Mature Cerebral Cortex Research Articles

Astrocytes Transplanted during Early Postnatal Development Integrate, Mature, and Survive Long Term in Mouse Cortex.

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35-47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine.SIGNIFICANCE STATEMENT The mechanisms that enable astrocytes to acquire diverse molecular and structural properties remain to be better understood. In this study, we systematically analyzed the properties of cortical astrocytes following their transplantation to the early postnatal brain. We found that immature cortical astrocytes transplanted into cerebral cortex during early postnatal mouse development integrate and establish normal astrocytic properties, and show long-term survival in vivo (more than one year). In contrast, transplanted cortical astrocytes display reduced or altered ability to integrate into the more mature cerebral cortex or developing cerebellum, respectively. This study demonstrates the developmental potential of transplanted cortical astrocytes and provides an approach to tease apart cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms that determine the structural, molecular, and physiological phenotype of astrocytes.

Supernumerary neurons within the cerebral cortical subplate in autism spectrum disorders

Autism spectrum disorders (ASDs) involve alterations to cortical connectivity that manifest as reduced coordinated activity between cortical regions. The neurons of the cortical subplate are a major contributor to establishing thalamocortical, corticothalamic and corticocortical long-range connections and only a subset of this cell population survives into adulthood. Previous reports of an indistinct gray-white matter boundary in subjects with ASD suggest that the adjacent subplate may also show organizational abnormalities. Frozen human postmortem tissue samples from the parietal lobe (BA7) were used to evaluate white-matter neuron densities adjacent to layer VI with an antibody to NeuN. In addition, fixed postmortem tissue samples from frontal (BA9), parietal (BA7) and temporal lobe (BA21) locations, were stained with a Golgi-Kopsch procedure, and used to examine the morphology of these neuronal profiles. Relative to control cases, ASD subjects showed a large average density increase of NeuN-positive profiles of 44.7 percent. The morphologies of these neurons were consistent with subplate cells of the fusiform, polymorphic and pyramidal cell types. Lower ratios of fusiform to other cell types are found early in development and although adult ASD subjects showed consistently lower ratios, these differences were not significant. The increased number of retained subplate profiles, along with cell type ratios redolent of earlier developmental stages, suggests either an abnormal initial population or a partial failure of the apoptosis seen in neurotypical development. These results indicate abnormalities within a neuron population that plays multiple roles in the developing and mature cerebral cortex, including the establishment of long-range cortical connections.

How Do Electric Fields Coordinate Neuronal Migration and Maturation in the Developing Cortex?

During development the vast majority of cells that will later compose the mature cerebral cortex undergo extensive migration to reach their final position. In addition to intrinsically distinct migratory behaviors, cells encounter and respond to vastly different microenvironments. These range from axonal tracts to cell-dense matrices, electrically active regions and extracellular matrix components, which may all change overtime. Furthermore, migrating neurons themselves not only adapt to their microenvironment but also modify the local niche through cell-cell contacts, secreted factors and ions. In the radial dimension, the developing cortex is roughly divided into dense progenitor and cortical plate territories, and a less crowded intermediate zone. The cortical plate is bordered by the subplate and the marginal zone, which are populated by neurons with high electrical activity and characterized by sophisticated neuritic ramifications. Neuronal migration is influenced by these boundaries resulting in dramatic changes in migratory behaviors as well as morphology and electrical activity. Modifications in the levels of any of these parameters can lead to alterations and even arrest of migration. Recent work indicates that morphology and electrical activity of migrating neuron are interconnected and the aim of this review is to explore the extent of this connection. We will discuss on one hand how the response of migrating neurons is altered upon modification of their intrinsic electrical properties and whether, on the other hand, the electrical properties of the cellular environment can modify the morphology and electrical activity of migrating cortical neurons.

Developmental Cell Death in the Cerebral Cortex.

