Myelin-associated glycoprotein and myelin galactolipids stabilize developing axo-glial interactions.

We have analyzed mice that lack both the myelin-associated glycoprotein (MAG) and the myelin galactolipids, two glial components implicated in mediating axo-glial interactions during the myelination process. The single-mutant mice produce abnormal myelin containing similar ultrastructural abnormalities, suggesting that these molecules may play an overlapping role in myelin formation. Furthermore, the absence of the galactolipids results in a disruption in paranodal axo-glial interactions, and we show here that similar, albeit less severe, abnormalities exist in the developing MAG mutant. In the double-mutant mice, maintenance of axo-glial adhesion is significantly more affected than in the single mutants, supporting the overlapping function hypothesis. We also show that independently of MAG, galactolipids, and paranodal junctional components, immature nodes of Ranvier form normally, but rapidly destabilize in their absence. These data indicate that distinct molecular mechanisms are responsible for the formation and maintenance of axo-glial interactions.

A genetic basis for obsessive grooming.

Excessive grooming behaviors, cleansing rituals, and self-mutilation are important features of a range of neuropsychiatric diseases including obsessive compulsive (OC)-spectrum disorders. In this issue of Neuron, Greer and Capecchi (2002) report that Hoxb8 mutant mice exhibit this behavioral phenotype. These Hoxb8 mutants will be valuable in exploring the genetics and pathophysiology of OC-spectrum disorders as well as strategies for their treatment.

Hoxb8 is required for normal grooming behavior in mice.

Repertoires of grooming behaviors critical to survival are exhibited by most animal species, including humans. Genes that influence this complex behavior are unknown. We report that mice with disruptions of Hoxb8 show, with 100% penetrance, excessive grooming leading to hair removal and lesions. Additionally, these mice excessively groom normal cagemates. We have been unable to detect any skin or PNS abnormalities in Hoxb8 mutants. These observations suggest that the excessive, pathological grooming exhibited by these mice results from CNS abnormalities. Consistent with this interpretation, we demonstrate Hoxb8 expression in regions of the adult mouse CNS previously implicated in the control of grooming. The aberrant behavior observed in Hoxb8 mutants is not unlike that of humans suffering from the OC-spectrum disorder, trichotillomania. Interestingly, Hoxb8 is expressed in regions of the CNS known as the "OCD-circuit."

Rescue of ataxia and preplate splitting by ectopic expression of Reelin in reeler mice.

The gene mutated in reeler (reelin) encodes a protein secreted by neurons in the developing brain that controls laminar positioning of migrating cells in the CNS by an unknown mechanism. To investigate Reelin function, we used the nestin promoter to express Reelin ectopically in the ventricular zone and other brain regions in transgenic mice. In the presence of the endogenous protein, ectopic Reelin did not alter cell migration in the neocortex or the cerebellum. However, in the reeler background, ectopic Reelin induced tyrosine phosphorylation of Dab-1 in the ventricular zone and rescued some, but not all, of the neuroanatomic and behavioral abnormalities characteristic of reeler. These results indicate that Reelin does not function simply as a positional signal. Rather, it appears to participate in multiple events critical for neuronal migration and cell positioning.

Towards the comprehension of genetic mechanisms controlling brain morphogenesis.

Over the past few years, a huge amount of work has provided mouse mutants for many genes required for regionalization of the developing brain. This remarkable work now offers the opportunity of unmasking new and unexpected gene functions that underlie a complex network of molecular interactions.

Ovarian abnormalities in the staggerer mutant mouse.

Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rora(sg) gene underlying the cerebellar abnormalities of the staggerer mutant.

Circling behavior in the Ecl mouse is caused by lateral semicircular canal defects.

