Enhanced dizocilpine efficacy in heterozygous reeler mice relates to GABA turnover downregulation

Reelin synthesized by cortical GABAergic interneurons throughout the telencephalon is secreted into the extracellular matrix (ECM) and binds with nM affinity to integrin receptors located at dendritic spine postsynaptic densities and positively modulates Arc and other dendritic resident mRNAs translation, thereby facilitating the onset of synaptic plasticity and LTP consolidation.

Accordingly, the reelin haploinsufficient heterozygous reeler mice (HRM) express a marked decrease of cortical thickness, of cortical and hippocampal dendritic spine density, and of cortical GAD₆₇ expression. Behaviorally, HRM manifest a sensorimotor deficit, an exaggerated response to fear, and a deficit in olfactory discrimination learning. HRM and wild-type mice (WTM) were trained to retrieve to criterion palatable chocolate-flavored food pellets in an eight-arm radial maze. In 9–14 days of training HRM and WTM learned the task equally well committing only a few errors. However, HRM, when compared with WTM, show a greater cognitive impairment following the administration of dizocilpine. Also, HRM are more susceptible to the increased locomotion and stereotypic behavior elicited by dizolcipine. The enhanced dizocilpine susceptibility of HRM is not due to differences in pharmacokinetics because the levels of dizocilpine in cortices of HRM and WTM were virtually equal. We also failed to detect differences between HRM and WTM in glutamate brain content and in the rate of ¹³C-glucose incorporation into the glutamate brain pools. In contrast we found that the conversion index of glutamate into GABA (an indirect measurement of GABA turnover rate) is decreased in cortex, hippocampus and striatum of HRM when compared to WTM. Thus, HRM recapitulate several neurochemical and behavioral endophenotypes reminiscent of schizophrenia and these mice can be proposed as a relevant animal model for the study of pharmacological treatments aimed at alleviating the sensory-motor and cognitive dysregulation associated with schizophrenia.     

A qualitative and quantitative light microscopic study of the inferior olivary complex of normal, reeler, and weaver mutant mice

In the normal mouse (+/+; +/rl) cerebellar Purkinje cells (PCs) are aligned in a monolayer and provide the main targets for incoming olivocerebellar climbing fibers (CF). In the neurological mutants, homozygous reeler (rl/rl), homozygous weaver (wv/wv) and heterozygous weaver (wv/+), cerebellar abnormalities exist in which many PCs are either missing or displaced. Therefore, it is of interest of determine if the inferior olivary complex (IO) in these mutants is also abnormal. This report concerns results obtained from a light microscopic study of the inferior olivary complex. Counts of IO cells revealed apparent differences in the IO in homozygous reeler when compared to normal littermates. Whereas in the normal mouse there are approximately 37,000 IO cells and clearly defined olivary subdivisions, the IO of the homozygous reeler has a 22.6% reduction in IO cells (mean = 28,770) and indistinct borders between the major olivary subdivisions. With regard to the heterozygous and homozygous weaver, surprisingly the IO morphology and cell numbers are similar to that of the wildtype mouse even though the animals have only 86% (wv/+, mean = 158,155) and 72% (wv/wv, mean = 131,882), respectively, of the normal numbers of PCs (+/+, mean = 183,857). Purkinje cell counts revealed that the midline vermal region is the most affected area in the cerebellum in wv/+ and wv/wv whereas counts in the lateral hemisphere are near normal. The PC/IO ratio in the homozygous weaver is approximately 3:1 as compared to 5:1 in the wildtype mouse. Recent electrophysiological findings in wv/wv indicate that PCs are multiply innervated by CFs. Since a transient phase of multiple innervation is normal in the immature rat, the situation in the adult homozygous weaver may represent a retention of this immature state. A factor which may play a role in this is the loss of parallel fiber (PF)-PC synapses resulting from massive postnatal granule cell death. An hypothesis suggesting an intrinsic PC time-dependent mutant gene effect is presented to account for the differences in the loss of Purkinje cells between wv/wv and wv/+ and between different regions of the cerebellum.     