In spite of the high metabolic cost of cellular production, the brain contains only a fraction of the neurons generated during embryonic development. In the rodent cerebral cortex, a first wave of programmed cell death surges at embryonic stages and affects primarily progenitor cells. A second, larger wave unfolds during early postnatal development and ultimately determines the final number of cortical neurons. Programmed cell death in the developing cortex is particularly dependent on neuronal activity and unfolds in a cell-specific manner with precise temporal control. Pyramidal cells and interneurons adjust their numbers in sync, which is likely crucial for the establishment of balanced networks of excitatory and inhibitory neurons. In contrast, several other neuronal populations are almost completely eliminated through apoptosis during the first two weeks of postnatal development, highlighting the importance of programmed cell death in sculpting the mature cerebral cortex.

The effect of unilateral stroke on autonomic function in the term newborn.

The mature cerebral cortex has a topographically organized influence on reflex autonomic centers of the brainstem and diencephalon and sympathetic activation coming primarily from the right hemisphere and parasympathetic activation from the left. In the term newborn, the maturational status of this central autonomic system remains poorly understood. Sixteen term newborns admitted to Children's National with unilateral middle cerebral artery (MCA) strokes (n = 8 left, n = 8 right) had archived continuous electrocardiograph (EKG) signals available. We compared stroke laterality and severity with indices of autonomic function, as measured by heart rate variability. We performed both time- and frequency-domain analyses on the R-R interval (RRi) over 24h of continuous EKG data at around 7 days of age. Right MCA stroke significantly increased sympathetic tone, while left MCA stroke increased parasympathetic tone. Regardless of laterality, stroke severity was associated inversely with sympathetic tone and positively with parasympathetic tone. Surprisingly, injury to either insular region had no significant autonomic effect. Phenobarbital blood levels were positively associated with sympathetic tone and inversely related to parasympathetic tone. Based on these findings, it is difficult to reconcile the functional topography of the central autonomic system in term newborns with that currently proposed for the normal mature brain. Further investigation is clearly needed.

Distribution and Cellular Localization of KCC2 in the Ferret Neocortex

KCC2 (a brain-specific potassium-chloride cotransporter) affects development of the cerebral cortex, including aspects of neuronal migration and cellular maturation and differentiation. KCC2 also modulates chloride homeostasis by influencing the switch of GABA from depolarizing in young neurons to hyperpolarizing in mature neurons. We describe the expression pattern, regional distribution, and cellular colocalization of KCC2 in the ferret cortex in normal kits and those treated with methylazoxymethanol acetate (MAM). We earlier developed a model of impaired cortical development by injecting MAM during mid-cortical gestation, which briefly interferes with neuronal production and additionally results in increased levels of KCC2 at P0. Using immunohistochemistry, we show a shift in KCC2 expression during development, being high in the subplate at P0, repositioning into a subtle laminar pattern in the neocortex at P7-P14, and becoming homogeneous at P35. KCC2 colocalizes with neuronal markers in the developing and mature cerebral cortex of normal ferrets and those treated with MAM, but shows a differential pattern of expression at different ages and locates in distinct cellular compartments during development. Subcellular localization shows that KCC2 predominantly situates in the membrane fraction of neocortical samples. These findings reveal that KCC2 colocalizes differentially with neurons and its expression pattern alters during development.

Fezf2 expression in layer 5 projection neurons of mature mouse motor cortex.

The mature cerebral cortex contains a wide diversity of neuron phenotypes. This diversity is specified during development by neuron-specific expression of key transcription factors, some of which are retained for the life of the animal. One of these key developmental transcription factors that is also retained in the adult is Fezf2, but the neuron types expressing it in the mature cortex are unknown. With a validated Fezf2-Gfp reporter mouse, whole-cell electrophysiology with morphology reconstruction, cluster analysis, in vivo retrograde labeling, and immunohistochemistry, we identify a heterogeneous population of Fezf2(+) neurons in both layer 5A and layer 5B of the mature motor cortex. Functional electrophysiology identified two distinct subtypes of Fezf2(+) neurons that resembled pyramidal tract projection neurons (PT-PNs) and intratelencephalic projection neurons (IT-PNs). Retrograde labeling confirmed the former type to include corticospinal projection neurons (CSpPNs) and corticothalamic projection neurons (CThPNs), whereas the latter type included crossed corticostriatal projection neurons (cCStrPNs) and crossed-corticocortical projection neurons (cCCPNs). The two Fezf2(+) subtypes expressed either CTIP2 or SATB2 to distinguish their physiological identity and confirmed that specific expression combinations of key transcription factors persist in the mature motor cortex. Our findings indicate a wider role for Fezf2 within gene expression networks that underpin the diversity of layer 5 cortical projection neurons.

