Zika virus impairs the development of blood vessels in a mouse model of congenital infection.

Zika virus (ZIKV) is associated with brain development abnormalities such as primary microcephaly, a severe reduction in brain growth. Here we demonstrated in vivo the impact of congenital ZIKV infection in blood vessel development, a crucial step in organogenesis. ZIKV was injected intravenously in the pregnant type 2 interferon (IFN)-deficient mouse at embryonic day (E) 12.5. The embryos were collected at E15.5 and postnatal day (P)2. Immunohistochemistry for cortical progenitors and neuronal markers at E15.5 showed the reduction of both populations as a result of ZIKV infection. Using confocal 3D imaging, we found that ZIKV infected brain sections displayed a reduction in the vasculature density and vessel branching compared to mocks at E15.5; altogether, cortical vessels presented a comparatively immature pattern in the infected tissue. These impaired vascular patterns were also apparent in the placenta and retina. Moreover, proteomic analysis has shown that angiogenesis proteins are deregulated in the infected brains compared to controls. At P2, the cortical size and brain weight were reduced in comparison to mock-infected animals. In sum, our results indicate that ZIKV impairs angiogenesis in addition to neurogenesis during development. The vasculature defects represent a limitation for general brain growth but also could regulate neurogenesis directly.

Callosal axon arbors in the limb representations of the somatosensory cortex (SI) in the agouti (Dasyprocta primnolopha).

The present report compares the morphology of callosal axon arbors projecting from and to the hind- or forelimb representations in the primary somatosensory cortex (SI) of the agouti (Dasyprocta primnolopha), a large, lisencephlic Brazilian rodent that uses forelimb coordination for feeding. Callosal axons were labeled after single pressure (n = 6) or iontophoretic injections (n = 2) of the neuronal tracer biotinylated dextran amine (BDA, 10 kD), either into the hind- (n = 4) or forelimb (n = 4) representations of SI, as identified by electrophysiological recording. Sixty-nine labeled axon fragments located across all layers of contralateral SI representations of the hindlimb (n = 35) and forelimb (n = 34) were analyzed. Quantitative morphometric features such as densities of branching points and boutons, segments length, branching angles, and terminal field areas were measured. Cluster analysis of these values revealed the existence of two types of axon terminals: Type I (46.4%), less branched and more widespread, and Type II (53.6%), more branched and compact. Both axon types were asymmetrically distributed; Type I axonal fragments being more frequent in hindlimb (71.9%) vs. forelimb (28.13%) representation, while most of Type II axonal arbors were found in the forelimb representation (67.56%). We concluded that the sets of callosal axon connecting fore- and hindlimb regions in SI are morphometrically distinct from each other. As callosal projections in somatosensory and motor cortices seem to be essential for bimanual interaction, we suggest that the morphological specialization of callosal axons in SI of the agouti may be correlated with this particular function.

The organizational variability of the rodent somatosensory cortex.

Rodentia is the largest mammalian order, with more than 2,000 species displaying a great diversity of morphological characteristics and living in different ecological niches (terrestrial, semi-aquatic, arboreal and fossorial). Analysis of the organization of the somatosensory areas in six species of rodents allowed us to demonstrate that although these species share a similar neocortical blueprint with other eutherian mammals, important differences exist between homologous areas across different species, probably as a function of both lifestyle and peripheral sensory specializations typical of each species. We based this generalization on a phylogenetic comparison of the intrinsic organization of the primary somatosensory area (SI) across representatives of different rodent suborders. This analysis revealed considerable structural variability, including the differential expansion of cortical representation of specific body parts (cortical amplification) as well as the parcellation of areas into processing modules.

The ontogenesis of the forebrain commissures and the determination of brain asymmetries.

We have reviewed the organization and development of the interhemispheric projections through the forebrain commissures, especially those of the CC, in connection with the development of brain asymmetries. Analyzing the available data, we conclude that the developing CC plays an important role in the ontogenesis of brain asymmetries. We have extended a previous hypothesis that the rodent CC may exert a stabilizing effect over the unstable populational asymmetries of cortical size and shape, and that it participates in the developmental stabilization of lateralized motor behaviors.

Neuroanatomical effects of neonatal transection of the corpus callosum in hamsters.