Topographic and zonal organization of the olivocerebellar projection in the reeler mutant mouse

The organization of the olivocerebellar projection in the homozygous reeler mouse (rl/rl) was studied with the use of microinjections of 3H-leucine in different regions of the inferior olivary complex (IO) or horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) into medial, intermediate, or lateral regions of the reeler cerebellum. The purpose of this investigation was to determine the pattern of termination of olivocerebellar climbing fibers (CFs) in the cerebellum via an anterograde tracing technique, and to determine the topographic organization of the olivocerebellar projection via both anterograde and retrograde methods. The inferior olive injections were made via the ventral (i.e., retropharygeal) approach to the IO to minimize diffusion into other brainstem precerebellar nuclei and thus to ensure accurate well-restricted, injection sites. Labeled CF terminals were seen in both the superficial Purkinje cell (PC) layer (normally positioned PCs) and around PCs in the granular layer and central masses (ectopic PCs). The pattern of labeling is suggestive of orthogonal organization, in that vertical columns of cells are labeled. This is especially apparent in the medial PC group, where at least three bands are identified. Within an orthogonal band, CF terminals are seen around both superficial and deep Purkinje cells. Our data indicate that olivocerebellar topography is generally similar in reeler and normal mice despite severe abnormalities in target cell position in the reeler. The medial cerebellar region receives input from the caudal two-fifths of the medial accessory olive (MAO). The intermediate PC cluster receives input from more rostral portions of all three olivary divisions (MAO, principal olive [PO] and dorsal accessory olive [DAO] ), while rostral portions of MAO and PO project to the lateral cerebellum. These results indicate that the zonal organization of the olivocerebellar projection in the adult reeler exhibits a pattern generally similar to that seen in normal mice. This suggests that an afferent system can develop a normal organization despite having ectopic targets.     

Ambiguus motoneurons innervating laryngeal and esophageal muscles are malpositioned in the Reelin-deficient mutant rat,Shaking Rat Kawasaki

Conclusions. The present study confirmed that ambiguus motoneurons innervating intrinsic laryngeal and esophageal muscles are radially malpositioned in the brainstem of the Shaking Rat Kawasaki (SRK), a reelin-deficient mutant rat. Objectives. Ambiguus motoneurons innervating the striated muscles of the larynx and esophagus take a long migration from their original birth plate in the floor of the fourth ventricle to their final settlement in the ventral margin of the medulla oblongata. To examine whether the migration of ambiguus nucleus neurons is affected in SRK, we studied localization of ambiguus motoneurons of postnatal day 21 (P21) normal and SRK rats. Materials and methods. To label ambiguus motoneurons retrogradely, horseradish peroxidase (HRP) was injected into some laryngeal muscles including cricothyroid, thyroarytenoid and posterior cricoarytenoid muscles, and the cervical and abdominal esophageal muscles of the SRK and normal controls 2 days before sacrifice. Results. In the P21 normal rat, HRP-positive laryngeal and esophageal motoneurons were found in the nucleus ambiguus, whereas in the P21 SRK, they were scattered from the base of the fourth ventricle to the ventro-lateral margin of the medulla, suggesting that radial migration of ambiguus motoneurons from their birthplace to their final settlement is guided by Reelin protein.     

Reelin mRNA Expression During Mouse Brain Development

Using in situ hybridization, expression of the mRNA for reelin, the gene most probably responsible for the reeler trait, was studied during mouse brain development. from embryonic day 13 to maturity. The highest level of expression was found in Cajal-Retzius neurons, while a high signal was also seen in the olfactory bulb, the external granular layer of the cerebellum and, particularly at early developmental stages, in hypothalamic differentiation fields, tectum and spinal cord. A moderate to low level of expression was found in the septa1 area, striatal fields, habenular nuclei, some thalamic nuclei, particularly the lateral geniculate, the retina and some nuclei of the reticular formation in the central field of the medulla. Paradoxically, no reelin expression was detected in radial glial cells, the cortical plate, Purkinje cells, inferior olivary neurons and many other areas that are characteristically abnormal in reeler mutant mice. Together with other preliminary studies, the present observations suggest that the action of reelin is indirect, possibly mediated by the extracellular matrix. Most of the data can be explained by supposing that reelin is a cell-repulsive molecule which prevents migrating neurons from invading reelin-rich areas, and thus facilitates the deployment of radial glial cell processes and the formation of early architectonic patterns.     

