Laminar and areal expression of unc5d and its role in cortical cell survival

The UNC-5 family of netrin receptors is known to regulate axon guidance, cell migration, and cell survival. We have previously demonstrated that unc5d, one of the UNC-5 family member genes, is specifically expressed in layer 4 of the developing rat neocortex (Zhong Y, Takemoto M, Fukuda T, Hattori Y, Murakami F, Nakajima D, Nakayama M, Yamamoto N. 2004. Identification of the genes that are expressed in the upper layers of the neocortex. Cereb Cortex. 14:1144-1152). However, the role of UNC5D in cortical development is still unknown. In this study, we revealed that unc5d was highly expressed in the primary sensory areas of the mouse neocortex at around postnatal day 7. Netrin-4 was also found to be predominantly expressed in layer 4 of the sensory cortex and sensory thalamic nuclei. Cell surface binding assay showed that netrin-4 protein bound to UNC5D-expressing cells. An in vitro study further demonstrated that cell death of unc5d-expressing layer 4 cells was reduced by exogenous application of netrin-4 protein, whereas UNC5D is not sufficient to mediate the effect of netrin-4 in deep layer cells. Taken together, these results suggest that UNC5D is primarily expressed by layer 4 cells in the primary sensory areas of the developing neocortex and may mediate the effect of netrin-4 on cortical cell survival in a lamina-specific manner.     

Spaceflight induces changes in the synaptic circuitry of the postnatal developing neocortex

The establishment of the adult pattern of neocortical circuitry depends on various intrinsic and extrinsic factors, whose modification during development can lead to alterations in cortical organization and function. We report the effect of 16 days of spaceflight [Neurolab mission; from postnatal day 14 (P14) to P30] on the neocortical representation of the hindlimb synaptic circuitry in rats. As a result, we show, for the first time, that development in microgravity leads to changes in the number and morphology of cortical synapses in a laminar-specific manner. In the layers II/III and Va, the synaptic cross-sectional lengths were significantly larger in flight animals than in ground control animals. Flight animals also showed significantly lower synaptic densities in layers II/III, IV and Va. The greatest difference was found in layer II/III, where there was a difference of 344 million synapses per mm(3) (15.6% decrease). Furthermore, after a 4 month period of re-adaptation to terrestrial gravity, some changes disappeared (i.e. the alterations were transient), while conversely, some new differences also appeared. For example, significant differences in synaptic density in layers II/III and Va after re-adaptation were no longer observed, whereas in layer IV the density of synapses increased notably in flight animals (a difference of 185 million synapses per mm(3) or 13.4%). In addition, all the changes observed only affected asymmetrical synapses, which are known to be excitatory. These results indicates that terrestrial gravity is a necessary environmental parameter for normal cortical synaptogenesis. These findings are fundamental in planning future long-term spaceflights.     

Retinol-binding protein gene is highly expressed in higher-order association areas of the primate neocortex

The neocortex consists of histochemically, connectionally, and functionally distinguishable areas. Recently, molecular biological techniques have enabled us to find rare types of genes expressed in specific neocortical areas. We previously reported occ1 gene as preferentially expressed in the primary visual cortex (V1), using the differential display method. Here, by differential display, we found selective and strong expression of the serum retinol-binding protein (RBP) gene, in higher-order association areas. In V1, RBP mRNA was expressed only in the superficial part of layer II, but its expression increased, involving deeper layers, along the visual pathway. In visual association areas such as TE, RBP mRNA was strongly expressed in both supra- and infragranular layers. In primary auditory and somatosensory areas, as in V1, RBP expression was low, and restricted to the upper part of the supragranular layers. The laminar pattern of RBP expression is in marked contrast with that of occ1; and in early visual areas where both genes are expressed, these occur in distinct sublayers within the supragranular layers. In neonatal monkeys, the area-specific expression pattern of RBP was less distinct, suggesting that the characteristic expression of RBP in higher-order association areas is mainly established postnatally.     