Newborn hamsters were subjected to surgical transection of their corpora callosa under hypothermic anesthesia. After completion of their development, one group of animals had their brains prepared for cyto- and myeloarchitectonic analysis. Another group had a small pellet of polyacrylamide gel containing horseradish peroxidase (HRP) implanted in different cortical loci. All were perfused with fixatives and had their brains cut into serial sections. The operated brains showed the following anatomical features: (1) The corpus callosum was partially or totally absent; (2) an abnormal longitudinal bundle was present bilaterally underneath the white matter; and (3) except for the physical displacement of some medial structures, the general architecture of the brain appeared unchanged. Analysis of HRP material revealed that (1) the longitudinal bundle contains cortical fibers, of which at least some are commissural; (2) these cortical fibers display a topographic arrangement within the bundle. Results suggest that brain anatomy of "surgical" acallosal hamsters compares closely with that observed in mice with congenital defects of the corpus callosum, a spontaneous condition which also occurs in humans.

The organization of subcortical projections of the hamster's visual cortex.

The subcortical projections of the hamster's visual cortex were determined by use of injections of tritiated proline and heat lesions placed in different cortical loci. The brains were processed for autoradiography and silver impregnation of degenerating axons. Striate cortex was shown to project ipsilaterally to the dorsocaudal region of the caudate nucleus, a dorsolateral area within the thalamic reticular nucleus (RT), a laterodorsal region of the nucleus lateralis anterior (LA), the rostral half of nucleus lateralis posterior (LP), the whole territory of the dorsal (dLGN) and ventral (vLGN) geniculate nuclei, the anterior (PA) and posterior (PP) pretectal nuclei, the superior colliculus (SC), and the precerebellar pontine nuclei. In addition, the medial visual area (18b) was shown to project to a medial band of LA and part of the caudal half of LP, while the adjoining parietal cortex was seen to terminate in a lateral part of the caudate, a ventral band of LA, and the ventral half of rostral LP. Segregation of different cortical inputs was clear in LA, LP, caudate, and pons. The projections to dLGN, vLGN, SC, LP, and PA were retinotopically organized. Clear evidence of some topography was found within RT, PP, and the pons, although a consistent map could not be derived from the data.

Effects of prenatal irradiation on the development of cerebral cortex and corpus callosum of the mouse.

Defects of the cerebral cortex and corpus callosum of mice subjected prenatally to gamma irradiation were evaluated as a function of dose and of embryonic age at irradiation. Pregnant mice were exposed to a gamma source at 16, 17, and 19 days of gestation (E16, E17, and E19, respectively), with total doses of 2 Gy and 3 Gy, in order to produce brain defects on their progeny. At 60 postnatal days, the brains of the offspring were analyzed qualitatively and quantitatively and compared with those of nonirradiated animals. Mice irradiated at E16 were all acallosal. Those that were exposed to 2 Gy displayed an aberrant longitudinal bundle typical of other acallosals, but this was not the case in those irradiated with 3 Gy. The corpus callosum of animals irradiated at E17 with 3 Gy was pronouncedly hypotrophic, but milder effects were observed in the other groups. Quantitative analysis confirmed a dependence of callosal midsagittal area upon dose and age at irradiation, and, in addition, indicated an interaction between these variables. The neocortex of irradiated animals was hypotrophic: layers II-III were much more affected than layer V, and this was more affected than layer VI. Quantitative analysis indicated that this effect also depended on dose and age at irradiation and that it was due to a loss of cortical neurons. Furthermore, a positive correlation was found between the number of neurons within layers II-III, and V and the midsagittal area of the corpus callosum. Ectopic neurons were found in the white matter and in layer I of animals irradiated at E16 and E17, indicating that fetal exposure to ionizing radiation interfered with the migration of cortical neuroblasts.

Callosal neurons in the cingulate cortical plate and subplate of human fetuses.

Given the scarcity of data on the development of the cerebral cortex and its connections in man, four brains of human fetuses at 25, 26, 30, and 32 weeks postovulation were used to investigate the following: 1) the radial distribution of callosal neurons in the cingulate cortex at the immediate postmigratory period; 2) the existence of callosally projecting neurons in the cortical subplate; and 3) the dendritic morphology of developing callosal neurons. The carbocyanine dye (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) (DiI) was used as a fluorescent postmortem tracer for the identification and morphological description of callosal neurons, 4-6 months after the insertion of DiI crystals at the callosal midplane. Sixty-one completely labeled neurons were selected for microscopical analysis, drawn by use of a camera lucida and photographed. The main findings were the following: 1) the human cingulate cortex at 25-32 weeks postovulation contains callosally projecting neurons both in the cortical plate and in the subplate; 2) callosal cells in the plate are mostly spiny pyramids with somata distributed uniformly throughout the depth of the plate, irrespective of rostrocaudal position. They have well-differentiated basal dendrites and apical dendrites that consistently ramify within layer 1; 3) subplate callosal cells are smooth neurons of diverse dendritic morphology, distributed widely throughout the subplate depth. They were classified into four cell types according to the dendritic morphology: radially oriented, horizontally oriented, multipolars, and inverted pyramids. These findings extend to the human brain some of the evidence obtained in animals concerning the development of the cerebral cortex, especially those that are relevant to the formation of a transitory circuitry in the subplate.

