Identification and characterization of a Reeler domain containing protein in Procambarus clarkii provides new insights into antibacterial immunity in crustacean.

Crayfish, as an invertebrate, relies only on the innate immune system to resist external pathogens. In this study, a molecule containing a single Reeler domain was identified from red swamp crayfish Procambarus clarkii (named as PcReeler). Tissue distribution analysis showed that PcReeler was highly expressed in gills and its expression was induced by bacterial stimulation. Inhibiting the expression of PcReeler by RNA interference led to a significant increase in the bacterial abundance in the gills of crayfish, and a significant increase in the crayfish mortality. Silencing of PcReeler influenced the stability of the microbiota in the gills revealed by 16S rDNA high-throughput sequencing. Recombinant PcReeler showed the ability to bind microbial polysaccharide and bacteria and to inhibit the formation of bacterial biofilms. These results provided direct evidence for the involvement of PcReeler in the antibacterial immune mechanism of P. clarkii.

Normal connectivity of thalamorecipient networks in barrel equivalents of the reeler cortex.

The reeler mouse mutant has long served as a primary model to study the development of cortical layers, which is governed by the extracellular glycoprotein reelin secreted by Cajal-Retzius cells. Because layers organize local and long-range circuits for sensory processing, we investigated whether intracortical connectivity is compromised by reelin deficiency in this model. We generated a transgenic reeler mutant (we used both sexes), in which layer 4-fated spiny stellate neurons are labeled with tdTomato and applied slice electrophysiology and immunohistochemistry with synaptotagmin-2 to study the circuitry between the major thalamorecipient cell types, namely excitatory spiny stellate and inhibitory fast-spiking (putative basket) cells. In the reeler mouse, spiny stellate cells are clustered into barrel equivalents. In these clusters, we found that intrinsic physiology, connectivity, and morphology of spiny stellate and fast-spiking, putative basket cells does not significantly differ between reeler and controls. Properties of unitary connections, including connection probability, were very comparable in excitatory cell pairs and spiny stellate/fast-spiking cell pairs, suggesting an intact excitation-inhibition balance at the first stage of cortical sensory information processing. Together with previous findings, this suggests that thalamorecipient circuitry in the barrel cortex develops and functions independently of proper cortical lamination and postnatal reelin signaling.

Postnatal injection of Reelin protein into the cerebellum ameliorates the motor functions in reeler mouse.

Reelin is a large secreted protein important for brain development and functions. In both humans and mice, the lack of Reelin gene causes cerebellar hypoplasia and ataxia. Treatment against Reelin deficiency is currently unavailable. Here, we show that the injection of recombinant Reelin protein into the cerebellum of Reelin-deficient reeler mice at postnatal day 3 ameliorates the forelimb coordination and mice are noted to stand up along cage wall more frequently. A mutant Reelin protein resistant to proteases has no better effect than the wild-type Reelin. Such ameliorations were not observed when a mutant Reelin protein that does not bind to Reelin receptors was injected and the injection of Reelin protein did not ameliorate the behavior of Dab1-mutant yotari mice, indicating that its effect is dependent on the canonical Reelin receptor-Dab1 pathway. Additionally, a Purkinje cell layer in reeler mice was locally induced by Reelin protein injection. Our results indicate that the reeler mouse cerebellum retains the ability to react to Reelin protein in the postnatal stage and that Reelin protein has the potential to benefit Reelin-deficient patients.

Heterozygous Dab1 Null Mutation Disrupts Neocortical and Hippocampal Development.

Loss-of-function mutations in Reelin and DAB1 signaling pathways disrupt proper neuronal positioning in the cerebral neocortex and hippocampus, but the underlying molecular mechanisms remain elusive. Here, we report that heterozygous yotari mice harboring a single autosomal recessive yotari mutation of Dab1 exhibited a thinner neocortical layer 1 than wild-type mice on postnatal day (P)7. However, a birth-dating study suggested that this reduction was not caused by failure of neuronal migration. In utero electroporation-mediated sparse labeling revealed that the superficial layer neurons of heterozygous yotari mice tended to elongate their apical dendrites within layer 2 than within layer 1. In addition, the CA1 pyramidal cell layer in the caudo-dorsal hippocampus was abnormally split in heterozygous yotari mice, and a birth-dating study revealed that this splitting was caused mainly by migration failure of late-born pyramidal neurons. Adeno-associated virus (AAV)-mediated sparse labeling further showed that many pyramidal cells within the split cell had misoriented apical dendrites. These results suggest that regulation of neuronal migration and positioning by Reelin-DAB1 signaling pathways has unique dependencies on Dab1 gene dosage in different brain regions.

