Lurcher Mutant Mice Research Articles

Effects of chlordiazepoxide on the emotional reactivity and motor capacities in the cerebellar Lurcher mutant mice

It is now well accepted that besides its roles in motor control, the cerebellum is involved in non-motor functions with emotional aspects. Consistent with this view, several studies highlighted that the cerebellar Lurcher mutant mice (+/Lc), with motor impairments, also exhibited altered emotional reactivity, previously interpreted in term of behavioural disinhibition. In this study, we investigated the effects of a classical anxiolytic on such disinhibition. For that, behaviours of +/Lc and control (+/+) mice injected with NaCl or chlordiazepoxide (CDP 5 and 7.5 mg/kg) were evaluated in the elevated plus-maze test. The motor impact of the drug (7.5 mg/kg only) was also evaluated in the hole-board and unstable platform tests. Our results showed that, compared to the +/+ mice, CDP injection greatly influenced the anxious-related behaviours in the +/Lc mice by reducing their preference to the open areas in the elevated plus-maze test. Furthermore, we found that injection of CDP at the dose of 7.5 mg/kg aggravated motor coordination deficit, altered motor learning capabilities in the mutants and provoked equilibrium disturbances in the non-mutant mice in the unstable platform test. These results indicated that CDP was able to reduce behavioural disinhibition in the cerebellar +/Lc mice and were discussed in term of implication of the cerebellar connections into CDP-sensitive neural circuitries involved in both emotional and motor processes.

TRAIL-related death receptors in normal, Lurcher and weaver mutant mouse brain

In this study, we searched for murine analogues of the four death-receptor types (TRAIL-R1 to R4), targeted by the tumour necrosis factor related apoptosis inducing ligand (TRAIL), which were recently identified in the human brain. The expression of TRAIL-receptors in the normal murine brain was investigated using antibodies directed against different epitopes of the human TRAIL-receptors. Mouse mutants, in particular weaver and Lurcher with their well defined spatio-temporal patterns of neurodegeneration in the cerebellum, the inferior olive and the substantia nigra, were used as a model for investigating a potential contribution of TRAIL-receptors to the genetically determined cell death observed in these mutants. Although all antibodies used, recognized the respective human antigens, only the murine analogue of the human TRAIL-R2 epitope was also identified in the mouse brain. Antisera against human TRAIL-R1, TRAIL-R3 and TRAIL-R4 failed to reveal any other murine TRAIL-receptor analogue. In normal mice, TRAIL-R2 is not universally expressed throughout the brain but rather restricted to specific neuronal populations predominantly consisting of large neurons. In weaver, the spatial patterns and relative densities of TRAIL-R2 labelling were virtually identical to those seen in wild-types during the period of cell death in the cerebellum and the substantia nigra. In Lurcher, TRAIL-R2 expression in cerebellar granule cells and inferior olivary neurons was identical to that in wildtypes but significantly reduced in Purkinje cells undergoing degeneration. Thus, although TRAIL-R2 is found to be expressed in various cell types of the murine brain, cell death in weaver and Lurcher mutants is apparently not accompanied by an upregulation of TRAIL-receptors.

Oscillating Purkinje neuron activity causing involuntary eye movement in a mutant mouse deficient in the glutamate receptor delta2 subunit.

How failures in regulation of synaptic transmission in the mammalian CNS affect neuronal activity and disturb motor coordination is addressed. The mutant mouse deficient in the glutamate receptor delta2 subunit, specifically expressed in cerebellar Purkinje neurons, has defects in synaptic regulations such as synaptic plasticity, stabilization, and elimination of synaptic connections and shows failures in motor coordination and learning. In this study, the cause of motor discoordination of the delta2 mutant mouse was analyzed by comparing its motor control ability with those of the wild-type mouse and the lurcher mutant mouse, which loses all Purkinje neurons, the sole output neurons in the cerebellar cortex. Unexpectedly, the delta2 mutant mouse showed severer motor discoordination than the lurcher mouse without any cerebellar cortical outputs. The delta2 mutant mouse showed involuntary spontaneous eye movement with characteristic 10 Hz oscillation, which disappeared by ablation of the cerebellar flocculus, suggesting that the delta2 mutant cerebellar cortex outputs an abnormal signal. In vivo extracellular recordings of neuronal activity revealed that Purkinje neurons tended to fire clustered action potentials and complex spikes at approximately 10 Hz in the delta2 mutant mouse. A whole-cell patch-clamp recording from Purkinje neurons in cerebellar slices indicated that the clustered action potentials could be induced by climbing fiber activation. Taken together, our results suggest that the delta2 subunit deficiency produces the oscillating activity in Purkinje neurons by enhancing climbing fiber inputs, causing surplus movement and affecting motor control worse than no signal at all.

