Upregulation of adrenomedullin and its receptor components during cardiomyocyte hypertrophy induced by chronic inhibition of nitric oxide synthesis in rats

Adrenomedullin may provide a compensatory mechanism to attenuate left ventricular hypertrophy (LVH). Nitric oxide synthase inhibition, induced by chronic administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats, induces cardiac hypertrophy in some, but not all cases; there are few reports of direct assessment of cardiomyocyte parameters. The objective was to characterize hypertrophic parameters in left (LV) and right ventricular (RV) cardiomyocytes after administration of L-NAME to rats for 8 wk and to determine whether adrenomedullin and its receptor components were upregulated. After treatment with L-NAME (20 and 50 mg x kg(-1) x day(-1)), compared with nontreated animals, 1) systolic blood pressure increased (by 34.2 and 104.9 mmHg), 2) heart weight-to-body wt ratio increased 24.1% at the higher dose (P

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Angiotensin converting enzyme inhibition from birth reduces body weight and body fat in Sprague-Dawley rats

In vitro studies have demonstrated that angiotensin II (ANG II) induces adipocyte hyperplasia and hypertrophy. The aim of the present study was to determine the effect of angiotensin-converting enzyme inhibition on body weight, adiposity and blood pressure in Sprague–Dawley rats. From birth half of the animals (n = 15) were given water to drink, while the remainder were administered perindopril in their drinking water (2 mg/kg/day). Food intake, water intake and body weight were measured weekly. Blood pressure was measured by tail cuff plethysmography at 11-weeks. Body fat content and distribution were assessed using dual energy X-ray absorptiometry and Magnetic Resonance Imaging at 12 weeks. Animals administered with perindopril had a body fat proportion that was half that of controls. This was consistent with, but disproportionately greater than the observed differences in food intake and body weight. Perindopril treatment completely removed hypertension. We conclude that the chronic inhibition of ANG II synthesis from birth specifically reduces the development of adiposity in the rat.

Effects of chronic acetazolamide administration on gas exchange and acid-base control in pulmonary circulation in exercising horses

P>Reasons for performing study:Carbonic anhydrase (CA) catalyses the hydration/dehydration reaction of CO(2) and increases the rate of Cl- and HCO(3)- exchange between the erythrocytes and plasma. Therefore, chronic inhibition of CA has a potential to attenuate CO(2) output and induce greater metabolic and respiratory acidosis in exercising horses.Objectives:To determine the effects of Carbonic anhydrase inhibition on CO(2) output and ionic exchange between erythrocytes and plasma and their influence on acid-base balance in the pulmonary circulation (across the lung) in exercising horses with and without CA inhibition.Methods:Six horses were exercised to exhaustion on a treadmill without (Con) and with CA inhibition (AczTr). CA inhibition was achieved with administration of acetazolamide (10 mg/kg bwt t.i.d. for 3 days and 30 mg/kg bwt before exercise). Arterial, mixed venous blood and CO(2) output were sampled at rest and during exercise. An integrated physicochemical systems approach was used to describe acid base changes.Results:AczTr decreased the duration of exercise by 45% (P < 0.0001). During the transition from rest to exercise CO(2) output was lower in AczTr (P < 0.0001). Arterial PCO(2) (P < 0.0001; mean +/- s.e. 71 +/- 2 mmHg AczTr, 46 +/- 2 mmHg Con) was higher, whereas hydrogen ion (P = 0.01; 12.8 +/- 0.6 nEq/l AczTr, 15.5 +/- 0.6 nEq/l Con) and bicarbonate (P = 0.007; 5.5 +/- 0.7 mEq/l AczTr, 10.1 +/- 1.3 mEq/l Con) differences across the lung were lower in AczTr compared to Con. No difference was observed in weak electrolytes across the lung. Strong ion difference across the lung was lower in AczTr (P = 0.0003; 4.9 +/- 0.8 mEq AczTr, 7.5 +/- 1.2 mEq Con), which was affected by strong ion changes across the lung with exception of lactate.Conclusions:CO(2) and chloride changes in erythrocytes across the lung seem to be the major contributors to acid-base and ions balance in pulmonary circulation in exercising horses.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Photosynthetic responses of seven tropical seagrasses to elevated seawater temperature

