Abstract: Increasing evidence indicates that neural stem/progenitor cells (NSPCs) reside in many regions of the central nervous system (CNS), including the subventricular zone (SVZ) of the lateral ventricle, subgranular zone of the hippocampal dentate gyrus, cortex, striatum, and spinal cord. Using a murine model of cortical infarction, we recently demonstrated that the leptomeninges (pia mater), which cover the entire cortex, also exhibit NSPC activity in response to ischemia. Pial ischemia-induced NSPCs expressed NSPC markers such as nestin, formed neurosphere-like cell clusters with self-renewal activity, and differentiated into neurons, astrocytes, and oligodendrocytes, although they were not identical to previously reported NSPCs such as SVZ astrocytes, ependymal cells, oligodendrocyte precursor cells, and reactive astrocytes. In this study, we showed that leptomeningeal cells in the poststroke brain express the immature neuronal marker doublecortin as well as nestin. We also showed that these cells can migrate into the poststroke cortex. Thus, the leptomeninges may participate in CNS repair in response to brain injury.
Abstract: The transcription factors Satb2 (special AT-rich sequence binding protein 2) and Ctip2 (COUP-TF interacting protein 2) have been shown to be required for callosal and corticospinal axon growth respectively from subtypes of cerebral cortex projection neurons. In this study we investigated early stages of directed axon growth in the embryonic mouse cerebral cortex, and studied the possible correlation with the expression of Satb2 and Ctip2. Electroporation of an EYFP-expressing plasmid at embryonic day 13.5 to label developing projection neurons revealed that directed axon growth is first seen in radially migrating neurons in the intermediate zone (IZ), prior to migration into the cortical plate, as has been suggested previously. Onset of expression of SATB2 and CTIP2 was also observed in the IZ, correlating well with this stage of migration and initiation of axon growth. Immunohistochemical staining through embryonic and early postnatal development revealed a significant population of Satb2/Ctip2 co-expressing cells, while retrograde axon tracing from the corpus callosum at embryonic day 18.5 back-labelled many neurons with bi-directional axon processes. However, through retrograde tracing and simultaneous immunohistochemical staining we show that these bi-directional processes do not correlate with Satb2/Ctip2 co-expression. Our work shows that although expression of these transcription factors correlates well with the appearance of directed axon growth during cortical development, the transcriptional code underlying the bi-directional axonal projections of early neocortical neurons is not likely to be the result of Satb2/Ctip2 co-expression.
Abstract: In this issue of Neuron, Bernard et al. (2012) report microarray-based transcriptional profiling of individually isolated layers from several cortical areas in adult Rhesus monkeys (Macaca mulatta). The resulting molecular signatures of neocortical organization are compared with human and mouse.
Abstract: We are interested in the role of neural activity mediated through regulated vesicular release in the stopping and early branching of the thalamic projections in the cortex. Axon outgrowth, arrival at the cortical subplate, side-branch formation during the waiting period and cortical plate innervation of embryonic thalamocortical projections occurs without major abnormalities in the absence of regulated release in Snap25 (-/-) null mutant mice [Washbourne et al. (2002) Nat. Neurosci. 5:19-26; Molnár et al. (2002) J. Neurosci. 22:10313-10323]. The fact that Snap25 (-/-) null mutant mice die at birth limited our previous experiments to the prenatal period. We therefore investigated the behaviour of thalamic projections in co-culture paradigms by using heterochronic thalamic [embryonic day (E)16-E18] and cortical [postnatal day (P)0-P3] explants, in which the stopping and branching behaviour has been previously documented. Our current co-culture experiments established that thalamic projections from E16-E18 Snap25(+/+) or Snap25 (-/-) explants behaved in an identical fashion in P0-P3 Snap25 (+/+) cortical explants after 7 days in vitro. Thalamic projections from Snap25 (-/-) explants developed similar patterns of fibre ingrowth to the cortex, and stopped and formed branches at a similar depth in the Snap25(+/+) cortical slice as in control cultures. These results imply that thalamic projections can reach their ultimate target cells in layer 4, stop, and start to develop branches in the absence of regulated vesicular transmitter release from their own terminals.
Abstract: In this review we discuss recent advances in the understanding of corticothalamic axon guidance; patterning of the early telencephalon, the sequence and choreography of the development of projections from subplate, layers 5 and 6. These cortical subpopulations display different axonal outgrowth kinetics and innervate distinct thalamic nuclei in a temporal pattern determined by cortical layer identity and subclass specificity. Guidance by molecular cues, structural cues, and activity-dependent mechanisms contribute to this development. There is a substantial rearrangement of the corticofugal connectivity outside the thalamus at the border of and within the reticular thalamic nucleus, a region that shares some of the characteristics of the cortical subplate during development. The early transient circuits are not well understood, nor the extent to which this developmental pattern may be driven by peripheral sensory activity. We hypothesize that transient circuits during embryonic and early postnatal development are critical in the matching of the cortical and thalamic representations and forming the cortical circuits in the mature brain.
Abstract: Thalamocortical axons must cross a complex cellular terrain through the developing forebrain, and this terrain has to be understood for us to learn how thalamocortical axons reach their destinations. Selective fasciculation, guidepost cells and various diencephalic and telencephalic gradients have been implicated in thalamocortical guidance. As our understanding of the relevant forebrain patterns has increased, so has our knowledge of the guidance mechanisms. Our aim here is to review recent observations of cellular and molecular mechanisms related to: the growth of thalamofugal projections to the ventral telencephalon, thalamic axon avoidance of the hypothalamus and extension into the telencephalon to form the internal capsule, the crossing of the pallial-subpallial boundary, and the growth towards the cerebral cortex. We shall review current theories for the explanation of the maintenance and alteration of topographic order in the thalamocortical projections to the cortex. It is now increasingly clear that several mechanisms are involved at different stages of thalamocortical development, and each contributes substantially to the eventual outcome. Revealing the molecular and cellular mechanisms can help to link specific genes to details of actual developmental mechanisms.
Abstract: Previous studies of macaque and human cortices identified cytoarchitectonically distinct germinal zones; the ventricular zone inner subventricular zone (ISVZ), and outer subventricular zone (OSVZ). To date, the OSVZ has only been described in gyrencephalic brains, separated from the ISVZ by an inner fiber layer and considered a milestone that triggered increased neocortical neurogenesis. However, this observation has only been assessed in a handful of species without the identification of the different progenitor populations. We examined the Amazonian rodent agouti (Dasyprocta agouti) and the marmoset monkey (Callithrix jacchus) to further understand relationships among progenitor compartmentalization, proportions of various cortical progenitors, and degree of cortical folding. We identified a similar cytoarchitectonic distinction between the OSVZ and ISVZ at midgestation in both species. In the marmoset, we quantified the ventricular and abventricular divisions and observed similar proportions as previously described for the human and ferret brains. The proportions of radial glia, intermediate progenitors, and outer radial glial cell (oRG) populations were similar in midgestation lissencephalic marmoset as in gyrencephalic human or ferret. Our findings suggest that cytoarchitectonic subdivisions of SVZ are an evolutionary trend and not a primate specific feature, and a large population of oRG can be seen regardless of cortical folding.
Abstract: Rodents and primates both show considerable variation in the overall size, the radial and tangential dimensions, folding and subdivisions into distinct areas of their cerebral cortex. Our current understanding of brain development is based on a handful of model systems. A detailed comparative analysis of the cellular and molecular mechanisms that regulate neural progenitor production, cell migration, and circuit assembly can provide much needed insights into the working of neocortical evolution. From the limited comparative data currently available, it is apparent that the emergence and variation of the neuronal progenitor cells have led to the production of increased neuronal populations and the evolution of the cortex. Further diversification and compartmentalization of the germinal zone together with changing proportions of radial glia in the ventricular zone and various intermediate progenitors in the subventricular zone may have been the driving force behind increased cell numbers in larger brains both in rodents and primates. Radial and tangential migratory patterns are both present in rodents and primates, but in different proportions. There are apparent differences between mouse and human in the generation and elaboration of the interneuronal subtypes and also in gene expression patterns associated with the appearance of distinct cortical areas. The increased cortical dimensions and the formation of a more elaborate cortical architecture in primates require a larger and more compartmentalized transient subplate zone during development. More comparative analysis in rodent and primate species with large, small, and smooth and folded brains is needed to reveal the biological significance of the alterations in these cortical developmental programs.
Abstract: During embryonic and early postnatal development subplate neurons integrate into the developing intra- and extracortical circuitry in a dynamic fashion. They extend long-range projections to adjacent cortical regions, the contralateral hemisphere through the corpus callosum, and to subcortical structures through the internal capsule. Here we studied the developmental changes of the somatodendritic morphology of subplate neurons with specified projection target in the mouse. To do so we used carbocyanine dye tracing from the callosum, the internal capsule, or the primary somatosensory cortex. The morphology of subplate cells with projections to any of these targets is very diverse and includes pyramidal, multipolar, and neurogliaform cells. Here we demonstrate that a subpopulation of subplate cells in the mouse cortex undergoes significant changes in somatodendritic morphology during the critical period for experimental modification of the cytoarchitectonic development of the barrel cortex. Between P2 and P7 the mean maximal extent of the primary dendrite decreases significantly for subplate cells with an axon projecting through the internal capsule. Moreover, at P2 some subplate cells extend a primary dendrite to the marginal zone, whereas all dendrites of P7 subplate cells end in or below layer 4. Additionally, by tracing connections from multiple targets with different carbocyanine dyes we identified subplate cells with multiple long-range projections.
Abstract: Increasing evidence shows that neural stem/progenitor cells (NSPCs) can be activated in the nonconventional neurogenic zones such as the cortex following ischemic stroke. However, the precise origin, identity, and subtypes of the ischemia-induced NSPCs (iNSPCs), which can contribute to cortical neurogenesis, is currently still unclear. In our present study, using an adult mouse cortical infarction model, we found that the leptomeninges (pia mater), which is widely distributed within and closely associated with blood vessels as microvascular pericytes/perivascular cells throughout central nervous system (CNS), have NSPC activity in response to ischemia and can generate neurons. These observations indicate that microvascular pericytes residing near blood vessels that are distributed from the leptomeninges to the cortex are potential sources of iNSPCs for neurogenesis following cortical infarction. In addition, our results propose a novel concept that the leptomeninges, which cover the entire brain, have an important role in CNS restoration following brain injury such as stroke.
Abstract: Understanding how the human cerebral cortex evolved to its present complex state is a fascinating topic for neuroscience, genetics, bioinformatics and comparative biology. To gain further insights into the origins of the mammalian neocortex and to understand how the cortex evolved to be able to serve more complex cognitive functions, we study the development of various extant species. Our aim is to correlate cortical cell numbers and neuronal cell types with the elaboration of cortical progenitor populations and their modes of proliferation in different species. There are several progenitors, i.e. the ventricular radial glia, the subventricular intermediate progenitors and subventricular (outer) radial glia types, but the contribution of each to cortical layers and cell types through specific lineages is not fully understood. Recent comparisons of the proportions of these progenitors in various species during embryonic neurogenesis have revealed the elaboration and cytoarchitectonic compartmentalization of the germinal zone, with alterations in the proportions of various types that can be included among the intermediate progenitors. Across species, larger and more diverse intermediate progenitor populations correlate with brain size and cortical cell diversity. Understanding the molecular and cellular interactions regulating the divisions of these intermediate progenitors not only has implications for cortical evolution but also relates to stem cell biology and illuminates the pathomechanisms of several cortical developmental disorders.
