Complementary Distribution of Collagen Type IV and the Epidermal Growth Factor Receptor in the Rat Embryonic Telencephalon

We previously identified an interaction between collagen typeIV and the EGF receptor that regulates the differentiation ofa limbic cortical phenotype in vitro (ferri and Levitt, 1995).In the present study, we map the expression of the EGF receptorand collagen type IV in the embryonic telencephalon of the rat.At embryonic day (E) 11, the earliest age examined, both proteinsare coexpressed throughout the ventricular zone in the cerebralwall; this zone remains immunoreactive throughout corticogenesis(E14–E19). The cells comprising the subventricular zoneare the most intensely immunoreactive for the EGF receptor,although little collagen type IV is detected in this region.In contrast, postmitotic neurons that leave the proliferativezones are negative for the receptor. Moreover, during the peakof neuronal migration, the intermediate zone lacks collagentype IV immunoreactivity. Neurons that settle in the corticalplate once again exhibit EGF receptor immunoreactivity; thissame zone is devoid of collagen type IV. By E19, coexpressionof both proteins is evident only in the rostral extension ofthe subventricular zone, the pathway of migrating cells leadingto the olfactory bulb. The temporal and spatial overlap of theEGE receptor and collagen type IV in the cortical progenitorpool in vivo indicates that these molecules may participatein the initial decisions of neuronal differentiation. Theirmodified distribution during cortical maturation suggests achanging role for both proteins.     

Early postnatal migration and development of layer II pyramidal neurons in the rodent cingulate/retrosplenial cortex

The cingulate and retrosplenial regions are major components of the dorsomedial (dm) limbic cortex and have been implicated in a range of cognitive functions such as emotion, attention, and spatial memory. While the structure and connectivity of these cortices are well characterized, little is known about their development. Notably, the timing and mode of migration that govern the appropriate positioning of late-born neurons remain unknown. Here, we analyzed migratory events during the early postnatal period from ventricular/subventricular zone (VZ/SVZ) to the cerebral cortex by transducing neuronal precursors in the VZ/SVZ of newborn rats/mice with Tomato/green fluorescent protein-encoding lentivectors. We have identified a pool of postmitotic pyramidal precursors in the dm part of the neonatal VZ/SVZ that migrate into the medial limbic cortex during the first postnatal week. Time-lapse imaging demonstrates that these cells migrate on radial glial fibers by locomotion and display morphological and behavioral changes as they travel through the white matter and enter into the cortical gray matter. In the granular retrosplenial cortex, these cells give rise to a Satb2+ pyramidal subtype and develop dendritic bundles in layer I. Our observations provide the first insight into the patterns and dynamics of cell migration into the medial limbic cortex.     

Retinoic acid synthesis in the postnatal mouse brain marks distinct developmental stages and functional systems

Retinoic acid (RA) affects development and function of the brain, but little is known about how much is made locally and where it is distributed. To identify RA-sensitive neural processes, we mapped the RA-synthesizing retinaldehyde dehydrogenases (RALDHs) during postnatal brain formation of the mouse. High and stable RALDH expressions mark the basal ganglia, olfactory bulbs, hippocampus and auditory afferents as major sites of RA actions in the functional brain. During the early postnatal period, transient and very high RALDH3 expressions distinguish two developmental events: (i) the colonization of the nucleus accumbens and the olfactory bulbs by neuronal precursors and (ii) the maturation of selected parts of the cerebral cortex. In the cortex, RALDH3 is transiently activated in postmigratory layer II/III neurons during formation of their dendritic arbors and it is transported in their axons across the corpus callosum. RALDH3-expressing cortical regions include most of the limbic lobe, with strongest expression in the anterior cingulate cortex, medial and lateral secondary visual cortices, auditory cortical areas, the secondary motor cortex and some association areas. The transient cortical expression points to a brief RA-critical period during differentiation of the cortical network that serves in the coordination of sensory-motor activity with emotional and recently learned information.     

