Navigational deficits in weaver mutant mice

Weaver mutant mice have selective degeneration of cerebellar granule cells. In comparison to normal mice, the weaver mutants made more errors and took a longer time to reach a platform in a water-maze. Results are discussed in terms of a role for the cerebellum in spatial orientation.     

Acquired immobility response in weaver mutant mice

Weaver mutant mice, contrary to normal littermate controls, did not acquire the immobility response in a forced swimming task. In regard to swimming performance, the weaver mutants swam with less ability but with more vigor than normal mice. Results are discussed in terms of the role of the cerebellum in swimming behavior.     

Delayed spontaneous alternation in weaver mutant mice

Weaver mutant mice alternated above chance levels but less often than normal mice in a 2-trial spontaneous alternation test. When the inter-trial (or retention) interval was manipulated, the weaver mutants alternated above chance levels only at the shortest (15 s) but not at the longest (4 or 5 min) intervals, contrary to normal mice that alternated above chance levels at all 3 intervals. Results are discussed in terms of a role for the cerebellum in spatial working memory.     

Spontaneous alternation and exploration in weaver mutant mice

Weaver mutant mice engaged less in motor activity and hole poking. Weaver mice alternated spontaneously in a 4-trial but not a 2-trial test. These results are similar to those of a previous study with staggerer mutants, in that both are less active. However, weaver mutants are less affected in the spontaneous alternation measure.     

Distribution kinetics of 18F-DOPA in weaver mutant mice

Distribution kinetics of 18F-fluoro-dihydroxy phenylalanine (18F-DOPA) were studied with high-resolution micro-positron emission tomography (microPET) imaging and conventional methods in control wild-type mice, heterozygous weaver mutant mice, and homozygous weaver mutant mice. 18F-DOPA uptake was significantly increased in the CNS within 60 min in all the genotypes examined. Homozygous weaver mutant mice exhibited significantly reduced 18F-DOPA uptake in the region of interest (striatum) as compared to heterozygous weaver mutant mice and control wild-type mice. 18F-DOPA was de-localized in the kidneys of homozygous weaver mutant mice. The radioactivity was localized primarily in the liver and kidneys within 2 h and in the urinary bladder within 4 h. After 8 h, it could be detected neither by conventional nor by microPET imaging. Distribution kinetics of 18F-DOPA with microPET imaging correlated and confirmed the conventional observations. These data are interpreted to suggest that microPET imaging may provide an efficient, noninvasive, cost-effective procedure to study distribution kinetics of PET radiopharmaceuticals in rare genetically altered animals. Furthermore, this unique and noninvasive approach may expedite quality control and drug development for human applications.     

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.     

Translocation of cytochrome c during cerebellar degeneration in Lurcher and weaver mutant mice

Cytochrome c translocation from the inner mitochondrial membrane into the cytosol is the initial step of the intrinsic apoptotic pathway. As no evidence was ever presented for cytochrome c translocation during cerebellar degeneration in Lurcher (Lc/+) and weaver (wv/wv) mutant mice, we searched for the presence of such a process in cerebellar homogenates of mutant and wild-type mice from postnatal day (P)1 to P56. Here we present the first documented time course of cytochrome c translocation spanning the entire period of neurodegeneration in both mutant types. We identified cytochrome c with Western blotting and monitored cell loss in the cerebellum with Calbindin D-28k immunohistochemistry, Nissl-staining and morphometry. No cytochrome c translocation was ever detected in wild-types at any age investigated. Translocated cytochrome c appeared between P13 and P21 in Lc/+ and between P5 and P6 in wv/wv. These two intervals precisely coincide with the respective periods of maximal neuronal death in the cerebellum. Secondary translocation was also observed at a later stage between P42 and P49 in Lc/+ and from P22 onwards in wv/wv. Since no substantial neuronal loss has ever been observed in Lc/+ and wv/wv mutants at these postnatal ages, the delayed translocation may correspond to cytochrome c of extraneuronal, presumably glial origin. Observations of an increased expression of glial fibrillary acidic protein and sustained remodeling of the astrocytic network in the cerebellum of both mutants, long after the cessation of neuronal death make this assumption rather plausible.     

