeras, more granule cells survive than in staggerer chimeras or inbred mouse strains. Whereas the ratio of granule to Purkinje cells in staggerer chimeras or inbred mouse strains is constant across all target sizes, in +/Lc chimeras the ratio of granule cells to Purkinje cells increases as the number of target neurons decreases. It seems likely that the increased granule cell survival is somehow related to the delayed degeneration of the +/Lc fraction of target cells in the +/Lc chimeras. Among the possible explanations for the observed results, we favor the hypothesis that a trophic factor is produced in +/Lc chimeras in response to the deafferentation of Purkinje cells that is capable of rescuing granule cells from target-related cell death. Our preference is based, in part, on observations of the state of the dendritic tree of the wild-type Purkinje cells that survive in +/Lc chimeras (Caddy et al., 1986).     

Purkinje cell loss is a characteristic of essential tremor

This paper began as a letter to the editor, commenting on several methodological and conceptual problems with the paper by Rajput et al. [1]. We were asked by the editors to expand the paper to include a more general discussion of the role of the cerebellum in essential tremor (ET). The study of the neuropathological underpinnings of essential tremor (ET) is a relatively new undertaking. The purpose of this paper is three-fold. The first is to comment on methodological problems in a recently-published paper by Rajput et al., the major one being the small sample size of that study, which resulted in a Type II statistical error. Hence, one cannot conclude based on their data that there is no Purkinje cell (PC) loss in ET. Secondly, we comment on conceptual problems with that study, which suggested that PC loss might not be a featured characteristic of ET because it is also found in other disease states. We discuss why this is an erroneous conclusion. Our third purpose is to more broadly discuss the role of the cerebellum in ET, giving consideration to the wealth of clinical and postmortem data that have accumulated over recent years. In this discussion, we make the following points: (1) it is now generally recognized that ET is a disease of cerebellar systems dysfunction, (2) given the nature of the postmortem work, revealing the presence of several types of structural-anatomical changes within the cerebellum and absence of detectable changes in other brain regions, the most empirically-based explanation is that the primary problem in ET is in the cerebellum itself, (3) that the collection of cellular changes in the cerebellum in ET are also present in other cerebellar degenerations should add to rather than detract from the notion that ET is a disease of cerebellar degeneration.     

Regional differences in the Purkinje cells settled pattern: a comparative autoradiographic study in control and homozygous weaver mice

Data accumulated over the last 10 years have led to the popular hypothesis that neutrophils and other inflammatory cells play a prominent role in the neuropathology of cerebral ischemia. This hypothesis was derived from a large number of studies involving three general observations: (1) leukocytes, particularly neutrophils, are present in ischemic tissue at the approximate time that substantial neuronal death occurs; (2) neutropenia is sometimes associated with reduced ischemic damage; and (3) treatments that prevent leukocyte vascular adhesion and extravasation into the brain parenchyma can be neuroprotective. This review reexamines the literature to ascertain its support for a pathogenic role for neutrophils in ischemia-induced neuronal loss. To accomplish this goal, we employed several logical theorems of "cause-effect" relationships, as they pertain to leukocytes and ischemic brain damage. Since the majority of studies focused on neutrophils as the most likely pathogenic inflammatory cell, this review necessarily does so here. We reasoned that if neutrophils play an important pathogenic (i.e., cause-effect) role in the neuronal damage that follows a stroke, then one should expect to find clear evidence that: (1) neutrophils invade the ischemic area prior to terminal stage infarction, (2) greater numbers of early appearing neutrophils are accompanied by evidence of greater neuronal loss, and (3) dose-related inhibition of neutrophil trafficking or activity produces a corresponding decrease in the degree of brain damage following ischemia. This review of the literature reveals that the existing evidence does not readily support any of these predictions and that, therefore, it consistently falls short of establishing a clear cause-effect relationship between leukocyte recruitment and the pathogenesis of ischemia. While the available evidence does not necessarily rule out a potential pathogenic role of neutrophils and other leukocytes, it nevertheless does expose serious weaknesses in the existing studies intended to support that hypothesis. For this reason we also offer suggestions for additional experiments and the inclusion of control groups that, in the future, might provide more effective or conclusive tests of the hypothesis.     

