Identification of amacrine neurons with a glycinergic and GABAergic phenotype in the mouse retina.

The amacrine neurons in the mammalian retina comprise a large variety of cell types with distinct properties and functions that serve to integrate and modulate signals presented to output neurons. The majority of them use either glycine or GABA as inhibitory neurotransmitters and express the glycine transporter 1 (GlyT1) or glutamic acid decarboxylase (GAD67) and GABA transporters (GAT1 and GAT3), as a glycinergic or GABAergic marker respectively. We report here a novel subpopulation of amacrine neurons expressing both, GABAergic and glycinergic markers, in retinas from wild-type C57BL/6J mice and two transgenic lines. In retinal sections from the transgenic line expressing eGFP under the control of the glycine transporter 2, eGFP expression was exclusively found in cell bodies and dendrites of inhibitory amacrine neurons, identified for their immunoreactivity to syntaxin 1A. All of the glycinergic and a large portion of the GABAergic amacrine neurons contained eGFP; of these, 8-10% of GlyT1 positive neurons were also labeled either with GAD67, GAT1 or GAT3. These findings were confirmed in retinas from a wild-type and a mouse line expressing eGFP under the GAD67 promoter and two different anti-GlyT1 antibodies, showing the presence of a subpopulation with a dual phenotype. Moreover, eGFP-positive dendrites on both mouse lines were found juxtaposed to GlyR subunits and the scaffold protein gephyrin in several areas of the inner plexiform layer, demonstrating the glycinergic character of these neurons. This dual phenotype was also demonstrated in primary retina cultures, in which isolated neurons were positive for GlyT1 and GAD67 or GAT1/3. Altogether, these data provide compelling evidence of a subpopulation of dual inhibitory, glycinergic/GABAergic amacrine neurons. The co-release of both neurotransmitters may serve to strengthen the inhibition on ganglion cells under synaptic hyperexcitability.

Analysis of natural-occurring and synthetic sexual hormones in sludge-amended soils by matrix solid-phase dispersion and isotope dilution gas chromatography-tandem mass spectrometry.

A sensitive analytical method is presented for the simultaneous determination of four synthetic estrogens and six steroid hormones in sludge-amended soil. The method employs matrix solid-phase dispersion (MSPD) followed by isotope dilution gas chromatography-tandem mass spectrometry injecting a large volume sample (10µL) after trimethylsilyl derivatization, using the solvent vent mode. It affords good resolution, high sensitivity and reproducibility and freedom from interferences even from complex matrices as soil amended with sewage sludge. The limits of detection (LODs) ranged from 10 to 300pgg(-1) with testosterone and progesterone having the highest limits. Soil amended with sewage sludge was spiked at 2, 10, 25 and 50ngg(-1) and the recoveries after MSPD with acetonitrile:methanol (90:10, v/v), ranged from 80 to 110% with relative standard deviations ≤9%. The method was applied to the analysis of six soil samples collected from agricultural plots and forested fields that had been amended with sewage sludge using isotopically labeled surrogates. Three of the synthetic estrogens studied were found at least in one of the six samples analyzed and trans-androsterone and estrone were the only natural hormones detected, although at very low levels (≤0.4ngg(-1)).

Determination of selected organic contaminants in soil by pressurized liquid extraction and gas chromatography tandem mass spectrometry with in situ derivatization.

The determination of organic contaminants in soil is a real challenge due to the large number of these compounds with quite different physico-chemical properties. In the present work, an analytical method was developed for the simultaneous determination in soil of 40 organic contaminants belonging to different chemical classes: polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers, UV filters, parabens, bisphenols and triclosan. Soil was extracted by pressurized liquid extraction and the extracts, without the need of a clean-up step, were analyzed by gas chromatography-tandem mass spectrometry after in situ derivatization in the gas chromatographic system. In the pressurized liquid extraction step, two extraction cycles were performed with a mixture of ethyl acetate-methanol (90:10, v/v) at 80 °C. Recovery of these contaminants from soil samples spiked at levels ranging from 30 to 120 ng g(-1) was satisfactory for most of the compounds. The developed procedure provided detection method limits from 0.1 to 2.5 ng g(-1). The analysis of soil samples collected in different agricultural fields confirmed the presence of some of the studied contaminants. Polycyclic aromatic hydrocarbons were the main contaminants detected, parabens and polychlorinated biphenyls were also found but at relatively low concentration levels, 2-ethylhexyl salicylate was the UV filter that appeared most frequently at levels ranging from 17.2 to 43.4 ng g(-1) and triclosan was found in eight out of fourteen samples, at relatively low concentration levels (0.8-28.6 ng g(-1)).

Analysis of volatiles from Spanish honeys by solid-phase microextraction and gas chromatography-mass spectrometry.

Headspace solid-phase microextraction (SPME), followed by gas chromatography (GC)-mass spectrometry (MS) determination, has been used for the analysis of honey volatiles. Two SPME fibers were employed to study the composition of volatiles from various types of Spanish honeys. The best results were obtained with the Carboxen/PDMS fiber, using a homogenization time of 1 h at 70 degrees C and a sampling period of 30 min. A total of 35 compounds were detected, most of them identified by GC-MS and quantified using external standards. Differences in the composition of honey volatiles were obtained, and these results allowed the differentiation of honeys. However, further studies are necessary to confirm the utility of this technique as an alternative tool for the characterization of the floral origin of honeys.