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The cerebral cortex is the source of our most complex cognitive capabilities and a vulnerable target of many neurological and neuropsychiatric disorders. Transcriptomics offers a new approach to understanding the cortex at the level of its underlying genetic code, and rapid technological advances have propelled this field to the high-throughput study of the complete set of transcribed genes at increasingly fine resolution to the level of individual cells. These tools have revealed features of the genetic architecture of adult cortical areas, layers, and cell types, as well as spatiotemporal patterning during development. This has allowed a fresh look at comparative anatomy as well, illustrating surprisingly large differences between mammals while at the same time revealing conservation of some features from avians to mammals. Finally, transcriptomics is fueling progress in understanding the causes of neurodevelopmental diseases such as autism, linking genetic association studies to specific molecular pathways and affected brain regions.

The adolescent brain is more “plastic” than it will ever be again, capable of remarkable adaptability in light of the many social, physical, sexual, and intellectual challenges that this developmental phase brings. This is also a peak time for clinical onset of most mental illnesses (see the chart) ( 1 ). One in five adolescents have a mental illness that will persist into adulthood ( 2 ). Mental illnesses that emerge before adulthood impose a 10-fold higher cost than those that emerge later in life ( 3 ). Mental health costs are the highest single source of global economic burden in the world ( 4 ).

The morphology of neurons in the main divisions of the hippocampal complex allow the easy identification of granule cells in the dentate gyrus and pyramidal cells in the CA1 and CA3 regions of Ammon's horn. However, neurons in the CA2 subfield have been much more difficult to reliably identify. We have recently identified a set of genes whose expression is restricted to either the dentate gyrus, CA1, CA2, or CA3. Here we show that these genes have an essentially nonoverlapping distribution throughout the entire septotemporal extent of the hippocampus. 3-Dimensional reconstruction of serial sections processed for in situ hybridization of mannosidase 1, alpha (CA1), bcl-2-related ovarian killer protein (CA3), and Purkinje cell protein 4 (dentate gyrus+CA2) was used to define the boundaries of each subregion throughout the entire hippocampus. The boundaries observed for these three genes are recapitulated across a much larger set of genes similarly enriched in specific hippocampal subregions. The extent of CA2 defined on the basis of gene expression is somewhat larger than that previously described on the basis of structural anatomical criteria, particularly at the rostral pole of the hippocampus. These results indicate that, at least at the molecular level, there are robust, consistent genetic boundaries between hippocampal subregions CA1, CA2, CA3, and the dentate gyrus, allowing a redefinition of their boundaries in order to facilitate functional studies of different neuronal subtypes in the hippocampus.

In situ hybridization of mouse embryo whole mounts and sagittal sections revealed a tissue- and stage-specific expression pattern of the transcriptionally regulated serum and glucocorticoid-inducible protein kinase (sgk) during embryogenesis. Sgk expression is first observed at embryonic day 8.5 (E8.5) in the decidua and yolk sac, and then during developmental stages E9.5 through E12.5 this kinase is highly localized in the heart chamber, otic vesicle, blood vessels surrounding the somites, and lung buds. At the later stages of mouse embryogenesis, E13.5 through E16.5, sgk expression becomes highly concentrated in brain (choroid plexus), distal epithelium and the terminal bronchi/bronchioles, adrenal gland, liver, thymus and intestines, remains high in heart tissue, and is expressed at a low level in the other embryonic tissues.

Recent studies have proposed roles for neurotrophins in the formation and plasticity of ocular dominance columns as well as in the regulation of dendritic arborization in visual cortex of higher mammals. To assess potential roles for neurotrophins in these processes, we have examined the developmental expression of BDNF and NT-3 mRNA in the cat's visual system using in situ hybridization. BDNF and NT-3 mRNAs are dynamically regulated in many CNS structures during embryonic and postnatal development, and both mRNAs undergo striking developmental changes in laminar specificity and levels of expression within primary visual cortex during the critical period for ocular dominance column formation. Within visual cortex, BDNF mRNA is found in neurons in deep cortical layers (5 and 6) prior to eye opening, and in both deep and superficial layers (2 and 3) shortly afterwards. Within layer 4, the target of thalamocortical axons, BDNF mRNA is low initially and rises to high levels by the end of the critical period for ocular dominance column formation. NT-3 mRNA is first detectable in small stellate neurons at the base of layer 4 (4c) after eye opening, and levels decrease near the end of the critical period. BDNF and NT-3 mRNAs can be detected in the lateral geniculate nucleus at birth, and levels peak during the critical period. In both structures, BDNF mRNA expression is maintained into adulthood, while NT-3 is undetectable in the adult. The presence and dynamic regulation of these neurotrophins in visual structures is consistent with suggested roles for both of these neurotrophins in axonal and dendritic remodeling known to accompany the formation of ocular dominance columns.

