Neuronal differentiation in cultures of weaver (wv) mutant mouse cerebellum

In the present study we report for the first time a weaver (wv) gene dose effect on neuron survival and neurite formation in vitro. Dissociated cerebellar cells from postnatal 7- and 8-day-old normal ( + / + ), heterozygous weaver ( + /wv) and homozygous weaver (wv/wv) mice were cultured as monolayers on poly-L-lysine coated glass. Cell death occurred rapidly in wv/wv cultures. Cell counts showed that less than 20% of the total neurons and neuronal precursors (identified by “birthday” radiolabeling techniques) survived by Day 3. Cell death was less extensive in + /wv cultures with 65% of the total neurons and 80% of the precursors surviving by Day 3. In contrast to wv/wv cultures, younger neurons survive better than the total population in + /wv cultures. The impairment of neurite formation over the first week is also proportional to the number of mutant genes as shown by quantitation of (a) the percentage of cells with neurites; (b) the percentage of cells with neurites of a given length class with time; (c) the lengths of the longest processes formed per cell. The mean longest neurite lengths obtained by computer digitization at 6 days in vitro were 41.8, 26.8, and 9.0 μm for + / +, + /wv, and wv/wv granule cells, respectively.     

Endogenous serotonin release from the dopamine-deficient striatum of the weaver mutant mouse

In addition to an altered dopaminergic input, the striatum of the weaver mutant mouse (wv/wv) has increased serotonin tissue content and uptake compared to the wild-type mouse (+/+). To gain information regarding the functional status of serotonergic inputs to thewv/wv striatum, endogenous serotonin release fromwv/wv and +/+ striatum was measured under basal conditions as well as in the presence of fenfluramine or elevated concentrations of potassium (K⁺). Fractional basal release of serotonin from the +/+ striatum was significantly greater than that from thewv/wv striatum. In the presence of K⁺, evoked release (stimulated release minus basal release) was greater from the +/+ striatum than from thewv/wv striatum. In the presence of fenfluramine, evoked serotonin release was greater from thewv/wv striatum compared to the +/+ striatum. These data are consistent with the involvement of an additional transmitter(s) in modulating serotonin release to a greater extent in thewv/wv than the +/+ striatum. The data on fenfluramine-stimulated serotonin release suggest that the additional serotonin content found in thewv/wv striatum is in a releasable pool but that striatal serotonin release might be attenuated more inwv/wv than in +/+ mice.     

Ontogeny of the cell adhesion molecule L1 in the cerebellum of weaver and reeler mutant mice

The ontogeny of cell adhesion molecule L1 in cerebellum was quantitatively assessed in weaver and reeler mutant mice and in heterozygous litter-mate controls. In the latter the concentration and the amount of L1 both increased from the first postnatal week to become maximum at the second. In contrast, in the weaver and reeler neurologic mutant mice, L1 decreased steadily. The L1 concentration and the amount of L1 was lower in the cerebellum of homozygous mutant mice than in litter-mate controls. The findings are consistent with L1 being a component of axonal plasma membranes. However, no evidence was found of any direct effect of thewv andrl phenotypes on L1 expression.     

Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [³H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [³H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [³H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [³]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [³H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [³H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [³H]DA on the right. Similarly, the animals that were used for [³H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [³H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [³H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.Special issue dedicated to Professor Sidney Ochs     

Comparison of alterations in tyrosine hydroxylase,dopamine levels,and dopamine uptake in the striatum of the weaver mutant mouse

Previous reports have shown that among the markers for the nigro-striatal dopamine (DA) system measured in the striatum, dopamine uptake seems to be more severely affected than the others in the weaver mutant mouse. In the present study we examined DA levels, tyrosine hydroxylase (TH) activity, and high-affinity DA uptake to determine if the DA uptake is most affected when all the measurements are made in the same striatal homogenate in the same laboratory. We found that the DA uptake activity was most altered (93% lower) compared to DA levels (68% lower) and TH activity (64% lower). The DA uptake was so low in the weaver that we could not obtain reliable kinetic parameters. For TH activity we found that the Vmax was 36% lower while the Km forl-tyrosine was 92% higher in the weaver striatum. This lower affinity for substrate suggests that the TH enzyme itself may be altered in the nigro-striatal system of the weaver mutant mouse.Special issue dedicated to Dr. Morris H. Aprison.     

