Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

A Role for Glutamate Transporters in the Regulation of Insulin Secretion

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from beta-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in beta-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from beta-cells is modulated by the flux of glutamate through the secretory granules

Detection of EPO injections using a rapid lateral flow isoform test

Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7-21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10- to 20-fold higher analytical rate than what is possible today

Detection of EPO injections using a rapid lateral flow isoform test

Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7-21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10- to 20-fold higher analytical rate than what is possible today