Hippocampal NMDA receptors are important for behavioural inhibition but not for encoding associative spatial memories

The idea that an NMDA receptor (NMDAR)-dependent long-term potentiation-like process in the hippocampus is the neural substrate for associative spatial learning and memory has proved to be extremely popular and influential. However, we recently reported that mice lacking NMDARs in dentate gyrus and CA1 hippocampal subfields (GluN1^ΔDGCA1 mice) acquired the open field, spatial reference memory watermaze task as well as controls, a result that directly challenges this view. Here, we show that GluN1^ΔDGCA1 mice were not impaired during acquisition of a spatial discrimination watermaze task, during which mice had to choose between two visually identical beacons, based on extramaze spatial cues, when all trials started at locations equidistant between the two beacons. They were subsequently impaired on test trials starting from close to the decoy beacon, conducted post-acquisition. GluN1^ΔDGCA1 mice were also impaired during reversal of this spatial discrimination. Thus, contrary to the widely held belief, hippocampal NMDARs are not required for encoding associative, long-term spatial memories. Instead, hippocampal NMDARs, particularly in CA1, act as part of a comparator system to detect and resolve conflicts arising when two competing, behavioural response options are evoked concurrently, through activation of a behavioural inhibition system. These results have important implications for current theories of hippocampal function.     

Alveolar macrophages are necessary for the systemic inflammation of acute alveolar hypoxia.

Alveolar hypoxia (Fi(O(2)) 0.10) rapidly produces inflammation in the microcirculation of skeletal muscle, brain, and mesentery of rats. Dissociation between tissue Po(2) values and inflammation, plus the observation that plasma from hypoxic rats activates mast cells and elicits inflammation in normoxic tissues, suggest that the response to hypoxia is initiated when mast cells are activated by an agent released from a distant site and carried by the circulation. These experiments tested the hypothesis that this agent originates in alveolar macrophages (AM). Male rats were depleted of AM by tracheal instillation of clodronate-containing liposomes. Four days after treatment, AM recovered by bronchoalveolar lavage were <10% of control. Control rats received buffer-containing liposomes. As expected, alveolar hypoxia (Fi(O(2)) 0.10) in control rats increased leukocyte-endothelial adherence, produced degranulation of perivascular mast cells, and increased fluorescent albumin extravasation in the cremaster microcirculation. None of these effects was seen when AM-depleted rats were exposed to hypoxia. Plasma obtained from control rats after 5 min of breathing 10% O(2) elicited inflammation when applied to normoxic cremasters. In contrast, normoxic cremasters did not develop inflammation after application of plasma from hypoxic AM-depleted rats. Supernatant from AM cultured in 10% O(2) produced increased leukocyte-endothelial adherence, vasoconstriction, and albumin extravasation when applied to normoxic cremasters. Normoxic AM supernatant did not produce any of these responses. The effects of hypoxic supernatant were attenuated by pretreatment of the cremaster with the mast cell stabilizer cromolyn. These data support the hypothesis that AM are the source of the agent that initiates hypoxia-induced systemic inflammation by activating mast cells.     

Increased resting-state perfusion after repeated encoding is related to later retrieval of declarative associative memories

Electrophysiological studies in animals have shown coordinated reactivation of neuronal ensembles during a restricted time period of behavioral inactivity that immediately followed active encoding. In the present study we directly investigated off-line processing of associative memory formation in the human brain. Subjects' regional cerebral blood flow (rCBF) as a surrogate marker of neural activity during rest was measured by MR-based perfusion imaging in a sample of 14 healthy male subjects prior to (Pre2) and after (Post) extensive learning of 24 face-name associations within a selective reminding task (SR). Results demonstrated significant Post-Pre2 rCBF increases in hippocampal and temporal lobe regions, while in a control comparison of two perfusion scans with no learning task in-between (Pre2-Pre1) no differences in rCBF emerged. Post perfusion scanning was followed by a surprise cued associative recall task from which two types of correctly retrieved names were obtained: older names already correctly retrieved at least once during one of the SR blocks, and recent names acquired during the last SR block immediately prior to the Post scan. In the anterior hippocampus individual perfusion increases were correlated with both correct retrievals of older and recent names. By contrast, older but not recently learned names showed a significant correlation with perfusion increases in the left lateral temporal cortex known to be associated with long-term memory. Recent, but not older names were correlated with dopaminergic midbrain structures reported to contribute to the persistence of memory traces for novel information. Although the direct investigation of off-line memory processing did not permit concomitant experimental control, neither intentional rehearsal, nor substantial variations in subjects' states of alertness appear to contribute to present results. We suggest that the observed rCBF increases might reflect processes that possibly contribute to the long-term persistence of memory traces.     

