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.

] i increases in local cell clusters, and synchronous transients in cell pairs undergoing mitotic division (8). In the postnatal neocortex, coordinated [Ca 2ϩ ] i rises can occur in 3-100 adjacent neurons, termed neuronal domains (9). These spontaneous events are mediated by gap junctional communication (10) and appear to involve cell-to-cell diffusion of the second messenger inositol trisphosphate (9, 11). Neocortical domains apparently can occur independently of action potential activity, but their frequency can be increased by activation of metabotropic glutamate receptors (mGluR) (11) (19,20). Recent work has also shown that the frequency and duration of [Ca 2ϩ ] i oscillations can differentially influence the expression of specific genes (21-23). One known mechanism for the generation of [Ca 2ϩ ] i oscillations by cell-to-cell signaling involves activation of the mGluR5 subtype of the group I mGluRs, which are highly expressed in the immature neocortex (16, 24, 25).Activation of mGluR5 leads to the production of inositol trisphosphate by phospholipase C, which, in turn, leads to increases in [Ca 2ϩ ] i . Little is known, however, about the role that mGluR signaling plays in the physiology of developing neocortical neurons.To investigate the potential role of mGluR activation in generating spontaneous [Ca 2ϩ ] i oscillations in developing neocortical neurons, we performed a series of calcium imaging experiments using embryonic and postnatal mouse neocortical slices. We found that activation of the mGluR5 subtype of mGluRs leads to [Ca 2ϩ ] i oscillations in immature neurons, and that endogenous glutamate acting on group I mGluRs is responsible for spontaneous [Ca 2ϩ ] i oscillations in developing neocortex. MethodsTissue Preparation. Coronal slices of neocortex (300-400 m) were prepared from neonatal or embryonic Swiss-Webster mice (Taconic Farms) by using a Vibratome (Pelco, Redding, CA) as previously described (13). For embryonic slices, brains were embedded in 4% low-melting agarose (Fisher Scientific) in PBS with glucose (Life Technologies, Grand Island, NY) prior to slicing. Slices were prepared and incubated in standard artificial cerebrospinal fluid (ACSF), which contains 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 1 mM MgSO 4 , 2 mM CaCl 2 , 25 mM NaHCO 3 , and 20 mM glucose (pH 7.4), oxygenated with 95% O 2 ͞5% CO 2 . Following slice preparation, slices were incubated in ACSF at room temperature for 15 min prior to loading with the indicator dye.Fluo-3 Loading. Cells were loaded with the calcium indicator dye fluo-3 by bath application of fluo-3 acetoxymethyl ester (fluo-3-AM; Molecular Probes). Loading was performed for 1-4 hr at room temperature in ACSF containing 15 M fluo-3-AM, 0.0067% pluronic F-127 (Molecular Probes), and 0.33% DMSO (Sigma). The loading chamber was continuously oxygenated with 95% O 2 ͞5% CO 2 .Calcium Imaging. Measurements of relative changes in [Ca 2ϩ ] i were made using vital epifluorescence microscopy of mouse neocortical slices loaded with fluo-3. Fo...

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.

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 (Ca²⁺), 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 Ca²⁺ ([Ca²⁺]_i) fluctuations of cells within the intact neocortical VZ. Previous observations have demonstrated that similar patterns of spontaneous [Ca²⁺]_i increase occur in both proliferative and postmitotic cortical cells, suggesting that they may be mechanistically similar. Our results suggest that the changes in [Ca²⁺]_i in VZ cells and cortical plate neurons are likely triggered by different mechansims, and imply that similar changes in [Ca²⁺]_i may underlie different signaling events during distinct phases of neocortical development.

BackgroundNo head-to-head clinical trials have been published comparing guanfacine extended release (GXR) and atomoxetine (ATX): two nonstimulants approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). However, other study designs or methods could be used to indirectly compare these two medications. Matching-adjusted indirect comparison (MAIC) is a recent methodology that utilizes individual patient data (IPD) from clinical trials for one treatment and published aggregate data from another treatment to estimate the relative efficacy of both, providing rapid, reliable comparative efficacy results.ObjectiveThe aim of this study was to compare the efficacy of GXR and ATX for the treatment of ADHD using MAIC.Study DesignA systematic literature search was conducted to identify ATX and GXR trials published through December 2012. Studies were selected for MAIC analyses on the basis of having comparable trial characteristics and study designs. Summary data from selected ATX trials and IPD from selected GXR trials were used. MAIC methodology ensured comparable populations: target doses for the ‘base case’ comparison were selected on the basis of maximum effective dosage ranges from the US FDA-approved product labels (GXR 0.09–0.12 mg/kg/day, ATX 1.2 mg/kg/day for children and adolescents weighing ≤70 kg). Individuals from GXR trials were selected if they matched inclusion/exclusion criteria from selected ATX trials; selected GXR IPD were then re-weighted to match the published ATX trial mean baseline characteristics and placebo outcomes. Sensitivity analyses were conducted, examining different dosage ranges and repeating the analysis in a larger number of trials, allowing for larger and more heterogeneous trial populations.Main Outcome MeasureThe primary outcome measure was change in ADHD Rating Scale IV (ADHD-RS-IV) total score.ResultsUsing MAIC in the base case comparison, significantly greater reductions in mean (standard error; SE) ADHD-RS-IV total scores from baseline to end of study were observed in patients treated with GXR relative to ATX [−7.0 (2.2); p < 0.01]. Significantly greater reductions for GXR over ATX were also demonstrated for hyperactivity/impulsivity [−3.8 (1.2); p < 0.01] and inattention [−3.2 (1.3); p < 0.05] subscales of the ADHD-RS-IV. Similar results were observed in MAIC sensitivity analyses evaluating other dosage ranges and using more heterogeneous trial populations (e.g., larger randomized sample, broader subject weight range, additional trials). Mean (SE) decreases in ADHD-RS-IV total scores were greater for GXR relative to ATX when including IPD for those administered GXR at lower than target dosage (0.075–0.090 mg/kg/day) compared with ATX at target dosage (1.2 mg/kg/day), with a relative improvement of −6.0 (2.7) (p < 0.05). Reductions in ADHD-RS-IV total scores were also greater for GXR in another MAIC examining GXR at target dosage (0.09–0.12 mg/kg/day) and a broader range of ATX dosages (including three additional trials evaluating ATX ≥1.2 mg/kg/day); relative ...