[3H]GABA binding in the cerebellum of the reeler murine mutant

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [³H]GABA binding have been observed. This study shows decreased affinity of the Na⁺-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na⁺-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na⁺-dependent and Na⁺-independent binding sites was not significantly altered. The decreased affinity of the Na⁺-independent GABA binding and the increased affinity of the Na⁺-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.     

Extent of multiple innervation of purkinje cells by climbing fibers in the olivocerebellar system of weaver,reeler, and staggerer mutant mice

Multiple innervation of cerebellar Purkinje cells (PCs) by climbing fibers (CFs) has been described recently in adult weaver, reeler, and staggerer mutant mice, instead of the monoinnervation found in normal adults. In the present study, the extent of this multiple innervation was estimated by two methods, using both evoked and spontaneous activity of the olivocerebellar system. Concordant values were obtained: the mean number of CF collaterals per PC was between 3.5 and 4 in weaver and staggerer and close to 3.2 for the multiply innervated PCs of reeler mice. These values are of the same order of magnitude as those for the transient multiple innervation in developing rats (Mariani and Changeux, 1981a, b).     

Reelin in the extracellular matrix and dendritic spines of the cortex and hippocampus: a comparison between wild type and heterozygous reeler mice by immunoelectron microscopy

Reelin is a glycoprotein (～400 kDa) secreted by GABAergic neurons into the extracellular matrix of the neocortex and hippocampus as well as other areas of adult rodent and nonhuman primate brains. Recent findings indicate that the heterozygote reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania. These include (1) a downregulation of both reelin mRNA and the translated proteins, (2) a decrease in the number of dendritic spines in cortical and hippocampal neurons, (3) a concomitant increase in the packing density of cortical pyramidal neurons, and (4) an age-dependent decrease in prepulse inhibition of startle. Interestingly, the heterozygous reeler mouse does not exhibit the unstable gait or the neuroanatomy characteristic of the null mutant reeler mouse. Immunocytochemical studies of the expression of reelin in mice have been primarily limited to light microscopy. In this study we present new immunoelectron microscopy data that delineates the subcellular localization of reelin in the cortex and hippocampus of the wild-type mouse, and compares these results to reelin expression in the heterozygous reeler mouse. In discontinuous areas of cortical layers I and II and the inner blade area of the dentate gyrus of the wild type mouse, extracellular reelin is associated with dendrites and dendritic spine postsynaptic specializations. Similar associations have been detected in the CA1 stratum oriens and other areas of the hippocampus. In the hippocampus, reelin expression is more expansive and more widespread than in cortical layers I and II. In contrast, extracellular reelin immunoreactivity is greatly diminished in all areas examined in the heterozygous reeler mouse. However, some cell bodies of GABAergic neurons in the cortex and hippocampus demonstrate an increased accumulation of reelin in the Golgi and endoplasmic reticulum. We suggest that in the heterozygous reeler mouse a downregulation of reelin biosynthesis results in a decreased rate of secretion into the extracellular space. This inhibits dendritic spine maturation and plasticity and leads to dissociation of dendritic postsynaptic density integrity and atrophy of spines. We speculate that the haploinsufficient reeler mouse may provide a model for future studies of the role of reelin, as it may be related to psychosis vulnerability.     

The reeler gene: a clue to brain development and evolution.

Reeler mutant mice are characterized by profuse anomalies of cell positioning in the telencephalic and cerebellar cortices as well as by distinct malformations in non-cortical structures such as the inferior olive, the facial nerve nucleus and other brainstem nuclei. Studies of the embryonic development of these structures reveal that the early cell patterns formed by reeler neurons is consistently affected, so that the reeler gene plays an important role in the development of nerve cell patterns. Comparative studies of cortical development in reptiles suggest further that the mammalian type of cortical architectonics has been acquired progressively during brain evolution, and reveal some similarities in early cortical organization between reeler and reptilian, particularly chelonian, embryos, most notably the presence of an inverted gradient of cortical histogenesis. These observations point to a possible role of the reeler gene in cortical evolution. Although the factors responsible for the formation of neural cell patterns are largely unknown, most data point to the importance of cell-cell interactions. Cell-interaction molecules have probably been acquired during brain evolution and the reeler gene could act by perturbing, directly or indirectly, such cell interactions. The characterization and thus the cloning of the reeler gene is therefore important for our understanding of brain development. Recent data on the fine chromosomal mapping of the mutation prior to its positional cloning are reported.     