Molecular tunnels and boundaries for growing axons in the anterior commissure of hamster embryos.

We have analyzed the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), laminin (LN), tenascin (TN), and glial fibrillary acidic protein (GFAP) along the anterior commissure (AC) of hamster embryos (n=175; from embryonic day (E)12 to E16). Frozen sections were cut at different planes from embryonic brains between E12 and E16, treated for immunohistochemistry, and observed under epifluorescence microscopy. During the pre-crossing stage (E12-E13), CSPG was expressed as a sagittal stratum between the interhemispheric fissure and the prospective AC region. TN appeared rostral to the third ventricle and along the medial subventricular zone of the lateral ventricles. LN and FN both presented a faint expression, and GFAP was not detected. Although AC axons started crossing the midline region (E13.5-E14), CSPG, FN, LN, and, much less intensely, GFAP circumscribed the AC bundle, forming a tunnel through which AC fibers elongate. TN was no longer seen at the midplane but remained visible laterally. During the post-crossing stage (E14.5-E16), CSPG and TN were no longer seen at the midline, although both could be observed between the AC limbs, seeming to form boundaries for AC lateral growth. LN and FN were then absent near the AC bundle. During this late stage, GFAP expression became most intense, forming a distinct tunnel around the AC. We have shown that the expression of extracellular matrix molecules and GFAP follow a time- and space-regulated course related to AC development, plausibly representing influential factors for growth and guidance of commissural fibers.

Transient populations of presumptive macrophages in the brain of the developing hamster, as indicated by endocytosis of blood-borne horseradish peroxidase.

During postnatal development, clusters of cells associated with the mononuclear phagocytic system appear within the white matter of rodents and cats. We studied the distribution and morphology of these cells in the hamster's brain during the first 2 weeks after birth. In animals of different ages, horseradish peroxidase was injected into the heart. After 3-6 h survival, the animals were perfused with aldehydes and had their brains removed, cut and reacted. In another series, fixed brain sections from horseradish peroxidase-injected and non-injected animals were reacted for a non-specific esterase expressed by monocytes and macrophages. The horseradish peroxidase reaction-product was seen throughout the nervous tissue at the first postnatal day, appearing more concentrated in certain brain sectors from postnatal day 3 through 10, to finally become restricted to a few regions at postnatal day 16. Horseradish peroxidase-labeled cells appeared in increasing numbers from postnatal day 1 to 8, decreasing thereafter to disappear completely at postnatal day 16. Some labeled cells were roundish or elliptical with few, if any, processes; others had several clearly detectable processes. Horseradish peroxidase-labelled cells formed clusters within the dorsal subventricular zone, dorsal cortical white matter, corpus callosum and several other prosencephalic fiber tracts. The morphology of esterase-reactive cells was less clearly outlined but their distribution and relative density correlated with those of horseradish peroxidase-labeled cells. Also, many horseradish peroxidase-labeled cells were esterase-positive in most clusters. We conclude that (1) some cells in the developing brain selectively endocytose and accumulate blood-borne horseradish peroxidase in their cytoplasm, (2) these cells do not appear to be neurons but a particular cell type associated to the mononuclear phagocytic system and (3) they cluster transiently in particular sectors of the cortical and subcortical white matter during the first 2 weeks after birth.

Neurogenesis and development of callosal and intracortical connections in the hamster.

