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PubMed1986

Immunocytochemical studies on axonal transport in adrenergic and cholinergic nerves using cytofluorimetric scanning.

The axonal transport of adrenergic and cholinergic axonal organelles in rat sciatic nerve has been studied using a cytofluorimetric scanning (CFS) technique. This technique gives quantitative data on material which accumulates in a nerve relative to a crush, as well as morphological and morphometrical information about the accumulated axons in the nerve. One important advantage is that several substances can be measured in the same nerve segment, thus reducing the number of animals needed. The substances must be made fluorescent, and in this study we have investigated noradrenaline (NA), using formaldehyde induced fluorescence, and dopamine beta-hydroxylase (DBH), tyrosine hydroxylase (TH), neuropeptide Y (NPY) and two cholinergic vesicle components (a transmembrane glycoprotein and synapsin I) using indirect immunofluorescence. The antisera used for labelling immunoreactive material (IR) were produced in rabbit or goat (DBH). In adrenergic axons NA, DBH-IR and TH-IR accumulated with time after crushing the nerve as described earlier with biochemical techniques. After reserpine, the amounts of amine granules transported distally in the sciatic nerve initially fell, but recovered during day 2 after reserpine. At day 4 the amount of NA and DBH-IR which was transported distally in the axons was supranormal, 160% and 140% of control, respectively, but the level of NPY-IR was not increased, even falling to subnormal at day 4, indicating different mechanisms for regulating the synthesis of DBH and NPY which are suggested to co-exist in axonal adrenergic large dense core vesicles. In cholinergic motor axons organelles, recognized by rabbit-anti-cholinergic synaptic vesicles-antiserum (RASVA) and by anti-synapsin I-antiserum, are transported distally at a rapid rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic Fibers↗
PubMed1986

Primary culture of striatal neurons: a model of choice for pharmacological and biochemical studies of neurotransmitter receptors.

Striatal neurons were cultured from fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). A double coating of culture dishes with polyornithine and fetal calf serum was needed in order to obtain synaptic differentiation. Synaptic vesicles were present in axon terminals as well as in varicosities along extended axons. The presence of differentiated synapses was confirmed by the immunostaining of the preparation with synapsin I antibody. After 13 days in vitro synapsin I was present in axonal varicosities and particularly concentrated at contact points between axonal terminals and postsynaptic sites on adjacent axons or perikarya. On a surface of 429 mm2 on which 2211 cells were observed under phase contrast microscopy only 7% were stained with an antibody against GFAP (glial fibrillary acidic protein). One or two days after the formation of differentiated synapses (11 DIV), a Ca2+-dependent liberation of GABA was observed. These cultures are an excellent model for studying the coupling of some neurotransmitter receptors with an adenylate cyclase. In particular using this preparation we were able to demonstrate that dopamine (D2) and serotonin-(5-HT1) receptors are negatively coupled with an adenylate cyclase. These cultures are also an excellent model to study the coupling of some neurotransmitter receptors with inositol phosphate producing enzymes. We demonstrated for the first time that the quisqualate subtype of glutamate receptors is able to increase inositol phosphate production in striatal neurons.

Animals↗
PubMed1985

Nerve growth cones isolated from fetal rat brain. II. Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation.

Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation were examined in growth cone particles (GCPs) prepared from fetal rat brain. Several major proteins which specifically bind a photoactivatable analogue of cAMP are observed in GCPs and correspond to isoelectric variants of the regulatory subunits of the cAMP-dependent protein kinase described in adult brain. We found no evidence for differential compartmentalization of specific cAMP-binding proteins in subcellular fractions of fetal brain or within GCPs. cAMP-stimulated phosphoproteins of GCPs are similar to cAMP-dependent protein kinase substrates characterized in nerve terminals (synaptosomes) of adult brain and include the nerve terminal-specific protein, synapsin I. However, as shown in the companion paper (Katz, F., L. Ellis, and K. H. Pfenninger (1985) J. Neurosci. 5: 1402-1411), this synaptic phosphoprotein is not the major kinase substrate in the GCP fraction. The finding of synapsin I in a subcellular fraction prepared from fetal brain suggests that components of the mature nerve terminal are already present in fetal brain during neuronal sprouting and prior to synaptogenesis.

Animals↗
PubMed1984

Calcium/calmodulin-dependent protein phosphorylation in the nervous system of Aplysia.

