Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-Daspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the ␥-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.Information about the mechanisms of synaptic transmission and synaptic plasticity in the mammalian brain derives primarily from electrophysiological studies of excitatory synapses in the hippocampus. These synapses use the neurotransmitter glutamate, which can act on distinct subtypes of ionotropic and metabotropic receptors that frequently colocalize at individual synapses but appear to subserve distinct functions (1-3). Two major subtypes of ionotropic receptors, AMPA (␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and NMDA (Nmethyl-D-aspartate) receptors, have been found at virtually all excitatory synaptic connections in the mammalian brain. AMPA receptors (AMPARs) are heteromers of the homologous subunits GluR1-4 and mediate the bulk of synaptic transmission during basal neural activity (1-3). NMDA receptors (NMDARs) also exist as heteromers formed from the NR1 subunit and one or more NR2A-D subunits (1-3). Because of their voltage dependence and high calcium permeability, NMDARs are particularly important for triggering several different forms of synaptic plasticity, including longterm potentiation and long-term depression. When inappropriately activated during a variety of pathological conditions, NMDARs also contribute to neuronal injury and death.It has commonly been assumed that AMPARs and NMDARs are colocalized at individual synapses (1-4), although it is now clear that these receptor subtypes interact with different proteins at the synapse (5). The distinct molecular interactions and functions of these receptor subtypes raise the possibility that their surface expression at synapses may be independently regulated....

Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviruses. Second, we demonstrate a method to construct adenovirus vectors carrying recombinant genes in place of all of the viral genes, so-called gutless adenovirus vectors. The pivotal feature in each method is the use of a negatively selected adenovirus named psi5. We engineered a cis-acting selection into psi5 by flanking its packaging site with loxP sites. When psi5 was grown in cells making a high level of Cre recombinase, the packaging site was deleted by recombination and the yield of psi5 was reduced to 5% of the wild-type level. To make a new E1-substituted virus, we used psi5 as a donor virus and recombined it with a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgrows psi5 in the presence of Cre recombinase. To make a gutless virus, we used psi5 as a helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and packaging. We found that a loxP site next to the packaging site of the gutless virus was necessary to neutralize homologous recombination between psi5 and the gutless viruses within their packaging domains.

No abstract

Anti-Hib PS antibodies bearing Hibld-1 were present in at least 85% of subjects immunized with either free Hib PS or Hib PS coupled to diphtheria toxoid (Hib PS-DT), and comprised on the average 60% ofthe total vaccine-induced serum anti-Hib PS. Hibld-1 expression was not related to age at vaccination inasmuch as infants, children, and adults had similar distributions of Hibld-1-positive anti-Hib PS after vaccination with Hib PS-DT. HibId-1 was expressed at a lower frequency and comprised a smaller fraction of the total anti-Hib PS antibody in adult preimmunization sera as compared to post-Hib PS immunization sera, suggesting that immunization preferentially Some ofthe results reported here were presented at thejoint meeting of the

Astrocytes in neuron-free cultures typically lack processes, although they are highly process-bearing in vivo. We show that basic fibroblast growth factor (bFGF) induces cultured astrocytes to grow processes and that Ras family GTPases mediate these morphological changes. Activated alleles of rac1 and rhoA blocked and reversed bFGF effects when introduced into astrocytes in dissociated culture and in brain slices using recombinant adenoviruses. By contrast, dominant negative (DN) alleles of both GTPases mimicked bFGF effects. A DN allele of Ha-ras blocked bFGF effects but not those of Rac1-DN or RhoA-DN. Our results show that bFGF acting through c-Ha-Ras inhibits endogenous Rac1 and RhoA GTPases thereby triggering astrocyte process growth, and they provide evidence for the regulation of this cascade in vivo by a yet undetermined neuron-derived factor.

A combination treatment of AAV2-hAADC with oral levodopa is a novel therapeutic approach that is being developed for late-stage Parkinson's disease. Biodistribution of AAV2-hAADC was assessed over a wide range of vector dose in 12 monkeys with parkinsonian syndrome, 6 months after intraputamenal infusion. Quantitative PCR (Q-PCR) from all the major neuroanatomical regions of the brain indicated a dose-dependent increase in vector DNA, with 99% being detected in the target site and other basal ganglia tissues. Within these tissues, the distribution varied widely between the putamen (PT) and the globus pallidus, and this was attributed to differences in vector transport. Q-PCR and immunocytochemistry were consistent with results reported earlier for various measures of transgene expression including aromatic L-amino acid decarboxylase (AADC) activity assays, behavioral response, and in vivo imaging with positron emission tomography (PET). Outside of the brain, trace amounts of vector DNA were detected in the spleens of animals in the two highest dose groups, but not in any other peripheral tissue, blood, or cerebrospinal fluid. Some increase in neutralizing antibody titers to adeno-associated virus type-2 (AAV2) capsid protein was observed in monkeys that received high doses of AAV2-hAADC or control AAV2-GFP. This study further validates convection-enhanced delivery (CED) as the preferred method of viral vector delivery to the brain, and supports a Phase I clinical testing of AAV2-hAADC in humans with Parkinson's disease.

ABSTRACT. We examined the ability of severe combined immunodeficient (=ID) mice-human peripheral blood leukocyte (PBL) chimeras to respond to immunization with Haemophilus influenza b polysaccharide (Hib PS) vaccines. Two to 3 wk after PBL engraftment, human-PBL-SCID mice, prepared with PBL from one of five adult donors, were immunized with free or protein-conjugated Hib PS. Antibody to Hib PS was quantitated in preimmunization and postimmunization sera. Before immunization, anti-Hib PS antibody was detectable (>lo ng/mL) in three of 40 mice. Of the 37 human-PBL-SCID mice not having detectable serum antibody before immunization, 31 produced 220 ng/mL (22-fold increase) anti-Hib PS antibody 2 to 3 wk after immunization. Both free and proteinconjugated forms of Hib PS were immunogenic. Geometric mean anti-Hib PS antibody levels ranged from 50 to 139 ng/mL. Vaccine-induced anti-Hib PS antibodies frequently expressed HibId-1, a cross-reactive idiotype that predominates the in vivo human antibody response to Hib PS. However, among mice engrafted with PBL from a single donor, the HibId-1 distribution was highly skewed, suggesting that clonally distinct B cells were being stimulated in individual mice. These findings indicate that human PBL transplanted into SCID mice are functionally responsive to Hib PS antigenic challenge. This system may serve as a useful model for studying the regulation and cellular requirements for human polysaccharide immunity. (Pediatr Res 32: 132-135,1992)

No abstract