Specification of excitatory neurons in the developing cerebral cortex: progenitor diversity and environmental influences.

The mature cerebral cortex harbors a heterogeneous population of glutamatergic neurons, organized into a highly intricate histological architecture. Classically, this mixed population of neurons was thought to be generated sequentially from a seemingly homogenous group of progenitors under the influence of external cues. This view, however, has been challenged in the last decade by evidences pointing to the existence of fate-restricted neuronal progenitors in the developing neocortex. Here, we review classical studies using cell transplantation, retroviral labeling and cell culture, as well as new data from genetic fate-mapping analysis, to discuss the lineage relationships between neocortical progenitors and subclasses of excitatory neurons. We also propose a temporal model to conciliate the existence of fate-restricted progenitors alongside multipotent progenitors in the neocortex. Finally, we discuss evidences for a critical period of plasticity among post mitotic excitatory cortical neurons when environmental influences could change neuronal cell fate.

Formation of cortical inhibition in ontogenesis

Analysis of literature data about development of cortical inhibition allows suggesting that synaptical inhibition of cerebral cortex neurons begins to be realized almost simultaneously with beginning of their generation of background activity. From the moment of the appearance of inhibitory processes there have been functioning all kinds of cortical inhibition. Thereby, the main mechanisms realizing cortical inhibition in the mature cerebral cortex begin functioning from the moment of its inclusion in ontogenesis.

Dysfunction of KCNK Potassium Channels Impairs Neuronal Migration in the Developing Mouse Cerebral Cortex

Development of the cerebral cortex depends partly on neural activity, but the identity of the ion channels that might contribute to the activity-dependent cortical development is unknown. KCNK channels are critical determinants of neuronal excitability in the mature cerebral cortex, and a member of the KCNK family, KCNK9, is responsible for a maternally transmitted mental retardation syndrome. Here, we have investigated the roles of KCNK family potassium channels in cortical development. Knockdown of KCNK2, 9, or 10 by RNAi using in utero electroporation impaired the migration of late-born cortical excitatory neurons destined to become Layer II/III neurons. The migration defect caused by KCNK9 knockdown was rescued by coexpression of RNAi-resistant functional KCNK9 mutant. Furthermore, expression of dominant-negative mutant KCNK9, responsible for the disease, and electrophysiological experiments demonstrated that ion channel function was involved in the migration defect. Calcium imaging revealed that KCNK9 knockdown or expression of dominant-negative mutant KCNK9 increased the fraction of neurons showing calcium transients and the frequency of spontaneous calcium transients. Mislocated neurons seen after KCNK9 knockdown stayed in the deep cortical layers, showing delayed morphological maturation. Taken together, our results suggest that dysfunction of KCNK9 causes a migration defect in the cortex via an activity-dependent mechanism.

Pancortins interact with amyloid precursor protein and modulate cortical cell migration