An afterdischarge in the bag cell neurons of Aplysia was previously shown to be associated with calcium entry into these cells and with changes in the phosphorylation state of at least two bag cell proteins (BC-I and BC-II). We have now investigated the role of calcium plus calmodulin (Ca/CaM) in the control of phosphorylation of Aplysia nervous system proteins, including those of the bag cell neurons. In cell-free preparations of Aplysia CNS, we demonstrated Ca/CaM-stimulated protein phosphorylation that could be inhibited by the calmodulin-blocking drugs R24571 , trifluoperazine, chlorpromazine, and W7 . A number of substrate proteins for Ca/CaM-dependent protein phosphorylation with Mr values from 17,000 to 310,000 were consistently observed in homogenates of the Aplysia CNS. In the bag cells, we found that a major substrate for Ca/CaM-dependent protein phosphorylation was the bag cell-specific, Mr = 21,000 protein (BC-II). BC-I (Mr = 33,000), on the other hand, appeared not to be a substrate for a Ca/CaM-dependent protein kinase. We found that there are a minimum of two Ca/CaM-dependent protein kinases in the Aplysia nervous system. These enzymes were distinguished on the basis of their subcellular distribution and their ability to phosphorylate distinct sites on synapsin I, an exogenous neuronal protein from vertebrates. Phosphorylation by one of these kinases (calmodulin kinase I) was on a site recovered in an Mr = 10,000 proteolytic fragment of synapsin I, and phosphorylation by the other (calmodulin kinase II) was on a site recovered in an Mr = 30,000 fragment. The predominant enzyme in the Aplysia CNS, as in the mammalian nervous system, was calmodulin kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Calmodulin-dependent protein kinase and associated substrates in Torpedo electric organ.

Calcium plus calmodulin (Ca2+/CaM)-dependent protein kinase activity was demonstrated in subcellular fractions from Torpedo californica electric organ. A protein kinase activity dependent on Ca2+/CaM was purified about 200-fold from electric organ cytosol using DEAE-cellulose and CaM-affinity chromatography. The most effective exogenous substrates for this enzyme were the synapse-specific protein Synapsin I (Protein I) and histone f3. Phosphorylase b, skeletal muscle myosin light chains, casein, phosvitin, histone f2b, and G-substrate were relatively poorly phosphorylated by Torpedo CaM-protein kinase. Thus, the enzyme differs in its substrate specificity from known cyclic nucleotide-dependent protein kinases, myosin light chain kinase and phosphorylase kinase. The Km for ATP was 15-20 microM; for Synapsin I, 0.8 microM; and for CaM, 85 nM. Two major endogenous substrates (Mr = 62,000 and 54,000) for CaM-protein kinase co-purified with the enzyme through the CaM-affinity column step. These two substrates, as well as the enzyme, were present in other subcellular fractions in addition to the cytosol, including crude membranes and purified synaptic vesicles. A third major substrate (Mr = 39,000) could be separated from the enzyme during purification and appeared to be localized primarily in the cytosol. CaM-protein kinase increased the phosphorylation of both serine and threonine residues in endogenous substrates. In contrast to previous reports, no evidence for Ca2+/CaM-dependent phosphorylation of any subunit of the acetylcholine receptor was obtained.

Animals↗
PubMed1995

Melatonin binding sites and their role in seasonal reproduction.

The pineal gland has a major role in the translation of scotophase duration into a hormonal signal by the hormone melatonin. Animals such as sheep, goats and deer use the seasonal variation of this signal to coordinate reproductive behaviour with the environment. Despite intensive research over more than 30 years the site(s) of action of melatonin and the resultant intracellular responses are still not clear. This review discusses recent work that has localized the site of action of melatonin in sheep using administration into the hypothalamus in vivo as well as studies on putative melatonin receptors in the pars tuberalis and brain. There is clear evidence that melatonin acting at the level of the pars tuberalis is involved in the seasonal regulation of prolactin secretion, but the evidence for involvement of the pars tuberalis in seasonal reproduction is not compelling. Localized administration of melatonin to the sheep brain revealed that areas anatomically distinct from the pars tuberalis, the ventromedial and arcuate nuclei, simulated seasonal reproductive changes in rams and ewes. Recent studies on brain melatonin binding sites in our laboratory have shown that an antagonist of tissue transglutaminase, Bacitracin, as well as substrates for the enzyme inhibit binding of melatonin to brain membranes. As a working hypothesis, we propose that pineal melatonin secretion alters seasonal reproduction by interactions with a neural transglutaminase at the synapse of neurones involved in the control of GnRH secretion. Synaptic transglutaminase is implicated in the control of the release of neurotransmitter via the synaptic vesicle associated protein, synapsin 1; activation of transglutaminase results in the covalent modification of synapsin 1 such that vesicles are not released from the cytoskeleton. Seasonal variation in the duration of melatonin secretion may result in similar variations in the duration of suppression and activation of transglutaminase. The resultant changes in transmitter release may then be responsible for the seasonal neuronal plasticity previously observed in GnRH neurones.

