Neonatal Exposure to Lipopolysaccharide Promotes Neurogenesis of Subventricular Zone Progenitors in the Developing Neocortex of Ferrets.

Lipopolysaccharide (LPS) is a natural agonist of toll-like receptor 4 that serves a role in innate immunity. The current study evaluated the LPS-mediated regulation of neurogenesis in the subventricular zone (SVZ) progenitors, that is, the basal radial glia and intermediate progenitors (IPs), in ferrets. Ferret pups were subcutaneously injected with LPS (500 µg/g of body weight) on postnatal days (PDs) 6 and 7. Furthermore, 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU) were administered on PDs 5 and 7, respectively, to label the post-proliferative and proliferating cells in the inner SVZ (iSVZ) and outer SVZ (oSVZ). A significantly higher density of BrdU single-labeled proliferating cells was observed in the iSVZ of LPS-exposed ferrets than in controls but not in post-proliferative EdU single-labeled and EdU/BrdU double-labeled self-renewing cells. BrdU single-labeled cells exhibited a lower proportion of Tbr2 immunostaining in LPS-exposed ferrets (22.2%) than in controls (42.6%) and a higher proportion of Ctip2 immunostaining in LPS-exposed ferrets (22.2%) than in controls (8.6%). The present findings revealed that LPS modified the neurogenesis of SVZ progenitors. Neonatal LPS exposure facilitates the proliferation of SVZ progenitors, followed by the differentiation of Tbr2-expressing IPs into Ctip2-expressing immature neurons.

Neurogenesis of Subventricular Zone Progenitors in the Premature Cortex of Ferrets Facilitated by Neonatal Valproic Acid Exposure.

The present study evaluated the neurogenesis of neonatal valproic acid (VPA) exposure on subventricular zone progenitors of the developing cerebral cortex in ferrets. VPA was injected at a dose of 200 µg/g of body weight into ferret infants on postnatal days 6 and 7. Two different thymidine analogues, 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU), were injected with a 48 h interval to label proliferating cells before and after VPA exposure. Two hours after BrdU injection, BrdU single- and EdU/BrdU double-labeled cells, but not EdU single-labeled cells, were significantly denser in both the inner and outer subventricular zones of VPA-exposed infants than in control infants. Notably, more than 97% of BrdU single- and EdU/BrdU double-labeled cells were immunopositive for Pax6, a stable marker for basal radial glia (bRG), in both groups. In contrast, the percentage of cells positively immunostained for Cux1, a postmitotic marker for upper-layer cortical neurons, in both EdU single- and BrdU single-labeled cells, was significantly higher in VPA-exposed infants than in control infants. These findings suggest that neonatal VPA exposure facilitates bRG proliferation, including self-renewal, followed by their differentiation into upper layer cortical neurons in the premature cortex of ferrets.

Male prevalent enhancement of leftward asymmetric development of the cerebellar cortex in ferrets (Mustela putorius).

The present study was conducted in MRI-based volumetry to characterize the sexual dimorphism of the cerebellum in young adult ferrets. High spatial resolution 3D anatomical MRI at 7-tesla were acquired ex vivo from fixed cerebella of 90-day-old male and female ferrets. The 3D morphology and topology of cerebellar structures were reproduced well by volume-rendered images obtained from MRI. Volume of the whole cerebellum was significantly larger in males than in females. The cerebellar cortex was further divided into five transverse domains: the anterior zone (AZ; lobules I-V), central zone anterior (lobule VI), central zone posterior (CZp; lobule VII), posterior zone (PZ; lobules VIII-IXa) and nodular zone (NZ; lobules IXb -X). Significantly greater volumes in males than in females were detected bilaterally in the AZ, CZp, and NZ, and leftward in PZ. Notably, the significant volume asymmetry was detected leftward in the CZp of males. By asymmetry quotient analysis, the counterclockwise torque asymmetry of the cerebellum was revealed, and it was more striking in males than in females. The present results suggest that sexual dimorphism of the ferret cerebellum is characterized by enhancing the leftward laterality in the CZp in males, forming the distinctive counterclockwise torque asymmetry.

