Moreover, there is considerable controversy concerning the effects of thiazolidinedione PPARγ agonists on the two major diseases of the gastrointestinal track: colorectal cancer and inflammatory bowel disease. We will undertake to review both historical and recently published data with a view toward summarizing what is presently known about the roles of PPARγ in both physiological and pathological processes in the gastrointestinal epithelium.Although the highest levels of PPAR From the standpointof pathology, PPARγ has been implicated in both transformation andinflammation in the gut. However, there are conflicting data concerning theefficacy and even the safety of PPARγ agonists in clinical management ofgastrointestinal cancer and inflammation. We will undertake in this review tosummarize both historical and recent data that relate to three questions: Whatis the physiological role of PPARγ in gastrointestinal epithelial cells? Is PPARγ a colon cancer suppressor? And what is therole of PPARγ in inflammation?Peroxisome proliferator-activated receptor-gamma PPARγ, NRC3 has beagonists , the rolal cells . High-leγ is expressed in epithelial cells of both thelarge and small intestines [γ at more or less uniform abundance from theduodenum to the caecum [γ are expressed in the proximal colon, withsomewhat lower levels observed in the mid and distal colon [γ in the body are observed in highlydifferentiated lumenal epithelial cells of the proximal colon. It has also beenobserved that PPARγ is induced when Caco2 cells undergodifferentiation in culture [γ is associated with differentiation of colonicepithelial cells. However, a careful analysis of PPARγ expression in the colon suggests that thisconclusion is correct only within a limited context [γ than lumenal epithelial cells from the samecrypts. Furthermore, PPARγ expression in the distal colon is more or lessuniform throughout the crypts, with slightly higher levels of expressionobserved in the less differentiated basal crypt cells, rather than the morehighly differentiated lumenal epithelial cells [γ is associated with differentiation of colonicepithelial cells. It is, however, the case that all colonic epithelial cellsexpress significant levels of PPARγ. This is an important point, since some of theeffects of PPARγ are manifest in the transit amplifying cellsthat support renewal of the colonic epithelium. Whether PPARγ is expressed in colonic stem cells remains anopen question.PPARtestines , 4. Relae caecum . Very hial colon , 6. The  context . Basal eal cells , 7. Thusγ expression is observed in the epithelium ofthe small intestine. Transit amplifying cells within the crypts of Lieberkühnexpress little or no PPARγ [γ is associated with differentiated function,and it has been reported that PPARγ collaborates with Hic5 to promotedifferentiation of embryonic small intestinal epithelial cells [Asignificantly different pattern of PPARno PPARγ , 9. Instal cells .γ in differentiated function of gut epithelialcells have been hindered by the lack of good cellular models to study themechanisms of action of PPARγ in culture. There are no nontransformedcolonic epithelial cell lines, and primary colonic epithelial cells have provedto be difficult, if not impossible, to maintain in culture for even a fewhours. There are several nontransformed epithelial cell lines derived from therat embryonic small bowel , but these cells are derived from proliferative crypt cells and, likethe transit amplifying cells from which they were derived, these cells expressno PPARγ. It has been possible to engineer RIE1 cellsto express PPARγ, thereby modeling the transition that occursat the crypt/villus junction [γ in such cells results in irreversiblewithdrawal form the cell cycle, promotes motility, and reduces cellularadhesion.Effortsto understand the role of PPARjunction , 11. Actγ targets in such cells fall into four major functionalcohorts [γ in other tissues [γ target genes was ontologically linked tosignal transduction. The data suggest that there is extensive crosstalk betweenPPARγ and other signaling pathways within intestinalepithelial cells. A third cohort of genes was linked to proliferation,consistent with the observation that activation of PPARγ within these cells results in inhibition ofculture growth and irreversible withdrawal from the cell cycle. Somewhatsurprisingly, the forth functional cohort of PPARγ target genes was ontologically linked tocellular motility and adhesion. Such processes have not been generally thoughtof as linked to activation of nuclear receptors. Nevertheless, activation ofPPARγ in intestinal epithelial cells potentlyinduces cellular motility, through a mechanism that involves Rho family GTPasesand MAPK activation [γ regulates intestinal epithelial cell motilityprovides a very important clue into the potential physiological role of thisreceptor in the gastrointestinal epithelium.Genomicprofiling indicates that PPARcohorts . The lar tissues –16. A setivation . Renewalγtargets in colonic epithelial cells isolated from thiazolidinedione-treatedmice indicates that, in general, similar processes are regulated in epithelialcells from the colon and small intestine [γ activation to metabolism, signal transduction,and migration/motility. In contrast to the results obtained with intestinalepithelial cells in culture, no proliferative cohort of PPARγ target genes was identified in colonicepithelial cells isolated from thiazolidinedione-treated mice. This result wasunanticipated since such drugs significantly inhibit BrdU incorporation intoboth proximal and distal colonic epithelial cells. Failure to detect a cohortof proliferation-related PPARγ target genes in vivo is probably attributable to the fact that only a smallsubpopulation of cells is involved in proliferation in the colonic epithelium,such that the contribution of RNA from such cells is diluted by the much largerpostmitotic population. Genomic studies of this sort are, therefore, useful foranalysis of PPARγ effect on the differentiated, postmitoticepithelial cells; but such studies are unlikely to reveal much informationabout the effects of PPARγ on proliferative colonic epithelial cells,which are presumably the targets for transformation.Genomicanalysis of PPARntestine . Major oγ expression and distribution are very differentin the proximal and distal colonic epithelium, implying that this receptor mayhave different functions in the proximal and distal colon. Genomic analysisindicates that the majority of PPARγ target genes are expressed in both proximaland distal epithelial cells [γ in these tissues. However, a subset of PPARγ target genes is restricted to the proximalcolon, and a second subset is expressed predominantly in the distal colon.Intriguingly, the proximal PPARγ target genes are all induced bythiazolidinediones, whereas the distal target genes are all repressed. Thesignificance of this observation is unknown at this time. However, theobservation that PPARγ represses genes that are differentiallyexpressed in the distal colon is consistent with the hypothesis that PPARγ may suppress differentiated function in thattissue. This hypothesis tends to contradict a large number of observations tothe effect that PPARγ promotes differentiated function, andadditional studies are needed to confirm this unanticipated observation.PPARal cells , suggestγ in nontransformed gut epithelial cellshas significantly impaired our ability to understand the role of this receptorin gut physiology. Nevertheless, genomic analyses of genetically engineeredintestinal epithelial cells and colonic epithelial cells isolated fromthiazolidinedione-treated mice have provided important clues. The data suggestthat PPARγ is a potent metabolic regulator in gutepithelial cells. This suggestion is obviously consistent with what we knowabout PPARγ in mesenchymal cells. There is a strongsuggestion that PPARγ is involved in extensive crosstalk with othersignal transduction pathways, suggesting that this receptor plays an importantrole in integrating the physiological response to a wide variety ofextracellular signals in vivo.Finally, both genomic and cellular data indicate that PPARγ plays a very important role in regulatingcellular motility, which is one of the major differentiated functions ofintestinal epithelial cells. The challenge at this time is to put theinformation we have into a physiological context and to use these data tounderstand the role of PPARγ in malignant transformation ofgastrointestinal epithelial cells.Insummary, the lack of appropriate cellular models to study the mechanism ofaction of PPARγ as a colon cancer suppressor is based upon arelatively small number of observations, and not all of these observations areconsistent with each other. The most compelling data come from studies usingthe azoxymethane (AOM)-treated rodent model, which has been widely studied as amodel of sporadic colon carcinogenesis. Two initial studies in AOM-treated ratsused troglitazone, a relatively weak PPARγ agonist [γ suppressed AOM-mediated colon tumor formationin mice [γ inhibits some very early step intransformation of colonic epithelial cells in AOM-treated rodents.The evidence in support of PPAR agonist , 18. The agonist . This wain mice . It has in mice , 20. OveIncontrast to the data cited above, two reports have concluded thatthiazolidinediones induce caecal tumors in mice , 22. Theγ field has revolved around two high-profilepapers that reported increased colon tumor formation in APC+/Min mice treated with thiazolidinediones [+/Min micetreated with either troglitazone or rosiglitazone. The companion paper reporteda slight, but significant, increase in the number of colon tumors in APC+/Min mice treated with troglitazone. No increase in tumor size was observed in thesecond report, which was, therefore, not entirely consistent with the first.Moreover, two subsequent reports failed to reproduce the effect ofthiazolidinediones in APC+/Δ1309 or APC+/Min mice [γ had no affect on tumor number or size in APC+/Min mice [γ promotes tumor formation in mice that containactivating germ line mutations in the Wnt/APC/β-catenin pathway. It has recently been reportedthat biallelic knockout of PPARγ in colonic epithelial cells promotes tumorformation in APC+/Min mice [γ is, in fact, acting to suppress tumorformation in the Min mouse. On the whole, the evidenceno longer supports the hypothesis that activation of PPARγ promotes tumor formation in mice with germline APC mutations.Themajor controversy in the PPARnediones , 24. OneMin mice , 26. It Min mice , an obseMin mice , indicatγ agonists in pre-established tumors inAOM-treated mice [β-catenin signaling pathway [γ under these