In vivo imaging of neural circuit formation in the neonatal mouse barrel cortex

Higher brain function in mammals primarily relies on complex yet sophisticated neuronal circuits in the neocortex. In early developmental stages, neocortical circuits are coarse. Mostly postnatally, the circuits are reorganized to establish mature precise connectivity, in an activity-dependent manner. These connections underlie adult brain function. The rodent somatosensory cortex (barrel cortex) contains a barrel map in layer 4 (L4) and has been considered an ideal model for the study of postnatal neuronal circuit formation since the first report of barrels in 1970. Recently, two-photon microscopy has been used for analyses of neuronal circuit formation in the mammalian brain during early postnatal development. These studies have further highlighted the mouse barrel cortex as an ideal model. In particular, the unique dendritic projection pattern of barrel cortex L4 spiny stellate neurons (barrel neurons) is key for the precise one-to-one functional relationship between whiskers and barrels and thus an important target of studies. In this article, I will review the morphological aspects of postnatal development of neocortical circuits revealed by recent two-photon in vivo imaging studies of the mouse barrel cortex and other related works. The focus of this review will be on barrel neuron dendritic refinement during neonatal development.     

Subbarrel Patterns in Somatosensory Cortical Barrels Can Emerge from Local Dynamic Instabilities

Complex spatial patterning, common in the brain as well as in other biological systems, can emerge as a result of dynamic interactions that occur locally within developing structures. In the rodent somatosensory cortex, groups of neurons called “barrels” correspond to individual whiskers on the contralateral face. Barrels themselves often contain subbarrels organized into one of a few characteristic patterns. Here we demonstrate that similar patterns can be simulated by means of local growth-promoting and growth-retarding interactions within the circular domains of single barrels. The model correctly predicts that larger barrels contain more spatially complex subbarrel patterns, suggesting that the development of barrels and of the patterns within them may be understood in terms of some relatively simple dynamic processes. We also simulate the full nonlinear equations to demonstrate the predictive value of our linear analysis. Finally, we show that the pattern formation is robust with respect to the geometry of the barrel by simulating patterns on a realistically shaped barrel domain. This work shows how simple pattern forming mechanisms can explain neural wiring both qualitatively and quantitatively even in complex and irregular domains.     

Intrinsic and extrinsic regulators of developmental timing: from miRNAs to nutritional cues

A fundamental challenge in biology is to understand the reproducibility of developmental programs between individuals of the same metazoan species. This developmental precision reflects the meticulous integration of temporal control mechanisms with those that specify other aspects of pattern formation, such as spatial and sexual information. The cues that guide these developmental events are largely intrinsic to the organism but can also include extrinsic inputs, such as nutrition or temperature. This review discusses the well-characterized developmental timing mechanism that patterns the C. elegans epidermis. Components of this pathway are conserved, and their links to developmental time control in other species are considered, including the temporal patterning of the fly nervous system. Particular attention is given to the roles of miRNAs in developmental timing and to the emerging mechanisms that link developmental programs to nutritional cues.     

Postnatal Development of Mouse “Whisker” Thalamus: Ventroposterior Medial Nucleus (VPM), Barreloids,and Their Thalamocortical Relay Neurons