Degeneration of pontine mossy fibres during cerebellar development in weaver mutant mice

In weaver mutant mice, substitution of an amino acid residue in the pore region of GIRK2, a subtype of the G-protein-coupled inwardly rectifying K+ channel, changes the properties of the homomeric channel to produce a lethal depolarized state in cerebellar granule cells and dopaminergic neurons in substantia nigra. Degeneration of these types of neurons causes strong ataxia and Parkinsonian phenomena in the mutant mice, respectively. On the other hand, the mutant gene is also expressed in various other brain regions, in which the mutant may have effects on neuronal survival. Among these regions, we focused on the pontine nuclei, the origin of the pontocerebellar mossy fibres, projecting mainly into the central region of the cerebellar cortex. The results of histological analysis showed that by P9 the number of neurons in the nuclei was reduced in the mutant to about one half and by P18 to one third of those in the wild type, whereas until P7 the number were about the same in wild-type and weaver mutant mice. Three-dimensional reconstruction of the nuclei showed a marked reduction in volume and shape of the mutant nuclei, correlating well with the decrease in neuronal number. In addition, DiI (a lipophilic tracer dye) tracing experiments revealed retraction of pontocerebellar mossy fibres from the cerebellar cortex after P5. From these results, we conclude that projecting neurons in the pontine nuclei, as well as cerebellar granule cells and dopaminergic neurons in substantia nigra, strongly degenerate in weaver mutant mice, resulting in elimination of pontocerebellar mossy fibres during cerebellar development.     

Topology of ionotropic glutamate receptors in brains of heterozygous and homozygous weaver mutant mice.

In weaver mice, mutation of a G-protein inwardly rectifying K(+) channel leads to a cerebellar developmental anomaly characterized by granule and Purkinje cell loss and, in addition, degeneration of dopaminergic neurons. To evaluate other deficits, ionotropic glutamate receptors sensitive to N-methyl-D-aspartate (NMDA), amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA) were examined by autoradiography with [(3)H]MK-801, [(3)H]AMPA, and [(3)H]KA. These surveys were carried out in selected areas of cerebral cortex, hippocampus and related limbic regions, basal ganglia, thalamus, hypothalamus, brainstem, and cerebellum from heterozygous (wv/+) and homozygous (wv/wv) weaver mutants, and compared to wild-type (+/+) mice. In wv/+ and wv/wv mutants, NMDA receptor levels were lower in cortical areas, septum, hippocampus, subiculum, neostriatum, nucleus accumbens, superior colliculus, and in the cerebellar granular layer. Densities of KA receptors were lower in cortical areas, hippocampus, limbic system structures, neostriatum, nucleus accumbens, thalamus and hypothalamus, superior and inferior colliculi, and cerebellar cortex of wv/wv mutants. Levels of AMPA receptors in the weaver were higher than in +/+ mice, particularly in somatosensory and piriform cortices and periaqueductal gray of wv/+, and in somatosensory cortex, CA1 field of Ammon's horn and cerebellar granular layer of wv/wv. Abnormal developmental signals, aberrant cellular responses, or a distorted balance between neurotransmitter interactions may underlie such widespread and reciprocal glutamate receptor alterations, while in the case of cerebellar cortex, NMDA receptors are lacking due to a massive disappearance of cerebellar granule cells and some loss of Purkinje neurons.     

Neural cell adhesion molecule and D3 protein in the cerebellum of weaver mutant mice

Weaver (wv/wv) mutant mice are characterized by extensive granule cell degeneration and can therefore be used as a model for brain degeneration that does not involve blood-brain barrier damage. The ontogeny of the neural cell adhesion molecule (NCAM) and the neuronal antigen D3 protein were investigated in cerebellum and forebrain of weaver mutant mice up to post-natal day 60. In the forebrain the concentration of both proteins was virtually unchanged. In cerebellum, in contrast, the concentration of D3 decreased markedly whereas that of NCAM remained unchanged. Similar findings were obtained at post-natal day 30 in the cerebellum of other neurologic mutants, namely the staggerer (sg/sg) and reeler (rl/rl). At this age the concentration of a synaptic vesicle marker, synaptophysin. was severely decreased in the cerebellum of all three mutants. The concentration of neuron-specific enolase was less affected, whereas the concentrations of the glial markers glutamine synthetase and glial fibrillary acidic protein were both increased. The concentration ratio, which probably reflects the ongoing rate of synaptic remodelling, was increased during the whole ontogeny of weaver mutant mice as compared with heterozygous controls. At post-natal day 30, the ratio was increased by 180% in weaver, by 170% in staggerer and by 60% in reeler cerebellum. These findings lend further support to the usefulness of the ratio as a marker of neural plasticity and synaptic remodelling in both animals and man.     

