Inflammatory microglia are glycolytic and iron retentive and typify the microglia in APP/PS1 mice.

Microglia, like macrophages, can adopt inflammatory and anti-inflammatory phenotypes depending on the stimulus. In macrophages, the evidence indicates that these phenotypes have different metabolic profiles with lipopolysaccharide (LPS)- or interferon-γ (IFNγ)-stimulated inflammatory cells switching to glycolysis as their main source of ATP and interleukin-4 (IL-4)-stimulated cells utilizing oxidative phosphorylation. There is a paucity of information regarding the metabolic signatures of inflammatory and anti-inflammatory microglia. Here, we polarized primary microglia with IFNγ and show that the characteristic increases in tumor necrosis factor-α (TNFα) and nitric oxide synthase 2 (NOS2) were accompanied by increased glycolysis and an increase in the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3, an enzyme that plays a significant role in driving glycolysis. These changes were associated with increased expression of ferritin and retention of iron in microglia. Significantly, retention of iron in microglia increased TNFα expression and also increased glycolysis suggesting that increased intracellular iron concentration may drive the metabolic and/or inflammatory changes. Analysis of microglia prepared from wildtype mice and from transgenic mice that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1; APP/PS1) revealed genotype-related increases in glycolysis, accompanied by increased PFKFB3, and an increase in the expression of ferritin. The data indicate a distinct metabolic signature of inflammatory microglia from APP/PS1 mice that are also distinguishable by their iron handling profiles.

Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-ß and cognitive function in APP/PS1 mice.

Activation of the inflammasome is implicated in the pathogenesis of an increasing number of inflammatory diseases, including Alzheimer's disease (AD). Research reporting inflammatory changes in post mortem brain tissue of individuals with AD and GWAS data have convincingly demonstrated that neuroinflammation is likely to be a key driver of the disease. This, together with the evidence that genetic variants in the NLRP3 gene impact on the risk of developing late-onset AD, indicates that targetting inflammation offers a therapeutic opportunity. Here, we examined the effect of the small molecule inhibitor of the NLRP3 inflammasome, MCC950, on microglia in vitro and in vivo. The findings indicate that MCC950 inhibited LPS+Aß-induced caspase 1 activation in microglia and this was accompanied by IL-1ß release, without inducing pyroptosis. We demonstrate that MCC950 also inhibited inflammasome activation and microglial activation in the APP/PS1 mouse model of AD. Furthermore, MCC950 stimulated Aß phagocytosis in vitro, and it reduced Aß accumulation in APP/PS1 mice, which was associated with improved cognitive function. These data suggest that activation of the inflammasome contributes to amyloid accumulation and to the deterioration of neuronal function in APP/PS1 mice and demonstrate that blocking assembly of the inflammasome may prove to be a valuable strategy for attenuating changes that negatively impact on neuronal function.

Impaired performance of female APP/PS1 mice in the Morris water maze is coupled with increased Aß accumulation and microglial activation.

BACKGROUND: Alzheimer's disease (AD) is characterized by progressive neuronal loss and cognitive decline. Epidemiological studies suggest that the risk of AD is higher in women even when data are adjusted for age. OBJECTIVE: We set out to compare changes in 9-month-old male and female mice which overexpress amyloid precursor protein (APP) with presenilin (PS1; APP/PS1 mice) and to evaluate whether any changes were coupled with deficits in spatial learning. METHODS: APP/PS1 mice were assessed for their ability to learn in the Morris water maze and Aß burden assessed by Congo Red and Aß triple ultrasensitive assay. Neuroinflammatory changes were examined in brain tissue along with expression of Aß-generating and Aß-degrading enzymes. RESULTS: A deficit in reversal phase learning in the Morris water maze was observed in female mice and was paralleled by evidence of increased accumulation of Aß, microglial activation and expression of IL-1ß. Accumulation of Aß was coupled with an increase in expression of BACE-1 and a decrease in insulin-degrading enzyme (IDE). CONCLUSION: The results indicate that the observed impairment in spatial memory in female APP/PS1 mice correlated with increased Aß burden and the changes in Aß may have occurred as a result of enhanced BACE-1 and decreased IDE expression.

Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip.

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants.

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.

Bland dyskaryosis: a new pitfall in liquid-based cytology.

OBJECTIVE: To describe our experience in recognizing an unusual presentation of severe dyskaryosis at two large cytology centres using ThinPrep liquid-based cytology (LBC). LBC has been introduced in England following successful pilot studies. It is clear that LBC improves visualization and preservation of cells, and that sensitivity for high-grade dyskaryosis is at least as good as for conventional cytology, and may be better. Several variants of high-grade dyskaryosis have been described on conventional cytology, including small and pale cell dyskaryosis. These are also seen on LBC. We are reporting a new variant of dyskaryosis which in our experience is seen only in LBC specimens prepared by the Thinprep method, which we have named bland dyskaryosis. This has not to our knowledge been previously described. Bland dyskaryosis is characterized by cells with a high nuclear/cytoplasmic ratio, nuclear hyperchromasia and is present in groups with a chaotic architecture. The chromatin pattern appears bland at low power screening examination. On high power examination, however, the chromatin pattern can be seen to be subtly abnormal. Nuclear membranes are smooth. These changes mimic endocervical cells or immature squamous metaplasia at low power. METHOD: Identification and description of cytological appearances observed in routine practice and correlation with histological diagnosis. CONCLUSION: The features of bland dyskaryosis should be disseminated through teaching activities. Recognition of this previously undescribed variant will prevent false negative reporting of LBC samples.

Infiltrating macrophages contribute to age-related neuroinflammation in C57/BL6 mice.

The recognized role of neuroinflammation in the age-related deterioration of neuronal function highlights the importance of understanding the factors that control microglial activation. Microglia, as the immune cells of the brain, are the arbiters of the inflammatory profile in the brain. Normally they are maintained in a quiescent state by means of ligand-receptor interactions with neurons, within a prevailing anti-inflammatory microenvironment. The evidence indicates that, as the ageing process continues, microglia become activated, shift towards an inflammatory phenotype and alter the milieu in the brain. Although there has been progress in identifying factors that contribute to age-related microglial activation, our understanding remains incomplete. Here we report that there was an age-related increase in circulating inflammatory cytokines, accompanied by microglial activation. Neutrophils, and to a greater extent, macrophages, infiltrate the brain with age, perhaps as a result of increased chemokine expression in the brain, specifically CXCL1 and CCL2. We sought to determine whether macrophages might trigger microglial activation and the evidence shows that conditioned medium obtained from interferon-γ (IFNγ)-stimulated macrophages potently activated microglia. The data suggest that infiltrating macrophages may be one factor that contributes to age-related microglial activation.

Docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: a lipidomic analysis.

The age-related decline in cognitive function has been associated with biochemical changes that can be attenuated following n-3 polyunsaturated fatty acid treatment. Dietary supplementation with docosahexaenoic acid (DHA) has been shown to reverse age-related changes in synaptic function. Here, lipidomic analyses were undertaken to examine changes in lipid classes and phospholipid species in cortical tissue of young (2-4 months) and aged (20-22 months), control- and DHA-treated (10mg daily) rats following treatment for 8 weeks, aiming to explore the mechanism of DHA action. Dietary supplementation normalised the age-related decrease in unsaturation index, reduced the levels of arachidonic acid-containing phospholipids in both young and aged animals, and gave rise to production of new phosphatidylserine and phosphatidylinositol species. These findings suggest that DHA may mediate some of its effects through alterations in the membrane lipid composition that can consequently affect the production of pro-inflammatory mediators and signalling molecular species.

Age-related impairment in long-term potentiation in hippocampus: a role for the cytokine, interleukin-1 beta?

