Differences in DNA methylation of insulin-like growth factor 2 and cadherin 13 in patients with preeclampsia.

OBJECTIVES: In a previous mass spectrometry study of our research group, 25 proteins were found to be differentially expressed in cerebrospinal fluid of patients with preeclampsia compared to controls. The objective of the current study was to investigate DNA methylation of the genes encoding for the former mentioned proteins in an independent dataset. STUDY DESIGN: In a nested case-control study of the Rotterdam Periconceptional Cohort, placental tissue, umbilical cord white blood cells and human umbilical vein endothelial cells (HUVEC) were obtained of 13 patients with early-onset preeclampsia, 16 patients with late-onset preeclampsia and 83 normotensive controls (27 patients with fetal growth restriction, 20 patients with spontaneous preterm birth and 36 uncomplicated pregnancies). DNA methylation of 783 CpGs in regions of 25 genes was measured. MAIN OUTCOME MEASURES: DNA methylation of selected candidate genes in early- and late-onset preeclampsia compared to fetal growth restriction, spontaneous preterm birth and uncomplicated controls. RESULTS: From the 783 CpGs of the 25 selected genes, 15 CpGs were differentially methylated between early-onset preeclampsia and spontaneous preterm birth (3.80 E-5 ≤ p ≤ 0.036). Four CpGs were differentially methylated between early-onset preeclampsia and fetal growth restriction (0.0002 ≤ p ≤ 0.037) and 13 CpGs were differentially methylated between early onset preeclampsia and uncomplicated controls (0.0001 ≤ p ≤ 0.04). CONCLUSION: Differences in DNA methylation were found in placental tissue, umbilical cord white blood cells and HUVEC of patients with early onset preeclampsia compared to (un)complicated controls, but not in patients with late-onset preeclampsia. The genes showing the largest differential methylation encode insulin-like growth factor 2 binding protein and receptor and cadherin 13.

Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues.

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.

Hemodynamic effects of intravenous nicardipine in severely pre-eclamptic women with a hypertensive crisis.

OBJECTIVE: Nicardipine permits rapid control of blood pressure in women with severe pre-eclampsia (PE) and hypertensive crisis. Our objective was to investigate its maternal and fetal hemodynamic effects. METHODS: Ten severely pre-eclamptic pregnant women who required intravenous nicardipine for severe hypertension were included in this prospective observational trial. Maternal macrocirculation was assessed by transthoracic echocardiography. Maternal microcirculatory perfusion was examined sublingually with the sidestream dark field imaging technique. Fetal hemodynamics were assessed by Doppler examinations of the uteroplacental and fetal circulations. Maternal cardiac output, total vascular resistance, mitral E/A ratio and capillary heterogeneity index, uterine artery pulsatility index and fetal cerebroplacental ratio were considered primary outcomes. Paired measurements, obtained before administration of nicardipine infusion and after stabilization of blood pressure, were compared. RESULTS: Administration of nicardipine significantly reduced the mean arterial blood pressure (median difference, 26 mmHg; P = 0.002) and total vascular resistance (median difference, 791 dynes × s/cm(5) ; P = 0.002) in all included women. This induced a reflex tachycardia with consequent increase in cardiac output of 1.55 L/min (P = 0.004). There were no significant changes in the other determinants of maternal or fetal hemodynamic parameters. CONCLUSIONS: Nicardipine effectively reduces blood pressure through selective afterload reduction that triggers an increase in cardiac output, without affecting maternal diastolic function, or microcirculatory, uteroplacental or fetal perfusion. This hemodynamic response is uniform and predictable. Fetomaternal cardiovascular profiling can be achieved by combining transthoracic echocardiography with obstetric Doppler.

Efficacy of DL-methionine hydroxy analogue-free acid in comparison to DL-methionine in growing male white Pekin ducks.

The present study was performed to assess the bioefficacy of DL-methionine hydroxy analogue-free acid (MHA) in comparison to DL-methionine (DLM) as sources of methionine for growing male white Pekin ducks in the first 3 wk of life. For this aim, 580 1-day-old male ducks were allocated into 12 treatment groups and received a basal diet that contained 0.29% of methionine, 0.34% of cysteine and 0.63% of total sulphur containing amino acids or the same diet supplemented with either DLM or MHA in amounts to supply 0.05, 0.10, 0.15, 0.20, and 0.25% of methionine equivalents. Ducks fed the control diet without methionine supplement had the lowest final body weights, daily body weight gains and feed intake among all groups. Supplementation of methionine improved final body weights and daily body weight gains in a dose dependent-manner. There was, however, no significant effect of the source of methionine on all of the performance responses. Evaluation of the data of daily body weight gains with an exponential model of regression revealed a nearly identical efficacy (slope of the curves) of both compounds for growth (DLM = 100%, MHA = 101%). According to the exponential model of regression, 95% of the maximum values of daily body weight gain were reached at methionine supplementary levels of 0.080% and 0.079% for DLM and MHA, respectively. Overall, the present study indicates that MHA and DLM have a similar efficacy as sources of methionine for growing ducks. It is moreover shown that dietary methionine concentrations of 0.37% are required to reach 95% of the maximum of daily body weight gains in ducks during the first 3 wk of life.