Repetitively burst-spiking neurons in reeler mice show conserved but also highly variable morphological features of layer Vb-fated "thick-tufted" pyramidal cells.

Reelin is a large extracellular glycoprotein that is secreted by Cajal-Retzius cells during embryonic development to regulate neuronal migration and cell proliferation but it also seems to regulate ion channel distribution and synaptic vesicle release properties of excitatory neurons well into adulthood. Mouse mutants with a compromised reelin signaling cascade show a highly disorganized neocortex but the basic connectional features of the displaced excitatory principal cells seem to be relatively intact. Very little is known, however, about the intrinsic electrophysiological and morphological properties of individual cells in the reeler cortex. Repetitive burst-spiking (RB) is a unique property of large, thick-tufted pyramidal cells of wild-type layer Vb exclusively, which project to several subcortical targets. In addition, they are known to possess sparse but far-reaching intracortical recurrent collaterals. Here, we compared the electrophysiological properties and morphological features of neurons in the reeler primary somatosensory cortex with those of wild-type controls. Whereas in wild-type mice, RB pyramidal cells were only detected in layer Vb, and the vast majority of reeler RB pyramidal cells were found in the superficial third of the cortical depth. There were no obvious differences in the intrinsic electrophysiological properties and basic morphological features (such as soma size or the number of dendrites) were also well preserved. However, the spatial orientation of the entire dendritic tree was highly variable in the reeler neocortex, whereas it was completely stereotyped in wild-type mice. It seems that basic quantitative features of layer Vb-fated RB pyramidal cells are well conserved in the highly disorganized mutant neocortex, whereas qualitative morphological features vary, possibly to properly orient toward the appropriate input pathways, which are known to show an atypical oblique path through the reeler cortex. The oblique dendritic orientation thus presumably reflects a re-orientation of dendritic input domains toward spatially highly disorganized afferent projections.

Granule cell dispersion in two mouse models of temporal lobe epilepsy and reeler mice is associated with changes in dendritic orientation and spine distribution.

Temporal lobe epilepsy is characterized by hippocampal neuronal death in CA1 and hilus. Dentate gyrus granule cells survive but show dispersion of the compact granule cell layer. This is associated with decrease of the glycoprotein Reelin, which regulates neuron migration and dendrite outgrow. Reelin-deficient (reeler) mice show no layering, their granule cells are dispersed throughout the dentate gyrus. We studied granule cell dendritic orientation and distribution of postsynaptic spines in reeler mice and two mouse models of temporal lobe epilepsy, namely the p35 knockout mice, which show Reelin-independent neuronal migration defects, and mice with unilateral intrahippocampal kainate injection. Granule cells were Golgi-stained and analyzed, using a computerized camera lucida system. Granule cells in naive controls exhibited a vertically oriented dendritic arbor with a small bifurcation angle if positioned proximal to the hilus and a wider dendritic bifurcation angle, if positioned distally. P35 knockout- and kainate-injected mice showed a dispersed granule cell layer, granule cells showed basal dendrites with wider bifurcation angles, which lost position-specific differences. Reeler mice lacked dendritic orientation. P35 knockout- and kainate-injected mice showed increased dendritic spine density in the granule cell layer. Molecular layer dendrites showed a reduced spine density in kainate-injected mice only, whereas in p35 knockouts no reduced spine density was seen. Reeler mice showed a homogenous high spine density. We hypothesize that granule cells migrate in temporal lobe epilepsy, develop new dendrites which show a spread of the dendritic tree, create new spines in areas proximal to mossy fiber sprouting, which is present in p35 knockout- and kainate-injected mice and loose spines on distal dendrites if mossy cell death is present, as it was in kainate-injected mice only. These results are in accordance with findings in epilepsy patients.