The abnormal cerebellar organization of Weaver and reeler mice does not affect the cellular distribution of three neuronal mRNAs

We used in situ hybridization of 35S-labeled antisense RNAs to study the cellular distribution of three neuronal mRNAs. We compared the expression of these RNAs in cerebellar Purkinje neurons in wild-type (C57Bl-6J) mice and in two mutants (Weaver and reeler) known to have abnormal cerebellar morphologies. In normal mice, GAD mRNA is present in four sets of neurons in the cerebellar cortex while calbindin mRNA is present only in Purkinje neurons. Proenkephalin mRNA is present in Golgi II neurons as well as in a set of neurons in the deep part of the molecular layer. Despite the dramatic differences in structural organization and inputs of Purkinje neurons in the cerebella of adult Weaver and reeler mice, the expression of these RNAs appears unchanged. These results support the hypothesis that Purkinje cell cytodifferentiation proceeds autonomously after its inception in early embryonic life.     

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.     

Cerebellar plasma membrane proteins and their antisera

Plasma membranes have been isolated from neonatal through adult cerebella by a sequence of differential centrifugation, aqueous two-phase polymer fractionation and density gradient centrifugation. The protein composition of cerebellar membranes from various aged mice was compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Increases in the relative amount of membrane proteins with molecular weights (X 10(-3)) of 400, 340, 270, 220, 54, 44, and 9.5 were most pronounced, while a protein of 66,000 Mr disappeared between birth and Day 25. The relationship of these proteins and others to specific cell types in the cerebellum was examined by preparing membrane fractions from isolated granule and Purkinje cells, as well as from the cerebella of neurological mutant mice: reeler, weaver, staggerer, and nervous. In addition, those membrane proteins on the surface of dissociated cerebellar cells were identified by lactoperoxidase-catalyzed iodination, while glycoproteins were identified by galactose oxidase treatment and NaB3H4 reduction. Rabbit antisera were prepared toward those SDS-PAGE membrane proteins which appeared cell specific or developmentally regulated. Sera from these rabbits were used with indirect immunoperoxidase and immunofluorescence to stain frozen sections of mouse cerebellum and dissociated cerebellar cell cultures. In tissue sections antiserum toward the 400,000 Mr protein (p400) and antiserum p14.7 gave strong reactions with Purkinje cells while anti-p130 reacted preferentially with Purkinje cell somas, anti-p220 stained small cells in the internal granule layer and anti-p30 displayed a coarse, grainy staining of the granule and molecular layers, characteristic of a synaptic localization. Only anti-p220 and anti-p130 bound to freshly dissociated cells or cultured cerebellar cells. Large phase-bright cells in the cultures bound antiserum p130. Anti-p220 reacted specifically with a subpopulation of small round viable cells that bound tetanus toxin and decreased in number from 9% at Day 3 to 0.5% of the cells by Day 11, suggestive of granule neurons.     

Differential development of vesicular glutamate transporters in brain: an in vitro study of cerebellar granule cells

The cerebellar granule cells have been extensively used for studies on metabolism, neurotransmission and neurotoxicology, since they can easily be grown in cultures. However, knowledge about the development of different proteins essential for synaptic transmission in these cells is lacking. This study has characterized the developmental profiles of the vesicular glutamate transporters (VGLUTs) and the synaptic vesicle proteins synapsins and synaptophysin in cerebellar granule cells and in co-cultures containing both granule cells and astrocytes. The protein levels of VGLUT2 decreased by approximately 70% from days 2 to 7 in vitro, whereas the levels of VGLUT1 increased by approximately 95%. Protein levels of synapsin I, synapsin IIIa and synaptophysin showed a developmental pattern similar to VGLUT1 while synapsin II and VGLUT3 were absent. The mRNA expressions of VGLUT1 and VGLUT2 were in accordance with the protein levels. The results indicate both that cerebellar granule cells are mature at approximately 7 days in vitro, and that the up-regulation of VGLUT1 and down-regulation of VGLUT2 in cerebellar granule cells are both independent of surrounding astrocytes and neuronal input. The results of this study are discussed in relation to general developmental profiles of VGLUTs in other brain regions.     

