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At least 919 records · Page 51Linked to original sources
PubMed1998

A murine model of HIV encephalitis: xenotransplantation of HIV-infected human neuroglia into SCID mouse brain.

The degree of neuronal damage in HIV encephalitis (HIVE) and the mechanisms leading to it are not known. Post-mortem human studies provide limited information concerning pathogenesis, and there are few animal models of HIVE. We have developed a murine model of HIVE based on HIV-infected human brain tissues grafted within the host CNS milieu. HIV-infected blood-derived macrophages were cocultured with and incorporated within second trimester human fetal brain neuroglia. Mixed neuronal-glial aggregates were injected into the striatum of SCID mice where they survived for more than 6 months. Cellular proliferation and differentiation were determined by immunohistochemical staining for proliferating cell nuclear antigen and cellular markers. Synapse formation was seen by immunocytochemistry for synapsin and by electron microscopy. Virus was detected by immunohistochemical staining for HIV gp41. Based on the long-term survival of human neuroglial xenografts containing HIV infected macrophages, we believe that this model will support the study of chronic HIV-associated neurodegeneration and the testing of various CNS targeted therapeutic interventions.

AIDS Dementia Complex↗
PubMed1999

SNARE complex at the ribbon synapses of cochlear hair cells: analysis of synaptic vesicle- and synaptic membrane-associated proteins.

Neurotransmitters are released via exocytosis of synaptic vesicles involving a fusion complex consisting of a set of highly conserved proteins, which form a multiprotein complex resulting in the docking of synaptic vesicles at the site of release. There are three major differences between cochlear hair cell synapses and CNS synapses: (i) hair cells have a specialized structure, the synaptic ribbon, to which synaptic vesicles are attached; (ii) hair cells can maintain high and sustained release of neurotransmitter; and (iii) hair cells lack synaptophysin and synapsin. These differences suggest that an unconventional mechanism of neurotransmitter release may be involved at ribbon synapses. In this study we used different and complementary approaches to determine whether or not ribbon-containing hair cells of the cochlea express any component of the core fusion complex found in conventional synapses. Syntaxin 1, the synaptic membrane synaptosome-associated protein (SNAP)-25 and vesicle-associated membrane protein (VAMP or synaptobrevin) were found to be present in the organ of Corti of both rat and guinea-pig, as shown by reverse transcription polymerase chain reaction and Western blotting. In situ hybridization and immunocytochemistry showed mRNA and protein expression, respectively, in both inner and outer hair cells. Synaptotagmins I and II, generally considered to play major roles in neurotransmitter release at central synapses, were not detected in the organ of Corti.

Animals↗
PubMed2000

Bone morphogenetic protein-7 enhances dendritic growth and receptivity to innervation in cultured hippocampal neurons.

Members of the bone morphogenetic protein (BMP) family of growth factors are present in the central nervous system during development and throughout life. They are known to play an important regulatory role in cell differentiation, but their function in postmitotic telencephalic neurons has not been investigated. To address this question, we examined cultured hippocampal neurons following treatment with bone morphogenetic protein-7 (BMP-7, also referred to as osteogenic protein-1). When added at the time of plating, BMP-7 markedly stimulated the rate of dendritic development. Within 1 day, the dendritic length of BMP-7-treated neurons was more than twice that of controls. By three days the dendritic arbors of BMP-7-treated neurons had attained a level of branching similar to that of 2-week-old neurons cultured under standard conditions. Several findings indicate that BMP-7 selectively enhances dendritic development. While dendritic length was significantly increased in BMP-7-treated neurons, the length of the axon was not. In addition, the mRNA encoding the dendritic protein MAP2 was significantly increased by BMP-7 treatment, but the mRNA for tubulin was not. Finally, BMP-7 did not enhance cell survival. Because dendritic maturation is a rate-limiting step in synapse formation in hippocampal cultures, we examined whether BMP-7 accelerated the rate at which neurons became receptive to innervation. Using two separate experimental paradigms, we found that the rate of synapse formation (assessed by counting synapsin I-positive presynaptic vesicle clusters) was increased significantly in neurons that had been exposed previously to BMP-7. Because BMP-7 and related BMPs are expressed in the hippocampus in situ, these factors may play a role in regulating dendritic branching and synapse formation in both development and plasticity.

Animals↗
PubMed2002

Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarray.