The epistatic circler mouse (Ecl mouse) is a preexisting mutant, which displays a circling phenotype and hyperactivity. It has been shown that the circling phenotype in this mutant results from a complex inheritance pattern, but the vestibular pathology has not been analyzed. The present study deals with the morphological and functional basis responsible for the circling behavior in the Ecl mouse. Morphological examination of the inner ears revealed a bilateral malformation of the horizontal (lateral) semicircular canal and duct. No cochlear abnormalities were detected, and auditory brainstem response (ABR) measurements indicated that the auditory system is not affected. Investigation of the vestibuloocular reflex (VOR) in Ecl mice showed that their horizontal VOR on stimulation is virtually absent, which correlates with the morphological findings.

Abnormal dispersion of a purkinje cell subset in the mouse mutant cerebellar deficient folia (cdf).

Purkinje cells of different molecular phenotypes subdivide the cortex of the cerebellum both rostrocaudally into parasagittal bands and mediolaterally into transverse zones. Superimposed on the Purkinje cell compartmentation, the cerebellar cortex is pleated into a reproducible array of lobes and lobules. During cerebellar development, Purkinje cell bands are formed through the rostrocaudal dispersal of embryonic clusters, triggered primarily by a Reelin-dependent signaling pathway. In the naturally occurring mouse mutant cerebellar deficient folia (cdf), there is a failure of Purkinje cell dispersion that results in widespread Purkinje cell ectopia in the adult. The ectopia is restricted primarily to that subset of Purkinje cells that does not express zebrin II/aldolase C and that forms ectopic clusters in among the cerebellar nuclei. Most Purkinje cells that express zebrin II are located normally in a monolayer. Thus, the cerebellum of cdf mutants has a failure of Purkinje cell dispersion that is confined primarily to a zebrin II-negative (zebrin II(-)) subpopulation. Despite the Purkinje cell ectopia, the parasagittal band organization of the cerebellum is still clear. The shortening of the cortex is distributed evenly over all lobules, with the result that transverse expression boundaries are relocated with respect to the lobules and fissures. The number of Purkinje cells in the cdf/cdf cerebellum is similar to the number in littermate controls. Therefore, it appears that the lesion in cdf results in the failure of a zebrin II(-) Purkinje cell subset to disperse either due to a cell intrinsic defect or due to an abnormal interaction between the Purkinje cells and either granule cells or afferent inputs.

Ectopic corticospinal tract and corticothalamic tract neurons in the cerebral cortex of yotari and reeler mice.

Reeler and yotari mice, which are mutant for Reelin or Dab1, respectively, show disorders of cerebral cortical lamination. We injected horseradish peroxidase (HRP) into the upper lumbar enlargement to label corticospinal tract (CST) neurons and wheat germ agglutinin-conjugated HRP (WGA-HRP) into the ventral lateral nucleus of the thalamus to label corticothalamic tract (CTT) neurons in both 19-day-old yotari and reeler mice with the aim of discovering whether or not they show differences in the distribution pattern of layer V or layer VI neurons. Similar injections of tracers were made in normal controls. HRP-labeled CST neurons, which were exclusively distributed in layer V of the normal cortex, were radially scattered in the cortex of both mutants, but those in reeler were more deeply distributed than in yotari. WGA-labeled CTT neurons, which were mainly located in layer VI in the normal cortex, were superficially distributed just beneath the pia mater in both reeler and yotari cortex. The present quantitative study shows that the distribution pattern of layer V neurons, but not layer VI neurons, differs between reeler and yotari mice, suggesting that the Reelin and Dab1 proteins may play different roles in the migration and cell positioning of layer V neurons.

Selective Purkinje cell ectopia in the cerebellum of the weaver mouse.

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

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.

Callosal axon guidance defects in p35(-/-) mice.