circumstances had a profoundinhibitory effect on tumor progression. This effect was most strikinglyapparent in the development of carcinoma insitu, which was detected in about 1/3 of the control tumors but wasnever observed in thiazolidinedione-treated tumors. Since formation ofcarcinoma in situ involves invasion of the surrounding stoma, it is tempting tospeculate that this observation indicates that PPARγ inhibits invasion in vivo, consistent with several reports that indicate thatinvasion by human colon cancer cell lines in culture is inhibited by PPARγ [γ in pre-established tumors had no significanteffect on BrdU incorporation, consistent with the lack of any significanteffect on tumor size. This observation is at variance with several reports thatthiazolidinediones inhibit proliferation of human colon cancer cell lines inculture and in xenografts [γ on established tumors may be due to inhibitionof tumor progression, rather than inhibition of tumor growth.Arecent report describes the effects of PPARted mice . Such tu pathway . Thiazol pathway . Howeverby PPARγ , 29. Notnografts . The sigγ suppresses colon carcinogenesis in mice. Theprimary effect appears to be inhibition of some early stage in transformation.The ability of PPARγ to block early stage transformation ispresumably due to some effect on normal colonic epithelial cells, whichemphasizes the importance of understanding the functions of this receptor inthe normal colonic epithelium. It is also clear that whereas PPARγ inhibits proliferation of normal intestinalepithelial cells, this response is attenuated early in transformation. Althoughthe antiproliferative effects of PPARγ appear to be lost in cells that have eithergerm line or somatic mutations in β-catenin signaling, the data indicate that thisreceptor still retains the ability to inhibit tumor progression, at least inAOM-induced tumors. Finally, we submit that there is very little solid evidencethat PPARγ promotes colon carcinogenesis under anypharmacologically relevant conditions.Onbalance, the data seem unambiguously clear in one respect: PPARγ as a suppressor of colon carcinogenesis inrodents is beyond question, but the evidence that PPARγ is a colon cancer suppressor in humans is notso compelling. Although an early report indicated that loss-of-functionmutations in PPARγ were common in colon cancer [γ is associated with colon cancer risk [γ2, which is expressed at relatively low levelsin the colonic epithelium [γ isoform, PPARγ1 which differs in N-terminal sequence fromPPARγ2. PPARγ expression is reduced in ulcerative colitis [γ expression increases the likelihood oftransformation in the human colon. However, the evidence in support of such aconclusion is not very strong. Finally, a small phase II trial in whichtroglitazone was used to treat patients with late stage metastatic colon cancerproduced no objective response [γ has little or no effect on growth ofestablished colon tumors in AOM-treated mice [Therole of PPARn cancer , this cln cancer . It has cer risk . Howeverithelium , 4, and  colitis  and acro colitis , two con colitis  to posturesponse . One migted mice .γ in humans comes from studies of establishedhuman colon cancer cell lines [γ. Such observations raise two importantquestions: why are some colon cancer cell lines resistant to PPARγ agonists? And to what extent are the effectsof thiazolidinediones dependent upon PPARγ expression and/or activity? Both of thesequestions are significant in terms of the use of thiazolidinediones aschemotherapeutic agents in colon cancer.Thebest evidence for a tumor suppressive role of PPARll lines –35, 43. γ agonists in colon cancer is as a preventiveagent. Clearly, PPARγ is preventive in mouse models of sporadiccolon cancer. However, the legal climate in USA at this time does not favor thedevelopment of chemopreventive drugs, particularly those that may havecardiovascular side effects [γ agonists. Patients with large numbers of ACFare at increased risk for development of subsequent colon cancers [γ agonists might result in a decrease in ACFsand a subsequent reduction in colon cancer risk. From a clinical standpoint, itis therefore important to ascertain if activation of PPARγ in humans suppresses ACF formation, as hasbeen observed in AOM-treated mice.Themost compelling case for pharmacological application of PPAR effects . These c cancers –47, and γ agonists have chemopreventive efficacy. Ourinitial attempts to collect data on time to formation of a second polyp inpatients who are taking thiazolidinediones indicates that there are great manyconfounding variables, which limit the power of such an analysis and mayaccount for the fact that few comprehensive anaylses of the effects of PPARγ agonists on colon cancerincidence have been reported [Finally,it is the case that several million individuals are taking thiazolidinedionesfor management of type II diabetes. It would seem that an epidemiologicalanalysis of these individuals might, once and for all, determine if PPARreported .γ suppresses some early stage in transformationof colonic epithelial cells. We submit that the balance of evidence isinconsistent with the hypothesis that PPARγ promotes colon carcinogenesis under anycircumstances. Although it is unlikely that PPARγ agonists will ever be developed aschemopreventive agents, it is plausible that we may, by studying the mechanismof action of PPARγ, elucidate downstream effectors that may bechemopreventive targets. Furthermore, it is possible that PPARγ agonists may have clinical applicability inprophylactic management of individuals who are at increased risk of coloncancer due to large numbers of ACFs.Insummary, we conclude that both genetic and pharmacological data indicate thatPPARγ has become a potential pharmacologicaltarget for treatment of inflammatory diseases of the colon, particularlyulcerative colitis (UC). PPARγ is highly expressed in both colonicepithelial cells and macrophages critical for innate immunity and guthomeostasis. Over the past decade, invitroand in vivo studies have defined an anti-inflammatory role formacrophage and epithelial PPARγ in regulating colonic inflammation.Studies utilizing in vitro cellculture models have established that thiazolidinedione PPARγ agonists can reduce NFκB activation and inflammatory geneexpression in colonic epithelial cells [γ has been confusing. Theanti-inflammatory effects of thiazolidinediones vary among studies due todifferences in cell models, the concentration, duration, and type ofthiazolidinedione  used, as well as the context ofinflammation studied . More itrations , 63. Desγ comes from landmark studies indicating that heterozygous PPARγ deficient mice were more susceptible toDSS- and TNBS-induced colitis [γ expression in certain cell types of thecolon plays an anti-inflammatory role. Recent studies elaborated on thesefindings by showing that mice deficient in PPARγ expression in epithelial cells andmacrophages displayed increased proinflammatory gene expression andsusceptibility to DSS colitis [γ expression in at least two cell types,epithelial and macrophage, can protect against at least one model of acutecolitis (DSS). Questions remain regarding the importance of macrophage andepithelial PPARγ in other models of acute and chroniccolitis. For example, what is the role of macrophage or epithelial PPARγ in a chronic colitis model driven byT-cells? Likewise, little is known about the role of dendritic and T-cellspecific expression of PPARγ in colitis, as PPARγ knockout animals currently do not existfor these cell types. Experimental models of colitis can be initiated bydistinct mechanisms and driven by infiltration of different cell types, bothepithelial and immune [γ in controlling gut inflammation.Furthermore, the importance of tissue specific PPARγ expression may depend largely on themodel of colitis examined, emphasizing the need to utilize multiple models toaccurately represent manifestations of human colitis. Collectively, knockoutanimals have confirmed a potential protective role of PPARγ in colitis but additional research isstill required to understand fully how tissue specific PPARγ expression influences differentmanifestations of colonic inflammation.Perhaps the strongest evidence for ananti-inflammatory role of PPAR colitis , 65. DSS colitis , 67. Thed immune –70. Giveγ expression protects against the developmentof colitis in several animal models, it seems logical that PPARγ might be a good target for treatment ofgut inflammation. In reality, the preventative and therapeutic efficacy oftargeting PPARγ with thiazolidinediones for treatmentof colitis is debatable. It remains unclear what phase, if any, would be bestfor targeting PPARγ with thiazolidinediones for treatmentof colitis: during the initiation, low grade, moderate, high grade, orremission phases of colitis. Our knowledge is largely based on animal studiesin which acute preventive doses of thiazolidinediones were administered beforethe initiation of colitis. When given acutely (0–3 days) beforeinducing stimuli  [−/− mice) [γ action is to suppress the initiation ofcolitis, and suggest that thiazolidinediones may be a useful chemopreventativeagent for the treatment of colitis. However, given their potentialcardiovascular side effects, it is unlikely that thiazolidinediones would beapproved as a chronic preventive agent for the management of gut inflammation.Moreover, the effectiveness of long term-preventative thiazolidinedionetreatment on suppressing gut inflammation remains to be fully established. Arecent report indicates that long-term treatment of mice with rosiglitazoneexacerbated DSS-induced colitis [Based on the evidence that (a) thiazolidinedionescan suppress inflammatory gene expression in vitro and (b) PPARor TNBS) , 71–73o/− mice) , thiazol colitis , raising−/− model of colitis provided no value [γ expression and/or activity thatcoincides with the level of inflammation. Indeed, PPARγ levels have been shown to bedownregulated in epithelial cells [γ in the colon [κB drives down PPARγ expression in response to lipopolysaccharide,thereby obviating the actions of PPARγand promoting an inflammatory state in macrophages [The alternative scenario is that thiazolidinedionesalone or in combination with anti-inflammatory/immunosuppressive drugs may beused to therapeutically target active inflammation. However, data from humanand animal studies regarding the usefulness of therapeutic doses of thiazolidinedionesin the treatment of colitis are inconsistent. When given after the initiationof DSS colitis, several studies found that thiazolidinediones had little or novalue in improving colitis symptoms , 65, 73.no value . Similarno value , howeverno value , 77, 78.no value . Howeveral cells  and macrrophages .