Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia.

Consistent with recent reports indicating that neurons differentiated in vitro from human-induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain, gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two independent discovery-based approaches-microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses-to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. These reproducible NPC phenotypes were identified through scalable assays that can be applied to expanded cohorts of SZ patients, making them a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

Specification of cerebral cortical areas.

How the immense population of neurons that constitute the human cerebral neocortex is generated from progenitors lining the cerebral ventricle and then distributed to appropriate layers of distinctive cytoarchitectonic areas can be explained by the radial unit hypothesis. According to this hypothesis, the ependymal layer of the embryonic cerebral ventricle consists of proliferative units that provide a proto-map of prospective cytoarchitectonic areas. The output of the proliferative units is translated via glial guides to the expanding cortex in the form of ontogenetic columns, whose final number for each area can be modified through interaction with afferent input. Data obtained through various advanced neurobiological techniques, including electron microscopy, immunocytochemistry, [3H]thymidine and receptor autoradiography, retrovirus gene transfer, neural transplants, and surgical or genetic manipulation of cortical development, furnish new details about the kinetics of cell proliferation, their lineage relationships, and phenotypic expression that favor this hypothesis. The radial unit model provides a framework for understanding cerebral evolution, epigenetic regulation of the parcellation of cytoarchitectonic areas, and insight into the pathogenesis of certain cortical disorders in humans.

Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition.

Autoradiographic evidence after injection of tritiated thymidine indicates that cell position in the laminae of the monkey visual cortex is systematically related to time of cell orgin. The earliest-formed neurons, destined for the deepest stratum, arise at about embryonic day 45, and the last ones, destined for the outermost cell stratum, form at about day 102; cells of intervening layers are generated at intervening times. No neocortical neurons are produced in the last two prenatal months or after birth. Compared to cortical neurons in rodents, those in the monkey arise earlier, and the "inside-out" relation of cell position to time of origin is more rigid.

Limits of neurogenesis in primates.

Systematic analysis of autoradiograms prepared from postpubertal rhesus monkeys given single and multiple injections of tritium-labeled thymidine and killed 3 days to 6 years later displayed a slow turnover of glial cells but failed to reveal any radiolabeled neurons. Therefore, unlike neurons of some nonprimate species, all neurons of the rhesus monkey brain are generated during prenatal and early postnatal life. A stable population of neurons in primates, including humans, may be important for the continuity of learning and memory over a lifetime.

Modulation of neuronal migration by NMDA receptors.

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor is essential for neuronal differentiation and establishment or elimination of synapses in a developing brain. The activity of the NMDA receptor has now been shown to also regulate the migration of granule cells in slice preparations of the developing mouse cerebellum. First, blockade of NMDA receptors by specific antagonists resulted in the curtailment of cell migration. Second, enhancement of NMDA receptor activity by the removal of magnesium or by the application of glycine increased the rate of cell movement. Third, increase of endogenous extracellular glutamate by inhibition of its uptake accelerated the rate of cell migration. These results suggest that NMDA receptors may play an early role in the regulation of calcium-dependent cell migration before neurons reach their targets and form synaptic contacts.

Selective role of N-type calcium channels in neuronal migration.

Analysis of neuronal migration in mouse cerebellar slice preparations by a laser scanning confocal microscope revealed that postmitotic granule cells initiate their migration only after the expression of N-type calcium channels on their plasmalemmal surface. Furthermore, selective blockade of these channels by addition of omega-conotoxin to the incubation medium curtailed cell movement. In contrast, inhibitors of L- and T-type calcium channels, as well as those of sodium and potassium channels, had no effect on the rate of granule cell migration. These results suggest that N-type calcium channels, which have been predominantly associated with neurotransmitter release in adult brain, also play a transient but specific developmental role in directed migration of immature neurons before the establishment of their synaptic circuits.

Overproduction and elimination of retinal axons in the fetal rhesus monkey.

Quantitative electron microscopic analysis reveals 2.85 million retinal axons in fetal rhesus monkeys--a number that is more than twice the 1.2 million present in the adult. More than 1 million supernumerary optic axons are eliminated before birth, simultaneously with the segregation of inputs from the two eyes into separate layers of the lateral geniculate nucleus. Selective elimination of optic axons may not only play a role in the segregation of binocular visual connections but, secondarily, may establish the ratio of crossed and uncrossed retinogeniculate projections.

Cell proliferation without neurogenesis in adult primate neocortex.

A recent assertion that new neurons are continually added to the neocortex of adult macaque monkeys has profound implications for understanding the cellular mechanisms of higher cognitive functions. Here we searched for neurogenesis in adult macaques by using immunofluorescent triple labeling for the DNA-replication indicator, bromodeoxyuridine (BrdU), and neuronal and glial cell markers. Although numerous BrdU-labeled cells were distributed throughout the cerebral wall, including the neocortex, these were identified as nonneuronal cells; evidence for newly generated neurons was limited to the hippocampus and olfactory bulb. Thus, our results do not substantiate the claim of neurogenesis in normal adult primate neocortex.

Contact-dependent inhibition of cortical neurite growth mediated by notch signaling.