Stress and anxious-related behaviors in Lurcher mutant mice

Blood corticosterone levels (CORT) were measured before and after the completion of the elevated +-maze test in cerebellar Lurcher mutant and control mice. Consistent with the existence of a much more pronounced activation of the hypothalamo–pituitary–adrenal (HPA) system in the mutants, our results showed that while basal CORT were similar in mutants and controls, the surge of this stress indicator was enhanced in the Lurcher mice after completion of a behavioral test of anxiety. In contrast, at the behavioral level, we also observed that Lurcher exhibited significantly reduced anxiety related indices; they spent a significant greater amount of time in the aversive places of the apparatus and entered them more frequently than non mutant mice. It is proposed that rather than less anxious, the Lurcher mice are less inhibited than controls when placed in anxiogenic situation and that such poor inhibition could be causally related to changes in HPA system regulation. The overall patterns of our behavioral and endocrinological results thereby provided the evidence that cerebellar circuitry is involved in producing changes in physiological and behavioral stress-related emotional responses.

Loss of pro-apoptotic BH3-only Bcl-2 family member Bim does not protect mutant Lurcher mice from neurodegeneration.

Lurcher (lc) mice have a semi-dominant mutation in the gene encoding the delta2 glutamate receptor (GRID2). The resulting constitutive activity of this receptor in heterozygous +/lc (grid(+/lc)) and homozygous (grid(lc/lc)) mice leads to the death of all cerebellar Purkinje cells and most afferent granule neurons. Some studies have indicated that the death of Purkinje cells occurs by apoptosis, and the secondary loss of granule neurons has been shown to require the pro-apoptotic Bcl-2 family member Bax. The BH3-only protein Bim has been shown to contribute to cytokine withdrawal-induced apoptosis of sympathetic neurons and to be responsible for the kidney degeneration in mice lacking the pro-survival protein Bcl-2. Because Bim is expressed strongly in cerebellar Purkinje cells, we have examined whether it has a role in their death in mutant Lurcher mice. Our studies show that Bim deficiency does not modify the Lurcher phenotype, ruling out an indispensable role for Bim in this neurodegenerative disease.

Development of early motor learning and topical motor skills in a model of cerebellar degeneration

The development of motor learning and topical motor skills has been studied during the first month of life in neurodeficient Lurcher mutant mice derived from C3H strain. The examination of motor functions was conducted using four methods when animals were consecutively placed on: a horizontal wire, a rotating cylinder, a bridge and slanting ladder (on the rotating cylinder and slanting ladder in three different starting positions). A total of 150 animals were used, half being heterozygous mutants (+/Lc), the remaining consisting of their healthy littermates, homozygous wild-type mice (+/+). Some animals of both types were confronted with the tasks in four testing days without practice while the others were trained in all of these methods one time daily at the age of 3, 6, 9, 12, 15, 20, 25 and 30 days. The results of the research are expressed as a percentage of reaching criteria in the trained and untrained mice of both types (+/Lc, +/+), respectively on days 15, 20, 25, and 30. Because of their motor handicap, Lurcher mutants showed significantly worse results than normal mice in almost all of the tests. The most interesting observations of the study were obtained on the rotating cylinder and the slanting ladder tests where mainly untrained mutants were unable to cope promptly with the different starting position and failed. However, in 30-day-old Lurchers the effect of learning was relatively better in some tests when compared with the wild-type of mice. Experiments showed that, in spite of progressive cerebellar degeneration, the ability of motor learning in Lurcher mutants survived to a certain degree. The worsening of results after frequent training in both trained Lurchers and normal mice aged 20 days as compared with untrained ones is discussed within the context of the “overtraining reversal effect” and other possible hypotheses.

Quantitative organization of neurotransmitters in the deep cerebellar nuclei of the Lurcher mutant.