Neuronal precursor cell migration in the developing mammalian brain is a complex process requiring the coordinated interaction of numerous proteins. We have recently shown that amyloid precursor protein (APP) plays a role in migration into the cortical plate through its interaction with two cytosolic signaling proteins, disabled 1 (DAB1) and disrupted in schizophrenia 1 (DISC1). In order to identify extracellular factors that may signal through APP to regulate migration, we performed an unbiased mass spectrometry-based screen for factors that bind to the extracellular domain of APP in the rodent brain. Through this screen, we identified an interaction between APP and pancortins, proteins expressed throughout the developing and mature cerebral cortex. Via co-immunoprecipitation, we show that APP interacts with all four of the mammalian pancortin isoforms (AMY, AMZ, BMY, BMZ). We demonstrate that the BMZ and BMY isoforms of pancortin can specifically reduce β-secretase- but not α-secretase-mediated cleavage of endogenous APP in cell culture, suggesting a biochemical consequence of the association between pancortins and APP. Using in utero electroporation to overexpress and knock down specific pancortin isoforms, we reveal a novel role for pancortins in migration into the cortical plate. Interestingly, we observe opposing roles for alternate pancortin isoforms, with AMY overexpression and BMZ knock down both preventing proper migration of neuronal precursor cells. Finally, we show that BMZ can partially rescue a loss of APP expression and that APP can rescue effects of AMY overexpression, suggesting that pancortins act in conjunction with APP to regulate entry into the cortical plate. Taken together, these results suggest a biochemical and functional interaction between APP and pancortins, and reveal a previously unidentified role for pancortins in mammalian cortical development.

Maternal immune activation alters behavior in adult offspring, with subtle changes in the cortical transcriptome and epigenome

Maternal immune activation during prenatal development, including treatment with the viral RNA mimic, polyriboinosinic–polyribocytidilic acid (poly IC), serves as a widely used animal model to induce behavioral deficits reminiscent of schizophrenia and related disease. Here, we report that massive cytokine activation after a single dose of poly IC in the prenatal period is associated with lasting working memory deficits in adult offspring. To explore whether dysregulated gene expression in cerebral cortex, contributes to cognitive dysfunction, we profiled the cortical transcriptome, and in addition, mapped the genome-wide distribution of trimethylated histone H3-lysine 4 (H3K4me3), an epigenetic mark sharply regulated at the 5′ end of transcriptional units. However, deep sequencing-based H3K4me3 mapping and mRNA profiling by microarray did not reveal significant alterations in mature cerebral cortex after poly IC exposure at embryonic days E17.5 or E12.5. At a small set of genes (including suppressor of cytokine signaling Socs3), H3K4me3 was sensitive to activation of cytokine signaling in primary cultures from fetal forebrain but adult cortex of saline- and poly IC-exposed mice did not show significant differences. A limited set of transcription start sites (TSS), including Disrupted-in-Schizophrenia 1 (Disc1), a schizophrenia risk gene often implicated in gene–environment interaction models, showed altered H3K4me3 after prenatal poly IC but none of these differences survived after correcting for multiple comparisons. We conclude that prenatal poly IC is associated with cognitive deficits later in life, but without robust alterations in epigenetic regulation of gene expression in the cerebral cortex.

Expression of Nurr1 during rat brain and spinal cord development

Nurr1 is a member of the nuclear receptor superfamily and is a regulatory factor of differentiation, migration and maturation of mesencephalic dopaminergic neurons. The present study was designed to observe the dynamic changes in the protein expression of Nurr1 and the relationship between Nurr1 and proliferating cell nuclear antigen (PCNA) during rat brain and spinal cord development. And we also investigated the significance of Nurr1 in differentiation and migration of nerve cells. Paraffin-embedded sections, immunohistochemistry, immunohistochemical double staining and Western blot techniques were used. The results demonstrate that the presence of Nurr1-positive cells increased during embryo development and that these cells slowly migrated to locations far from the lateral ventricle. In postnatal rats, the presence of Nurr1-positive cells surrounding the lateral ventricle decreased markedly. The expression of Nurr1 in the cerebral cortex peaked at postnatal days 1–5 (P1–P5) and then decreased as the cells matured, becoming rare in the mature cerebral cortex. As the cells matured, a staircase-shaped migration of Nurr1-positive cells from dorsal areas to ventral areas of the spinal cord could be observed. As maturation continued, the presence of Nurr1-positive cells in the spinal cord decreased, and no Nurr1-positive cells were found in the mature spinal cord. The comparative observation of Nurr1 and PCNA showed that the two proteins were expressed in different regions and in different cells. Nurr1 was confined to differentiated and migrating immature cells and was not present in proliferating cells. We suggest that Nurr1 may play a regulatory role in the differentiation, migration and maturation of nerve cells in the rat brain and spinal cord.