γ expression and activation in epithelialand immune cell types in the control of colonic inflammation, it remainsunclear whether targeting PPARγ with thiazolidinediones will be aneffective strategy for treating gut inflammation. While animal models suggest afavorable role of thiazolidinediones in chemoprevention, the efficacy andsafety of long-term use of thiazolidinediones as preventative agents ormaintenance therapy in UC patients remains to be assessed. Likewise, the use ofthiazolidinediones as therapeutic agents for treatment of colitis iscontroversial. It remains unclear during what clinical phase of inflammation thiazolidinedioneswould be most effective for treating UC patients or which patients would bemost likely to benefit from treatment. Moreover, there is considerable concernregarding whether the adverse effects of thiazolidinediones would outweigh thepotential benefit for patients with UC. In general, the majority of studies arein agreement that thiazolidinediones may be better exploited for therapeutictreatment of mild-moderate active colitis rather than during severeinflammation. Our understanding of the preventative and therapeutic potentialof targeting PPARγ with thiazolidinediones is largelylimited by several factors, including the lack of (a) experimental usage ofdistinct models of colitis, (b) understanding of tissue specific roles of PPARγ in different models of colonicinflammation, (c) understanding of factors affecting thiazolidinedione efficacy, (d) understanding of the mechanism of the anti-inflammatoryactions of PPARγ, and (e) understanding of the adverse effects of short- and long-termuse of thiazolidinediones during inflammation. Additional animal and clinicalstudies should resolve these discrepancies and provide further insight into theappropriate use, preventative or therapeutic, of thiazolidinediones fortreatment of gut inflammation.Insummary, although the current data supports a role for PPAR


Elevated serum homocysteine is associated with an increased risk of cardiovascular disease (CVD). This may reflect a reduced systemic remethylation capacity, which would be expected to cause decreased genomic DNA methylation in peripheral blood leukocytes (PBL).P = 0·01). Global DNA methylation was significantly elevated in men with a history of CVD or its predisposing conditions at baseline (P = 0·03) but not in women (P = 0·53). Fifty-two subjects  who were negative for these CVD/predisposing conditions at baseline acquired one or more of these conditions by the time of their follow-up I interviews, which took place on average about 5·8 years post-enrollment. Global DNA methylation levels of the 22 incident cases in men were intermediate  relative to the 56 male subjects who remained free of CVD/predisposing conditions at follow-up  and the 51 male subjects with a diagnosis of CVD or predisposing conditions reported at baseline (highest AS 184) (P for trend = 0.0008) No such association was observed in women (P = 0.91). Baseline body mass index was positively associated with AS in both men and women (P = 0·007).We examined the association between prevalence of CVD  and its predisposing conditions  and PBL global genomic DNA methylation as represented by ALU and Satellite 2 (AS) repetitive element DNA methylation in 286 participants of the Singapore Chinese Health Study, a population-based prospective investigation of 63,257 men and women aged 45–74 years recruited during 1993–1998. Men exhibited significantly higher global DNA methylation [geometric mean ): 159 ] than women [133 ] (Our findings indicate that elevated, not decreased, PBL DNA methylation is positively associated with prevalence of CVD/predisposing conditions and obesity in Singapore Chinese. MTHFR) genotype as independent predictors of plasma homocysteine in Singapore Chinese Cardiovascular disease (CVD) is the leading cause of death in most countries The subjects were participants of the Singapore Chinese Health Study, a population-based prospective cohort study of Chinese men and women, aged 45–74 years at baseline. They belonged to the two major Chinese dialect groups in Singapore (Hokkien and Cantonese) and lived in government housing estates where 86% of all residents in Singapore resided during the period of enrollment At recruitment, each participant completed an in-person interview using a structured questionnaire that requested information about demographic characteristics, height and weight, use of tobacco, usual physical activity, medical history, and family history of cancer. The questionnaire included a validated semi-quantitative food frequency section listing 165 food items commonly consumed in the study population, from which average daily intake of calories and roughly 100 nutrients and non-nutritive ingredients per subject were computed using the Singapore Food Composition Table Between 1994 and 1998, a random 3% sample of cohort participants was recontacted for donation of blood and urine specimens. The 286 subjects in the current study represented the first accrued participants of this biospecimen subcohort sample)/((METHYLATED GENE/CONTROL REACTION)SssI-Reference)*100, in which “METHYLATED GENE” refers the methylation measurement at a particular locus such as ALU or SAT2 and “CONTROL REACTION” refers to the methylation-independent measurement using the Alu-based control reaction. The composite methylation measurements of ALU (ALU-M2) and SAT2 (SAT2-M1) were used for MethyLight-based estimates of genomic 5-methylcytosine content DNA was extracted from peripheral blood leukocytes collected from the 286  study subjects. Sodium bisulfite conversion of genomic DNA was conducted. The samples used in our study were stored at −30°C to minimize DNA degradation and (methyl)cytosine deamination. Methylation levels of repetitive elements were determined using MethyLight technology as described previously P values reported in this paper. Since length of sample storage may have an influence on our data, we repeated all analyses with length of sample storage (months) as an additional covariate in the GLM models. No material changes to the results are noted. Since serum homocysteine may be a confounder in the examination of AS in relation to BMI and CVD, it was entered as a covariate to the appropriate GLM models in addition to age, gender and BMI. All statistical computations were conducted using the statistical program SAS, version 6·12 . All P values reported are two-tailed and statistical significance was defined as P<0·05.Global methylation as assessed by the AS index showed a markedly skewed distribution toward high values. This deviation from normality was largely corrected via a logarithmic transformation of the actual values of AS. Thus, geometric means (as opposed to arithmetic means) of AS and their corresponding 95% confidence intervals were presented. We decided on a more conservative, non-parametric approach to formal statistical testings of the data. Therefore, we employed the generalized linear modeling (GLM) methods on ranked values of the AS index, as opposed to its logarithmically transformed values, in calculating all P = 0·02; Men had significantly higher AS values within each age group (P = 0·045) significantly higher mean AS  compared to those without such a history  (P = 0·055) relative to those without such histories . This relationship between prevalence of myocardial infarction, stroke or their predisposing medical conditions and global DNA methylation was principally observed in men (P = 0·03). To further delineate whether this gender effect and CVD status were correlated, we analyzed the AS index by gender stratified by CVD status. The gender effect was mostly coming from the subgroup of subjects with a history of CVD at baseline  and/or stroke at baseline showed a borderline ] . Subject-null aortic DNA compared with controls MTHFR and TYMS, in relation to AS levels. Genotypes of MTHFR (P = 0.03) but not TYMS (P = 0.24) were significantly associated with AS levels ] showed elevated AS compared to those with BMI below 24 kg/m2 .The constellation of positive associations between global PBL DNA methylation and prevalence of CVD or its risk factors suggests a potential relationship between metabolic syndrome S levels . MeanwhiP = 0.008) but not female (P = 0.91) subjects exhibited an association between AS levels and status of CVD/predisposing conditions at baseline and at followup/death (2 and above) and a physician-diagnosed history of myocardial infarction, stroke, hypertension and/or diabetes . The mean time interval between the two interviews (baseline and follow-up I) was 5·8 years  among the 52,325 participants of the Follow-up I Survey. We identified 47 subjects who were free of a history of myocardial infarction, stroke, hypertension, and/or diabetes at recruitment but had developed at least one of these conditions during follow-up. In addition, we identified from death certificate reviews that five subjects had died of one of these listed conditions as of December 31, 2004. Meanwhile, 133 subjects were still negative for CVD or its predisposing conditions by the time of their follow-up interviews. Male , who found that vascular disease patients with elevated plasma tHcy and AdoHcy concentrations and low plasma AdoMet/AdoHcy ratios had lower levels of genomic DNA methylation r = 0.47; r = 0.54, respectively). Moreover, it has been observed that imprinted gene H19 is hypermethylated, not hypomethylated, in brain and aorta of hyperhomocysteinemic mice, although the effect of hyperhomocysteinemia on H19 DMD methylation was tissue-specific in these mice We present here the results of a population-based prospective cohort study of risk factors for CVD. This study was initiated to ascertain whether PBL DNA methylation could serve as a stable measure of systemic methyl group supply, analogous to the use of glycated forms of hemoglobin to provide measures of long-term mean blood glucose levels. However, we did not observe the anticipated correlations between PBL DNA methylation and plasma folate and homocysteine, dietary folate and the B vitamins, and folate metabolizing genotypes such as 2Although we did not find a link between lipid metabolites In summary, this is the first report of an association between global DNA methylation assessed by ALU/SAT2 methylation Table S1Geometric means (95% CI) of AS index by serum homocysteine at baseline and gender, stratified by CVD status.(0.05 MB DOC)Click here for additional data file.Table S2Quartile cut-points of plasma homocysteine, B vitamins and cholesterols.(0.04 MB DOC)Click here for additional data file.