Reelin dorsal horn neurons co-express Lmx1b and are mispositioned in disabled-1 mutant mice.

Mice missing either Reelin or Disabled-1 (Dab1) exhibit dorsal horn neuronal positioning errors and display heat hypersensitivity and mechanical insensitivity. Reelin binds its receptors, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, leading to the recruitment and phosphorylation of Dab1 and activation of downstream pathways that regulate neuronal migration. Previously, we reported that 70% of Dab1 laminae I-II neurons co-expressed LIM-homeobox transcription factor 1-beta (Lmx1b). Here, we asked whether Reelin-expressing dorsal horn neurons co-express Lmx1b, are mispositioned in dab1 mutants, and contribute to nociceptive abnormalities. About 90% of Reelin-labeled neurons are Lmx1b-positive in laminae I-II, confirming that most Reelin and Dab1 neurons are glutamatergic. We determined that Reelin-Lmx1b and Dab1-Lmx1b dorsal horn neurons are separate populations, and together, comprise 37% of Lmx1b-positive cells within and above the Isolectin B4 (IB4) layer in wild-type mice. Compared to wild-type mice, dab1 mutants have a reduced area of laminae I-II outer (above the IB4 layer), more Reelin-Lmx1b neurons within the IB4 layer, and fewer Reelin-Lmx1b neurons within the lateral reticulated area of lamina V and lateral spinal nucleus. Interestingly, both Reelin- and Dab1-labeled dorsal horn neurons sustain similar positioning errors in mutant mice. After noxious thermal and mechanical stimulation, Reelin, Lmx1b, and Reelin-Lmx1b neurons expressed Fos in laminae I-II and the lateral reticulated area in wild-type mice and, therefore, participate in nociceptive circuits. Together, our data suggest that disruption of the Reelin-signaling pathway results in neuroanatomical abnormalities that contribute to the nociceptive changes that characterize these mutant mice.

Quantitative Three-Dimensional Reconstructions of Excitatory Synaptic Boutons in the "Barrel Field" of the Adult "Reeler" Mouse Somatosensory Neocortex: A Comparative Fine-Scale Electron Microscopic Analysis with the Wild Type Mouse.

Synapses are key structural determinants for information processing and computations in the normal and pathologically altered brain. Here, the quantitative morphology of excitatory synaptic boutons in the "reeler" mutant, a model system for various neurological disorders, was investigated and compared with wild-type (WT) mice using high-resolution, fine-scale electron microscopy (EM) and quantitative three-dimensional (3D) models of synaptic boutons. Beside their overall geometry, the shape and size of presynaptic active zones (PreAZs) and postsynaptic densities (PSDs) forming the active zones and the three pools of synaptic vesicles (SVs), namely the readily releasable pool (RRP), the recycling pool (RP), and the resting pool, were quantified. Although the reeler mouse neocortex is severely disturbed, no significant differences were found in most of the structural parameters investigated: the size of boutons (~3 µm2), size of the PreAZs and PSDs (~0.17 µm2), total number of SVs, and SVs within a perimeter (p) of 10 nm and p20 nm RRP; the p60 nm, p100 nm, and p60-p200 nm RP; and the resting pool, except the synaptic cleft width. Taken together, the synaptic organization and structural composition of synaptic boutons in the reeler neocortex remain comparably "normal" and may thus contribute to a "correct" wiring of neurons within the reeler cortical network.

Mispositioned Neurokinin-1 Receptor-Expressing Neurons Underlie Heat Hyperalgesia in Disabled-1 Mutant Mice.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.

Structural and Synaptic Organization of the Adult Reeler Mouse Somatosensory Neocortex: A Comparative Fine-Scale Electron Microscopic Study of Reeler With Wild Type Mice.