Tracking of Internal Granular Progenitors Responding to Valproic Acid in the Cerebellar Cortex of Infant Ferrets.

Internal granular progenitors (IGPs) in the developing cerebellar cortex of ferrets differentiate towards neural and glial lineages. The present study tracked IGPs that proliferated in response to valproic acid (VPA) to determine their fate during cerebellar cortical histogenesis. Ferret kits were used to administer VPA (200 µg/g body weight) on postnatal days 6 and 7. EdU and BrdU were injected on postnatal days 5 and 7, respectively, to label the post-proliferative and proliferating cells when exposed to VPA. At postnatal day 20, when the external granule layer was most expanded, EdU- and BrdU-single-labeled cells were significantly denser in the inner granular layer of VPA-exposed ferrets than in controls. No EdU- or BrdU-labeling was found in Purkinje cells and molecular layer interneurons. Significantly higher percentages of NeuN and Pax6 immunostaining in VPA-exposed ferrets revealed VPA-induced differentiation of IGPs towards granular neurons in BrdU-single-labeled cells. In contrast, both EdU- and BrdU-single-labeled cells exhibited significantly greater percentages of PCNA immunostaining, which appeared in immature Bergman glia, in the internal granular layer of VPA-exposed ferrets. These findings suggest that VPA affects the proliferation of IGPs to induce differentiative division towards granular neurons as well as post-proliferative IGPs toward differentiation into Bergmann glia.

MRI-based morphometric characterizations of sexual dimorphism of the cerebrum of ferrets (Mustela putorius).

The present study aimed to characterize cerebral morphology in young adult ferrets and its sexual dimorphism using high-field MRI and MRI-based morphometry. Ex vivo short TR/TE (typical T1-weighted parameter setting for conventional MRI) and T2W (long TR/TE) MRI with high spatial resolution at 7-tesla could visualize major subcortical and archicortical structures, i.e., the caudate nucleus, lentiform nucleus, amygdala and hippocampus. In particular, laminar organization of the olfactory bulb was identifiable by short TR/TE-MRI. The primary and secondary sulci observable in the adult ferret were distinguishable on either short TR/TE- or T2W-MRI, and the cortical surface morphology was reproduced well by 3D-rendered images obtained by short TR/TE-MRI. The cerebrum had a significantly lower volume in females than in males, which was attributed to region-specific volume reduction in the cerebral cortex and subcortical white matter in females. A sexual difference was also detected, manifested by an overall reduction in normalized signal ratios of short TR/TE-MRI in all cerebral structures examined in females than in males. On the other hand, an alternating array of higher and lower short TR/TE-MRI intensity transverse zones throughout the cortex, which was reminiscent of the functional cortical areas, was revealed by maximum intensity projection (MIP) in 3D. The normalized signal ratio of short TR/TE-MRI, but not T2W-MRI in the cortex, was negatively correlated with the density of myelin-basic protein immunoreactive fibers (males, r=-0.440; females, r=-0.481). The present results suggest that sexual differences in the adult ferret cerebrum are characterized by reduced volumes of the cerebral cortex and subcortical white matter in females, and by overall reductions in physiochemical characteristics, as obtained by short TR/TE-MRI, in females. It should be noted that short TR/TE-MRI-based MIP delineated functional cortical areas related to myeloarchitecture in 3D. Such an approach makes possible conventional investigation of the functional organization of the cerebral cortex and its abnormalities using high-field MRI.

Differential staining patterns of immunohistochemical markers for neurogenesis staging in the premature cerebellum of ferrets and mice.

Immunohistochemical staining patterns of markers for neurogenesis staging were compared at the identical stage of cerebellar histogenesis between ferrets (aged 20 days) and mice (aged 10 days). Proliferating cell nuclear antigen (PCNA) immunostaining was observed largely in the granular precursors of the external granular layer (EGL) in both ferrets and mice. PCNA-immunostaining was also found in brain lipid-binding protein-immunopositive cells in the internal granular layer and was more abundant in ferrets than in mice. Paired box 6 immunostaining appeared largely in the EGL granular precursors in mice, whereas it emerged in the migrating/differentiating granular precursors in ferrets. These findings revealed that the types and neurogenesis stages of the EGL granular precursors detected by immunohistochemical markers differed between ferrets and mice.