The Lurcher mutant mouse is characterized by a primary selective loss of Purkinje cells, leading to the near total apoptotic death of these neurons. In contrast to the subsequent massive secondary degeneration of the granule cells and the inferior olivary neurons, only mild degeneration occurs in the deep cerebellar nuclei (DCN). However, it is not known to what extent the different populations of DCN neurons-glutamatergic principal projection neurons, gamma-aminobutyric acid (GABA)-ergic inferior olivary projection neurons, and glycinergic neurons-are affected in their neurotransmitter composition. To answer this question we studied the neurotransmitter contents (glutamate, GABA, and glycine) of DCN neurons and the size of synaptic boutons immunohistochemically on serial semithin sections in both Lurcher and wild-type mice. Applying the physical dissector counting method, our results confirmed the mild degeneration (a reduction by 20%) of large glutamatergic neurons and a more pronounced degeneration of GABAergic (by 42%) and glycinergic neurons (by 45%). On the other hand, an analysis of neurons colabeled for both GABA and glycine, revealed that this specific colabeling increased in the Lurcher mutant (by 40%). In addition, both the GABA-immunolabeled (IL) (by 56%) and the glycine-IL (by 45%) synaptic boutons showed an increase in diameter in the mutant. The density of these boutons showed a decrease of 30% each. In summary, the increase in the number of neurons colabeled for GABA and glycine, together with the increase in the size of the inhibitory synaptic boutons, could help in providing the minimum inhibition needed to maintain a residual "cerebellar" functionality in the Lurcher DCN.

Morphology of Adrenal Gland and Lymph Organs is Impaired in Neurodeficient Lurcher Mutant Mice

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Behavioral and biochemical effects of l-tryptophan and buspirone in a model of cerebellar atrophy

The Lurcher mutant mouse can be considered an adequate model of autosomal dominant spinocerebellar atrophy because of the severe degeneration of its cerebellar cortex and inferior olive. The purpose of this study was to determine whether the motor coordination deficits of Lurcher mutants could be improved after chronic administration of the serotonin (5-hydroxytryptamine; 5-HT) precursor, l-tryptophan, or of the 5-HT 1A agonist, buspirone. During these treatments, the mice were submitted to behavioral evaluations using the coat hanger and the rotorod tests, as well as an inclined screen and a vertical grid test. At the end of treatments, 5-HT and 5-hydroxindole-3-acetic acid (5-HIAA) were measured in six brain regions. On the coat hanger test, administration of l-tryptophan accelerated movements along the horizontal bar by 44%, while buspirone increased the time spent on the apparatus by 11%. Neither drug had an effect on climbing ability or on the time spent on a rotating beam. Administration of l-tryptophan increased 5-HIAA levels in frontal cortex, neostriatum, thalamus, brainstem, cerebellum and spinal cord, but elevated 5-HT only in neostriatum, brainstem and cerebellum. In contrast, buspirone led to 5-HT increases in cerebellum and augmented 5-HIAA in the spinal cord. The modest test-specific improvements are consistent with some of the clinical data concerning 5-HT pharmacotherapy in patients suffering from cerebellar atrophy.