Hippocampal CA3 NMDA receptors are crucial for memory acquisition of one-time experience

Lesion and pharmacological intervention studies have suggested that in both human patients and animals the hippocampus plays a crucial role in the rapid acquisition and storage of information from a novel one-time experience. However, how the hippocampus plays this role is poorly known. Here, we show that mice with NMDA receptor (NR) deletion restricted to CA3 pyramidal cells in adulthood are impaired in rapidly acquiring the memory of novel hidden platform locations in a delayed matching-to-place version of the Morris water maze task but are normal when tested with previously experienced platform locations. CA1 place cells in the mutant animals had place field sizes that were significantly larger in novel environments, but normal in familiar environments relative to those of control mice. These results suggest that CA3 NRs play a crucial role in rapid hippocampal encoding of novel information for fast learning of one-time experience.     

Modeling the acquisition of counting with an associative network

In the acquision of counting by children, there are three interesting phenomena (Fuson et al. 1982): (1) the number word sequence produced by children can be divided into three distinct portions, called the conventional, stable nonconventional, and unstable portions; (2) irregular number words such as fifteen are omitted more often than regular ones such as fourteen, sixteen, and seventeen; and (3) initially the number word sequence is in a recitation form, rather than in the form of an associative chain of separable serial elements. Our paper at first analyzes these phenomena from the viewpoint of associative memory by assuming the number word sequences are made up of many associative relationships between the number words. This assumption is not contradictory to the third phenomenon described above, because the associative relationships are not confined only to those between the serial number words. On the basis of these anaylses, an associative network model, HAPS proposed by one of the authors (Hirai 1983), is extended so that it can mimic some aspects of the learning of sequence which involves the above three phenomena. The learning and production of sequence by the network are simulated on a digital computer, and the results show that the three phenomena can be observed in the performance of the network.     

Sleep supports selective retention of associative memories based on relevance for future utilization

An outstanding question is whether memory consolidation occurs passively or involves active processes that selectively stabilize memories based on future utility. Here, we differentially modulated the expected future relevance of two sets of picture-location associations after learning. Participants first studied two sets of picture-location associations. After a baseline memory test, they were instructed that only one set of associations would be retested after a 14-hour delay. For half of the participants, this test-retest delay contained a night of sleep; for the other half the delay included a normal working day. At retest, participants were re-instructed and against their expectations tested on both sets of associations. Our results show that post-learning instruction about subsequent relevance selectively improves memory retention for specific associative memories. This effect was sleep-dependent; it was present only in the group of subjects for which the test-retest delay contained sleep. Moreover, time spent asleep for participants in this sleep group correlated with retention of relevant but not irrelevant associations; participants who slept longer forgot fewer associations from the relevant category. In contrast, participants that did not sleep forgot more relevant than irrelevant associations across the test-retest delay. In summary, our results indicate that it is possible to modulate the retention of selected memories after learning with simple verbal instructions on their future relevance. The finding that this effect depends on sleep demonstrates this state's active role in memory consolidation and may have utility for educational settings.     

Basal forebrain cholinergic neurons are necessary for estrogen to enhance acquisition of a delayed matching-to-position T-maze task