Animals↗
PubMed1994

p145, a major Grb2-binding protein in brain, is co-localized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation.

Three major rat brain proteins were recently found to bind the SH3 domains of Grb2: synapsin I, dynamin, and a novel 145-kDa protein (p145) (McPherson, P. S., Czernik, A. J., Chilcote, T. J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J., and De Camilli, P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6486-6490). We have now used antibodies raised against p145 which had been purified by Grb2 affinity chromatography and SDS-polyacrylamide gel electrophoresis to characterize this protein. p145 is neuron-specific and co-distributes with dynamin in subcellular fractions of rat brain. Both proteins are partially recovered in soluble and particulate fractions. By immunofluorescence, p145 is closely co-localized with dynamin, which we show here to be highly concentrated in nerve terminals. In contrast to synapsin I, which is highly enriched in a purified synaptic vesicle fraction, both p145 and dynamin are de-enriched in this fraction. However, both proteins are found on membranes which are immunoisolated with antibodies directed against the synaptic vesicle membrane protein synaptophysin, suggesting that dynamin and p145 are localized in part on organelles which represent intermediate stages in the reformation of synapatic vesicles during recycling. Finally, we have determined that p145, like dynamin, is a phosphoprotein which undergoes dephosphorylation in response to nerve terminal depolarization. These findings suggest that p145 participates with dynamin in synaptic vesicle endocytosis and recycling.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Calcium/calmodulin-dependent protein kinase I. cDNA cloning and identification of autophosphorylation site.

Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was previously purified from bovine brain (Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262, 7273-7281) based on its ability to phosphorylate the synaptic vesicle protein, synapsin I at site 1. The cDNA for this protein kinase has now been cloned from both a rat and a bovine brain cDNA library and the complete amino acid sequence of rat CaM kinase I determined. The rat cDNA encoded a protein of 331 amino acids with a calculated M(r) of 37,545, and the encoded kinase was expressed in bacteria as a glutathione S-transferase fusion protein. The resulting fusion protein was purified by Sepharose-CaM affinity chromatography and shown to be totally dependent on Ca2+ and CaM for activity. Furthermore, the purified kinase phosphorylates synapsin I at the same site (site 1) as the endogenous brain enzyme. CaM kinase I is homologous to other known protein kinases and contains all nine invariant amino acids conserved in the catalytic domain of this class of enzymes. CaM kinase I was most identical to CaM kinase II both in the catalytic domain and in a short region at the COOH-terminal that is predicted to be the calmodulin-binding domain. CaM kinase I appeared to be encoded by a single gene. RNase protection assays detected the mRNA encoding CaM kinase I in all tissues examined. High concentrations of the kinase mRNA were found in all regions of the brain with frontal cortex showing the greatest level. CaM kinase I was autophosphorylated in a Ca2+/CaM-dependent manner at a threonyl residue (Thr-177) which is located at a position equivalent to that of the threonyl residue (Thr-197) autophosphorylated in cAMP-dependent protein kinase.

Adrenal Glands↗
PubMed1998

Suppression of acute experimental allergic encephalomyelitis by intraperitoneal administration of synaptosomal antigens.