Abstract: The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and - when barrier integrity is impaired - in the pathology of many common CNS disorders such as Alzheimer's disease, Parkinson's disease and stroke.
Abstract: There is currently a debate about the evolutionary origin of the earliest generated cortical preplate neurons and their derivatives (subplate and marginal zone). We examined the subplate with murine markers including nuclear receptor related 1 (Nurr1), monooxygenase Dbh-like 1 (Moxd1), transmembrane protein 163 (Tmem163), and connective tissue growth factor (Ctgf) in developing and adult turtle, chick, opossum, mouse, and rat. Whereas some of these are expressed in dorsal pallium in all species studied (Nurr1, Ctgf, and Tmem163), we observed that the closely related mouse and rat differed in the expression patterns of several others (Dopa decarboxylase, Moxd1, and thyrotropin-releasing hormone). The expression of Ctgf, Moxd1, and Nurr1 in the oppossum suggests a more dispersed subplate population in this marsupial compared with mice and rats. In embryonic and adult chick brains, our selected subplate markers are primarily expressed in the hyperpallium and in the turtle in the main cell dense layer of the dorsal cortex. These observations suggest that some neurons that express these selected markers were present in the common ancestor of sauropsids and mammals.
Abstract: In the mammalian cortex, neurons and glia form a patterned structure across six layers whose complex cytoarchitectonic arrangement is likely to contribute to cognition. We sequenced transcriptomes from layers 1-6b of different areas (primary and secondary) of the adult (postnatal day 56) mouse somatosensory cortex to understand the transcriptional levels and functional repertoires of coding and noncoding loci for cells constituting these layers. A total of 5,835 protein-coding genes and 66 noncoding RNA loci are differentially expressed ("patterned") across the layers, on the basis of a machine-learning model (naive Bayes) approach. Layers 2-6b are each associated with specific functional and disease annotations that provide insights into their biological roles. This new resource (http://genserv.anat.ox.ac.uk/layers) greatly extends currently available resources, such as the Allen Mouse Brain Atlas and microarray data sets, by providing quantitative expression levels, by being genome-wide, by including novel loci, and by identifying candidate alternatively spliced transcripts that are differentially expressed across layers.
Abstract: It has been well established that maternal inflammation during pregnancy alters neurological function in the offspring, but its impact on cortical development and long-term consequences on the cytoarchitecture is largely unstudied. Here we report that lipopolysaccharide-induced systemic maternal inflammation in C57Bl/6 mice at embryonic Day 13.5 of pregnancy, as early as 8 h after challenge, caused a significant reduction in cell proliferation in the ventricular zone of the developing cerebral cortex, as revealed by quantification of anti-phospho-Histone H3 immunoreactivity and bromodeoxyuridine pulse labelling. The angle of mitotic cleavage, determined from analysis of haematoxylin and eosin staining, cyclin E1 gene expression and the pattern of β-catenin immunoreactivity were also altered by the challenge, which suggests a change from symmetric to asymmetric division in the radial progenitor cells. Modifications of cortical lamination and gene expression patterns were detected at post-natal Day 8 suggesting prolonged consequences of these alterations during embryonic development. Cellular uptake of proteins from the cerebrospinal fluid was observed in brains from lipopolysaccharide-treated animals in radial progenitor cells. However, the foetal blood-brain barrier to plasma proteins remained intact. Together, these results indicate that maternal inflammation can disrupt the ventricular surface and lead to decreased cellular proliferation. Changes in cell density in Layers IV and V at post-natal Day 8 show that these initial changes have prolonged effects on cortical organization. The possible shift in the fate of progeny and the resulting alterations in the relative cell numbers in the cerebral cortex following a maternal inflammatory response shown here will require further investigation to determine the long-term consequences of inflammation on the development of neuronal circuitry and behaviour.
Abstract: Progress in understanding development of the brain has already started to explain the patterns of neurogenesis, progenitor subtypes, combinatorial transcription codes both for cell types and regional specification of the cerebral cortex, migratory routes taken by cells and axons and establishment of neural connectivity. This progress has mostly been experimental and based on murine studies. Although the mouse is a superb model for exploiting the modern tools of molecular genetics, allowing us to monitor and modulate selected groups of neurons, it has several limitations. The human brain has unique qualities; and there are strong clinical reasons for wanting to improve understanding of its developmental properties. Some developmental steps are considered exclusively in human; other features are not necessarily unique but certainly much more apparent in human than in other species ( Clowry et al., 2010). Indeed, some fundamental recent discoveries were made in humans.
Abstract: The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription-polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells.
Abstract: The development of the mammalian neocortex relies heavily on subplate. The proportion of this cell population varies considerably in different mammalian species. Subplate is almost undetectable in marsupials, forms a thin, but distinct layer in mouse and rat, a larger layer in carnivores and big-brained mammals as pig, and a highly developed embryonic structure in human and non-human primates. The evolutionary origin of subplate neurons is the subject of current debate. Some hypothesize that subplate represents the ancestral cortex of sauropsids, while others consider it to be an increasingly complex phylogenetic novelty of the mammalian neocortex. Here we review recent work on expression of several genes that were originally identified in rodent as highly and differentially expressed in subplate. We relate these observations to cellular morphology, birthdating, and hodology in the dorsal cortex/dorsal pallium of several amniote species. Based on this reviewed evidence we argue for a third hypothesis according to which subplate contains both ancestral and newly derived cell populations. We propose that the mammalian subplate originally derived from a phylogenetically ancient structure in the dorsal pallium of stem amniotes, but subsequently expanded with additional cell populations in the synapsid lineage to support an increasingly complex cortical plate development. Further understanding of the detailed molecular taxonomy, somatodendritic morphology, and connectivity of subplate in a comparative context should contribute to the identification of the ancestral and newly evolved populations of subplate neurons.
Abstract: Blood vessels play a critical role in regulating neural stem cell proliferation and migration. We show here that blood vessels became progressively aligned in the direction of the rostral migratory stream (RMS) from embryonic day 14 to postnatal day 4. Dividing cells revealed by phosphohistone H3+ immunoreactivity were statistically closer to isolectin B4+ blood vessels than predicted by chance in the emerging RMS. The close proximity of blood vessels and H3+ cells was consistent regardless of the age of the RMS and was strikingly similar to the embryonic cerebral cortex. In contrast to the adult RMS, we found no evidence for preferential juxtaposition of migratory doublecortin-positive neuroblasts and vasculature in the neonatal RMS. Our work provides an important framework for understanding the precise mechanism behind regulation of proliferation.
Abstract: We investigated the expression and role of the dopamine receptor 3 (D3R) in postnatal mouse subventricular zone (SVZ). In situ hybridization detected selective D3R mRNA expression in the SVZ. Fluorescence activated cell sorting (FACS) of adult SVZ subtypes using hGFAP-GFP and Dcx-GFP mice showed that transit amplifying progenitor cells and niche astrocytes expressed D3R whereas stem cell-like astrocytes and neuroblasts did not. To determine D3R's role in SVZ neurogenesis, we administered U-99194A, a D3R preferential antagonist, and bromodeoxyuridine in postnatal mice. In vivo D3R antagonism decreased the numbers of newborn neurons reaching the core and the periglomerular layer of the olfactory bulb. Moreover, it decreased progenitor cell proliferation but did not change the number of label-retaining (stem) cells, commensurate with its expression on transit amplifying progenitor cells but not SVZ stem cell-like astrocytes. Collectively, this study suggests that dopaminergic stimulation of D3R drives proliferation via rapidly amplifying progenitor cells to promote murine SVZ neurogenesis.
Abstract: The subplate is a largely transient zone containing precocious neurons involved in several key steps of cortical development. The majority of subplate neurons form a compact layer in mouse, but are dispersed throughout a much larger zone in the human. In rodent, subplate neurons are among the earliest born neocortical cells, whereas in primate, neurons are added to the subplate throughout cortical neurogenesis. Magnetic resonance imaging and histochemical studies show that the human subplate grows in size until the end of the second trimester. Previous microarray experiments in mice have shown several genes that are specifically expressed in the subplate layer of the rodent dorsal cortex. Here we examined the human subplate for some of these markers. In the human dorsal cortex, connective tissue growth factor-positive neurons can be seen in the ventricular zone at 15-22 postconceptional weeks (PCW) (most at 17 PCW) and are present in the subplate at 22 PCW. The nuclear receptor-related 1 protein is mostly expressed in the subplate in the dorsal cortex, but also in lower layer 6 in the lateral and perirhinal cortex, and can be detected from 12 PCW. Our results suggest that connective tissue growth factor- and nuclear receptor-related 1-positive cells are two distinct cell populations of the human subplate. Furthermore, our microarray analysis in rodent suggested that subplate neurons produce plasma proteins. Here we demonstrate that the human subplate also expresses α2zinc-binding globulin and Alpha-2-Heremans-Schmid glycoprotein/human fetuin. In addition, the established subplate neuron marker neuropeptide Y is expressed superficially, whereas potassium/chloride co-transporter (KCC2)-positive neurons are localized in the deep subplate at 16 PCW. These observations imply that the human subplate shares gene expression patterns with rodent, but is more compartmentalized into superficial and deep sublayers. This increased complexity of the human subplate may contribute to differential vulnerability in response to hypoxia/ischaemia across the depth of the cortex. Combining knowledge of cell-type specific subplate gene expression with modern imaging methods will enable a better understanding of neuropathologies involving the subplate.
Abstract: Cell polarity plays a key role in the development of the central nervous system (CNS). Interestingly, disruption of cell polarity is seen in many cancers. ASPP2 is a haplo-insufficient tumor suppressor and an activator of the p53 family. In this study, we show that ASPP2 controls the polarity and proliferation of neural progenitors in vivo, leading to the formation of neuroblastic rosettes that resemble primitive neuroepithelial tumors. Consistent with its role in cell polarity, ASPP2 influences interkinetic nuclear migration and lamination during CNS development. Mechanistically, ASPP2 maintains the integrity of tight/adherens junctions. ASPP2 binds Par-3 and controls its apical/junctional localization without affecting its expression or Par-3/aPKC lambda binding. The junctional localization of ASPP2 and Par-3 is interdependent, suggesting that they are prime targets for each other. These results identify ASPP2 as a regulator of Par-3, which plays a key role in controlling cell proliferation, polarity, and tissue organization during CNS development.