Development of Purkinje cell somatic spines in the weaver mouse

Summary The cerebellar cortices of weaver mice and their normal littermates ranging in age from six to thirteen days after birth were examined with the electron microscope. The initial development of spines, both somatic and dendritic, are the same in both the weaver and its normal littermates despite the extreme paucity of parallel fibers in the former.     

Selective Purkinje cell ectopia in the cerebellum of the weaver mouse

The adult mouse cerebellar vermis consists of four transverse zones, each of which is further subdivided into parasagittal stripes. In the adult weaver (wv/wv) mouse, the zebrin II expression pattern in the cerebellar vermis is abnormal, consistent with the absence of a central zone (approximately lobules VI/VII). Because the small, heat shock protein HSP25 is a constitutive marker of parasagittal bands of Purkinje cells in the caudal central zone and the nodular zone (approximately lobules IX/X), we used HSP25 immunocytochemistry to show that the patterning abnormalities in wv/wv reflect selective Purkinje cell ectopia rather than the absence of the central zone. A specific HSP25-immunopositive Purkinje cell ectopia within the central zone was identified. Symmetrical clusters of HSP25-immunopositive Purkinje cells, which presumably would have formed the parasagittal stripes in the wild type, are present ectopically on either side of the midline in wv/wv. In contrast, in the nodular zone, HSP25-immunopositive Purkinje cells form a near-monolayer and are organized into parasagittal stripes. We therefore conclude that specific Purkinje cell clusters in the wv/wv cerebellum fail to disperse and that this ectopia contributes to the topographical abnormalities.     

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

Naturally occurring mutations are valuable tools for the study of neural development, especially when used in conjunction with the techniques of chimeric mouse production. In this study we examine the response of Purkinje cell dendrites to the altered developmental environment found in the lurcher in equilibrium with wild-type chimera. Lurcher (+/Lc) is an autosomal dominant gene that causes the cell-autonomous degeneration of all Purkinje cells of +/Lc genotype. Thus, in +/Lc in equilibrium with +/+ chimeras, only wild-type Purkinje cells survive to maturity. The number of these survivors can vary from less than 10,000 to greater than 100,000. Previous work has shown that the final ratio of presynaptic granule cells to postsynaptic Purkinje cells is increased in lurcher chimeras. On average, therefore, one might expect that each remaining Purkinje cell would experience an increased supply of afferents, and our hypothesis was that dendritic growth and/or sprouting might occur as a result. This proved incorrect and, indeed, the Purkinje cells in the lurcher chimeras show changes of a predominantly atrophic nature. Unusual morphologies are found, including variable branching density, failure of the distal dendrite to reach the pial surface, loss of isoplanarity, and the frequent appearance of large caliber, primary or secondary dendritic branches ending abruptly in "stub ends." Quantitative analysis of Golgi-Cox impregnated material reveals that in lurcher chimeras the Purkinje cell dendritic arbor is reduced by more than 60% compared to wild-type animals. We present possible explanations for this finding and consider several potential implications.     

The fine structure of the Purkinje cell and its afferents in lurcher chimeric mice

Lurcher is an autosomal dominant mutation in the mouse. Heterozygote (+/Lc) animals lose 100% of their cerebellar Purkinje cells during the first postnatal month. Aggregation chimeras made between +/Lc and wild-type embryos have been used to demonstrate that this neuronal cell death is a cell autonomous property of the +/Lc Purkinje cells. In lurcher chimeras, all +/Lc PCs die while wild-type Purkinje cells survive in the numbers expected. Although they are normal in number, previous work from our laboratories has shown that when the genetically wild-type Purkinje cells are present in the mosaic environment of the lurcher chimeric mouse they develop a very unusual morphology. Their dendritic trees are small, and the caliber of their dendrites is increased. This paper examines the fine structure of these unusual cells as well as their afferent fibers. Purkinje cell somas in the lurcher chimera have an increased number of lysosomes and the rough endoplasmic reticulum is improperly configured. In the majority of the Purkinje cell dendrites the organelles are disorganized; it is not certain whether this is a cause or a consequence of the increase in dendritic caliber previously reported. Presynaptic fibers have been examined and, while all classes of expected synapses can be observed, the numbers of synaptic profiles visible in any one thin section are reduced. Climbing fiber terminations on the Purkinje cells were smaller than normal with a greatly diminished number of constituent vesicles. Unexpectedly, we found unusual morphologies among the Bergmann glial fibers and the presence of unusual (or ectopic) astrocytic like glial cells near the pial surface. These changes in turn were accompanied by an increase in the number of glial-like fibers near the pia in some parts of the chimeric cerebellar cortex. The results are discussed in light of our knowledge of other mutant mice, and a hypothesis is put forward to explain some of our results.     