The developmental time-course of callosal and ipsilateral corticocortical projections was studied in embryonic and postnatal hamsters, from the time of neurogenesis until the appearance of adult patterns. Callosal neurogenesis was determined by combining the incorporation of [3H]thymidine injected on specific embryonic days with retrograde labelling of callosal neurons in the adult animal. The development of both callosal and corticocortical projections was studied by the transport of wheat germ agglutinin conjugated to horseradish peroxidase. Despite a significant radial disperson of postmigratory neurons born on the same day, it was found that the birthdates of callosally-projecting neurons in the frontal cortex were not restricted to a short period of time, but extended between embryonic days 13 and 15. This period covers the neurogenesis of cells in cortical layers III-V. Elongation of callosal axons (and possibly also of corticocortical fibres) started a couple of days before birth in the frontal cortex, and continued through the first postnatal days. After a "waiting period" of a few days, axons from both sets of projections were seen innervating restricted target sectors of the cortex. The zones of origin of these projections were initially exuberant, but were subsequently trimmed to overlap completely with the corresponding terminal fields. It is concluded that callosal and ipsilateral corticocortical projections undergo similar sequences of ontogenetic stages, suggesting that the development of neocortical connectivity as a whole may be governed by one and the same set of rules.

Quality improvement practices in clinical and anatomic pathology services. A College of American Pathologists Q-probes study of the program characteristics and performance in 580 institutions.

Participants of the College of American Pathologists Q-Probes program described their quality improvement practices for clinical and anatomic pathology. In 580 institutions, the median time required for a median of 12 indicators of quality was 40 hours/month, with the number of indicators and the time spent directly dependent on bed size (P = .0001). The overwhelming majority of participants reported benefit from their quality improvement programs in terms of patient outcomes, as a management tool, and for risk management. Six indicators in clinical pathology and four indicators in anatomic pathology were used in more than 75% of laboratories, whereas an additional seven indicators in clinical pathology and five in anatomic pathology were used in more than 50% of laboratories. The authors conclude that quality improvement practices are similar among laboratories, and irrespective of increasing regulatory requirements, pathologists and senior laboratory personnel spend large amounts of time for activities that they believe improve the quality of services rendered.

Bicommissural neurones in the cerebral cortex of developing hamsters.

The trajectory, developmental time course, and origin of callosal fibres that recross through the anterior commissure were studied in developing hamsters, using carbocyanines in fixed brains on different ages. The bicommissural fibres were found in hamsters from E15 through P7, but disappeared after P7. By double labelling it was found that the neurones of origin of these bicommissural fibres were located in the lateral cortex within the region where the callosal zone of origin overlaps that of the anterior commissure. From these experiments, it was concluded that the axons of a group of cells in the lateral cortex of developing rodents are branched and grow transiently through both the callosum and the anterior commissure.

Lateralization of rotational behavior in developing and adult hamsters.

Rotational asymmetries were studied in developing and adult hamsters, and compared to verify if early lateralization is a predictor of the animals' later performance. Animals were divided into two groups: group I (GI) was tested from P46 (P1 = day of birth) to P62, and group II (GII) was tested daily from P2 to P60. They were placed in a cylindric arena for 5 min under video recording, and their 90 degree right and left displacements were counted and normalized. Since adult animals of both groups did not differ significantly in the distribution of asymmetries, the data were pooled together: 38.8% were non-lateralized (NL), 39.4% were right-rotators (RR), and 21.8% were left-rotators (LR). No significant difference was discerned between males and females. Distribution of asymmetries in GII animals between P2 and P10 showed a predominance of lateralized (64.7% RR and 21.5% LR) over NL animals (13.8%). The proportion of pups that maintained their classification into adulthood was only 46%, and the kappa coherence coefficient for these data was only 0.09. We conclude that: (1) most adults are lateralized, RR being more frequent; (2) the proportion of lateralized adults is not significantly altered by early testing; (3) there is no significant difference between males and females; (4) most developing hamsters are lateralized, right-rotators being more frequent; and (5) the animals' early classification is not a good predictor of their preference as adults.

Ontogenesis of lateralized rotational behavior in hamsters: a time series study.

This is a longitudinal study of the postnatal development of lateralized rotational behavior. Hamsters (n = 75) were tested for spontaneous rotational behavior in cylindrical arenas, from P2 (P1 = day of birth) to P60. A daily laterality index was calculated for each animal, of which the averages and standard deviations were used to follow the animals' lateralized behavior. A strong variability between and within animals appeared throughout development, with a tendency to the right side in most animals, which declined after the first postnatal week. No oscillatory cycles were identified. To study patterns of development, the series were divided into four periods and the animals were separated into five groups. The laterality indexes of all four periods were significantly different between the groups. A total of 79% of the animals showed consistent behavior along development: either a preference to one side (20% left, 26% right), or no preference at all (33%). The remaining animals changed preference during development. Only a few animals remained strongly lateralized throughout the 60 days, most of them showing a slight, non-significant preference after P10. Results suggest an ontogenetic decrease in lateralization of this behavior that could in part be explained by the maturation of an interhemispheric regulatory system.