Four-factor prothrombin complex concentrate reverses apixaban-associated bleeding in a rabbit model of acute hemorrhage.

BACKGROUND: Apixaban is a direct factor Xa inhibitor approved for the treatment and prevention of thromboembolic disease. There is a lack of data regarding its reversal in cases of acute bleeding or prior to emergency surgery that needs addressing. OBJECTIVES: This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®) /Kcentra(®) , CSL Behring) can effectively reverse apixaban-associated bleeding in an in vivo rabbit model and evaluated the correlations between in vivo hemostasis and in vitro coagulation parameters. METHODS: For dose-finding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800-1600 µg kg(-1) ) and, following a standardized kidney incision, volume of blood loss and time to hemostasis were measured. In a subsequent study phase, anesthetized rabbits were treated with apixaban 1200 µg kg(-1) followed by 4F-PCC (6.25-100 IU kg(-1) ), and the effects on the same bleeding parameters were assessed. In parallel, coagulation parameters were monitored. RESULTS: Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration. Preincision treatment with 4F-PCC resulted in a statistically significant reversal in bleeding time (all doses) and volume (doses ≥ 12.5 IU kg(-1) ). Of the coagulation parameters measured, thrombin generation initiated using the RD reagent (phospholipids only) was the most sensitive to in vivo measures of 4F-PCC's hemostatic efficacy, although some correlations were also observed for prothrombin time and whole blood clotting time. CONCLUSIONS: In this rabbit model of acute hemorrhage, 4F-PCC showed potential for reversing the bleeding effects of apixaban. Clinical data in apixaban-treated patients are needed to confirm these results.

The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation.

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been suggested that inflammation associated with the development of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, we investigated the hypothesis that feeding grape seed and grape marc meal extract (GSGME) as a plant extract rich in flavonoids attenuates inflammation and ER stress in the liver of dairy cows. Two groups of cows received either a total mixed ration as a control diet or the same total mixed ration supplemented with 1% of GSGME over the period from wk 3 prepartum to wk 9 postpartum. Dry matter intake during wk 3 to 9 postpartum was not different between the 2 groups. However, the cows fed the diet supplemented with GSGME had an increased milk yield and an increased daily milk protein yield. Cows supplemented with GSGME moreover had a significantly reduced mRNA abundancy of fibroblast growth factor (FGF) 21, a stress hormone induced by various stress conditions, in the liver in wk 1 and 3 postpartum. In contrast, mRNA abundances of a total of 3 genes involved in inflammation and 14 genes involved in ER stress response, as well as concentrations of triacylglycerols and cholesterol, in liver samples of wk 1 and 3 postpartum did not differ between the 2 groups. Overall, this study shows that supplementation of GSGME did not influence inflammation or ER stress in the liver but increased milk yield, an effect that could be due to effects on ruminal metabolism.

The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.

Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

Assessment of the ability of the Privigen® purification process to deplete thrombogenic factor XIa from plasma.

BACKGROUND AND OBJECTIVES: Activated clotting factor FXI (FXIa) has been postulated to play a significant role in thromboembolic events potentially associated with the administration of intravenous immunoglobulin. The purpose of this study was to demonstrate that thrombogenic agents, in particular FXIa and FXI, are depleted or inactivated in Privigen(®) . MATERIALS AND METHODS: The ability of the purification process to deplete FXIa from plasma was studied. All steps of the Privigen(®) production were investigated for potential activation of FXI to FXIa with spiking experiments. RESULTS: Privigen(®) contains no procoagulant activity as determined by FXIa chromogenic assay, non-activated partial thromboplastin time (NaPTT) and thrombin generation assays (TGA, FXIa-like activity). The coagulation times were >200 s in the NaPTT test. FXIa was below the detection limit of 0·14 ng/ml (chromogenic assay) and below the quantification limit of 0·2 ng/ml (TGA). FXIa spiking experiments showed that the analytical methods used can detect traces of procoagulant activity in immunoglobulin samples. FXIa spiking and kinetic experiments during the octanoic acid fractionation step showed that a substantial reduction in FXIa specific activity (by ≥99·9% within 40 min of octanoic acid incubation) was reached already at an early stage of the manufacturing process. These results were confirmed in vivo: in a modified Wessler test, no thrombus was reported. CONCLUSION: The Privigen(®) manufacturing process has the capability to remove thrombogenic factors: octanoic acid precipitation, designed to remove a variety of contaminants during immunoglobulin purification, also removes almost all FXIa from plasma and further purification steps do not activate FXI.

