Delayed and isoform-specific effect of NMDA exposure on neural cell adhesion molecules in hippocampus.

Brief stimulation of N-methyl-D-aspartate (NMDA) receptors has been shown to generate proteolytic fragments from the extracellular domain of neural cell adhesion molecules (NCAMs). In the present study, hippocampal slice cultures were used to demonstrate that such brief stimulation is followed by a delayed increase in the 180-kDa isoform NCAM-180. The slices were exposed to NMDA for 30 s followed by rapid quenching with the antagonist AP5. Immunoassays of the experimental samples indicated that concentrations of NCAM-180 were elevated above matched controls 2-3 h after the NMDA exposure, but not at earlier or later time points. This effect was isoform-specific as concentrations of the 140-kDa NCAM species were not found to increase. Interestingly, similar selectivity was evident with prolonged infusions of NMDA where, in contrast to the effect of brief stimulation, NCAM-180 content was reduced to 50% while levels of NCAM-140 were unchanged. Together with previous findings, the data indicate that the synaptic chemistries activated by NMDA differentially regulate NCAM-180 at the translation level and by localized activation of proteases.

The development of multiple domains of child and adolescent self-concept: a cohort sequential longitudinal design.

The development of child and adolescent self-concept was examined as a function of the self-concept domain, social/developmental/educational transitions, and gender. In two overlapping age cohorts of public school students (Ns = 936 and 984), five dimensions of self-concept were evaluated every 6 months in a manner that spanned grades 3 through 11 (representing the elementary, middle, and high school years). Domains of self-concept included academic competence, physical appearance, behavioral conduct, social acceptance, and sports competence. Structural equation modeling addressed questions about the stability of individual differences over time. Multilevel modeling addressed questions about mean-level changes in self-concept over time. Significant effects emerged with regard to gender, age, dimension of self-concept, and educational transition.

Acute effects of sildenafil (viagra) on blue-on-yellow and white-on-white Humphrey perimetry.

OBJECTIVE: To study the effects of sildenafil on blue-on-yellow and white-on-white Humphrey visual field (HVF). MATERIALS AND METHODS: Healthy subjects, ages 20 to 38 years, were prospectively randomized to active drug (n = 5) or placebo (n = 3) groups. Blue-on-yellow and white-on-white HVF testing was performed before and 1 hour after masked dosing of sildenafil 200 mg or placebo. Changes in mean deviation (MD) were compared between groups. RESULTS: Three of three placebo and four of five sildenafil subjects had no change on HVF. One of five sildenafil subjects had a decrease in MD of 17.9 dB and 4.7 dB on blue-on-yellow and white-on-white HVF testing, respectively. This subject reported more systemic side effects than other subjects. CONCLUSIONS: Sildenafil has no effect on HVF testing in most persons; however, sildenafil caused an acute abnormality of HVF testing in one subject, who experienced pronounced non-visual systemic symptoms; this effect was greater on blue-on-yellow than white-on-white HVF.

Peripheral administration of a serine protease inhibitor blocks kindling.

An inhibitor of tissue plasminogen activator (tPA) was tested for its effects on the rapid kindling induced by a series of afterdischarges (ADs) triggered in hippocampus over a 3-h period. Rats injected with vehicle prior to the session had prolonged ADs in tests carried out 10 days later. This was not the case for animals treated with the inhibitor. These findings support the hypothesis that activity-driven proteolysis contributes importantly to the production of long-lasting physiological changes.

Are the discrepancies between self- and others' appraisals of competence predictive or reflective of depressive symptoms in children and adolescents: a longitudinal study, Part II.

Two cohorts of children and adolescents (who started 6th grade in 1993 and 1996), parents, teachers, and peers participated in a 4-wave, 2-year, longitudinal study of perceived competence and depressive symptoms. The authors assessed children's tendencies to underestimate their competence (discrepant self-appraisals) relative to the appraisals of significant others. We also assessed the degree to which self-appraisals reflected the evaluations of others (reflective self-appraisals). Domains of competence were academic competence, physical appearance, behavioral conduct, social acceptance, and athletic competence. Cross-sectional analyses indicated that depressive symptoms correlated with reflective and discrepant self-appraisals. Longitudinal analyses revealed that reflective and discrepant self-appraisals predicted subsequent depressive symptoms and that depressive symptoms predicted discrepant but not reflective self-appraisals. Clinical implications of the findings are discussed.

