Exposure to complex environments results in more sparse representations of space in the hippocampus.

The neural circuitry mediating sensory and motor representations is adaptively tuned by an animal's interaction with its environment. Similarly, higher order representations such as spatial memories can be modified by exposure to a complex environment (CE), but in this case the changes in brain circuitry that mediate the effect are less well understood. Here, we show that prolonged CE exposure was associated with increased selectivity of CA1 "place cells" to a particular recording arena compared to a social control (SC) group. Furthermore, fewer CA1 and DG neurons in the CE group expressed high levels of Arc protein, a marker of recent activation, following brief exposure to a completely novel environment. The reduced Arc expression was not attributable to overall changes in cell density or number. These data indicate that one effect of CE exposure is to modify high-level spatial representations in the brain by increasing the sparsity of population coding within networks of neurons. Greater sparsity could result in a more efficient and compact coding system that might alter behavioural performance on spatial tasks. The results from a behavioural experiment were consistent with this hypothesis, as CE-treated animals habituated more rapidly to a novel environment despite showing equivalent initial responding.

Patterning of retinoic acid signaling and cell proliferation in the hippocampus.

The nuclear receptor ligand retinoic acid (RA) has been identified as an endogenous regulatory factor in the hippocampus, acting on pyramidal neurons and granule neuron progenitors, but almost nothing is known about the distribution of RA itself in the hippocampus. This study describes the source of RA for the rodent hippocampus in the meninges via the key RA synthetic enzyme retinaldehyde dehydrogenase 2 (RALDH2). Diffusion of RA from the meninges potentially creates a gradient of RA across the infrapyramidal and suprapyramidal blades of the dentate gyrus, enhanced by the expression of the RA catabolic enzyme Cyp26B1 between the blades, and an infrapyramidal and suprapyramidal blade difference is evident in RA-regulated transcription. This asymmetry may contribute to some of the physiological and molecular differences between the blades, including a disparity in the rates of cell proliferation in the subgranular zone of the two blades through RA inhibition of cell proliferation. Such differences can be altered by either the application of excess RA, its effect dependent on the relative position along the septotemporal axis, or change in RA signaling through mutation of retinol binding protein, while the capacity of RA to inhibit proliferation of cells in the dentate gyrus is demonstrated using in vitro slice culture. Use of synthetic and catabolic enzymes in the hippocampus to create differing zones of RA concentration parallels the mechanisms used in the developing brain to generate patterns of RA-regulated transcription.

Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain.

Prevailing notions of cerebral vascularization imply that blood vessels sprout passively into the brain parenchyma from pial vascular plexuses to meet metabolic needs of growing neuronal populations. Endothelial cells, building blocks of blood vessels, are thought to be homogeneous in the brain with respect to their origins, gene expression patterns and developmental mechanisms. These current notions that cerebral angiogenesis is regulated by local environmental signals contrast with current models of cell-autonomous regulation of neuronal development. Here we demonstrate that telencephalic angiogenesis in mice progresses in an orderly, ventral-to-dorsal gradient regulated by compartment-specific homeobox transcription factors. Our data offer new perspectives on intrinsic regulation of angiogenesis in the embryonic telencephalon, call for a revision of the current models of telencephalic angiogenesis and support novel roles for endothelial cells in brain development.

Retinoic acid influences neuronal migration from the ganglionic eminence to the cerebral cortex.

The ganglionic eminence contributes cells to several forebrain structures including the cerebral cortex, for which it provides GABAergic interneurons. Migration of neuronal precursors from the retinoic-acid rich embryonic ganglionic eminence to the cerebral cortex is known to be regulated by several factors, but retinoic acid has not been previously implicated. We found retinoic acid to potently inhibit cell migration in slice preparations of embryonic mouse forebrains, which was reversed by an antagonist of the dopamine-D(2) receptor, whose gene is transcriptionally regulated by retinoic acid. Histone-deacetylase inhibitors, which amplify nuclear receptor-mediated transcription, potentiated the inhibitory effect of retinoic acid. Surprisingly, when retinoic acid signalling was completely blocked with a pan-retinoic acid receptor antagonist, this also decreased cell migration into the cortex, implying that a minimal level of endogenous retinoic acid is necessary for tangential migration. Given these opposing effects of retinoic acid in vitro, the in vivo contribution of retinoic acid to migration was tested by counting GABAergic interneurons in cortices of adult mice with experimental reductions in retinoic acid signalling: a range of perturbations resulted in significant reductions in the numerical density of some GABAergic interneuron subpopulations. These observations suggest functions of retinoic acid in interneuron diversity and organization of cortical excitatory-inhibitory balance.

