Alfonso Fairén

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Different origins and developmental histories of transient neurons in the marginal zone of the fetal and neonatal rat cortex.

Two major classes of early‐born neurons are distinguished during early corticogenesis in the rat. The first class is formed by the cortical pioneer neurons, which are born in the ventricular neuroepithelium all over the cortical primordium. They appear at embryonic day (E) 11.5 in the lateral aspect of the telencephalic vesicle and cover its whole surface on E12. These cells, which show intense immunoreactivity for calbindin and calretinin, are characterized by their large size and axonal projection. They remain in the marginal zone after the formation of the cortical plate; they project first into the ventricular zone, and then into the subplate and the internal capsule. Therefore, these cells are the origin of the earliest efferent pathway of the developing cortex. Pioneer neurons are only present in prenatal brains. The second class is formed by subpial granule neurons, which form the subpial granular layer (SGL), previously considered to be found exclusively in the human cortex. SGL neurons are smaller than pioneer neurons. They are generated in a transient compartment of the retrobulbar ventricle between E12 and E14, and we propose the hypothesis that they invade the marginal zone, through tangential subpial migration, at different moments of fetal life. SGL neurons contain calbindin, calretinin, and gamma‐aminobutyric acid (GABA), but the GABA‐immunoreactive group becomes inconspicuous before birth. The extracellular matrix‐like glycoprotein reelin, a molecule crucial for cortical lamination, is prenatally expressed by SGL neurons; postnatally, it is present in both Cajal‐Retzius cells and subpial pyriform cells, both derivatives of SGL cells. In the rat, Cajal‐Retzius cells are horizontal neurons that remain only until the end of the first postnatal week. They are located in layer I at a critical distance of approximately 20 μm from the pial surface and express reelin and, only occasionally, calretinin. Subpial pyriform cells coexpress reelin and calretinin and remain in layer I longer than Cajal‐Retzius cells. Both pioneer neurons and subpial granule neurons are specific to the cortex. They mark the limit between the rudimentary cerebral cortex and olfactory bulb in the rat during early corticogenesis. J. Comp. Neurol. 397:493–518, 1998.

Non-synaptic dendritic spines in neocortex.

A long-held assumption states that each dendritic spine in the cerebral cortex forms a synapse, although this issue has not been systematically investigated. We performed complete ultrastructural reconstructions of a large (n=144) population of identified spines in adult mouse neocortex finding that only 3.6% of the spines clearly lacked synapses. Nonsynaptic spines were small and had no clear head, resembling dendritic filopodia, and could represent a source of new synaptic connections in the adult cerebral cortex.

Cortical Cells That Migrate Beyond Area Boundaries: Characterization of an Early Neuronal Population in the Lower Intermediate Zone of Prenatal Rats

Studies of the early development of the mammalian cerebral cortex have revealed that the earliest generated neurons that form the primordial plexiform layer (also called preplate or marginal zone) distribute among layer I and layer VII (subplate). By means of bromodeoxyuridine labelling of cells becoming postmitotic, we have found evidence that, in the rat, an additional group of neurons of the primordial plexiform layer remains in the close vicinity of the ventricular zone. This finding, in line with the proposal by Marín‐Padilla (Z Anat. Entwicklungsgesch., 134, 117‐145, 1971), implies that the primordial plexiform layer suffers a tripartition after the formation of the cortical plate and of the intermediate zone (the latter soon becomes the embryonic white matter). Thus, primordial plexiform layer derivatives are in layer I, layer VII (subplate) and in the lower part of the embryonic white matter. This early generated neuronal population is also revealed with an antibody that recognizes the larger (67 kDa) isoform of glutamic acid decarboxylase (Kaufman et al., Science, 232, 1138‐1140, 1986). This is in accord with the earlier finding of a GABA‐containing cell population showing a similar spatiotemporal distribution. The early generated neurons of the embryonic white matter migrate tangentially and, in early postnatal animals, are found as interstitial cells in the medial regions of the subcortical white matter and at the midline in the corpus callosum. At caudal levels, similar cells invade the subpyramidal strata of the developing hippocampus. This tangential migration might explain the tangential dispersion of neural cell clones described in recent studies of cell lineage in the cerebral cortex.