Seizure induced synthesis of fibronectin is rapid and age dependent: implications for long-term potentiation and sprouting.

Extracellular matrix proteins are induced by activity in adult brain but the time course of these responses, and hence the possibility of their involvement in use-dependent synaptic plasticity, is not known. To evaluate this issue, the influence of seizures on fibronectin expression was evaluated in the adult and developing hippocampus. In adult rats, kainic acid-induced seizures increased fibronectin mRNA and immunoreactivity (ir) by about 1 h after the first behavioral seizure. In situ hybridization analysis indicated that fibronectin mRNA was increased in broadly distributed glial cells as well as within discrete neuronal populations that normally express this transcript. Western blots demonstrated that increased fibronectin-ir was evident in both soluble and non-soluble fractions at the same time point. Immunocytochemical colocalization confirmed that fibronectin-ir was indeed elevated in broadly distributed glial fibrillary acidic protein-ir astroglia. Seizures had no detectable effect on fibronectin-ir in the hippocampus of nine day old rats. Together with previous results, the above findings suggest that intense physiological activity triggers a 'matrix response' (i.e., release proteases, activate integrins, secrete matrix proteins) that is sufficiently rapid to participate in the consolidation of long-term potentiation (LTP). The absence of such reactions in the immature hippocampus is in accord with the hypothesis that matrix proteins generated by mature astroglia impose temporal and spatial limitations on axonal remodeling.

Proteolysis of cell adhesion molecules by serine proteases: a role in long term potentiation?

Tissue plasminogen activator (tPA), a serine protease endogenous to hippocampal neurons, is shown to recognize a highly conserved sequence in the extracellular domain of cell adhesion molecules (CAMs). When added to brain homogenates, tPA generated a CAM fragment similar in size to that produced in hippocampal slices by brief periods of NMDA receptor stimulation. The serine protease inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride blocked the effects of tPA with an approximately 50% suppression at 250 microM. The inhibitor at this concentration had no evident effect on synaptic responses but caused long term potentiation to decay back to baseline over a 1 h period. These results suggest that extracellular breakdown of cell adhesion molecules initiated by NMDA receptors and mediated by serine proteases contributes to the formation of stable potentiation.

Activation of NMDA receptors stimulates extracellular proteolysis of cell adhesion molecules in hippocampus.

Recent work indicates that treatments which block adhesion receptors prevent the stabilization of long term potentiation (LTP). The experiments reported here show that brief stimulation of hippocampal NMDA receptors, a triggering event for LTP induction, results in the extracellular proteolysis of two or more members of the Cell Adhesion Molecule (CAM) family. This effect is rapid, occurs at a consensus serine protease site, and is selective to NMDA receptors. It is also found in vivo after kainic acid induced seizures. Cleavage of adhesive connections could be an early step in the formation of new synaptic configurations.

The relationship between adhesion molecules and neuronal plasticity.

1. It is presently widely assumed that structural reorganization of synaptic architectures subserves the functional gains that define certain neuronal plasticities. 2. While target molecules thought to participate in such morphological dynamics are not well defined, growing evidence suggests a pivotal role for cell adhesion molecules. 3. Herein, brief discussions are presented on (i) the history of how adhesion molecules became implicated in plasticity and memory processes, (ii) the general biology of some of the major classes of such molecules, and (iii) the future of the adhesion molecule/plasticity relationship.

Beta-amyloid increases cathepsin D levels in hippocampus.

A previous study established that beta-amyloid(1-42) is sequestered and retained intact for extended periods by select populations of neurons in cultured hippocampal slices. The present experiment tested if this effect is accompanied by increases in cathepsin D, a characteristic feature of lysosomal dysfunction and one that has been implicated in key aspects of brain aging in humans. Slices incubated with beta-amyloid(1-42) (15-30 microM) for 6 days had 56% greater concentrations of cathepsin D than controls. Scrambled peptides had no effect. The amyloid-induced increase was additive with that produced by submaximal concentrations of an inhibitor of cathepsins B and L but occluded that caused by chloroquine. This pattern of results (1) indicates that the uptake of amyloid results in lysosomal dysfunction and (2) suggests that perturbations of intralysosomal pH may contribute to this effect.