Abstract: BACKGROUND: Long considered to be the building block of life, it is now apparent that protein is only one of many functional products generated by the eukaryotic genome. Indeed, more of the human genome is transcribed into noncoding sequence than into protein-coding sequence. Nevertheless, whilst we have developed a deep understanding of the relationships between evolutionary constraint and function for protein-coding sequence, little is known about these relationships for non-coding transcribed sequence. This dearth of information is partially attributable to a lack of established non-protein-coding RNA (ncRNA) orthologs among birds and mammals within sequence and expression databases. RESULTS: Here, we performed a multi-disciplinary study of four highly conserved and brain-expressed transcripts selected from a list of mouse long intergenic noncoding RNA (lncRNA) loci that generally show pronounced evolutionary constraint within their putative promoter regions and across exon-intron boundaries. We identify some of the first lncRNA orthologs present in birds (chicken), marsupial (opossum), and eutherian mammals (mouse), and investigate whether they exhibit conservation of brain expression. In contrast to conventional protein-coding genes, the sequences, transcriptional start sites, exon structures, and lengths for these non-coding genes are all highly variable. CONCLUSIONS: The biological relevance of lncRNAs would be highly questionable if they were limited to closely related phyla. Instead, their preservation across diverse amniotes, their apparent conservation in exon structure, and similarities in their pattern of brain expression during embryonic and early postnatal stages together indicate that these are functional RNA molecules, of which some have roles in vertebrate brain development.
Abstract: The major lineages of mammals (Eutheria, Metatheria, and Monotremata) diverged more than 100 million years ago and have undergone independent changes in the neocortex. We found that adult South American gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii) possess a significantly lower number of cerebral cortical neurons compared with the mouse (Mus musculus). To determine whether the difference is reflected in the development of the cortical germinal zones, the location of progenitor cell divisions was examined in opossum, tammar wallaby, and rat. The basic pattern of the cell divisions was conserved, but the emergence of a distinctive band of dividing cells in the subventricular zone (SVZ) occurred relatively later in the opossum (postnatal day [P14]) and the tammar wallaby (P40) than in rodents. The planes of cell divisions in the ventricular zone (VZ) were similar in all species, with comparable mRNA expression patterns of Brn2, Cux2, NeuroD6, Tbr2, and Pax6 in opossum (P12 and P20) and mouse (embryonic day 15 and P0). In conclusion, the marsupial neurodevelopmental program utilizes an organized SVZ, as indicated by the presence of intermediate (or basal) progenitor cell divisions and gene expression patterns, suggesting that the SVZ emerged prior to the Eutherian-Metatherian split.
Abstract: Many specifically human psychiatric and neurological conditions have developmental origins. Rodent models are extremely valuable for the investigation of brain development, but cannot provide insight into aspects that are specifically human. The human brain, and particularly the cerebral cortex, has some unique genetic, molecular, cellular and anatomical features, and these need to be further explored. Cortical expansion in human is not just quantitative; there are some novel types of neurons and cytoarchitectonic areas identified by their gene expression, connectivity and functions that do not exist in rodents. Recent research into human brain development has revealed more elaborated neurogenetic compartments, radial and tangential migration, transient cell layers in the subplate, and a greater diversity of early-generated neurons, including predecessor neurons. Recently there has been a renaissance of the study of human brain development because of these unique differences, made possible by the availability of new techniques. This review gives a flavour of the recent studies stemming from this renewed focus on the developing human brain.
Abstract: Much of the original historical data behind the greatest discoveries in neuroscience are now lost. However, a recently rediscovered box of histological slides belonging to Sir Charles Sherrington, a pioneer in spinal cord and motor control research, has survived at the University of Oxford since 1936. Sherrington coined the term 'synapse', developed the concept of inhibition in neuronal function, demonstrated the integration of sensory and motor actions of the nervous system, and examined the synaptic activity of single neurons and their integration into neuronal circuits. Here, we explore Sherrington's lifetime of discoveries, with reference to histological specimens from his box of slides.
Abstract: BACKGROUND: Laser capture microdissection enables the isolation of single cells or small cell groups from histological sections under direct microscopic observation. Combined with quantitative PCR or microarray, it is a very powerful approach for studying gene expression profiles in discrete cell populations. The major challenge for such studies is to obtain good quality RNA from small amounts of starting material. RESULTS: We have developed a simple, flexible, and low-cost method for simultaneously producing RNA from discrete cell groups in embryonic day 15 mouse brain. In particular, we have optimized the following key steps in the procedure: staining, cryosectioning, storage of sections and harvesting of microdissected cells. We obtained the best results when staining 20 mum-thick sections with 1% cresyl violet in 70% ethanol and harvesting the microdissected tissue in RNA stabilization solution. In addition, we introduced three stop-points in the protocol which makes the tedious process of laser capture microdissection more flexible, without compromising RNA quality. CONCLUSION: Using this optimized method, we have consistently obtained RNA of high quality from all four simultaneously microdissected cell groups. RNA integrity numbers were all above 8, and long cDNA fragments (> 1.2 kb) were successfully amplified by reverse transcription PCR from all four samples. We conclude that RNAs isolated by this method are well suited for downstream quantitative PCR or microarray studies.
Abstract: The transcription factor Pax6 has been implicated in neocortical neurogenesis in vertebrates, including humans. Analyses of the role of Pax6 in layer formation and cognitive abilities have been hampered by perinatal lethality of Pax6 mutants. Here, we generated viable mutants exhibiting timed, restricted inactivation of Pax6 during early and late cortical neurogenesis using Emx1-Cre and hGFAP-Cre lines, respectively. The disruption of Pax6 at the onset of neurogenesis using Emx1-Cre line resulted in premature cell cycle exit of early progenitors, increase of early born neuronal subsets located in the marginal zone and lower layers, and a nearly complete absence of upper layer neurons, especially in the rostral cortex. Furthermore, progenitors, which accumulated in the enlarged germinal neuroepithelium at the pallial/subpallial border in the Pax6 mutants, produced an excess of oligodendrocytes. The inactivation of Pax6 after generation of the lower neuronal layers using hGFAP-Cre line did not affect specification or numbers of late-born neurons, indicating that the severe reduction of upper layer neurons in Pax6 deficiency is mostly attributable to a depletion of the progenitor pool, available for late neurogenesis. We further show that Pax6(fl/fl);Emx1-Cre mutants exhibited deficiencies in sensorimotor information integration, and both hippocampus-dependent short-term and neocortex-dependent long-term memory recall. Because a majority of the morphological and behavior disabilities of the Pax6 mutant mice parallel abnormalities reported for aniridia patients, a condition caused by PAX6 haploinsufficiency, the Pax6 conditional mutant mice generated here represent a valuable genetic tool to understand how the developmental cortical disruption can lead to a human behavior abnormality.
Abstract: The establishment of connectivity between specific thalamic nuclei and cortical areas involves a dynamic interplay between the guidance of thalamocortical axons and the elaboration of cortical areas in response to appropriate innervation. We show here that Sema6A mutants provide a unique model to test current ideas on the interactions between subcortical and cortical guidance mechanisms and cortical regionalization. In these mutants, axons from the dorsal lateral geniculate nucleus (dLGN) are misrouted in the ventral telencephalon. This leads to invasion of presumptive visual cortex by somatosensory thalamic axons at embryonic stages. Remarkably, the misrouted dLGN axons are able to find their way to the visual cortex via alternate routes at postnatal stages and reestablish a normal pattern of thalamocortical connectivity. These findings emphasize the importance and specificity of cortical cues in establishing thalamocortical connectivity and the spectacular capacity of the early postnatal cortex for remapping initial sensory representations.
Abstract: There is evidence for interaction between the developing circulatory and nervous systems. Blood vessels provide a supporting niche in regions of adult neurogenesis. Here we present a systematic analysis of vascular development in the embryonic murine cortex and demonstrate that dividing cells, including Tbr2-positive intermediate progenitor cells, are closer to the vasculature than expected from a random distribution. To examine whether neurites of the newly generated embryonic neurons find blood vessels as an attractive and permissive substrate, we overlayed green fluorescent protein (GFP)-labeled dissociated cortical progenitors on embryonic organotypic cortical slice cultures with labeled vasculature. Our observations of neurites extending toward and along labeled blood vessels support the notion of vascular-neuronal interactions. The altered cortical layering had no obvious effect on the vascular patterns within the cortical plate (CP) in shaking rat Kawasaki (SRK) and the reeler mutant mouse at the ages studied (E19 and P3). It appears that similarly to other neurogenic regions in the adult, the embryonic "vascular niche" might influence neural progenitor cells during telencephalic neurogenesis, neuronal migration, and neurite extension, but the laminar phenotype of cell classes within the CP has limited influence on the developing vasculature.
Abstract: The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.
Abstract: The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor-related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.
Abstract: In the Golli-tau-eGFP (GTE) transgenic mouse the reporter gene expression is largely confined to the layer of subplate neurons (SPn), providing an opportunity to study their intracortical and extracortical projections. In this study, we examined the thalamic afferents and layer IV neuron patterning in relation to the SPn neurites in the developing barrel cortex in GTE mouse at ages embryonic day 17 (E17) to postnatal day 14 (P14). Serotonin transporter immunohistochemistry or cytochrome oxydase histochemistry was used to reveal thalamic afferent patterning. Bizbenzimide staining identified the developing cytoarchitecture in coronal and tangential sections of GTE brains. Enhanced green fluorescent protein (GFP)-labelled neurites and thalamic afferents were both initially diffusely present in layer IV but by P4-P6 both assumed the characteristic periphery-related pattern and became restricted to the barrel hollows. This pattern gradually changed and by P10 the GFP-labelled neurites largely accumulated at the layer IV-V boundary within the barrel septa whereas thalamic afferents remained in the hollows. To investigate whether this reorganisation is dependent on sensory input, the whiskers of row 'a' or 'c' were removed at P0 or P5 and the organisation of GFP-labelled neurites in the barrel cortex was examined at P6 or P10. In the contralateral region corresponding to row 'a' or 'c' the lack of hollow to septa rearrangement of the GFP-labelled neurites was observed after P0 row removal at P10 but not at P6. Our findings suggest a dynamic, sensory periphery-dependent integration of SPn neurites into the primary somatosensory cortex during the period of barrel formation.
Abstract: Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.
Abstract: The neurons of the mammalian neocortex are organised into six layers. By contrast, the reptilian and avian dorsal cortices only have three layers which are thought to be equivalent to layers I, V and VI of mammals. Increased repertoire of mammalian higher cognitive functions is likely a result of an expanded cortical surface area. The majority of cortical cell proliferation in mammals occurs in the ventricular zone (VZ) and subventricular zone (SVZ), with a small number of scattered divisions outside the germinal zone. Comparative developmental studies suggest that the appearance of SVZ coincides with the laminar expansion of the cortex to six layers, as well as the tangential expansion of the cortical sheet seen within mammals. In spite of great variation and further compartmentalisation in the mitotic compartments, the number of neurons in an arbitrary cortical column appears to be remarkably constant within mammals. The current challenge is to understand how the emergence and elaboration of the SVZ has contributed to increased cortical cell diversity, tangential expansion and gyrus formation of the mammalian neocortex. This review discusses neurogenic processes that are believed to underlie these major changes in cortical dimensions in vertebrates.
Abstract: Transcription factor Pax6 exerts a prominent rostrolateral(high) to caudomedial(low) expression gradient in the cortical progenitors and have been implicated in regulation of area identity in the mammalian cortex. Herein, we analyzed the role of Pax6 in molecular arealization and development of thalamocortical connections in the juvenile cortex-specific conditional Pax6 knock-out mice (Pax6cKO). Using a set of molecular markers of positional identity (Id2, Cadherin6, COUP-TF1, RZRbeta, and EphA7), we show that, in the juvenile Pax6cKO, the relative size of caudal cortical areas (putative visual and somatosensory) are mildly enlarged, whereas the rostral domain (putative motor) is severely reduced. Despite the rostral shift of graded expression of areal markers, the distribution of area-specific thalamocortical and corticofugal projections appear normal in the Pax6cKO. This indicates that change of the size of cortical areas is not accompanied by a change in cortical identity. We show furthermore that, despite a severe depletion of supragranular cortical layers and accumulation of cells along the pallial-subpallial boundary, thalamocortical fibers establish a periphery-related pattern of the somatosensory cortex in normal position in Pax6cKO. Our findings indicate that Pax6 expression gradients in cortical progenitors do not directly impart thalamocortical or corticofugal areal identity.
