Cajal-Retzius Research Articles

Generation of Cajal–Retzius neurons in mouse forebrain is regulated by transforming growth factor β-Fox signaling pathways

The generation of Cajal–Retzius (CR) neurons is restricted to discrete sites in the telencephalon. Most of these sites do not express Foxg1, a transcription factor that inhibits transforming growth factor (TGF)β-dependent upregulation of p21. We tested the hypothesis that TGFβ signaling triggers CR neurogenesis in Foxg1-deficient zones through p21 induction. In Foxg1 +/+ mice, p21 (a) was expressed in select cycling cells in CR neuron-producing areas and (b) was co-localized in newly generated CR neurons. Zones of CR neuronal production and p21 expression were expanded in the forebrains of Foxg1 Cre/Cre mice. Manipulation of TGFβ signaling in explants from cortical hems of wild-type mice altered p21 expression and the production of CR neurons. Furthermore, despite continued TGFβ activity, p21 immunoreactivity diminished in CR neurons with distance from their generation site. This implicated a second pathway controlling p21 expression. We provide evidence that Foxo3a, which has been shown to translocate into the nucleus to act as a transcriptional co-activator of TGFβ-dependent upregulation of p21, is strategically expressed to be involved in controlling p21 expression in CR neurons. Specifically, Foxo3a was nuclear in p21+/reelin+ cells in sites of CR neuronal generation, however, nuclear Foxo3a immunoreactivity was absent in p21−/reelin+ cells distal from sites of CR neurogenesis. Thus, TGFβ and Foxo3a may work in concert to regulate expression of p21 during CR neuronal generation.

Kinetic Properties of Cl−Uptake Mediated by Na+-Dependent K+-2Cl−Cotransport in Immature Rat Neocortical Neurons

GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity. Although it is well accepted that depolarizing GABA actions are caused by an elevated intracellular Cl- concentration ([Cl-]i), the mechanisms of Cl- accumulation in immature neurons are still a matter of debate. Using patch-clamp, microfluorimetric, immunohistochemical, and molecular biological approaches, we studied the mechanism of Cl- uptake in Cajal-Retzius (CR) cells of immature [postnatal day 0 (P0) to P3] rat neocortex. Gramicidin-perforated patch-clamp and 6-methoxy-N-ethylquinolinium-microfluorimetric measurements revealed a steady-state [Cl-]i of approximately 30 mM that was reduced to values close to passive distribution by bumetanide or Na+-free solutions, suggesting a participation of Na+-K+-2Cl- cotransport isoform 1 (NKCC1) in maintaining elevated [Cl-]i. Expression of NKCC1 was found in CR cells on the mRNA and protein levels. To determine the contribution of NKCC1 to [Cl-]i homeostasis in detail, Cl- uptake rates were analyzed after artificial [Cl-]i depletion. Active Cl- uptake was relatively slow (47.2 +/- 5.0 microM/s) and was abolished by bumetanide or Na+-free solution. Accordingly, whole-cell patch-clamp recordings revealed a low Cl- conductance in CR cells. The low capacity of NKCC1-mediated Cl- uptake was sufficient to maintain excitatory GABAergic membrane responses, however, only at low stimulation frequencies. In summary, our results demonstrate that NKCC1 is abundant in CR cells of immature rat neocortex and that the slow Cl- uptake mediated by this transporter is sufficient to maintain high [Cl-]i required to render GABA responses excitatory.

Cajal–Retzius cells and subplate neurons differentially express vesicular glutamate transporters 1 and 2 during development of mouse cortex