Synaptic Chemistry Associated with Aberrant Neuronal Development in the Reeler Mouse

Abstract: Pre- and postsynaptic neurochemical markers for several afferent and intrinsic neuronal systems were measured in the mouse mutant, reeler. In the neocortex of the reeler, the relative positions of the polymorphic and pyramidal cells were inverted but this was not associated with alterations in the content/mg protein of synaptic markers for noradrenergic [tyrosine hydroxylase (TH), norepinephrine (NE), NE uptake], cholinergic [choline acetyltransferase (ChAT), quinuclidinyl benzilate (QNB) binding], γ-aminobutyric acid (GABA)ergic (glutamate decarboxylase, GABA uptake, GABA receptors, GABA) or glutamatergic (glutamate uptake, receptors, glutamate) neurons. The laminar distributions of the hippocampal neurons were disrupted and associated with mild hypoplasia; consistent with this alteration, the content/mg protein of some GABAergic (GABA uptake) and glutamatergic (glutamate receptors) markers were slightly increased. The reeler cerebellum was characterized not only by misalignment of neurons but also by a marked loss of granule cells. Commensurate with the degree of cerebellar hypoplasia, the total amount of glutamate content, [³H]l-glutamate uptake activity, [³H]muscimol, and [³H]QNB ligand binding were reduced in the reeler cerebellum. In contrast, presynaptic markers for the noradrenergic (TH, NE) climbing fibers and the cholinergic (ChAT) mossy fibers were significantly increased/mg protein but their total content/cerebellum was near normal. Our data support suggestions that cerebellar granule cells use glutamate as their neurotransmitter and contain GABA and cholinergic receptors. The findings also suggest that misplaced cortical and cerebellar neurons retain normal neurochemical characteristics and that the morphologic alterations do not markedly affect the quantitative development of aminergic afferent systems.     

Determinants of nerve cell patterns during development: a review

The aim of the work reviewed is to define some of the mechanisms which are implicated in the control of neural cell pattern formation in the developing central nervous system. This question was examined by studies of brain embryonic development in normal and reeler mutant mice, which are characterized by profuse architectonic anomalies. The adult reeler phenotype is characterized by extreme abnormalities of cell positioning in the telencephalic and cerebellar cortices as well as by distinct architectonic anomalies in non-cortical structures such as the inferior olive, the facial nerve nucleus and other brainstem nuclei. Studies of the embryonic development of these structures reveal that neurons are generated at the normal time and migrate along normal pathways. Moreover, the processes of directional axonal growth, differentiation of class-specific features of neurons and glia, and synaptogenesis appear unaffected by the reeler mutation. In all instances, however, the early cell patterns formed by reeler neurons is consistently less regular than in normal embryos. These data indicate that brain architectonics does not exclusively result from the maturation of cells, neurites and connections, but is also contingent upon a specific stabilization of early neurons at the end of migration. One may infer that the presence of a normal allele at the reeler locus is necessary for this stabilization to occur normally, or that it is submitted to genetic control. Although the factor(s) responsible for the stable configuration of neural cell patterns are still unknown, several hypotheses can be considered. There is ample evidence against the role of diffusible factors, mesodermal components and afferent fiber systems. So far, most data point to the importance of cell-cell interactions which can be of three types: homophilic (neuronal-neuronal), heterophilic (neuronal-glial), or both. The cell-interaction mechanism could have been acquired during brain evolution of the mammalian lineage and the reeler gene could act by perturbing, directly or indirectly, these cell interactions. A better definition of the mechanisms responsible for the organization of nerve cell patterns is central to our understanding of brain development in normal as well as in pathological states. By following the example of recent successful research on invertebrate brain development, we believe that the genetic approach to this important question is a valuable one.     

