Branchiogenic motoneurons innervating facial, masticatory, and esophageal muscles show aberrant distribution in the reeler-phenotype mutant rat, Shaking Rat Kawasaki

Shaking Rat Kawasaki (SRK) is an autosomal recessive mutant rat that is characterized by cerebellar ataxia. Although previous studies indicated many points of similarity between this mutant rat and the reeler mouse, nonlaminated structures such as the facial nucleus have not been studied in this mutant rat. Nissl-stained sections through the brainstem showed that the cytoarchitecture of the facial, motor trigeminal, and ambiguus nuclei was abnormal in SRK, especially in the lateral cell group of the facial nucleus and the compact formation of the ambiguus nucleus. To examine whether orofacial motoneurons are also malpositioned in the SRK rat, horseradish peroxidase (HRP) was injected into the facial, masticatory, and abdominal esophageal muscles of the SRK rats and normal controls to label facial, trigeminal, and ambiguus motoneurons, respectively. HRP-labeled facial, trigeminal, and ambiguus motoneurons of the SRK rat were distributed more widely than those of their normal counterparts, as in the case of the reeler mouse, with the one exception that labeled facial motoneurons innervating the nasolabial muscle were distributed more widely in the ventrolateral-to-dorsomedial direction in comparison with those of the reeler mutant. These data demonstrate that nonlaminated structures in the brainstem of the SRK rat are affected severely, as is the case in the reeler mutant mouse.     

Neonatla vestibular stimulation and mating in cerebellar mutants

Two cerebellar mutants, staggerer and reeler, and their congenic nonmutants were used in this experiment. Experimental animals were subjected to intense rotational stimulation on a tilted plane during the first 3 weeks of life, while controls were left nonstimulated. The capacity for mating, as evidenced by vaginal plugs or the occurrence of pregnancy, was assayed during two periods: between 36 and 89 days of age (Experiment A) and between 90 and 120 days of age (Experiment B). During Experiment A the mutants as well as the normals were caged inter se with partners of the opposite sex. During Experiment B the animals were caged with intact, sexually experienced partners. The animals were examined daily for evidence of mating. During Experiment A, only 3 of the 89 couples participating in this study showed evidence of mating. During Experiment B, the number of males of both strains which had mated increased significantly. The staggerer females showed a relatively high level of mating activity, whether stimulated or not. The reeler females, in contrast, rarely mated, although early stimulation significantly increased the level of sexual efficiency. The majority of the normal males and females mated, whether stimulated or not. It was concluded that massive motor-sensory stimulation in infancy, improving gait and body balance in staggerer and reeler mice, may also improve mating efficiency.     

Central noradrenaline metabolism in cerebellar ataxic mice

Central noradrenaline (NA) metabolism was investigated in 4 types of ataxic mutant mice, weaver, reeler, staggerer and rolling mouse Nagoya (RMN) and hypocerebellar mice experimentally produced by neonatal treatment with cytosine arabinoside (ara-C). As neurochemical markers for synthesis, steady-state level and turnover of NA, we measured tyrosine hydroxylase (TH) activity, NA and total (free + conjugated) 3-methoxy-4-hydroxyphenylethyleneglycol (total MHPG) concentrations in the cerebellum, cerebral cortex and spinal cord. In the weaver and staggerer whose cerebellar weight loss was severe (40 and 21% of controls, respectively), the 3 neurochemical markers were all increased in the cerebellum, and a similar increase was observed in other regions. In the reeler, the 3 markers were also markedly increased in the cerebellum, but no similar pattern of increase was observed in other regions. In the RMN whose cerebellum showed the least weight loss (76% of control), only TH activity and MHPG concentration were increased in the cerebellum and spinal cord but not in the cerebral cortex. In ara-C-treated mice, the neurochemical markers were also increased in the cerebellum, but to a lesser extent in other regions. These results suggest that, although total contents and total activity of these markers per whole cerebellum were significantly decreased, the central NA metabolism was basically enhanced throughout the CNS of the cerebellar ataxic mice irrespective of their cause. The degree and extent of this enhancement appeared to be correlated with the degree of the cerebellar weight loss.     

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.     