In the light of the various neurobiological effects of glutamate in brain development, although some embryonic cells are a probable source of glutamate involved in the development of precursor cells and/or immature neurons, little is known about when and where glutamate plays its crucial roles during corticogenesis. To investigate these roles, we focused on the developmental expression of vesicular glutamate transporter (VGLUT)1 and VGLUT2, which are regarded as the best markers for verifying glutamatergic neuron identity, especially the spatiotemporal distributions of their transcripts and proteins in the developing mouse cortex and hippocampus. In situ hybridization studies revealed that VGLUT1 mRNA is expressed in preplate and marginal zone cells at embryonic day (E)10 and in subplate cells by E13, whereas VGLUT2 mRNA is expressed in preplate and marginal zone cells at E10 and in cells of the subventricular zone by E13. Reverse transcriptase-polymerase chain reaction analysis detected full-length VGLUT1 and VGLUT2 gene transcripts in the embryonic brain. By dual labeling combined with immunostaining for microtubule-associated protein 2 (MAP2) or reelin, we showed that MAP2-positive preplate and marginal zone neurons and subplate neurons express VGLUT1, while reelin-positive preplate and marginal zone cells and MAP2-negative subventricular zone cells express VGLUT2. The present study is the first to provide morphologically reliable evidence showing that Cajal-Retzius cells and subplate neurons are glutamatergic, and that the two cells differentially express VGLUT1 and VGLUT2, respectively, as the specific transport system of glutamate in some events orchestrated by these cells during the cortical development of mice.

Distribution patterns of estrogen receptor α and β in the human cortex and hippocampus during development and adulthood

The expression of estrogen receptors (ERs) in the developing and adult human brain has not been clearly established, although estrogens are crucial for neuronal differentiation, synapse formation, and cognitive functions. By using immunohistochemistry, we have studied the distribution of ER alpha and ER beta in human cerebral cortex and hippocampus from early prenatal stages to adult life. ER alpha was detected in the cortex at 9 gestational weeks (GW), with a high expression in proliferating zones and the cortical plate. The staining intensity decreased gradually during prenatal development but increased again from birth to adulthood. In contrast, ER beta was first detected at 15 GW in proliferating zones, and at 16/17 GW, numerous ER beta immunopositive cells were also observed in the cortical plate. ER beta expression persisted in the adult cortex, being widely distributed throughout cortical layers II-VI. In addition, from around 15 GW to adulthood, ER alpha and ER beta were expressed in human hippocampus mainly in pyramidal cells of Ammon's horn and in the dentate gyrus. Western blotting and immunohistochemistry in the adult cerebral cortex and hippocampus revealed lower protein expression of ER alpha compared with ER beta. Double immunostaining showed that during fetal life both ERs are expressed in neurons as well as in radial glia, although only ER alpha is expressed in the Cajal-Retzius neurons of the marginal zone. These observations demonstrate that the expression of ER alpha and ER beta displays different spatial-temporal patterns during human cortical and hippocampal development and suggest that both ERs may play distinct roles in several processes related to prenatal brain development.

Developmental Downregulation of Excitatory GABAergic Transmission in Neocortical Layer I via Presynaptic Adenosine A1 Receptors

Layer I of the developing cortex contains a dense GABAergic fiber plexus. These fibers provide excitatory inputs to Cajal-Retzius (CR) cells, the early born neurons in layer I. CR cells possess an extensive axonal projection and form synaptic contacts with excitatory, presumably pyramidal, neurons before birth. Interestingly, activity of CR cells declines during the first postnatal week, but mechanism(s) underlying this phenomenon is not yet known. Here we recorded inhibitory postsynaptic currents (IPSCs) in CR cells at postnatal day (P) 1-2 and P5-7. Blockade of adenosine A(1) receptors (A(1)Rs) increased the amplitude of evoked IPSCs (eIPSCs) and decreased paired-pulse ratio at P5-7 but not at P1-2. A(1)R activation decreased the mean eIPSC amplitude at P5-7, but failed to affect eIPSCs at P1-2. Ecto-adenosine triphosphatase (ATPase) inhibition completely abolished the A(1)R-mediated effects suggesting that extracellular ATP is the main source of adenosine. Because A(1)R blockade did not affect the median miniature IPSC amplitude, our results demonstrate that adenosine reduces gamma-aminiobutyric acid (GABA) release probability via presynaptic A(1)Rs at P5-7. As neuronal activity in layer I can depolarize pyramidal neurons influencing thereby glutamatergic synaptogenesis in the lower cortical layers, postnatal weakening of GABAergic transmission by adenosinergic system might reflect a developmental downregulation of this excitatory drive when glutamatergic synapses are formed.