Ageing is associated with impairments in a variety of biological functions, one of the most striking might be considered to be the impairment in cognitive function. This impairment probably relates to the vulnerability of the hippocampus to the ageing process, since several cognitive functions rely on the integrity of this brain area. Analysis of the mechanisms underlying the effect of ageing on hippocampal function has focused to a great extent on analysis of age-related changes in long-term potentiation (LTP) in hippocampus of experimental animals. LTP is a remarkable form of synaptic plasticity which is characterized by a persistent increase in synaptic efficacy following tetanic stimulation of an afferent pathway to one of the hippocampal subfields. On the basis of its properties, LTP has been proposed as a biological substrate for learning and/or memory. There is general agreement that aged rats exhibit an impaired ability to sustain LTP but there is no agreement on the underlying cause of this deficit. In this review, the evidence which suggests that age-related changes in membrane composition, triggered by oxidative changes, might significantly contribute to the impairment in LTP, is considered. These findings are supported by recent data which indicates that dietary supplementation with the antioxidant vitamins, E and C, reversed the age-related impairment in LTP, in parallel with reversing the age-related decrease in alpha-tocopherol concentration and the age-related increase in lipid peroxidation. The possibility that the proinflammatory cytokine, interleukin-1 beta, triggers several age-related changes in hippocampus is considered and, based on the evidence presented, the hypothesis that interleukin-1 beta plays a significant role in ageing in the hippocampus is proposed.

Mutational analysis of the transforming growth factor beta receptor type II gene in human ovarian carcinoma.

In the present study, we evaluated a series of sporadic ovarian carcinomas for mutations within the entire coding region of TbetaR-II. Using reverse transcription-PCR and "Cold" single-strand conformational polymorphism analysis, 6 of 24 samples (25%) were found to contain code-altering mutations in TbetaR-II: (a) four mutations resulting in amino acid substitutions in the highly conserved serine/threonine kinase domain; (b) one mutation resulting in a conservative amino acid change in the transmembrane domain; and (c) a 1-bp insertion in the polyadenylic acid microsatellite region resulting in a reading frameshift. In addition, six cases (25%) exhibited a common bp substitution (C-->T at nucleotide 1322) in both tumor and patient-matched normal tissues. This is the first report of such TbetaR-II mutations in primary human ovarian carcinomas. Immunohistochemical analysis demonstrated a loss of expression of TbetaR-II in 5 of 22 available tumors (23%; 4 of which also had mutations in the coding region) and decreased expression of TbetaR-II in 10 of 22 available tumors (44%; 1 of which had a mutation in the coding region). Thus, the loss or decreased expression of TbetaR-II seems to be a common event in sporadic ovarian carcinomas, and mutational inactivation, due to either frameshift mutations in the polyadenylic acid microsatellite region or point mutations in conserved functional domains, is one mechanism by which this occurs.

Effect of duration of exposure to verapamil on vincristine activity against multidrug-resistant human leukemic cell lines.

Verapamil sensitizes multidrug-resistant cell lines to various heterocyclic anticancer drugs by inhibition of energy-dependent release of drug, presumably by interaction with membrane glycoproteins involved in drug efflux. This work assessed verapamil sensitization of human multidrug-resistant lymphocytic and myeloid leukemic cell lines (CEM/VLB100, HL-60/AR) to vincristine during exposures of short duration (4 h). When cells were transferred to drug-free medium immediately after simultaneous 4-h exposures to vincristine and verapamil, the antiproliferative activity of vincristine was not altered in CEM/VLB100 cells and was only moderately increased in HL-60/AR cells. In contrast, when cells were transferred to verapamil-containing medium, vincristine activity was greatly increased against both CEM/VLB100 and HL-60/AR cells. Verapamil enhanced accumulation and inhibited release of [3H]vincristine by CEM/VLB100 and HL-60/AR cells, indicating that the sensitization was due to an increase in cell-associated vincristine after transfer of cells to vincristine-free medium. Slot blot analysis of cellular RNA with the pMDR1 probe revealed high levels of expression of the mdr1 gene in CEM/VLB100 cells but no detectable expression in HL-60/AR cells. Consistent with this finding, polypeptides (Mr 170,000 to 180,000) that were recognized by a monoclonal antibody (C219) against P-glycoprotein were greatly overexpressed in CEM/VLB100 cells, but were expressed at low levels, if at all, in HL-60/AR cells. These results demonstrate the importance of duration of exposure to verapamil in reversing multidrug resistance, not only in cells that overexpress P-glycoprotein but also in cells, such as HL-60/AR, that express little, if any, P-glycoprotein.