Morphological and metabolic changes in the cortex of mice lacking the functional presynaptic active zone protein bassoon: a combined 1H-NMR spectroscopy and histochemical study

Mice lacking functional presynaptic active zone protein Bassoon are characterized by an enlarged cerebral cortex and an altered cortical activation pattern. This morphological and functional phenotype is associated with defined metabolic distortions as detected by a metabonomic approach using high-field (14.1 T) high-resolution 1H-nuclear magnetic resonance spectroscopy (MRS) in conjunction with statistical pattern recognition. Within the cortex but not in the cerebellum, concentrations of N-acetyl aspartate, glutamine, and glutamate are significantly reduced, whereas the majority of all other detectable low molecular metabolites are unchanged. The reduction of the neuron-specific metabolite N-acetyl aspartate in the cortex coincides with a significant decrease in neuronal density in cortical layer V. Comparing the neuron with glia cell densities across the cortex reveals cortex layer-dependent alterations in the ratio between both cell types. Whereas the ratio shifts significantly toward neurons in the cortical input layers IV, the ratio is reversed in cortical layer V. Consequently, the previously observed altered neuronal activation pattern in the cortex is reflected not only in defined cytoarchitectural anomalies but also in metabolic disturbances in the glutamine-glutamate and N-acetyl aspartate metabolism.     

Selective expression of doublecortin and LIS1 in developing human cortex suggests unique modes of neuronal movement

The genes doublecortin (DCX) and LIS1 are required for proper cortical neuronal migration and differentiation in humans. Here, we study the expression pattern of the encoded proteins of these genes in developing human brain. LIS1 stained virtually all migrating neurons throughout periods of development. Initially, DCX extensively overlapped with Reelin in early preplate stage in radially oriented columns of cells in the ventricular zone, whereas at later stages, the majority of DCX-positive cells were horizontally oriented. During the cortical plate stage, two opposite patterns of DCX expression were found: in radially oriented apical processes, presumably of pyramidal cells in the cortical plate, and in non-radially oriented mono- or bipolar neurons with migratory morphologies in the deep compartments of the cerebral wall. The extensive co-localization of DCX and Calretinin in non-radially oriented neurons suggested a non-pyramidal phenotype. These cells assumed a more vertical orientation upon entering the subplate. In addition, DCX was expressed by cells in the subpial granular layer and by Cajal-Retzius cells. In a 19 week human fetal cortex with a LIS1 mutation, the number of Reelin-expressing Cajal-Retzius cells was reduced and their morphology was abnormal. DCX was expressed by cells in all regions, but in extremely low numbers, suggesting that LIS1 deficiency adversely affects the migration and differentiation of DCX- and Reelin-positive neurons.     

Role of integrins in the development of the cerebral cortex

Spatial and temporal changes in expression and function of integrin receptors in the developing cerebral wall parallel neurogenesis, radial glial differentiation, neuronal migration and the emergence of neuronal layers in the cerebral cortex. The distinct outcomes of integrin and extracellular matrix ligand mutations underscore the dynamic role they play in these processes during corticogenesis. The changing patterns of adhesive interactions mediated by integrins and their ligands across the cerebral wall during embryogenesis may set in motion developmental programs needed for progressive acquisition of different neuronal or glial phenotypes in the cerebral cortex. Here we discuss the role of integrins during cortical layer formation.     

Disruption of neuronal migration and radial glia in the developing cerebral cortex following ablation of Cajal-Retzius cells