This study uses chlorophyll a fluorescence to examine the effect of environmentally relevant (1-4 h) exposures of thermal stress (35-45 [deg]C) on seagrass photosynthetic yield in seven tropical species of seagrasses. Acute response of each tropical seagrass species to thermal stress was characterised, and the capacity of each species to tolerate and recover from thermal stress was assessed. Two fundamental characteristics of heat stress were observed. The first effect was a decrease in photosynthetic yield (Fv / Fm) characterised by reductions in F and Fm'. The dramatic decline in Fv / Fm ratio, due to chronic inhibition of photosynthesis, indicates an intolerance of Halophila ovalis, Zostera capricorni and Syringodium isoetifolium to ecologically relevant exposures of thermal stress and structural alterations to the PhotoSystem II (PSII) reaction centres. The decline in Fm' represents heat-induced photoinhibition related to closure of PSII reaction centres and chloroplast dysfunction. The key finding was that Cymodocea rotundata, Cymodocea serrulata, Halodule uninervis and Thalassia hemprichii were more tolerant to thermal stress than H. ovalis, Z. capricorni and S. isoetifolium. After 3 days of 4 h temperature treatments ranging from 25 to 40 [deg]C, C. rotundata, C. serrulata and H. uninervis demonstrated a wide tolerance to temperature with no detrimental effect on Fv / Fm' qN or qP responses. These three species are restricted to subtropical and tropical waters and their tolerance to seawater temperatures up to 40 [deg]C is likely to be an adaptive response to high temperatures commonly occurring at low tides and peak solar irradiance. The results of temperature experiments suggest that the photosynthetic condition of all seagrass species tested are likely to suffer irreparable effects from short-term or episodic changes in seawater temperatures as high as 40-45 [deg]C. Acute stress responses of seagrasses to elevated seawater temperatures are consistent with observed reductions in above-ground biomass during a recent El Nino event.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

L-NAME Treatment Enhances Exercise-induced Content of Myocardial Heat Shock Protein 72 (Hsp72) in Rats

Background/Aim: Nitric oxide (NO) modulates the expression of the chaperone Hsp72 in the heart, and exercise stimulates both NO production and myocardial Hsp72 expression. The main purpose of the study was to investigate whether NO interferes with an exercise-induced myocardial Hsp72 expression. Methods: Male Wistar rats (70-100 days) were divided into control (C, n= 12), L-NAME-treated (L, n= 12), exercise (E, n= 13) and exercise plus L-NAME-treated (EL, n= 20) groups. L-NAME was given in drinking water (700 mg. L(-1)) and the exercise was performed on a treadmill (15-25 m.min(-1), 40-60 min. day(-1)) for seven days. Left ventricle (LV) protein Hsp content, NOS and phosphorylated-NOS (p-NOS) isoforms were measured using Western blotting. The activity of NOS was assayed in LV homogenates by the conversion of [(3)H] L-arginine to [(3)H] L-citrulline. Results: Hsp72 content was increased significantly (223%; p < 0.05) in the E group compared to the C group, but exercise alone did not alter the NOS content, p-NOS isoforms or NOS activity. Contrary to our expectation, L-NAME enhanced (p < 0.05) the exercise-induced Hsp72 content (EL vs. C, L and E groups = 1019%, 548% and 457%, respectively). Although the EL group had increased stimulatory p-eNOS(Ser1177) (over 200%) and decreased inhibitory p-nNOS(Ser852) (similar to 50%) compared to both the E and L groups (p < 0.05), NOS activity was similar in all groups. Conclusions: Our results suggest that exercise-induced cardiac Hsp72 expression does not depend on NO. Conversely, the in vivo L-NAME treatment enhances exercise-induced Hsp72 production. This effect may be due to an increase in cardiac stress. Copyright (C) 2011 S. Karger AG, Basel

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats

Objective: In this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. Materials and methods: Oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. Results: In NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. Conclusions: In addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney. (C) 2010 Elsevier Ltd. All rights reserved.

Antifibrotic Effect of Tamoxifen in a Model of Progressive Renal Disease

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-beta 1 and plasminogen activator inhibitor-1, and with a significant reduction in a-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1 beta- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-beta 1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-beta 1, suggesting that it may have therapeutic use in CKD treatment.