Enzymatic methylation of microsomal metabolites of benzo(a)pyrene.

These studies suggest that the microsomal metabolism of benzo(a)pyrene (BP) produces metabolites which can be methylated by the catechol-o-methyltransferase (COMT)/S-adenosylmethionine (SAM) enzyme/donor combination. Induced microsomes converted 12 to 15% of substrate BP to polar products. Approximately 0.06% of substrate BP was recovered as COMT/SAM-reactive substances. In tests for specificity, COMT/SAM was found to react with catechols, but not with dihydrodiols, quinones, a phenol, an epoxide, or 1,4-hydroquinone. Organic extracts of COMT/[14C]SAM incubations with BP were fractionated by high-performance liquid chromatography. The appearance of radiolabeled chromatographic bands required the presence of substrate BP, microsomes, and COMT/[14C]SAM. When the Ames mutagenesis assay was supplemented with COMT/SAM, a 36% reduction was observed in the number of revertant colonies induced by the microsomal oxidation of BP. In contrast, the mutagenic properties of 2-aminofluorene were not affected by COMT/SAM. These observations indicate that COMT/SAM does not generally inhibit mixed-function oxidase activity but rather reacts with substances which are activated by ring oxygenations.

Dietary antioxidant supplementation reverses age-related neuronal changes.

Evidence suggests that reactive oxygen species in brain may play a role in the development of age-related neuronal impairments, and that the increase in the concentration of the proinflammatory cytokine, interleukin-1beta (IL-1beta), in aged brain tissue, may also be a contributory factor. In this study, we have analyzed changes in enzymatic and nonenzymatic antioxidant levels, in parallel with interleukin-1beta concentration, in cortical tissue prepared from young and aged rats. We report that there was an age-related increase in the activity of superoxide dismutase without concomitant changes in the activity of catalase or glutathione peroxidase and an age-related decrease in the concentrations of alpha-tocopherol and ascorbate. These observations, coupled with age-related increases in lipid peroxidation and interleukin-1beta concentration, are consistent with a compromised antioxidant defense in cortex of aged rats, a proposal supported by the finding that these changes were not observed in cortical tissue prepared from rats fed on a diet supplemented with alpha-tocopherol and ascorbate for 12 weeks.

Regulation of intracellular Ca2+ concentration by interleukin-1beta in rat cortical synaptosomes: an age-related study.

The pro-inflammatory cytokine interleukin-1beta (IL-1beta) is released by cells during injury and stress, and increased neuronal expression of IL-1beta is a feature of age-related neurodegeneration. We have recently reported that IL-1beta has a biphasic effect on the K+-induced rise in intracellular Ca2+ concentration ([Ca2+]i) in cortical synaptosomes, exerting an inhibitory effect on the K+-induced rise in [Ca2+]i at lower (3.5 ng/mL) concentrations and a stimulatory effect on the K+-induced rise in [Ca2+]i at higher (100 ng/mL) concentrations. In the present study, we observed that the K+-induced rise in [Ca2+]i was inhibited to a similar extent by the lower concentration of IL-1beta in cortical synaptosomes prepared from young (3-month-old), middle-aged (12-month-old) and aged (24-month-old) rats. In contrast, cortical synaptosomes prepared from the aged rats exhibited an increased susceptibility to the higher concentration of IL-1beta, resulting in a marked elevation in [Ca2+]i. We propose that the age-related increase in neuronal concentration of IL-1beta promotes a dramatic elevation in [Ca2+]i following membrane depolarization, thereby altering Ca2+ homeostasis and exacerbating neuronal vulnerability to excitotoxicity.

Increased IL-1beta in cortex of aged rats is accompanied by downregulation of ERK and PI-3 kinase.