Evidence for a cell-specific action of Reelin in the spinal cord

Reelin, the extracellular matrix protein missing in reeler mice, plays an important role in neuronal migration in the central nervous system. We examined the migratory pathways of phenotypically identified spinal cord neurons to determine whether their positions were altered in reeler mutants. Interneurons and projection neurons containing choline acetyltransferase and/or NADPH diaphorase were studied in E12.5-E17.5 reeler and wild-type embryos, and their final locations were assessed postnatally. While three groups of dorsal horn interneurons migrated and differentiated normally in reeler mice, the migrations of both sympathetic (SPNs) and parasympathetic preganglionic neurons (PPNs) were aberrant in the mutants. Initially reeler and wild-type SPNs were detected laterally near somatic motor neurons, but by E13.5, many reeler SPNs had mismigrated medially. Postnatally, 79% of wild-type SPNs were found laterally, whereas in reeler, 92% of these neurons were positioned medially. At E13.5, both reeler and wild-type PPNs were found laterally, but by E14.5, reeler PPNs were scattered across the intermediate spinal cord while wild-type neurons correctly maintained their lateral location. By postnatal day 16, 97% of PPNs were positioned laterally in wild-type mice; in contrast, only 62% of PPNs were found laterally in mutant mice. In E12.5-E14.5 wild-type mice, Reelin-secreting cells were localized along the dorsal and medial borders of both groups of preganglionic neurons, but did not form a solid barrier. In contrast, Dab1, the intracellular adaptor protein thought to function in Reelin signaling, was expressed in cells having positions consistent with their identification as SPNs and PPNs. In combination, these findings suggest that, in the absence of Reelin, both groups of autonomic motor neurons migrate medially past their normal locations, while somatic motor neurons and cholinergic interneurons in thoracic and sacral segments are positioned normally. These results suggest that Reelin acts in a cell-specific manner on the migration of cholinergic spinal cord neurons.     

An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.     

Some monotreme milk "whey" and blood proteins

1. Electrophoretic studies are made of mature phase milk "whey" proteins and blood serum proteins of echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus). The echidna milk bands are designated A-M, those of platypus A-G. Some of the proteins are isolated and characterized. 2. Echidna band A protein has some similarity to high cystine "whey" proteins. Band E protein (apparent Mr 21,000) may be a beta-lactoglobulin-like protein. Band M is lysozyme. Band C is serum albumin. Bands G-K are transferrins. 3. Platypus milk bands A, C, D, F and G are isolated. Bands F and G are transferrins. 4. Lactose synthase and lytic activities are examined.     

Galactosyltransferase Defects in Reeler Mouse Brains

Galactosyltransferase activities were examined in the cerebellum, cerebral cortex, and brain stem of reeler and wild-type mice. Galactosyltransferase assays were optimal for all required substrates, linear with incubation time, and proportional to protein concentration. In brain areas affected by the reeler mutation (i.e., cerebral cortex and cerebellum), galactosylation of both endogenous and exogenous glycoprotein acceptors was greatly reduced in reeler relative to controls. On the other hand, glycosylation of endogenous glycolipids was low, and equal between reeler and wild-type. Galactosyltransferase activities were similar, though not identical, in reeler and wild-type brain stems, which are phenotypically normal in reeler mice. Glucosyltransferase, beta-galactosidase, beta-N-acetylglucosaminidase, acid phosphatase, and lactate dehydrogenase specific activities were all unaffected in reeler cerebella, while galactosyltransferase activity was 52% of control. Inhibition of either UDPgalactose hydrolysis or beta-galactosidase had no effect on galactosyltransferase activity. The spectrum or galactosyltransferase deficiencies in reeler suggests that this enzyme is associated with the development of young granule cells.     

Glutamate Dehydrogenase in Cerebellar Mutant Mice: Gene Localization and Enzyme Activity in Different Tissues

Many similarities of both the inheritance pattern and the neuropathology can be observed between olivopontocerebellar atrophies, or so-called multiple system atrophies (MSAs), and murine cerebellar mutations like Purkinje cell degeneration, nervous, staggerer, weaver, and reeler. Our study aimed to test whether the glutamate dehydrogenase (GDH) deficiency observed in some MSA patients could be found also in any of the murine mutants. GDH activity was assayed in several organs of these mutants, and no general deficiency was detected. By contrast, the level was found to be elevated in the cerebellum. The GDH gene was localized on mouse chromosome 14 and does not map close to any known neurological mutation in the mouse. We conclude, for the moment, that none of these cerebellar mutant mice can be considered as an animal model for GDH-deficient MSA.     