The exuberant growth of neurites during development becomes markedly reduced as cortical neurons mature. In vitro studies of neurons from mouse cerebral cortex revealed that contact-mediated Notch signaling regulates the capacity of neurons to extend and elaborate neurites. Up-regulation of Notch activity was concomitant with an increase in the number of interneuronal contacts and cessation of neurite growth. In neurons with low Notch activity, which readily extend neurites, up-regulation of Notch activity either inhibited extension or caused retraction of neurites. Conversely, in more mature neurons that had ceased their growth after establishing numerous connections and displayed high Notch activity, inhibition of Notch signaling promoted neurite extension. Thus, the formation of neuronal contacts results in activation of Notch receptors, leading to restriction of neuronal growth and a subsequent arrest in maturity.

Transient synapses in the embryonic primate spinal cord.

Electron microscopic and tritiated thymidine autoradiographic analysis of the embryonic spinal cord in the rhesus monkey reveals considerable rearrangement of cellular and synaptic relationships in the posterior (sensory) quadrant during early developmental stages. This remodeling involves the death of an entire population of neurons that received synapses from sensory afferent axons and the possible relocation of these afferents upon subsequently generated viable substantia gelatinosa neurons.

Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex.

Synapses develop concurrently and at identical rates in different layers of the visual, somatosensory, motor, and prefrontal areas of the primate cerebral cortex. This isochronic course of synaptogenesis in anatomically and functionally diverse regions indicates that the entire cerebral cortex develops as a whole and that the establishment of cell-to-cell communication in this structure may be orchestrated by a single genetic or humoral signal. This is in contrast to the traditional view of hierarchical development of the cortical regions and provides new insight into the maturation of cortical functions.

The changing view of neural specificity.

The generation of specific patterns of neuronal connections has usually been regarded as a central problem in neurobiology. The prevailing view for many years has been that these connections are established by complementary recognition molecules on the pre- and postsynaptic cells (the chemoaffinity theory). Experimental results obtained in the past decade, however, indicate that the view that axon guidance and synaptogenesis proceed according to restrictive chemical markers is too narrow. Although a more rigid plan may prevail in some invertebrates, the formation of specific connections in vertebrates also involves competition between axon terminals, trophic feedback between pre- and postsynaptic cells, and modification of connections by functional activity.

Interdigitation of contralateral and ipsilateral columnar projections to frontal association cortex in primates.

The combined use of two anterograde axonal transport methods reveals that in the prefrontal association cortex of macaque monkeys, associational projections from the parietal lobe of one hemisphere interdigitate with callosal projections from the opposite frontal lobe, forming adjacent columns 300 to 750 micrometers wide. The finding of separate and alternating ipsilateral and contralateral inputs in the frontal association cortex opens up new possibilities for the functional analysis of this large but unexplored area of the primate brain.

D1 dopamine receptors in prefrontal cortex: involvement in working memory.

The prefrontal cortex is involved in the cognitive process of working memory. Local injections of SCH23390 and SCH39166, selective antagonists of the D1 dopamine receptor, into the prefrontal cortex of rhesus monkeys induced errors and increased latency in performance on an oculomotor task that required memory-guided saccades. The deficit was dose-dependent and sensitive to the duration of the delay period. These D1 antagonists had no effect on performance in a control task requiring visually guided saccades, indicating that sensory and motor functions were unaltered. Thus, D1 dopamine receptors play a selective role in the mnemonic, predictive functions of the primate prefrontal cortex.

Alpha 2-adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates.

This study provides evidence that the alpha 2-adrenergic receptor agonist clonidine ameliorates the cognitive deficits exhibited by aged nonhuman primates through drug actions at alpha 2 receptors. Furthermore, pharmacological profiles in animals with lesions restricted to the dorsolateral prefrontal cortex indicate that this area may be the site of action for some of clonidine's beneficial effects. These results demonstrate that alpha-adrenergic systems contribute to cognitive function and suggest a new strategy for treating memory disorders in aged humans.

Processing of the notch ligand delta by the metalloprotease Kuzbanian.

Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands.

Dissociation of object and spatial processing domains in primate prefrontal cortex.

Areas and pathways subserving object and spatial vision are segregated in the visual system. Experiments show that the primate prefrontal cortex is similarly segregated into object and spatial domains. Neurons that code information related to stimulus identity are dissociable, both by function and region, from those that code information related to stimulus location. These findings indicate that the prefrontal cortex contains separate processing mechanisms for remembering "what" and "where" an object is.

Reversal of antipsychotic-induced working memory deficits by short-term dopamine D1 receptor stimulation.

Chronic blockade of dopamine D2 receptors, a common mechanism of action for antipsychotic drugs, down-regulates D1 receptors in the prefrontal cortex and, as shown here, produces severe impairments in working memory. These deficits were reversed in monkeys by short-term coadministration of a D1 agonist, ABT 431, and this improvement was sustained for more than a year after cessation of D1 treatment. These findings indicate that pharmacological modulation of the D1 signaling pathway can produce long-lasting changes in functional circuits underlying working memory. Resetting this pathway by brief exposure to the agonist may provide a valuable strategy for therapeutic intervention in schizophrenia and other dopamine dysfunctional states.

Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein.

The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.

Areal segregation of face-processing neurons in prefrontal cortex.

A central issue in cognitive neuroscience concerns the functional architecture of the prefrontal cortex and the degree to which it is organized by sensory domain. To examine this issue, multiple areas of the macaque monkey prefrontal cortex were mapped for selective responses to visual stimuli that are prototypical of the brain's object vision pathway-pictorial representations of faces. Prefrontal neurons not only selectively process information related to the identity of faces but, importantly, such neurons are localized to a remarkably restricted area. These findings suggest that the prefrontal cortex is functionally compartmentalized with respect to the nature of its inputs.