Motor skills and motor learning in Lurcher mutant mice during aging

Motor learning abilities on the rotorod and motor skills (muscular strength, motor coordination, static and dynamic equilibrium) were investigated in three-, nine-, 15- and 21-month-old Lurcher and control mice. Animals were subjected to motor training on the rotorod before being subjected to motor skills tests. The results showed that control mice exhibited decrease of muscular strength and specific equilibrium impairments in static conditions with age, but were still able to learn the motor task on the rotorod even in old age. These results suggest that, in control mice, efficiency of the reactive mechanisms, which are sustained by the lower transcerebellar loop (cerebello-rubro-olivo-cerebellar loop), decreased with age, while the efficiency of the proactive adjustments, which are sustained by the upper transcerebellar loop (cerebello-thalamo-cortico-ponto-cerebellar loop), did not. In spite of their motor deficits, Lurcher mutants were able to learn the motor task at three months, but exhibited severe motor learning deficits as soon as nine months. Such a deficit seems to be associated with dynamic equilibrium impairments, which also appeared at nine months in these mutants. By two months of age, degeneration of the cerebellar cortex and the olivocerebellar pathway in Lurcher mice has disrupted both lower and upper transcerebellar loops. Disruption of the lower loop could well explain precocious static equilibrium deficits. However, in spite of disruption of the upper loop, motor learning and dynamic equilibrium were preserved in young mutant mice, suggesting that either deep cerebellar nuclei and/or other motor structures involved in proactive mechanisms needed to maintain dynamic equilibrium and to learn motor tasks, such as the striatopallidal system, are sufficient. The fact that, in Lurcher mutant mice, motor learning decreased by the age of nine months suggests that the above-mentioned structures are less efficient, likely due to degeneration resulting from precocious and focused neurodegeneration of the cerebellar cortex. From this behavioral approach of motor skills and motor learning during aging in Lurcher mutant mice, we postulated the differential involvement of two transcerebellar systems in equilibrium maintenance and motor learning. Moreover, in these mutants, we showed that motor learning abilities decreased with age, suggesting that the precocious degeneration of the cerebellar Purkinje cells had long-term effects on motor structures which are not primarily affected. Thus, from these results, Lurcher mutant mice therefore appear to be a good model to study the pathological evolution of progressive neurodegeneration in the central nervous system during aging.

Influence of the Lurcher mutation on myosin heavy chain expression in skeletal and cardiac muscles.

This study evaluated the influence of Lurcher mutation, characterized by degeneration of cerebellar Purkinje cells, granule cells, and inferior olive neurons, on cardiac and skeletal muscles: one respiratory (diaphragm, Dia), three masticatory (anterior temporalis, AT; masseter superficialis, MS and anterior digastric, AD), one hind limb (soleus, S), entire tongue (T), and one cardiac (ventricle, V) muscles. Body and muscle weight, muscle protein content, and myosin heavy chain (MHC) isoforms relative expression were then compared in Lurcher mutant mice vs. normal, according to sex. Male body weight was always greater than female one, but there was no specific muscle difference in females, except for T relative weight which was greater in normal females. Muscle protein concentration was greater in normal males except for AD and T in which it was lower. Lurcher mutant mice showed a reduced whole body growth but no specific muscle atrophy (except in male AT), and a global decrease in muscle protein content which made muscles more fragile (except in female Dia and male T, in which it was greater). Lurcher mutation induced a global reduction of muscle protein concentration whereas a general influence of sex could not be disclosed. Concerning MHC relative composition, all the muscles were fast-twitch: Dia, AT, MS, AD, S, and T predominantly expressed the fast type 2 MHC isoforms, except female S, whereas V contained only MHC alpha, also a fast MHC. Female muscles were slower than male ones and classification of muscles in terms of shortening velocity was comparable in normal male and female. In other respects, male Lurcher mutant muscles were slower and consequently more fatigue resistant than normal, except T which became faster and less fatigue resistant. On the contrary, in female mutants, only the Dia was slower than normal one, MS and AD were comparable to normal ones and finally, AT, S, and T were faster than normal ones. It should be noted that a developmental MHC (neonatal) was present in Lurcher AD. Motor control, which influences muscle structure, is altered in Lurcher mutant and could be one of the causal factor of the fast-to-slow MHC switches observed in some mutant muscles. It seems therefore that cerebellar Purkinje cells, granule cells, and inferior olive neurons are very important in maintaining the structural integrity of both cardiac and skeletal muscle, and their degeneration is accompanied by important muscles modifications. J. Cell. Biochem. Suppl. 36: 222-231, 2001.

Treadmill Performance of Mice with Cerebellar Lesions:: 2. Lurcher Mutant Mice

The sensorimotor skills of a spontaneous mouse mutant with cerebellar cortical atrophy, Lurcher, were examined on either a fast or a slow treadmill inclined at one of three slopes, requiring forward movements in order to avoid footshocks. During the early part of acquisition, Lurcher mutants had lower latencies before falling on either treadmill than normal mice, but not during a retention test. For both Lurcher mutants and controls, the amount of time spent walking as a function of time spent on the belt increased with an increase in belt speed. Lurcher mutants had higher walking time/total time ratios on the slow but not on the fast treadmill. It is concluded that cerebellar cortical degeneration impaired the time course of acquisition but not long-term retention of the treadmill task.

