Developmental regulation of CPG15 expression in Xenopus.

Mechanisms controlling dendritic arbor formation affect the establishment of neuronal circuits. Candidate plasticity gene 15 (CPG15) is a glycosylphosphatidyl inositol (GPI)-linked activity-induced protein that has been shown to function as an intercellular signaling molecule that can promote the morphological and physiological development of the Xenopus retinotectal system. A thorough understanding of CPG15 function requires knowledge of the spatiotemporal expression of the endogenous protein. We therefore cloned Xenopus cpg15 and used RNA in situ hybridization and immunohistochemistry to determine the pattern of CPG15 expression. cpg15 mRNA and CPG15 protein are first detectable in the developing spinal cord and become widespread as development proceeds. CPG15 is expressed in sensory regions of the brain, including the visual, auditory, and olfactory systems. Within the retina, CPG15 is only expressed in retinal ganglion cells. CPG15 protein is concentrated in axon tracts, including retinal axons. These data support a model in which CPG15 expressed in retinal ganglion cells is trafficked to retinal axons, where it modulates postsynaptic dendritic arbor elaboration, and synaptic maturation.

Molecular analysis of developmental plasticity in neocortex.

Gene expression studies indicate that during activity-dependent developmental plasticity, N-methyl-D-aspartate receptor activation causes a Ca(2+)-dependent increase in expression of transcription factors and their downstream targets. The products of these plasticity genes then operate collectively to bring about the structural and functional changes that underlie ocular dominance plasticity in visual cortex. Identifying and characterizing plasticity genes provides a tool for molecular dissection of the mechanisms involved. Members of second-messenger pathways identified in adult plasticity paradigms and elements of the transmission machinery are the first candidate plasticity genes tested for their role in activity-dependent developmental plasticity. Knockout mice with deletions of such genes have allowed analyzing their function in the context of different systems and in different paradigms. Studies of mutant mice reveal that activity-dependent plasticity is not necessarily a unified phenomenon. The relative importance of a gene can vary with the context of its expression during different forms of plasticity. Forward genetic screens provide additional new candidates for testing, some with well-defined cellular functions that provide insight into possible plasticity mechanisms.

Dynamic regulation of cpg15 during activity-dependent synaptic development in the mammalian visual system.

During visual system development, neural activity regulates structural changes in connectivity including axonal branching and dendritic growth. Here we have examined a role for the candidate plasticity gene 15 (cpg15), which encodes an activity-regulated molecule that can promote dendritic growth, in this process. We report that cpg15 is expressed in the cat visual system at relatively high levels in the lateral geniculate nucleus (LGN) but at very low levels in its synaptic target, layer 4 of the visual cortex. Prenatally, when cpg15 mRNA in the LGN is most abundant, expression is insensitive to action potential blockade by tetrodotoxin. Postnatally, activity regulation of cpg15 emerges in the LGN coincident with development of ocular dominance columns in the visual cortex. cpg15 can be detected in layers 2/3 and 5/6 of visual cortex postnatally, and expression in layers 2/3 is activity-regulated during known periods of activity-dependent plasticity for these layers. Localization and regulation of cpg15 expression in the visual system are consistent with a presynaptic role for CPG15 in shaping dendritic arbors of target neurons during activity-dependent synaptic rearrangements, both in development and adulthood.

Promotion of dendritic growth by CPG15, an activity-induced signaling molecule.

Activity-independent and activity-dependent mechanisms work in concert to regulate neuronal growth, ensuring the formation of accurate synaptic connections. CPG15, a protein regulated by synaptic activity, functions as a cell-surface growth-promoting molecule in vivo. In Xenopus laevis, CPG15 enhanced dendritic arbor growth in projection neurons, with no effect on interneurons. CPG15 controlled growth of neighboring neurons through an intercellular signaling mechanism that requires its glycosylphosphatidylinositol link. CPG15 may represent a new class of activity-regulated, membrane-bound, growth-promoting proteins that permit exquisite spatial and temporal control of neuronal structure.

Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms.

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions: cpg2--a dystrophin-like cytoskeletal protein; cpg4--a heat-shock protein: cpg16--a protein kinase; cpg20--a transcription factor; cpg21--a dual-specificity MAP-kinase phosphatase; and cpg30 and cpg38--two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of the cpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

A set of genes expressed in response to light in the adult cerebral cortex and regulated during development.

Activity-dependent plasticity is thought to underlie both formation of appropriate synaptic connections during development and reorganization of adult cortical topography. We have recently cloned many candidate plasticity-related genes (CPGs) induced by glutamate-receptor activation in the hippocampus. Screening the CPG pool for genes that may contribute to neocortical plasticity resulted in the identification of six genes that are induced in adult visual cortical areas in response to light. These genes are also naturally induced during postnatal cortical development. CPG induction by visual stimulation occurs primarily in neurons located in cortical layers II-III and VI and persists for at least 48 hr. Four of the visually responsive CPGs (cpg2, cpg15, cpg22, cpg29) are previously unreported genes, one of which (cpg2) predicts a "mini-dystrophin-like" structural protein. These results lend molecular genetic support to physiological and anatomical studies showing activity-dependent structural reorganization in adult cortex. In addition, these results provide candidate genes the function of which may underlie mechanisms of adult cortical reorganization.