Topographic distribution of dopamine uptake,choline uptake,choline acetyltransferase,and GABA uptake in the striata of weaver mutant mice

The topographic distribution of dopamine (DA) uptake, choline uptake, choline acetyltransferase (ChAT) activity and GABA uptake within the striata of weaver mutant mice and control mice was determined. Uptake of [³H]dopamine, [³H]choline and [¹⁴C]GABA, as well as ChAT activity were determined in samples prepared from the dorsolateral, dorsomedial, ventrolateral and ventromedial portions of the striatum. In 45–60 day old control mice, dopamine uptake was homogeneously distributed throughout the striatum. On the other hand, striata from weaver mice exhibited an uneven distribution with the ventral aspects having greater uptake activity than the dorsal regions. Thus, although the ventral portion of the striatum is less severely affected than the dorsal portion, all areas of the striatum exhibited significantly reduced uptake rates. In 9 and 12 month old mice, choline uptake was higher in lateral than medial zones of the striatum of both genotypes and no differences were observed between genotypes. GABA uptake was higher in the ventral striatum than in the dorsal striatum but again no differences were found between weaver and control mice. The results of this study indicate that the entire weaver striatum is severely deficient in its ability to recapture dopamine and thus is functionally compromised. The results also indicate that the striatal cholinergic and GABAergic interneurons are not directly or indirectly affected by the weaver gene.Special ïssue dedicated to Dr. Morris H. Aprison     

Age-Dependent Alterations in Dopamine Content, Tyrosine Hydroxylase Activity, and Dopamine Uptake in the Striatum of the Weaver Mutant Mouse

Abstract: Mice of different ages and homozygous or heterozygous for the weaver gene ( wv ) were used to study the time course for the effect of the weaver gene on several striatal dopaminergic parameters. Dopamine uptake was decreased in the homozygous weaver at all ages examined. The deficit in uptake at the earliest age studied, postnatal day 3, was approximately 50% and increased to greater than 70% at older ages. In control mice, dopamine uptake reached a maximum by postnatal day 22, but in homozygous weaver mice, development of uptake activity was curtailed by postnatal day 7. Dopamine content and tyrosine hydroxylase activity were significantly decreased in the homozygous weaver at all ages studied except postnatal days 7 and 10. The magnitude of the deficit in dopamine content ranged from approximately 40% at postnatal days 3 and 5 to about 70% in adults (6 months to 1 year of age). The magnitude of the deficit in tyrosine hydroxylase activity ranged from 40 to 70%. In general, no major differences between heterozygotes and controls were observed for any of the dopaminergic parameters investigated. The results of the present investigation indicate that neurochemical alterations can be observed in the striata of weaver mice as early as postnatal day 3 and raise the possibility that the striatal dopamine transporter may be an early target of the weaver mutation.     

cDNA approaches to isolation of the mouse mutant weaver gene

The mouse autosomal recessive mutant gene weaver (wv) results in abnormalities in cerebellum, substantia nigra and testis. Although a subtracted cDNA library prepared by removing P31 (wv/wv) sequences from a P1 (wv/+) library should contain mainly nonrepetitive neonatal sequences, unfortunately, repetitive sequences still appear during screening. Two clones, one repetitive, the other not, are used to illustrate the problems encountered in attempting to isolate the weaver gene from a subtracted cDNA library.Special issue dedicated to Dr. Sidney Ochs.     