Hormonal and morphological study of the pituitaries in reeler mice

Reelin is a neuronal glycoprotein that plays a crucial role in brain layer formation during prenatal development. The reeler mutant mouse lacks Reelin, leading to abnormalities in the neuronal layering of cerebral cortex and cerebellum, producing ataxia, tremor and abnormal locomotion. Reeler mice are reported to have growth retardation and most of them are sterile or unable to bring up their newborns. Since the brain is one of the main regulator of pituitary hormone secretion and no information was reported regarding pituitary function and structure in these mutant mice, we studied pituitary endocrine activity and morphology in reeler mice. Mice were classified in three groups as reeler homozygote (RHM), reeler heterozygote (RHT) or control (CO). Pituitary hormone blood levels were assessed by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA). Animals and their pituitaries were weighted and pituitaries were studied by histology, immunohistochemistry and electron microscopy. Results showed statistically significant differences in body weight and in adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) blood levels between the three groups. In contrast, growth hormone (GH) blood levels showed a high individual variation and no decrease in reeler groups compared with CO. Morphological studies revealed no differences in pituitary cell types except that somatotrophs appeared to be slightly smaller in RHM and RHT. Although it seems that pituitary hypofunction is not responsible for growth retardation, more studies are needed to obtain a deeper insight into the endocrine status of these mutant mice to elucidate the cause of their low body weight and reproductive behaviour.     

Ganglioside patterns during cerebral development in the normal and reeler mouse

Gangliosides were extracted from cerebral tissue of reeler and normal mice, and analyzed by two-dimensional thin-layer chromatography at embryonic and postnatal ages. The ganglioside pattern changed substantially as development proceeded in the telencephalon of both reeler and normal mice, but was the same at any given age for both conditions, despite the marked histological differences between reeler and normal samples. These results indicate that abnormal ganglioside patterns do not result from the reeler mutation at early stages of brain development, and that the cell misalignment characteristic of the reeler phenotype involves molecules other than gangliosides.     

Reelin expression during embryonic brain development in Crocodylus niloticus

The expression of reelin mRNA and protein was studied during embryonic brain development in the Nile crocodile Crocodylus niloticus, using in situ hybridization and immunohistochemistry. In the forebrain, reelin was highly expressed in the olfactory bulb, septal nuclei, and subpial neurons in the marginal zone of the cerebral cortex, dorsal ventricular ridge, and basal forebrain. At early stages, reelin mRNA was also detected in subventricular zones. In the diencephalon, the ventral lateral geniculate nuclei and reticular nuclei were strongly positive, with moderate expression in the habenula and focal expression in the hypothalamus. High expression levels were noted in the retina, the tectum, and the external granule cell layer of the cerebellum. In the brainstem, there was a high level of signal in cochleovestibular, sensory trigeminal, and some reticular nuclei. No expression was observed in the cortical plate or Purkinje cells. Comparison with reelin expression during brain development in mammals, birds, turtles, and lizards reveals evolutionarily conserved, homologous features that presumably define the expression profile in stem amniotes. The crocodilian cortex contains subpial reelin-positive cells that are also p73 positive, suggesting that they are homologous to mammalian Cajal-Retzius cells, although they express the reelin gene less intensely. Furthermore, the crocodilian cortex does not contain the subcortical reelin-positive cells that are typical of lizards but expresses reelin in subventricular zones at early stages. These observations confirm that reelin is prominently expressed in many structures of the embryonic brain in all amniotes and further emphasize the unique amplification of reelin expression in mammalian Cajal-Retzius cells and its putative role in the evolution of the cerebral cortex.     

Location of preganglionic neurons is independent of birthdate but is correlated to reelin-producing cells in the spinal cord

Many studies suggest that during neuronal development the birthdate of a neuron appears to have significant consequences for its ultimate location and identity. Our past study shows that sympathetic preganglionic neurons (SPN) in mice lacking the reelin gene settle in abnormal positions in the spinal cord. In the present study we determined that birthdate is not a factor contributing to the abnormal position of SPN in reeler. In both normal and reeler mice the period of neurogenesis of SPN was similar, and the final location of SPN in the spinal cord was independent of birthdate. Additionally, we have identified at least two types of ventral interneurons, V1 and V2, that are involved in the production of Reelin and the positioning of SPN in the spinal cord.