LIM homeodomain factor Lhx5 is required for normal development and migration of Cajal–Retzius cells

LIM homeodomain factor Lhx5 is required for normal development and migration of Cajal–Retzius cells

Expression pattern of stop lacZ reporter gene in adult and developing mouse brain

Stable tubulin-only polypeptide (STOP) proteins are microtubule-associated proteins responsible for microtubule stabilization in neurons. STOP null mice show apparently normal cerebral anatomy but display synaptic defects associated with neuroleptic-sensitive behavioral disorders. STOP null mice have therefore been proposed as an animal model for the study of schizophrenia. In the present study, the expression pattern of STOP gene in developing and adult brain has been examined by using lacZ gene inserted in the STOP locus, as a reporter gene. beta-Galactosidase (beta-gal) immunostaining was confined to neuronal cells and projections. Strong labeling was observed in the whole olfactory system, cortical layer VII, hippocampus, hypothalamus, cerebellum, habenula, fasciculus retroflexus, and interpeduncular nucleus in adults. Additionally, ventral thalamic nucleus, clusters of positive cells in striatum, and Cajal-Retzius cells of cortical layer I were labeled in young mice. The strong expression of STOP lacZ reporter gene observed in brain is confined to areas that may be involved in the schizophrenia-related symptoms observed in STOP-deficient mice.

The tracing study of developing entorhino-hippocampal pathway

The entorhino-hippocampal pathway is the major excitatory input from neurons of the entorhinal cortex on both ipsilateral and contralateral hippocampus/dentate gyrus. This fiber tract consists of the alvear path, the perforant path and a crossed commissural projection. In this study, the histogenesis and development of the various subsets of the entorhino-hippocampal projection have been investigated. DiI, DiO, Fast Blue tracing and calretinin immunocytochemistry as well as were carried out with pre and postnatal rats at different developmental stages. The alvear path and the commissural pathway start to develop as early as embryonic day E16, while the first perforant afferents reach the stratum lacunosum-moleculare of the hippocampus at E17 and at outer molecular layer of the denate gyrus at postnatal day 2. Retrograde tracing with DiI identifies entorhinal neurons in layer II–IV as the developmental origin of the entorhino-hippocampal pathway. Furthermore, calretinin immunocytochemistry revealed transitory Cajal-Retzius cells in the stratum lacunosum-moleculare of the hippocampus from E16. DiI labeling of entorhinal cortex fibers and combined calretinin-immunocytochemistry reveal a close relationship between Cajal-Retzius cells and entorhinal afferents. This temporal and spatial relationship suggests that Cajal-Retzius cell serves as a guiding cue for entorhinal afferents at early cortical development.

Breaches of the pial basement membrane and disappearance of the glia limitans during development underlie the cortical lamination defect in the mouse model of muscle‐eye‐brain disease

Neuronal overmigration is the underlying cellular mechanism of cerebral cortical malformations in syndromes of congenital muscular dystrophies caused by defects in O-mannosyl glycosylation. Overmigration involves multiple developmental abnormalities in the brain surface basement membrane, Cajal-Retzius cells, and radial glia. We tested the hypothesis that breaches in basement membrane and the underlying glia limitans are the key initial events of the cellular pathomechanisms by carrying out a detailed developmental study with a mouse model of muscle-eye-brain disease, mice deficient in O-mannose beta31,2-N-acetylglucosaminyltransferase 1 (POMGnT1). The pial basement membrane was normal in the knockout mouse at E11.5. It was breached during rapid cerebral cortical expansion at E13.5. Radial glial endfeet, which comprise glia limitans, grew out of the neural boundary. Neurons moved out of the neural boundary through these breaches. The overgrown radial glia and emigrated neurons disrupted the overlying pia mater. The overmigrated neurons did not participate in cortical plate (CP) development; rather they formed a diffuse cell zone (DCZ) outside the original cortical boundary. Together, the DCZ and the CP formed the knockout cerebral cortex, with disappearance of the basement membrane and the glia limitans. These results suggest that disappearance of the basement membrane and the glia limitans at the cerebral cortical surface during development underlies cortical lamination defects in congenital muscular dystrophies and a cellular mechanism of cortical malformation distinct from that of the reeler mouse, double cortex syndrome, and periventricular heterotopia.