Localization of ApoER2, VLDLR and Dab1 in radial glia: groundwork for a new model of reelin action during cortical development

The reelin signaling pathway regulates laminar positioning of radially migrating neurons during cortical development. It has been suggested that reelin secreted by Cajal-Retzius cells in the marginal zone could provide either a stop or an attractant signal for migratory neurons expressing reelin receptors, but the proposed models fail to explain recent experimental findings. Here we provide evidence that the reelin receptor machinery, including the lipoprotein receptors ApoER2 and VLDLR along with the cytoplasmic adaptor protein Dab1, is located in radial glia precursors whose processes span the entire cortical wall from the ventricular zone to the pial surface. Moreover, in reeler mice, defective in reelin, decreased levels of Dab1 in the ventricular zone correspond to an accumulation of the protein in radial end-feet beneath the pia matter. Our results support that neural stem cells receive a functional reelin signal. They are also consistent with a working model of reelin action, according to which reelin signaling on the newborn neuron-inherited radial process regulates perikaryal translocation and positioning.

Distribution of calbindin and parvalbumin in the developing somatosensory cortex and its primordium in the rat: an immunocytochemical study

SummaryImmunocytochemical techniques were used to analyze the distribution of the calcium-binding proteins calbindin and parvalbumin during the pre- and postnatal development of the rat somatosensory cortex. Calbindin occurs in most early differentiated neurons that form the primordial plexiform layer at embryonic day 14. This expression in transient; during the perinatal period, calbindin becomes immunologically undetectable within the structures derived from the primordial plexiform layer, i.e., the prospective layers I and VIb. Immunoreactive neurons are also absent from adult layers I and VIb. Calbindin is also detected in a second population of neurons which, from embryonic day 18 onwards, distributes diffusely within the cortical plate. Some neurons of this population show morphological traits of immaturity, while others show complete dendritic arborization. The definitive pattern of distribution of calbindin-immunoreactive neurons is achieved by postnatal day 22. Infragranular layers contain intensely-immunoreactive cells whose numerical density decreases during postnatal development, whereas in supragranular layers similar neurons are interspersed among numerous faintly-stained neurons.Parvalbumin is detected for the first time at postnatal day 6, within a small group of neurons located in cortical layer V, and extends afterwards through the whole thickness of the cerebral cortex. At this same postnatal stage, groups of immunoreactivepuncta are also found in layer IV of the somatosensory cortex; these puncta increase in density progressively and, at embryonic day 13, immunoreactive cells appear also grouped at this level. At this postnatal age, parvalbumin immunostaining delineates the somatosensory map in cortical layer IV. From this stage to adulthood, the number of immunoreactive neurons increases in the whole thickness of the somatosensory cortex. Barrels in layer IV become less distinct as immunoreactive cells and processes invade the septa. Layer IV in the adult somatosensory cortex appears more densely populated by parvalbumin immunoreactive neurons and puncta than in the surrounding areas.

Subpallial origin of a population of projecting pioneer neurons during corticogenesis

Pyramidal neurons of the mammalian cerebral cortex are generated in the ventricular zone of the pallium whereas the subpallium provides the cortex with inhibitory interneurons. The marginal zone contains a subpial stream of migratory interneurons and two different classes of transient neurons, the pioneer neurons provided with corticofugal axons, and the reelin-expressing Cajal–Retzius cells. We found in cultured slices that the medial ganglionic eminence provides the reelin-negative pioneer neurons of the marginal zone. Pioneer neurons sent long projection axons that went through the cortical plate and reached the subplate and the lateral border of the lateral ganglionic eminence. In the cultured slices, pioneer neurons were functionally mature: they displayed a voltage-gated sodium current, expressed functional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and showed γ-aminobutyric acid type A (GABAA) postsynaptic events that were modulated by presynaptic AMPA receptors. Pioneer neurons expressed the adhesion molecules L1 and TAG-1; the latter has been reported to control tangential migrations to the neocortex [Denaxa, M., Chan, C.-H., Schachner, M., Parnavelas, J. & Karagogeos, D. (2001) Development (Cambridge, U.K.) 128, 4635–4644], and we show here that the pioneer neurons of the marginal zone are the cellular substrate of such a function. Finally, we show that, in early corticogenesis, reelin controls both the tangential migration of cortical interneurons toward the cortical plate and the tangential migration of pioneer neurons toward the marginal zone.