Mice lacking p35, an activator of cdk5 in the central nervous system (CNS), exhibit defects in a variety of CNS structures, most prominently characterized by a disruption in the laminar structure of the neocortex (Chae et al., 1997). In addition, alterations of certain axonal fiber tracts are found in the cortex of p35 mutant mice. Notably, the corpus callosum appears bundled at the midline, but dispersed lateral to the midline. Tracer injection experiments in adult p35 mutant mice reveal that projecting cortical axons fail to assimilate into the corpus callosum, and take oblique paths to the midline. After crossing the midline, cortical axons defasciculate prematurely from the corpus callosum and take similarly oblique paths through the cortex. This callosal phenotype is not detected in reeler mice, which also exhibit defects in cortical lamination, suggesting that the lack of fasciculation of callosal axons is not an inherent manifestation of a disruption of cortical lamination. The embryonic callosal axon tract is defasciculated before crossing the midline, suggesting that axon guidance may be affected during embryonic development of the corpus callosum. In addition, embryonic thalamocortical afferents also exhibit a defasciculated phenotype. These results suggest that defective axonal fasciculation and guidance may be primary responses to the loss of p35 in the cortex. Furthermore, this study postulates a role for the p35/cdk5 kinase in molecular signaling pathways necessary for proper guidance of selective axons during embryonic development.

Elongation of hair cell stereocilia is defective in the mouse mutant whirler.

The recessive mouse mutant whirler (wi) shows no response to sound and exhibits circling and head-tossing behaviour, indicative of both auditory and vestibular dysfunction. The wi mutation maps genetically to mouse chromosome 4. We examined the organ of Corti of whirler mutants to explore the possibility that the wi mutation affects sensory hair cells. Scanning electron microscopy (SEM) reveals that the specialised microvilli (stereocilia) that are projected by the sensory hair cells and are vital for sound transduction are abnormal in wi homozygotes. Specifically, wi homozygous inner hair cell (IHC) stereocilia are approximately half the length of equivalent stereocilia in heterozygous littermates. They are arranged normally into ranks, but the gradation in height and width of stereocilia in adjacent ranks is less prominent in wi homozygotes. Analysis of IHC stereocilia during the course of their development shows that, by embryonic day 18.5, mutant stereocilia are already significantly shorter than those in controls. Mutant stereocilia elongate at a normal rate, at least until postnatal day 1, but prematurely stop elongating between postnatal days 1 and 4. Stereocilia length then decreases. At postnatal day 15, outer hair cell (OHC) stereocilia in wi homozygotes appear short and are arranged in a rounded, "U" shape rather than the normal "W" or "V" shape. Eventually, both IHCs and OHCs degenerate. We show that the whirler locus encodes a protein(s) required for the elongation and maintenance of IHC and OHC stereocilia.

Lack of Reelin causes malpositioning of nigral dopaminergic neurons: evidence from comparison of normal and Reln(rl) mutant mice.

The reeler gene (Reln(rl), formerly rl) product Reelin controls neuronal migration and positioning and thereby plays a key role in brain development. Mutation of Reln leads to widespread disruption of laminar cortical regions and ectopia in some brainstem nuclei. In the embryonic striatum of normal mice, a substantial expression of reelin mRNA has been documented; however, the anomalous positioning of neurons in the basal ganglia of reeler mice remains to be studied. We provide first evidence for a potential role of Reelin in the developmental formation of the substantia nigra. In reeler mutant mice lacking Reelin, dopaminergic neurons destined for the substantia nigra fail to migrate laterally and become anomalously clustered just lateral to the ventral tegmental area. Their axons appear to project to striatal patches forming "dopamine islands." Results from the normal mice show that, at the midembryonic stage, Reelin identified with CR-50 is highly concentrated in the ventral mesencephalon, where nigral dopaminergic neurons are in progress to migrate laterally to their eventual position of the adult brain. A combination of CR-50 labeling and anterograde axonal tracing provided evidence that embryonic striatal neurons may supply the ventral portion of the mesencephalon with Reelin through their axonal projections. We hypothesize that Reelin plays a role in the positioning of nigral dopaminergic neurons and that it can act as an environmental cue at a remote site far from its birthplace via a transaxonal delivery system.

Axonal secretion of Reelin by Cajal-Retzius cells: evidence from comparison of normal and Reln(Orl) mutant mice.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.

Neurobiological effects of a null mutation depend on genetic context: comparison between two hotfoot alleles of the delta-2 ionotropic glutamate receptor.