Intraseptal injections of the selective cholinergic immunotoxin 192 IgG-saporin (SAP) were performed to determine whether basal forebrain cholinergic neurons are necessary for hormone-mediated enhancement of acquisition in a delayed matching-to-position (DMP) T-maze task. The DMP task is a simple spatial learning task. Studies have shown that continuous estradiol replacement enhances acquisition of the DMP task in young ovariectomized rats and that long-term treatment with either estradiol or estradiol + progesterone can prevent a deficit in DMP acquisition in old rats. In the present study, continuous estradiol replacement significantly enhanced acquisition of the DMP task by non-SAP-treated, ovariectomized rats. In contrast, neither continuous estradiol nor weekly administration of estradiol + progesterone significantly enhanced acquisition of the DMP task in rats that received intraseptal injections of either a high dose (1.0 microg) or a low dose (0.22 microg) of SAP. Animals that reached criterion were significantly impaired by rotating the maze 180 degrees regardless of treatment, suggesting that animals in all groups used extramaze cues to at least some degree to solve the task. SAP-treated animals were slightly more sensitive to increasing the intertrial delay than non-SAP-treated controls, suggesting that the SAP lesions produced a modest deficit in spatial working memory. Immunohistochemistry confirmed the loss of cholinergic neurons in specific regions of the basal forebrain of SAP-treated animals. In addition, DMP acquisition correlated significantly with ChAT activity in the hippocampus and frontal cortex. The data suggest that basal forebrain cholinergic projections are necessary for hormone-mediated enhancement of DMP acquisition.     

CD4+ T lymphocytes are not necessary for the acute rejection of vascularized mouse lung transplants

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4(+) T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4(+) T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4(+) T cell-mediated allorecognition pathways. CD4(+) T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4(+) T cells, but does not delay acute rejection when CD4(+) T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.     

Casein as a necessary factor in the production of stimulatory material for associative growth of lactic streptococci

Strains of Streptococcus cremoris KH and HC produced material that was stimulatory for S. cremoris R₆ in milk and in the dialyzable fraction of milk, but not in the dialysate fraction of milk, lactic acid whey, or lactose broth. The addition of casein to these latter media permitted the production of this stimulatory material to occur. Tryptone, peptone, and yeast extract could not be substituted for casein in producing the stimulatory material or in initiating associative growth in the lactic acid whey. The minimum concentration of casein required appeared to be from 2.0 to 2.5%.     

Microtubules are necessary for lamellae retraction of Vero cells

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.     

FGF signals from the nasal pit are necessary for normal facial morphogenesis

Fibroblast growth factors (FGFs) are required for brain, pharyngeal arch, suture and neural crest cell development and mutations in the FGF receptors have been linked to human craniofacial malformations. To study the functions of FGF during facial morphogenesis we locally perturb FGF signalling in the avian facial prominences with FGFR antagonists, foil barriers and FGF2 protein. We tested 4 positions with antagonist-soaked beads but only one of these induced a facial defect. Embryos treated in the lateral frontonasal mass, adjacent to the nasal slit developed cleft beaks. The main mechanisms were a block in proliferation and an increase in apoptosis in those areas that were most dependent on FGF signaling. We inserted foil barriers with the goal of blocking diffusion of FGF ligands out of the lateral edge of the frontonasal mass. The barriers induced an upregulation of the FGF target gene, SPRY2 compared to the control side. Moreover, these changes in expression were associated with deletions of the lateral edge of the premaxillary bone. To determine whether we could replicate the effects of the foil by increasing FGF levels, beads soaked in FGF2 were placed into the lateral edge of the frontonasal mass. There was a significant increase in proliferation and an expansion of the frontonasal mass but the skeletal defects were minor and not the same as those produced by the foil. Instead it is more likely that the foil repressed FGF signaling perhaps mediated by the increase in SPRY2 expression. In summary, we have found that the nasal slit is a source of FGF signals and the function of FGF is to stimulate proliferation in the cranial frontonasal mass. The FGF independent regions correlate with those previously determined to be dependent on BMP signaling. We propose a new model whereby, FGF-dependent microenvironments exist in the cranial frontonasal mass and caudal maxillary prominence and these flank BMP-dependent regions. Coordination of the proliferation in these regions leads ultimately to normal facial morphogenesis.     

Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion

Current models for membrane fusion in diverse biological processes are focused on the local action of fusion proteins present in the contact zone where the proteins anchored in one membrane might interact directly with the other membrane. Are the fusion proteins outside of the contact zone just bystanders? Here we assess the role of these "outsider" proteins in influenza virus hemagglutinin-mediated fusion between red blood cells and either hemagglutinin-expressing cells or viral particles. To selectively inhibit or enhance the actions of hemagglutinin outsiders, the antibodies that bind to hemagglutinin and proteases that cleave it were conjugated to polystyrene microspheres too large to enter the contact zone. We also involved hemagglutinin outsiders into interactions with additional red blood cells. We find the hemagglutinin outsiders to be necessary and sufficient for fusion. Interfering with the activity of the hemagglutinin outsiders inhibited fusion. Selective conversion of hemagglutinin outsiders alone into fusion-competent conformation was sufficient to achieve fusion. The discovered functional role of fusion proteins located outside of the contact zone suggests a tempting analogy to mechanisms by which proteins mediate membrane fission from outside of the fission site.     