Development of Forward and Feedback Connections between Areas V1 and V2 of Human Visual Cortex

We have determined the sequence in which forward connectionsbetween visual cortical areas V1 and V2, and feedback connectionsbetween V2 and V1 develop in humans. For this purpose Dii wasinjected into V1 and V2 of postmortem brains of different pre-and postnatal ages. The laminar distribution of labeled fibersand cell bodies In V1 and V2 Indicates that forward and feedbackconnections emerge shortly before birth. The development ofboth pathways proceeds over several postnatal months such thatthe laminar termination pat tern of forward connections appearsrelatively mature before feedback connections reach their matureform. At 31 weeks of gestation both forward and feedback connectionsoriginate exclusively from deep-layer neu rons, which extendaxons in deep layers only. By 9 d postnatal, forward connectionsfrom V1 to V2, n ad dition to layers 5 and 6, also arise fromneurons in layer 4B of V1. At this stage for the first timeforward fibers enter layer 4 at the topographically appropriatelocation of V2. At 9 d postnatal most feedback fibers from V2still occupy deep layers of Vi but many, through inter stitialgrowth, elaborate vertical sprouts at regular in tervals alongthe length of horizontal axons. As feedback connections mature,distal segments of horizontal axons are pruned beck to branchpoints and fibers assume L-shaped configurations. By 1 weeksof age forward fibers from V1 enter V2 through deep and superficiallayers and provide input to layers 3 and 4. At this stage feedbackfibers from V2 have entered layer 4B of V1. By 4 months of ageforward connections have assumed all the laminar characteristicsof mature connections; that is, they arise from layers 2/3,48, 5, and 6 of V1, and terminate In layers 3 and 4 of V2. Insharp contrast, at 4 months of age feedback connections to V1are still immature, showing terminations In layers 4B, 5, and6 but no input to layer 2/3. The protracted postnatal emergence of feedback con nectionsis similar to that of local long-range connec tions within layer2/3 of V1 (Burkhalter at al., i993). Since both of thsse circuitsare thought to provide in formation about the context in whichobjects are seen, it is interesting to speculate that the lateonset of texture segmentation in infants (Atkinson and Braddick.1992; Sireteanu and Rieth, 1992) may be related to the postnatal maturation of specific Intracortical circuits.     

Cortical structure predicts the pattern of corticocortical connections

Cortical areas are linked through pathways which originate and terminate in specific layers. The factors underlying which layers are involved in specific connections are not well understood. Here we tested whether cortical structure can predict the pattern as well as the relative distribution of projection neurons and axonal terminals in cortical layers, studied with retrograde and anterograde tracers. We used the prefrontal cortices in the rhesus monkey as a model system because their laminar organization varies systematically, ranging from areas that have only three identifiable layers, to those that have six layers. We rated each prefrontal area based on the number and definition of its cortical layers (level 1, lowest; level 5, highest). The structural model accurately predicted the laminar pattern of connections in approximately 80% of the cases. Thus, projection neurons from a higher-level cortex originated mostly in the upper layers and their axons terminated predominantly in the deep layers (4-6) of a lower-level cortex. Conversely, most projection neurons from a lower- level area originated in the deep layers and their axons terminated predominantly in the upper layers (1-3) of a higher-level area. In addition, the structural model accurately predicted that the proportion of projection neurons or axonal terminals in the upper to the deep layers would vary as a function of the number of levels between the connected cortices. The power of this structural model lies in its potential to predict patterns of connections in the human cortex, where invasive procedures are precluded.      

Excitatory amino acid, GABA(A), and GABA(B) binding sites in human striate cortex.

Receptor autoradiography was used to study the laminar distribution of excitatory amino acid, GABA(A), and GABA(B) binding sites in human striate cortex. Binding sites for all these receptor subtypes were found within striate cortex, but there were marked differences in the laminar distribution of binding sites. NMDA binding sites were most dense in layers 1-4C, with highest density in layer 4C and lower levels in layers 5 and 6. Among non-NMDA binding sites, alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid binding sites had highest levels in layers 1-3, intermediate levels in layers 5 and 6, and lowest levels in layers 4B and 4C. Kainate and metabotropic binding sites were more uniformly distributed across cortical laminae, with a trend toward highest kainate binding in layers 5 and 6. GABA(A)/benzodiazepine binding sites had highest levels in layers 2, 3, and 4C, with intermediate levels in 4B and lowest levels in layers 1, 5, and 6. GABA(B) binding sites were uniformly distributed across laminae. There was no evidence of a "columnar" or "blob" pattern of any binding site within any of the laminae. With the exception of kainate, metabotropic excitatory amino acid, and GABA(B) binding sites, the laminar distribution of binding sites within striate cortex was different than that seen in adjacent visual cortex.     