Escherichia coli guaB promoter (PguaB) regulates transcription of two genes, guaB and guaA, that are required for the synthesis of guanosine 5′-monophosphate (GMP), a precursor for the synthesis of guanine nucleoside triphosphates. Transcription from PguaB increases as a function of increasing cellular growth rate, and this is referred to as growth rate-dependent control (GRDC). Here we investigated the role of the factor for inversion stimulation (FIS) in the regulation of this promoter. The results showed that there are three binding sites for FIS centred near positions −11, +8 and +29 relative to the guaB transcription start site. Binding of FIS to these sites results in repression of PguaB in vitro but not in vivo. Deletion of the fis gene results in increased PguaB activity in vivo, but GRDC of PguaB is maintained.The  Escherichia coli guaB promoter (PguaB) regulates transcription of two genes, guaB and guaA, which together constitute the guaBA operon. The guaB and guaA genes encode inosine 5′-monophosphate (IMP) dehydrogenase and guanosine 5′-monophosphate (GMP) synthetase, respectively, and are required for the biosynthesis of GMP from the common purine nucleotide precursor, IMP (guaB is regulated by the cAMP receptor protein (CRP) and a putative CRP binding site is centred near position −117.5 relative to the guaB transcription start site  α subunit (αCTD)  regulates transcription by binding to highly degenerate 15 bp DNA sequences , extending from positions −253 to +36 relative to the guaB transcription start site [PguaB (−253 to +36)] (guaB (−253 to +10) promoter , which contains the same upstream end point as the full-length promoter but has a downstream end point at +10, and the PguaB (−69 to +36) promoter , which has the same downstream end point as the full-length promoter but has an upstream end point at position −69 relative to the guaB transcription start site. Fusions were carried on λ prophage and were constructed using a system based on λimm21  and PguaB (−133 to +36), respectively. pUCG-253 is a derivative of pUC19 containing PguaB (−253 to +36). The plasmid pRLG770 has been described previously (guaB (−253 to +36), PguaB (−133 to +36), PguaB (−59 to +36) and PguaB (−37 to +36) were constructed previously (guaB (−133 to +21), PguaB (−133 to +1) and PguaB (−253 to +10), respectively.All strains were derivatives of the to +36)] , the Pgueviously . pRLG770EcoRI–XhoI DNA fragment in pBSG-253 was purified following electrophoresis in a 6 % acrylamide gel (XhoI) end with [γ-32P]ATP  and subsequently purified according to a published procedure (EcoRI–XhoI fragment in pBSG-133 was labelled similarly at the upstream end. Labelled DNA fragment (4 nM) was incubated at room temperature for 30 min in a volume of 20 μl containing 20 mM HEPES (pH 8.0), 5 mM MgCl2, 50 mM potassium glutamate, 1 mM DTT, 20 μg ml−1 sonicated calf thymus DNA  and 5 % (v/v) glycerol, in the absence or presence of purified FIS protein. Purified FIS was a generous gift from T. Gaal and R. L. Gourse (University of Wisconsin–Madison). DNase I footprinting and DNA fragment separation were performed exactly as described previously (The rocedure . The Ecoeviously . FootpriguaB (−253 to +36) was amplified by PCR from pUCG-253, using primers pUC19(for) (5′-ACGTTGTAAAACGACGGCCAG-3′) and pUC19(rev) (5′-GCGCGGATCCATGACCATGATTACGCCAAGCT-3′). A DNA fragment containing the rrnB P1 promoter with FIS site I (positions −87 to +50 relative to the rrnB P1 transcription start site) and an rrnB P1 promoter derivative that did not contain a FIS site  were PCR amplified from plasmids pRLG1616 and pRLG4720, respectively, using a forward primer with the sequence 5′-GTATCACGAGGCCCT-3′ and reverse primer RLG1620 (5′-GCGCTACGGCGTTTCACTTC-3′), both of which are vector-specific  mmol−1, MP Biomedicals] and DNA polymerase I Klenow fragment. Labelled DNA  was incubated at room temperature for 30 min in a volume of 10 μl containing 20 mM HEPES (pH 8.0), 5 mM MgCl2, 50 mM potassium glutamate, 1 mM DTT, 10 % (v/v) glycerol and 20 μg ml−1 sonicated calf thymus DNA , in the absence or presence of different concentrations of FIS. Samples were loaded (under tension) onto a 6 % acrylamide gel (37.5 : 1 acrylamide : bis acrylamide) containing 7.5 % (v/v) glycerol while running at ∼15 V cm−1, and gels were run for ∼1 h at 4 °C. Radiolabelled DNA was visualized using a FujiFilm FLA3000 phosphorimager.A DNA fragment containing PguaB fragments , and reactions were allowed to proceed for 20 min.Multiple-round transcription reactions were performed as described previously, using supercoiled pRLG770 derivatives containing Pragments . As a coguaB-lacZ transcriptional fusion were employed in the measurement of promoter activity in vivo. Cells were inoculated from dense starter cultures into media that supported different cellular growth rates, as described previously (A/g)(a/t)(a/t)(T/A)(t/a)(t/a)(T/c)(g/a)nnC-3′] contains five highly conserved positions (underlined) that are the most significant for the recruitment of FIS  or the rrnB P1 fragment by EMSA was 50 nM. Increasing the FIS concentration to 300 nM resulted in the formation of three different complexes between FIS and PguaB ] was radiolabelled at the downstream end, and DNase I footprinting was performed in the presence or absence of purified FIS. The results show that increasing the concentration of FIS up to 500 nM resulted in increased protection at two sites centred near positions −11 and +8  ] was radiolabelled at the upstream end for DNase I footprinting. The results show that, in addition to the protection observed at FIS sites 1 and 2, a third site (FIS site 3) centred near position +29 was also bound by FIS. FIS sites 2 and 3 were identified by comparison with the consensus as being more likely to recruit FIS   and shorter derivatives  (end points as indicated). Addition of FIS to the transcription reaction resulted in ∼8–10-fold repression of transcription from PguaB (−253 to +36). Under the same conditions, there was no repressive effect of FIS on transcription from the rrnB P1 promoter ] did not lead to any significant change in the degree of repression afforded by FIS, confirming that putative FIS sites I–IV do not play a role in the regulation of PguaB by FIS. Deletion of the PguaB UP element [PguaB (−37 to +36)] gave rise to an undetectable level of transcripts from PguaB in the presence of FIS, which meant that the fold repression afforded by FIS could not be determined. Deletion of FIS site 3 [i.e. PguaB (−133 to +21)] led to decreased repression by FIS (approximately sixfold repression), and deletion of both FIS site 2 and FIS site 3 [i.e. PguaB (−133 to +1)] gave rise to an approximately twofold repression by FIS  retains FIS site 1 and is still subject to some degree of repression by FIS, these results indicate that FIS sites 1–3 each contribute to repression of PguaB, and that FIS site 1 can function independently of the other FIS sites. A PguaB derivative harbouring a deletion of FIS site 3 and deletion of the downstream six bases of FIS site 2 [i.e. PguaB (−253 to +10)] was subject to an approximately fourfold repression by FIS, indicating that FIS site 2 had not been completely inactivated  was measured in a wild-type strain background and in a strain that harboured a deletion in the fis gene. To determine whether FIS site 3 is important for GRDC of PguaB, activity of PguaB (−253 to +10) was also analysed. In exponentially growing wild-type E. coli cells, the activity of PguaB (−253 to +36) increased approximately twofold with every doubling of the growth rate as, shown previously  was higher than in the wild-type at all growth rates and was more pronounced at faster growth rates (i.e. an approximately twofold increase in activity was observed at the fastest growth rate)  activity occurred for every doubling of the growth rate in comparison to a 1.84-fold increase for PguaB (−253 to +36)  or that FIS sites 1–3 may not contribute to repression of PguaB in vivo under the conditions employed, and therefore the observed FIS-dependent repression is indirect.GRDC of PguaB varies with the growth phase, transcription from a PguaB derivative with an upstream end point of −69 that contained all three experimentally determined FIS sites [i.e. PguaB (−69 to +36)] was measured at different stages of growth in a wild-type strain, and in a strain that harboured a deletion in the fis gene. This promoter derivative was chosen as it lacks the putative CRP site centred near position −117.5 . The increase in activity in a fis strain over the corresponding part of the growth curve was less marked (i.e. there was a ∼16 % increase in promoter activity). Upon entry into stationary phase, there was a gradual decrease in the promoter activity in both strain backgrounds , are protected by FIS in DNase I footprinting experiments. One of the other two candidate FIS sites overlaps the UP element, and the remaining site is located at a distance upstream of the core promoter elements (centred at −213). Neither of these sites recruits FIS as judged by DNase I footprinting. Putative FIS sites I–IV also do not bind FIS. Interestingly, we show that a site centred at position −11, which was not identified as a likely FIS site, also recruits FIS. This suggests that although bioinformatic analyses are useful when searching for sites that are bound by FIS, they may not be useful in identifying some FIS binding sites that exhibit a weak match to the consensus.A previous study identified four adjacent putative FIS binding sites (FIS sites I–IV) located upstream of the UP element at P for FIS . By empltyrT promoter, FIS binding to sites II and III  cooperatively affects the binding of FIS to site I centred at −71 bp upstream of the transcription start site . Interestingly, at PguaB, FIS sites 2 and 3 are positioned 22 bp apart (centre to centre) which will also place them on the same face of the DNA helix and a similar distance apart as FIS sites I and II at the tyrT promoter. However, the centres of FIS sites 1 and 2 at PguaB are 18 bp apart, making it unlikely that they are located on the same face of the DNA. Therefore, it is possible that FIS binds cooperatively to sites 2 and 3, but it appears less likely that occupancy of sites 2 and/or 3 stimulates binding of FIS to site 1.At the guaB ∼8–10-fold in vitro. Deletion of FIS site 3 results in partial relief of repression, and deletion of FIS sites 2 and 3 together further relieves repression in vitro. The residual FIS-mediated repression of the guaB promoter fragment containing the +1 downstream end point is likely to occur through interactions with site 1, and would suggest that binding of to site 1 does not require cooperative interactions with FIS dimers bound to adjacent sites. The binding of FIS to site 1 is likely to sterically hinder the recruitment of RNAP to PguaB, as observed at the crp1 promoter .Consistent with the location of functional FIS sites, we show that FIS represses transcription from PguaB in vitro, evidence for direct repression by FIS in vivo was not obtained . This is consistent with the results of a chromatin immunoprecipitation (ChIP)-chip analysis carried out under similar conditions, in which FIS binding at PguaB was not detected ) or absence [PguaB (−69 to +36)] of the putative CRP site centred at −117.5. This rules out the possibility that the effect of deleting fis on guaB promoter activity is mediated by changes in CRP abundance [FIS has been shown to modulate transcription of crp (guaB in a fis background occurs as a result of altered regulation of PguaB by a transcription factor other than CRP, for example H-NS or HU (guaB repression that occurs upon deleting FIS sites 2 and 3 is masked in vivo through an alternative compensatory regulatory mechanism. A less likely explanation, in view of the poor match to the consensus FIS binding site, is that FIS binding to site 1 mediates full FIS-mediated repression in vivo.Although our results demonstrate that FIS represses transcription from PguaB UP element, and sequences located further upstream, are required for GRDC of PguaB (guaB (guaB is subject to growth phase-dependent control. However, although levels of FIS protein are also subject to growth phase-dependent control, it does not appear to play an important role in growth phase-dependent control at PguaB. Thus, the physiological role of FIS at the guaB promoter remains to be elucidated.A previous study has shown that the Pof PguaB . Our res


Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1–43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1–43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity. Current research on synaptic transmission seeks to understand the complex regulation of individual synapses within neural networks. Especially the modification of synaptic connections as a crucial element of structural plasticity and long-term memory has been in the focus of intense research Besides these structural considerations, functional parameters of synapse populations such as spontaneous activity Interestingly, morphological features have been shown to be relevant for the modification of individual synaptic connections. For example, the release probability between neighbouring synapses is highly correlated in vivo measurements To extend the link between morphological observations and synaptic function, in this study the release characteristics of individual synapses were investigated with regard to their spatial arrangement. This was made possible by the fact that with fluorescence microscopy other than with electron microscopy the number of vesicles in a terminal can be assessed in a highly parallelized approach  All animals were handled in strict accordance with good animal practice as defined by the guidelines of the State of Bavaria, and all animal work was approved by the Kollegiales Leitungsgremium of the Franz-Penzoldt Zentrum, Erlangen (reference number TS-1/10).−1) cells were triturated mechanically and plated in MEM medium, supplemented with 10% fetal calf serum and 2% B27 Supplement . If required, neurons were transfected with synaptopHluorin under control of a synapsin promoter in vitro.Hippocampal neuronal cultures were prepared from one to four days old Wistar rats . Briefly, newborn rats were sacrificed by decapitation in accordance with the guidelines of the State of Bavaria. Hippocampi were removed from the brain and transferred into ice cold Hank's salt solution, and the dentate gyrus was cut away. After digestion with trypsin .Experiments were conducted at room temperature on a Nikon TI-Eclipse inverted microscope equipped with a 60×, 1.2 NA water immersion objective and Perfect Focus System™. Fluorescent dyes were excited by a Nikon Intensilight C-HGFI through excitation filters centred at 482 nm and 640 nm using dichroic longpass mirrors (cut-off wavelength 500 nm and 650 nm), respectively. The emitted light passed emission band-pass filters ranging from 500 nm–550 nm and 650 nm–700 nm, respectively  and was projected onto a cooled EM-CCD camera ; 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione  and 50 µM D-amino-5-phosphonovaleric acid  were added to prevent recurrent activity.Cover slips were placed into a perfusion chamber (volume = 500 µl) containing extracellular medium containing (in mM): 144 NaCl, 2.5 KCl, 2.5 CaClRecycled synaptic vesicles were labelled with FM 1–43 . To stain the total recycling pool nerve terminals were loaded with 1200 action potentials (APs) at 40 Hz For the anti-synaptotagmin-1-cypHer experiments cultured dispersed hippocampal neurons were incubated for 0.5 h to 1 h with 0.6 µg of CypHer™5E labelled anti-synaptotagmin1 antibody (αSyt1-cypHer)  in extracellular medium to stain synaptic boutons. To identify active synapses, cells were electrically stimulated with 1200 AP at 40 Hz. Images were recorded with 2000 ms exposure time at a framerate of 0.33 Hz.In the synaptopHluorin experiments boutons were stimulated with 600 APs at 30 Hz. Images were recorded with 100 ms exposure time at a framerate of 0.5 Hz. For all experiments camera binning was 2×2 and resulting image stacks were converted into tagged image file format (TIFF).tot the mean of five values before the onset of the stimulus was subtracted from the mean of five values after the total destain period. The absolute fluorescence decrease ΔF (FM 1–43 and anti-synaptotagmin-1-cypHer) or increase (synaptopHluorin) was calculated as the difference of the mean of three (anti-synaptotagmin-1-cypHer) or five (FM 1–43 and synaptopHluorin) values before the first stimulation and the mean of three or five values after the end of the first stimulus with 600 APs or 1200 APs, respectively. The number of neighbours was determined by counting the detected spots in a circular region with a diameter of 30 pixels (pixel size sp = 267 nm at binning 2×2), which correspond to a diameter of 8 µm, around each spot . The mean background determined from the intensity histogram of recorded image stacks was subtracted spot see  or approWhen a continuous distribution is described, it might be useful to introduce the terms “high values”, “low values” and “average values” in order to emphasize differences in their individual properties. Accordingly, we discriminated between “synapses with large recycling pool sizes” and “synapses with small recycling pool sizes” that we defined as synapses with the highest or lowest 10% of recycling vesicle numbers as well as “synapses with average recycling pool sizes” (median +/− 5%) respectively.ρ between two parameters was calculated. To clarify the statistical results obtained from Wilcoxon rank sum test and Spearman's rank correlation coefficient ρ, we used the effect size dd between the first and the last bin by: Statistical analysis was performed by MATLAB . Differences in the mean or median of data points e.g. see  were tes2) around each detected synapse (see 2 and 100 µm2 (data not shown). In analogy with the positively skewed distribution of the recycling pool size and release probabilities of synapses Complete staining of the recycling vesicle pool using styryl dye FM 1–43 apse see . The resNext we wanted to clarify if synapses localized in clusters exhibited common or different synaptic features compared to boutons surrounded by low numbers of synaptic neighbours. For better inter-experimental comparison we replaced arbitrary fluorescent units with the number of synaptic vesicles in the recycling pool. This was achieved by determining the fluorescence of a single vesicle by performing subtotal staining experiments with 20 AP at 10 Hz  as described When analyzing the recycling pool size and the number of neighbours of each synapse, we observed that synapses located within synapse clusters contained significantly more recycling vesicles . In prinρ = 0.97±0.03, p<0.01) and was limited by the maximal recycling pool size, and thus saturation was observed with an increasing number of neighbours (see d of 4.76) d = 2.30; Spearman's ρ = 0.95, p<0.001). We found that synapse clusters consisted of synapses with large recycling pools, which is not necessarily identical to the finding that synapses with large recycling pools are surrounded by a high number of neighbours.A considerable variance in the maximal number of neighbours as well as the recycling pool size was found in each experiment, which is likely due to the individual outgrowth of neurons on each coverslip. The relation between recycling pool size and number of neighbours, however, was conserved in all individual experiments . The mean value of the recycling pool size (137 vesicles) was also similar to previously published data (127 vesicles) in a typical hippocampal tissue culture d = 2.49; Spearman's ρ = 0.51, p<0.001, p is continuous and the histogram was skewed positively with a median of 0.19. A fit with Γ density function We first tested if the positively skewed recycling pool size distribution In order to exclude that synapse clusters of vesicle rich synapses are a phenomenon of styryl-dye labelling we tested sets of experiments in which we had used an antibody based functional labelling system as well as overexpression experiments for their compatibility with the results from FM-dye labelling.For live-cell antibody labelling of functional terminals we used a monoclonal antibody directed against the intra-vesicular domain of synaptotagmin-1. Synaptotagmin-1 becomes accessible on the cell surface after synaptic vesicle exocytosis and can be labelled with an antibody, which is then internalized when the vesicles are retrieved We found that synaptic terminals marked by αSyt1-CypHer were heterogeneously distributed in hippocampal neuronal cultures . Here thIn an overexpression approach we chose to transfect hippocampal neurons with synaptopHluorin (spH) which also can be used to define functional synaptic boutons The release characteristics of individual synapses provide the basis for network phenomena such as long-term memory formation and learning. The regulation of the individual synapses parameters has therefore been investigated intensively. For example, synaptic release probability and synapse size increased after complete block of fast voltage-gated sodium channels with tetrodotoxin Besides these functional parameters, activity-dependent changes of the spatial distribution of synapses within networks were also analysed. The number of functional excitatory synapses in the CA3 area of the developing hippocampus was found to be increased after blockade of spontaneous network activity These results show the diverse reactions of neural networks following stimulation and non-stimulation. In either case, the integrated individual synaptic changes in groups of functionally related neurons result in network plasticity. This network plasticity implies synaptic rewiring and synaptic weight changes in vivo recordings. Even though these studies did not specifically focus on synapse distribution, beautiful examples can be found in their figures: the mere visual impression is sufficient to spot a heterogeneity in acute hippocampal slice preparations . B, Correlation of exocytosed quanta with recycling pool size during stimulation with 600 APs at 30 Hz and distribution of recycling pool size for this measurement series . The mean value of the recycling pool size (137 vesicles) is indicated by the red dotted line. C, Distribution of fused vesicles and release probabilities following stimulation of 20 APs. The histogram is well fitted by Γ  with λ = 8.32 (dotted line). D, Correlation between synapse diameter and recycling pool size . The mean and standard deviation of the synapse diameter was 882.09 nm±249.92 nm. Data was grouped in bins and averaged. With increasing synapse diameter the recycling pool size increases . Error bars indicate standard deviations for synapse diameter and recycling pool size, respectively.(1.25 MB DOC)Click here for additional data file.Figure S2Fluorescence difference image of images before and after complete destain (twofold stimulation with 900 APs at 30 Hz). Synaptic vesicles were loaded with the styryl dye FM 1–43 . This difference-image was used to generate image in (0.97 MB DOC)Click here for additional data file.