The putative excitatory and inhibitory cell classes within the mouse primary visual cortex V1 have different functional properties as studied using recording microelectrode. Excitatory neurons show high selectivity for the orientation angle of moving gratings while the putative inhibitory neurons show poor selectivity. However, the study of selectivity of the genetically identified interneurons and their subtypes remain controversial. Here we use novel Cre-driver and reporter mice to identify genetic subpopulations in vivo for two photon calcium dye imaging: Wfs1(+) / Gad1(-) mice that labels layer 2/3 excitatory cell population and Pvalb(+) / Gad1(+) mice that labels a genetic subpopulation of inhibitory neurons. The cells in both mice were identically labeled with a tdTomato protein, visible in vivo, using a Cre-reporter line. We found that the Wfs1(+) cells exhibited visual tuning properties comparable to the excitatory population, i.e. high selectivity and tuning to the angle, direction and spatial frequency of oriented moving gratings. The functional tuning of Pvalb(+) neurons was consistent with previously reported narrow spiking interneurons in microelectrode studies, exhibiting poorer selectivity than the excitatory neurons. This study demonstrates the utility of Cre-transgenic mouse technology in selective targeting of subpopulations of neurons and makes them amenable to structural, functional and connectivity studies.

Sleep deprivation (SD) leads to a suite of cognitive and behavioral impairments, and yet the molecular consequences of SD in the brain are poorly understood. Using a systematic immediate-early gene (IEG) mapping to detect neuronal activation, the consequences of SD were mapped primarily to forebrain regions. SD was found to both induce and suppress IEG expression (and thus neuronal activity) in subregions of neocortex, striatum, and other brain regions. Laser microdissection and cDNA microarrays were used to identify the molecular consequences of SD in seven brain regions. In situ hybridization (ISH) for 222 genes selected from the microarray data and other sources confirmed that robust molecular changes were largely restricted to the forebrain. Analysis of the ISH data for 222 genes (publicly accessible at http://sleep.alleninstitute.org) provided a molecular and anatomic signature of the effects of SD on the brain. The suprachiasmatic nucleus (SCN) and the neocortex exhibited differential regulation of the same genes, such that in the SCN genes exhibited time-of-day effects while in the neocortex, genes exhibited only SD and waking (W) effects. In the neocortex, SD activated gene expression in areal-, layer-, and cell type-specific manner. In the forebrain, SD preferentially activated excitatory neurons, as demonstrated by double-labeling, except for striatum which consists primarily of inhibitory neurons. These data provide a characterization of the anatomical and cell type-specific signatures of SD on neuronal activity and gene expression that may account for the associated cognitive and behavioral effects.

SummaryGenetic strategies for perturbing activity of selected neurons hold great promise for understanding circuitry and behavior. Several such strategies exist, but there has been no direct demonstration of reversible inactivation of mammalian neurons in vivo. We previously reported quickly reversible inactivation of neurons in vitro using expression of the Drosophila allatostatin receptor (AlstR). Here, adeno-associated viral vectors are used to express AlstR in vivo in cortical and thalamic neurons of rats, ferrets, and monkeys. Application of the receptor's ligand, allatostatin (AL), leads to a dramatic reduction in neural activity, including responses of visual neurons to optimized visual stimuli. Additionally, AL eliminates activity in spinal cords of transgenic mice conditionally expressing AlstR. This reduction occurs selectively in AlstR-expressing neurons. Inactivation can be reversed within minutes upon washout of the ligand and is repeatable, demonstrating that the AlstR/AL system is effective for selective, quick, and reversible silencing of mammalian neurons in vivo.