Topological Link-Vertex Analysis of the growth of Purkinje cell dendritic trees in normal, reeler, and weaver mice

The growth of Purkinje cell dendritic trees in normal, reeler, and weaver mice has been defined by using Link-Vertex Analysis. Growth probably occurs in three phases in normal and cortically located reeler trees. Phase I, completed by 7 days postnatum (dpn), establishes a rudimentary tree of 100 segments by random terminal branching. Phase II lasts from 7 to 20 dpn when some 690 and 450 segments are generated in normal and cortical reeler trees respectively. Phase II is initiated by an inductive stimulus mediated by a finite number of parallel fibres. Thereafter, dendritic trees develop a similar topology in normal and cortical reeler Purkinje cells through random interactions between parallel fibres and dendritic growth cones. We have defined this process with the aid of computer simulation techniques. Interactions appear to be restricted to a narrow growth front occupied by the highest centrifugally ordered terminals behind which adhesions occur at a greatly reduced frequency. The cortical reeler tree thus has fewer segments than normal because fewer parallel fibres are available, but it is surprisingly normal in most other respects. Phase III is a period of remodelling and extends from 20 dpn into adulthood when high-ordered terminals are eroded and middle-ordered terminals are added, with no change in total segment number in both normal and cortical reeler trees. Weaver and deeply placed reeler Purkinje cell dendritic trees are not influenced by parallel fibres. Accordingly, their growth is arrested at the end of Phase I, when both types of mutant tree have generated 100 segments by unrestrained random terminal branching. In the absence of parallel fibres, Phase II is not induced and remodelling, characteristic of Phase III, does not occur.     

Glial cell line-derived neurotrophic factor protects midbrain dopamine neurons from the lethal action of the weaver gene: a quantitative immunocytochemical study.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and repair midbrain dopamine neurons in vivo using animal models created with neurotoxins. The weaver mouse (wv/wv) has natural and spontaneous midbrain dopaminergic cell death which gives a unique opportunity to examine the effects of GDNF. The present study was designed to investigate a possible neuroprotective role by GDNF for midbrain dopamine neurons in the wv/wv. Weaver pups were given 1 microl injections on postnatal day 1. The wv/wv placebo group received a single unilateral injection into the right lateral ventricle of phosphate buffered saline (PBS) while the GDNF treated wv/wv mice received either 1.0 microg/microl or 10.0 microg/microl GDNF in PBS. All mice were sacrificed on postnatal day 20 and their brains were processed for tyrosine hydoxylase (TH) immunocytochemistry. When compared to the placebo group, the 1 microg GDNF group showed significantly less cell death on the injection side, but the contralateral side showed no significant sparing of TH neurons. The combined counts from both sides show significantly more TH staining neurons in the 1 microg GDNF group compared to placebo. When compared to placebo-injected controls, the 10 microg GDNF treated group showed significantly more TH staining neurons on the injected side, contralateral side, and combined. The results demonstrate that GDNF does protect weaver dopaminergic midbrain neurons from the lethal action of the weaver gene and the effect is positively correlated to dosage.     