Amyloid beta protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxyterminal fragments of the amyloid precursor protein.

A critical issue concerning Alzheimer's disease is its selectivity, which leads to cellular degeneration in certain brain areas but not in others, and whether this pathogenic selectivity involves products of the amyloid precursor protein (APP). Here, we show that the amyloid beta protein Abeta1-42 is accumulated gradually and is retained intact by field CA1, but not by other subdivisions, of organotypic hippocampal slice cultures. In contrast, the slightly shorter Abeta1-40 peptide was not sequestered selectively. Sequestration of Abeta1-42 was followed by the build-up of carboxyterminal fragments of the endogenous precursor protein that were identified by immunoprecipitation. Unlike the peptide uptake, this induction appeared to be stochastic at the cellular level. In addition, the APP fragments were distributed more broadly within the CA1 pyramidal neurons than the sequestered Abeta1-42, and they appeared to be localized to synaptic terminals in the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of the subfield CA3. Concentrations of synaptophysin, a presynaptic marker, decreased as the number of neurons producing amyloidogenic species increased. These results indicate that exogenous Abeta1-42 sets into motion a sequence that involves 1) selective uptake of the peptide by vulnerable cells at risk in Alzheimer's disease, 2) markedly enhanced production of amyloidogenic precursor material, and 3) slow deterioration of central synapses.

Mannose-specific lectins modulate ligand binding to AMPA-type glutamate receptors.

Binding of [3H]AMPA was increased above control levels in rat brain membranes that had been incubated with concanavalin A (Con A) or a lectin from Lens culinaris (LC), both of which bind mannose residues. This did not occur with any of six lectins with other specificities. The magnitude of the increased binding varied from 15% in cortex to 70% in hippocampus and decreased significantly between 3 weeks and 6 months of age. Succinylated Con A was without effect and neither Con A nor LC increased binding to solubilized AMPA receptors. Increases in binding were not obtained in membranes purified from HEK293 cell lines expressing homomeric AMPA receptors. This indicates that mannose specific lectins may enhance binding by cross-linking AMPA receptors to each other or to proteins that are specific to brain. Con A has been reported to reduce glutamate receptor desensitization with higher efficacy at kainate than at AMPA receptors; the increase in binding reported here appears to be unrelated to such effects because (1) it was not affected by drugs that block desensitization and (2) [3H]kainate binding was reduced rather than increased by Con A. These observations suggest that AMPA receptor kinetic properties not involving desensitization are influenced by extracellular interactions between the receptors and other transmembrane proteins.

Variants of the receptor/channel clustering molecule gephyrin in brain: distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain.

The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino-terminal domain of gephyrin (GN(N)) and 2) its invariant carboxy-terminus (GN(C)). Both antibodies recognized an antigen with the expected molecular weight of 93-95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti-GN(N) was markedly distinct from that labeled by anti-GN(C), a difference consistent with carboxy-terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy-terminal gephyrin fragments of 48-50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis.

Sialic acid residues indirectly modulate the binding properties of AMPA-type glutamate receptors.

Manipulations that disrupt the extracellular interactions of neural cell adhesion molecules (NCAMs) block the formation of stable long-term potentiation (LTP) but do not reverse already established potentiation. Several studies have implicated a change in AMPA-type glutamate receptors as being responsible for the expression of LTP but there are no evident links between NCAMs and the receptors. NCAMs are major carriers of sialic acid residues in the brain and removal of these with neuraminidase markedly affects the binding properties of the adhesion molecules. Therefore, the present study tested if neuraminidase treatment produces a change in AMPA receptors. Preincubation of cortical membranes with the enzyme for 15 min at 37 degrees C caused a approximately 5% reduction in the apparent sizes of NCAMs 140 and 180 but had no detectable influence on the sizes of various glutamate receptor subunits. The same treatment resulted in a 20 +/- 1% increase in the binding of [3H]AMPA with no apparent effect on binding to NMDA-type glutamate receptors or to high affinity kainate receptors. In membranes from the hippocampus, neuraminidase induced a 30 +/- 2% increase in binding which Scatchard analyses showed to be due to an increase in receptor affinity. Finally, neuraminidase had no effect on either the binding properties of solubilized AMPA receptors or on AMPA receptors stably expressed in a non-neuronal cell line. These results: (i) demonstrate that modulation of the extracellular environment can influence the binding properties of AMPA receptors, (ii) indicate that sialic acid residues in the extracellular compartment of synapses exert a significant and indirect influence on AMPA receptors and, (iii) suggest a route whereby NCAMs and LTP could be linked.