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

Metastability and dynamics of stem cells: from direct observations to inference at the single cell level

Organismal development, homeostasis, and pathology are rooted in inherently probabilistic events. From gene expression to cellular differentiation, rates and likelihoods shape the form and function of biology. Processes ranging from growth to cancer homeostasis to reprogramming of stem cells all require transitions between distinct phenotypic states, and these occur at defined rates. Therefore, measuring the fidelity and dynamics with which such transitions occur is central to understanding natural biological phenomena and is critical for therapeutic interventions.

While these processes may produce robust population-level behaviors, decisions are made by individual cells. In certain circumstances, these minuscule computing units effectively roll dice to determine their fate. And while the 'omics' era has provided vast amounts of data on what these populations are doing en masse, the behaviors of the underlying units of these processes get washed out in averages.

Therefore, in order to understand the behavior of a sample of cells, it is critical to reveal how its underlying components, or mixture of cells in distinct states, each contribute to the overall phenotype. As such, we must first define what states exist in the population, determine what controls the stability of these states, and measure in high dimensionality the dynamics with which these cells transition between states.

To address a specific example of this general problem, we investigate the heterogeneity and dynamics of mouse embryonic stem cells (mESCs). While a number of reports have identified particular genes in ES cells that switch between 'high' and 'low' metastable expression states in culture, it remains unclear how levels of many of these regulators combine to form states in transcriptional space. Using a method called single molecule mRNA fluorescent in situ hybridization (smFISH), we quantitatively measure and fit distributions of core pluripotency regulators in single cells, identifying a wide range of variabilities between genes, but each explained by a simple model of bursty transcription. From this data, we also observed that strongly bimodal genes appear to be co-expressed, effectively limiting the occupancy of transcriptional space to two primary states across genes studied here. However, these states also appear punctuated by the conditional expression of the most highly variable genes, potentially defining smaller substates of pluripotency.

Having defined the transcriptional states, we next asked what might control their stability or persistence. Surprisingly, we found that DNA methylation, a mark normally associated with irreversible developmental progression, was itself differentially regulated between these two primary states. Furthermore, both acute or chronic inhibition of DNA methyltransferase activity led to reduced heterogeneity among the population, suggesting that metastability can be modulated by this strong epigenetic mark.

Finally, because understanding the dynamics of state transitions is fundamental to a variety of biological problems, we sought to develop a high-throughput method for the identification of cellular trajectories without the need for cell-line engineering. We achieved this by combining cell-lineage information gathered from time-lapse microscopy with endpoint smFISH for measurements of final expression states. Applying a simple mathematical framework to these lineage-tree associated expression states enables the inference of dynamic transitions. We apply our novel approach in order to infer temporal sequences of events, quantitative switching rates, and network topology among a set of ESC states.

Taken together, we identify distinct expression states in ES cells, gain fundamental insight into how a strong epigenetic modifier enforces the stability of these states, and develop and apply a new method for the identification of cellular trajectories using scalable in situ readouts of cellular state.

Early maternal deprivation reduces prepulse inhibition and impairs spatial learning ability in adulthood: No further effect of post-pubertal chronic corticosterone treatment

Prolonged maternal deprivation leads to long-term alterations in hypothalamic–pituitary–adrenal (HPA) axis activity, disturbances of auditory information processing and neurochemical changes in the adult brain, some of which are similar to that observed in schizophrenia. Here we report the adult behavioural effects of maternal deprivation (12 h on postnatal days 9 and 11) in Wistar rats on paradigms of auditory information processing (prepulse inhibition), sensitivity to dopamimetics (amphetamine-induced hyper-locomotion) and cognition (T-maze delayed alternation and Morris water-maze). In addition, we examined the long-lasting effect of chronic 21-day corticosterone treatment during the post-pubertal period (i.e., postnatal days 56–76) on each of these behavioural paradigms in maternally deprived and control rats. Behavioural testing commenced 2 weeks after the termination of corticosterone treatment. Maternal deprivation led to a significant reduction in PPI and impaired spatial learning ability in adulthood, but did not affect the behavioural response to amphetamine. Post-pubertal chronic corticosterone treatment did not have any major long-lasting effects on any of the behavioural measures in either maternally deprived or control rats. Our findings further support maternal deprivation as an animal model of specific aspects of schizophrenia.