Microcirculation in women with severe pre-eclampsia and HELLP syndrome: a case-control study.

OBJECTIVE: To compare microcirculatory perfusion in women with severe pre-eclampsia against that in healthy pregnant women, and secondly in women with severe pre-eclampsia with or without HELLP syndrome (haemolysis, elevated liver enzymes, and low platelets). DESIGN: Case-control study. SETTING: University Hospital Rotterdam, the Netherlands. POPULATION: Twenty-three women with severe pre-eclampsia and 23 healthy pregnant controls, matched for maternal and gestational age. Out of the 23 women with severe pre-eclampsia, ten presented with HELLP syndrome. METHODS: Microcirculation was analysed sublingually by a non-invasive sidestream dark-field imaging device (SDF). MAIN OUTCOME MEASURES: Perfused vessel density (PVD), microcirculatory flow index (MFI), and heterogeneity index (HI) were calculated for both small vessels (∅ < 20 µm; capillaries) and non-small vessels (∅ > 20 µm; venules and arterioles). RESULTS: There were no significant differences between women with severe pre-eclampsia and healthy controls. Women with pre-eclampsia and HELLP syndrome showed a reduced PVD (P = 0.045), MFI (P = 0.008), and increased HI (P = 0.002) for small vessels, as compared with women with pre-eclampsia but without HELLP syndrome. CONCLUSIONS: Sidestream dark-field is a novel, promising technique in obstetrics that permits the non-invasive evaluation of microcirculation. We did not observe major differences in sublingual microcirculatory perfusion between women with severe pre-eclampsia and healthy pregnant controls. In women with severe pre-eclampsia, the presence of HELLP syndrome is characterised by impaired capillary perfusion.

Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice.

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Learning chronobiology by improving Wikipedia.

Although chronobiology is of growing interest to scientists, physicians, and the general public, access to recent discoveries and historical perspectives is limited. Wikipedia is an online, user-written encyclopedia that could enhance public access to current understanding in chronobiology. However, Wikipedia is lacking important information and is not universally trusted. Here, 46 students in a university course edited Wikipedia to enhance public access to important discoveries in chronobiology. Students worked for an average of 9 h each to evaluate the primary literature and available Wikipedia information, nominated sites for editing, and, after voting, edited the 15 Wikipedia pages they determined to be highest priorities. This assignment (http://www.nslc.wustl.edu/courses/Bio4030/wikipedia_project.html) was easy to implement, required relatively short time commitments from the professor and students, and had measurable impacts on Wikipedia and the students. Students created 3 new Wikipedia sites, edited 12 additional sites, and cited 347 peer-reviewed articles. The targeted sites all became top hits in online search engines. Because their writing was and will be read by a worldwide audience, students found the experience rewarding. Students reported significantly increased comfort with reading, critiquing, and summarizing primary literature and benefited from seeing their work edited by other scientists and editors of Wikipedia. We conclude that, in a short project, students can assist in making chronobiology widely accessible and learn from the editorial process.

Reversal of dabigatran anticoagulation by prothrombin complex concentrate (Beriplex P/N) in a rabbit model.

BACKGROUND: One limitation of the direct thrombin inhibitor dabigatran is the lack of specific antidotes that allow acute bleeding events to be managed or urgent interventional procedures performed. Prothrombin complex concentrates (PCCs) have served as a standard treatment for the reversal of coumarin anticoagulation. OBJECTIVES: This study was designed to determine in an animal model whether a PCC (Beriplex P/N) can effectively reverse the effects of dabigatran. An additional objective was to evaluate markers of dabigatran-associated bleeding diathesis. METHODS: Anesthetized rabbits were treated with 0.4 mg kg(-1) dabigatran followed by PCC doses of 20, 35 or 50 IU kg(-1) or placebo. After a standardized kidney incision, volume of blood loss and time to hemostasis were determined. RESULTS: From an initial mean of 29 mL, blood loss progressively declined by 5.44 mL with a 95% confidence interval (CI) of 2.21-8.67 mL per 10 IU kg(-1) increment in PCC dose (P = 0.002). At a PCC dose of 50 IU kg(-1) blood loss was fully normalized. Increasing PCC doses shortened the median time to hemostasis from 20.0 to 5.7 min (P < 0.001). The rate of hemostasis was nearly trebled with each 10 IU kg(-1) increment in PCC dose (rate ratio, 2.89; CI, 1.64-5.09). CONCLUSIONS: In this animal study, PCC showed potential as an agent for reversing the effects of dabigatran. Further investigation is warranted.