Cajal-Retzius cells express vesicular glutamate transporter 2 (VGLUT2) during the mouse corticogenesis

Cajal-Retzius cells express vesicular glutamate transporter 2 (VGLUT2) during the mouse corticogenesis

Dendritic and spinal pathology in the acoustic cortex in Alzheimer's disease: morphological and morphometric estimation by Golgi technique and electron microscopy

Conclusions. The morphological and morphometric estimation of the dendrites and the dendritic spines in the acoustic cortex in Alzheimer's disease revealed substantial alterations of the dendritic arborization and marked loss of the dendritic spines. This may be related to communication impairment even in early cases of Alzheimer's disease. Objectives. Alzheimer's disease is characterized by progressive loss of memory, impairment of judgment, and decline in communication and speech eloquence. In the present study we attempted to describe the morphological and morphometric alterations of the dendrites and the dendritic spines in the acoustic cortex in early cases of Alzheimer's disease, in order to approach the communication impairment of patients suffering from Alzheimer's disease, from the neuropathological point of view. Materials and methods. We studied the acoustic cortex in 22 cases of Alzheimer's disease by Golgi technique and electron microscopy. Results. The morphological and morphometric estimation of the acoustic cortex revealed loss of Cajal-Retzius cells in layer I, as well as an impressive abbreviation of the dendritic fields associated with loss of dendritic spines in all layers of the cortex. Numerous distorted, dystrophic and degenerated dendritic spines were also seen, which were intermixed with a considerable number of giant spines. The dendritic and spinal alterations were closely associated with mitochondrial alterations.

Comparative aspects of p73 and Reelin expression in Cajal-Retzius cells and the cortical hem in lizard, mouse and human

Cajal-Retzius (CR) cells of the mammalian neocortex co-express the extracellular matrix protein Reelin and p73, a transcription factor involved in cell death and survival. Most neocortical CR cells derive from the cortical hem, with minor additional sources. We analyzed the distribution of Reelin and p73 immunoreactive (ir) neurons in the telencephalon of Lacerta galloti from early embryonic stages to hatching. Numerous Reelin-ir cells appeared in the pallial MZ from the preplate stage onward. Conversely, p73-ir cells were rare in the pallial preplate and not observed in the cortical plate. Subpallial p73-ir cells spread from the septum and the telencephalic–diencephalic boundary to the pial surface of the basal forebrain and amygdala, respectively, where they co-expressed Reelin and p73. A small group of Reelin/p73-ir CR cells appeared in a rudimentary cortical hem at the interface of the medial cortex and choroid plexus. Comparison with early embryonic stages of mice and humans showed similar foci of p73-ir cells in the septum and at the telencephalic–diencephalic boundary and revealed an increasing prominence of the cortical hem, in parallel with increasing numbers of neocortical Reelin/p73 positive CR cells, which attain highest differentiation in the human brain. Our data show that Reelin-expression in the pallium is evolutionarily conserved and independent of a cortical hem, and suggest that p73 in the cortical hem may be involved in the evolutionary increase in number and complexity of the mammalian neocortical CR cells.

Microarray Analysis of the Fetal Hippocampus in the Emx2 Mutant

Deficiency in the transcription factor Emx2 causes a specific alteration of hippocampal development, which has been well analyzed morphologically. We are currently using microarrays and in situ hybridization to characterize gene expression in the Emx2 mutant hippocampus. In this report on our preliminary results for the fetal stage, we identify a group of genes for most of which a putative relation to Emx2 pathways has not been previously recognized. Some candidates are development genes or are involved in functional maturation, and show expression in the hippocampal plate and/or developing dentate gyrus. A second class of candidates label neuronal, glial or vascular structures in the outer marginal zone, and likely represent markers for cell populations specifically absent in the mutant. Our results point at pathways and processes altered in the mutant, particularly the Notch and chemokine pathways, the processes of cell migration, axonal guidance and angiogenesis, and the relation of pia and Cajal-Retzius cells with hippocampal morphogenesis.