The effect of a synaptosomal fraction isolated from bovine brain was examined on acute experimental allergic encephalomyelitis (EAE) in Wistar rats. Intraperitoneal administration of the animals with low doses of saline-soluble synaptosomal antigens 10 and 3 days previous to the active induction of the disease was an effective way of suppressing EAE. This treatment diminished the incidence and severity of EAE, reverted the appearance of central nervous system histological and biochemical alterations, and produced changes in the autoimmune humoral response against the encephalitogenic myelin basic protein. The phenomenon observed by treatment with synaptosomal fraction is similar to the previously described suppression mediated by myelin antigens. Taking into account that affinity-purified antibodies and T lymphocytes specific for myelin basic protein can also recognize several neuronal proteins, among them the specific synaptosomal protein synapsin I, can be suggested that antigen-driven bystander suppression could be a mechanism by which synaptosomal proteins suppress the response against myelin antigens.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2000

Rabies virus entry at the neuromuscular junction in nerve-muscle cocultures.

Early events in rabies virus entry into neurons were investigated in chick spinal cord-muscle cocultures. Rabies virus (CVS strain) was adsorbed to the surface of cells in the cold. At times up to 10 min of warming to 37 degrees C, virus was most intensely localized to dense swellings on the myotube surface. Texas Red-labeled alpha-bungarotoxin, which binds to nicotinic acetylcholine receptors, colocalized precisely with virus at the densities identifying these regions as neuromuscular junctions. Rabies virus also colocalized in the junctions with synapsin I, a marker for synaptic vesicles. The endosome tracers Lucifer Yellow, Texan Red-dextran, and rhodamine-wheat germ agglutinin were added to the cultures at the end of the virus adsorption period and the cultures were warmed. At 10 min, rabies virus and tracers colocalized at neuromuscular junctions and nerve terminals. At 30 min, rabies virus and tracers showed more intense fluorescence over nerve fibers and nerve cell bodies. At 60 min, nerve terminals, nerve fibers, and nerve cell bodies showed intense fluorescence and colocalization for rabies virus and tracers. LysoTracker Red, a marker for acidic compartments, colocalized with rabies virus at nerve-muscle contacts. These findings show that in nerve-muscle cocultures, the neuromuscular junction is the major site of entry into neurons. Colocalization of virus and endosome tracers within nerve terminals indicates that virus resides in an early endosome compartment, some of which are acidified. The progressive increase of virus and tracers in nerve fibers and nerve cell bodies over time is consistent with retrograde transport of endocytosed virus from the motor nerve terminal.

Animals↗
PubMed2002

Putative neurohemal release zones in the stomatogastric nervous system of decapod crustaceans.

The stomatogastric nervous system (STNS) of decapod crustaceans has long been used to study the modulation of small neural circuits. Profiles in the sheath of the nerves and ganglia of the STNS, which contain only dense-core vesicles, have been described in electron microscopical studies (Friend [1976] Cell Tissue Res. 175:369-380; Kilman and Marder [1997] Soc Neurosci Abstr. 23:477; Skiebe and Ganeshina [2000] J Comp Neurol 420:373-397). These profiles resemble those found in neurohemal organs and suggest the presence of neurohemal release zones in the STNS. To map these putative neurohemal release zones, a combination of two antibodies was used in the present study. A synapsin antibody recognizing vesicle proteins of clear vesicles was combined with a synaptotagmin antibody recognizing vesicle proteins of clear and dense-core vesicles. Exclusive synaptotagmin-like staining, therefore, indicated the regions with only dense-core vesicles. Such a staining was found in a mesh in the perineural sheath of nerves in the STNS of all three species investigated. In the crayfish Cherax destructor and the lobster Homarus americanus, the stained mesh was located in the sheath of nerves connecting all four ganglia of the STNS, whereas in the crab Cancer pagurus it was found on different nerves, which are more directly exposed to the hemolymph in this species. Exclusive synaptotagmin-like staining was also found in a putative neurohemal release zone in the sheath of the circumoesophageal connectives and the postoesophageal commissure in C. destructor. These data suggest that an important source of modulation of the networks and the muscles of the stomach is a compartmentalized release of neurohormones from zones in the STNS.

Animals↗
PubMed2003

Ecological, evolutionary, and functional correlates of sensilla number and glomerular density in the olfactory system of decapod crustaceans.