Induction of cerebellar cortical neurogenesis immediately following valproic acid exposure in ferret kits.

Introduction: Valproic acid (VPA) is an anticonvulsant/antiepileptic drug that regulates neurogenesis. Its effects vary depending on the timing of exposure and the types of neural progenitors involved. Neonatal exposure to VPA causes autism spectrum disorder-like behaviors in some mammalian species, including ferrets. Ferrets experience the cerebellar cortical histogenesis during early postnatal period. However, no studies have evaluated the effect of VPA on cerebellar corticohistogenesis. The present study aimed to determine the effects of VPA exposure on the developing cerebellar cortex in ferret kits with a particular focus on the cortical neurogenesis. Methods: The experimental kits each received an intraperitoneal injection of VPA, 200 µg/g body weight, on postnatal days 6 and 7. EdU and BrdU were administered on postnatal days 5 and 7, respectively, to label cells proliferating prior to and following exposure to VPA. Results: We found that 2 h post BrdU injection, BrdU-labeled cells were abundantly distributed in the internal granular layer (IGL), whereas EdU-labeled cells were primarily relegated to the inner pre-migratory zone of the external granular layer (EGL). The density of BrdU-single-labeled cells was significantly lower in the EGL and significantly higher in the IGL of the VPA-exposed group, as compared to the control group. Immunostaining for doublecortin, a marker of immature neurons, was observed in BrdU-single-labeled cells in the IGL of the VPA-exposed group, which was significantly higher than that observed in the control group. EdU-single-labeled cells that had proliferated prior to VPA exposure were also detected in the IGL. While the cell density remained unchanged, significant changes were observed in the proportions of EdU-single-labeled cells immunostained with marker antigens; higher proportion of PCNA immunostaining, but lower proportion of S100 immunostaining in the VPA-exposed group compared to the control group. Discussion: These findings suggest the presence of progenitors in the IGL of the developing cerebellar cortex in ferret kits. We called them "internal granular progenitors." The progenitors may proliferate in response to VPA, leading the differentiated lineage more toward neurons than to glial cells. Thus, VPA may facilitate the differentiative division of internal granular progenitors to produce cerebellar granular neurons.

Quantitative assessment of central nervous system disorder induced by prenatal X-ray exposure using diffusion and manganese-enhanced MRI.

Prenatal radiation exposure induces various central nervous system (CNS) disorders depending on the dose, affected region and gestation period. The goal of this study was to assess noninvasively a CNS development disorder induced by prenatal X-ray exposure using quantitative manganese-enhanced MRI (MEMRI) as well as apparent diffusion coefficient (ADC) and transverse relaxation time (T(2)) maps in comparison with immunohistological staining. The changes in ΔR(1) (increase in the longitudinal relaxation rate (R(1)) from before and after MnCl(2) administration.) induced by the Mn(2+) contrast agent were evaluated in the CNS of normal and prenatally irradiated rats. ADC and T(2) were also compared with the histological results obtained using hematoxylin and eosin (to estimate cell density), activated caspase-3 (apoptotic cells) and glial fibrillary acidic protein (proliferation of astrocytes/astroglia). We found the following: (i) the decreased Mn(2+) uptake (indicated by a smaller ΔR(1)) for radiation-exposed rats was predominantly correlated with a decrease in cell viability (apoptotic cytopathogenicity) and CNS cell density after prenatal radiation exposure; (ii) the longer T(2) and ADC were associated with a decrease in CNS cell density and apoptotic alteration after radiation exposure. In addition to the slight proliferation of astroglia (+58%), there was a substantial decrease in cell density (-78%) and an excessive increase in apoptotic cells (+613%) in our prenatal radiation exposure model. The results suggest that MEMRI in the prenatal X-ray exposure model predominantly reflected the decrease in cell density and viability rather than the proliferation of astroglia. In conclusion, quantitative MEMRI with ADC/T(2) mapping provides objective information for the in vivo assessment of cellular level alterations by prenatal radiation exposure, and has the potential to be used as a standard approach for the evaluation of the cellular damage of radiotherapy.