Abstract: The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.
Abstract: The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.
Abstract: Building the brain is like erecting a house of cards. The early connections provide the foundation of the adult structure, and disruption of these may be the source of many developmental flaws. Cerebral cortical developmental disorders (including schizophrenia and autism) and perinatal injuries involve cortical neurons with early connectivity. The major hindrance of progress in understanding the early neural circuits during cortical development and disease has been the lack of reliable markers for specific cell populations. Due to the advance of powerful approaches in gene expression analysis and the utility of models with reporter gene expressions in specific cortical cell types, our knowledge of the early cortical circuits is rapidly increasing. With focus on the sub-plate, layer VI and layer V projection neurons, we shall illustrate the progress made in the understanding of their neurochemical properties, physiological characteristics and their integration into the early intracortical and extracortical circuitry. This field benefited from recent developments in mouse genetics in generating models with subtype specific gene expression patterns, powerful cell dissection and separation methods combined with microarray analysis. The emergence of cortical cell type specific biomarkers will not only help neuropathological diagnosis, but will also eventually reveal the causal relations in the pathogenesis of various cortical developmental disorders.
Abstract: The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic beta-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.
Abstract: Embryonic germinal zones of the dorsal and ventral telencephalon generate cortical neurons during the final week of gestation in rodent and during several months in human. Whereas the vast majority of cortical interneurons originate from the ventral telencephalon, excitatory neurons are locally generated within the germinal zone of the dorsal telencephalon, the future cerebral cortex, itself. However, a number of studies have described proliferating cells external to the ventricular and subventricular germinal zones in the developing dorsal telencephalon. In this study, we performed a comprehensive cell density analysis of such 'extra-ventricular proliferating cells' (EVPCs) during corticogenesis in rat and human using a mitotic marker anti-phospho-histone H3. Subsequently, we performed double-labelling studies with other mitotic and cell type specific markers to undertake phenotypic characterisation of EVPCs. Our findings show: (1) the densities of extra-ventricular H3-positive (H3+) cells were surprisingly similar in preplate stage rat and human; (2) extra-ventricular proliferation continues during mid-and late corticogenesis in rat and in early fetal human cortex; and (3) extra-ventricular cells appear to be mitotic precursors as they are not immunoreactive for a panel of early post-mitotic and cell type-specific markers, although (4) a subset of EVPCs are proliferating microglia. These data suggest that some aspects of early corticogenesis are conserved between rodent and human despite marked differences in the duration of neurogenesis and the anatomical organisation of the developing cerebral cortex.
Abstract: The mammalian neocortex consists of six layers. By contrast, the reptilian and avian cortices have only three, which are believed to be equivalent to layers I, V and VI of mammals. In mammals, the majority of cortical cell proliferation occurs in the ventricular and subventricular zones, but there are a small number of scattered individual divisions throughout the cortex. Neurogenesis in the cortical subventricular zone is believed to contribute to the supragranular layers. To estimate the proportions of different forms of divisions in reptiles and birds, we examined the site of proliferation in embryonic turtle (stages 18-25) and chick (embryonic days 8-15) brains using phospho-histone H3 (a G2 and M phase marker) immunohistochemistry. In turtle, only few scattered abventricular H3-immunoreactive cells were found outside the ventricular zone; the majority of the H3-immunoreactive cells were located in the ventricular zone throughout the entire turtle brain. Ventricular zone cell proliferation peaks at stages 18 and 20, before an increase of abventricular proliferation at stages 23 and 25. In turtle cortex, however, abventricular proliferation at any given stage never exceeded 17.5+/-2.47% of the total division and the mitotic profiles did not align parallel to the ventricular zone. Phospho-histone H3 immunoreactivity in embryonic chick brains suggests the lack of subventricular zone in the dorsal cortex, but the presence of subventricular zone in the ventral telencephalon. We were able to demonstrate that the avian subventricular zone is present in both pallial and subpallial regions of the ventral telencephalon during embryonic development, and we characterize the spatial and temporal organization of the subventricular zone. Comparative studies suggest that the subventricular zone was involved in the laminar expansion of the cortex to six layers in mammals from the three-layered cortex found in reptiles and birds. Within mammals, the number of neurons in a cortical column appears to be largely constant; nevertheless, there are considerable differences between the germinal zones in mammalian species. It is yet to be determined whether these elaborations of the subventricular zone may have contributed to cell diversity, tangential expansion or gyrus formation of the neocortex and whether it might have been the major driving force behind the evolution of the six-layered neocortex in mammals.
Abstract: Origin, timing and direction of neuronal migration during brain development determine the distinct organization of adult structures. Changes in these processes might have driven the evolution of the forebrain in vertebrates. GABAergic neurons originate from the ganglionic eminence in mammals and migrate tangentially to the cortex. We are interested in differences and similarities in tangential migration patterns across corresponding telencephalic territories in mammals and reptiles. Using morphological criteria and expression patterns of Darpp-32, Tbr1, Nkx2.1 and Pax6 genes, we show in slice cultures of turtle embryos that early cohorts of tangentially migrating cells are released from the medial ganglionic eminence between stages 14 and 18. Additional populations migrate tangentially from the dorsal subpallium. Large cohorts of tangentially migrating neurons originate ventral to the dorsal ventricular ridge at stage 14 and from the lateral ganglionic eminence from stage 15. Release of GABAergic cells from these regions was investigated further in explant cultures. Tangential migration in turtle proceeds in a fashion similar to mammals. In chimeric slice culture and in ovo graft experiments, the tangentially migrating cells behaved according to the host environment - turtle cells responded to the available cues in mouse slices and mouse cells assumed characteristic migratory routes in turtle brains, indicating highly conserved embryonic signals between these distant species. Our study contributes to the evaluation of theories on the origin of the dorsal cortex and indicates that tangential migration is universal in mammals and sauropsids.
Abstract: Innervation of the neocortex by the thalamus is dependent on the precise coordination of spatial and temporal guidance cues. In this issue of Cell, work by López-Bendito et al.(2006) reveals that tangentially migrating cells within the ventral telencephalon are essential for axonal navigation between the thalamus and the neocortex, a process apparently mediated by Neuregulin-1/ErbB4 short- and long-range signaling.
Abstract: The earliest generated cells of the mammalian cerebral cortex form the preplate layer (PPL). The subsequently born cortical plate (CP) cells split this layer into the superficial layer I (LI) and the deep subplate (SP). The cellular and molecular mechanisms that underlie this event are unclear. To investigate the role of the cyclin-dependent kinase 5 (Cdk5) and its activator p35 in preplate splitting, we used Nissl staining, carbocyanine dye tracing, cell birthdating, and immunohistochemistry for calretinin (CalR) in p35 and Cdk5 knockout mice. Our data demonstrated changes in early cortical lamination and aberrant thalamic axon trajectories in these mice. Specifically, LI was thicker, and cell-dense and thalamic axons did not accumulate in the SP layer before invading the CP. Instead, they grew past the SP and more superficial cortical layers and coursed obliquely toward the pial surface. This behavior has been previously observed in reeler mice and suggests a defect in PPL splitting. CalR immunohistochemistry and bromo-deoxyuridine birthdating confirmed the abnormality in position of the earliest generated cortical cells of mutants. These observations suggest that the p35/Cdk5 pathway plays a role in preplate splitting in addition to regulating layer formation.
Abstract: The nature of cerebral cortical circuitry has been increasingly clarified by markers for the identification of precise cell types with specific morphology, connectivity and distinct physiological properties. Molecular markers are not only helpful in dissecting cortical circuitry, but also give insight into the mechanisms of cortical neuronal specification and differentiation. The two principal neuronal types of the cerebral cortex are the pyramidal and GABAergic cells. Pyramidal cells are excitatory and project to distant targets, while GABAergic neurons are mostly inhibitory non-pyramidal interneurons. Reliable markers for specific subtypes of interneurons are available and have been employed in the classification and functional analysis of cortical circuitry. Until recently, cortical pyramidal neurons have been considered a homogeneous class of cells. This concept is now changing as the powerful tools of molecular biology and genetics identify molecular tags for subtypes of pyramidal cells such as: Otx-1 [Frantz, G.D., Bohner, A.P., Akers, R.M., McConnell, S.K., 1994. Regulation of the POU domain gene SCIP during cerebral cortical development. J. Neurosci. 14, 472-485; Weimann, J.M., Zhang, Y.A., Levin, M.E., Devine, W.P., Brulet, P., McConnell, S.K., 1999. Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 24, 819-831]; SMI-32, N200 and FNP-7 [Voelker, C.C., Garin, N., Taylor, J.S., Gahwiler, B.H., Hornung, J.P., Molnár, Z., 2004. Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of layer V pyramidal neurons in vivo and in vitro. Cereb. Cortex 14, 1276-1286]; ER81 [Hevner, R.F., Daza, R.A., Rubenstein, J.L., Stunnenberg, H., Olavarria, J.F., Englund, C., 2003. Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons. Dev. Neurosci. 25 (2-4), 139-151; Yoneshima, H., Yamasaki, S., Voelker, C., Molnár, Z., Christophe, E., Audinat, E., Takemoto, M., Tsuji, S., Fujita, I., Yamamoto, N., 2006. ER81 is expressed in a subpopulation of layer 5 projection neurons in rodent cerebral cortices. Neuroscience, 137, 401-412]; Lmo4 [Bulchand, S., Subramanian, L., Tole, S., 2003. Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex. Dev. Dyn. 226, 460-469; Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207-221]; CTIP2 [Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207-221]; Fez1 [Molyneaux, B.J., Arlotta, P., Hirata, T., Hibi, M., Macklis, J.D., 2005. Fez1 is required for the birth and specification of corticospinal motor neurons. Neuron 47 (6), 817-831; Chen, B., Schaevitz, L.R., McConnell, S.K., 2005. Fez1 regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc. Natl. Acad. Sci. U.S.A. 102 (47), 17184-17189]. These genes outline the numerous subtypes of pyramidal cells and are increasingly refining our previous classifications. They also indicate specific developmental programs operate in cell fate decisions. This review will describe the progress made on the correlation of these markers to each other within a specific subtype of layer V neurons with identified, stereotypic projections. Further work is needed to link these data with observations on somatodendritic morphology and physiological properties. The integrated molecular, anatomical and physiological characterisation of pyramidal neurons will lead to a much better appreciation of functional cortical circuits.
Abstract: We describe a distinctive, widespread population of neurons situated beneath the pial surface of the human embryonic forebrain even before complete closure of the neural tube. These 'predecessor' cells include the first neurons seen in the primordium of the cerebral cortex, before the onset of local neurogenesis. Morphological analysis, combined with the study of centrosome location, regional transcription factors and patterns of mitosis and neurogenesis, indicates that predecessor cells invade the cortical primordium by tangential migration from the subpallium. These neurons, described here for the first time, precede all other known cell types of the developing cortex.