Studies were performed to determine the effects of acute and chronic voluntary periods of exercise on the expression of hippocampal genes. RNAs from rodents exposed to a running wheel for 3, 7 and 28 days were examined using a microarray with 1176 cDNAs expressed primarily in the brain. The expression of selected genes was quantified by Taqman RT-PCR or RNase protection assay. The largest up-regulation was observed in genes involved with synaptic trafficking (synapsin I, synaptotagmin and syntaxin); signal transduction pathways (Ca2+/calmodulin-dependent protein kinase II, CaM-KII; mitogen-activated/extracellular signal-regulated protein kinase, MAP-K/ERK I and II; protein kinase C, PKC-delta) or transcription regulators (cyclic AMP response element binding protein, CREB). Genes associated with the glutamatergic system were up-regulated (N-methyl-d-aspartate receptor, NMDAR-2A and NMDAR-2B and excitatory amino acid carrier 1, EAAC1), while genes related to the gamma-aminobutyric acid (GABA) system were down-regulated (GABAA receptor, glutamate decarboxylase GAD65). Brain-derived neurotrophic factor (BDNF) was the only trophic factor whose gene was consistently up-regulated at all timepoints. These results, together with the fact that most of the genes up-regulated have a recognized interaction with BDNF, suggest a central role for BDNF on the effects of exercise on brain plasticity. The temporal profile of gene expression seems to delineate a mechanism by which specific molecular pathways are activated after exercise performance. For example, the CaM-K signal system seems to be active during acute and chronic periods of exercise, while the MAP-K/ERK system seems more important during long-term exercise.

Animals↗
PubMed1994

Glycolipids isolated from Aplysia kurodai can activate cyclic adenosine 3',5'-monophosphate-dependent protein kinase from rat brain.

Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonoglycosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGal beta 1-->3GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2](2-aminoethylphosphonyl-->6)Glc beta 1-->4Glc beta 1-->1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 microM. Activation of cAMP-kinase was maximal with 250 microM SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.

Animals↗
PubMed1997

Occurrence of two types of secretory vesicles in the human neuroblastoma SH-SY5Y.

Western blot analysis showed that the human neuroblastoma SH-SY5Y expresses the proteins synaptotagmin l, synaptobrevin, synapsin-l, rab3a, syntaxin, SNAP-25, NSF, alpha-SNAP, and munc-18, which have been implicated in the movement, docking, and fusion of vesicles during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins I and II, synaptotagmin l, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine-beta-hydroxylase on sucrose-D2O gradient fractions indicates the presence of a population of noradrenaline-containing large dense-cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48-kDa synaptophysin antigen absent from the large dense-cored vesicles. Immunocytochemical experiments show that not all of the vesicles that express synaptotagmin l contain secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH-SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarization-evoked release of noradrenaline from SH-SY5Y occurs by LDCV exocytosis. This enhances the value of SH-SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated.

Antibody Specificity↗
PubMed1998

Role of calcineurin in Ca2+-induced release of catecholamines and neuropeptides.

Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2+-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2+-induced release of [3H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca2+/calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles.

Animals↗
PubMed1998

Brain beta-spectrin phosphorylation: phosphate analysis and identification of threonine-347 as a heparin-sensitive protein kinase phosphorylation site.

Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain beta-spectrin yielded a stoichiometry of 3.2 +/- 0.18 mol of PO4/mol of beta-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO4/mol of spectrin heterodimer, respectively. The 32P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [32P]orthophosphate showed phosphorylation of only beta-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on beta-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated beta-spectrin showed that [32P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an alpha-chymotryptic 32P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338-348 on the beta1 repeat of beta-spectrinG (betaSPIIa) gene. These data suggest that phosphorylation of Thr347, which is localized on the presumptive synapsin I binding domain of beta-spectrinG, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.

Amino Acid Sequence↗
PubMed2001

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

Alternative Splicing↗
PubMed2002

Gene expression analysis of spontaneously hypertensive rat cerebral cortex following transient focal cerebral ischemia.

Identification of novel modulators of ischemic neuronal death helps in developing new strategies to prevent the stroke-induced neurological dysfunction. Hence, the present study evaluated the gene expression changes in rat cerebral cortex at 6 and 24 h of reperfusion following transient middle cerebral artery occlusion (MCAO) by GeneChip analysis. Transient MCAO resulted in selective increased mRNA levels of genes involved in stress, inflammation, transcription and plasticity, and decreased mRNA levels of genes which control neurotransmitter function and ionic balance. In addition to a number of established ischemia-related genes, many genes not previously implicated in transient focal ischemia-induced brain damage [suppressor of cytokine signaling (SOCS)-3, cAMP responsive element modulator (CREM), cytosolic retinol binding protein (CRBP), silencer factor-B, survival motor neuron (SMN), interferon-gamma regulatory factor-1 (IRF-1), galanin, neurotrimin, proteasome subunit RC8, synaptosomal-associated protein (SNAP)-25 A and B, synapsin 1a, neurexin 1-beta, ras-related rab3, vesicular GABA transporter (VGAT), digoxin carrier protein, neuronal calcium sensor-1 and neurodap] were observed to be altered in the ischemic cortex. Real-time PCR confirmed the GeneChip results for several of these transcripts. SOCS-3 is a gene up-regulated after ischemia which modulates inflammation by controlling cytokine levels. Antisense knockdown of ischemia-induced SOCS-3 protein expression exacerbated transient MCAO-induced infarct volume assigning a neuroprotective role to SOCS-3, a gene not heretofore implicated in ischemic neuronal damage.