Target-Related and Intrinsic Neuronal Death in Lurcher Mutant Mice Are Both Mediated by Caspase-3 Activation

The Lurcher (Lc) mutation in the delta2 glutamate receptor gene leads to the presence of a constitutive inward current in the cerebellar Purkinje cells of Lurcher heterozygous mice and to the postnatal degeneration of these neurons. In addition, cerebellar granule cells and olivary neurons of Lc/+ mice die as an indirect effect of the mutation after the loss of their target Purkinje cells. The apoptotic nature of Lc/+ Purkinje cell death remains controversial. To address this question, we studied the involvement of caspase-3, a key effector of apoptosis, in the neurodegenerative processes occurring in Lc/+ cerebellum. Several antibodies recognizing different regions of caspase-3 were used in immunoblotting and immunohistochemical experiments. We demonstrate that pro-caspase-3 is specifically upregulated in the dying Lc/+ Purkinje cells, but not in granule cells and olivary neurons, suggesting that different death-inducing signals trigger variant apoptotic pathways in the CNS. The subcellular localization of pro-caspase-3 was shown to be cytoplasmic and mitochondrial. Active caspase-3 as well as DNA fragmentation was found in numerous granule cells and some Purkinje cells of the Lc/+ cerebellum. Thus, caspase-3 activation is involved in both the direct and indirect neuronal death induced by the Lurcher mutation, strongly supporting the idea that the Lc/+ Purkinje cell dies by apoptosis.

Differential effects of l-trytophan and buspirone on biogenic amine contents and metabolism in Lurcher mice cerebellum

The effects of serotoninergic stimulation on monoamines were studied in the heterozygous Lurcher ( Lc/+) mutant mouse, a model of human cerebellar ataxia. Wild type (+/+) and Lc/+ mice were treated for 40 days with l-tryptophan or buspirone, and serotonin (5-HT), dopamine (DA), noradrenaline (NA) and their main metabolites were measured in the cerebellum. In +/+ mice, only buspirone increased concentrations of 5-HT metabolites. In the hypoplastic Lc/+ cerebellum, indoleamines were higher, and increased further after both treatments. The 5-HT turnover index was increased in +/+ mice by buspirone, while in Lc/+ mutants it increased after l-tryptophan but was decreased by buspirone, indicating that in the mutants nerve terminals synthesize and accumulate 5-HT, but may not utilize it efficiently. Catecholamine contents remained unchanged in +/+ mice, but in Lc/+ mutants with higher endogenous NA, l-tryptophan further increased NA and 3,4-dihydroxy-phenylacetic acid (DOPAC), and buspirone augmented NA, DA and DOPAC levels.

Role of an enriched environment on the restoration of behavioral deficits in Lurcher mutant mice.

Lurcher mutant mice, characterized by massive degeneration of the cerebellar cortex, and normal littermate controls were reared from birth either in standard conditions or in an enriched environment. The effects of this manipulation on motor functions, landmark water maze learning, exploration, and anxiety were evaluated at 3 months of age. Under standard conditions, Lurcher mutants were impaired in comparison to controls on tests of sensorimotor function and had altered exploratory tendencies. The enriched housing improved the motor coordination of Lurcher mutants and decreased the number of trials before reaching criterion in the landmark water maze. In addition to its effects in Lurcher mutants, enriched rearing also increased some behavioral abilities in normal mice. It is hypothesized that enriched housing altered brain morphology or neurochemistry in both normal and cerebellar-damaged animals.

Effects of Dopaminergic Agents and of an NMDA Receptor Antagonist on Motor Coordination in Lurcher Mutant Mice

Lurcher mutant mice, characterized by an ataxic gait and olivocerebellar degeneration, were evaluated for motor coordination in the coat-hanger test after peripheral injections of two doses of dextromethorphan, a noncompetitive N-methyl- d-aspartate receptor antagonist, l-dopa/carbidopa, and SKF 77434, a dopamine D 1 receptor agonist. There was an improvement in the distance traveled on the suspended horizontal string after 25 and 50 mg/kg of dextromethorphan and 37.5 mg/kg of l-dopa/carbidopa, but not after SKF 77434. None of the drugs reduced movement times or increased latencies before falling. These results indicate that NMDA receptor antagonism or stimulation of some dopaminergic mechanisms partially improve genetically determined cerebellar ataxia in mice.