Neural selective activation and temporal regulation of a mammalian GAP-43 promoter in zebrafish.

Neurons throughout the vertebrate nervous system selectively activate the gene for a growth cone component, GAP-43, during embryonic development, and then decrease its expression abruptly as they form synapses. Distal interruption of mature axons in the central nervous system (CNS) of fish and amphibians, but not in the mammalian CNS reverses the developmental down-regulation of GAP-43 expression. To explore functional conservation and divergence of cis-acting elements that regulate expression of the GAP-43 gene, we studied activation, in transgenic zebrafish embryos, of mammalian GAP-43 genomic sequences fused to a marker gene. The DNA fragments containing the GAP-43 promoter, including a short fragment of 386 base pairs, were preferentially activated in the embryonic fish nervous system at times when extensive neuronal differentiation and neurite outgrowth take place. After 2 days of development, expression of the mammalian transgenes was specifically downregulated in the fish spinal cord but increased in more rostral regions of the CNS. This expression pattern was well correlated with the regulation of the endogenous fish GAP-43 gene revealed by in situ hybridization. Elements of the mammalian gene located a substantial distance upstream of the minimal promoter directed additional expression of the marker gene in a specific set of non-neural cells in zebrafish embryos. Our results indicate that cis-acting elements of the GAP-43 gene, and signaling pathways controlling these elements during embryonic development, have been functionally conserved in vertebrate evolution.

Numerous candidate plasticity-related genes revealed by differential cDNA cloning.

Plasticity is a property of the nervous system that allows it to modify its response to an altered input. This capacity for change suggests that there are molecular mechanisms in neurons that can couple stimuli to long-term alterations in phenotype. Neuronal excitation elicits rapid transcriptional activation of several immediate-early genes, for example c-fos, c-jun and zif268. Many immediate-early genes encode transcription factors that control expression of downstream genes whose products are believed to bring about long-term plastic changes. Here we use a highly sensitive differential complementary DNA cloning procedure to identify genes that may participate in long-term plasticity. We cloned 52 cDNAs of genes induced by the glutamate analogue kainate in the hippocampus dentate gyrus. The number of these candidate plasticity-related genes (CPGs) is estimated to be 500-1,000. One of the cloned CPGs (16C8), encoding a protease inhibitor, is induced by a stimulus producing long-term potentiation and during dentate gyrus development; a second, cpg1, is dependent on activation of the NMDA (N-methyl-D-aspartate) receptor for induction and encodes a new small, dentate-gyrus-specific protein. Seventeen of the cloned CPGs encode known proteins, including six suggesting that strong neuronal activation leads to de novo synthesis of vesicular and other synaptic components.

A neural-specific GAP-43 core promoter located between unusual DNA elements that interact to regulate its activity.

In an effort to identify cis-acting elements that respond to signals controlling different stages of neural differentiation, we have analyzed the promoter and surrounding regulatory sequences of the rat GAP-43 gene. Expression of this gene is both neural specific and, within neurons, strongly modulated by signals related to axon integrity. Expression analysis in cell lines and primary rat cortical cultures demonstrates that neural-selective gene expression can be directed by a 386 base pair GAP-43 promoter fragment that contains canonical TATA and CCAAT box consensus sequences. A short region of homology with other neural-specific genes, identified upstream of the core promoter, is not essential for selective expression in neuronal cells. Within cortical cell cultures, expression is strongly modulated by two interacting elements on either side of the promoter, each of which contains a sequence with the potential to adopt an unusual DNA conformation. While each of these flanking elements reduces expression when added alone to the core promoter, each counteracts the negative influence of the other when both elements are present.

The beta-adrenergic receptor survives solubilization in deoxycholate while forming a stable association with the agonist.

Agonist, but not antagonist, protects the beta-adrenergic receptor from inactivation during solubilization in deoxycholate. Protection is apparently due to locking of the agonist in the receptor, a high affinity interaction induced or stabilized by this detergent. The guanyl nucleotide-binding protein which normally interacts with the receptor to induce high affinity binding of the hormone is apparently not required in this agonist-specific locking process. It is therefore possible that deoxycholate mimics the effect of the guanyl nucleotide-binding protein on the conformation of the hormone-occupied receptor. The experiments further show that the receptor in deoxycholate is quite stable for days, provided that it is occupied by the agonist. Removal of deoxycholate brings about the release of the locked agonist and the return of the receptor to the dynamic functional state.