Organization of cerebellar cortex secondary to deficit of granule cells in weaver mutant mice

This study deals with some consequences of the early postnatal abnormalities of cerebellar Bergmann glial fibers and granule cell neurons. (1) Cerebellar size is mildly reduced in heterozygous weaver (+/wv) mice and markedly reduced in homozygotes (wv/wv), but the pattern of fissures is essentially normal. Comparison with other mutants displaying small cerebella suggests that cell proliferation rate in the external granular layer is a key deteminant of cerebellar cortical folding. (2) Mossy fiber terminals differentiate on schedule despite the reduced number and abnormal positions of granule cells. However, many of them enter the modified molecular layer, and as noted especially in noninbred wv/wv mice one to two years old, form synapses with dendrites of aberrant granule cells. Where granule cells are absent, mossy fibers form more than the normal number of synapses with dendrites of Golgi type II neurons. (3) Purkinje cells are only mildly affected by the disorder of neighbouring cells. Their dendrites grow abnormally into the territory occupied by external granule cells, reach the external surface, and may turn inward. They form few tertiary branches. Dendritic spines are present in profusion and show membrane thickenings akin to normal postsynaptic elements. Although they receive no axonal contacts, the spines persist, enveloped by glial processes, for at least two years. Apart from the absence of parallel fiber contacts, afferent and intrinsic axons form the normal classes of synaptic connections with Purkinje cells. (4) Interneurons of the molecular layer are generated on schedule. At the time of their earliest recognition, they reside in the external granular layer, where they receive synaptic contacts from climbing fibers and other interneurons. In the absence of parallel fibers, interneurons differentiate in situ but their dendrites are abortive and randomly oriented. Growth of their dendrites, in contrast to that of Purkinje cell dendrites, appears to be markedly influenced by the organization of the local cellular milieu.     

Effect of chronic treatment with riluzole on the nigrostriatal dopaminergic system in weaver mutant mice

The effects of a chronic treatment with the anti-glutamate and sodium channel modulating neuroprotective agent riluzole on the degeneration of dopamine-containing neurons were studied in the brain of weaver mutant mice. In these animals, as in Parkinson's disease, dopaminergic neurons of the nigro-striatal pathway undergo spontaneous and progressive cell death. Homozygous weaver mice were orally treated twice a day with either 8 mg/kg riluzole or placebo for 2 months. Quantification of tyrosine-hydroxylase and dopamine-transporter axonal immunostaining in the striatum revealed that riluzole significantly increased the density of striatal dopaminergic nerve terminals. These results suggest that riluzole protects dopaminergic processes in the weaver mice and/or promotes their neuroplasticity.     

Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice

Golgi, electronmicroscopic and autoradiographic analyses of the developing cerebellar cortex were carried out in homozygous wild-type (+/+), heterozygous (+/wv), and homozygous weaver (wv/wv) littermates. At 3 to 15 days after birth, when granule cells normally migrate from the external granular layer inward to their adult position, the somas of postmitotic granule cells in +/+ animals move along straight elongated Bergmann glial fibers which span the molecular layer. As early as postnatal day 3 and increasingly for about the next ten days, some Bergmann fibers in the +/wv cerebellum are enlarged, irregular in caliber, electronlucent and may contain dense bodies and vacuoles. Some granule cells contiguous with the altered Bergmann fibers have round rather than elongated somas, while some have lost contact with Bergmann fibers and appear to degenerate several days after their genesis. Most other granule cells migrate slowly but do eventually reach the granular layer, so that the pathology is less striking in adult than in developing heterozygotes. In wv/wv cerebella, radially-aligned Bergmann fibers appear reduced in number during the developmental period, and few of them display normal cytological features. Few normally migrating granule cells were observed. Most granule cells fail to form axons and die in the external granular layer close to their site of genesis. While the primary genetic defect is unknown, the Bergmann glial disorder appears to precede the migration failure of granule cells and this in turn, along with impaired axon genesis, appears to be a factor leading to granule cell death.     

Novel effects of MPTP: MAO-B unrelated opioidergic activity in mice

The presence of opioidergic activity after i.p. injection of N-methyl-4-phenyl tetrahydropyridine (MPTP) has been investigated in albino mice by studying analgesia and the Straub reaction. MPTP (6.25-25 mg kg-1) produced a dose-related analgesic response and Straub reaction. These effects of MPTP were effectively antagonized by prior naloxone treatment but remained unaffected after the MAO-B inhibitor deprenyl. MPTP thus possesses significant opioidergic activity and this, unlike its neurotoxic actions, does not appear to be dependent on oxidative conversion to MPP+ (1-methyl-4-phenyl pyridinium).     