Immunohistochemical characterization of postnatal changes in cerebellar cortical cytoarchitectures in ferrets.

We immunohistochemically characterized postnatal changes in cerebellar cortical cytoarchitectures in ferrets using markers for cerebellar cortical neurons and glial cells. Although 10 lobules of the vermis were already observed on postnatal day (PD) 4, Purkinje cells were still arrayed into two to three layers. Purkinje cells were aligned in a monolayer by PD 10 and formed mature shapes on PD 42 by developing their dendritic arbors. Parvalbumin immunostaining revealed relatively slower maturation of Purkinje cells in the Lobule X cortex than in other lobules. Basket and stellate cells emerged in the molecular layer on PDs 21 and 42, respectively. Rosette-like arranged glutamate decarboxylase 65 and 67-positive puncta were observed in the inner granular layer (IGL) on PD 21. Proliferating cell nuclear antigen immunostaining appeared in the outer zone of the external granular layer (EGL) containing progenitors of granular neurons on PDs 4-21. Bergmann glial processes extending vertically through the molecular layer and EGL were visible with GFAP immunostaining on PD 10 and thereafter. Their somata, aligned in the Purkinje cell layer, showed immunopositivity to Sox2 already on PD 4 and subsequently to S100 protein on PD 10. Sox2-positive cells were found sparsely in the IGL. Few of them were NeuN positive on PD 90, predicting the possibility of adult neurogenesis. These immunohistochemical results revealed that ferrets underwent cerebellar cortical histogenesis during their postnatal life in sequences. Relatively slow development or maturation of the ferret cerebellum was revealed by the timing of the monolayer alignment and morphological maturation of Purkinje cells.

Neonatal valproic acid exposure produces altered gyrification related to increased parvalbumin-immunopositive neuron density with thickened sulcal floors.

Valproic acid (VPA) treatment is associated with autism spectrum disorder in humans, and ferrets can be used as a model to test this; so far, it is not known whether ferrets react to developmental VPA exposure with gyrencephalic abnormalities. The current study characterized gyrification abnormalities in ferrets following VPA exposure during neonatal periods, corresponding to the late stage of cortical neurogenesis as well as the early stage of sulcogyrogenesis. Ferret pups received intraperitoneal VPA injections (200 µg/g of body weight) on postnatal days (PD) 6 and 7. BrdU was administered simultaneously at the last VPA injection. Ex vivo MRI-based morphometry demonstrated significantly lower gyrification index (GI) throughout the cortex in VPA-treated ferrets (1.265 ± 0.027) than in control ferrets (1.327 ± 0.018) on PD 20, when primary sulcogyrogenesis is complete. VPA-treated ferrets showed significantly smaller sulcal-GIs in the rostral suprasylvian sulcus and splenial sulcus but a larger lateral sulcus surface area than control ferrets. The floor cortex of the inner stratum of both the rostral suprasylvian and splenial sulci and the outer stratum of the lateral sulcus showed a relatively prominent expansion. Parvalbumin-positive neuron density was significantly greater in the expanded cortical strata of sulcal floors in VPA-treated ferrets, regardless of the BrdU-labeled status. Thus, VPA exposure during the late stage of cortical neurogenesis may alter gyrification, primarily in the frontal and parietotemporal cortical divisions. Altered gyrification may thicken the outer or inner stratum of the cerebral cortex by increasing parvalbumin-positive neuron density.

The Proliferation of Dentate Gyrus Progenitors in the Ferret Hippocampus by Neonatal Exposure to Valproic Acid.