Distinct distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 M(R) antigen (GR53) in brain tissue.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

Stable maintenance of glutamate receptors and other synaptic components in long-term hippocampal slices.

Cultured hippocampal slices retain many in vivo features with regard to circuitry, synaptic plasticity, and pathological responsiveness, while remaining accessible to a variety of experimental manipulations. The present study used ligand binding, immunostaining, and in situ hybridization assays to determine the stability of AMPA- and NMDA-type glutamate receptors and other synaptic proteins in slice cultures obtained from 11 day postnatal rats and maintained in culture for at least 4 weeks. Binding of the glutamate receptor ligands [3H]AMPA and [3H]MK-801 exhibited a small and transient decrease immediately after slice preparation, but the binding levels recovered by culture day (CD) 5-10 and remained stable for at least 30 days in culture. Autoradiographic analyses with both ligands revealed labeling of dendritic fields similar to adult tissue. In addition, slices at CD 10-20 expressed a low to high affinity [3H]AMPA binding ratio that was comparable with that in the adult hippocampus (10:1). AMPA receptor subunits GluR1 and GluR2/3 and an NMDA receptor subunit (NMDAR1) exhibited similar postcutting decreases as that exhibited by the ligand binding levels, followed by stable recovery. The GluR4 AMPA receptor subunit was not evident during the first 10 CDs but slowly reached detectable levels thereafter in some slices. Immunocytochemistry and in situ hybridization techniques revealed adult-like labeling of subunit proteins in dendritic processes and their mRNAs in neuronal cell body layers. Long-term maintenance was evident for other synapse-related proteins, including synaptophysin, neural cell adhesion molecule isoforms (NCAMs), and an AMPA receptor related antigen (GR53), as well as for certain structural and cytoskeletal components (e.g., myelin basic protein, spectrin, microtubule-associated proteins). In summary, following an initial and brief depression, many synaptic components were expressed at steady-state levels in long-term hippocampal slices, thus allowing the use of such a culture system for investigations into mechanisms of brain synapses.

Induction of beta-amyloid-containing polypeptides in hippocampus: evidence for a concomitant loss of synaptic proteins and interactions with an excitotoxin.

Long-term cultures of brain slices were used to test if the lysosomotropic agent chloroquine induces beta-amyloid-related peptides in hippocampus and if such effects are accompanied by other manifestations of brain aging. Chloroquine administration resulted in the appearance of a carboxyl-terminal fragment of the beta-amyloid precursor protein (APP); the 27-kDa antigen was detectable after 24 h, increased rapidly for 6-10 days, and was eliminated upon drug washout. Immunocytochemical analyses showed that beta-amyloid immunoreactivity accumulated in the perikarya of pyramidal neurons, primarily in the form of punctate bodies. These effects were accompanied by a correlated loss (and recovery) of the presynaptic marker synaptophysin and by a delayed reduction of postsynaptic glutamate receptors, while cytoskeletal proteins were unchanged. Acute administration of chloroquine had no evident effects on synaptic responses but prolonged applications caused a decrease in the maximum amplitude of field potentials. Finally, a brief pretreatment with the excitotoxin kainic acid had little effect with regard to APP fragments or synaptophysin, but altered the events following from a subsequent infusion of chloroquine. Buildup of the 27-kDa APP fragment and loss of synaptophysin were more rapid and, more importantly, did not reverse upon washout of chloroquine. These findings indicate that lysosomal dysfunction in hippocampus results in the accumulation of a particular APP fragment and suggest that this event, or a variable correlated with it, is linked to the loss of synaptic proteins. They also raise the possibility that certain aspects of brain aging reflect a synergism between lysosomal disturbances and excitotoxicity.