Cortical neurons are generated within the proliferative layer and follow a strict 'inside-out' gradient of migration and positioning, which determines the characteristic layering and pattern of neural connections in the adult cerebral cortex. Thus, directional migration of postmitotic neuroblasts towards layer I and regulation of the radial glia phenotype subserving cortical migration are central issues in corticogenesis. Recent studies showing that the gene disrupted in the reeler mutation--reelin--is expressed in Cajal-Retzius cells have indicated a role for these pioneer neurons in cortical migration. We show here that ablation of Cajal-Retzius cells in layer I by local application of domoic acid in newborn mice arrests migration of the late-generated neurons, destined to cortical layers II-III, that have been labeled by 5-bromodeoxyuridine injections administered at E16. In addition, degeneration of Cajal-Retzius cells in newborn mice dramatically decreases the number of radial glial apical processes identified by nestin-immunostaining, but increases the number of maturing glial fibrillary acidic protein-positive astrocytes. These findings support an essential role for Cajal-Retzius cells in neuronal migration and corticogenesis, by regulating the identity and function of radial glia and the radial glia-to-astrocyte transformation.     

Characterization of the growth of cultured cortical neurons on sections of adult rat cortex.

Cultured neurons isolated from the embryonic day 18 (E18) rat cortex were plated onto cryostat sections of adult CNS and peripheral nerve. The ability of these sections to support neuronal attachment and neurite extension was compared to that of neurons on the adjacent poly-L-lysine (PLL)-coated glass coverslips. A quantitative analysis of the data demonstrated that sections of cerebral cortex provide a good substrate for neuronal attachment and growth of neurites, similar to that of the PLL-coated glass coverslips. Sections of sciatic nerve demonstrated a decreased ability to support neuronal attachment and the growth of processes as compared to sections of cortex. Sections of optic nerve supported limited neuronal attachment, and the few neurons that were present on top of the sections were devoid of processes. Control experiments showed that the ability of the cortical sections to support the attachment and growth of cultured neurons was due to the molecular composition of the sections and not to influences of the tissue culture environment. The interactions of neurons with the sections of CNS cortex were partially dependent on a member of the beta 1 integrin family of extracellular matrix receptors present on cultured neurons. This in vitro system allowed for the definition of some of the molecular and the cellular interactions that may occur between growing axons and the environment of the adult cortex.     

Early expression of GABA-containing neurons in the prefrontal and visual cortices of rhesus monkeys.

Light and electron microscopic immunohistochemistry was used to examine the time of emergence and distribution of GABA-containing cells in an association (prefrontal) and primary sensory (visual) region of the telencephalon at progressive fetal and postnatal stages of cortical maturation in the rhesus monkey. Thirty fetuses and six postnatal monkeys were examined beginning at embryonic day 41 (E41), the start of cortical neurogenesis, to birth (E165) and proceeding to maturity (greater than 5 years of age). The emergence and major developmental modifications in the distribution of immunoreactive neurons in both areas examined were nearly identical. GABA-immunolabeled neurons were present throughout the full thickness of the cerebral wall, including the cortical plate and the developmentally transient marginal, subplate, and ventricular zones, as early as E41. An important and surprising result was that a subset of bipolar migrating neurons in the subplate zone also contained GABA at these early stages. GABA-containing neurons in the ventricular and subventricular zones disappeared after E100, when neurogenesis is completed. In contrast, the number of immunoreactive multipolar and bipolar neurons within the subplate zone diminished between E100 and E131. By the first postnatal week, the distribution and density of GABA-containing neurons in the cortex appeared qualitatively similar to that observed in mature monkeys. The early appearance of GABA in cortical neurons and its expression by a population of migrating neurons suggest that a subset of cortical neurons may be committed to a transmitter phenotype independent of synaptic interactions and prior to attaining their adult positions in the maturing cerebral cortex.     

Synaptogenesis in layer I of the human cerebral cortex in the first half of gestation

The formation of synapses is among the most important steps in neuronal differentiation and the establishment of neuronal circuits. To establish baseline data about the time of onset, density and the course of synaptic formation in different regions of the human cerebral cortex before birth, synaptogenesis in layer I was examined by electron microscopy in fetuses ranging in age from 6 to 24 gestational weeks. Synapses were first observed in the primordial plexiform layer (marginal zone) in both the lateral and medial cerebral walls between the 6th and 7th gestational week, before the formation of the cortical plate. The density of synapses increased rapidly after the formation of the cortical plate, increasing by 37% between 12 and 14 weeks. Synaptogenesis proceeded at the same rate in the lateral and occipital cortex during this period. Further, with one exception, the insular region, synaptic density was comparable in prospective areas of prefrontal, motor, visual, temporal and cingulate cortex in a group of fetuses at midgestation (20 weeks). The results are consistent with a synchronous course of synaptogenesis of the neocortex.      