Abstract: This review aims to provide examples of how both comparative and genetic analyses contribute to our understanding of the rules for cortical development and evolution. Genetic studies have helped us to realize the evolutionary rules of telencephalic organization in vertebrates. The control of the establishment of conserved telencephalic subdivisions and the formation of boundaries between these subdivisions has been examined and the very specific alterations at the striatocortical junction have been revealed. Comparative studies and genetic analyses both demonstrate the differential origin and migratory pattern of the two basic neuron types of the cerebral cortex. GABAergic interneurons are mostly generated in the subpallium and a common mechanism governs their migration to the dorsal cortex in both mammals and sauropsids. The pyramidal neurons are generated within the cortical germinal zone and migrate radially, the earliest generated cell layers comprising preplate cells. Reelin-positive Cajal-Retzius cells are a general feature of all vertebrates studied so far; however, there is a considerable amplification of the Reelin signalling with cortical complexity, which might have contributed to the establishment of the basic mammalian pattern of cortical development. Based on numerous recent observations we shall present the argument that specialization of the mitotic compartments may constitute a major drive behind the evolution of the mammalian cortex. Comparative developmental studies have revealed distinct features in the early compartments of the developing macaque brain, drawing our attention to the limitations of some of the current model systems for understanding human developmental abnormalities of the cortex. Comparative and genetic aspects of cortical development both reveal the workings of evolution.
Abstract: During development, several populations of progenitor cells in the dorsal telencephalon generate a large variety of neurons which acquire distinct morphologies and physiological properties and serve distinct functions in the mammalian cortex. This paper reviews recent work that has identified (i) key molecules involved in the specification and differentiation of cortical neurons, (ii) novel genes which distinguish distinct subsets of cortical progenitors and may be involved in the diversification of cortical neurons present in different cortical layers, and (iii) mechanisms involved in the generation of different projection neuronal subtypes in the well-studied model of layer 5 of the rodent cortex.
Abstract: Laminar organization is a fundamental cytoarchitecture in mammalian CNS and a striking feature of the neocortex. ER81, a transcription factor, has recently been utilized as a marker of cells in the layer 5 of the neocortex. We further pursued the distribution of ER81 to investigate the identity of the ER81-expressing cells in the brain. Er81 transcript was expressed in a subset of pyramidal cells that were scattered throughout the entire width of layer 5. In the rat cortex, Er81 transcripts were first detected in the ventricular zone at E15, remained expressed in putative prospective layer 5 neurons during infant and juvenile stages. The ER81-expressing subpopulation in adult layer 5 neurons did not segregate with the phenotypes of the projection targets. By retrograde labeling combined with immunohistochemistry or reverse transcription-polymerase chain reaction analysis, we found ER81 expression in nearly all of the layer 5 neurons projecting to the spinal cord or to the superior colliculus, while in only one-third of the layer 5 neurons projecting to the contralateral cortex. Er81 was also detected in layer 5 neurons in a P2 Japanese macaque monkey but not in adult monkey cortices. These findings suggest that a neuron class defined by a molecular criterion does not necessarily segregate with that defined by an anatomical criterion, that ER81 is involved in cell differentiation of a subset of layer 5 projection neurons and that this mechanism is conserved among rodents and primates.
Abstract: The cortex receives its major sensory input from the thalamus via thalamocortical axons, and cortical neurons are interconnected in complex networks by corticocortical and callosal axons. Our understanding of the mechanisms generating the circuitry that confers functional properties on cortical neurons and networks, although poor, has been advanced significantly by recent research on the molecular mechanisms of thalamocortical axonal guidance and ordering. Here we review recent advances in knowledge of how thalamocortical axons are guided and how they maintain order during that process. Several studies have shown the importance in this process of guidance molecules including Eph receptors and ephrins, members of the Wnt signalling pathway and members of a novel planar cell polarity pathway. Signalling molecules and transcription factors expressed with graded concentrations across the cortex are important in establishing cortical maps of the topography of sensory surfaces. Neural activity, both spontaneous and evoked, plays a role in refining thalamocortical connections but recent work has indicated that neural activity is less important than was previously thought for the development of some early maps. A strategy used widely in the development of corticocortical and callosal connections is the early overproduction of projections followed by selection after contact with the target structure. Here we discuss recent work in primates indicating that elimination of juvenile projections is not a major mechanism in the development of pathways feeding information forward to higher levels of cortical processing, although its use is common to developing feedback pathways.
Abstract: Donald Hebb postulated the existence of a mechanism of activity-dependent transcription and synaptic modification almost 60 years ago. While the details of this process are still unclear, a new study by Ince-Dunn et al. in this issue of Neuron indicates that NeuroD2, a calcium-regulated transcription factor, plays a central role in thalamocortical synaptic maturation.
Abstract: Transient synapse formation between thalamic axons and subplate neurons is thought to be important in thalamocortical targeting. Shaking rat Kawasaki (SRK), having reversed cortical layering similarly observed in reeler mouse, provides an interesting model system to test this idea. The spatial and temporal pattern of excitation was investigated using optical recording with voltage-sensitive dyes in thalamocortical slice preparations from SRK. At postnatal day 0 (P0), a strong optical response was elicited within the superplate of the SRK in the cell layer corresponding to subplate in wild-type (WT) rats. By P3, this response rapidly descended into deep cortical layers comprised of layer IV cells, as identified with 5-bromo-2'-deoxyuridine birthdating at embryonic day 17. During the first 3 postnatal days, both the subplate and cortical plate responses were present, but by P7, the subplate response was abolished. Tracing individual axons in SRK revealed that at P0-P3, a large number of thalamocortical axons reach the superplate, and by P7-P10, the ascending axons develop side branches into the lower or middle cortical layers. Synaptic currents were also demonstrated in WT subplate cells and in SRK superficial cortical cells using whole-cell recording. These currents were elicited monosynaptically, because partial AMPA current blockade did not modify the latencies. These results suggest that the general developmental pattern of synapse formation between thalamic axons and subplate (superplate) neurons in WT and SRK is very similar, and individual thalamic arbors in cortex are considerably remodeled during early postnatal development to find layer IV equivalent neurons.
Abstract: 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.
Abstract: Previous studies have shown that layer V pyramidal neurons projecting either to subcortical structures or the contralateral cortex undergo different morphological and electrophysiological patterns of development during the first three postnatal weeks. To isolate the determinants of this differential maturation, we analyzed the gene expression and intrinsic membrane properties of layer V pyramidal neurons projecting either to the superior colliculus (SC cells) or the contralateral cortex (CC cells) by combining whole cell recordings and single-cell RT-PCR in acute slices prepared from postnatal day (P) 5-7 or P21-30 old mice. Among the 24 genes tested, the calcium channel subunits alpha1B and alpha1C, the protease Nexin 1, and the calcium-binding protein calbindin were differentially expressed in adult SC and CC cells and the potassium channel subunit Kv4.3 was expressed preferentially in CC cells at both stages of development. Intrinsic membrane properties, including input resistance, amplitude of the hyperpolarization-activated current, and action potential threshold, differed quantitatively between the two populations as early as from the first postnatal week and persisted throughout adulthood. However, the two cell types had similar regular action potential firing behaviors at all developmental stages. Surprisingly, when we increased the duration of anesthesia with ketamine-xylazine or pentobarbital before decapitation, a proportion of mature SC cells, but not CC cells, fired bursts of action potentials. Together these results indicate that the two populations of layer V pyramidal neurons already start to differ during the first postnatal week and exhibit different firing capabilities after anesthesia.
Abstract: Intrinsic genetic differences and extrinsic influences both contribute to the formation of functional and anatomical areas within the mammalian cerebral cortex. Using subtractive hybridisation and differential screening, we demonstrated that plasticity-related gene-3 (PRG-3) is differentially expressed between the dorsal and lateral cortex at P6. PRG-3 is one of the key enzymes that are involved in the metabolism of phospholipids, such as LPA and S1P, in the nervous system. Here, for the first time, we report the mRNA expression pattern of PRG-3 in the developing and adult mouse cerebral cortex.
Abstract: We used a combination of immunohistochemistry and carbocyanine dye tracing to study neurons and their processes in the human embryonic forebrain, 4-7 weeks after conception, before the onset of synaptogenesis. We discovered a widespread network of precocious MAP2 (microtubule-associated protein 2)-immunoreactive cells, with long, nonaxonal processes, before the appearance of the cortical plate and the establishment of thalamocortical connectivity. Dye tracing revealed that the processes of these precocious cells form tangential links between intermediate zones of the thalamus, ganglionic eminence, hypothalamus, and cortical preplate. The spatiotemporal distribution and morphology of the precocious neurons in the cortical preplate suggest that they are generated outside the cerebral wall rather than in the local ventricular zone. The first thalamocortical axons and axons of preplate cells extend across diencephalo-telencephalic and striatocortical boundaries before the arrival of the first cortical plate neurons. Precocious cells may provide initial communication between subdivisions of the embryonic brain as well as guidance cues for navigation of growing axons and/or transverse neuronal migration.
Abstract: To identify the origin and track the migratory pathway of specific subpopulations of GABAergic interneurons, we studied tangential migration in a recently developed GAD65-GFP transgenic mouse strain. First, we used immunohistochemical methods to characterize the expression of specific neurochemical markers in the GAD65-GFP neurons. Then, organotypic cultures were used in combination with birth-dating studies to determine the time of generation, place of origin and migratory route of these cells. From E14 to E15, the highest density of GAD65-GFP cells was seen in the lower intermediate zone; however, at later stages more GAD65-GFP cells were observed in the subventricular zone. Migratory GAD65-GFP cells express GAD65, but not calretinin or reelin. Surprisingly, only 4% were calbindin immunopositive. At P21, GAD65-GFP cells were found predominantly in layers II-III and expressed calretinin and neuropeptide Y. Remarkably, almost all cholecystokinin-positive but very few parvalbumin-positive neurons expressed GFP. In vitro studies demonstrated that the caudal ganglionic eminence gives rise to a large proportion of GAD65-GFP interneurons and in vivo birth-dating experiments showed that GAD65-GFP interneurons in supragranular layers are born at late embryonic development. Taken together these results support the idea that the destination layer of GABAergic interneurons is closely linked to their place of origin and time of generation.
Abstract: There are two main types of layer V pyramidal neurons in rat cortex. Type I neurons have tufted apical dendrites extending into layer I, produce bursts of action potentials and project to subcortical targets (spinal cord, superior colliculus and pontine nuclei). Type II neurons have apical dendrites, which arborize in layers II-IV, do not produce bursts of action potentials and project to ipsilateral and contralateral cortex. The specific expression of different genes and proteins in these two distinct layer V neurons is unknown. To distinguish between distinct subpopulations, fluorescent microspheres were injected into subcortical targets (labeling type I neurons) or primary somatosensory cortex (labeling type II neurons) of adult rats. After transport, cortical sections were processed for immunohistochemistry using various antibodies. This study demonstrated that antigens recognized by SMI-32, N200 and FNP-7 antibodies were only expressed in subcortical (type I)--but not in contralateral (type II)--projecting neurons. NR1, NR2a/b, PLCbeta1, BDNF, NGF and TrkB antigens were highly expressed in all neuronal subpopulations examined. Organotypic culture experiments demonstrated that the development of neurofilament expression and laminar specificity does not depend on the presence of the subcortical targets. This study suggests specific markers for the subcortical projecting layer V neuron subpopulations.