Inhibitory synapse cover on the somata of excitatory neurons in macaque monkey visual cortex

Electron microscopy was used in macaque monkey cortical area V1 to investigate what factors might determine the proportion of somatic membrane covered by inhibitory type 2 synapses. In a sample of 4654 excitatory neurons, synapse cover did not correlate consistently with cell variety (pyramid or spiny stellate), soma size, synaptic apposition length or thalamic input. There were significant differences in somatic synapse cover per layer, but the pattern of differences in cover among layers differed significantly between animals, suggesting that laminar environment alone is not a generally applicable determinant of amount of inhibitory synapse cover. The pattern of cover for cells in different layers was, however, similar between the two hemispheres of an individual monkey. Measures of inhibitory synapse cover on four sets of pyramidal neurons in layers 5 and 6, each with different efferent projection targets, showed that the sets differed significantly from other cells in their respective layers, and differed significantly from each other. These findings demonstrate that there is unique circuitry for different subsystems within single layers of cortex and provide a rationale for the rich variety of cortical GABAergic interneurons within single layers.     

Differential gene expression between sensory neocortical areas: potential roles for Ten_m3 and Bcl6 in patterning visual and somatosensory pathways

Adult neocortical areas are characterized by marked differences in cytoarchitecture and connectivity that underlie their functional roles. The molecular determinants of these differences are largely unknown. We performed a microarray analysis to identify molecules that define the somatosensory and visual areas during the time when afferent and efferent projections are forming. We identified 122 molecules that are differentially expressed between the regions and confirmed by quantitative polymerase chain reaction 95% of the 20 genes tested. Two genes were chosen for further investigation: Bcl6 and Ten_m3. Bcl6 was highly expressed in the superficial cortical plate corresponding to developing layer IV of somatosensory cortex at postnatal day (P) 0. This had diminished by P3, but strong expression was found in layer V pyramidal cells by P7 and was maintained until adulthood. Retrograde tracing showed that Bcl6 is expressed in corticospinal neurons. Ten_m3 was expressed in a graded pattern within layer V of caudal cortex that corresponds well with visual cortex. Retrograde tracing and immunostaining showed that Ten_m3 is highly expressed along axonal tracts of projection neurons of the developing visual pathway. Overexpression demonstrated that Ten_m3 promotes homophilic adhesion and neurite outgrowth in vivo. This suggests an important role for Ten_m3 in the development of the visual pathway.     

Laminar development of receptive fields,maps and columns in visual cortex: the coordinating role of the subplate

How is development of cortical maps in V1 coordinated across cortical layers to form cortical columns? Previous neural models propose how maps of orientation (OR), ocular dominance (OD), and related properties develop in V1. These models show how spontaneous activity, before eye opening, combined with correlation learning and competition, can generate maps similar to those found in vivo. These models have not discussed laminar architecture or how cells develop and coordinate their connections across cortical layers. This is an important problem since anatomical evidence shows that clusters of horizontal connections form, between iso-oriented regions, in layer 2/3 before being innervated by layer 4 afferents. How are orientations in different layers aligned before these connections form? Anatomical evidence demonstrates that thalamic afferents wait in the subplate for weeks before innervating layer 4. Other evidence shows that ablation of the cortical subplate interferes with the development of OR and OD columns. The model proposes how the subplate develops OR and OD maps, which then entrain and coordinate the development of maps in other lamina. The model demonstrates how these maps may develop in layer 4 by using a known transient subplate-to-layer 4 circuit as a teacher. The model subplate also guides the early clustering of horizontal connections in layer 2/3, and the formation of the interlaminar circuitry that forms cortical columns. It is shown how layer 6 develops and helps to stabilize the network when the subplate atrophies. Finally the model clarifies how brain-derived neurotrophic factor (BDNF) manipulations may influence cortical development.     