In VivoIntracellular Recording Suggests That Gray Matter Astrocytes in Mature Cerebral Cortex and Hippocampus Are Electrophysiologically Homogeneous

Previous anatomical and in vitro electrophysiology studies suggest that astrocytes are heterogeneous in physiology, morphology, and biochemical content. However, the extent to which this diversity applies to in vivo conditions is largely unknown. To characterize and classify the physiological and morphological properties of cerebral cortical and hippocampal astrocytes in the intact brain, we performed in vivo intracellular recordings from single astrocytes using anesthetized mature rats. Astrocytes were classified based on their glial fibrillary acidic protein (GFAP) immunoreactivity and cell body locations. We analyzed morphometric measures such as the occupied volume and polarity, as well as physiological characteristics such as the mean membrane potential. These measurements did not show obvious segregation into subpopulations, suggesting that gray matter astrocytes in the cortex and hippocampus are composed of a homogeneous population in mature animals. The membrane potential of astrocytes in both cortex and hippocampus fluctuated within a few millivolts in the presence of spontaneous network activity. These membrane potential fluctuations of an astrocyte showed a significant variability that depended on the local field potential state and cell body location. We attribute the variability of the membrane potential fluctuations to local potassium concentration changes due to neuronal activity.

Untangling the cortex: Advances in understanding specification and differentiation of corticospinal motor neurons

The mature cerebral cortex contains a staggering variety of projection neuron subtypes, and a number of complementary studies have recently begun to define their identity and embryonic origin. Among the different types of cortical projection neurons, subcerebral projection neurons, including corticospinal motor neurons (CSMN), have been extensively studied and some of the molecular controls over their differentiation have been elucidated. Here, we first provide an overview of the approaches used to purify and molecularly profile neuronal populations of the neocortex and, more broadly, of the central nervous system (CNS). Next, we specifically review recent progress in understanding the genes that define and control development of the CSMN population. Finally, we briefly discuss the relevance of this work to current questions regarding the mechanisms of the establishment of projection neuron subtype identity in the neocortex and its implications to direct the differentiation of CSMN for therapeutic benefit.

Reelin and apolipoprotein E receptor 2 in the embryonic and mature brain: effects of an evolutionary change in the apoER2 gene

In the mature cerebral cortex of higher vertebrates, neurons are arranged in layers, each layer containing neurons of the same functional class. The cortical layering pattern is laid down during development by migration of young post-mitotic neurons along glial fibres to their correct positions in the cortical plate. The mechanics of whole-cell movement are well understood, but there is still uncertainty as to how a migrating neuron is instructed to leave its glial support when it reaches its destination. An intraneuronal signalling pathway initiated by reelin and containing apolipoprotein E receptor 2 (apoER2) is essential for normal cortical layering, and there is strong evidence that detachment of a migrating neuron is brought about by reelin-dependent downregulation of alpha3 integrin. But there remains the problem of how the reelin signal is switched on at a position in the cortex appropriate for each class of neuron. ApoER2 of placental mammals contains an amino acid sequence that is encoded in a separate exon in the apoER2 gene and is required for normal memory and spatial learning. The separate exon is not present in marsupials, birds or reptiles. The addition of this exon to the evolving apoER2 gene may have contributed to the success of placental mammals.

Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex

In the developing brain, gradients are commonly used to divide neurogenic regions into distinct functional domains. In this article, we discuss the functions of morphogen and gene expression gradients in the assembly of the nervous system in the context of the development of the cerebral cortex. The cerebral cortex is a mammal-specific region of the forebrain that functions at the top of the neural hierarchy to process and interpret sensory information, plan and organize tasks, and to control motor functions. The mature cerebral cortex is a modular structure, consisting of anatomically and functionally distinct areas. Those areas of neurons are generated from a uniform neuroepithelial sheet by two forms of gradients: graded extracellular signals and a set of transcription factor gradients operating across the field of neocortical stem cells. Fgf signaling from the rostral pole of the cerebral cortex sets up gradients of expression of transcription factors by both activating and repressing gene expression. However, in contrast to the spinal cord and the early Drosophila embryo, these gradients are not subsequently resolved into molecularly distinct domains of gene expression. Instead, graded information in stem cells is translated into discrete, region-specific gene expression in the postmitotic neuronal progeny of the stem cells.

Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex

Radial glial cells play a critical role in the construction of mammalian brain by functioning as a source of new neurons and by providing a scaffold for radial migration of new neurons to their target locations. Radial glia transform into astrocytes at the end of embryonic development. Strategies to promote functional recovery in the injured adult brain depend on the generation of new neurons and the appropriate guidance of these neurons to where they are needed, two critical functions of radial glia. Thus, the competence to regain radial glial identity in the adult brain is of significance for the ability to promote functional repair via neurogenesis and targeted neuronal migration in the mature brain. Here we show that the in vivo induction of the tyrosine kinase receptor, ErbB2, in mature astrocytes enables a subset of them to regain radial glial identity in the mature cerebral cortex. These new radial glial progenitors are capable of giving rise to new neurons and can support neuronal migration. These studies indicate that ErbB2 signaling critically modulates the functional state of radial glia, and induction of ErbB2 in distinct adult astrocytes can promote radial glial identity in the mature cerebral cortex.

β‐dystrobrevin, a kinesin‐binding receptor, interacts with the extracellular matrix components pancortins

The dystrobrevins (alpha and beta) are components of the dystrophin-associated protein complex (DPC), which links the cytoskeleton to the extracellular matrix and serves as a scaffold for signaling proteins. The precise functions of the beta-dystrobrevin isoform, which is expressed in nonmuscle tissues, have not yet been determined. To gain further insights into the role of beta-dystrobrevin in brain, we performed a yeast two-hybrid screen and identified pancortin-2 as a novel beta-dystrobrevin-binding partner. Pancortins-1-4 are neuron-specific olfactomedin-related glycoproteins, highly expressed during brain development and widely distributed in the mature cerebral cortex of the mouse. Pancortins are important constituents of the extracellular matrix and are thought to play an essential role in neuronal differentiation. We characterized the interaction between pancortin-2 and beta-dystrobrevin by in vitro and in vivo association assays and mapped the binding site of pancortin-2 on beta-dystrobrevin to amino acids 202-236 of the beta-dystrobrevin molecule. We also found that the domain of interaction for beta-dystrobrevin is contained in the B part of pancortin-2, a central region that is common to all four pancortins. Our results indicate that beta-dystrobrevin could interact with all members of the pancortin family, implying that beta-dystrobrevin may be involved in brain development. We suggest that dystrobrevin, a motor protein receptor that binds kinesin heavy chain, might play a role in intracellular transport of pancortin to specific sites in the cell.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. I. Cerebral cortex

Signaling through G-protein-coupled receptors is modulated by a family of regulator of G protein signaling (RGS) proteins that have been implicated in several neurological and psychiatric disorders. Defining the detailed expression patterns and developmental regulation of RGS proteins has been hampered by an absence of antibodies useful for mapping. We have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express GFP under the control of endogenous regulator of G-protein signaling 4 (RGS4) enhancer elements. This report focuses on expression patterns in the developing and mature cerebral cortex. Based on reporter distribution, RGS4 is expressed by birth in neurons across all cortical domains, but in different patterns that suggest region- and layer-specific regulation. Peak expression typically occurs before puberty, with complex down-regulation by adulthood. Deep and superficial neurons, in particular, vary in their patterns across developmental age and region and, in primary sensory cortices, layer IV neurons exhibit low or no expression of the GFP reporter. These data suggest that altering RGS4 function will produce a complex neuronal phenotype with cell- and subdomain-specificity in the cerebral cortex.