Abstract: We analysed the laminar distribution of transmembrane currents from embryonic (E) day 17 until adulthood after selective thalamic stimulation in slices of rat forebrain to study the development of functional thalamocortical and cortico-cortical connections. At E18 to birth a short-latency current sink was observed in the subplate and layer 6, which was decreased, but not fully abolished in a cobalt containing solution or after the application of glutamate receptor blockers (APV and DNQX). This indicated that embryonic thalamic axons were capable of conducting action potentials to the cortex and some of them had already formed functional synapses there. Between birth and P3, when thalamic axons were completing their upward growth, a sink gradually appeared more superficially in the dense cortical plate and synchronously, a current source aroused in layer 5. Both sinks and sources completely disappeared after blocking synaptic transmission. The adult-like distribution of CSDs became apparent after P7. The component in layer 6 cannot be blocked completely after this age suggesting antidromic activation. This study demonstrated that cells of the lowest layers of the cortex received functional thalamic input before birth and that thalamocortical axons formed synapses with more superficial cells as they grew into the cortical plate.
Abstract: 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.
Abstract: Thalamic axons, which carry most of the information from the sensory environment, are amongst the first projections to reach the cerebral cortex during embryonic development. It has been proposed that the scaffold of early generated cells in the ventral thalamus, internal capsule and preplate play a pivotal role in their deployment through sharp gene expression boundaries. These ideas were recently evaluated in various strains of mutant mice. In Tbr1, Gbx2, Pax6 KO both thalamic and corticofugal projections fail to traverse the striatocortical junction. In both Emx2 and Pax6 KO brains, the misrouted thalamic afferents are accompanied by displacements of the pioneering projections from the internal capsule. Regardless of their altered route, thalamic afferents in the reeler and L1 KO mice seem to be able to redistribute themselves on the cortical sheet and establish normal periphery-related representation in the somatosensory cortex. Early neural activity delivered through the thalamic projections is thought to be involved in the realignment process of thalamic axons at the time of their accumulation in the subplate layer. However, axonal growth and the early topographic arrangement of thalamocortical fiber pathways appear normal in the Snap25 KO, where action potential mediated synaptic vesicle release is disrupted. We therefore suggest that intercellular communication mediated by constitutive secretion of transmitters or growth factors might play a dominant role during early thalamocortical development.
Abstract: The deep layers of the mammalian cerebral cortex contain pyramidal neurons that project predominantly to subcortical targets. To understand the mechanisms that determine the identity of deeper layer neurons, a PCR based subtractive hybridisation was performed to isolate genes that are specifically expressed during the specification of these neurons. One of the genes we isolated was the rat homologue of the mouse Slap-1. SLAP-1 is an adaptor protein containing SH2-SH3 domains and it participates in the signalling of Receptor Tyrosine Kinases. In situ hybridisation studies have shown that Slap-1 is not substantially expressed before E17.At later stages, it is specifically and selectively expressed by deeper layer neurons and by neurons of layers II/III in the developing cortex. The specific timing and location of its expression, suggests that this gene may play a role in the differentiation of these neurons.
Abstract: The transcription factor PAX6 has been implicated in forebrain patterning, cerebral cortical arealization and in development of thalamocortical connections. Using a Pax6/lacZ knockout mouse, in which the endogenous Pax6 expression is reflected by beta-galactosidase activity, we have studied the consequences of the loss of Pax6 function on thalamocortical (TCA) and corticofugal axon (CFA) pathfinding during the period of embryonic day (E) 14.5 to E18.5. Carbocyanine dye tracing in Pax6 heterozygotes (Pax6(+/-)) and Pax6 wild-type (Pax6(+/+)) brains revealed that CFAs and TCAs temporarily arrested their growth at E14.5 at the border of the beta-galactosidase-positive region at the pallial/subpallial boundary (PSPB), before they continued towards their targets. However, in Pax6 homozygous (Pax6(-/-)) embryos, CFAs and TCAs were unable to encounter each other at the PSPB and reach their final targets. Instead of crossing the PSPB, they had the tendency to descend into the ventral pallium in large aberrant fascicles. In addition, cells with a presumptive guide-post function, which are normally situated in the ventral thalamus, internal capsule and hypothalamus, were more dispersed in the hypothalamus and ventral pallium. These pathfinding defects were confirmed by immunohistochemistry for L1 and TAG1, markers of the early axonal connections. The aberrant development of axonal connections in absence of Pax6 function appear to be related to ultrastructural defects of cells along the PSPB, as well as to a failure of axonal guidance molecule expression, including Sema3c and Sema5a.
Abstract: To elucidate the formation of early thalamocortical synapses we recorded optical images with voltage-sensitive dyes from the cerebral cortex of prenatal rats by selective thalamic stimulation of thalamocortical slice preparations. At embryonic day (E) 17, thalamic stimulation elicited excitation that rapidly propagated through the internal capsule to the cortex. These responses lasted less than 15 ms, and were not affected by the application of glutamate receptor antagonists, suggesting that they might reflect presynaptic fiber responses. At E18, long-lasting (more than 300 ms) responses appeared in the internal capsule and in subplate. By E19, long-lasting responses increased in the cortical subplate. By E21, shortly before birth, the deep cortical layers were also activated in addition to the subplate. These long-lasting responses seen in the internal capsule and subplate were blocked by the antagonist perfusion, but the first spike-like responses still remained. The laminar location of the responses was confirmed in the same slices by Nissl staining and subplate cells were labeled by birthdating with bromodeoxyuridine at E13. Our results demonstrate that there is a few days delay between the arrival of thalamocortical axons at the subplate at E16 and the appearance of functional thalamocortical synaptic transmission at E19. Since thalamocortical connections are already functional within the subplate and in the deep cortical plate at embryonic ages, prenatal thalamocortical synaptic connections could influence cortical circuit formation before birth.
Abstract: Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.
Abstract: Embryological development is uniquely positioned to illuminate both hodology in adult brains and its inherited genetic bases. The lateral corner of the lateral ventricle in mammals is a particularly crucial region where cell migration patterns, transiently formed connections, axonal growth kinetics, and fasciculation patterns are complex and interactive. Based on hodology, the sauropsid anterior dorsal ventricular ridge (ADVR) has been proposed as the homologue on a one-to-one basis of the mammalian lateral neocortex (LNC), the basolateral amygdalar complex (BLA), or the claustrum-endopiriform nucleus (CE). Data on gene expression patterns during development have indicated ADVR homology with parts of the latter two structures rather than with LNC. Collothalamic nuclei (the set of dorsal thalamic nuclei that receive their predominant input from the midbrain roof) project to part of BLA and to LNC. Recent findings demonstrate a complex pattern of mutually overlapping but noncongruent gene expression territories and collothalamic projections, which suggests a new, collopallial field hypothesis that the ADVR is homologous as a field to all three structures LNC, BLA, and CE. This hypothesis accounts for current hodological and developmental data as well as for lack of a CE in monotremes and for an abnormal subcortical lamina of gray matter that results from a genetic abnormality in humans.
Abstract: The claustrum, which comprises the claustrum proper and the endopiriform nucleus, is generally thought to be present in all mammals. Some previous reports of its possible absence in monotremes have appeared in the literature, but the question of its presence or absence in this clade has not been formally addressed. Whether monotremes have a claustrum is of some importance for formulating and evaluating hypotheses relating to the evolution of the structures in the lateral sector of the pallium across amniotes. Archival sets of sections through the brains of the platypus and the short-beaked echidna were examined and included material stained for seven different histochemical and immunohistochemical protocols. No cytoarchitectonically distinct claustrum could be identified in this material for either monotreme. We thus conclude that if monotremes have any cell population that is homolgous to the claustrum of therian mammals, it is entirely cryptic. A claustrum might have been present in ancestral mammals and lost in the monotreme clade, or it might have been gained at the origin of therian mammals. Nonetheless, its absence as a cytoarchitectonically discrete and identifiable structure in monotremes fails to support homology of the claustrum of therian mammals with any single part of the sauropsid pallium.
Abstract: The dorsal ventricular ridge (DVR) of reptiles is one of two regions of the reptilian telencephalon that receives input from the dorsal thalamus. Although studies demonstrate that two visual thalamic nuclei, the dorsal lateral geniculate and rotundus, send afferents to the dorsal cortex and DVR, respectively, relatively little is known about physiologic representations. The present study determined the organization of the visual recipient region of the iguana DVR. Microelectrode mapping techniques were used to determine the extent, number of subdivisions, and retinotopy within the visually responsive region of the anterior DVR (ADVR). Visually responsive neurons were restricted to the anterior two thirds of the ADVR. Within this region, two topographically organized subdivisions were determined. Each subdivision contained a full representation of the visual field and could be distinguished from the other by differences in receptive field properties and reversals in receptive field progressions across their mutual border. A third subdivision of the ADVR, in which neurons are responsive to visual stimulation is also described; however, a distinct visuotopic representation could not be determined for this region. This third region forms a shell surrounding the lateral, dorsal, and medial aspects of the topographically organized subdivisions. These results demonstrate that there are multiple physiologic subdivisions in the thalamic recipient zone of the ADVR of the iguana. Comparisons to the ADVR of other reptiles are made, homologies to ectostriatial regions of the bird are proposed, and the findings are discussed in relation to telencephalic organization of other vertebrates.
Abstract: Embryology is the interface of genetic inheritance and phenotypic expression in adult forms, and as such is uniquely positioned to illuminate both. Embryonic cell migration pattern, transient connectivity, axonal growth kinetics and fasciculation patterns can clearly be substantially impacted at the striatocortical junction, which appears to be critical for telencephalic development. Similarly, the big questions concerning pallial evolution in amniotes all involve the pivotal region at the pallial-subpallial boundary, an area where complex developmental cross-currents may be involved in the specification of multiple structures that are thus related to each other. We review some of the positions based on recent genetic data and/or hodology, then suggest that comparative studies of intervening, embryological events may resolve some of the apparent conflicts and illuminate the evolutionary scenario. We propose a new hypothesis, the collopallial field hypothesis, which specifies that the anterior dorsal ventricular ridge of sauropsids and a set of structures in mammals--the lateral neocortex, basolateral amygdalar complex, and claustrum-endopiriform nucleus formation--are homologous to each other as derivatives of a common embryonic field. We propose that in mammals the laterally lying collopallium splits, or differentiates, into deep (claustroamygdalar) and superficial (neocortical) components, whereas in sauropsids, this split does not occur.