Effect of Purkinje cell loss on cerebellar gangliosides in nervous mutant mice

The distribution of cerebellar gangliosides was studied in adult (73 ± 2 days) nervous (nr/nr) mutant mice which lose 50–90% of their Purkinje cells. This neuronal loss is associated with significant reductions in cerebellar weight and ganglioside concentration. The cerebellar dry weights (mg) and the ganglioside concentrations (μg N-acetylneuraminic acid per 100 mg dry weight) in nr/nr mice and age-matched normal littermates (+/?) are 7.4 ± 0.3 mg and 13.2 ± 0.4 mg; and 411.7 ± 4.8 μg and 438.5 ± 2.1 μg, respectively. Abnormalities were also observed for the concentration of certain ganglioside species. Most notably, GT1a is significantly reduced by 42%, and GD3 is significantly increased by 29% in the nr/nr mice compared to the +/? mice. The nr/nr mice also express a slight but significant reduction in GT1b. No ganglioside abnormalities were observed between the nr/nr and +/? mice in cerebral cortex. We previously found reduced cerebellar GT1a content in other mutants that also lose Purkinje cells, i.e., sg/sg, pcd/pcd, and Lc/+. GT1a is not reduced, however, in wv/wv mice that lose mostly granule cells. The findings in nr/nr mice are therefore consistent with our hypothesis that GT1a is enriched in Purkinje cells. GD1a, which is enriched in mature granule cells, is not reduced in the nr/nr mice. Since we previously found that GD3 is a good marker for reactive glia in neurological disease, the elevated GD3 concentration in the nr/nr mice indicates a mild gliosis. Our findings with nr/nr and the other neurological mutants indicate that gangliosides can be useful as cell-surface markers for monitoring changes in the cytoarchitecture of the mouse cerebellum.     

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.     

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 mutants. No specific [125I]human IGF-II binding was observed in the cerebellar cortex of weaver mutants. These studies suggest that specific IGF-II receptor sites are located on the granule cells of the cerebellum.     

Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene

Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells.     

Diverse effects of Purkinje cell loss on deep cerebellar and vestibular nuclei neurons in Purkinje cell degeneration mutant mice: A possible compensatory mechanism

The genetic defect in the Purkinje cell degeneration (PCD) mutant mouse completely disrupts the cerebellar corticonuclear connection through intrinsic action on the final integrating unit of the cerebellar cortex, the Purkinje cell (PC). The postsynaptic target neurons of the PC in the deep cerebellar nuclei (DCN) and the vestibular nuclei (VN) are denervated by this PC loss by more than two-thirds of their total γ-aminobutyric acid (GABA)-ergic innervation. This massive disinhibition should be reflected in an increased and thus electrophysiologically detectable activity of the respective neurons. To address this question, we performed extracellular recordings of PCD mutant and corresponding wild-type VN neurons under sinusoidal vestibular stimulation. The response amplitudes (neuronal response to sinusoidal rotation) of VN neurons in PCD mutant mice showed a decrease rather than the expected increase. The same was true for the mean resting rate, whereas the phase relationships were unaffected for the most part. This finding is a clear indication of compensatory reactions in the VN that substitute quantitatively for the lost PC inhibition. The expression of the calcium-binding protein parvalbumin (Parv) is assumed to correlate with the physiological activity of neurons, and Parv is localized predominantly in inhibitory neurons. Because inhibitory inter- or projecting neurons are also largely denervated by the PC loss, Parv immunocytochemistry also was performed. In wild-type mice, only very few Parv-immunopositive (Parv+) somata were present in the VN, and none were present in the DCN. In PCD mutant mice, a substantial number of Parv+ neuronal somata were visible in the VN, and even more were visible in the DCN. This increase in Parv+ somata in PCD mutant mice is closely related temporally and spatially to the extent of denervation caused by the PCD. Parv+ neuronal somata are first visible in the dentate nucleus at postnatal day (P) 24 and appear in the other cerebellar and VN up to P29. Direct double labeling of Parv and GABA and of Parv and glycine reveals that the large majority of Parv+ neurons colocalize GABA, glycine, or both inhibitory transmitters. These results show that neurons that are postsynaptic to cerebellar PC develop diverse physiological and biochemical reactions in the course of genetically determined PCD. These mechanisms are likely to contribute to the phenotypically mild motor disturbances observed in PCD mutant mice. J. Comp. Neurol. 384:580–596, 1997. © 1997 Wiley-Liss, Inc.