Hotfoot is a mutant mouse with an ataxic phenotype which has been shown to be due to a mutation in the Grid2 gene. In this paper, we compare molecular, morphological, electrophysiological and behavioral features of two Grid2 alleles: Grid2(ho-4J) and Grid2(ho-Nancy). We first show that these two mutations are deletions in the open reading frame of the gene and that no GRID2 protein is detectable in extracts of mutant cerebella, suggesting that the two alleles are null-like mutations. Morphological and electrophysiological analyses reveal no obvious differences between the two strains: both strains showed the naked Purkinje dendritic spines and mismatch between the length of the presynaptic active zone and postsynaptic differentiation characteristic of the hotfoot mutation; and the same low level (20%) of multiple climbing fiber innervation of Purkinje cells was found in both strains. Only differences in motor behavior were found between the two strains. The Grid2(ho-4J) mouse shows more severe ataxia that the Grid2(ho-Nancy) mouse and, although both strains show a clear capacity to improve their performance of a motor task with training, the Grid2(ho-4J) performance remains very poor whereas Grid2(ho-Nancy) mice approach control levels. The only difference between the two strains is their genetic background. Our results show that the genetic background must be taken into account when analyzing sensorimotor performances of mutant mice.

Biochemical and anatomical evidence for specialized voltage-dependent calcium channel gamma isoform expression in the epileptic and ataxic mouse, stargazer.

Inherited forms of ataxia and absence seizures in mice have been linked to defects in voltage-dependent calcium channel subunits. However, a correlation between the sites of neuronal dysfunction and the impact of the primary lesion upon calcium channel subunit expression or function has not been clearly established. For example, the mutation in stargazer mice has pleiotropic consequences including synaptic alterations in cerebellar granule cells, hippocampal CA3/mossy fibers, and cortical neurons in layer V that, presumably, lead to ataxia and seizures. Genetic analysis of stargazer mice determined that the defective gene encodes a protein expressed in brain (gamma2) with limited homology to the skeletal muscle L-type calcium channel gamma1 subunit. Although additional gamma isoforms have been subsequently identified primarily in neural tissue, little was known about the proteins they encode. Therefore, this study explored the distribution and biochemical properties of gamma2 and other gamma isoforms in wild-type and stargazer brain. We cloned human gamma2, gamma3, and gamma4 isoforms, produced specific anti-peptide antibodies to gamma isoforms and characterized both heterologously expressed and endogenous gamma. We identified regional specificity in the expression of gamma isoforms by western analysis and immunohistochemistry. We report for the first time that the mutation in the stargazer gene resulted in the loss of gamma2 protein. Furthermore, no compensatory changes in the expression of gamma3 or gamma4 protein were evident in stargazer brain. In contrast to other voltage-dependent calcium channel subunits, gamma immunostaining was striking in that it was primarily detected in regions highly enriched in excitatory glutamatergic synapses and faintly detected in cell bodies, suggesting a role for gamma in synaptic functions. Sites of known synaptic dysfunction in stargazer (the hippocampal CA3 region, dentate gyrus, and cerebellar molecular layer) were revealed as relying primarily upon gamma2, as total gamma isoform expression was dramatically decreased in these regions. Electron microscopy localized anti-gamma antibody immunostaining to dendritic structures of hippocampal mossy fiber synapses, with enrichment at postsynaptic densities. To assess the association of native gamma with voltage-dependent calcium channel or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, gamma isoforms (gamma2, gamma3 and gamma4) were detergent solubilized from mouse forebrain. Antibodies against a highly conserved C-terminal epitope present in gamma2, gamma3 and gamma4 immunoprecipitated voltage-dependent calcium channel subunits (alpha1B), providing the first in vivo evidence that gamma and voltage-dependent calcium channels form stable complexes. Furthermore, both anti-gamma2 antibodies and anti-alpha1B antibodies independently immunoprecipitated the AMPA receptor subunit, GluR1, from mouse forebrain homogenates. In summary, loss of gamma2 immunoreactivity in stargazer is precisely localized so as to contribute to previously characterized synaptic defects. The data in this paper provide compelling evidence that gamma isoforms form complexes in vivo with voltage-dependent calcium channels as well as AMPA receptors, are selectively and differentially expressed in neuronal processes, and localize primarily to dendritic structures in the hippocampal mossy fiber region.