Ageing is accompanied by a myriad of changes, which lead to deficits in synaptic function and recent studies have identified an increase in concentration of the proinflammatory cytokine, interleukin-1beta (IL-1beta), as a factor which significantly contributes to deterioration of cell function. Here, we consider that increased IL-1beta concentration and upregulation of IL-1beta-induced cell signalling cascades may be accompanied by downregulation of survival signals, perhaps as a consequence of decreased neurotrophins-associated signalling. The data indicate that increased IL-1beta concentration was coupled with downregulation of ERK and phosphoinositide-3 kinase (PI-3 kinase) in cortical tissue prepared from aged rats. These changes could not be attributed to decreased concentration of NGF or BDNF but the evidence suggested that they may be a consequence of an age-related change in the anti-inflammatory cytokine, IL-4. Significantly, treatment of aged rats with eicosapentaenoic acid reversed the age-related increases in IL-1beta and IL-1beta-induced signalling and also the age-related changes in IL-4, ERK and PI-3 kinase.

Interleukin-1 beta inhibits glutamate release in hippocampus of young, but not aged, rats.

The proinflammatory cytokine, interleukin-1, is synthesized in neuronal and glial cells and is released in response to stress/injury. IL-1 exerts profound effects on the central nervous system, which include an inhibitory effect on synaptic activity in hippocampus, a brain area expressing a high density of IL-1 receptors. We report that IL-1 beta has an inhibitory effect on KCl-stimulated release of glutamate and KC1-stimulated [45Ca] influx in synaptosomes prepared from hippocampus of 4-month-old rats. These effects were inhibited by the endogenous receptor antagonist, IL-1ra, and by the phospholipase A2 (PLA2) inhibitor, quinacrine, suggesting that IL-1 receptor activation is coupled to PLA2. An inhibitory effect of IL-1 beta on protein kinase C activity was also observed. KC1-induced calcium-dependent release and calcium influx, and protein kinase C activity were significantly decreased in hippocampal synaptosomes prepared from 22-month-old compared to 4-month-old animals. In contrast to the inhibitory effect of IL-1 beta in synaptosomes prepared from young adult animals, no effect was observed on release, calcium influx, or protein kinase C activity in synaptosomes prepared from aged animals. We report that there is an age-related increase in expression of IL-1 beta in hippocampus and propose that this change may underlie the attenuated responses to IL-1 beta in hippocampus of aged animals.

Age-related changes in LTP and antioxidant defenses are reversed by an alpha-lipoic acid-enriched diet.

Among the age-related changes identified in rat hippocampus are impairments in LTP and glutamate release. These deficits have been coupled with decreased arachidonic acid concentration. In this study we compared LTP and glutamate release in groups of aged and young rats fed for 8 weeks on a control diet or on a diet enriched in alpha-lipoic acid. Dietary supplementation in aged rats restored hippocampal arachidonic acid concentration to levels observed in tissue prepared from young rats. We observed that aged rats that received the experimental diet sustained LTP in perforant path-granule cell synapses in a manner indistinguishable from young rats whereas the age-related impairment in glutamate release was reversed in synaptosomes prepared from dentate gyrus obtained from these rats. The evidence presented supports the hypothesis that the alpha-lipoic acid-enriched diet has antioxidant properties, because the age-related increase in superoxide dismutase activity and decrease in alpha-tocopherol concentration were reversed. The finding that the age-related increase in interleukin-1 (IL-1)beta concentration was also reversed suggests a possible role for this cytokine in ageing.

Long-term Activation of Phosphoinositide Turnover Associated with Increased Release of Amino Acids in the Dentate Gyrus and Hippocampus Following Classical Conditioning in the Rat.