Prenatal and neonatal exposure to valproic acid (VPA) is associated with human autism spectrum disorder (ASD) and can alter the development of several brain regions, such as the cerebral cortex, cerebellum, and amygdala. Neonatal VPA exposure induces ASD-like behavioral abnormalities in a gyrencephalic mammal, ferret, but it has not been evaluated in brain regions other than the cerebral cortex in this animal. This study aimed to facilitate a comprehensive understanding of brain abnormalities induced by developmental VPA exposure in ferrets. We examined gross structural changes in the hippocampus and tracked proliferative cells by 5-bromo-2-deoxyuridine (BrdU) labeling following VPA administration to ferret infants on postnatal days (PDs) 6 and 7 at 200 µg/g of body weight. Ex vivo short repetition time/time to echo magnetic resonance imaging (MRI) with high spatial resolution at 7-T was obtained from the fixed brain of PD 20 ferrets. The hippocampal volume estimated using MRI-based volumetry was not significantly different between the two groups of ferrets, and optical comparisons on coronal magnetic resonance images revealed no differences in gross structures of the hippocampus between VPA-treated and control ferrets. BrdU-labeled cells were observed throughout the hippocampus of both two groups at PD 20. BrdU-labeled cells were immunopositive for Sox2 (>70%) and almost immunonegative for NeuN, S100 protein, and glial fibrillary acidic protein. BrdU-labeled Sox2-positive progenitors were abundant, particularly in the subgranular layer of the dentate gyrus (DG), and were denser in VPA-treated ferrets. When BrdU-labeled Sox2-positive progenitors were examined at 2 h after the second VPA administration on PD 7, their density in the granular/subgranular layer and hilus of the DG was significantly greater in VPA-treated ferrets compared to controls. The findings suggest that VPA exposure to ferret infants facilitates the proliferation of DG progenitors, supplying excessive progenitors for hippocampal adult neurogenesis to the subgranular layer.

Zebrin II expressing Purkinje cell phenotype-related and -unrelated cerebellar abnormalities in Cav2.1 mutant, rolling mouse Nagoya.

Rolling mouse Nagoya is an ataxic mutant mouse that carries a mutation in a gene encoding for the alpha 1A subunit of the voltage-gated P/Q-type Ca2+ channel (Cav2.1). This report summarizes our studies and others concerning cerebellar abnormalities in rolling mice based on chemical neuroanatomy. While there are no obvious cerebellar deformations in this mutant mouse, the altered functions of Purkinje cells can be revealed as a reduced expression of type 1 ryanodine receptor (RyR1) in all Purkinje cells uniformly throughout the cerebellum, and as an ectopic expression of tyrosine hydroxylase (TH) in the Purkinje cell subsets with the zebrin II-immunopositive phenotype. As the mutated Cav2.1 channel is expressed at uniform levels in all Purkinje cells, its copresence with RyR1 staining suggests that a Cav2.1 channel dysfunction links with the expression of RyR1 in Purkinje cells of rolling mice. However, an ectopic expression of TH in the Purkinje cells is topologically related to the projection of corticotrophin-releasing factor-immunopositive climbing fibers rather than expression of the mutated Cav2.1 channel. On the other hand, increased levels of serotonin (5-HT) in 5-HTergic fibers were revealed immunohistochemically in Purkinje cells of the vermis of rolling cerebellum. Thus, to determine whether or not cerebellar abnormalities are related to Purkinje cell populations revealed by zebrin II expression is essential for enhancing our understanding of the pathogenesis of hereditary cerebellar ataxic mutants such as rolling mice.

Striking pattern of Purkinje cell loss in cerebellum of an ataxic mutant mouse, tottering.

Tottering mouse is an ataxic mutant that carries a mutation in a gene encoding for the apha1A subunit of P/Q-type Ca2+ channel (Cav2.1). This study revisited to examine whether a Purkinje cell loss occurred in the cerebellum of tottering mice. In tottering mice, Calbindin D-28k negative gaps were apparent in the vermis but not in the hemisphere. Calbindin D-28k immunofluorescence with DAPI staining demonstrated the absence of Purkinje cells in the Calbindin D-28k negative gaps. The Purkinje cell loss seemed to be observed prominently in the zebrin II negative compartments of the anterior vermis, but in the zebrin II positive compartments of the posterior vermis. Quite consistent with the histopathological observations, quantitation of the density of Calbindin D-28k and zebrin II immunopositive Purkinje cells in the tottering cerebellum revealed that the Purkinje cells were selectively lost in the zebrin II immunonegative compartments of the lobules I and II but in the zebrin II immunopositive compartments in the lobule IX. Those results predict that the susceptibility to the Cav2.1 gene defect is different among Purkinje cell phenotypes of the tottering cerebellum rather than the expression pattern of mutated Cav2.1 channels. This may result in the reproducible parasagittal pattern of Purkinje cell loss.