Glutamate transporters in the spinal cord of the wobbler mouse.

We studied the role of glutamate excitotoxicity in motor neuron degeneration in the wobbler mouse (wr/wr), a model of amyotrophic lateral sclerosis and spinal muscular atrophies. Choline acetyltransferase (ChAT) activity was decreased in the cervical spinal cord and in the muscles innervated by nerves originating in this region of wobbler mice, but no differences were found in the lumbar spinal cord and in the hindleg muscles. Glial fibrillar acid protein (GFAP), a marker of reactive gliosis, was significantly higher in the cervical spinal cord of wobbler mice aged 4 weeks than in controls and the differences were more marked at 12 weeks; no differences were found in the lumbar spinal cord. In spite of this selective degeneration of motor neurons (resulting in strong decrease in the neuronal glutamate transporter EAAC1) and reactive gliosis in the cervical spinal cord, the levels of the glial glutamate transporter proteins GLT-1 and GLAST were similar in wobbler and control mice. Plasma concentrations of excitatory amino acids were no different at any time examined. Our results exclude the involvement of decrease in glutamate GLT 1 transporter in the motor neuron degeneration in wobbler mice.

Callosal commissural neurons of Dab1 deficient mutant mouse, yotari.

The yotari mouse is an autosomal recessive mutant mouse, caused by mutation of disabled homolog 1 (Dab1) gene. The mutant mouse is recognized by unstable gait and tremor and by early deaths around at the time of weaning. The cytoarchitectures of cerebeller and cerebral cortices and hippocampal formation of the yotari mouse are abnormal. These malformations strikingly resemble those of reeler mouse. In the present study we examined the callosal commissural (CC) neurons of yotari, reeler and normal mice with the injection of recombinant adenovirus into the frontal area 1 (Fr1) to find some possible phenotypes specific for the yotari mouse. The distribution pattern of CC neurons of the yotari was similar to that of the reeler: retrogradely labeled CC neurons were seen throughout all depths of the contralateral Fr1. However, the present statistical analysis revealed that the difference of the mean intracortical position of the CC neurons between the yotari and the reeler is significantly different (Student's t-test), suggesting that the phenotype of the yotari is clearly different from that of the reeler.

Developmental expression of the GIRK family of inward rectifying potassium channels: implications for abnormalities in the weaver mutant mouse.

G-protein-gated inward rectifying potassium channels (GIRKs) are a newly identified gene family. These gene products are thought to form functional channels through the assembly of heteromeric subunits. Recently, it has been demonstrated that a point mutation in the GIRK2 gene, one of the GIRK family members, is the cause of the neurological and reproductive defects observed in the weaver (wv) mutant mouse. The mechanism(s) by which a single amino acid substitution in GIRK2 protein leads to the severe phenotypes in the wv / wv mouse is not fully understood. However, it implicates the importance of GIRK channels in neuronal development. To characterize the mRNA expression patterns of GIRK1-3 during mouse brain development we have used in situ hybridization analyses. We found that the expression of all three genes showed developmental regulation. In most areas that showed expression, the levels of GIRK1-3 transcripts reached their peak at around postnatal day 10 (P10). In general, GIRK1 showed the least fluctuation in its levels of expression during development, while dynamic changes were found with the levels of GIRK2 and GIRK3 transcripts. GIRK3 becomes the predominant inward rectifying K+-channel in the brain at later postnatal ages. In the CNS regions affected in the wv / wv mouse, GIRK2 is the predominant inward rectifying channel that is expressed. This suggests that the presence of the other subtypes are able to compensate for the mutated GIRK2 channel in weaver neurons that survive.