Glycoproteins required for entry are not necessary for egress of pseudorabies virus

In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono- or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.     

Antiviral antibodies are necessary for control of simian immunodeficiency virus replication

To better define the role of B cells in the control of pathogenic simian immunodeficiency virus (SIV) replication, six rhesus monkeys were depleted of B cells by intravenous infusion of rituximab (anti-CD20) 28 days and 7 days before intravaginal SIVmac239 inoculation and every 21 days thereafter until AIDS developed. Although the blood and tissues were similarly depleted of B cells, anti-SIV immunoglobulin G (IgG) antibody responses were completely blocked in only three of the six animals. In all six animals, levels of viral RNA (vRNA) in plasma peaked at 2 weeks and declined by 4 weeks postinoculation (PI). However, the three animals prevented from making an anti-SIV antibody response had significantly higher plasma vRNA levels through 12 weeks PI (P = 0.012). The remaining three B-cell-depleted animals made moderate anti-SIV IgG antibody responses, maintained moderate plasma SIV loads, and showed an expected rate of disease progression, surviving to 24 weeks PI without developing AIDS. In contrast, all three of the B-cell-depleted animals prevented from making anti-SIV IgG responses developed AIDS by 16 weeks PI (P = 0.0001). These observations indicate that antiviral antibody responses are critical in maintaining effective control of SIV replication at early time points postinfection.     

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.     

Sphingolipids are necessary for nicotinic acetylcholine receptor export in the early secretory pathway

The nicotinic acetylcholine receptor (AChR) is the prototype ligand-gated ion channel, and its function is dependent on its lipid environment. In order to study the involvement of sphingolipids (SL) in AChR trafficking, we used pharmacological approaches to dissect the SL biosynthetic pathway in CHO-K1/A5 cells heterologously expressing the muscle-type AChR. When SL biosynthesis was impaired, the cell surface targeting of AChR diminished with a concomitant increase in the intracellular receptor pool. The SL-inhibiting drugs increased unassembled AChR forms, which were retained at the endoplasmic reticulum (ER). These effects on AChR biogenesis and trafficking could be reversed by the addition of exogenous SL, such as sphingomyelin. On the basis of these effects we propose a 'chaperone-like' SL intervention at early stages of the AChR biosynthetic pathway, affecting both the efficiency of the assembly process and subsequent receptor trafficking to the cell surface.     

Thyroid hormones are necessary for the metamorphosis of tarpon Megalops cyprinoides leptocephali

This study investigates the effects of thyroxine (T₄), triiodothyronine (T₃) and thiourea (TU) on the metamorphosis of tarpon Megalops cryprinoides leptocephali. TU is an anti-thyroid hormone drug that inhibits the production of T₄ and T₃ in the thyroid tissue. Fully grown tarpons leptocephali were collected at the river mouth and, in the laboratory, were immediately treated with 100 ppb T₄, 10 ppb T_3, or 300 ppm TU. The appropriate concentrations were validated in a preliminary dose response experiment. Morphological and physiological characteristics that indicate metamorphic processes were measured every 2 days. T₄ and T₃ slightly speeded up the metamorphosis of tarpons compared with the control group. The experimental treatments produced accelerated reductions in length, increases in head/body ratio, swimbladder development, and loss of body water and sodium. In contrast, TU treatment caused metamorphic stasis with complete inhibition of metamorphosis between days 6 and 8. Thyroid hormone treatment stimulated fast otolith growth while TU treatment stopped otolith growth between days 6 and 9. Leptocephali in T₄, T₃ and control groups completed metamorphosis in 10-14 days, but TU-treated tarpons remained in the metamorphic leptocephalus stage more than 22 days. In addition, the inhibition of leptocephalus metamorphosis by 300 ppm TU can be reversed in the presence of 10 ppb T₃. These results indicate that thyroid hormones are involved in regulating the metamorphosis of leptocephali.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type