Bcl-2, Bax and Bcl-x expression in neuronal apoptosis: a study of mutant weaver and lurcher mice

We investigated the expression of the apoptosis modulating proteins Bcl-2, Bax and Bcl-x in the cerebellum of mutant lurcher and weaver mice. Lurcher Purkinje cells and weaver germinal (granule neuron progenitor) cells both die via apoptosis during the postnatal cerebellar development. No significant changes in the expression patterns were detected prior to the actual cell death process. Instead apoptotic lurcher Purkinje cells exhibited increased Bax and Bcl-x expression, while surviving cells had an expression pattern similar to that of healthy littermates. Increased Bax expression was also found in apoptotic weaver germinal cells, while no change of Bcl-x expression was detected. Bcl-2 was expressed at low levels in cerebellar neurons and no loss of Bcl-2 was evident. The observed expression patterns of Bcl-2, Bax and Bcl-x protein in apoptotic lurcher and weaver neurons support the hypothesis that the execution of neuronal apoptosis involves increased expression of Bax, which could represent a general mechanism in diverse neurodegenerative processes. Received: 13 July 1997 / Revised, accepted: 27 February 1998     

CYP 2E1 mutant mice are resistant to DDC-induced enhancement of MPTP toxicity

In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may have a detoxifying role.     

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.     

Plasticity of GABAergic terminals in Deiters'' nucleus of weaver mutant and normal mice: a quantitative light microscopic study

This study reports on the developmental changes in size and the average density of GABAergic axonal boutons bordering on the somata of large neurons in the dorsal part of the lateral vestibular nucleus (Deiters' nucleus) in normal and mutant mice. Weaver mutants, PCD mutants and the corresponding wild types were used to test for size alterations and differences in the number of GABA-immunopositive terminals. Hemicerebellectomized animals were examined in addition. Quantification of bouton profile size was performed from 30-microns-thick vibratome and 0.5-micron Araldite-embedded semi-thin sections immunoreacted for GABA from 7 days postnatally up to an age of 9 months. Terminal density was determined at the 5-6 month stage from semi-thin sections only. Morphometric analysis over the lifetime of normal animals (B6CBA) revealed a progressive increase in the size of bouton profiles, which peaked at 5-6 months and reached sizes of 2-3 microns2. In weaver mutants a parallel development in terminal size was found to be present, but the size of the largest terminals exceeded those of the controls by 75-100%, reaching 3-6 microns2 with the same time course. PCD mutants, with an almost total absence of Purkinje cells had, on the contrary, small bouton profiles that reached a maximum of only 2 microns2. The hemicerebellectomized animals responded with decreased bouton profile size ipsilaterally. The terminal numbers per unit membrane length were surprisingly similar in wild types and weaver mutants, despite a reduction in Purkinje cells of almost 50% in the weaver anterior lobe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced MPTP neurotoxicity in striatum of the mutant mouse tottering

The effects of MPTP treatment (4 × 10 mg/kg, 2-h intervals) on in vivo striatal binding of (+)-α-[³H]dihydrotetrabenazine ((+)-[³H]DTBZ) to the vesicular monoamine transporter type 2 (VMAT2) were examined in wild type (+,+) and tottering (tg/tg) mice of the C57BL/6J strain. The tottering mutant has been previously characterized as having hyperinnervation of noradrenergic terminals in the brain, with increased concentrations of norepinephrine and increased numbers of VMAT2 binding sites. In wild-type mice, MPTP caused a significant decrease in specific striatal (+)-[³H]DTBZ binding in both males (-71%) and females (-57%), consistent with dopaminergic terminal losses. In the tottering mice, the neurotoxic effects of MPTP were diminished, with smaller losses of (+)-[³H]DTBZ binding observed both in males (-45%) and females (-26%). These results are consistent with the hypothesis that vesicular storage (as a result of hyperinnervation) offers neuroprotection toward MPTP toxicity, although the confounding effects of increases in norepinephrine concentrations or changes in calcium ion channel function (both also characteristics of the tottering mutant) cannot be ruled out. The tottering mutant does, however, offer another animal model to examine the biochemical features responsible for MPTP toxicity. Synapse 30:205–210, 1998. © 1998 Wiley-Liss, Inc.     

Differential effects of deprenyl and MPTP on catecholamines and activity in BALB/c mice.

The present study examined the effects of deprenyl (1.5 mg kg-1) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg kg-1) on dopamine (DA), norepinephrine (NE) and motor activity in BALB/c mice. Results indicate that pre-treatment with deprenyl injections protected against MPTP's toxicity on DA, NE and motor function. However, if deprenyl was given 3 or 7 days after the MPTP injection (for 3 days in succession), it could not reverse MPTP's toxicity. However, if deprenyl was given for an extended period of 7 days, it reversed MPTP's toxicity on NE, but not on DA and behaviour. If deprenyl was given soon after MPTP each day for 10 days, it protected against MPTP's toxicity on DA and NE, but not on locomotor activity. These results suggest that only prior and repeated deprenyl injection has a satisfactory protective effect against MPTP's neurotoxicity.