A-type inhibition, but it remains unclear how desynchronization occurs. We hypothesize that slow inhibition plays a key role in desynchronizing projection neurons. Because synaptic noise is believed to be the dominant factor that limits neuronal reliability, we consider a computational model of the antennal lobe in which a population of oscillatory neurons interact through unreliable GABAA and GABAB inhibitory synapses. From theoretical analysis and extensive computer simulations, we show that transmission failures at slow GABAB synapses make the neural response unpredictable. Depending on the balance between GABAA and GABAB inputs, particular neurons may either synchronize or desynchronize. These findings suggest a wiring scheme that triggers stimulus-specific synchronized assemblies. Inhibitory connections are set by Hebbian learning and selectively activated by stimulus patterns to form a spiking associative memory whose storage capacity is comparable to that of classical binary-coded models. We conclude that fast inhibition acts in concert with slow inhibition to reformat the glomerular input into odor-specific synchronized neural assemblies.It has been proposed that synchronized neural assemblies in the antennal lobe of insects encode the identity of olfactory stimuli. In response to an odor, some projection neurons exhibit synchronous firing, phase-locked to the oscillations of the field potential, whereas others do not. Experimental data indicate that neural synchronization and field oscillations are induced by fast GABA A fundamental question in computational neuroscience is to understand how interactions between neurons underlie sensory coding and information storage. In the first relay of the insect olfactory system, odorant stimuli trigger synchronized activities in neuron populations. Synchronized assemblies may arise as a consequence of inhibitory coupling, because they are disrupted when inhibition is pharmacologically blocked. Using computational modelling, we studied the role of inhibitory, noisy interactions in producing stimulus-specific synchrony. So far, experimental data and modelling studies indicate that fast inhibition induces neural synchrony, but it remains unclear how desynchronization occurs. From theoretical analysis and computer simulations, we found that slow inhibition plays a key role in desynchronizing neurons. Depending on the balance between fast and slow inhibitory inputs, particular neurons may either synchronize or desynchronize. The complementary roles of the two synaptic time scales in the formation of neural assemblies suggest a wiring scheme that produces stimulus-specific inhibitory interactions and endows inhibitory sub-circuits with properties of binary memories. The primary olfactory center of insects, the antennal lobe (AL), is a network of excitatory projection neurons (PNs) interconnected via inhibitory local neurons (LNs). Such excitatory-inhibitory architectures are known to produce network oscillations A receptors. Neural synchronization and field potential oscillations are lost when GABAA inhibition is pharmacologically blocked by local injection of picrotoxin into the AL of the locust B receptors has been shown in the Drosophila AL B postsynaptic potentials present a much slower decay rate than the ones produced by GABAA inhibition. Interestingly, little evidence for oscillation and synchronization has been found in DrosophilaB inhibition is pharmacologically blocked It is now clear that PN synchrony results from the interplay with GABAergic LNs, and more specifically from ionotropic GABAA-type inhibition synchronizes whereas slow GABAB-type inhibition desynchronizes. Previous theoretical studies have shown that inhibitory networks synchronize  that displays type 2 excitability B-type inhibition. Evidence in favour of this hypothesis is provided by in vitro recordings in MCs B inhibition is pharmacologically blocked. Application of a GABAB antagonist in the Drosophila AL, however, shows just the opposite  and another with slow GABAB synapses (τGABA = 100 ms). Since chemical synapses are believed to be quite unreliable Pfailure is taken into account. Rasterplots in A and F ∼10 Hz with GABAB). As revealed by Equation A-1  of the PN population at the n-th cycle can be expressed as a simple linear recursive relationk〉 = N(1−Pfailure) and kσ2+NPfailure (1−Pfailure) are the mean and variance in the number k of inhibitory synaptic events received by the PNs at each cycle. Note that the mathematical analysis in Pfailure = 1. σ2(n) given by Equation 1 to the one obtained from simulations (see n = 3 cycles (300 ms with GABAB versus 150 ms with GABAA). This stable state does not depend on initial conditions  is attributable to variance in the amount of received inhibition kσ2. This variance comes from the presence of synaptic failures in our model (or from heterogeneous connectivity as we will see later). Because σ is proportional to the decay time constant of the inhibitory synapse, slow inhibition amplifies synaptic noise and leads to unpredictible firings. This finding can be noted in σ>10 ms with slow GABAB synapses for Pfailure≥0.5. In contrast, variable inhibition is especially tolerated with fast GABAA synapses since σ<5 ms for any value of Pfailure. Equation 2 also holds for extended AL models taking into account lateral excitation between PNs  of the PN population at the n-th cycle isλ is the time constant of the exponential release distribution  = σ2(n−1) = σasyn2 andσ2 is the spike time jitter obtained in the case of synchronous GABA release and is simply given by Equation 2. Asynchronous release accentuates temporal dispersion by adding the extra term λ2/(〈k〉−1). asyn2 to the one obtained from simulations for different values of λ2. For the simulations, we considered a network of N = 100 neurons coupled all-to-all with fast GABAA synapses . For λ = 0 ms (synchronous release), we have σasyn = σ = 1 ms . We observe that σasyn2 increases linearly with λ2, as predicted by Equation 4. From Equation 4, σasyn = 10 ms when λ = 70 ms, which is the same level of temporal dispersion as the one obtained with synchronous release and slow GABAB synapses . The loss of spike-timing precision is thus achieved with asynchronous release, despite fast GABAA synapses. Actually, the asynchronous synaptic events sum gradually over time so as to produce a resulting inhibition which decays with a time constant approximately equal to λ . With GABAA-type inhibition, the phase-locking probability decreases in a nonlinear way. More important is the presence of a plateau for Pfailure<0.7 which maintains a high probability of synchrony despite unreliable synapses. In contrast, the phase-locking probability decreases linearly with Pfailure for GABAB-type inhibition. Thus, a small amount of synaptic noise on GABAB synapses is sufficient to degrade synchronization in homogeneous networks.A classical approach for measuring synchrony is to consider that a spike occuring at time k of inhibition very different than the mean inhibition 〈k〉 fire far away from the population and, thus, are not synchronized. Synchronized neurons are those for which k≈〈k〉. In the following, we analytically quantify the conditional probability that particular neurons receiving k inhibitory synaptic events fire in synchrony with the neuronal population. A lower bound on this conditional probability was previously derived in In heterogeneous networks, the number of inhibitory inputs differs from one cell to another. Is the heterogeneity in connectivity sufficient to break synchrony in the absence of synaptic failure? As seen in σ2 is given by Equation 2. We have checked numerically that Equation 6 is a good candidate for the phase-locking probability. A and GABAB type inhibition, the phase-locking probability is an inverted U-function centered on the inhibition 〈k〉 received on average by the neurons. The existence of this inverted U-function does not depend on a specific choice for ε . Therefore, variable inhibition received on slow GABAB synapses leads to desynchronization. In contrast, variable inhibition is especially tolerated with fast GABAA synapses because the synchronization window is broader.Here A or GABAB on synchrony has been studied in isolation. We now consider a network of N = 100 neurons coupled with both fast and slow inhibition. A probability of synaptic failure (Pfailure = 0.5 and 0.0) is considered and two patterns of connectivity are taken into account: global  and heterogeneous (neurons are randomly connected with 0.5 probability). A and GABAB conductances ag and bg. In the absence of synaptic failure and network heterogeneity, the synchronized state , the line demarcating the synchronous state in ag/bg≈25 (σ<5 ms when ag/bg>25).In the previous sections, the effect of GABAre = 0.5  as well re = 0.0 . Thus, nA and GABAB inputs differs from one cell to another and thus some neurons exhibit synchronized activity while others do not. If neural assemblies do play a role in sensory representation, then the identities of the synchronized neurons would be reproducible across repeated trials and would be altered by changing the pattern of connections. To test this hypothesis, we performed repeated simulations with two different networks (A and B). A or GABAB blocked. The state of a PN at each oscillatory cycle is represented as a bit 1 or 0 depending on whether its firing is synchronized or not. At each oscillatory cycle, the stimulus is thus characterized as a point in a multidimensional space, where each dimension corresponds to the binary state of a given PN. A and GABAB inhibition. These two clusters are almost linearly separable. They overlap, however, when GABAA or GABAB is blocked. These observations indicate that GABAA and GABAB are both needed to create specific assemblies of synchronized neurons.In heterogenous networks, the number of GABAA and GABAB connectivity. In the AL of the honeybee, the GABAergic network is functionnally organized to reflect correlations between glomeruli Drosophila, inhibitory LNs present specificity in their odor responses M binary patterns iμξ ∈ {0,1}, we consider, for simplicity, that the GABAB network is global and that the GABAA network is trained using clipped Hebbian learning :ijJ = 1 if presynaptic neuron j is connected to postsynaptic neuron i with a fast GABAA type synapse and ijJ = 0 otherwise. A connectivity trained from a single pattern. The PNs in the antennal lobe do not inhibit each other directly but they do so via local neurons. Inhibitory LNs receive direct synaptic input from olfactory receptors A connection in our model is functionally active between neurons j and i when both ijJ = 1 (connection set by Equation 7) and jξ = 1 (reflecting the fact that a putative LN associated with this connection is activated by input jξ). A GABAB connection is functionally active between neurons j and i only when jξ = 1 . A and GABAB connections activated by input pattern ξ (noisy version of training pattern μξ). As seen previously, the relative number of GABAA and GABAB inputs modulate the degree of synchrony. In B inhibition whereas the other PNs synchronize. If state 1 or 0 is assigned to synchronized or desynchronized neurons respectively, then the original training pattern μξ is retrieved.In the previous sections, we have shown that synchronized neural assemblies are triggered by GABATo illustrate the functioning of the spiking associative memory, we used the learning rule (7) to train the GABAergic network with the three black-and-white images ‘0’, ‘1’ and ‘2’ depicted in N neurons and provide computer simulations confirming our estimate. In the simulations of the spiking associative memory, the peak conductance values for GABAA and GABAB have been adjusted according to ag/bg = 25  obtained analytically in An important metric of spiking associative memories is capacity. In other words, how many patterns can be stored and retrieved reliably by considering phase-locked neurons? We present a simple analysis that leads to an estimate of the capacity for a network of αc given by Equation 9 to the one estimated numerically for our spiking associative memory . The period of the network oscillation increases linearly with τGABA . Our work differs from previous theoretical studies in which the stimuli are encoded by transient synchrony, i.e., the subset of synchronized neurons changes over time A input. The most active LNs inhibit the others and may even suppress their activity due to strong LN-LN inhibition. These active LNs, however, increase their adaptation current, which makes subsequent firing harder. Such a fatigue mechanism leads to a complex time-varying