We followed developmental changes in “barreloid” thalamocortical relay cell (TCR) dendritic arbors between postnatal day 5 (P5; birth = P0) and adulthood. Single neurons in 150- to 250-μm coronal or oblique slices through the somatosensory thalamus in mice of different postnatal ages were injected with lucifer yellow (LY) under direct visualization. Filled cells in the ventroposterior medial nucleus (VPM) were imaged with a confocal microscope, and rendered and analyzed on a computer workstation with special-purpose software. The whisker representation in the thalamus, as revealed by the pattern of barreloids, was demonstrated by oblique illumination of the slices and/or later cytochrome oxidase (CO) staining. VPM cross-sectional area trebles from P5 to adulthood. Barreloids (single-whisker representations) are well delineated in unstained sections until P10-P11; thereafter, barreloids can only be recognized with difficulty with the CO stain. Thalamocortical relay cell (TCR) somal volumes increase rapidly in the first 2 weeks. The number of primary dendrites does not change, nor does the length of the primary dendritic segments, from P5 to adulthood; however, distal dendritic segments elongate and increase in number. Dendritic arbors are confined on P5 to single barreloids; in adults they extend to adjacent barreloids. The postnatal transformation of dendritic arbors by process growth to adjacent barreloids is mainly completed by P18. A change in the developmental role of these cells, from instructing whisker pattern formation to integrating sensory information from more than one whisker, thus occurs after the whisker pattern in the barrel cortex is established. It coincides with the age at which animals are known to begin exploratory whisking behaviors. The mechanism appears to be by growth and remodeling of distal dendrites rather than by oriented growth and regression, as has been reported for stellate cells in cortical whisker barrels     

Barrels III: Proceedings of a Satellite Symposium of the 1990 Society for Neuroscience Meeting

On October 27 and 28, 1990, approximately 100 somatosensory neurobiologists met in St. Louis, Missouri to discuss the current state of inquiry into the organization of the somatosensory system, emphasizing those portions of the system devoted to processing of inputs from digitized cutaneous organs, such as the rodent mystacial vibrissae. Given the homeomorphic relationship between the vibrissae and cortical and subcortical barrels, a large number of laboratories now employ this model to ask fundamental questions about central processing of sensory inputs, mechanisms controlling topographic pattern formation, and substrates for injury-induced neuronal reorganization. The focus of the third annual Barrels Symposium (Barrels III) was on behavioral aspects of the whisker sense, cholinergic regulation of cortical modules, and genetic and peripheral determinants of barrel development.     

Temporal control of self‐organized pattern formation without morphogen gradients in bacteria

Diverse mechanisms have been proposed to explain biological pattern formation. Regardless of their specific molecular interactions, the majority of these mechanisms require morphogen gradients as the spatial cue, which are either predefined or generated as a part of the patterning process. However, using Escherichia coli programmed by a synthetic gene circuit, we demonstrate here the generation of robust, self‐organized ring patterns of gene expression in the absence of an apparent morphogen gradient. Instead of being a spatial cue, the morphogen serves as a timing cue to trigger the formation and maintenance of the ring patterns. The timing mechanism enables the system to sense the domain size of the environment and generate patterns that scale accordingly. Our work defines a novel mechanism of pattern formation that has implications for understanding natural developmental processes.     

BDNF and trkB mRNA Expression in Neurons of the Neonatal Mouse Barrel Field Cortex: Normal Development and Plasticity after Cauterizing Facial Vibrissae

Development of the central somatosensory system is profoundly modulated by the sensory periphery. Cauterization of facial whiskers alters the segregation pattern of barrels in rodents only during a few days just after birth (critical period). Although a molecular basis of the segregation of barrel neurons and the critical period for the anatomical plasticity observed in layer IV barrel neuron is not clear yet, the accumulating evidence suggests that neurotrophins modulate synaptic connections including central nervous system. In this study, we showed by in situ hybridization that mouse barrel side neurons express brain-derived neurotrophic factor (BDNF) mRNA and both catalytic and non-catalytic forms of trkB mRNA. Cautery of row C vibrissae on the right side of the face within 24 h after birth (post natal day 0, PND0) reduced the expression of BDNF and trkB mRNA from the division region between the contralateral row C barrels at PND7. The vibrissae in row A, C, and E were cauterized at PND0 followed by quantitative RT-PCR for BDNF and trkB mRNA with total RNA isolated from the barrel region at PND7. The result showed that BDNF, but not trkB, mRNA was increased several-fold in the contralateral barrel region. These data suggest that the expression of BDNF mRNA is differentially regulated between injured barrels and actively innervated barrels. The differential expression of the mRNA encoding neurotrophins and their receptors may be important in regulating the injury-dependent re-segregation of barrels.     