Neuronal Clones in the Cerebral Cortex Show Morphological and Neurotransmitter Heterogeneity during Development

The mammalian cerebral cortex, although a structure of greatcomplexity, is characterized by a high degree of organizationwhere the proportions, spatial relationships, and propertiesof the various cell types are rigidly controlled. The mechanismsresponsible for the creation of such a rigid distribution ofcell types are not known. Lineage studies in adult rats havesuggested that each of the cortical progenitor cells liningthe telencephalic ventricles during embryonic development givesrise to progeny of the same phenotype (homogeneous clones).However, the possibility that homogeneous clones are the resultof complex processes affecting both the final number and thephenotype of clonally related cells during development has notbeen investigated. In the present study, we followed the developmentof cortical cell lineages labeled with retroviral injectionsat embryonic day (E) 16 in rats of 7, 14, or 21 d of age usingelectron microscopy and immunocytochemistry for the neurotransmittersglutamate and GABA. We found that a significant number of corticalclones at postnatal day (P) 7 and P14, and fewer at P21, showedmixed pyramidal/nonpyramidal cell composition. We sometimesobserved that "mixed" neuronal clones contained cells immunoreactivefor both glutamate and GABA. In the general population of corticalcells, "bireactive" neurons represented 3.7% of all neuronsat P7, 1.8% at P14, and 0.6% in adult rats. Although the changein the composition of neuronal clones between the third weekof postnatal life and adulthood may be due to changes in thephenotype of some developing neurons, we would like to suggestthat it is probably due to selective neuronal cell death.     

Developmental Changes Revealed by Immunohistochemical Markers in Human Cerebral Cortex

The developing human cerebral cortex is distinguished by a particularlywide subplate, a transient zone in which crucial cell-cell interactionsoccur. To further understand the role of the subplate in humanbrain development, we have studied the immunohistochemical expressionof certain neuronal (GAP-43, MAP-2 parvalbumin) and astroglial(vimentin, GFAP} markers in the developing visual cortex fromgestational ages of 14 weeks to 9 months post-term. At 14–22weeks, immunoreactivity to GAP-43, a protein involved in axonaloutgrowth, was most prominent in the subplate and marginal zoneneuropil and in the fibers of the radiations running near theventricular zone; at 22–42 weeks, GAP-43 immunoreactivefibers were observed in the maturing cortical plate. Immunoreactivityfor the microtubule-associated protein MAP-2 was present inthe differentiating cortical plate at 14 weeks, but at 22–42weeks was most prominent in the somata and dendrites of differentiatedneurons, particularly the Cajal-Retzius neurons of the marginalzone, in neurons of the subplate and in those forming corticallayer 5. Parvalbumin immunoreactivity did not appear until 26weeks, when stained neurons were in a sparse band of cells inlayer 6 and upper subplate. Vimentin and GFAP did not staindifferentiated neuronal cells. Vimentin immunoreactivity appearedearly in neuroepithelial and radial glial cells, decreasingafter 35 weeks, with a concomitant increase in GFAP immunoreactivityin radial glial and maturing astrocytic cells. Our results showthat despite the greater complexity of the developing humanneocortex, molecular markers are expressed in spatial and temporalpatterns similar to those observed in non-human primates, carnivoresand rodents. These protein markers should prove useful in developmentalstaging, and in providing a framework in which to examine congenitaldisorders of cerebral development.     

