Adaptation to synaptic inactivity in hippocampal neurons

In response to activity deprivation, CNS neurons undergo slow adaptive modification of unitary synaptic transmission. The changes are comparable in degree to those induced by brief intense stimulation, but their molecular basis is largely unknown. Our data indicate that prolonged AMPAR blockade acts through loss of Ca2+ entry through L-type Ca2+ channels to bring about an increase in both vesicle pool size and turnover rate, as well as a postsynaptic enhancement of the contribution of GluR1 homomers, concentrated at the largest synapses. The changes were consistent with a morphological scaling of overall synapse size, but also featured a dramatic shift toward synaptic drive contributed by the Ca2+-permeable homomeric GluR1 receptors. These results extend beyond "synaptic homeostasis" to involve more profound changes that can be better described as "metaplasticity".     

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.     

alpha- and betaCaMKII. Inverse regulation by neuronal activity and opposing effects on synaptic strength

We show that alpha and betaCaMKII are inversely regulated by activity in hippocampal neurons in culture: the alpha/beta ratio shifts toward alpha during increased activity and beta during decreased activity. The swing in ratio is approximately 5-fold and may help tune the CaMKII holoenzyme to changing intensities of Ca(2+) signaling. The regulation of CaMKII levels uses distinguishable pathways, one responsive to NMDA receptor blockade that controls alphaCaMKII alone, the other responsive to AMPA receptor blockade and involving betaCaMKII and possibly further downstream effects of betaCaMKII on alphaCaMKII. Overexpression of alphaCaMKII or betaCaMKII resulted in opposing effects on unitary synaptic strength as well as mEPSC frequency that could account in part for activity-dependent effects observed with chronic blockade of AMPA receptors. Regulation of CaMKII subunit composition may be important for both activity-dependent synaptic homeostasis and plasticity.     

Stimulation of the antigen receptor on WEHI-231 B lymphoma cells results in a voltage-independent increase in cytoplasmic calcium

WEHI-231, a lymphoma-derived murine B cell line, responded to anti-IgM antibodies by increasing the concentration of free calcium in the cytoplasm from 140 nM to 590 nM within 15 sec. This is very similar to the response observed previously in normal B cells (Pozzan et al., 1982, J. Cell Biol. 94:335). Only antibodies specific for mIgM stimulated this response; control antibodies had no effect. In addition, anti-IgM did not stimulate a response by a mutant with a greatly decreased amount of membrane IgM. The relationship of this increase in cytoplasmic calcium to the plasma membrane potential was examined. Anti-IgM did not cause a rapid depolarization of the cells, suggesting that a voltage-dependent calcium channel was not responsible for the calcium increase. Furthermore, experimental depolarization of WEHI-231 cells did not cause a calcium influx, and the calcium increase caused by anti-IgM was not greatly affected by previous depolarization or by prevention of depolarization. These experiments argue strongly that the increase in cytoplasmic calcium was not mediated by a depolarization-activated calcium channel, such as the one found in cardiac muscle and in some neurons. Indeed, a significant portion of the initial increase in cytoplasmic calcium was due to the release of calcium from internal stores, suggesting the involvement of a soluble mediator. Examination of these internal storage sites in permeabilized cells revealed that inositol 1,4,5-trisphosphate could induce the release of calcium. These results are consistent with the hypothesis that the calcium increase in B cells stimulated by anti-IgM is caused by breakdown of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol trisphosphate, with the latter compound mediating calcium mobilization.     

Identification of High-Risk Plaques by MRI and Fluorescence Imaging in a Rabbit Model of Atherothrombosis

IntroductionThe detection of atherosclerotic plaques at risk for disruption will be greatly enhanced by molecular probes that target vessel wall biomarkers. Here, we test if fluorescently-labeled Activatable Cell Penetrating Peptides (ACPPs) could differentiate stable plaques from vulnerable plaques that disrupt, forming a luminal thrombus. Additionally, we test the efficacy of a combined ACPP and MRI technique for identifying plaques at high risk of rupture.ConclusionsOur targeted fluorescence ACPP probes distinguished disrupted plaques from stable plaques with high sensitivity and specificity. The combination of anatomic, MRI-derived predictors for disruption and ACPP uptake can further improve the power for identification of high-risk plaques and suggests future development of ACPPs with molecular MRI as a readout.     

Overexpression of the NR2A subunit in the forebrain impairs long‐term social recognition and non‐social olfactory memory

Animals must recognize and remember conspecifics and potential mates, and distinguish these animals from potential heterospecific competitors and predators. Despite its necessity, aged animals are known to exhibit impaired social recognition memory. As the brain ages, the ratio of NR2A:NR2B in the brain increases over time and has been postulated to underlie the cognitive decline observed during the aging process. Here, we test the hypothesis that an increased NR2A:NR2B subunit ratio underlies long‐term social recognition memory. Using transgenic overexpression of NR2A in the forebrain regions, we investigated the ability of these mice to learn and remember male and female conspecifics, mice of another strain and animals of another rodent species, the rat. Furthermore, due to the importance of olfaction in social recognition, we tested the olfactory memory in the NR2A transgenic mice. Our series of behavioral experiments revealed significant impairments in the NR2A transgenic mice in long‐term social memory of both male and female conspecifics. Additionally, the NR2A transgenic mice are unable to recognize mice of another strain or rats. The NR2A transgenic mice also exhibited long‐term memory impairments in the olfactory recognition task. Taken together, our results provide evidence that an increased NR2A:NR2B ratio in the forebrain leads to reduced long‐term memory function, including the ethologically important memories such as social recognition and olfactory memory .     

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births^1,2, 60-80% of all miscarriages^3,4, 10% of stillbirths^2,5, 13% of individuals with congenital heart disease⁶, 3-6% of infertility cases², and in many patients with developmental delay and birth defects⁷. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance^8,9.  Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents^10-13.Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy''s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)^14,15.     

genetic overexpression of NR2B subunit enhances social recognition memory for different strains and species

The ability to learn and remember conspecifics is essential for the establishment and maintenance of social groups. Many animals, including humans, primates and rodents, depend on stable social relationships for survival. Social learning and social recognition have become emerging areas of interest for neuroscientists but are still not well understood. It has been established that several hormones play a role in the modulation of social recognition including estrogen, oxytocin and arginine vasopression. Relatively few studies have investigated how social recognition might be improved or enhanced. In this study, we investigate the role of the NMDA receptor in social recognition memory, specifically the consequences of altering the ratio of the NR2B:NR2A subunits in the forebrain regions in social behavior. We produced transgenic mice in which the NR2B subunit of the NMDA receptor was overexpressed postnatally in the excitatory neurons of the forebrain areas including the cortex, amygdala and hippocampus. We investigated the ability of both our transgenic animals and their wild-type littermate to learn and remember juvenile conspecifics using both 1-hr and 24-hr memory tests. Our experiments show that the wild-type animals and NR2B transgenic mice preformed similarly in the 1-hr test. However, transgenic mice showed better performances in 24-hr tests of recognizing animals of a different strain or animals of a different species. We conclude that NR2B overexpression in the forebrain enhances social recognition memory for different strains and animal species.