Follow-up study of subventricular zone progenitors with multiple rounds of cell division during sulcogyrogenesis in the ferret cerebral cortex.

The subventricular zone (SVZ) of the developing cerebral cortex appears transiently during cortical neurogenesis and is known as the second proliferative zone that contains intermediate progenitor cells and self-renewable neuronal stem cells-the so-called basal radial glia (bRG). The present study attempted to track the differentiation and migration dynamics of SVZ progenitors undergoing multiple cell divisions at the late stage of neurogenesis in a course of sulcogyrogenesis in the ferret, a gyrencephalic mammal. Ferret pups were given a 5-ethynyl-2'-deoxyuridine (EdU) injection on postnatal day (PD) 5 followed by a 5-bromo-2'-deoxyuridine (BrdU) injection on PD 7. The 48 h interval between EdU and BrdU injections covered the minimum times for the first and second S-phase of self-renewing bRG. Two h after BrdU injection, EdU/BrdU-double labeled cells were found in the inner or outer SVZ (iSVZ and oSVZ), more than 80% of which were Sox2-positive. Furthermore, 95.8% of EdU/BrdU-double labeled Sox2-positive progenitors in the iSVZ and 84.2% in the oSVZ were also Pax6-positive, defining these progenitors as bRG. On PD 20, all EdU/BrdU-double labeled cells were NeuN-immunopositive, and more than 60% of these were parvalbumin-immunopositive. EdU/BrdU-double labeled neurons were distributed densely in the superficial portion of the outer cortical stratum. Cluster analysis divided the gyral and sulcal regions into higher and lower density groups, respectively, based on the diversity of the cortical density of EdU/BrdU-double labeled neurons. The higher density group included the gyral and sulcal regions of the prefrontal, parietooccipital and/or cingulate cortex, corresponding to cortical regions associated with evolutionary expansion. Although a limited population of neurons within a narrow time window of cortical neurogenesis was tracked, the present findings suggest that neurons derived from bRG at the late stage of neurogenesis express parvalbumin during corticohistogenesis. Due to the diversity of sulcogyral distributions, neurons derived from bRG may be implicated in evolutionary cortical expansion.

Spatial distribution of corticotropin-releasing factor immunopositive climbing fibers in the mouse cerebellum: analysis by whole mount immunohistochemistry.

This study examined the spatial organization of corticotropin-releasing factor (CRF) immunopositive climbing fibers in the mouse cerebellum by whole mount immunohistochemistry. A striking pattern of parasagittal stripes of CRF staining was revealed. Cryosections of whole mount CRF stained cerebellum showed that anti-CRF immunostaining is restricted to climbing fibers in the molecular layer and does not penetrate deeper into the granular layer. The array of CRF stripes was reminiscent of zebrin II immunopositive Purkinje cell stripes in the anterior vermis and the hemispherical lobules. However, a direct comparison of the two distributions showed that the CRF-defined parasagittal stripes and transverse zones in the posterior vermis are different from those defined by the expression of zebrin II: in particular, CRF immunostaining revealed a transverse boundary between lobules VIb and VII and the presence of four CRF-immunopositive climbing fiber stripes in lobule VIII. Furthermore, an array of CRF stripes was seen in lobule X, the flocculus and the paraflocculus, despite uniform zebrin II expression in these areas. In these cases some, but not all, CRF-immunopositive stripes shared boundaries with Purkinje cell stripes that were visualized by the expression of heat shock protein 25. The results reveal a reproducible pattern of CRF-immunopositive climbing fiber innervation in the mouse cerebellum that bears a complex relationship to the stripes delineated by Purkinje cell compartmentation antigens.

Prenatal Irradiation-Induced Hippocampal Abnormalities in Rats Evaluated Using Manganese-Enhanced MRI.