Glutamic acid: A strong candidate as the neurotransmitter of the cerebellar granule cell

Free amino acids and cholinergic enzymes were investigated in the cerebellum of reeler and weaver mice in an attempt to identify the neurotransmitter characteristic of the granule cell population and to clarify any neurotransmitter abnormalities of their pre- and postsynaptic neurons induced by their depletion. The data indicate that glutamic acid may be the neurotransmitter of the granule cells. Pre- and postsynaptic neurotransmitter activity seemed not to be markedly altered in cerebellar granule cell dysgenesis.     

Estimation of genetic distances between “reeler” and nearby loci on mouse chromosome 5

Genetic distances between reeler (gene symbol rl) and the adjacent loci Pgy-1, Sor, and En-2 on proximal chromosome 5 were estimated using a backcross panel between rlOrl/rlOrl BALB/c and C57 mice. Pgy-1 and Sor are located approximately 7 cM away from and centromeric to reeler, whereas En-2 is located distally, approximately 8 cM from reeler. Backcrosses between rlOrl/rlOrl BALB/c and mice with the T31H translocation showed that the breakpoint is located less than 2 cM from reeler. Together with previous work, these observations suggest the following gene order: Cen-Sor/Pgy-1-rl-T31H-En-2-other loci.     

Dab1 (Disable homolog-1) reelin adaptor protein is overexpressed in the olfactory bulb at early postnatal stages

Dab1 mediates reelin signalling and plays critical roles in early brain development such as the stereotypical positioning of neurons in the brain. The olfactory bulb undergoes a prominent layering reorganization, but shows not apparent differences between wild type and reeler in the layer organization. Therefore, an accurate regional and cellular simultaneous analysis of these molecules becomes essential to clarify the role played by Dab1 upon Reelin effect. The present study reveals a strong and consistent Dab1 mRNA and protein expressions, throughout the olfactory bulb layers in both wild type and reeler mice. In addition, noteworthy is the pattern of Dab1 location within cell nuclei in both strains. Furthermore, a temporal increment of Dab1 expression levels is detected from P0 to P15 in both strains, being the protein quantity higher in reeler than in wild type mice. Altogether, our results revealed that Reln acts directly from projection neurons via the production of different Reln fragments. Changes in the pattern of Dab1 expression could reflect an alternative Reln function in postnatal and adult stages, besides a possible regulation of Dab1 by other molecules distinct to Reln.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Congruence of vascular network remodeling and neuronal dispersion in the hippocampus of reelin-deficient mice

In the hippocampus, neurons and fiber projections are strictly organized in layers and supplied with oxygen via a vascular network that also develops layer-specific characteristics in wild-type mice, as shown in the present study for the first time in a quantitative manner. By contrast, in the reeler mutant, well known for its neuronal migration defects due to the lack of the extracellular matrix protein reelin, emerging layer-specific characteristics of the vascular pattern were found to be remodeled during development of the dentate gyrus. Remarkably, in the first postnatal week, when a granule cell layer was still discernable in the reeler dentate gyrus, also the reeler vascular pattern resembled wild type. Thus, at postnatal day 6, unbranched microvessels traversed the granule cell layer and bifurcated when reaching the subgranular zone. Only after the first postnatal week vascular network remodeling in the reeler dentate gyrus became apparent, when the proportion of dispersed granule cells increased. Hence, vessel bifurcation frequency decreased in the maturing reeler dentate gyrus, but increased in wild type, resulting in significant differences (approx. 100%; p < 0.01) between adult wild type and reeler. Moreover, layer-specific vessel bifurcation frequencies disappeared in the maturing reeler dentate gyrus. Finally, a wild type-like vascular pattern was also found in the dentate gyrus of mice deficient for the reelin receptor very low density lipoprotein receptor (VLDLR), precluding a requirement of VLDLR for normal vascular pattern formation in the dentate gyrus. In sum, our findings show that vascular network remodeling in the reeler dentate gyrus is closely linked to the progression of granule cell dispersion.