competition between LNs that may depend on which LNs win the competition first. In contrast, the neural assemblies created by our mechanism are stable and do not depend on the initial state of the network, synchronized or not. Although LNs have not been used explicitly in our model, we propose another potential role for inhibitory local neurons (see below). Another difference with Our AL model converges onto assemblies of synchronized neurons triggered by the GABAergic network . The relDrosophila AL Modelling early olfactory systems as a network of neurons coupled with inhibition is not uncommon, see for example B connectivity is fixed and global and that the GABAA connectivity is trained according to the Hebbian axiom “cells that fire together, wire together”. We showed that lateral GABAA connections set by Hebbian learning endow the spiking network with properties of binary associative memories  is the received synaptic current and Iext = I+Iinj−Ith is a constant external current. I represents a driving current, Iinj is an injected current, and Ith denotes the rheobase, i.e., the minimal current required for repetitive firing. The QIF neuron fires as soon as V reaches the threshold Vth. Right after the spike, V is reset to the value Vreset.PNs are modelled as quadratic integrate-and-fire (QIF) neurons Iext<0 there are two fixed points. The stable ones defines the resting potentialIn the absence of synaptic current, the QIF neuron presents two distinct regimes depending on the sign of the external current. When Iext>0 the QIF neuron fires regularly and the firing frequency scales as θ-neuron C = 0.143 nF, TV = −41.18 mV, q = 9.29×10−4 mS V−1, Ith = 0.527 nA, Vth = 30 mV and Vreset = −70 mV. From Equation 11, Vrest = −65 mV when Iext = −Ith.The unstable one is the threshold above which the neuron fires a single spike. When Isyn in Equation 10 results from the integration of GABAergic currents over the dendritic tree. At each synapse, GABAI (in nA) is given byE is the reversal potential of the synapse (E = −70 mV for GABAA and −95 mV for GABAB) and g is the peak synaptic conductance in µS. The GABAA peak conductance ag is in the range (0.25–1.2)×10−3 µS and the GABAB peak conductance bg is in the order of 0.06×10−3 µS it are the times of the synaptic events and τGABA is the synaptic time decay (τGABA = 10 ms for GABAA and 100 ms for GABAB). The Heaviside function H ensures causality.The synaptic current it = ift+Δ where the ift are the firing times of the presynaptic neuron and Δ = 5 ms is the propagation delay. When asynchronous release is considered, each pre-synaptic spike triggers a number of GABAergic post-synaptic events. These events are triggered asynchronously, according to an exponential distribution of standard deviation λ. The probability that a presynaptic spike at time ift produces a post-synaptic event at time it is described by:Inhibitory interneurons may release transmitters synchronously or asynchronously N = 10×10 neurons corresponding to these odor-responding PNs. We take I = 0.75 nA in Equation 10 so that, without synaptic coupling, PNs are oscillators firing at the same frequency (about 40 Hz). In the network, PNs are coupled directly via GABAergic synapses. Inhibitory LNs are not modelled explicitly because of the lack of experimental data concerning the functionning of LNs and because of LN diversity. We consider two types of inhibitory synapses, GABAA and GABAB, and a probability of synaptic failure . The network was programmed in C and simulated with a fourth-order Runge-Kutta integration method with a time step of 50 µs. The initial network condition corresponds to a completely desynchronized neuronal population. This is obtained from the following procedure. The firing times T of the neurons are given by integrating Equation 10 with Isyn = 0 from their initial membrane potentials V(0) to the firing threshold V(I) = VthNot all the PNs fire in the presence of an odor. In the Locust for example, about 100 PNs (out of 830) are activated by the presentation of an odor Tmax is obtained when V(0) = Vreset. This firing time equation is then solved for V(0)The maximum firing time V(0) for firing times T taken randomly between 0 and Tmax. This initialization procedure of the PNs ensures firing times uniformly distributed over .The above equation provides initial membrane potentials σ is similar to the one in The estimation procedure for the spike time jitter T̅ of the neuronal population is computed and the phase-locking probability is obtained by counting the relative number of spikes falling into a bin of ±ε ms around the mean firing time T̅ . Each individual pattern however elicits a specific sub-network of GABAA and GABAB coupling  are considered as active bits and the remaining (1−f)N neurons (with higher σ) are inactive bits. All the stored patterns are considered to be retrieved reliably if the mean overlap between stored and retrieved patterns exceeds 0.9. The above procedure is repeated with a larger number of stored patterns until the patterns can no longer be retrieved reliably. Each storage capacity estimated in The critical storage capacity Text S1Spike time jitter of the PN population.(0.04 MB PDF)Click here for additional data file.Figure S1The synaptic parameters control the period of the network oscillation. Period of the network oscillation versus parameters of the GABAergic synapses (time constant and synaptic conductance).(0.02 MB PDF)Click here for additional data file.Figure S2AL model with PN-PN excitatory connections.(0.03 MB PDF)Click here for additional data file.Figure S3AL model with inhibitory LNs.(0.38 MB PDF)Click here for additional data file.Video S1Phase-locked activity of the spiking associative memory for noisy ‘0’ pattern. The neurons which correspond to black and white pixels in the original training pattern are classified as foregrounds and backgrounds, respectively. The network is simulated for 1 sec of biological time. The LFP, computed as the average of the PNs' membrane potentials,oscillate at ≍ 25 Hz. At each cycle, foreground and background neurons, firing within a temporal window of ? ms around the peak of the LFP, are shown as red and blue pixels, respectively.(8.30 MB AVI)Click here for additional data file.Video S2Phase-locked activity of the spiking associative memory for noisy ‘1’ pattern. Conventions are similar to (8.30 MB AVI)Click here for additional data file.Video S3Phase-locked activity of the spiking associative memory for noisy ‘2’ pattern. Conventions are similar to (8.30 MB AVI)Click here for additional data file.


Photoreceptive, melanopsin-expressing retinal ganglion cells (mRGCs) encode ambient light (irradiance) for the circadian clock, the pupillomotor system, and other influential behavioral/physiological responses. mRGCs are activated both by their intrinsic phototransduction cascade and by the rods and cones. However, the individual contribution of each photoreceptor class to irradiance responses remains unclear. We address this deficit using mice expressing human red cone opsin, in which rod-, cone-, and melanopsin-dependent responses can be identified by their distinct spectral sensitivity. Our data reveal an unexpectedly important role for rods. These photoreceptors define circadian responses at very dim “scotopic” light levels but also at irradiances at which pattern vision relies heavily on cones. By contrast, cone input to irradiance responses dissipates following light adaptation to the extent that these receptors make a very limited contribution to circadian and pupillary light responses under these conditions. Our data provide new insight into retinal circuitry upstream of mRGCs and optimal stimuli for eliciting irradiance responses. ROpn1mw) mice reveal rod, cone, and melanopsin phases to NIF vision ► Rods drive circadian responses to very low irradiances (scotopic threshold) ► Circadian responses to light in the photopic range can be rod driven ► Light adaptation limits the influence of cones on NIF vision► Red cone knockin ( The mammalian eye extracts at least two fundamentally different sorts of information from the light environment. Its most widely appreciated role is to provide a spatial map of the visual scene. However, equally important is its ability to encode irradiance, the ambient level of light. This irradiance information defines a variety of nonimage forming (NIF) behavioral and physiological responses to light. These include synchronization of endogenous circadian clocks to light:dark (LD) cycles (photoentrainment) and regulation of pupil size, pineal melatonin production, sleep propensity, and aspects of gross physiology.Recent years have seen the discovery and elucidation of a new class of retinal photoreceptor seemingly dedicated to irradiance detection. This comprises a small number of directly photosensitive retinal ganglion cells  expressiThe presence of mRGCs would appear to free the retina's most abundant photoreceptors (rods and cones) from contributing to irradiance pathways. However, mRGCs receive synaptic input from the outer retina  and, whiThe discovery of mRGCs has seen an application in the field of lighting design, with evidence that brighter and/or “bluer” lights can improve health and well-being for a number of at risk populations in both clinical and field settings . One of ROpn1mw) carrying a human red cone opsin coding sequence in place of their native m-cone opsin  results in a substantial, long-wavelength shift in the spectral sensitivity of those cones that would ordinarily express m-cone opsin . In elengths B. We foupe, mice A and 2B.mwR mice C. By conmwR mice D. This s in mice , indicat in mice .12 photons/cm2/s phenocopies the loss of bright-light responses in melanopsin knockout mice (rd/rd cl) mice failed to respond to even the brightest 650 nm stimuli (not shown), while their threshold for a measurable pupillary response at 500 nm was, as previously reported  of irradiance. This impressive dynamic range cannot be explained by a simple linear summation of cone- and melanopsin- dependent responses. Rather it implies a “winner takes all” arrangement in which cones define responses at irradiances below melanopsin threshold, but have little impact on pupil size once melanopsin is activated. Together these data reveal that, under these conditions, the combined action of cones and melanopsin can drive graded decreases in pupil size over at least 6 decimal orders . As expected, 500 nm stimuli induced marked phase delays whose magnitude was irradiance dependent (ROpn1mw mice showed very poor sensitivity to 650 nm. Significant phase delays were elicited only by bright (>1013 photons/cm2/s) stimuli, representing an increase in threshold irradiance of at least 1000× compared to 500 nm. This is much greater than that predicted for a response driven by red cones (The same (M1) class of mRGC is responsible for routing rod and cone input to both the circadian clock and that portion of the pretectum responsible for the PLR  lacking critical elements of rod , only two out of six −/− Gnat1−/−Opn4 animals unambiguously entrained with a phase angle equivalent to that observed in wild-type controls  or long- (644 nm) wavelength light. Both wavelengths effectively lengthened τ  of mouse circadian rhythms lengthens according to Aschoff's rule . To detethened τ . Howeverthened τ , suggestOur pupillometry experiments confirm that cones can drive NIF responses in vivo . FurtherROpn1mw mice. We found that 5 min of exposure to 1013 photons/cm2/s of 644 nm light greatly reduced pupil responses to a subsequent 650 nm test stimulus of equivalent irradiance (15 photons/cm2/s 644 nm) rendered the pupil entirely refractory to irradiances (1014 photons/cm2/s 650 nm) that drove large constrictions when dark-adapted  for between 5 min and 1 hr induced a shift in sensitivity, but importantly did not reduce the ability of melanopsin to drive large pupil responses to irradiances equivalent to that of the adapting light , “melanopsin only” (rd/rd cl), and “melanopsin-less” (−/−Opn4) genotypes . Alternative possibilities include direct connections from rods to a subset of cone ON bipolars , or direrd/rd cl mice show circadian phase shifts to 15 min stimuli at least 10-fold dimmer than this  subtype of mRGC drives both circadian and pupillary responses studied here , our finROpn1mw JNOpn1mw according to the rules of the International Committee on Standardized Genetic Nomenclature for Mice) colony were a generous gift of J. Nathans . In females heterozygous for the knockin allele, X-inactivation ensures separate populations of mid- and long-wavelength sensitive cones. However, in these experiments