Inducible and reversible NR1 knockout reveals crucial role of the NMDA receptor in preserving remote memories in the brain

Long-term storage of information is a hallmark feature of the brain, yet routine turnover of synaptic receptors appears to be intrinsically paradoxical to this capability. To investigate how the brain preserves its delicate synaptic efficacies, we generated inducible and reversible knockout mice in which the NMDA receptor can be temporarily switched off in the forebrain specifically during the storage stage. Retention of 9-month contextual and cued fear memories is severely disrupted by prolonged, but not transient, loss of the NMDA receptor that occurs 6 months after initial training and at least 2 months prior to memory retrieval. Normal learning and memory function in subsequent tasks following the 9-month retention tests suggest that the observed retention deficits did not result from recall or performance impairment. Thus, our study reveals a hitherto unrecognized role of the NMDA receptor in dynamically maintaining the long-term synaptic stability of memory storage circuits in the brain.     

A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of approximately 10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.     

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Prolonged culture of normal chorionic villus cells yields ICF syndrome-like chromatin decondensation and rearrangements

Untreated cultures from normal chorionic villus (CV) or amniotic fluid-derived (AF) samples displayed dramatic cell passage-dependent increases in aberrations in the juxtacentromeric heterochromatin of chromosomes 1 or 16 (1qh or 16qh). They showed negligible levels of chromosomal aberrations in primary culture and no other consistent chromosomal abnormality at any passage. By passage 8 or 9, 82 +/- 7% of the CV metaphases from all eight studied samples exhibited 1qh or 16qh decondensation and 25 +/- 16% had rearrangements in these regions. All six analyzed late-passage AF cultures displayed this regional decondensation and recombination in 54 +/- 16 and 3 +/- 3% of the metaphases, respectively. Late-passage skin fibroblasts did not show these aberrations. The chromosomal anomalies resembled those diagnostic for the ICF syndrome (immunodeficiency, centromeric region instability, and facial anomalies). ICF patients have constitutive hypomethylation at satellite 2 DNA (Sat2) in 1qh and 16qh, generally as the result of mutations in the DNA methyltransferase gene DNMT3B. At early and late passages, CV DNA was hypomethylated and AF DNA was hypermethylated both globally and at Sat2. DNMT1, DNMT3A, or DNMT3B RNA levels did not differ significantly between CV and AF cultures or late and early passages. The high degree of methylation of Sat2 in late-passage AF cells indicates that hypomethylation of this repeat is not necessary for 1qh decondensation. Sat2 hypomethylation may nonetheless favor 1qh and 16qh anomalies because CV cultures, with their Sat2 hypomethylation, displayed 1qh and 16qh decondensation and rearrangements at significantly lower passage numbers than did AF cultures. Also, CV cultures had much higher ratios of ICF-like rearrangements to heterochromatin decondensation in chromosomes 1 and 16. These cultures may serve as models to help elucidate the biological consequences of cancer-associated satellite DNA hypomethylation.     

Molecular cytogenetic analysis of inv dup(15) chromosomes, using probes specific for the Prader-Willi/Angelman syndrome region: clinical implications.

Twenty-seven cases of inverted duplications of chromosome 15 (inv dup [15]) were investigated by FISH with two DNA probes specific for the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region on proximal 15q. Sixteen of the marker chromosomes displayed two copies of each probe, while in the remaining 11 markers no hybridization was observed. A significant association was found between the presence of this region and an abnormal phenotype (P < .01). This is the largest study to date of inv dup(15) chromosomes, that uses molecular cytogenetic methods and is the first to report a significant association between the presence of a specific chromosomal region in such markers and an abnormal phenotype.     

A quantitative resolution of the spectra of a membrane potential indicator,diS-C3-(5), bound to cell components and to red blood cells

Summary Cationic cyanine dyes have been widely used to measure electrical potentials of red blood cells and other membrane preparations. A quantitative analysis of the binding of the most extensively studied of these dyes, diS-C₃-(5), to red blood cells and their constituents is presented here. Absorption spectra were recorded for the dye in suspensions of isolated red cell membranes and in solutions of cell lysate. The dependence of the spectra on the concentrations of dye and cell constituents shows that the dye binds to these membranes as monomers with an absorbance maximum at 670 nm instead of 650 nm as for free aqueous dye and that the dye binds to oxyhaemoglobin partly as monomer but primarily as dimer, with absorbance maxima ca. 670 and 595 nm, respectively. Quantitative estimates are derived for all binding constants and extinction coefficients. These estimates are applied to suspensions of whole cells to predict the dye binding, absorbance spectra, and calibration curves of binding and fluorescencevs. membrane voltage. Satisfactory agreement is found with binding and absorbance data for whole cells at zero membrane potential and with the binding and fluorescence data reported by Hladky and Rink (J. Physiol. (London) 263:287, 1976) for cells driven to positive and negative potentials using valinomycin. The marked tendency of oxyhaemoglobin to bind dye as dimer is not shared by some other proteins tested, including deocyhaemoglobin and oxymyoglobin.     

Viability of Rhizobium bacteroids.

Bacteroids prepared from nodules of soybean and bean were tested for viability. Contrary to the prevailing view that bacteroids are nonviable, it was found that bacteroids averaged 90% viability, irrespective of Rhizobium strain, nodule age, or nodule environment.     

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.     

Nitric oxide acts as a retrograde messenger during long-term potentiation in cultured hippocampal neurons

We examined long-term potentiation (LTP) at synapses between hippocampal neurons in dissociated cell culture following presynaptic, postsynaptic, or extracellular application of a nitric oxide (NO) scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts a retrograde messenger during LTP.     

Trisomy 1q43 syndrome: a consistent phenotype with macrocephaly, characteristic face, developmental delay and cardiac anomalies

Trisomy 1q43 syndrome: a consistent phenotype with macrocephaly, characteristic face, developmental delay and cardiac anomalies: Patients with trisomy (1)(q42-qter) present with psychomotor retardation, macrocephaly, occasional presence of facial capillary naevi, cardio-vascular anomalies and small size for gestational age. We report on a girl with the same pattern of malformations, who has pure trisomy 1 q43: duplication of the region (1) (q43) and the translocation of the terminal region of the other chromosome 1 to the derivative 1, narrowing down the critical region for the characteristic traits of severe developmental delay, macrocephaly and congenital cardiac malformations.