Expression of SDF-1 and CXCR4 during Reorganization of the Postnatal Dentate Gyrus

Previous studies have demonstrated that stromal cell-derived factor 1 (SDF-1) is crucial for early dentate development; however, the mouse mutants for this chemokine and its only receptor, CXCR4, are neonatally lethal, making conclusions about the role of these molecules in postnatal development difficult to sustain. Previous expression analyses have used single labeling, but the distribution of CXCR4 is complex and to determine the cell types expressing CXCR4 requires multiple marker labeling. In this study, we examined the distribution of SDF-1 and CXCR4 mRNAs during the first postnatal weeks, combining these markers with several other cell-type-specific markers. We found that SDF-1 has three sites of expression: (1) continuation of prenatal expression in the meninges; (2) expression in Cajal-Retzius cells occupying the molecular layer of the upper and lower blades of the dentate, and (3) the maturing dentate granule neurons themselves. The timing of expression in these three sites corresponds to alterations in the distribution of the primary cell types expressing CXCR4 during the same periods, notably the expression of CXCR4 in radial-glial-like GFAP-expressing dentate precursors and immature dentate granule neurons. Taken together, our data suggest potential ongoing roles for SDF-1/CXCR4 signaling in the dentate gyrus during the early postnatal period that will be tested in the future with more precise genetic approaches.

Origins and migratory routes of murine Cajal‐Retzius cells

The first layer that appears in the cortical neuroepithelium, the preplate, forms in the upper part of the cortex immediately below the pial surface. In mice, this layer exists between embryonic days (E) 10 and 13, and it hosts different cell populations. Here, we have studied the first cell population generated in the preplate, the Cajal-Retzius cells. There is considerable confusion regarding these cells with respect to both their site of generation and the migratory routes that they follow. This perhaps is due largely to the different opinions that exist regarding their characterization. We have studied the site of origin of these cells, their migratory routes, and the molecular markers that may distinguish them by injecting tracers into early embryos, culturing them in toto for 24 hours, and then performing immunohistochemistry. We found that the Cajal-Retzius cells are most likely generated in the cortical hem by comparing with other cortical or extracortical origins. These cells are generated mainly at E10 and E11, and they subsequently migrate tangentially to cover the whole cortical mantle in 24 hours. From their site of origin in the medial wall of the telencephalon, they spread in a caudorostral direction, following an oblique migratory path toward the lateral part of the neuroepithelium. Prior to the splitting of the preplate, a percentage of the Cajal-Retzius cells that can be distinguished by the expression of reelin do not contain calretinin. Furthermore, there were no early-migrating neurons that expressed calbindin.

N‐ethylmaleimide increases release probability at GABAergic synapses in layer I of the mouse visual cortex

The sulphydryl alkylating agent N-ethylmaleimide (NEM) has been often used as an uncoupler of pertussis toxin-sensitive G-proteins. However, the effects of NEM on gamma-aminobutyric acid (GABA)ergic synaptic transmission remain controversial. Using the whole-cell patch-clamp technique, GABA(A) receptor-mediated postsynaptic currents (IPSCs) have been recorded from Cajal-Retzius (CR) cells in layer I of the neonatal mouse visual cortex. NEM increased the frequencies of both spontaneous and miniature IPSCs (mIPSCs) without an effect on the median mIPSC amplitudes or mIPSC kinetics. The NEM actions on mIPSCs did not depend on the extracellular Ca(2+), Ca(2+) release from intracellular stores, adenylyl cyclase and protein kinase A activities. NEM increased the mean amplitudes of evoked IPSCs and strongly decreased the paired-pulse ratio. The size of the readily releasable pool of presynaptic vesicles (RRP) was estimated using a high-frequency stimulation protocol. The RRP size was not affected by NEM. In addition, NEM significantly decreased the latency between the stimulus and the onset of GABA release. These results suggest that NEM selectively increases GABA release probability. At postnatal day 2, mIPSCs were observed only in about 30% of CR cells. NEM application revealed, however, that more than 90% of CR cells receive GABAergic inputs. Therefore, NEM seems to be a useful tool to verify the existence of 'silent' GABAergic synapses.

Cajal–Retzius cells switch from expressing γ‐less to γ‐containing GABAA receptors during corticogenesis