The release of amino acids and the hydrolysis of inositol phospholipids were examined in parallel in three hippocampal areas following classical conditioning. Paired or unpaired tone(CS) - shock(US) presentations were given to animals engaged in a previously acquired food-motivated lever-pressing task. Conditioned suppression of lever-pressing was the behavioural measure of conditioning. Twenty-four hours after the last conditioning session, the dentate gyrus and areas CA3 and CA1 of the hippocampus were removed bilaterally from conditioned and pseudoconditioned animals, and slices cut and stored in liquid nitrogen for subsequent analysis. Crude synaptosomal pellets were prepared to investigate: (i) potassium-stimulated release of preloaded [3H]glutamate and [14C]aspartate in the presence and absence of extracellular Ca2+; (ii) [3H]inositol labelling of phosphoinositides and inositol phosphates; and (iii) [14C]arachidonic acid labelling of 1,2-diacylglycerol (1,2-DG). Potassium-stimulated, Ca2+-dependent release of [3H]glutamate in synaptosomes prepared from the dentate gyrus and area CA3 was significantly greater in conditioned animals than in pseudoconditioned animals. In area CA1, K+-stimulated, Ca2+-dependent release of [14C]aspartate was significantly increased in conditioned animals. These results confirm in synaptosomes, and extend to a period of 24 h our previous report of an increased release of transmitter in the dentate gyrus and hippocampus associated with classical conditioning. In parallel with the increased release of amino acids, learning was associated with a significant increase in labelling of phosphoinositides and inositol phosphates by [3H]inositol and a significant increase in labelling of 1,2-DG by [14C]arachidonic acid in the three hippocampal areas examined. It is suggested that a long-lasting presynaptic activation of inositol lipid metabolism may contribute to the learning-dependent increase in the capacity of hippocampal terminals to release transmitter and hence to the maintenance of a neurochemical trace which may, at least in part, underlie lasting changes in synaptic function built up during associative learning.

Time-related Changes in Basal Phosphoinositide Turnover after Induction of Long-term Potentiation in the Dentate Gyrus are Blocked by Commissural Stimulation.

We have examined basal phosphoinositide turnover in synaptosomes obtained from the dentate gyrus of anaesthetized rats in which long-term potentiation was induced unilaterally in perforant path-granule cell synapses. Relative to the unpotentiated side, [3H]myo-inositol labelling of inositol phosphates was significantly enhanced 45 min and 3 h after induction of long-term potentiation, but reduced after 2.5 min. Similarly, [14C]arachidonic acid labelling of 1,2-diacylglycerol was increased 45 min and 3 h after induction of long-term potentiation, but reduced after 2.5 min. In a second series of experiments, induction of long-term potentiation was blocked by stimulation of the commissural projection to granule cells. In synaptosomes prepared from this tissue, there was no difference in phosphoinositide turnover between tetanized and control sides at any of the three post-tetanic intervals. We conclude that in the dentate gyrus, long-term potentiation is associated with an increase in phosphoinositide turnover which is established between 2.5 min and 45 min post-tetanus and which persists for at least 3 h.

Analysis of the mechanisms underlying the age-related impairment in long-term potentiation in the rat.

The process of ageing is common to all organisms but despite its universality, understanding of the cellular and molecular changes which accompany ageing is poor. With the increase in the ageing population, the need for systematic study of the process becomes a priority but the development of a systematic approach to the problem requires the development of a testable theory of ageing. While several theories of ageing have been proposed /336/, between which there is at least some overlap, no generally-accepted theory has been identified. In relation to the brain, four interrelated theories of ageing have received significant attention; these are (1) the membrane hypothesis, (2) the free radical hypothesis, (3) the calcium hypothesis and (4) the glucocorticoid theory. The overlap between these hypotheses is such that a causal relationship between them is very likely with age-related changes in membrane composition triggered by changes in free radical production, while changes in membrane composition are likely to account for changes in membrane function including age-related changes in calcium homeostasis. The glucocorticoid theory could equally well be termed the stress hypothesis of ageing, which in the hippocampus at least, might trigger age-related changes in free radical production. This review is not intended to be a balanced appraisal of each theory but emphasizes aspects of the membrane hypothesis of ageing and discusses some relevant aspects of the other hypotheses. Attention is focussed on analysis of the biochemical changes which might underlie age-related changes in hippocampal function, particularly maintenance of long-term potentiation (LTP), and these changes are discussed in the context of the four hypotheses of ageing.