One of the features common among olfactory systems for vertebrate and invertebrate species is the division of the primary processing area into distinct clumps of synaptic neuropil, called glomeruli. The olfactory glomeruli appear to serve as functional units of olfaction and are the location of the primary processing between chemosensory afferents and second-order neurons. Although glomeruli are found across all phyla, their numbers and size appear to be characteristic for each species, giving rise to the speculation that there is a relationship between glomerular number and function. It has been hypothesized, for example, that animals with more glomeruli may be able to resolve a wider range of odors. Crustacean species are distributed among freshwater, marine, and terrestrial habitats in arctic, temperate, and tropical climates. They also exhibit a variety of lifestyles and behaviors in which olfaction may play a dominant role. Feeding, for example, ranges from carnivorous, through subaquatic and terrestrial omnivorous scavenging, to filter feeding. Mating and territorial behaviors also are known to involve chemical signals. The current study examines glomerular numbers in the olfactory lobes of 17 crustacean species from six of the seven taxa now included in the reptantian decapods. Estimates of the glomerular numbers were obtained from the analysis of sectioned material treated immunocytochemically with an antibody against synapsin that labels proteins contained in neuronal terminals. The numbers of glomeruli found in the different species were then compared with the volume of the glomerular neuropil, numbers of olfactory sensilla, life styles, habitat, and phylogenetic affinities. The picture that emerges from these correlations is that the decapod crustaceans have exploited various strategies in the construction of their olfactory systems in which the problems of size, sensitivity, and selectivity have all interacted. We find a continuum across the groups ranging from those that favor a high convergence of receptor neurons onto a few glomeruli to those that share a small number of receptor neurons among many glomeruli. The potential functional consequences of these differences are discussed.

Animals↗
PubMed2003

Heterogeneous expression of SNAP-25 and synaptic vesicle proteins by central and peripheral inputs to sympathetic neurons.

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple-labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome-associated protein of 25 kDa (SNAP-25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP-25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20-50 nM for 2 hours in vitro) generated significant cleavage of SNAP-25 and produced similar dose- and time-dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP-25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP-25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP-25 and other synaptic proteins.

Adrenergic Fibers↗
PubMed2004

F-actin at identified synapses in the mushroom body neuropil of the insect brain.

The distribution of f-actin stained by fluorescent phalloidin was investigated in the brain of several insect species, with a special focus on the mushroom body. For localizing f-actin in identified neurons and at synapses, additional staining with fluorescent dextrans and anti-synapsin I immunostaining was employed. Intense f-actin staining was consistently found in synaptic complexes of the mushroom body calyces (calycal microglomeruli [MG]). These MG contain a central core of presynaptic boutons, predominantly belonging to deutocerebral cholinergic excitatory projection neurons, which are surrounded by a shell of numerous Kenyon cell (KC) dendritic tips. In the cricket Gryllus bimaculatus, high-resolution confocal laser scanning imaging revealed colocalization of f-actin with KC dendritic spine parts within MG. Although presynaptic boutons appear to be mainly devoid of f-actin-phalloidin fluorescence, there appears to be an accumulation of f-actin in KC dendritic spines synaptically contacting the boutons. Electron microscopy of boutons and dextran-stained KC dendrites revealed their pre- and postsynaptic sites, with KCs being strictly postsynaptic elements. Their subsynaptic membrane appositions are considered to be associated with f-actin. Focal accumulation of f-actin in the dendritic tips of KCs was found to be a general feature of MG, with either spheroidal or indented boutons of different sizes, as encountered in the mushroom bodies of the cricket, honey bee, ant, and fruit fly. The structural similarities of calycal MG and f-actin accumulation in KC dendrites with cerebellar microglomeruli are considered comparatively. The accumulation of f-actin in KC dendrites is discussed in view of mushroom body plasticity and its potential role in learning and memory formation.

Actins↗
PubMed2005

Differential regulation of GABA(A) receptor and gephyrin postsynaptic clustering in immature hippocampal neuronal cultures.