Cajal-Retzius cells are implicated in regulating neuronal migration and lamination during corticogenesis. In rodents, Cajal-Retzius cells are transient, being prevalent in the marginal zone of the embryonic neocortex and declining over the first two postnatal weeks. While studies have examined in postnatal neocortex the properties of GABA(A) receptors in Cajal-Retzius cells, less is known about their disposition at embryonic stages. Here, we combined patch-clamp electrophysiology and single-cell mRNA profiling to probe the expression of GABA(A) receptors in Cajal-Retzius cells. In embryonic neocortical slices, GABA elicited GABA(A) receptor-mediated current responses that were diazepam-insensitive and inhibited by Zn(2+), a pharmacological profile consistent with expression of gamma-less GABA(A) receptor isoforms. Non-Cajal-Retzius cells in the same embryonic slices, on the other hand, were robustly potentiated by diazepam and were insensitive to Zn(2+), typical of gamma-containing GABA(A) receptor isoforms, as were Cajal-Retzius cells in the postnatal neocortex. Single-cell mRNA profiling and immunohistochemistry confirmed expression of GABA(A) receptor gamma subunit transcript and protein, respectively, in individual reelin-expressing cells in the postnatal cortex but not in their embryonic counterparts. We conclude that Cajal-Retzius cells express gamma-less GABA(A) receptors at embryonic stages and switch to expressing gamma-containing GABA(A) receptor isoforms during postnatal neocortical development.

Neurobiology of Schizophrenia

With its hallucinations, delusions, thought disorder, and cognitive deficits, schizophrenia affects the most basic human processes of perception, emotion, and judgment. Evidence increasingly suggests that schizophrenia is a subtle disorder of brain development and plasticity. Genetic studies are beginning to identify proteins of candidate genetic risk factors for schizophrenia, including dysbindin, neuregulin 1, DAOA, COMT, and DISC1, and neurobiological studies of the normal and variant forms of these genes are now well justified. We suggest that DISC1 may offer especially valuable insights. Mechanistic studies of the properties of these candidate genes and their protein products should clarify the molecular, cellular, and systems-level pathogenesis of schizophrenia. This can help redefine the schizophrenia phenotype and shed light on the relationship between schizophrenia and other major psychiatric disorders. Understanding these basic pathologic processes may yield novel targets for the development of more effective treatments.

Stromal-Derived Factor-1 (CXCL12) Regulates Laminar Position of Cajal-Retzius Cells in Normal and Dysplastic Brains

Normal brain development requires a series of highly complex and interrelated steps. This process presents many opportunities for errors to occur, which could result in developmental defects in the brain, clinically referred to as malformations of cortical development. The marginal zone and Cajal-Retzius cells are key players in cortical development and are established early, yet there is little understanding of the factors resulting in the disruption of the marginal zone in many types of cortical malformation syndromes. We showed previously that treatment with methylazoxymethanol in rats causes marginal zone dysplasia with displacement of Cajal-Retzius cells to deeper cortical layers. Here we establish that loss of activity of the chemokine stromal-derived factor-1 (SDF1) (CXCL12), which is expressed by the leptomeninges, is necessary and sufficient to cause marginal zone disorganization in this widely used teratogenic animal model. We also found that mice with mutations in the main receptor for SDF1 (CXCR4) have Cajal-Retzius cells displaced to deeper cortical layers. Furthermore, by inhibiting SDF1 signaling in utero by intraventricular injection of a receptor antagonist, we establish that SDF1 signaling is required for the maintenance of Cajal-Retzius cell position in the marginal zone during normal cortical development. Our data imply that cortical layering is not a static process, but rather requires input from locally produced molecular cues for maintenance, and that complex syndromes of cortical malformation as a result of environmental insults may still be amenable to explanation by interruption of specific molecular signaling pathways.

Meninges control tangential migration of hem-derived Cajal-Retzius cells via CXCL12/CXCR4 signaling

Cajal-Retzius cells are critical in the development of the cerebral cortex, but little is known about the mechanisms controlling their development. Three focal sources of Cajal-Retzius cells have been identified in mice-the cortical hem, the ventral pallium and the septum-from where they migrate tangentially to populate the cortical surface. Using a variety of tissue culture assays and in vivo manipulations, we demonstrate that the tangential migration of cortical hem-derived Cajal-Retzius cells is controlled by the meninges. We show that the meningeal membranes are a necessary and sufficient substrate for the tangential migration of Cajal-Retzius cells. We also show that the chemokine CXCL12 secreted by the meninges enhances the dispersion of Cajal-Retzius cells along the cortical surface, while retaining them within the marginal zone in a CXCR4-dependent manner. Thus, the meningeal membranes are fundamental in the development of Cajal-Retzius cells and, hence, in the normal development of the cerebral cortex.