Effects of L-tryptophan on indoleamines and catecholamines in discrete brain regions of wild type and Lurcher mutant mice.

The biochemical parameters of the serotoninergic system were examined in wild type mice and Lurcher mutants after chronic treatment (40 days) with the serotonin (5-HT) precursor L-tryptophan (50 mg/kg; i.p.). Tissue contents in 5-HT, dopamine and noradrenaline, as well as some of their metabolites, were measured in frontal cortex, neostriatum, thalamus, brainstem, cerebellum and spinal cord by high-performance liquid chromatography. The tissue levels were used as a biochemical index of the function of the monoamine innervations in this animal model of cerebellar ataxia. The results show that Lurcher mutants retain higher concentrations of L-tryptophan and total indoleamines, but that 5-HT is probably stored in a non-releasable compartment. In the particular case of the hypoplastic cerebellum, the reorganization of 5-HT nerve terminals leads to an accrued indoleamine synthesis, indicating that the Lurcher mutants can accumulate 5-HT, but do not utilize it efficiently in synaptic transmission.

Serotonin innervation of Lurcher mutant mice: basic data and manipulation with a combination of amantadine, thiamine and l-tryptophan

The Lurcher (Lc/+) mutant mouse is characterized by a considerable atrophy of the cerebellum due to a massive loss of cerebellar Purkinje and granule cells, as well as of neurons from the inferior olivary nucleus. In this study the effects of a therapeutic combination of amantadine, thiamine and L-tryptophan on the serotonin (5-HT) innervation was assessed in Lurcher mice by autoradiography, using [ 3H]citalopram to label 5-HT transporters. In wild type mice as well as in both saline-treated and drug-treated Lurcher mutants, [ 3H]citalopram binding remained unchanged in forebrain and brainstem regions. In the cerebellum, labelling of deep cerebellar nuclei (CBnuc) was about twofold higher than in the cortex (CBctx). In saline-treated Lurcher mutants compared to wild type mice, the densities of [ 3H]citalopram were 98% higher in CBctx, and 180% higher in CBnuc. In CBctx of drug-treated Lurcher mutants, transporter densities were 89% higher than in the wild type, but did not differ from the saline-treated Lurcher. In the CBnuc of the drug-treated Lurcher mutants, [ 3H]citalopram binding was 50% higher than in the saline-treated Lurcher group, and 320% higher than in wild type mice. The results show that 5-HT transporters, already upregulated in the CBnuc of Lurcher mutant mice, can be further increased by a pharmacological treatment, possibly altering the availability of 5-HT in some of its target areas.

Role of the cerebellum in exploration behavior

Compared to +/+ mice, Lurcher (+/Lc) mutant mice whose cerebellar cortex is lacking almost all Purkinje cells and granule cells, exhibit a low level of exploration; this deficit is not due to a low level of activity but to both a decreased motivation to explore a novel environment and to spatial deficits. The characteristics of exploration in cerebellectomized +/+ and +/Lc mice suggest that the cerebellum is involved not only in cognitive but also in motivational processes.

Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice.

Lurcher mutant mice are characterized by massive degeneration of cerebellar Purkinje cells and granule cells and by deficits in motor coordination. Regional brain variations of cytochrome oxidase (CO) activity were analyzed to identify those brain regions with abnormal metabolic activity as a secondary consequence of the cerebellar atrophy and to establish the relationship between CO activity and motor deficits. Lurcher mutants had higher CO activity in all three cerebellar deep nuclei than normal littermate controls of the same background strain. Higher CO activity was also found in Lurcher mutants in brain regions directly connected to the cerebellum, such as the lateral vestibular nucleus, the cochlear nucleus, the red nucleus, the ventrolateral thalamus, the dorsal raphe, the interpeduncular nucleus, and the inferior colliculus. By contrast, there was a sharp decrease in CO activity in the inferior olive. As for brain regions not directly connected to the cerebellum, higher CO activity was observed in the trigeminal motor nucleus and the CA1 molecular layer of the hippocampus, which highlights probable transsynaptic alterations as a secondary consequence of cerebellar atrophy. A positive correlation between CO activity in the red nucleus and latencies before falling in two motor-coordination tests indicates that a compensatory increase of metabolic activity in a cerebellar efferent region is associated with improved behavior.