Targets of the nuclear factor I regulon involved in early and late development of postmitotic cerebellar granule neurons.

Recent studies have shown that the nuclear factor I (NFI) family controls multiple stages of the postmitotic differentiation of cerebellar granule neurons (CGNs). Regulation of cell-cell signaling is an integral part of this NFI program, which involves expression of the cell adhesion molecules N cadherin and ephrin B1 throughout postmitotic CGN development. Here, we identify two additional downstream targets of NFI that are involved in extracellular CGN interactions. The cell adhesion molecule Tag-1 is highly enriched in CGNs undergoing parallel fiber formation and is down-regulated prior to onset of radial migration. We found that Tag-1 expression was strongly reduced by NFI dominant repression in immature primary CGNs and in the cerebella of E18 Nfib-null mice. Transient transfection and chromatin immunoprecipitation suggested that the Tag-1 gene is directly regulated by NFI. Furthermore, functional, Nfi knockout and chromatin immunoprecipitation studies implicated Wnt7a as a direct target of NFI in maturing CGNs. Wnt7a is secreted by developing CGNs and is required for maturation of mossy fiber-CGN synaptic rosettes. Consistent with this, synapsin I was greatly reduced within the internal granule cell layer of P17 Nfia-null mice. These findings indicated that NFI controls CGN postmitotic maturation through a combination of extracellular signaling molecules that operate either continuously to regulate multiple stages of development (N cadherin and ephrin B1) or primarily at early (Tag-1) or late (Wnt7a) maturation steps. They also illustrate the importance of NFI as a critical link between cell-intrinsic mechanisms and cell-cell interactions in the development of the mouse cerebellum.

Cdk5 regulates the phosphorylation of tyrosine 1472 NR2B and the surface expression of NMDA receptors.

NMDA receptors (NMDARs) are a major class of ionotropic glutamate receptors that can undergo activity-dependent changes in surface expression. Clathrin-mediated endocytosis is a mechanism by which the surface expression of NR2B-containing NMDA receptors is regulated. The C terminus of the NMDA receptor subunit NR2B contains the internalization motif YEKL, which is the binding site for the clathrin adaptor AP-2. The tyrosine (Y1472) within the YEKL motif is phosphorylated by the Src family of kinases and this phosphorylation inhibits the binding of AP-2 and promotes surface expression of NMDA receptors. Cdk5 is a serine/threonine kinase that has been implicated in synaptic plasticity, learning, and memory. Here we demonstrate that inhibition of Cdk5 results in increased phosphorylation of Y1472 NR2B at synapses and decreased binding of NR2B to beta2-adaptin, a subunit of AP-2, thus blocking the activity-dependent endocytosis of NMDA receptors. Furthermore, we show that inhibition of Cdk5 increases the binding of Src to postsynaptic density-95 (PSD-95), and that expression of PSD-95 facilitates the phosphorylation of Y1472 NR2B by Src. Together, these results suggest a model in which inhibition of Cdk5 increases the binding of Src to PSD-95 and the phosphorylation of Y1472 NR2B by Src, which results in decreased binding of NR2B to AP-2, and NR2B/NMDAR endocytosis. This study provides a novel molecular mechanism for the regulation of the surface expression of NR2B-containing NMDA receptors and gives insight into the Cdk5-dependent regulation of synaptic plasticity.

Nuclear factor I coordinates multiple phases of cerebellar granule cell development via regulation of cell adhesion molecules.