Cortical Target Depletion and Ingrowth of Geniculocortical Axons: Implications for Cortical Specification

During the early development of the neocortex, thalamocorticalaxons arrive potentially in time to instruct migrating corticalneurons in several aspects of local differentiation, such asnumber of layer IV neurons and efferent connectivity. Migrationof layer IV neurons into the cortical plate just precedes thalamocorticalinvasion, suggesting that these neurons could cue or tropicallydirect thalamic ingrowth. To explore the interactions of layerIV neurons and their thalamocortical input, we administereda mitotic inhibitor methylazoxymethanol acetate (MAM) intraperitoneallyto timed pregnant hamsters on E14 when layer IV neurons arenormally being generated in striate cortex. Reduced numbersof cortical neurons overall, the absence of small-diameter granuleneurons, and the absence of the zone of reduced density of callosallyprojecting neurons suggest that neither the depletion of layerIV cells in the ventricular zone nor thalamic afferents in thesubplate or cortical plate respecify the later generated cohortof neurons (presumptive layer II/III neurons) to acquire morphologicaland connectional properties of layer IV. Dil injections intothe dorsal lateral geniculate nucleus (LGd) of animals fromembryonic (E15) and postnatal (P7) ages show that the finalposition of thalamic axons with respect to layer V is not affectedby the absence of E14 neurons. In the normal visual cortex,geniculocortical axons have begun their arborization in theirpresumptive target layer in the upper cortex immediately belowthe undifferentiated cortical plate on P4, while in MAM animals,this process occurs 1 d later. The extent and density of arborizationis much reduced in the thinner cortex of the MAM animals. Wethus find no evidence for instruction of migrating neurons bythalamocortical axons to assume the layer IV phenotype; if instructiondoes occur, it must take place in a very restricted time window.Thalamic axons can also find their laminar position in the absenceof cells of this phenotype.     

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.     

Blockade of GABA(B) receptors alters the tangential migration of cortical neurons

To better understand the role of neurotransmitter receptors in neuronal differentiation and maturation a detailed knowledge of their identity, location and function in the plasma membrane of specific neuronal populations during development is required. Combining pre-embedding immunocytochemistry with cell tracking in embryonic brain slice cultures we show that virtually all neurons (approximately 98%) migrating through the lower intermediate zone (LIZ) on their way from the medial ganglionic eminence to the cerebral cortex, express GABA(B)R1. Blockade of GABA(B)Rs with a specific antagonist, CGP52432, resulted in a concentration-dependent accumulation of these tangentially migrating neurons in the ventricular/sub-ventricular zones (VZ/SVZ) of the cortex and fewer cells were observed in the cortical plate/marginal zone (CP/MZ) and LIZ. Moreover, they had significantly shorter leading processes compared with similar migrating cells in control slices. Electrophysiological recording in LIZ and CP cells revealed no direct effect of either CGP52432 or the GABA(B)R agonist, baclofen, on resting membrane properties suggesting that the effect of CGP52432 on migration might be mediated through a metabotropic action or the regulation of release of factors controlling migration. These results suggest that GABA(B)Rs have an important modulatory role in the migration of cortical interneurons.     