The reeler mouse has been widely used to study various aspects of cortico- and synaptogenesis, but also as a model for several neurological and neurodegenerative disorders. In contrast to development, comparably little is known about the neuronal composition and synaptic organization of the adult reeler mouse neocortex, in particular at the fine-scale electron microscopic level, which was investigated here and compared with wild type (WT) mice. In this study, the "barrel field" of the adult reeler and WT mouse somatosensory neocortex is used as a model system. In reeler the characteristic six-layered structure is no longer existent, but replaced by a conglomerate of neurons organized in homologous clusters with maintained morphological identity and heterologous clusters between neurons and/or oligodendrocytes. These clusters are loosely scattered throughout the neocortical mass between the pial surface and the white matter. In contrast to WT, layer 1 (L1), if existent, seems to be diluted into the volume of the neocortical mass with no clear boundary. L1 also contains clusters of migrated or persistent neurons, oligodendro- and astrocytes. As in WT, myelinated and unmyelinated axons were found throughout the neocortical mass, but in reeler they were organized in massive fiber bundles with a high fiber packing density. A prominent and massive thalamocortical projection traverses through the neocortical mass, always accompanied by numerous "active" oligodendrocytes whereas in WT no such projections were found and "silent" oligodendrocytes were restricted to the white matter. In the adult reeler mouse neocortex, synaptic boutons terminate on somata, dendritic shafts, spines of different types and axon initial segments with no signs of structural distortion and/or degeneration, indicating a "normal" postsynaptic innervation pattern of neurons. In addition, synaptic complexes between boutons and their postsynaptic targets are tightly ensheathed by fine astrocytic processes, as in WT. In conclusion, the neuronal clusters may represent a possible alternative organization principle in adult reeler mice "replacing" layer formation. If so, these homologous clusters may represent individual "functional units" where neurons are highly interconnected and may function as the equivalent of neurons integrated in a cortical layer. The structural composition and postsynaptic innervation pattern of neurons by synaptic boutons provide the structural basis for the establishment of a functional although altered cortical network in the adult reeler mouse.

Developmental abnormality contributes to cortex-dependent motor impairments and higher intracortical current requirement in the reeler homozygous mutants.

The motor deficit of the reeler mutants has largely been considered cerebellum related, and the developmental consequences of the cortex on reeler motor behavior have not been examined. We herein showed that there is a behavioral consequence to reeler mutation in models examined at cortex-dependent bimanual tasks that require forepaw dexterity. Using intracortical microstimulation, we found the forelimb representation in the motor cortex was significantly reduced in the reeler. The reeler cortex required a significantly higher current to evoke skeletal muscle movements, suggesting the cortical trans-synaptic propagation is disrupted. When the higher current was applied, the reeler motor representation was found preserved. To elucidate the influence of cerebellum atrophy and ataxia on the obtained results, the behavioral and neurophysiological findings in reeler mice were reproduced using the Disabled-1 (Dab1) cKO mice, in which the Reelin-Dab1 signal deficiency is confined to the cerebral cortex. The Dab1 cKO mice were further assessed at the single-pellet reach and retrieval task, displaying a lower number of successfully retrieved pellets. It suggests the abnormality confined to the cortex still reduced the dexterous motor performance. Although possible muscular dysfunction was reported in REELIN-deficient humans, the function of the reeler forelimb muscle examined by electromyography, morphology of neuromuscular junction and the expression level of choline acetyltransferase were normal. Our results suggest that the mammalian laminar structure is necessary for the forepaw skill performance and for trans-synaptic efficacy in the cortical output.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.

The Functioning of a Cortex without Layers.

A major hallmark of cortical organization is the existence of a variable number of layers, i.e., sheets of neurons stacked on top of each other, in which neurons have certain commonalities. However, even for the neocortex, variable numbers of layers have been described and it is just a convention to distinguish six layers from each other. Whether cortical layers are a structural epiphenomenon caused by developmental dynamics or represent a functionally important modularization of cortical computation is still unknown. Here we present our insights from the reeler mutant mouse, a model for a developmental, "molecular lesion"-induced loss of cortical layering that could serve as ground truth of what an intact layering adds to the cortex in terms of functionality. We could demonstrate that the reeler neocortex shows no inversion of cortical layers but rather a severe disorganization that in the primary somatosensory cortex leads to the complete loss of layers. Nevertheless, the somatosensory system is well organized. When exploring an enriched environment with specific sets of whiskers, activity-dependent gene expression takes place in the corresponding modules. Precise whisker stimuli lead to the functional activation of somatotopically organized barrel columns as visualized by intrinsic signal optical imaging. Similar results were obtained in the reeler visual system. When analyzing pathways that could be responsible for preservation of tactile perception, lemniscal thalamic projections were found to be largely intact, despite the smearing of target neurons across the cortical mantle. However, with optogenetic experiments we found evidence for a mild dispersion of thalamic synapse targeting on layer IV-spiny stellate cells, together with a general weakening in thalamocortical input strength. This weakening of thalamic inputs was compensated by intracortical mechanisms involving increased recurrent excitation and/or reduced feedforward inhibition. In conclusion, a layer loss so far only led to the detection of subtle defects in sensory processing by reeler mice. This argues in favor of a view in which cortical layers are not an essential component for basic perception and cognition. A view also supported by recent studies in birds, which can have remarkable cognitive capacities despite the lack of a neocortex with multiple cortical layers. In conclusion, we suggest that future studies directed toward understanding cortical functions should rather focus on circuits specified by functional cell type composition than mere laminar location.