Boundaries defined by adhesion molecules during development of the cerebral cortex: the J1/tenascin glycoprotein in the mouse somatosensory cortical barrel field

The distribution of the 200/220 KDa J1 glycoprotein (J1-200/220), within the developing vibrissae-related barrel field of the mouse somatosensory cortex, was studied by immunocytochemistry using a monoclonal antibody. J1-200/220, a member of the L2/HNK-1 family of adhesion molecules, also appears to be the mouse homologue of tenascin. J1/tenascin-positive barrel-like structures are visible in the somatosensory cortex between 24 and 48 hr after birth, with the molecule present in prospective barrel boundaries. Immunoelectronmicroscopy reveals labeling that is associated with glial and neuronal plasma membranes, as well as glial end-feet on blood vessels. A possible major source of J1/tenascin expression at this time is astrocyte precursor cells and radial glia. In the putative astrocyte precursor cells, immunolabeling was observed within organelles including the Golgi apparatus. At P6-7 J1/tenascin is most prevalent within prospective interbarrel septae. J1/tenascin-positive barrel boundaries are barely visible on P9 and not observed on P16. The findings indicate that J1/tenascin represents a major component of previously described "hidden" boundaries that we have seen during development using other methodologies. The expression of adhesion molecule-rich boundaries during the critical stages of barrel field formation indicates roles for such molecules during specific cerebral cortical pattern formation events.     

What Choline Metabolism Can Tell Us About the Underlying Mechanisms of Fetal Alcohol Spectrum Disorders

The consequences of fetal exposure to alcohol are very diverse and the likely molecular mechanisms involved must be able to explain how so many developmental processes could go awry. If pregnant rat dams are fed alcohol, their pups develop abnormalities characteristic of fetal alcohol spectrum disorders (FASD), but if these rat dams were also treated with choline, the effects from ethanol were attenuated in their pups. Choline is an essential nutrient in humans, and is an important methyl group donor. Alcohol exposure disturbs the metabolism of choline and other methyl donors. Availability of choline during gestation directly influences epigenetic marks on DNA and histones, and alters gene expression needed for normal neural and endothelial progenitor cell proliferation. Maternal diets low in choline alter development of the mouse hippocampus, and decrement memory for life. Women eating low-choline diets have an increased risk of having an infant with a neural tube or orofacial cleft birth defect. Thus, the varied effects of choline could affect the expression of FASD, and studies on choline might shed some light on the underlying molecular mechanisms responsible for FASD.     

Cytotactin and its proteoglycan ligand mark structural and functional boundaries in somatosensory cortex of the early postnatal mouse

The expression of the extracellular matrix molecules cytotactin, which is synthesized by glia, and cytotactin-binding (CTB) proteoglycan, which is synthesized by neurons, was examined in the developing brain of the mouse, specifically in the cortical barrel field, using highly specific polyclonal antibodies to the purified molecules. Both molecules appeared early in the development of the cortex but were excluded from the centers of the developing barrels at the time of entry and arborization of thalamocortical axons. Of the two major forms of cytotactin (220 and 200 kDa), the larger form predominated during development of the mouse brain and also predominated in mixed neuron-glia cultures but not in pure glial cultures. Both cytotactin and CTB proteoglycan were recognized by various lectins that have been shown in other studies to demarcate the barrel field: both molecules were recognized by lentil lectin and concanavalin A and CTB proteoglycan was also recognized by peanut and wheat germ agglutinins. The HNK-1 carbohydrate antigen, present on cytotactin, CTB proteoglycan, and other adhesion molecules, was also found in the barrel walls and diminished in the barrel hollows. Cytotactin and CTB proteoglycan were preferentially expressed in barrel walls through P12. After this time, their expression became uniform even though the histological pattern of barrel walls and hollows was maintained. The fusion of a row of barrels which results from peripheral damage to a row of whiskers was accompanied by the loss of patterned expression of both molecules following electrocauterization of a row of whisker follicles at P1.5. We conclude that activity from the periphery is important not only to development of anatomical pattern but also of the molecular pattern and that the expression of both glial and neuronal proteins can respond to such activity. The results are consistent with previous studies showing that incoming thalamocortical axons play a primary role in barrel field formation. They also suggest that both the migration of cortical neurons on glia and the refinement of the mapping between the peripheral whisker field and its cortical representation may depend upon the distribution of substrate adhesion molecules.     