Distribution and morphology of callosal commissural neurons within the motor cortex of normal and reeler mice

Retrograde transport of horseradish peroxidase was used to examine the cells of origin of the callosal commissural fibers (CC neurons) in the primary motor cortex of normal and reeler mice. Quantitative analysis of the intracortical, laminar distribution, and dendritic orientation of CC neurons was performed in conjunction with qualitative observation of their morphology. For comparison, similar quantitative data were obtained for the cells of origin of the corticospinal tract (CST) of normal and reeler mice from materials described previously by Terashima et al. ('83). In the normal mouse, CC neurons are distributed in a bilaminar pattern such that the largest number of cells are located in supragranular layers II and III and in infragranular layer V. The majority of CC neurons are normal (upright) pyramids, although a few in the upper zone of layer VI are inverted pyramidal cells. In the reeler mutant, CC neurons are found in all cortical layers, but two-thirds are situated in the lower half of the cortex. On the basis of the celL shape and orientation of the apical dendrite, CC neurons of the reeler were classified into six morphological types: (1) typical pyramidal, (2) inverted pyramidal, (3) tumbled, (4) hook-shaped, (5) polymorphic, and (6) simple. The apical dendrites of the CC neurons in all layers of the cortex of the reeler mouse are randomly oriented; no direct relationship between the intracortical position of the soma and orientation of the apical dendrite was found. In contrast, CST neurons in the reeler mutant are concentrated in the outer third of the cortex, and there is a relationship between the laminar distribution of these cells and the alignment of their dendrites with respect to the pial surface: the apical dendrites of CST neurons lie in superficial layers tend to be oriented obliquely, whereas those of CST neurons in the deeper of cortex most often are oriented vertically, i.e, toward the pial surface. Quantitative analysis revealed that the relative intracortical positions of CC and CST neurons are reversed in the reeler mutant although both populations exhibited greater laminar disposition, and as a consequence, there is more intermingling of the two cell groups in the reeler than in the normal mouse. Thus, the present study suggests that the normal cytoarchitectonics of the primary motor cortex are inverted in the reeler mutant mouse.     

Cell-type-specific consequences of Reelin deficiency in the mouse neocortex, hippocampus, and amygdala

The disrupted cortical lamination phenotype in reeler mice and subsequent identification of the Reelin signaling pathway have strongly informed models of cortical development. We describe here a marker-based phenotyping approach to reexamine the cytoarchitectural consequences of Reelin deficiency, using high-throughput histology and newly identified panels of highly specific molecular markers. The resulting cell-type-level cytoarchitectural analysis revealed novel features of abnormal patterning in the male reeler mouse not obvious with less specific markers or histology. The reeler cortex has been described as a rough laminar inversion, but the data presented here are not compatible with this model. The reeler cortex is disrupted in a more complex fashion, with some regions showing a mirror-image laminar phenotype. Major rostrocaudal and cell-type-specific differences in the laminar phenotype between cortical areas are detailed. These and similar findings in hippocampus and amygdala have implications for mechanisms of normal brain development and abnormalities in neurodevelopmental disorders.     

Impaired nerve regeneration in reeler mice after peripheral nerve injury

Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up-regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin ( reeler ) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild-type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild-type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration.     

Lis1 reduction causes tangential migratory errors in mouse spinal cord

Mutations in human LIS1 cause abnormal neuronal migration and a smooth brain phenotype known as lissencephaly. Lis1^+/? (Pafah1b1) mice show defective lamination in the cerebral cortex and hippocampal formation, whereas homozygous mutations result in embryonic lethality. Given that Lis1 is highly expressed in embryonic neurons, we hypothesized that sympathetic and parasympathetic preganglionic neurons (SPNs and PPNs) would exhibit migratory defects in Lis1^+/? mice. The initial radial migration of SPNs and PPNs that occurs together with somatic motor neurons appeared unaffected in Lis1^+/? mice. The subsequent dorsally directed tangential migration, however, was aberrant in a subset of these neurons. At all embryonic ages analyzed, the distribution of SPNs and PPNs in Lis1^+/? mice was elongated dorsoventrally compared with Lis1^+/+ mice. Individual cell bodies of ectopic preganglionic neurons were found in the ventral spinal cord with their leading processes oriented along their dorsal migratory trajectory. By birth, Lis1^+/? SPNs and PPNs were separated into distinct groups, those that were correctly, and those incorrectly positioned in the intermediate horn. As mispositioned SPNs and PPNs still were detected in P30 Lis1^+/? mice, we conclude that these neurons ceased migration prematurely. Additionally, we found that a dorsally located group of somatic motor neurons in the lumbar spinal cord, the retrodorsolateral nucleus, showed delayed migration in Lis1^+/? mice. These results suggest that Lis1 is required for the dorsally directed tangential migration of many sympathetic and parasympathetic preganglionic neurons and a subset of somatic motor neurons. J. Comp. Neurol. 520:1198–1211, 2012. © 2011 Wiley Periodicals, Inc.     