Synaptogenesis in the Prefrontal Cortex of Rhesus Monkeys

Since the turn of the century, the prefrontal association areasof the cerebral cortex have been thought to be among the lastregions of the cortical mantle to develop. We have examinedthe course of synaptogenesis in the macaque prefrontal cortexby quantitative electron microscopic analysis in 25 rhesus monkeysranging in age from embryonic day 47 (E47) to 20 years of age.A series of overlapping electron micrographs spanning the wholecortical thickness in each animal provided data on the number,the proportion, and the density of synapses per unit area (N_A)and per unit volume (N_V) of neuropil. The tempo and kinetics of synapse formation in prefrontal cortexclosely resemble those described for sensory and motor areas,particularly during the stages of synapse acquisition and overproduction(Rakic et al., 1986). In young embryos, we describe a precorticalphase (E47-E78), when synapses are found only above and below,but not within, the cortical plate. Following that, there isan early cortical phase, from E78 to E104, during which synapsesaccumulate within the cortical plate, initially exclusivelyon dendritic shafts. The next rapid phase of synaptogenesisbegins at 2 months before birth and ends approximately at 2months after birth, culminating with a mean density of 750 millionsynapses per cubic micrometer. This accumulation is largelyaccounted for by a selective increase in axospine synapses inthe supragranular layers. The period of explosive synaptic densityis followed by a protracted plateau stage that lasts from 2months to 3 years of age when synaptic density remains relativelyconstant. The final period of decline, from 3 years throughover 20 years of age, is marked by a slight but statisticallysignificant decline in synaptic density. Concurrent recruitment of synapses with that of sensory andmotor areas supports the concept that the initial establishmentof cortical circuitry is governed by general mechanisms commonto all areas, independent of their specific functional domain.The finding that synaptic density is relatively stable fromearly adolescence through puberty (the plateau period) is indicativeof the importance, in primates, of a consistent and high synapticdensity during the formative years when learning experiencesare most intense.     

Increased neuronal production,enlarged forebrains and cytoarchitectural distortions in beta-catenin overexpressing transgenic mice

beta-Catenin can function in the decision of neural precursors to proliferate or differentiate during mammalian neuronal development and may regulate cerebral cortical size by controlling the generation of neural precursor cells. Mice expressing high levels of a stabilized beta-catenin transgene in neural precursors develop enlarged brains with expanded precursor populations, increased cerebral cortical surface area, and folds resembling sulci and gyri of higher mammals present at birth. Here we report the effects in adult mice expressing lower levels of the same stabilized beta-catenin transgene in neural precursors. Adult transgenic animals develop enlarged forebrains with thin cerebral cortices with increased surface area, expanded subventricular zones with subcortical aggregations of neurons and enlarged, distorted hippocampi. The brains from transgenic mice also show apparent arrest of neuronal migration and dramatic disorganization of the layering of the cerebral cortex. These findings suggest that beta-catenin can cause expansion of the precursor pool resulting in increased neuronal production and greater brain size and suggest a crucial role for beta-catenin in neuronal migration and cortical lamination.     

Cholinergic action on cortical glial cells in vivo

This study aims at understanding complex interactions betweencortical neurons, glia and blood supply developing during thetransition from slow-wave sleep to wakefulness. In spite ofessential advances from in vitro and culture preparations, thebasic mechanisms of glial interactions with their cellular andionic environment had remained uninvestigated in vivo. Herewe approach this issue by performing simultaneous intracellularrecordings of cortical neurons and glia, together with measurementsof cerebral blood flow (CBF), extracellular K⁺ concentrationsand local field potentials in both anesthetized (ketamine–xylazine)and naturally behaving cats. Under anesthesia, cortical activationwas elicited with electric stimulation of cholinergic nuclei(pedunculopontine tegmental in the brainstem and/or nucleusbasalis in the basal forebrain). Iontophoretic application ofacetylcholine on the recorded cells was also used. In the vastmajority of cases (>80%) glial cells were hyperpolarizedduring electric stimulation or spontaneous activation. Thisresult was also obtained in all cases where iontophoresis wasused or when glutamatergic kainate/quisqualate receptors wereblocked with 6-cyano-7-nitroquinoxaline-2,3-dione. The glialhyperpolarization was associated with steady neuronal depolarization,increased CBF, lower extracellular K⁺ concentration, increasedmembrane resistance, decreased membrane capacitance and persistentpositive DC field potentials. In some cases of cortical activation(<20%), glial cells displayed sustained depolarizing potentials,in parallel with neuronal depolarization, decreased CBF andmore negative DC field potentials. The above-mentioned effectsof cholinergic activation were blocked by the muscarinic antagonistscopolamine. We propose that the glial response to cholinergicactivation results from the balance between the direct hyperpolarizingaction of acetylcholine and the depolarizing modulation of glutamatefrom the neighboring neurons, in addition to the modulationof the interglial communication pathway and/or the ionic trafficacross blood vessels.