we used exclusively hemizygous males (referred to here as ROpn1mw) or female homozygous knockins (R/ROpn1mw), neither of which retain a copy of the native mouse m-cone opsin and thus lack mid-wavelength sensitive cones.Experiments were in compliance with relevant laws and institutional guidelines at the University of Manchester and Johns Hopkins University. Founders for our  to as R  and L [J as R [L ; Opn1mwtmax 498 or 644 nm, half-bandwidth ≤37 nm). There was a standard 10 s gap between removal from this adapting light and application of the test stimulus during which the animals were in darkness.Pupillometry was conducted as previously described  on unane−/− Gnat1−/−Opn4 mice, animals were exposed to a 12 hr:12 hr LD cycle. One group of mice were exposed to 1 m W/cm2  for 21 days before being released into constant darkness (DD). To explore the irradiance dependence of entrainment of −/− Gnat1−/−Opn4 mice, a second group of animals were first exposed to the same lighting regime at 235 μW/cm2, followed by log unit decreases in irradiance every 2 weeks down to 0.2 μW/cm2. The irradiance at which animals free-run was defined as the point at which the period of their activity rhythm differed from 24 hr. To measure phase shifts, mice were first housed for at least 14 days under a 12 hr:12 hr LD cycle  before being released into DD. Light pulses at CT16 (4 hr after activity onset) were administered after 7 days in DD and animals were left for a further 10 days. Regression lines through the time of activity onset before and after the light pulse were used to calculate the phase shift. Light stimuli were applied in a specialized chamber with full internal reflectance using light from either a Xe arc source  or, when higher intensities were required, an LED source . This apparatus allows near full-field illumination, but some complexity in the visual scene is unavoidable because animals are freely moving. For constant light experiments, diffuse near monochromatic light  was applied using custom-built LED arrays (Philips LumiLED) supported above the home cage. In the case of LED sources, the effective photon flux for red cone, rod, or melanopsin photoreceptors was determined by calculating the photon flux at each wavelength, corrected according to the spectral efficiency function for that pigment, and integrating across wavelengths.Circadian rhythms were assessed by monitoring wheel-running activity using standard methods. Briefly, male mice were singly housed with free access to a running wheel, revolutions of which were monitored using either the Chronobiology Kit  or Clocklab . To determine the limits of entrainment for p in photons/cm2/s) according to the formula: ϕp = P × λ × 5.03 × 1015; where p = power in W and λ = wavelength.Light measurements employed an optical power meter  and a spectrometer  as appropriate. In the case of near monochromatic stimuli, we were able to convert these power measurements to photon flux .To facilitate comparisons with other publications, these can also be expressed as scotopic cd/m3 scotopic td. 2) can be assumed for simplicity, while acknowledging that this may overestimate retinal illumination by >10× if the pupil were fully constricted. This gives a conservative estimate of 16 cd/m2 (or 6 × 1012 photons/cm2/s for stimuli near 500 nm) as a lower limit for significant rod opsin bleach in our experiments. Fifty percent bleach would then require irradiances at least 10× greater than this.In view of the evidence that rods drive circadian responses at relatively high irradiances , it is iNote that these measurements and conversions will provide an accurate approximation of corneal irradiance in the phase shifting and pupillometry experiments for which a Ganzfeld stimulus was employed, but less so for the LL study in which light was applied to the subject's home cage.max ≈ 480 nm (rd/rd cl mice following pretreatment with 500 nm (Indications that melanopsin may employ an intrinsic photoreversal mechanism for bleach recovery raise the possibility that its photosensitivity is increased by prior exposure to long-wavelength light. A consensus on this question has not been reached (see ≈ 480 nm , and thi≈ 480 nm A, but beh 500 nm E, we do


Several aspects of perception, particularly those pertaining to vision, are closely linked to the occipital alpha (α) rhythm. However, how the α rhythm relates to the activity of neurons that convey primary visual information is unknown. Here we show that in behaving cats, thalamocortical neurons in the lateral geniculate nucleus (LGN) that operate in a conventional relay-mode form two groups where the cumulative firing is subject to a cyclic suppression that is centered on the negative α rhythm peak in one group and on the positive peak in the other. This leads to an effective temporal framing of relay-mode output and results from phasic inhibition from LGN interneurons, which in turn are rhythmically excited by thalamocortical neurons that exhibit high-threshold bursts. These results provide a potential cellular substrate for linking the α rhythm to perception and further underscore the central role of inhibition in controlling spike timing during cognitively relevant brain oscillations. The EEG alpha (α) (8–13 Hz) rhythm is intimately associated with many basic aspects of perception . For exaA key brain area in both the transmission of visual information and the generation of the α rhythm is the primary visual thalamus or dorsal lateral geniculate nucleus (LGN) . In thisIn cortical circuits the timing of principal cell firing during cognitively relevant brain oscillations is largely determined by the coordinated activity of various types of inhibitory interneurons . RecentlDuring natural wakefulness EEG recordings in freely moving cats regularly exhibited α rhythm episodes  that were synchronized with local field oscillations (LFOs) in the LGN . As noteIn the remaining 67% of TC neurons (n = 28 of 42), we observed single spikes only, i.e., tonic or relay-mode firing A. In mosTo gain an insight into the overall output that would be generated by ensembles of α-rhythm-correlated relay-mode TC neurons, we constructed cumulative spike timing histograms, with respect to the LFO, for the two groups of these cells. As would be expected given that neurons in each group share a common phase at which the minimum amount of firing occurs but show no communal point in the oscillation cycle where maximum firing takes place B, these To test our proposal that inhibitory thalamic neurons play a key role in shaping the output of TC neuron firing during α rhythms , we analDuring low-frequency (∼7–12 Hz) cortical oscillations in the somatosensory system of rats that occur in quiet waking and immobility and which are often considered to be the analog of the human mu (μ) rhythm in this species , neurons in the TRN generate robust, rhythmic bursting in register with individual LFP waves   and/or m2 test: p > 0.5 for both cases). Notably, despite the general effects of mGluR activation on TC neurons being similar to those of activating mAChRs, but again consistent with in vivo α rhythms being primarily supported by a cholinergic drive  or positive  LFO peak, the cumulative firing in each group was again characterized by histograms that exhibited an essentially flat profile except for a brief suppression of firing near the negative D, top oric drive , pharmacic drive .Having established that during cholinergically induced in vitro α rhythms the output of spontaneously active relay-mode TC neurons is equivalent to that observed in vivo, we further scrutinized the dynamics of individual relay-mode TC neurons in this condition in order to dissect the cellular events that lead to a differential, cyclic modulation of firing. Hyperpolarization of relay-mode TC neurons below action potential threshold revealed subthreshold membrane potential fluctuations that were phase related to local network activity (n = 12) A. MoreovA receptor antagonist SR95531  (10 μM) (n = 5) confirmed that they were genuine GABAergic IPSPs  recorded in this manner exhibited unambiguous IPSPs  that (1) occurred with clear rhythmicity   displayed mainly one or two events per oscillation cycle that occurred primarily close to the negative LFO peak  , GBZ (10 μM) abolished correlations between relay-mode firing and ongoing α activity  A and 5B. n = 13) C, and (3 n = 10) C and 5E, n = 10) D and 5F.We next turned our attention to identifying the source of phasic inhibition that is present in relay-mode TC neurons during Cch-induced α activity. We reasoned that because these cells receive LFO-correlated rhythmic IPSPs, they must be the recipients of an inhibitory pathway that originates in the rhythmic HT bursting cell network but which must ultimately be administered by either neurons in PGN or local GABAergic interneurons in the LGN . To inve+/QX314-filled electrodes (p > 0.1)  . Extracep > 0.1) C.+/QX314-filled electrodes (p > 0.1)  were slightly more depolarized (≤50 pA), their pattern of firing could be transformed into one consisting of rhythmic bursts of up to 10 action potentials  C. These p > 0.1) D, and thThe two types of firing observed for LGN interneurons in vitro are clearly comparable with the output of putative interneurons recorded during naturally occurring α rhythms in vivo C. IndeedImportantly, in all LGN interneurons recorded in vitro, the combined application of the AMPA/kainate receptor antagonist NBQX (20 μM) and the NMDA receptor antagonist APV (50 μM) not only blocked EPSPs but also eliminated all spontaneous firing (n = 8) D. Thus, Because of the synchrony between HT bursting TC neurons and presumed interneurons observed in vivo D, and siFinally, although burst firing at depolarized membrane potentials is an intrinsic property of LGN interneurons   is an intrinsic phenomenon that is primarily reliant on Cachannels , (2) canchannels , (3) canto burst , and (4)to burst  or isolato burst  EPSPs, aPhasic inhibition onto TC neurons is also a key component in generating thalamic spindle oscillations . HoweverDespite the definitive status of the α rhythm as a wake-related EEG hallmark, its preeminence in the eyes closed condition and its classical suppression upon eye opening have led to a common belief that it represents an idling of visual cortical areas. However, a long history of experimental observations from humans not only shows that the α rhythm also occurs abundantly when the eyes are open, but also that it is closely linked to the timing of certain types of perceptual events  such as All in vivo and in vitro experiments were carried out in accordance with the guidelines of the local ethical committees, the UK Animals (Scientific Procedure) Act, 1986 and the Hungarian Act of Animal Care and Experimentation , which conforms to the European Community regulations (86/609/). All efforts were made to minimize the suffering and number of animals used in each experiment.Surgery for chronic implantation was carried out as described previously . Briefly2 and NO2 (2:1) and 2.5% halothane, a wide craniotomy was performed, and the brain was removed. Sagittal slices of the LGN-PGN were prepared and maintained as described previously  artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl (134), KCl (2), KH2PO4 (1.25), MgSO4 (1), CaCl2 (2), NaHCO3 (16), and glucose (10). Extracellular single-unit and field recordings were performed using glass pipettes filled with 0.5 M NaCl (resistance: 1–5 MΩ) connected to a Neurolog 104 differential amplifier . Independently mounted intracellular recordings, using the current-clamp technique, were performed with standard-wall glass microelectrodes filled with 1M potassium acetate (KAc) or 2M ceasium acetate and 50 mM QX314 (resistance: 80–120 MΩ), and in some cases 2% biocytin or neurobiotin, and connected to an Axoclamp-2A amplifier  operating in bridge mode. Additional information relating to the acquisition and analysis of in vitro electrophysiological data is given in the Young adult cats (1–1.5 kg) were deeply anaesthetized with a mixture of Oeviously . For rec


Plasma CEA values and serum γ glutamyl transpeptidase activities have been compared in control subjects and 109 patients with colorectal carcinoma and 35 with non-malignant hepatic disease. CEA values alone differentiate hepatic metastases from non-malignant hepatic disease with a high degree of certainty. While CEA may be elevated with metastases, irrespective of site, γGT is elevated mainly in association with hepatic metastases. The combination of CEA and γGT is helpful in identifying hepatic metastases and in their differentiation from local recurrences or metastases to other sites.