Dopamine release is impaired in a mouse model of DYT1 dystonia

Early onset torsion dystonia, the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein torsinA. This form of dystonia is referred to as DYT1. We have used a transgenic mouse model of DYT1 dystonia [human mutant-type (hMT)1 mice] to examine the effect of the mutant human torsinA protein on striatal dopaminergic function. Analysis of striatal tissue dopamine (DA) and metabolites using HPLC revealed no difference between hMT1 mice and their non-transgenic littermates. Pre-synaptic DA transporters were studied using in vitro autoradiography with [(3)H]mazindol, a ligand for the membrane DA transporter, and [(3)H]dihydrotetrabenazine, a ligand for the vesicular monoamine transporter. No difference in the density of striatal DA transporter or vesicular monoamine transporter binding sites was observed. Post-synaptic receptors were studied using [(3)H]SCH-23390, a ligand for D(1) class receptors, [(3)H]YM-09151-2 and a ligand for D(2) class receptors. There were again no differences in the density of striatal binding sites for these ligands. Using in vivo microdialysis in awake animals, we studied basal as well as amphetamine-stimulated striatal extracellular DA levels. Basal extracellular DA levels were similar, but the response to amphetamine was markedly attenuated in the hMT1 mice compared with their non-transgenic littermates (253 +/- 71% vs. 561 +/- 132%, p < 0.05, two-way anova). These observations suggest that the mutation in the torsinA protein responsible for DYT1 dystonia may interfere with transport or release of DA, but does not alter pre-synaptic transporters or post-synaptic DA receptors. The defect in DA release as observed may contribute to the abnormalities in motor learning as previously documented in this transgenic mouse model, and may contribute to the clinical symptoms of the human disorder.     

The role of dopamine in motor symptoms in the R6/2 transgenic mouse model of Huntington's disease

In both Huntington's disease (HD) patients and genetic mouse models of HD, there is a pre-symptomatic loss of dopamine (DA) receptors, suggesting that dysfunctional dopaminergic neurotransmission may be involved in early HD presentation. However, the role of DA in HD symptoms is not fully understood. In this study, we examined the possibility that dysfunctional dopaminergic neurotransmission contributes to the progressive decline in motor function of a transgenic mouse model of HD (R6/2 line). We found that R6/2 mice display an age-dependent abnormal behavioural response to (+)-methamphetamine (METH) and a dose-dependent increase in sensitivity to METH toxicity compared with wild-type (WT) mice. R6/2 mice also showed an attenuated response to cocaine, indicating that DA release may be compromised. Striatal DA levels were reduced in R6/2 mice by 9 weeks of age. Replacement of DA by chronic treatment with laevodopa (L-DOPA, administered as Sinemet) caused short-term improvements in activity and rearing behaviour, and abolished abnormal spontaneous hindlimb grooming. However, long-term treatment with L-DOPA had deleterious effects on survival and rotarod performance of R6/2 mice. These results suggest that dysfunctional DA neurotransmission contributes to phenotype development in R6/2 mice and thus also may be important in symptom progression in HD.     

Genetic and behavioral characterization of a Kmt2d mouse mutant,a new model for Kabuki Syndrome

The recessive mutant mice bate palmas (bapa) ‐ claps in Portuguese arose from N‐ethyl‐N‐nitrosourea mutagenesis. A single nucleotide, T > C, change in exon 13, leading to a Thr₁₂₈₉Ala substitution, was identified in the lysine (K)‐specific methyltransferase 2D gene (Kmt2d) located on chromosome 15. Mutations with a loss‐of‐function in the KMT2D gene on chromosome 12 in humans are responsible for Kabuki syndrome (KS). Phenotypic characterization of the bapa mutant was performed using a behavioral test battery to evaluate the parameters related to general activity, the sensory nervous system, the psychomotor system, and the autonomous nervous system, as well as to measure motor function and spatial memory. Relative to BALB/cJ mice, the bapa mutant showed sensory and psychomotor impairments, such as hypotonia denoted by a surface righting reflex impairment and hindquarter fall, and a reduction in the auricular reflex, suggesting hearing impairment. Additionally, the enhanced general activity showed by the increased rearing and grooming frequency, distance traveled and average speed possibly presupposes the presence of hyperactivity of bapa mice compared with the control group. A slight motor coordination dysfunction was showed in bapa mice, which had a longer crossing time on the balance beam compared with BALB/cJ controls. Male bapa mice also showed spatial gait pattern changes, such as a shorter stride length and shorter step length. In conclusion, the bapa mouse may be a valuable animal model to study the mechanisms involved in psychomotor and behavior impairments, such as hypotonia, fine motor coordination and hyperactivity linked to the Kmt2d mutation.     