Cortico-cortical connections reorganize in hamsters after neonatal transection of the callosal bridge.

Neonatal hamsters were subjected to transection of the callosal bridge. Later examination of the brains showed complete or partial absence of the corpus callosum and an anomalous bilateral longitudinal bundle of fibers. In addition, aberrant commissural fibers were seen to connect heterotopically the parietal cortex with the contralateral frontal cortex through a callosal remnant over the septum, and the olfactory cortex with the opposite frontal and parietal cortices through the anterior commissure.

Dose-dependent occurrence of the aberrant longitudinal bundle in the brains of mice born acallosal after prenatal gamma irradiation.

Two groups of pregnant mice were gamma-irradiated at the 16th gestation day with doses of 2 or 3 Gy. All litters were born acallosal, but while the 3-Gy mice showed a severely hypotrophic neocortex without the aberrant longitudinal bundle typical of early disconnected rodents, in the 2-Gy group the cortex was less deranged and the aberrant bundle appeared consistently underneath the white matter.

Retinofugal projections in the opossum, An anterograde degeneration and radioautographic study.

A study of anterograde degeneration and anterograde transport was undertaken in the opossum primary optic system in order to clarify several points regarding fiber organization and patterns of terminal fields. Through the radioautographic technique of axon tracing, it was demonstrated that the accessory optic system follows the generalized scheme of Hayhow, consisting of two fascicles the three terminal nuclei.

Receptive field properties of single units in the opossum striate cortex.

On the basis of their trigger-features, 98 units out of 127 recorded in striate cortex of immobilized opossums, under forced breathing of a nitrous oxide/oxygen mixture, were classified into 5 receptive field groups. Group 1 units (20/127) responding to small stationary spots were shown to be made up of regions of opposite response type and mutual antagonism, separate by linear boundaries. The optimal discharge was elicited by a stimulus configuration consisting of rectilinear regions of opposite contrast positioned and oriented in the visual field so as not to elicit antagonism while maximizing the overlap with regions responsive to that contrast. To edges in motion these units were shown to be made up of light and dark discharge centers, the locations of which could not be predicted from the map to stationary spots. In addition to position and orientation, direction was another important stimulus parameter. Group 2 units (34/127) had uniform requirements of stimulus orientation, direction of motion or both, througout the receptive field. Width was rarely a significant variable. Three subgroups were detected: orientation selective, directional selective and orientation-direction sensitive. Group 3 units (18/127) required stopped stimuli. In most instances (14/18) this property was attributed to a suppressive surround with relatively non-specific stimulus requirements. Oriented and non-oriented responsive receptive fields were observed. Group 3 units with no surround (4/18) responded best to properly positioned and oriented wedges, usually of 90 degrees. Group 4 units (24/127) had uniform fields with little stimulus specificity and were often responsive to diffuse light. Although not sensitive to stimulus orientation and direction, motion was frequently a requisite for optimal responses. Group 5 receptive fields (2/127) had concentrically arranged regions of distinct response type which displayed mutual antagonism. No sensitivity to orientation or direction was detected. Twenty-nine units remained unclassified. Other group distinctions were the relatively higher spontaneous activity of group 4 units and the large field sizes encountered among groups 1 and 4 when compared to group 2. Based on their properties and receptive field type distribution, we propose that striate receptive fields in the opossum have a similar organization to those of other mammals.

Myelination of the cerebral commissures of the hamster, as revealed by a monoclonal antibody specific for oligodendrocytes.

Myelination of the cerebral commissures of the hamster was studied by immunostaining with a monoclonal antibody (Rip) specific for oligodendrocytes. Immunostained, preensheathing cells were first observed in the anterior commissure on P6 (P1 = day of birth). By P8, immunopositive oligodendrocytes and myelinated fibers clustered around some of them were detected within the posterior limb of the anterior commissure, ventrally at the rostral half of the callosum, and in the hippocampal commissure. On P12, all the commissures had myelinated fibers throughout their extent, but the callosum and the hippocampal commissure exhibited higher densities of myelinated fibers rostrally. Between P15 and P22, the pattern of myelination approached that of the adult. In the context of other developmental events, myelination of the corpus callosum and of the anterior commissure is a late event, occurring predominantly after stabilization of axon number, either at the end of the progressive accretion of axons, as in the anterior commissure, or after the selective elimination of callosal projections.