Novel IgCAM, MDGA1, expressed in unique cortical area- and layer-specific patterns and transiently by distinct forebrain populations of Cajal-Retzius neurons

The laminar and area patterning of the mammalian neocortex are two organizing principles that define its functional architecture. Members of the immunoglobulin (Ig) superfamily of cell adhesion molecules influence neural development by regulating cell adhesion, migration, and process growth. Here we describe the dynamic expression of the unique Ig-containing cell adhesion molecule, MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1), during forebrain development in mice and compare it with other markers. We show that MDGA1 is a layer-specific marker and an area-specific marker, being expressed in layers 2/3 throughout the neocortex, but within the primary somatosensory area (S1), MDGA1 is also uniquely expressed in layers 4 and 6a. Comparisons with other markers, including cadherins, serotonin, cytochrome oxidase, ROR beta, and COUP-TF1, reveal unique features of patterned expression of MDGA1 within cortex and S1 barrels. Further, our findings indicate that at earlier stages of development, MDGA1 is expressed by Reelin- and Tbr1-positive Cajal-Retzius neurons that originate from multiple sources outside of neocortex and emigrate into it. At even earlier stages, MDGA1 is expressed by the earliest diencephalic and mesencephalic neurons, which appear to migrate from a MDGA1-positive domain of progenitors in the diencephalon and form a "preplate." These findings show that MDGA1 is a unique marker for studies of cortical lamination and area patterning and together with recent reports suggest that MDGA1 has critical functions in forebrain/midbrain development.     

Interference with the development of early generated neocortex results in disruption of radial glia and abnormal formation of neocortical layers

Early generated layers of neocortex are important factors in forming the subsequent architecture of the cerebral cortex. To further explore the role of early generated cortex, we disrupted formation of an early generated cohort of cells by intraperitoneal injections of the mitotic inhibitor methylazoxymethanol (MAM) into pregnant ferrets timed to coincide with generation of subplate neurons in the ventricular zone. Our studies demonstrate that if early development of the neocortex is interrupted by injection of MAM during embryogenesis (on embryonic day 24 or 28; E24 or E28), a distinct laminar pattern fails to form properly in the parietal cortex. A reduced number of MAP2-positive cells were observed in the region of the subplate when compared with the number of MAP2-positive cells found in normal animals. Interference with the superficial neocortical layers that form later during development (on embryonic day 33) by appropriately timed MAM injections does not result in a severely disrupted laminar pattern. The interrupted laminar pattern that arises after early MAM injections coincides with distorted radial glial cells (identified by immunoreactivity to the intermediate filament protein, vimentin), which occur after early, but not late, MAM injections. Further analysis suggests that interference with early development of neocortex leads to premature differentiation of radial glial cells into astrocytes, as demonstrated by the presence of glial fibrillary acidic protein (GFAP). Experiments involving injections of the thymidine analog, bromodeoxyuridine (BRDU), demonstrated that 4 days after E24 MAM injection cells are generated and migrate into the thin cortical plate. By E38, however, cells continue to be generated in animals treated with MAM on E24 but do not reach their normal positions in the cortical plate. In addition, immunoreactivity using the CR50 antibody, which identifies presumptive Cajal-Retzius cells present in layer 1, demonstrates that the CR50-positive cells, normally precisely located in the outer portion of layer 1, are distributed in disarray throughout the thickness of the neocortex and intermediate zone in early MAM-treated animals, but not in those treated with MAM injections later during gestation. These findings are consistent with the idea that early generated layers are important in providing factors that maintain the environment necessary for subsequent neuronal migration and formation of neocortical layers.     