Disabled-1 dorsal horn spinal cord neurons co-express Lmx1b and function in nociceptive circuits.

The Reelin-signaling pathway is essential for correct neuronal positioning within the central nervous system. Mutant mice with a deletion of Reelin, its lipoprotein receptors, or its intracellular adaptor protein Disabled-1 (Dab1), exhibit nociceptive abnormalities: thermal (heat) hyperalgesia and reduced mechanical sensitivity. To determine dorsal horn alterations associated with these nociceptive abnormalities, we first characterized the correctly positioned Dab1 neurons in wild-type and mispositioned neurons in Reelin-signaling pathway mutant lumbar spinal cord. Using immunofluorescence, we found that 70% of the numerous Dab1 neurons in Reln+/+ laminae I-II and 67% of those in the lateral reticulated area and lateral spinal nucleus (LSN) co-express the LIM-homeobox transcription factor 1 beta (Lmx1b), an excitatory glutamatergic neuron marker. Evidence of Dab1- and Dab1-Lmx1b neuronal positioning errors was found within the isolectin B4 terminal region of Reln-/- lamina IIinner and in the lateral reticulated area and LSN, where about 50% of the Dab1-Lmx1b neurons are missing. Importantly, Dab1-Lmx1b neurons in laminae I-II and the lateral reticulated area express Fos after noxious thermal or mechanical stimulation and thus participate in these circuits. In another pain relevant locus - the lateral cervical nucleus (LCN), we also found about a 50% loss of Dab1-Lmx1b neurons in Reln-/- mice. We suggest that extensively mispositioned Dab1 projection neurons in the lateral reticulated area, LSN, and LCN and the more subtle positioning errors of Dab1 interneurons in laminae I-II contribute to the abnormalities in pain responses found in Reelin-signaling pathway mutants.

Intracortical Network Effects Preserve Thalamocortical Input Efficacy in a Cortex Without Layers.

Layer IV (LIV) of the rodent somatosensory cortex contains the somatotopic barrel field. Barrels receive much of the sensory input to the cortex through innervation by thalamocortical axons from the ventral posteromedial nucleus. In the reeler mouse, the absence of cortical layers results in the formation of mispositioned barrel-equivalent clusters of LIV fated neurons. Although functional imaging suggests that sensory input activates the cortex, little is known about the cellular and synaptic properties of identified excitatory neurons of the reeler cortex. We examined the properties of thalamic input to spiny stellate (SpS) neurons in the reeler cortex with in vitro electrophysiology, optogenetics, and subcellular channelrhodopsin-2-assisted circuit mapping (sCRACM). Our results indicate that reeler SpS neurons receive direct but weakened input from the thalamus, with a dispersed spatial distribution along the somatodendritic arbor. These results further document subtle alterations in functional connectivity concomitant of absent layering in the reeler mutant. We suggest that intracortical amplification mechanisms compensate for this weakening in order to allow reliable sensory transmission to the mutant neocortex.

Reelin and Neuropsychiatric Disorders.