Abstract: This study is concerned with the role of impulse activity and synaptic transmission in early thalamocortical development. Disruption of the gene encoding SNAP-25, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor complex required for regulated neuroexocytosis, eliminates evoked but not spontaneous neurotransmitter release (Washbourne et al., 2002). The Snap25 null mutant mouse provides an opportunity to test whether synaptic activity is required for prenatal neural development. We found that evoked release is not needed for at least the gross formation of the embryonic forebrain, because the major features of the diencephalon and telencephalon were normal in the null mutant mouse. However, half of the homozygous mutants showed undulation of the cortical plate, which in the most severely affected brains was accompanied by a marked reduction of calbindin-immunoreactive neurons. Carbocyanine dye tracing of the thalamocortical fiber pathway revealed normal growth kinetics and fasciculation patterns between embryonic days 17.5 and 19. As in normal mice, mutant thalamocortical axons reach the cortex, accumulate below the cortical plate, and then start to extend side-branches in the subplate and deep cortical plate. Multiple carbocyanine dye placements in the cortical convexity revealed normal overall topography of both early thalamocortical and corticofugal projections. Electrophysiological recordings from thalamocortical slices confirmed that thalamic axons were capable of conducting action potentials to the cortex. Thus, our data suggest that axonal growth and early topographic arrangement of these fiber pathways do not rely on activity-dependent mechanisms requiring evoked neurotransmitter release. Intercellular communication mediated by constitutive secretion of transmitters or growth factors, however, might play a part.
Abstract: Emx2 knockout mice appear to show a shift in the areal identity in the cerebral cortex , which is matched with altered distribution of thalamocortical projections (Bishop et al. [2000] Science 288:344-349; Mallamaci et al. [2000] Nat Neurosci. 3:679-686) [corrected]. We have examined the early establishment of these projections to understand how the altered Emx2 expression results in changes in their cortical targeting. We used carbocyanine dye tracing to visualize thalamocortical and corticofugal projections as well as immunohistochemistry for L1 and TAG-1, respective markers of the two axonal systems, in wild-type, heterozygote, and null mutant for Emx2 at embryonic (E) ages ranging from E13.5 to E18.5. These tracing studies demonstrated that, in Emx2 knockout mice, a large proportion of early thalamocortical projections were misrouted at the border between the diencephalon and telencephalon. This abnormality was associated with displaced connectivity of the internal capsule cells at the diencephalic-telencephalic junction. Interestingly, most of the aberrant thalamic projections compensated for the ventral entry to the telencephalon and still ascended to the cortex. Although this early targeting abnormality is associated with the altered Emx2 expression pattern in the cortex, it most probably occurs independently from it, and is related to earlier guidance defects at the diencephalic-telencephalic boundary. These defects might result in the altered and delayed arrival of thalamic projections to the cortex and, thus, contribute to the shifted thalamocortical matching previously observed in the Emx2 knockout mice.
Abstract: Most parts of the brain are conserved across reptiles and birds (sauropsids) and mammals. Two major qualitative differences occur in the upper part, or pallium, of the telencephalon, the most rostral part of the brain. Mammals have a six-layered neocortex and also exhibit a different morphological organization in the lateral half, or sector, of their pallium than do sauropsids. These differences of lateral pallial construction may derive from small but crucial differences in migration patterns of neuronal precursors generated at or above the corner of the lateral ventricle, the corticostriatal junction (CS). Sauropsids have a large structure, the dorsal ventricular ridge, that is proliferated from this region, and its anterior part (ADVR) receives ascending projections from the dorsal thalamus. Mammals have multiple structures in this same region-the lateral part of neocortex, amygdala, and claustrum-endopiriform formation. We propose here that, as the degree of development of structures that form the deeper tier of the pallium varies across the stages of embryology and across phylogeny, mutations may have occurred during evolution at the origin of mammals that had profound consequences for the fate of neural populations generated in the region of the CS and its neighboring pallial germinal zone.
Abstract: Perturbation of the homeostasis between proteases and their inhibitors has been associated with lesion-induced or degenerative neuronal changes. Protease nexin-1 (PN-1), a secreted serine protease inhibitor, is constitutively expressed in distinct neuronal cell populations of the adult CNS. In an earlier study we showed that transgenic mice with ectopic or increased expression of PN-1 in postnatal neurons have altered synaptic transmission. Here these mice are used to examine the impact of an extracellular proteolytic imbalance on long-term neuronal function. These mice develop disturbances in motor behavior from 12 weeks on, with some of the histopathological changes described in early stages of human motor neuron disease, and neurogenic muscle atrophy in old age. In addition, sensorimotor integration, measured by epicranial multichannel recording of sensory evoked potentials, is impaired. Our results suggest that axonal dysfunction rather than cell death underlies these phenotypes. In particular, long projecting neurons, namely cortical layer V pyramidal and spinal motor neurons, show an age-dependent vulnerability to PN-1 overexpression. These mice can serve to study early stages of in vivo neuronal dysfunction not yet associated with cell loss.
Abstract: Thalamocortical projections in mammals must travel through a considerable portion of the newly formed subdivisions of the embryonic forebrain. They descend through the ventral thalamus, advance in the internal capsule amongst cells which already possess dorsal thalamic projections, traverse the striatocortical junction, and then reach the cerebral cortex by associating with subplate cells and their early corticofugal fibers. The interactions of the thalamocortical projections with early generated, largely transient cells of these regions are believed to play a crucial role in their deployment. These ideas are supported by recent work on reeler and other strains of mutant mice. While we are beginning to understand the basic pattern of the cellular and molecular interactions employed in mammalian thalamocortical development, comparative developmental studies hold the promise to reveal the underlying logic of these steps and the evolutionary origin of the mammalian cerebral cortex.
Abstract: The general patterns of early thalamocortical development follow a similar sequence in all mammals. Thalamocortical projections descend through the ventral thalamus, advance in the internal capsule amongst cells which already possess dorsal thalamic projections, then reach the cerebral cortex by associating with subplate cells and their early corticofugal projections. Initially, the thalamic projections pause in the internal capsule and subplate layer. The interactions of the thalamocortical projections with the early generated, largely transient cells of the subplate, marginal zone, internal capsule and ventral thalamus are believed to play a crucial role in the organized deployment of thalamic projections and establishing a functional cortical architecture. Selective fasciculation, contact guidance and release of neurotrophic factors are thought to play roles in the development of thalamocortical projections. These ideas are obtaining support from recent work on reeler and other strains of mice. The evolutionary origin of these largely transient cells and the overlying logic of early developmental steps are not understood. The behaviour of the thalamocortical and corticothalamic projections at the corticostriatal junction is particularly puzzling. The comparison of early forebrain development in mammals and reptiles is beginning to reveal highly conserved cellular and molecular interactions during early thalamocortical development and to reveal homologies between telencephalic subdivisions.
Abstract: The aim of our study is to understand the development of the earliest connections in the mammalian pallium by documenting the distribution of cells and fibres labelled from the dorsal and ventral thalamus, internal capsule, perirhinal, and dorsal cortex during the period between embryonic day (E) 14 and 17 by using carbocyanine dye tracing in fixed embryonic rat brains. Dye placed in the thalamus of E14 brains backlabels cells in the thalamic reticular nucleus and within the primitive internal capsule. Both anterograde and retrograde tracing confirmed that the first corticofugal projections reach the internal capsule by E14. At E15-E16, after the first cortical plate cells have migrated into the lateral cortex, some cells of the cortical plate and subplate and marginal zone, are backlabelled from the internal capsule, but still not from the dorsal thalamus, even with very long incubation periods. Crystal placement into the perirhinal cortex at E14-E15 labels numerous cells within the internal capsule, whereas no such cells are revealed from dorsal cerebral cortex until E17, suggesting that internal capsule cells establish early connections with the perirhinal and ventral but not dorsal cortex. We propose that the growth of axons from cortex to dorsal thalamus is delayed in two regions: first from E14-E15 at the lateral entrance of the internal capsule and then, from E16, closer to the thalamus, probably within the thalamic reticular nucleus. Subplate projections reach the proximity of the diencephalon at an early stage, but they might never enter the dorsal thalamus.
Abstract: Neuronal heterotopia are seen in various pathologies and are associated with intractable epilepsy. We examined brain tissue from four children with subcortical or periventricular nodular heterotopia of different aetiologies: one with severe epilepsy following focal brain trauma at 17 weeks gestation, one with hemimegalencephaly and intractable epilepsy, one with focal cortical dysplasia and intractable epilepsy, and one dysmorphic term infant with associated hydrocephalus and polymicrogyria. The connectivity of nodules was investigated using histological and carbocyanine dye (DiI) tracing techniques. DiI crystal placement adjacent to heterotopic nodules revealed numerous DiI-labelled fibres within a 2-3 mm radius of the crystals. Although we observed labelled fibres closely surrounding nodules, the majority did not penetrate them. Placement of DiI crystals within nodules also identified a limited number of projections out of the nodules and in one case there was evidence for connectivity between adjacent nodules. The cellular and neurochemical composition of nodules was also examined using immunohistochemistry for calretinin and neuropeptide Y (NPY), which are normally expressed in GABAergic cortical interneurons. Within heterotopic nodules from all cases, numerous calretinin-positive neurons were identified, along with a few cell bodies and many processes positive for NPY. Calretinin-positive neurons within nodules were less morphologically complex than those in the cortex, which may reflect incomplete differentiation into an inhibitory neuronal phenotype. There were also abnormal clusters of calretinin-positive cells in the overlying cortical plate, indicating that the migratory defect which produces heterotopic nodules also affects development of the cortex itself. Thus, heterotopic nodules consisting of multiple neuronal cell types are associated with malformation in the overlying cortical plate, and have limited connectivity with other brain regions. This abnormal development of connectivity may affect neuronal maturation and consequently the balance of excitation and inhibition in neuronal circuits, leading to their epileptogenic potential.
Abstract: Two visual areas, V1 and V2 (first and second visual areas), appear to be present in the posterior neocortex of all eutherian mammals investigated so far. However, previous studies have not established whether an area homologous to V2 also exists in metatherian mammals (marsupials). Using electrophysiological techniques, we mapped the visual receptive fields of neurons in the striate and peristriate cortices of the northern quoll, an Australian marsupial. We found that neurons in a 2-mm-wide strip of cortex rostrolateral to V1 form a single, relatively simple representation of the complete contralateral hemifield. This area resembles V2 of eutherians in several respects: (i) neurons in the medial half of the peristriate area represent the lower visual quadrant, whereas those in the lateral half represent the upper visual quadrant; (ii) the vertical meridian of the visual field is represented adjacent to V1, while the visual field periphery is represented along the lateral and rostrolateral borders of the peristriate area; (iii) there is a marked anisotropy in the representation, with a larger magnification factor parallel to the V1 border than perpendicular to this border; and (iv) receptive fields of multiunit clusters in the peristriate cortex are much larger than those of cells in V1 at comparable eccentricities. The cortex immediately rostral and lateral to V2 did not respond to visual stimulation under our recording conditions. These results suggest that V1 and V2 together form a 'core' of homologous visual areas, likely to exist in all therian mammals.