Neural stem/progenitor cells in the adult brain are able to generate both the brain's major cell types: glial cells, which are non-neuronal, and the active nerve cells or neurons. Neurons are produced in just two regions of the brain. Lie et al. have now identified a protein family that instructs the adult neural stem cells to produce neurons, rather than glial cells. The signal molecule Wnt3 is shown to be crucial for the production of neurons in the adult hippocampus, a region believed to be involved in learning and memory formation. Ultimately these studies may help develop therapies to repair brain damage caused by disease or trauma. The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus1. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population1. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development2. We also show that the Wnt/β-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.

DNA methylation-mediated epigenetic regulation plays critical roles in regulating mammalian gene expression, but its role in normal brain function is not clear. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, has been shown to bind methylated gene promoters and facilitate transcriptional repression in vitro . Here we report the generation and analysis of MBD1 -/- mice. MBD1 -/- mice had no detectable developmental defects and appeared healthy throughout life. However, we found that MBD1 -/- neural stem cells exhibited reduced neuronal differentiation and increased genomic instability. Furthermore, adult MBD1 -/- mice had decreased neurogenesis, impaired spatial learning, and a significant reduction in long-term potentiation in the dentate gyrus of the hippocampus. Our findings indicate that DNA methylation is important in maintaining cellular genomic stability and is crucial for normal neural stem cell and brain functions.

Genetic methods for neuronal silencing have great promise for allowing selective inactivation of specific cell types within complex neural systems. Present methods, however, are limited in their reversibility by the slow time scale (days) of transcriptional regulation. We report the rapid and reversible inactivation of mammalian cortical neurons expressing the insect G-protein-coupled receptor AlstR (Drosophila allatostatin receptor) [corrected] after application of its peptide ligand allatostatin (AL). The onset and reversal of inactivation could be achieved rapidly, within minutes. Moreover, the effects of AL were selective for AlstR-transfected neurons. The AlstR/AL system is therefore a promising genetic method for selective and quickly reversible silencing of neuronal activity.

The hippocampus consists of distinct anatomic regions that have been demonstrated to have different biological functions. To explore the molecular differences between hippocampal subregions, we performed transcriptional profiling analysis by using DNA microarray technology. The cRNA derived from the CA1, CA3, and dentate gyrus regions of the hippocampus and from spinal cord was hybridized to Affymetrix high-density oligo arrays. This systematic approach revealed sets of genes that were expressed specifically in subregions of the hippocampus corresponding to predefined cytoarchitectural boundaries, which could be confirmed by in situ hybridization and Real Time quantitative polymerase chain reaction. The relative enrichment and absence of genes in the hippocampal subregions support the conclusion that there is a molecular basis for the previously defined anatomic subregions of the hippocampus and also reveal genes that could be important in defining the unique functions of the hippocampal subfields. J. Comp. Neurol. 441:187–196, 2001. © 2001 Wiley-Liss, Inc.

The neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a candidate retrograde signaling molecule for geniculocortical axons during the formation of ocular dominance columns. Here we examined whether neuronal activity can regulate BDNF mRNA in eye-specific circuits in the developing cat visual system. Dark-rearing throughout the critical period for ocular dominance column formation decreases levels of BDNF mRNA within primary visual cortex, whereas short-term (2 d) binocular blockade of retinal activity with tetrodotoxin (TTX) downregulates BDNF mRNA within the lateral geniculate nucleus (LGN) and visual cortical areas. Brief (6 hr to 2 d) monocular TTX blockade during the critical period and also in adulthood causes downregulation in appropriate eye-specific laminae in the LGN and ocular dominance columns within primary visual cortex. Monocular TTX blockade at postnatal day 23 also downregulates BDNF mRNA in a periodic fashion, consistent with recent observations that ocular dominance columns can be detected at these early ages by physiological methods. In contrast, 10 d monocular TTX during the critical period does not cause a lasting decrease in BDNF mRNA expression in columns pertaining to the treated eye, consistent with the nearly complete shift in physiological response properties of cortical neurons in favor of the unmanipulated eye known to result from long-term monocular deprivation. These observations demonstrate that BDNF mRNA levels can provide an accurate “molecular readout” of the activity levels of cortical neurons and are consistent with a highly local action of BDNF in strengthening and maintaining active synapses during ocular dominance column formation.