The aim of this study was to characterize hippocampal abnormalities in rats after prenatal x-ray irradiation using manganese-enhanced MRI (MEMRI). All radiation-exposed rat brains showed a reduced volume with prominent dilatation of lateral ventricles. Moreover, MEMRI-enhanced areas within the hippocampus were reduced in volumes by approximately 25% of controls, although the entire volume of hippocampus was decreased by approximately 50% of controls. MEMRI signals were enhanced strongly in the hilus and granular layer of the dentate gyrus (DG) and the pyramidal layer and infrapyramidal region of the CA3 region, and moderately along the CA1/2 pyramidal cell layer in the control rats. In radiation-exposed rats, MEMRI signals in the CA1/2 regions disappeared due to disrupting their laminar organization, although strong MEMRI signals were sustained in the DG and CA3 regions. Histopathological examinations in radiation-exposed rats revealed disorganizations of the DG granule cell layer and the CA3 pyramidal cell layer with reducing the cell density. The CA1/2 pyramidal cell layer was disrupted by invading ectopic cell mass. Neural cell adhesion molecule (NCAM)-positive fiber bundles were sustained in radiation-exposed rats, although they distributed aberrantly in the suprapyramidal CA3 region with a slight reduction of NCAM staining. Furthermore, glial components consisted largely by astrocytes and minor by microglia were densely distributed in the DG rather than in other hippocampal regions, and their density radiation-exposed rats. In conclusion, MEMRI signal enhancements could delineate different neuronal and/or glial components among hippocampal regions. We characterized microstructures of the deformed hippocampus as well as its macrostructures in a prenatally radiation-exposed rat model using in vivo MEMRI. The present findings provide advantageous information for detecting nondestructively hippocampal deformations in neurodevelopmental disorders.

Isotropic 25-Micron 3D Neuroimaging Using ex vivo Microstructural Manganese-Enhanced MRI (MEMRI).

MRI observations following in vivo administration of Mn2+ [manganese (Mn)-enhanced MRI, MEMRI] have been used as an excellent morphological and functional MRI tool for in vivo preclinical studies. To detect brain three-dimensional (3D) microstructures, we improved the ex vivo MEMRI method for mouse brains after in vivo Mn administration and obtained high-resolution MRIs using a cryogenic radiofrequency (RF) coil. Male C57BL/6 mice (n = 8) were injected with 50 mM MnCl2 intravenously and MEMRIs of the brain were acquired in vivo after 24 h, followed by perfusion fixation with a 4% paraformaldehyde (PFA) solution. High-resolution 25-µm isotropic MRIs were successfully acquired from the extracted brain tissue and could identify the brain microstructures, especially in the hippocampus [the pyramidal cell layer through CA1-3 and the dentate gyrus (DG) granular layers (GLs)], cell layers of cerebellum, three sub-regions of the deep cerebellar nucleus, and white matter (WM) structures [e.g., the fasciculus retroflexus (fr) and optic tract in the thalamus]. The following technical conditions were also examined: (i) the longitudinal stability of Mn-enhanced ex vivo tissue after in vivo administration; and (ii) the effects of mixing glutaraldehyde (GA) with the fixative solution for the preservation of in vivo MEMRI contrast. Our results indicate that ex vivo MEMRI observations made shortly after fixation maintain the contrast observed in vivo. This research will be useful for non-destructive whole-brain pathological analysis.

Spatial learning and expression of neural cell adhesion molecule L1 in rats X-irradiated prenatally.

The present study was designed to present evidence to clarify the relationships between learning ability, neuronal cell adhesion molecule L1 expression and hippocampal structural changes in the rat model received X-irradiation at an embryonic stage (E15). Water maze task indicated that all of the irradiated rats failed to learn the task in the whole training procedure. Their latency to the platform and swimming distance were significant differences from those sham-treated controls. Histological studies showed that the hippocampal ectopias induced by X-rays in the CA1 were involved in the spatial learning impairment, in which they hampered normal processes in learning development and transmission of information. Number, size and positions of the ectopias in the dorsal parts of the hippocampus were confirmed to be related to degrees of spatial learning impairment. On the other hand, L1 expression in the hippocampus was examined with Western blot analysis. The results indicated a lower content of L1 in the irradiated rats. A decrease in L1 might be one of reasons to cause disorganization of the septohippocampal pathways. These findings suggest some mechanisms of spatial learning impairment can be attributed to the formation of the hippocampal ectopias and redaction of L1 following prenatal exposure to X-irradiation.