Neurons derived from radial glial cells establish radial units in neocortex.

The neocortex of the adult brain consists of neurons and glia that are generated by precursor cells of the embryonic ventricular zone. In general, glia are generated after neurons during development, but radial glia are an exception to this rule. Radial glia are generated before neurogenesis and guide neuronal migration. Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete. Although the lineage relationships of cortical neurons and glia have been explored, the clonal relationship of radial glia to other cortical cells remains unknown. It has been suggested that radial glia may be neuronal precursors, but this has not been demonstrated in vivo. We have used a retroviral vector encoding enhanced green fluorescent protein to label precursor cells in vivo and have examined clones 1-3 days later using morphological, immunohistochemical and electrophysiological techniques. Here we show that clones consist of mitotic radial glia and postmitotic neurons, and that neurons migrate along clonally related radial glia. Time-lapse images show that proliferative radial glia generate neurons. Our results support the concept that a lineage relationship between neurons and proliferative radial glia may underlie the radial organization of neocortex.

An excitatory GABAergic plexus in developing neocortical layer 1.

Layer 1 of the developing rodent somatosensory cortex contains a dense, transient GABAergic fiber plexus. Axons arising from the zona incerta (ZI) of the ventral thalamus contribute to this plexus, as do axons of intrinsic GABAergic cells of layer 1. The function of this early-appearing fiber plexus is not known, but these fibers are positioned to contact the apical dendrites of most postmigratory neurons. Here we show that electrical stimulation of layer 1 results in a GABA(A)-mediated postsynaptic current (PSC) in pyramidal neurons. Gramicidin perforated patch recording demonstrates that the GABAergic layer 1 synapse is excitatory and can trigger action potentials in cortical neurons. In contrast to electrical stimulation, activation of intrinsic layer 1 neurons with a glutamate agonist fails to produce PSCs in pyramidal cells. In addition, responses can be evoked by stimulation of layer 1 at relatively large distances from the recording site. These findings are consistent with a contribution of the widely projecting incertocortical pathway, the only described GABAergic projection to neonatal cortex. Recording of identified neonatal incertocortical neurons reveals a population of active cells that exhibit high frequencies of spontaneous action potentials and are capable of robustly activating neonatal cortical neurons. Because the fiber plexus is confined to layer 1, this pathway provides a spatially restricted excitatory GABAergic innervation of the distal apical dendrites of pyramidal neurons during the peak period of cortical synaptogenesis.

Extrinsic GABAergic innervation of developing neocortical layer 1 in organotypic slice co-cultures.

Afferents from the zona incerta (ZI) of the ventral thalamus contribute to the dense, transient gamma-aminobutyric acid (GABA)ergic fiber plexus in layer 1 of the developing rodent somatosensory cortex. Incertocortical axons contact the distal apical dendrites of postmigratory cortical pyramidal cells. Although recent work has shown that these GABAergic incertocortical fibers are likely to provide widespread fast synaptic excitation of pyramidal cells in layers 2-6 during peak periods of cortical synaptogenesis, little is known about the mechanisms by which these axons project to the neocortex and are confined to layer 1. Here we characterize organotypic slice co-cultures in which a region of embryonic diencephalon containing the ZI is maintained adjacent to a region of embryonic somatosensory cortex. Diencephalic explants from transgenic mice expressing enhanced green fluorescent protein (EGFP) enabled direct visualization of diencephalocortical connections. Isochronic co-cultures exhibited diencephalocortical fiber ingrowth immunoreactive for both GABA and the presynaptic vesicle-associated protein synaptophysin that was restricted to neocortical layer 1. This pattern of lamina-specific diencephalocortical ingrowth occurred irrespective of placement of the afferent explant, and persisted in the absence of action potential activity and GABA(A) receptor activation. Heterochronic co-cultures containing older cortex demonstrated that the cortical explants remain permissive for lamina-specific ingrowth through the first postnatal week. Organotypic slice cocultures provide a system in which to study the mechanisms underlying the layer 1-specific ingrowth of extrinsic GABAergic inputs to the perinatal neocortex.

Calcium dynamics of neocortical ventricular zone cells.

Cell-cell signaling within the neocortical ventricular zone (VZ) has been shown to influence the proliferation of VZ precursor cells and the subsequent differentiation and fate of postmitotic neurons. Calcium (Ca(2+)), a ubiquitous second messenger implicated in the regulation of many aspects of development, may play a role in these signaling events. Accordingly, we have examined the spatiotemporal patterns of spontaneous intracellular free Ca(2+) ([Ca(2+)](i)) fluctuations of cells within the intact neocortical VZ. Previous observations have demonstrated that similar patterns of spontaneous [Ca(2+)](i) increase occur in both proliferative and postmitotic cortical cells, suggesting that they may be mechanistically similar. Our results suggest that the changes in [Ca(2+)](i) in VZ cells and cortical plate neurons are likely triggered by different mechansims, and imply that similar changes in [Ca(2+)](i) may underlie different signaling events during distinct phases of neocortical development.

Endogenous activation of metabotropic glutamate receptors in neocortical development causes neuronal calcium oscillations.

Oscillations in intracellular free calcium concentration ([Ca(2+)](i)) occur spontaneously in immature neurons of the developing cerebral cortex. Here, we show that developing murine cortical neurons exhibit calcium oscillations in response to direct activation of the mGluR5 subtype of the group I metabotropic glutamate receptor (mGluR). In contrast, other manipulations that elicit [Ca(2+)](i) increases produce simple, nonoscillatory changes. Furthermore, we find that spontaneous oscillatory [Ca(2+)](i) activity is blocked by antagonists of group I mGluRs, suggesting a specific role for mGluR activation in the promotion of oscillatory [Ca(2+)](i) dynamics in immature cortical neurons. The oscillatory pattern of [Ca(2+)](i) increases produced by mGluR activation might play a role in the regulation of gene expression and the control of developmental events.