Autoradiographic localization ofβ1 andα1-adrenoceptors in the midbrain and forebrain of normal and reeler mutant mice

The distribution of alpha-1 and beta-1 adrenoceptors has been studied in the midbrain and forebrain of normal and reeler mutant mice, using autoradiographic visualization of radioiodinated HEAT and ICYP, respectively. All cortical structures and nuclear groups of the murine forebrain and midbrain bind ICYP and HEAT. For each ligand, there is substantial regional variation in binding density and these variations tend to observe boundaries between nuclei or cortical regions or the stratification of cortical regions. Regional variations in binding densities are generally different for ICYP and HEAT. Binding sites for ICYP are distributed densely throughout all fields of the neocortex (particularl, layersI–III > VI) and paleocortex, the striatum, pallidum, substantia nigra and superficial strata of the superior colliculus. Dense concentrations of binding sites for HEAT in cortical structures, by contrast, are limited to frontal (all layers except IV) and anterior cingulate regions of the neocortex and, as with ICYP, the stratum lacunosum-moleculaire of the regio superior of the hippocampal formation. In subcortical structures, again in contrast to the pattern with ICYP, binding density is greatest in the principal nuclei of the dorsal thalamus and the septal nuclei. the regional binding patterns of both ICYP and HEAT in the reeler brain are identical to those in the normal animal. Differential laminar binding patterns within the neocortex are approximately inverted in the two genotypes, however. Thus, binding of ICYP is densest in an inner zone of the mutant, but in the outer 3 layers of the normal neocortex. Binding of this ligand is of relatively lower density in an outer zone of the mutant and in the inner 3 layers of the normal neocortex. Similar inversions are characteristics of the laminar binding patterns of HEAT in the frontal, primary sensory and associational cortical regions of the two genotypes where densest binding is encountered superficially in reeler but at deeper levels of the normal neocortex.     

Rescue of the reeler phenotype in the dentate gyrus by wild-type coculture is mediated by lipoprotein receptors for Reelin and Disabled 1

Reelin is a positional signal for the lamination of the dentate gyrus. In the reeler mutant lacking Reelin, granule cells are scattered all over the dentate gyrus. We have recently shown that the reeler phenotype of the dentate gyrus can be rescued in vitro by coculturing reeler hippocampal slices with slices from wild-type hippocampus. Here we studied whether Reelin from other brain regions can similarly induce this rescue effect and whether it is mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR). We found that coculturing reeler hippocampal slices with slices from wild-type olfactory bulb, cerebellum, and neocortex rescued the reeler phenotype as seen before with hippocampal slices, provided that the Reelin-synthesizing cells of these regions were placed near the marginal zone of the reeler hippocampal slice. However, coculturing wild-type hippocampal slices with hippocampal slices from mutants deficient in ApoER2 and VLDLR did not rescue the reeler-like phenotype in these cultures. Similarly, no rescue of the reeler-like phenotype was observed in slices from mutants lacking Disabled 1 (Dab1), an adapter protein downstream of Reelin receptors. Conversely, reeler hippocampal slices were rescued by coculturing them with slices from Dab1(-/-) mutants or ApoER2(-/-)/VLDLR(-/-) mice. These findings show that Reelin from other brain regions can substitute for the loss of hippocampal Reelin and that rescue of the reeler phenotype observed in our coculture studies is mediated via lipoprotein receptors for Reelin and Dab1.