[Cancellation of IVF-ET cycles: poor prognosis, poor responder, or variability of the response to controlled ovarian hyperstimulation? An analysis of 142 cancellations]. / Annulations en fécondation in vitro : mauvais pronostic, mauvaises répondeuses, ou variabilité de la réponse à la stimulation ? Analyse de 142 annulations.

INTRODUCTION: This retrospective study aimed at analyzing IVF-ET management and outcome after cancellation of a first cycle for poor response. PATIENTS AND METHOD: One hundred and forty-two infertile patients were included in this observational study. After an overall analysis on the outcome of the second IVF-ET attempt, a sub-analysis was performed according to the presence or the absence of poor prognostic criteria defined as mentioned: patient age superior to 38 years old, antral follicle count (3-9 mm in diameter) inferior to 10 on cycle day 3 and day 3 serum AMH and FSH levels less than 1 ng/mL and more than 10 IU/mL, respectively. Main outcome measures were the cancellation rates, pregnancy and live birth rates. RESULTS: When a controlled ovarian stimulation was performed, patients with poor prognosis had higher cancellation rates (37.8% vs. 13.3%, P<0.004) and lower pregnancy and live birth rates (22.2% vs. 35.0%, P<0.05 and 11.1% vs. 26.1%, P<0.05, respectively) as compared to good prognosis women. CONCLUSION: The relatively high cancellation rate in patients with poor prognosis raises the question of the use of IVF modified natural cycle in this group.

The association of the Friesinger score and estimated glomerular filtration rate in an urban South Asian patient population.

AIM: The correlation between kidney function and coronary artery disease (CAD) severity as assessed by an angiographic score has not yet been studied in the South Asian population. We sought to estimate the association by performing a single-center, cross-sectional study. PATIENTS AND METHODS: The estimated glomerular filtration rate (eGFR) calculated by the CKD-EPI equation and the Friesinger score to quantify the severity of CAD were the primary endpoints in patients undergoing coronary angiograms. RESULTS: The mean eGFR was significantly lower in participants with a Friesinger score of > 5 compared to participants with a score of < 5 (73 vs. 86 ml/min/1.73 m(2) by MDRD). In univariate analysis, an eGFR of < 55 ml/min/1.73 m(2) was associated with a 9.5-fold increased odds of a higher Friesinger score compared to an eGFR >= 55 ml/min/1.73 m2 (p = 0.043), which was unchanged in multivariate analysis. In multivariate analysis, a 10 ml/min/1.73 m(2) decrease in eGFR was associated with a 1.63-fold increased odds of a higher score (95% CI 1·10 - 2.37, p = 0.042). Traditional risk factors such as a history of previous CAD, hypertension, and dyslipidemia remained predictors of a higher Friesinger score. CONCLUSION: Our study demonstrates that kidney function as assessed by eGFR is a significant independent predictor of severity of CAD as determined by the Friesinger score.

Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells.

The ability of two types of single walled carbon nanotubes (SWCNT), namely Arc Discharge (AD) and HiPco single walled carbon nanotubes, to induce an indirect cytotoxicity in A549 lung cells by means of medium depletion was investigated. The nanotubes were dispersed in a commercial cell culture medium and subsequently removed by centrifugation and filtration. Spectroscopic analysis confirmed the removal of the nanotubes and showed differing degrees of alteration of the composition of the medium upon the removal of the nanotubes. The ability to induce an indirect cytotoxic effect by altering the medium was evaluated using two endpoints, namely the Alamar Blue (AB) and the Clonogenic assay. Exposure of the A549 cells to the depleted medium which had previously contained carbonaceous nanoparticles, revealed significant cytotoxicity for both endpoints employed. The results presented demonstrate that single walled carbon nanotubes can induce an indirect cytotoxicity by alteration of cell culture medium (in which they have previously been dispersed) which potentially results in a false positive toxic effect being observed in cytotoxicity studies.

Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin alpha.

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus--Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin alpha, suggesting that both CPs are imported into the nucleus via an importin alpha-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons.

Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato-dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2- and VGLUT1-positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.