Effects of tree species diversity on a community of weaver spiders in a tropical forest plantation

The effects of producer diversity on predators have received little attention in arboreal plant communities, particularly in the tropics. This is particularly true in the case of tree diversity effects on web‐building spiders, one of the most important groups of invertebrate predators in terrestrial plant communities. We evaluated the effects of tree species diversity on the community of weaver spiders associated with big‐leaf mahogany (Swietenia macrophylla) in 19, 21 × 21‐m plots (64 plants/plot) of a tropical forest plantation which were either mahogany monocultures (12 plots) or polycultures (seven plots) that included mahogany and three other tree species. We conducted two surveys of weaver spiders on mahogany trees to evaluate the effects of tree diversity on spider abundance, species richness, diversity, and species composition associated with mahogany. Our results indicated that tree species mixtures exhibited significantly greater spider abundance, species richness, and diversity, as well as differences in spider species composition relative to monocultures. These results could be due to species polycultures providing a broader range of microhabitat conditions favoring spider species with different habitat requirements, a greater availability of web‐building sites, or due to increased diversity or abundance of prey. Accordingly, these results emphasize the importance of mixed forest plantations for boosting predator abundance and diversity and potentially enhancing herbivore pest suppression. Future work is necessary to determine the specific mechanisms underlying these patterns as well as the top‐down effects of increased spider abundance and species richness on herbivore abundance and damage.     

The development of mouse spinal cord in tissue culture

Summary Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells and, finally, the cytodifferentiation of neurons were observed. A preliminary account of this work was given at the Tissue Culture Association Meeting in 1977, and the Canadian Federation of Biological Societies Meeting in 1977 (1,2). This work was supported by Grant MT 4235 from the Medical Research Council of Canada.     

Social behavior in a genetic model of dopamine dysfunction at different neurodevelopmental time points

Impairments in social behavior characterize many neurodevelopmental psychiatric disorders. In fact, the temporal emergence and trajectory of these deficits can define the disorder, specify their treatment and signal their prognosis. The sophistication of mouse models with neurobiological endophenotypes of many aspects of psychiatric diseases has increased in recent years, with the necessity to evaluate social behavior in these models. We adapted an assay for the multimodal characterization of social behavior at different development time points (juvenile, adolescent and adult) in control mice in different social contexts (specifically, different sex pairings). Although social context did not affect social behavior in juvenile mice, it did have an effect on the quantity and type of social interaction as well as ultrasonic vocalizations in both adolescence and adulthood. We compared social development in control mice to a transgenic mouse model of the increase in postsynaptic striatal D2R activity observed in patients with schizophrenia (D2R‐OE mice). Genotypic differences in social interactions emerged in adolescence and appeared to become more pronounced in adulthood. That vocalizations emitted from dyads with a D2R‐OE subject were negatively correlated with active social behavior while vocalizations from control dyads were positively correlated with both active and passive social behavior also suggest social deficits. These data show that striatal dopamine dysfunction plays an important role in the development of social behavior and mouse models such as the one studied here provide an opportunity for screening potential therapeutics at different developmental time points.     