Development and pathology of the hyaloid, choroidal and retinal vasculature

During embryogenesis, the development and differentiation of the eye requires the concomitant formation of the neural/glial elements along with a dense vascular network. The adult neural retina is supported by two distinct vascular systems, the proper retinal vessels and the choroidal vessels. The two beds differ not only in their pattern of embryonic differentiation, but also in their function in the adult organism. The retinal vasculature has barrier properties similar to those observed in the brain, whereas the choroidal vessels display a highly fenestrated phenotype. The hyaloid vasculature is a transient embryonic vascular bed which is complete at birth in mammals and regresses contemporaneously with the formation of the retinal vasculature. The dependence of the retina on its blood supply makes it highly vulnerable to any vascular changes and indeed ocular diseases, such as proliferative retinopathy, age-related macular degeneration and the hyperplastic primary vitreous, which are associated with abnormalities of the different vascular beds of the eye. A number of factors have been implicated in developmental and pathological changes in vessel formation and regression, including fibroblast growth factors, platelet-derived endothelial growth factor and vascular endothelial growth factor, among others. The purpose of this review is to describe and discuss new insights into the mechanisms and molecular cues involved in the development of the normal and pathological vascular systems of the eye. The characterization of the molecules and cell-cell interactions involved in the formation, stabilization and regression of new vessels has led to the identification of potential control points for therapeutic intervention.     

A temporospatial map of adhesive molecules in the organ of corti of the mouse cochlea

In the nervous system, several classes of cell-surface and extracellular matrix molecules have been implicated in processes such as neural growth, fasciculation, pathfinding, target recognition and synaptogenesis, which require cell-to-cell or cell-to-substrate binding. In the developing mouse cochlea, little is known about the types of cell-surface and extracellular matrix molecules existing along the neural growth paths or their possible roles in development. Whole mount and sectioned cochlear tissue were immunolabeled for six different adhesive molecules - neural cell adhesion molecule (NCAM), polysialic acid (PSA), neural cell adhesion molecule L1, E-cadherin, syndecan-1 and tenascin-C. A temporospatial map of adhesive molecule distribution in the basal turns of the mouse cochlea was generated. Distributions of adhesive molecules were compared to each other and to the known progress of neural development in the region. This comparison demonstrated differences in the complements of adhesive molecules between the inner and outer hair cell regions, and variations in the expressions of adhesion molecules among different types of nerve fibers. In addition, developmental changes in the adhesive environment around and beneath the outer hair cells coincided with the known timing of the appearance of morphologically defined efferent synapses. These observations raise the possibility that molecular differences at the cell surface of inner and outer hair cells are one way that ingrowing neurites distinguish different environments to determine their growth routes and synaptic partners in the cochlea. In addition these observations demonstrate the potential for differential signaling of afferent and efferent innervation by altering the microenvironments in which synapses are formed.     

Patterning of the embryo: the first spatial decisions in the life of a mouse

Although in most species the polarity of the embryo takes its roots from the spatial patterning of the egg, mammals were viewed as an exception. This was because the anteroposterior polarity of the mouse embryo could not be seen until gastrulation, and no developmental cues were known that could define polarity at earlier stages. Why should we now re-consider this view? While mechanisms of axis formation in mammals could, in principle, be unique, the evolutionary conservation of numerous other developmental processes raises the question of why mammals would have evolved a different way or timing of organising their embryonic polarity. Indeed, recent evidence shows that well before the onset of gastrulation, the mouse embryo initiates asymmetric patterns of gene expression in its visceral endoderm. Although this extra-embryonic tissue does not contribute to the body itself, it is involved in axis formation. Other recent work has revealed that spatial distribution of cells in the visceral endoderm can be traced back to polarity present at the blastocyst stage. These insights have raised the possibility that embryonic polarity might also originate early during development of mammalian embryos. Indeed it now appears that there are at least two spatial cues that operate in the mouse egg to shape polarity of the blastocyst. One of these is at the animal pole, which is defined by the site of female meiosis, and another is associated with the position of sperm entry. In this review I discuss these recent findings, which have led to the recognition that mouse embryos initiate development of their polarity at the earliest stages of their life. This novel perspective raises questions about the nature of cellular and molecular mechanisms that could convert developmental cues in the zygote to axes of the blastocyst, and hence into polarity of the post-implantation embryo. It also brings to light the need to understand how such mechanisms could enable early mouse development to be so regulative.     