A central question is how various stages of neuronal development are integrated as a differentiation program. Here we show that the nuclear factor I (NFI) family of transcriptional regulators is expressed and functions throughout the postmitotic development of cerebellar granule neurons (CGNs). Expression of an NFI dominant repressor in CGN cultures blocked axon outgrowth and dendrite formation and decreased CGN migration. Inhibition of NFI transactivation also disrupted extension and fasciculation of parallel fibers as well as CGN migration to the internal granule cell layer in cerebellar slices. In postnatal day 17 Nfia-deficient mice, parallel fibers were greatly diminished and disoriented, CGN dendrite formation was dramatically impaired, and migration from the external germinal layer (EGL) was retarded. Axonal marker expression also was disrupted within the EGL of embryonic day 18 Nfib-null mice. NFI regulation of axon extension was observed under conditions of homotypic cell contact, implicating cell surface proteins as downstream mediators of its actions in CGNs. Consistent with this, the cell adhesion molecules ephrin B1 and N-cadherin were identified as NFI gene targets in CGNs using inhibitor and Nfi mutant analysis as well as chromatin immunoprecipitation. Functional inhibition of ephrin B1 or N-cadherin interfered with CGN axon extension and guidance, migration, and dendritogenesis in cell culture as well as in situ. These studies define NFI as a key regulator of postmitotic CGN development, in particular of axon formation, dendritogenesis, and migratory behavior. Furthermore, they reveal how a single transcription factor family can control and integrate multiple aspects of neuronal differentiation through the regulation of cell adhesion molecules.

Dopamine receptor activation modulates GABA neuron migration from the basal forebrain to the cerebral cortex.

GABA neurons of the cerebral cortex and other telencephalic structures are produced in the basal forebrain and migrate to their final destinations during the embryonic period. The embryonic basal forebrain is enriched in dopamine and its receptors, creating a favorable environment for dopamine to influence GABA neuron migration. However, whether dopamine receptor activation can influence GABA neuron migration is not known. We show that dopamine D1 receptor activation promotes and D2 receptor activation decreases GABA neuron migration from the medial and caudal ganglionic eminences to the cerebral cortex in slice preparations of embryonic mouse forebrain. Slice preparations from D1 or D2 receptor knock-out mouse embryos confirm the findings. In addition, D1 receptor electroporation into cells of the basal forebrain and pharmacological activation of the receptor promote migration of the electroporated cells to the cerebral cortex. Analysis of GABA neuron numbers in the cerebral wall of the dopamine receptor knock-out mouse embryos further confirmed the effects of dopamine receptor activation on GABA neuron migration. Finally, dopamine receptor activation mobilizes striatal neuronal cytoskeleton in a manner consistent with the effects on neuronal migration. These data show that impairing the physiological balance between D1 and D2 receptors can alter GABA neuron migration from the basal forebrain to the cerebral cortex. The intimate relationship between dopamine and GABA neuron development revealed here may offer novel insights into developmental disorders such as schizophrenia, attention deficit or autism, and fetal cocaine exposure, all of which are associated with dopamine and GABA imbalance.

A retinoic-acid critical period in the early postnatal mouse brain.

BACKGROUND: A normal supply of vitamin A, which regulates gene expression through its active form retinoic acid, is required by many organs; both excess and deficiency can be teratogenic. Very little is known about the role of retinoic acid in maturation of the mammalian forebrain. METHODS: As retinoic acid cannot be visualized directly, we mapped its actions in the forebrain with indirect morphologic methods and by applying retinoic acid overdoses to early postnatal mice. RESULTS: During this time, the morphologic indicators of retinoic acid actions are localized mainly in the limbic system and they undergo rapid changes. Retinoic acid overdoses can cause lasting behavioral abnormalities that point to disrupted limbic functions. In the anterior cingulate cortex, inhibitory interneurons are affected, and in the hippocampus, primarily the dentate gyrus is abnormal. CONCLUSIONS: Retinoic acid is involved in functional maturation of limbic regions of the forebrain with a critical stage early postnatally in mice, when their brains are particularly vulnerable to vitamin A perturbations. This developmental time in mice compares with the second trimester of gestation in humans, a stage when in genetically predisposed individuals the corresponding brain regions are known to pass through a period of increased susceptibility to environmental disturbances.

13-cis Retinoic acid (accutane) suppresses hippocampal cell survival in mice.