Proper neuronal migration and laminar formation during corticogenesis is essential for normal brain function. Disruption of these developmental processes is thought to be involved in the pathogenesis of some neuropsychiatric conditions. Especially, Reelin, a glycoprotein mainly secreted by the Cajal-Retzius cells and a subpopulation of GABAergic interneurons, has been shown to play a critical role, both during embryonic and postnatal periods. Indeed, animal studies have clearly revealed that Reelin is an essential molecule for proper migration of cortical neurons and finally regulates the cell positioning in the cortex during embryonic and early postnatal stages; by contrast, Reelin signaling is closely involved in synaptic function in adulthood. In humans, genetic studies have shown that the reelin gene (RELN) is associated with a number of psychiatric diseases, including Schizophrenia (SZ), bipolar disorder (BP) and autistic spectrum disorder. Indeed, Reln haploinsufficiency has been shown to cause cognitive impairment in rodents, suggesting the expression level of the Reelin protein is closely related to the higher brain functions. However, the molecular abnormalities in the Reelin pathway involved in the pathogenesis of psychiatric disorders are not yet fully understood. In this article, we review the current progress in the understanding of the Reelin functions that could be related to the pathogenesis of psychiatric disorders. Furthermore, we discuss the basis for selecting Reelin and molecules in its downstream signaling pathway as potential therapeutic targets for psychiatric illnesses.

Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum.

A mutation of the reln gene gives rise to the Reeler mouse (reln (-∕-)) displaying an ataxic phenotype and cerebellar hypoplasia. We have characterized the neurochemistry of postnatal (P0-P60) reln (-∕-) mouse cerebella with specific attention to the intervention of cell proliferation and apoptosis in the P0-P25 interval. Homozygous reln (-∕-) mice and age-matched controls were analyzed by immunofluorescence using primary antibodies against NeuN, calbindin, GFAP, vimentin, SMI32, and GAD67. Proliferation and apoptosis were detected after a single intraperitoneal BrdU injection and by the TUNEL assay with anti-digoxigenin rhodamine-conjugated antibodies. Quantitative analysis with descriptive and predictive statistics was used to calculate cell densities (number/mm(2)) after fluorescent nuclear stain (TCD, total cell density), labeling with BrdU (PrCD, proliferating cell density), or TUNEL (ApoCD, apoptotic cell density). By this approach we first have shown that the temporal pattern of expression of neuronal/glial markers in postnatal cerebellum is not affected by the Reeler mutation. Then, we have demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to reduction of cortical size and cellularity (TCD), and that TCD is, in turn, linked to quantitative differences in the extent of cell proliferation and apoptosis, as well as derangements in their temporal trends during postnatal maturation. Finally, we have calculated that PrCD is the most important predictive factor to determine TCD in the cerebellar cortex of the mutants. These results support the notion that, beside the well-known consequences onto the migration of the cerebellar neurons, the lack of Reelin results in a measurable deficit in neural proliferation.

The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse.

The Reeler heterozygous mice (reln(+/-)) are haplodeficient in the gene (reln) encoding for the reelin glycoprotein (RELN) and display reductions in brain/peripheral RELN similar to autistic or schizophrenic patients. Cytoarchitectonic alterations of the reln(+/-) brain may be subtle, and are difficult to demonstrate by current histological approaches. We analyzed the number and topological organization of the Purkinje neurons (PNs) in five vermal lobules - central (II-III), culmen (IV-V), tuber (VIIb), uvula (IX), and nodulus (X) - that process different types of afferent functional inputs in reln(+/+) and reln(+/-) adult mice (P60) of both sexes (n=24). Animals were crossed with L7GFP mice so that the GFP-tagged PNs could be directly identified in cryosections. Digital images from these sections were processed with different open source software for quantitative topological and statistical analyses. Diversity indices calculated were: maximum caliper, density, area of soma, dispersion along the XZ axis, and dispersion along the YZ axis. We demonstrate: i. reduction in density of PNs in reln(+/-) males (14.37%) and reln(+/-) females (17.73%) compared to reln(+/+) males; ii. that reln(+/-) males have larger PNs than other genotypes, and females (irrespective of the reln genetic background) have smaller PNs than reln(+/+) males; iii. PNs are more chaotically arranged along the YZ axis in reln(+/-) males than in reln(+/+) males and, except in central lobulus, reln(+/-) females. Therefore, image processing and statistics reveal previously unforeseen gender and genotype-related structural differences in cerebellum that may be clues for the definition of novel biomarkers in human psychiatric disorders.