Gephyrin is a postsynaptic scaffolding protein involved in clustering of glycine- and GABA(A) receptors at inhibitory synapses. The role of gephyrin in GABAergic synapses, the nature of its interactions with GABA(A) receptors, and the mechanisms of targeting to GABAergic synapses are largely unknown. To gain further insights into these questions, the formation of GABA(A) receptor and gephyrin clusters and their distribution relative to presynaptic terminals were investigated in immature cultures of embryonic hippocampal neurons using triple immunofluorescence staining. GABA(A) receptor clusters, labeled for the alpha2 subunit, formed independently of gephyrin clusters, and were distributed on neurites at constant densities, either extrasynaptically or, to a lesser extent, postsynaptically, apposed to synapsin-I-positive axon terminals. In contrast, gephyrin clusters were always associated with GABA(A) receptors and were preferentially localized postsynaptically. Their density increased linearly with the extent of innervation, which developed rapidly during the first week in vitro. These results suggested that GABA(A) receptor clustering is mediated by cell-autonomous mechanisms independent of synapse formation. Their association with gephyrin is dynamically regulated and may contribute to stabilization at postsynaptic sites. Labeling for vesicular glutamate transporters revealed that most synapses in these immature cultures were presumably glutamatergic, implying that postsynaptic GABA(A) receptor and gephyrin clusters initially were located in "mismatched" synapses. However, clusters appropriately localized in GABAergic synapses were distinctly larger and more intensely stained. Altogether, these results demonstrate that the targeting of GABA(A) receptor and gephyrin clusters to GABAergic synapses occurs secondarily and is regulated by presynaptic factors that are not essential for clustering.

Animals↗
PubMed2006

Developmental period for N-methyl-D-aspartate (NMDA) receptor-dependent synapse elimination correlated with visuotopic map refinement.

During a short perinatal interval, N-methyl-D-aspartate receptor (NMDAR) function is essential to a process in which spontaneous retinal waves focus retinal axon arbors in the superficial layers of the rodent superior colliculus (sSC). Here we provide evidence that this NMDAR-dependent axonal refinement occurs through elimination of uncorrelated retinal synapses arising from disparate loci, rather than stabilization of topographically appropriate inputs. The density of synaptic release sites within fluorescently labeled retinal terminals was counted in double-labeling experiments using confocal microscopy and antibodies against synaptophysin or synapsin-1. Chronic NMDAR blockade from birth increased retinal axon synapse density at postnatal days (P) 6, 8, and 10, suggesting that NMDAR currents reduce synapse density during the refinement period. With assay at P14, after focal arborization has been established, the effect disappeared. Conversely, chronic NMDA treatment, known to induce functional synaptic depression in the sSC, decreased retinocollicular synapse density at P14, but not earlier, during the refinement period (P8). Thus during the development of retinocollicular topographic order, there is a period when NMDAR activity predominantly eliminates retinal axon synapses. We were able to extend this period by using retinal lesions to reduce synaptic density in a defined zone. Synapse density on intact retinocollicular axons sprouting into this zone was increased by NMDAR blockade, even when examined at P14. Thus, the period of NMDAR-dependent synaptic destabilization is terminated by a factor related to the density and refinement of retinal arbors.

Animals↗
PubMed2001

Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development.

Neural tissue and smooth muscle appear early in the developing fetal lung, but little is known of their origin and subsequent distribution. To investigate the spatial and temporal distribution of nerves, ganglia, and airway smooth muscle during the early pseudoglandular stage, fetal mouse lungs at embryonic days (E) 11 to 14 were immunostained as whole-mounts and imaged by confocal microscopy. At E11, the primordial lung consisted of the future trachea and two budding epithelial tubules that were covered in smooth muscle to the base of the growing buds. The vagus and processes entering the lung were positive for the neural markers PGP 9.5 (protein gene product 9.5) and synapsin but no neurons were stained at this stage. An antibody to p75NTR revealed neural crest cells on the future trachea as well as in the vagus and in processes extending from the vagus to the lung. This finding indicates that even though neuronal precursors are already present at this stage, they are still migrating into the lung. By E12, neural tissue was abundant in the proximal part of the lung and nerves followed the smooth muscle-covered tubules to the base of the growing buds. At E13 and E14, a neural network of interconnected ganglia, innervated by the vagus, covered the trachea. The postganglionic nerves mainly followed the smooth muscle-covered tubules, but some extended out into the mesenchyme beyond the epithelial buds. Furthermore, we show in a model of cultured lung explants that neural tissue and smooth muscle persist and continue to grow and differentiate in vitro. By using fluorescent markers and confocal microscopy, we present the developing lung as a dynamic structure with smooth muscle and neural tissue in a prime position to influence growth and development.

Actins↗
PubMed2005

Increased utility in the CNS of a powerful neuron-specific tetracycline-regulatable adenoviral system developed using a post-transcriptional enhancer.

BACKGROUND: In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system. METHODS: The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP. RESULTS: The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression. CONCLUSIONS: We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies.

Adenoviridae↗