Animals↗
PubMed2003

Cloning, characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin-dependent protein kinase I gamma in the rat brain.

Ca2+/calmodulin-dependent protein kinase I (CaMKI), originally identified as a protein kinase phosphorylating synapsin I, has been shown to constitute a family of closely related isoforms (alpha, beta and gamma). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKI gamma 1 and -gamma 2. CaMKI gamma 1 and -gamma 2 contain an identical N-terminal catalytic domain with different C-terminal regions due to the deletion of the 425-bp nucleotide sequence of CaMKI gamma 1 in CaMKI gamma 2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM-dependent activity of CaMKI gamma 1 by the preincubation with Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), but no significant activation of CaMKI gamma 2. Northern blot analysis has demonstrated the predominant expression of CaMKI gamma in the brain. RT-PCR analysis has revealed similar expression patterns between CaMKI gamma 1 and CaMKI gamma 2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKI gamma mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108-15 cells, transfected CaMKI gamma 1 and -gamma 2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKI gamma may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.

Alternative Splicing↗
PubMed2003

Glutamate transport, glutamine synthetase and phosphate-activated glutaminase in rat CNS white matter. A quantitative study.

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was approximately 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15-20% of those in cortex; the activities of glutamine synthetase and phosphate-activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11-25% of cortical values, and activities of aspartate and alanine aminotransferases were 50-70% of cortical values. The glutamate level in fimbria and corpus callosum was 5-6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U-14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.

Animals↗
PubMedDate not supplied

Neurogenesis in the crustacean ventral nerve cord: homology of neuronal stem cells in Malacostraca and Branchiopoda?

In Insecta and malacostracan Crustacea, neurons in the ventral ganglia are generated by the unequal division of neuronal stem cells, the neuroblasts (Nbs), which are arranged in a stereotyped, grid-like pattern. In malacostracans, however, Nbs originate from ectoteloblasts by an invariant lineage, whereas Nbs in insects differentiate without a defined lineage by cell-to-cell interactions within the neuroectoderm. As the ventral ganglia in entomostracan crustaceans were thought to be generated by a general inward proliferation of ectodermal cells, the question arose as to whether neuroblasts in Euarthropoda represent a homologous type of stem cell. In the current project, neurogenesis in metanauplii of the entomostracan crustaceans Triops cancriformis Fabricius, 1780 (Branchiopoda, Phyllopoda) and Artemia salina Linné, 1758 (Branchiopoda, Anostraca) was examined by in vivo incorporation of the mitosis marker bromodeoxyuridine (BrdU) and compared to stem cell proliferation in embryos of the malacostracan Palaemonetes argentinus Nobili, 1901 (Eucarida, Decapoda). The developmental expression of synaptic proteins (synapsins) was studied immunohistochemically. Results indicate that in the ventral neurogenic zone of Branchiopoda, neuronal stem cells with cellular characteristics of malacostracan neuroblasts are present. However, a pattern similar to the lineage-dependent, grid-like arrangement of the malacostracan neuroblasts was not found. Therefore, the homology of entomostracan and malacostracan neuronal stem cells remains uncertain. It is now well established that during arthropod development, identical and most likely homologous structures can emerge, although the initiating steps or the mode of generation of these structures are different. Recent evidence suggests that adult Entomostraca and Malacostraca share corresponding sets of neurons so that the present report provides an example that those homologous neurons may be generated via divergent developmental pathways. In this perspective, it remains difficult at this point to discuss the question of common patterns of stem cell proliferation with regard to the phylogeny and evolution of Atelocerata and Crustacea.

Animals↗
PubMed2001

A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus.