The SCID/Beige mouse as a model to investigate protection against Yersinia pestis

In this study, we have shown that severe combined immunodeficient/beige mice reconstituted with hyperimmune Balb/c lymphocytes can be used as a model to demonstrate adoptive and passive protection against plague infection. Reconstitution of severe combined immunodeficient/beige mice was successful in nine out of ten mice as demonstrated by spleen colonisation and sustained circulating immunoglobulin titres. Furthermore, an increase in antibody titre was evident after a booster immunisation of reconstituted mice. Presence of circulating antibody correlated with protection against a systemic plague challenge and indicated that in reconstituted mice adoptive transfer of a functional immune system had occurred. The severe combined immunodeficient/beige mouse was also used to demonstrate passive protection against inhaled and systemic plague infection. The reconstituted severe combined immunodeficient/beige mouse model demonstrating protective immunity against plague will be further developed to identify the immune cell subsets responsible for this protection.     

Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction

Impaired maturation of nerve growth factor precursor (proNGF) and its accumulation has been reported in several neurodegenerative diseases, myocardial infarction and diabetes. To elucidate the direct impact of proNGF accumulation identified the need to create a transgenic model that can express fully mutated cleavage-resistant proNGF. Using Cre-Lox technology, we developed an inducible endothelial-specific proNGF transgenic mouse (proNGF^Loxp) that overexpresses GFP-conjugated cleavage-resistant proNGF123 when crossed with VE-cadherin-CreERT2 (Cre). Expression of proNGF, inflammatory mediators, NGF and VEGF was evaluated by PCR, Western blot and immunohistochemistry. EC-proNGF overexpression was confirmed using colocalization of anti-proNGF within retinal vasculature. EC-proNGF did not cause retinal neurotoxicity or marked glial activation at 4-weeks. Microvascular preparation from Cre-proNGF mice showed significant imbalance of proNGF/NGF ratio, enhanced expression of TNF-α and p75^NTR, and tendency to impair TrkA phosphorylation compared to controls. EC-proNGF overexpression triggered mRNA expression of p75^NTR and inflammatory mediators in both retina and renal cortex compared to controls. EC-proNGF expression induced vascular permeability including breakdown of BRB and albuminuria in the kidney without affecting VEGF level at 4-weeks. Histopathological changes were assessed after 8-weeks and the results showed that EC-proNGF triggered formation of occluded (acellular) capillaries, hall mark of retinal ischemia. EC-proNGF resulted in glomerular enlargement and kidney fibrosis, hall mark of renal dysfunction. We have successfully created an inducible mouse model that can dissect the contribution of autocrine direct action of cleavage-resistant proNGF on systemic microvascular abnormalities in both retina and kidney, major targets for microvascular complication.     

The Hemimelic extra toes mouse mutant: Historical perspective on unraveling mechanisms of dysmorphogenesis

Hemimelic extra toes (Hx) arose spontaneously as a dominant mutation in B10.D2/nSnJ mice in 1967. It specifically affects the appendicular skeleton, causing variable foreshortening of the tibia (radius) and preaxial polydactylism. Early anatomical studies revealed anterior overgrowth of the autopod, with decreased apoptosis and increased mitosis in the anterior apical ectodermal ridge and underlying mesenchyme; overextension of apoptosis in the central zeugopod accounted for hemimelia. The Hx mutant phenotype was coarsely mapped to mouse chromosome (Chr) 5 and closely linked to engrailed‐2 (En2) and Sonic hedgehog (Shh). This region is syntenic to human Chr 7q36 that harbors several dominant mutations affecting the hand. High‐resolution genome mapping identified the Hx mutation as a G → A base pair transition within Intron 5 of the murine Lmbr1 locus. The critical effect is on a multifunctional conserved regulatory element that acts as a limb‐specific, long‐distance cis‐acting enhancer of Shh expression. As such, the Hx mutant phenotype results from ectopic Shh signals at the anterior margin of the limb bud that directly or indirectly alter FGF4 signaling from the apical ectodermal ridge. Given significant advances in understanding of embryonic development in general and limb development in particular, this review article reveals how research that once attracted interest of teratologists has advanced across the decades to pinpoint a critical molecular lesion and reveal a potential mechanism of a specific malformation that is found commonly in experimental teratology. Birth Defects Research (Part C) 90:155–162, 2010. © 2010 Wiley‐Liss, Inc.