Use of the acne drug Accutane (13-cis retinoic acid, [13-cis RA]) has been associated with severe depression. This association has been considered controversial because no causative link has been found between 13-cis RA and this disorder. A recent hypothesis has suggested that atrophy of the hippocampus can result in depression. We now show, in a mouse model, that endogenous RA generated by synthetic enzymes in the meninges acts on hippocampal granule neurons, and chronic (3-week) exposure to a clinical dose of 13-cis RA may result in hippocampal cell loss. In humans this may be conjectured to be the mechanism by which Accutane contributes to depression.

Fructose metabolism in the cerebellum.

Under normal physiological conditions, the brain utilizes only a small number of carbon sources for energy. Recently, there is growing molecular and biochemical evidence that other carbon sources, including fructose, may play a role in neuro-energetics. Fructose is the number one commercial sweetener in Western civilization with large amounts of fructose being toxic, yet fructose metabolism remains relatively poorly characterized. Fructose is purportedly metabolized via either of two pathways, the fructose-1-phosphate pathway and/or the fructose-6-phosphate pathway. Many early metabolic studies could not clearly discriminate which of these two pathways predominates, nor could they distinguish which cell types in various tissues are capable of fructose metabolism. In addition, the lack of good physiological models, the diet-induced changes in gene expression in many tissues, the involvement of multiple genes in multiple pathways involved in fructose metabolism, and the lack of characterization of some genes involved in fructose metabolism have complicated our understanding of the physiological role of fructose in neuro-energetics. A recent neuro-metabolism study of the cerebellum demonstrated fructose metabolism and co-expression of the genes specific for the fructose 1-phosphate pathway, GLUT5 (glut5) and ketohexokinase (khk), in Purkinje cells suggesting this as an active pathway in specific neurons? Meanwhile, concern over the rapid increase in dietary fructose, particularly among children, has increased awareness about how fructose is metabolized in vivo and what effects a high fructose diet might have. In this regard, establishment of cellular and molecular studies and physiological characterization of the important and/or deleterious roles fructose plays in the brain is critical. This review will discuss the status of fructose metabolism in the brain with special reference to the cerebellum and the physiological roles of the different pathways.

Retinoic acid signaling and function in the adult hippocampus.

Retinoic acid (RA) is an essential growth factor, derived from vitamin A, which controls growth by activating specific receptors that are members of the nuclear receptor family of transcriptional regulators. Its function in control of growth and differentiation in the embryonic CNS has been extensively investigated, but a role for RA in the mature brain has only recently become apparent. Although the adult CNS has much less capacity for change compared to the embryonic CNS, a limited amount of flexibility, referred to as neural plasticity, still exists. It is these processes that RA influences in the adult brain, including long-term potentiation and neurogenesis. The hippocampus is a brain region dependent upon neural plasticity for its function in learning and memory, and this review focuses on the roles that RA may play in regulating these processes in the adult.

Defects in tangential neuronal migration of pontine nuclei neurons in the Largemyd mouse are associated with stalled migration in the ventrolateral hindbrain.

The LARGE gene encodes a putative glycosyltransferase that is required for normal glycosylation of dystroglycan, and defects in LARGE can cause abnormal neuronal migration in congenital muscular dystrophy (CMD). Previous studies have focused on radial migration, which is disrupted at least in part due to breaks in the basal lamina. Through analysis of precerebellar nuclei development in the Large(myd) mouse hindbrain, we show that tangential migration of a subgroup of hindbrain neurons may also be disrupted. Within the precerebellar nuclei, the pontine nuclei (PN) are severely disrupted, whereas the inferior olive (IO), external cuneate nuclei (ECN) and lateral reticular nuclei (LRN) appear unaffected. Large and dystroglycan are widely expressed in the hindbrain, including in the pontine neurons migrating in the anterior extramural migratory stream (AES). BrdU labeling and immunohistochemical studies suggest normal numbers of neurons begin their journey towards the ventral midline in the AES in the Large(myd) mouse. However, migration stalls and PN neurons fail to reach the midline, surviving as ectopic clusters of cells located under the pial surface dorsally and laterally to where they normally would finish their migration near the ventral midline. Stalling of PN neurons at this location is also observed in other migration disorders in mice. These observations suggest that glycan-dependent dystroglycan interactions are required for PN neurons to correctly respond to signals at this important migrational checkpoint.