GABA-A receptors regulate neocortical neuronal migration in vitro and in vivo

The cortical migration process depends on a number of trophic factors and on the activation of different voltage- and ligand-gated channels. We investigated the role of gamma-aminobutyric acid (GABA) type A receptors in the neuronal migration process of the newborn rat parietal cortex in vivo and in vitro. Local in vivo application of the GABA-A antagonist bicuculline methiodide (BMI) or the agonist muscimol via cortical surface Elvax implants induced prominent alterations in the cortical architecture when compared with untreated or sham-operated controls. BMI- and muscimol-treated animals revealed heterotopic cell clusters in the upper layers and a complete loss of the cortical lamination in the region underlying the Elvax implant. Immunocytochemical staining for glial fibrillary acidic protein, N-methyl-D-aspartate receptors, and GABA demonstrated that heterotopia was not provoked by glial proliferation and confirmed the presence of both glutamatergic and GABAergic neurons. In organotypic neocortical slices from embryonic day 18-19 embryos, application of BMI and to a lesser extent also muscimol induced an increase in the migration speed and an accumulation of neurons in the upper cortical layers. Spontaneous intracellular calcium ([Ca2+]i) oscillations in neocortical slices from newborn rats were abolished by BMI (5 and 20 microM) and muscimol (1 and 10 microM), indicating that both compounds interfere with [Ca2+]i signaling required for normal neuronal migration. Electrophysiological recordings from migrating neurons in newborn rat neocortical slices indicate that long-term application of muscimol causes a pronounced reduction (1 microM muscimol) or blockade (10 microM) in the responsiveness of postsynaptic GABA-A receptors due to a pronounced receptor desensitization. Our results indicate that modulation of GABA-A receptors by compounds acting as agonists or antagonists may profoundly influence the neuronal migration process in the developing cerebral cortex.     

Differential effects of endocannabinoids on glutamatergic and GABAergic inputs to layer 5 pyramidal neurons

Endocannabinoids are emerging as potent modulators of neuronal activity throughout the brain, and activation of the type-1 cannabinoid receptor (CB1R) reduces sensory-evoked cortical responses in vivo, presumably by decreasing excitatory transmission. In the neocortex, CB1R is differentially expressed across neocortical laminae, with highest levels of expression in layers 2/3 and 5. Although we have shown that cannabinoid signaling in layer 2/3 of somatosensory cortex targets both gamma-aminobutyric acid (GABA) and glutamate release, the predominant effect is a net increase in pyramidal neuron (PN) activity due to disinhibition. The role of endocannabinoid signaling in layer 5, the main output layer of the neocortex, remains unknown. We found that inducing activity in layer 5 PNs resulted in endocannabinoid-mediated depolarization-induced suppression of excitation (DSE), whereas the majority of inhibitory inputs were cannabinoid insensitive. Furthermore, in contrast to layer 2/3, the net effect of elevations in action potential firing of layer 5 PNs was an endocannabinoid-mediated decrease in PN spike probability. Interestingly, excitatory synaptic currents in layer 5 evoked by intralaminar stimulation were cannabinoid sensitive, whereas inputs evoked from layer 2/3 were insensitive, suggesting specificity of cannabinoid signaling across glutamatergic inputs. Thus, cannabinoids have differential effects on excitation and inhibition across cortical layers, and endocannabinoid signaling in layer 5 may serve to selectively decrease the efficacy of a subset of excitatory inputs.     

Activity-dependent regulation of synapse and dendritic spine morphology in developing barrel cortex requires phospholipase C-beta1 signalling

The phospholipase C-beta1 (PLC-beta1) signalling pathway, activated via metabotropic glutamate receptors (mGluRs), is implicated in activity-dependent development of the cerebral cortex, as both PLC-beta1 and mGluR5 knockout mice exhibit disrupted barrel formation in somatosensory cortex. To characterize the effects of this signalling system on development of synaptic circuitry in barrel cortex, we have examined neuronal ultrastructure, synapse formation and dendritic spine morphology in PLC-beta1 knockout mice. Qualitative ultrastructure of neurons and synapse density in layers 2-4 of barrel cortex were unchanged in PLC-beta1 knockout mice during development [postnatal day (P) 5] and in mature cortex (P19-21). We found a decrease in the proportion of synapses with symmetric morphology at P5 that was gone by P19-21, indicating a transient imbalance in excitatory and inhibitory circuitry. We also investigated dendritic spines by back-labelling layer 5 pyramidal neurons with carbocyanine. We observed normal dendritic spine densities on apical dendrites as they passed through layer 4 of barrel cortex, but spine morphology was altered in PLC-beta1 knockout mice at P9. These observations indicate that the PLC-beta1 signalling pathway plays a role in the development of normal cortical circuitry. Interrupting this regulation leads to changes in synapse and dendritic spine morphology, possibly altering post-synaptic integration of signal.     

Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons

Projection neurons destined for the cortical plate are generated sequentially from the proliferative ventricular and subventricular zones (VZ/SVZ) of the pallium. However, the respective contribution of both proliferative zones to the generation of cortical plate neurons is better established in humans and non-human primates than in rodents. We identified Cux2 as a new marker for murine cortical subpopulations and used it to provide new insights to the development of the mouse cortex. Cux2 is an orthologue of the Drosophila cut gene, which encodes a homeodomain protein involved in neuronal specification. During cortical development Cux2 identifies two subpopulations with different spatial origins, migratory behaviours and phenotypic characteristics: (i) a population of interneurons, which invades the pallium from the subpallium; and (ii) a neuronal population produced in the pallium around embryonic day 11.5, which divides in the SVZ and accumulates in the intermediate zone (IZ). Subsequently, Cux2 is a marker of upper cortical layers. Using different molecular markers and Pax6-deficient mice, we provide data that suggest a relationship between the early-determined Cux2-positive neuronal precursors in the SVZ/IZ and upper layer neurons. This suggests that laminar determination of upper cortical layer neurons occurs during the earliest stages of corticogenesis.     

Neurogenesis in the postnatal human epileptic brain

OBJECT: The normal adult human telencephalon does not reveal evidence of spontaneous neuronal migration and differentiation despite the robust germinal capacity of the subventricular zone (SVZ) astrocyte ribbon that contains neural stem cells. This might be because it is averse to accepting new neurons into an established neuronal network, probably representing an evolutionary acquisition to prevent the formation of anomalous neuronal circuits. Some forms of epilepsy, such as malformations of cortical development, are thought to be due to abnormal corticogenesis during the embryonic and early postnatal periods. The role of postnatal architectural reorganization and possibly postnatal neurogenesis in some forms of epilepsy in humans remains unknown. In this study the authors used resected specimens of epileptic brain to determine whether neurogenesis could occur in the diseased tissue. METHODS: The authors studied freshly resected brain tissue obtained in 47 patients who underwent neurosurgical procedures and four autopsies. Forty-four samples were harvested in patients who underwent resection for the treatment of pharmacoresistant epilepsy. RESULTS: Using organotypic brain slice preparations cultured with 5-bromodeoxyuridine (a marker for cell proliferation), immunohistochemistry, and cell trackers, the authors demonstrate the presence of spontaneous cell proliferation, migration, and neuronal differentiation in the adult human telencephalon that starts in the SVZ and progresses to the adjacent white matter and neocortex in human neocortical pathological structures associated with epilepsy. No cell migration or neuronal differentiation was found in the control group. CONCLUSIONS: The presence of spontaneous neurogenesis associated with some forms of human neocortical epilepsy may represent an erroneous and maladaptive mechanism for neuronal circuitry repair, or it may be an intrinsic part of the pathogenic process.     

Scheduling of monoaminergic neurotransmitter receptor expression in the primate neocortex during postnatal development.

Quantitative in vitro autoradiography was used to study the postnatal development of monoaminergic receptors (D1 and D2 dopaminergic, 5-HT1 and 5-HT2 serotonergic, and alpha 1, alpha 2, and beta noradrenergic sites) in the prefrontal, primary motor, somatosensory, and visual cortex of rhesus monkeys at birth and 1, 2, 4, 8, 12, 36, and 60 months of age. The density of all receptors studied increased rapidly within the first 2 postnatal months to levels as high as two times that recorded in the adults. After the fourth month, receptor density began a decline that subsided around the time of puberty. This course of developmental change was similar in all cortical layers and in all regions examined. However, the magnitude of the transient overproduction and eventual reduction in receptor density varied across the cortical layers and cytoarchitectonic areas in a manner specific to the individual receptor sites. Overall, cortical maturation was associated with the increased tendency of monoaminergic receptors to concentrate preferentially in the superficial cortical layers. The common developmental course of monoaminergic receptors in diverse cytoarchitectonic areas reveals an impressive coordination in the expression and regulation of these functionally relevant proteins in the cerebral cortex during infancy and adolescence.