Abstract: Explants of embryonic or postnatal rat cortex, organotypically cultured in serum-free medium, maintain their structural integrity and their upper layers continue to mature. Coculture of portions of embryonic thalamus with cortical slices taken at different ages reveals a temporal cascade of cortical signals. (1) Slices of occipital cortex taken at E19 or earlier stimulate axonal outgrowth from explants of embryonic lateral geniculate nucleus but do not allow the fibers to invade. (2) In cortical slices taken after E19 but before P2, thalamic axons enter the slice, from any direction, and extend radially across the entire depth of the cortical plate without branching or terminating. (3) In slices taken after P2, fibers slow down, arborize, and terminate in the maturing layer 4 of the cortex. If the thalamic explant is placed against the pial surface of the cortical slice, axons still enter and branch in the same layer. These findings imply that the developing cortex expresses a diffusible growth-promoting factor and then itself becomes growth permissive, and finally the maturing layer 4 expresses a "stop signal." In triple cocultures of one thalamic explant with a "choice" of two neighboring slices, thalamic axons will not invade slices of cerebellum but behave indistinguishably in response to slices from any region of the hemisphere. Thus the initial tangential distribution of the thalamic projection in vivo (which is achieved by about E16) is unlikely to be controlled by regional variation in signals produced by the cortex. When cortical slices were precultured alone for 7-14 days before the addition of an explant of embryonic thalamus for 4 further days of coculture, the pattern of innervation was more appropriate to the chronological age of the slice than the age at which it was first taken. Thus the timing of the cascade of cortical properties is at least partly intrinsically determined. This sequence of expression of these signals suggests that they play a part in vivo in controlling the outgrowth of thalamic fibers, their accumulation under the cortical plate, their invasion of the plate, and their arborization in layer 4.
Abstract: Theories of both cortical field development and cortical evolution propose that thalamocortical projections play a critical role in the differentiation of cortical fields (; ). In the present study, we examined how changing the size of the immature neocortex before the establishment of thalamocortical connections affects the subsequent development and organization of the adult neocortex. This alteration in cortex is consistent with one of the most profound changes made to the mammalian neocortex throughout evolution: cortical size. Removing the caudal one-third to three-fourths of the cortical neuroepithelial sheet unilaterally at an early stage of development in marsupials resulted in normal spatial relationships between visual, somatosensory, and auditory cortical fields on the remaining cortical sheet. Injections of neuroanatomical tracers into the reduced cortex revealed in an altered distribution of thalamocortical axons; this alteration allowed the maintenance of their original anteroposterior distribution. These results demonstrate the capacity of the cortical neuroepithelium to accommodate different cortical fields at early stages of development, although the anteroposterior and mediolateral relationships between cortical fields appear to be invariant. The shifting of afferents and efferents with cortical reduction or expansion at very early stages of development may have occurred naturally in different lineages over time and may be sufficient to explain much of the phenotypic variation in cortical field number and organization in different mammals.
Abstract: We are interested in similarities and conserved mechanisms in early development of the reptilian and mammalian thalamocortical connections. We set out to analyse connectivity in embryonic turtle brains (Pseudemys scripta elegans, between stages 17 and 25), by using carbocyanine dye tracing. From the earliest stages studied, labelling from dorsal and ventral thalamus revealed backlabelled cells among developing thalamic fibres within the lateral forebrain bundle and striatum, which had similar morphology to backlabelled internal capsule cells in embryonic rat (Molnár and Cordery, 1999). However, thalamic crystal placements did not label cells in the dorsal ventricular ridge (DVR) at any stage examined. Crystal placements into both dorsal and lateral cortex labelled cells in the DVR and, reciprocally, DVR crystal placements labelled cells in the dorsal and lateral cortices. Retrograde labelling revealed that thalamic fibres arrive in the DVR and dorsal cortex by stage 19. The DVR received projections from the nucleus rotundus and the dorsal cortex exclusively from the perirotundal complex (including lateral geniculate nucleus). Thalamic fibres show this remarkable degree of specificity from the earliest stage we could examine with selective retrograde labelling (stage 19). Our study demonstrates that axons of similar cells are among the first to reach dorsal and ventral thalamus in mammals and reptiles. Our connectional analysis in turtle suggests that some cells of the mammalian primitive internal capsule are homologous to a cell group within the reptilian lateral forebrain bundle and striatum and that diverse vertebrate brains might use a highly conserved pattern of early thalamocortical development.
Abstract: We determined the time-course and general pattern of thalamocortical development of Monodelphis domestica by tracing projections with carbocyanine dye in fixed postnatal brains between postnatal day 2 (P2) and P30. By P2, the first neurons have migrated to form the preplate of the lateral cortex and have sent out axons into the intermediate zone. By P3, fibers from the preplate of more dorsal cortex have entered the intermediate zone, and, by P5, they reach the primitive internal capsule. Crystal placements in the dorsal thalamus at P2-P3 reveal thalamic axons extending down through the diencephalon and growing out through the internal capsule among groups of back-labelled cells that already project into the thalamus. Thalamic axons arrive at the cortex after the arrival of cells of the true cortical plate has split the preplate into marginal zone and subplate. Axons from the ventral part of the dorsal thalamus reach the lateral cortex by P5: Dorsal thalamic fibers arrive at the extreme dorsal cortex by P9. The deeper layers of the cortex appear to mature relatively earlier in Monodelphis than in eutherian mammals, and the subplate becomes less distinct. Thalamic fibers and their side branches proceed into the cortex without an obvious period of waiting in the subplate, but they do not penetrate the dense cortical plate itself. Monodelphis could provide an excellent model species, because the development of its thalamocortical connections is entirely an extrauterine process: The period P0-P15 corresponds to that of E12-P0 in the rat.
Abstract: In the mutant mouse reeler, the tangential distribution of thalamocortical fibers is essentially normal, even though neurons of the cortical plate accumulate below the entire early-born preplate population (Caviness et al., 1998). This seems incompatible with the hypothesis that cells of the subplate (the lower component of the preplate in normal mammals) form an axonal scaffold that guides thalamic fibers and act as temporary targets for them (Blakemore and Molnár, 1990, Shatz et al., 1990). We used carbocyanine dyes to trace projections in wild-type and reeler mice between embryonic day 13 and postnatal day 3. Preplate formation and early extension of corticofugal fibers to form a topographic array are indistinguishable in the two phenotypes. So too are the emergence of thalamic axons in topographic order through the primitive internal capsule, their meeting with preplate axons, and their distribution over the preplate scaffold. Distinctive differences appear after the cortical plate begins to accumulate below the preplate of reeler, causing the preplate axons to form oblique fascicles, running through the cortical plate. Thalamic axons then pass through the plate within the same fascicles and accumulate in the "superplate" layer for approximately 2-3 d, before defasciculating and plunging down to terminate deep in the cortical plate, creating the curious "looping" pattern seen in the adult. Thus, thalamocortical innervation in reeler follows the same algorithm of development but in relation to the misplaced population of early-born neurons. Far from challenging the theory that preplate fibers guide thalamic axons, reeler provides strong evidence for it.
Abstract: We labeled axonal projections using carbocyanine dyes in the developing rat brain to study cellular interactions that might underlie the establishment of thalamocortical connectivity. By embryonic day 14 (E14), groups of neurons in the ventral diencephalon and the primitive internal capsule have established projections to the dorsal thalamus, and thalamic fibers pass in topographic order among them. Simultaneously, axons from the early-born cells in both subplate and marginal zone (i.e., the original cortical preplate) establish an ordered array that fills the intermediate zone. Thalamic axons and preplate fibers meet in the lateral part of the internal capsule (at E15 for occipital cortex and dorsolateral thalamus). Subsequently, selective labeling of corresponding thalamic and early corticofugal projections reveals thalamic fibers growing in association with early corticofugal axons, right up to the cortical subplate. A small carbocyanine crystal implanted at any point in the cortex shortly after the arrival of thalamic axons (E16 for the occipital cortex) labels a single, tight bundle containing both descending and ascending fibers, rather than two separate tracts, providing further evidence for intimate topographic association of the two axon systems. Crystals placed in a row, parasagittally or coronally along the hemisphere, reveal separate, topographically distributed, discrete fiber bundles throughout the pathway, leading to spatially ordered groups of back-labeled thalamic cells. These results indicate that the topography of thalamic axons is maintained throughout the pathway and that they reach the cortex by associating with the projections of a number of preexisting cells, including the preplate scaffold.
Abstract: We propose that a sequence of individually simple mechanisms influences the pattern of thalamocortical innervation, which itself contributes to the determination of regional differentiation of the neocortex. In co-culture, the cortex appears to exert a remote growth-promoting influence on thalamic axons from E15, becomes growth-permissive to axon invasion at about E20 and expresses a stop signal, causing termination in layer IV, from P2-3. This cascade of cortical signals may determine the timing of events in vivo. However, any part of the thalamus will innervate any region of the developing cortex in culture, without obvious preference, suggesting that the topographic distribution of thalamic fibres in vivo does not depend on regional chemospecificity. The initial extension of axons from the cortical preplate and the thalamus starts at about E14, and the topography of both may be influenced by their temporal sequences of outgrowth (chronotopy). The axon arrays meet in the basal telencephalon, after which the preplate scaffold may guide thalamic axons and ensure both their 'capture' within the subplate layer and the establishment of the waiting period. The unusual pattern of innervation in the Reeler mutant mouse supports the hypothesis that thalamic axons grow over preplate fibres to find the waiting compartment.
Abstract: A cascade of simple mechanisms influences thalamic innervation of the neocortex. The cortex exerts a remote growth-promoting influence on thalamic axons when they start to grow out, becomes growth-permissive when the axons begin to invade, and later expresses a 'stop signal', causing termination in layer 4. However, any part of the thalamus will innervate any region of developing cortex in culture, and the precise topographic distribution of thalamic fibres in vivo is unlikely to depend exclusively on regional chemoaffinity. The 'handshake hypothesis' proposes that axons from the thalamus and from early-born cortical preplate cells meet and intermingle in the basal telencephalon, whereafter thalamic axons grow over the scaffold of preplate axons, and become 'captured' for a waiting period in the subplate layer below the corresponding part of the cortex. The bizarre pattern of development of thalamic innervation in the mutant reeler mouse provides strong evidence that thalamic axons are guided by preplate axons.
Abstract: The mammalian cerebral cortex consists of many structurally and functionally specialized areas, with characteristic input from particular nuclei of the thalamus. Some localized external influence, such as the arrival of fibres from the appropriate thalamic nucleus before or around the time of birth, could trigger the emergence of committed cortical fields from an undifferentiated 'protocortex. The guidance of axons from each thalamic nucleus to its appropriate target area in the cortex could, then, be crucial in the regulation of cortical differentiation. Recently, Yamamoto et al. and Bolz et al. have demonstrated that cocultured explants of rat lateral geniculate nucleus and visual cortex can form layer-specific interconnections. We have now tested the possibility that each cortical area exerts a selective trophic influence on axons from its appropriate thalamic nucleus, and vice versa, by coculturing explants of different regions of the thalamus and cortex taken at various stages of development. Although thalamo-cortical and cortico-thalamic connections formed in vitro can be remarkably normal in many respects, they lack regional specificity.
Abstract: Progress in understanding development of the brain has already started to explain the patterns of neurogenesis, progenitor subtypes, combinatorial transcription codes both for cell types and regional specification of the cerebral cortex, migratory routes taken by cells and axons and establishment of neural connectivity. This progress has mostly been experimental and based on murine studies. Although the mouse is a superb model for exploiting the modern tools of molecular genetics, allowing us to monitor and modulate selected groups of neurons, it has several limitations. The human brain has unique qualities; and there are strong clinical reasons for wanting to improve understanding of its developmental properties. Some developmental steps are considered exclusively in human; other features are not necessarily unique but certainly much more apparent in human than in other species ( Clowry et al., 2010). Indeed, some fundamental recent discoveries were made in humans.