Semaphorin 3A-mediated axon guidance regulates convergence and targeting of P2 odorant receptor axons.

Semaphorins are known to play an important role in axon guidance of vertebrate olfactory sensory neurons to their targets in specific glomeruli of the olfactory bulb (OB). However, it is not clear how semaphorin-mediated guidance contributes to a systematic hierarchy of cues that govern the organization of this system. Because of the putative role that odorant receptor molecules such as P2 could play in establishing appropriate glomerular destinations for growing olfactory axons, we have also determined the spatial organization of P2 glomeruli in semaphorin 3A (Sema3A) mutant mice. First, in the postnatal OB of control and Sema3A(-/-) mice, we analysed the trajectories of olfactory axons that express the Sema3A receptor, neuropilin-1 (npn-1) and the positions of npn-1(+) glomeruli. Sema3A at the ventral OB midline guides npn-1(+) axons to targets in the lateral and medial OB. Absence of Sema3A permits many npn-1 axons to terminate aberrantly in the rostral and ventral OB. Second, in Sema3A(-/-) mice, many P2 axons are abnormally distributed throughout the ventral OB nerve layer and converge in atypical locations compared with littermate controls where P2 axons converge on stereotypically located lateral and medial glomeruli. In addition to their radically altered spatial distribution, P2 glomeruli in Sema3A(-/-) mice are significantly smaller and more numerous than in heterozygote littermates. These data show that Sema3A is an important repulsive olfactory guidance cue that establishes restricted npn-1(+) subcompartments in the olfactory bulb. Furthermore, Sema3A plays a key role in the convergence of axons expressing the odorant receptor P2 onto their appropriate targets.

Cocaine exposure decreases GABA neuron migration from the ganglionic eminence to the cerebral cortex in embryonic mice.

Recurrent exposure of the developing fetus to cocaine produces persistent alterations in structure and function of the cerebral cortex. Neurons of the cerebral cortex are derived from two sources: projection neurons from the neuroepithelium of the dorsal pallium and interneurons from the ganglionic eminence of the basal telencephalon. The interneurons are GABAergic and reach the cerebral cortex via a tangential migratory pathway. We found that recurrent, transplacental exposure of mouse embryos to cocaine from embryonic day 8 to 15 decreases tangential neuronal migration and results in deficits in GABAergic neuronal populations in the embryonic cerebral wall. GABAergic neurons of the olfactory bulb, which are derived from the ganglionic eminence via the rostral migratory pathway, are not affected by the cocaine exposure suggesting a degree of specificity in the effects of cocaine on neuronal migration. Thus, one mechanism by which prenatal cocaine exposure exerts deleterious effects on cerebral cortical development may be by decreasing GABAergic neuronal migration from the ganglionic eminence to the cerebral wall. The decreased GABA neuron migration may contribute to persistent structural and functional deficits observed in the exposed offspring.

Molecular structure of cytoplasmic dynein 2 and its distribution in neuronal and ciliated cells.

Cytoplasmic dynein is involved in a wide variety of cellular functions. In addition to the initially characterized form (MAP 1C/dynein 1), a second form of cytoplasmic dynein (dynein 2) has been identified and implicated in intraflagellar transport (IFT) in lower eukaryotes and in Golgi organization in vertebrates. In the current study, the primary structure of the full-length dynein 2 heavy chain (HC) was determined from cDNA sequence. The dynein 1 and dynein 2 sequences were similar within the motor region, and around the light intermediate chain (LIC)-binding site within the N-terminal stem region. The dynein 2 HC co-immunoprecipitated with LIC3, a homologue of dynein 1 LICs. Dynein 2 mRNA was abundant in the ependymal layer of the neural tube and in the olfactory epithelium. Antibodies to dynein 2 HC, LIC3 and a component of IFT particles strongly stained the ependymal layer lining the lateral ventricles. Both dynein 2 HC and LIC3 staining was also observed associated with connecting cilia in the retina and within primary cilia of non-neuronal cultured cells. These data support a specific role for dynein 2 in the generation and maintenance of cilia.