Perlecan controls neurogenesis in the developing telencephalon

BackgroundPerlecan is a proteoglycan expressed in the basal lamina of the neuroepithelium during development. Perlecan absence does not impair basal lamina assembly, although in the 55% of the mutants early disruptions of this lamina conducts to exencephaly, impairing brain development. The rest of perlecan-null brains complete its prenatal development, maintain basal lamina continuity interrupted by some isolated ectopias, and are microcephalic. Microcephaly consists of thinner cerebral walls and underdeveloped ganglionic eminences. We have studied the mechanisms that generate brain atrophy in telencephalic areas where basal lamina is intact.ResultsBrain atrophy in the absence of perlecan started in the ventral forebrain and extended to lateral and dorsal parts of the cortex in the following stages. First, the subpallial forebrain developed poorly in early perlecan-null embryos, because of a reduced cell proliferation: the number of cells in mitosis decreased since the early stages of development. This reduction resulted in a decreased tangential migration of interneurons to the cerebral cortex. Concomitant with the early hypoplasia observed in the medial ganglionic eminences, Sonic Hedgehog signal decreased in the perlecan-null floor plate basal lamina at E12.5. Second, neurogenesis in the pallial neuroepithelium was affected in perlecan deficient embryos. We found reductions of nearly 50% in the number of cells exiting the cell cycle at E12–E13. The labeling index, which was normal at this age, significantly decreased with advancing corticogenesis. Moreover, nestin+ or PCNA+ progenitors increased since E14.5, reaching up to about 150% of the proportion of PCNA+ cells in the wild-type at E17.5. Thus, labeling index reduction together with increased progenitor population, suggests that atrophy is the result of altered cell cycle progression in the cortical progenitors. Accordingly, less neurons populated the cortical plate and subplate of perlecan-null neocortex, as seen with the neuronal markers β-tubulin and Tbr1.ConclusionAs a component of the basal lamina, perlecan both maintains this structure and controls the response of the neuroepithelium to growth factors. Less mitotic cells in the early medial ganglionic eminences, and impaired cell cycle progression in the late neocortex, suggests insufficient recruitment and signaling by neurogenic morphogens, such as SHH or FGF2.

A simple and reliable method for correlative light and electron microscopic studies.

We describe in detail a simple method for flat-embedding that can be subsequently used in correlative light and electron microscopic studies. The method can be applied to any material suitable for electron microscopy and is especially useful for study of the synaptology and ultrastructural characteristics of immunocytochemically or morphologically identified neurons or their processes. We present here an example to show how accurately one can delineate the fine details of a complex axonal arborization impregnated with the Golgi method in the mouse cerebral cortex. Golgi-impregnated sections to be studied at the electron microscopic level are osmicated, dehydrated, infiltrated with Araldite resin, flat-embedded, and identified cells or processes photographed. Serial semi-thin sections (1-2 microns thick) are then cut with an ultramicrotome, examined with the light microscope, and the elements rephotographed. Selected semi-thin sections are then resectioned on the ultramicrotome at 60-70 nm and examined electron microscopically. This method allows the systematic and accurate localization of stained cells and processes throughout the successive steps of the procedure.

Transient c-fos expression accompanies naturally occurring cell death in the developing interhemispheric cortex of the rat

We have searched for the possible correlation of naturally occurring cell death with spontaneously enhanced c-fos expression in the developing cerebral cortex of normal Wistar albino rats. During the late prenatal and early postnatal period, cells with irregular contours and intracytoplasmic electron-dense granules (granule-containing cells) were apparent in the interhemispheric cortex, including the anterior cingulate and the retrosplenial cortices. These cells were loosely packed within the cortical layers derived from the cortical plate. Having excluded the possibility that these cells could be phagocytes by immunocytochemical experiments, we propose that they are cells in different phases of a process of autophagic degeneration and death. Images of extreme nuclear pyknosis were also apparent in identical locations. Cells showing immunoreactivity for c-Fos protein appeared in the same cortical areas. The immunoreactive cells were very abundant in the retrosplenial cortex, but were also present in the anterior cingulate cortex. These cells showed markedly irregular contours and large, densely immunoreactive intracytoplasmic inclusions; these images were similar to those of granule-containing cells revealed by conventional stains. The immunoreactivity for c-Fos protein was ephemeral, occurring exclusively during embryonic days 20 and 21, but granule-containing cells were observed for a longer period. The present results provide evidence, albeit indirect, that c-fos expression may occur in certain neural cells at the onset of a process of death by autophagia, and suggest a possible involvement of the proto-oncogene c-fos in certain forms of naturally occurring neuronal death.