Many forms of long-lasting behavioral and synaptic plasticity require the synthesis of new proteins. For example, long-term potentiation (LTP) that endures for more than an hour requires both transcription and translation. The signal-transduction mechanisms that couple synaptic events to protein translational machinery during long-lasting synaptic plasticity, however, are not well understood. One signaling pathway that is stimulated by growth factors and results in the translation of specific mRNAs includes the rapamycin-sensitive kinase mammalian target of rapamycin (mTOR, also known as FRAP and RAFT-1). Several components of this translational signaling pathway, including mTOR, eukaryotic initiation factor-4E-binding proteins 1 and 2, and eukaryotic initiation factor-4E, are present in the rat hippocampus as shown by Western blot analysis, and these proteins are detected in the cell bodies and dendrites in the hippocampal slices by immunostaining studies. In cultured hippocampal neurons, these proteins are present in dendrites and are often found near the presynaptic protein, synapsin I. At synaptic sites, their distribution completely overlaps with a postsynaptic protein, PSD-95. These observations suggest the postsynaptic localization of these proteins. Disruption of mTOR signaling by rapamycin results in a reduction of late-phase LTP expression induced by high-frequency stimulation; the early phase of LTP is unaffected. Rapamycin also blocks the synaptic potentiation induced by brain-derived neurotrophic factor in hippocampal slices. These results demonstrate an essential role for rapamycin-sensitive signaling in the expression of two forms of synaptic plasticity that require new protein synthesis. The localization of this translational signaling pathway at postsynaptic sites may provide a mechanism that controls local protein synthesis at potentiated synapses.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Overexpression of acid-sensing ion channel 1a in transgenic mice increases acquired fear-related behavior.

The acid-sensing ion channel 1a (ASIC1a) is abundantly expressed in the amygdala complex and other brain regions associated with fear. Studies of mice with a disrupted ASIC1 gene suggested that ASIC1a may contribute to learned fear. To test this hypothesis, we generated mice overexpressing human ASIC1a by using the pan-neuronal synapsin 1 promoter. Transgenic ASIC1a interacted with endogenous mouse ASIC1a and was distributed to the synaptosomal fraction of brain. Transgenic expression of ASIC1a also doubled neuronal acid-evoked cation currents. The amygdala showed prominent expression, and overexpressing ASIC1a enhanced fear conditioning, an animal model of acquired anxiety. These data raise the possibility that ASIC1a and H(+)-gated currents may contribute to the development of abnormal fear and to anxiety disorders in humans.

Acid Sensing Ion Channels↗
PubMed2004

Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development.

Recent studies have shown that the behavioral performance of adult honey bees is influenced by the temperature experienced during pupal development. Here we explore whether there are temperature-mediated effects on the brain. We raised pupae at different constant temperatures between 29 and 37 degrees C and performed neuroanatomical analyses of the adult brains. Analyses focused on sensory-input regions in the mushroom bodies, brain areas associated with higher-order processing such as learning and memory. Distinct synaptic complexes [microglomeruli (MG)] within the mushroom body calyces were visualized by using fluorophore-conjugated phalloidin and an antibody to synapsin. The numbers of MG were different in bees that had been raised at different temperatures, and these differences persisted after the first week of adult life. In the olfactory-input region (lip), MG numbers were highest in bees raised at the temperature normally maintained in brood cells (34.5 degrees C) and significantly decreased in bees raised at 1 degrees C below and above this norm. Interestingly, in the neighboring visual-input region (collar), MG numbers were less affected by temperature. We conclude that thermoregulatory control of brood rearing can generate area- and modality-specific effects on synaptic neuropils in the adult brain. We propose that resulting differences in the synaptic circuitry may affect neuronal plasticity and may underlie temperature-mediated effects on multimodal communication and learning.

Animals↗
PubMed2005

Generation of tissue-specific transgenic birds with lentiviral vectors.

Birds are of great interest for a variety of research purposes, and effective methods for manipulating the avian genome would greatly accelerate progress in fields that rely on birds as model systems for biological research, such as developmental biology and behavioral neurobiology. Here, we describe a simple and effective method for producing transgenic birds. We used lentiviral vectors to produce transgenic quails that express GFP driven by the human synapsin gene I promoter. Expression of GFP was specific to neurons and consistent across multiple generations. Expression was sufficient to allow visualization of individual axons and dendrites of neurons in vivo by intrinsic GFP fluorescence. Tissue-specific transgene expression at high levels provides a powerful tool for biological research and opens new avenues for genetic manipulation in birds.

Animals↗
PubMed1983

cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor.

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the gamma and delta subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the gamma and delta subunits of the receptor. cAMP activation of the endogenous phosphorylation of the gamma and delta subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the gamma and delta subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of (32)P per mol of receptor, respectively. The initial rates of phosphorylation of the gamma and delta subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the gamma and delta subunits had K(m) values of 4.0 and 3.3 muM and V(max) values of 2.7 and 2.1 mumol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.

Animals↗