Expression of the cystathionine beta synthase (CBS) gene during mouse development and immunolocalization in adult brain.

Hyperhomocysteinemia, caused by a lack of cystathionine beta synthase (CBS), leads to elevated plasma concentrations of homocysteine. This is a common risk factor for atherosclerosis, stroke, and possibly neurodegenerative diseases. However, the mechanisms that link hyperhomocysteinemia due to CBS deficiency to these diseases are still unknown. Early biochemical studies describe developmental and adult patterns of transsulfuration and CBS expression in a variety of species. However, there is incomplete knowledge about the regional and cellular expression pattern of CBS, notably in the brain. To complete the previous data, we used in situ hybridization and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. In the early stages of development, the Cbs gene was expressed only in the liver and in the skeletal, cardiac, and nervous systems. The expression declined in the nervous system in the late embryonic stages, whereas it increased in the brain after birth, peaking during cerebellar development. In the adult brain, expression was strongest in the Purkinje cell layer and in the hippocampus. Immunohistochemical analyses showed that the CBS protein was localized in most areas of the brain but predominantly in the cell bodies and neuronal processes of Purkinje cells and Ammon's horn neurons.     

Localization of the Extracellular Matrix Protein SC1 to Synapses in the Adult Rat Brain

Extracellular matrix molecules play important roles in neural developmental processes such as axon guidance and synaptogenesis. When development is complete, many of these molecules are down-regulated, however the molecules that remain highly expressed are often involved in modulation of synaptic function. SC1 is an example of an extracellular matrix protein whose expression remains high in the adult rat brain. Confocal microscopy revealed that SC1 demonstrates a punctate pattern in synaptic enriched regions of the cerebral cortex and cerebellum. Higher resolution analysis using electron microscopy indicated that SC1 localizes to synapses, particularly the postsynaptic terminal. SC1 was also detected in perisynaptic glial processes that envelop synapses. This work was supported by the National Science and Engineering Research Council of Canada.     

THE EFFECTS OF HYPERKETONEMIA ON GLYCOLYTIC INTERMEDIATES IN THE DEVELOPING RAT BRAIN

Abstract— Newborn rats from dams fed on a high fat diet developed increased ketonemia and significant hypertriglyceridemia i.e. "hyperketonemic pups". This perinatal metabolic stress led to an alteration in the developmental pattern of glycolytic intermediates in their brains.
In control rats, the concentration of glucose 6-phosphate (G6P) in the brain was high at birth, and gradually decreased to adult values by the third week of life. In contrast, the fructose-1,6-diphosphate (FDP) concentration was low at birth and increased thereafter. The lactate concentration was also high at birth but decreased to the adult level by the first day of life. In the brains of control pups, lactate and pyruvate concentrations remained relatively constant during the first 3 weeks of life.
In the brains of hyperketonemic pups, the concentration of G6P was the same as in the control animals at birth but decreased significantly during the first days of life. During early development the concentrations of FDP and pyruvate were significantly lower and the concentration of lactate, higher in the hyperketonemic pups as compared to the control group. The alteration in the concentration of these glycolytic intermediates in the brains of hyperketonemic pups indicated a change in the developmental pattern of glycolysis. The ratio of [lactate]/[pyruvate] also suggested an increased cytoplasmic redox potential in the brains of hyperketonemic pups during the first week of life.