High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses.

Synaptic plasticity involves remodeling of extracellular matrix. This is mediated, in part, by enzymes of the matrix metalloproteinase (MMP) family, in particular by gelatinase MMP-9. Accordingly, there is a need of developing methods to visualize gelatinolytic activity at the level of individual synapses, especially in the context of neurotransmitters receptors. Here we present a high-resolution fluorescent in situ zymography (ISZ), performed in thin sections of the alcohol-fixed and polyester wax-embedded brain tissue of the rat (Rattus norvegicus), which is superior to the current ISZ protocols. The method allows visualization of structural details up to the resolution-limit of light microscopy, in conjunction with immunofluorescent labeling. We used this technique to visualize and quantify gelatinolytic activity at the synapses in control and seizure-affected rat brain. In particular, we demonstrated, for the first time, frequent colocalization of gelatinase(s) with synaptic N-methyl-D-aspartic acid (NMDA)- and AMPA-type glutamate receptors. We believe that our method represents a valuable tool to study extracellular proteolytic processes at the synapses, it could be used, as well, to investigate proteinase involvement in a range of physiological and pathological phenomena in the nervous system.

Molecular techniques for detection of Tribolium confusum infestations in stored products.

The confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) is a stored-product pest that contaminates a wide range of food products, from flour and cereals to spices. The insect reduces food quality and is responsible for large economic losses every year. Although several methods for detection of stored-product pests are common and widely used, they are time-consuming and expensive. Therefore, establishing molecular methods of detection of stored-product pests could provide a useful alternative method. We have undertaken attempts to establish methods of detection of T. confusum based on molecular biology techniques of standard and real-time polymerase chain reaction (PCR). Total DNA of T. confusum and red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), used as a negative control, was isolated from insects and used as a template in standard and real-time PCR reactions. Specific primers have been designed on the basis of sequences of internal transcribed spacer (ITS) fragment of rDNA and subunit I of mitochondrial cytochrome oxidase of T. confusum available in the GenBank database. Standard PCR reactions with primers specific to the ITS fragment proved to be reliable and sensitive. Real-time PCR reactions with primers specific for mitochondrial DNA are considered to serve as a supplemental detection method for quantitative assessment of the infestation level.

A clarification for the subgenera of Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 2009, with respect to the International Code of Zoological Nomenclature.

The recent re-description of Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 2009 has inadvertently led to the description of an objective synonym within its subgenera nominal taxa. To resolve this issue, we have re-described both subgenera, and proposed a new substitute name for one subgenus, in line with the International Code of Zoological Nomenclature. Additionally we have confirmed the placement of two recently published Paramacrobiotus species, not included in the last revision, within the respective subgenera established herein.

Kinetic properties and adrenergic control of TREK-2-like channels in rat medial prefrontal cortex (mPFC) pyramidal neurons.

TREK-2-like channels were identified on the basis of electrophysiological and pharmacological tests performed on freshly isolated and enzymatically/mechanically dispersed pyramidal neurons of the rat medial prefrontal cortex (mPFC). Single-channel currents were recorded in cell-attached configuration and the impact of adrenergic receptors (α1, α2, ß) stimulation on spontaneously appearing TREK-2-like channel activity was tested. The obtained results indicate that noradrenaline decreases the mean open probability of TREK-2-like channel currents by activation of ß1 but not of α1- and α2-adrenergic receptors. Mean open time and channel conductance were not affected. The system of intracellular signaling pathways depends on the activation of protein kinase A. We also show that adrenergic control of TREK-2-like channel currents by adrenergic receptors was similar in pyramidal neurons isolated from young, adolescent, and adult rats. Immunofluorescent confocal scans of mPFC slices confirmed the presence of the TREK-2 protein, which was abundant in layer V pyramidal neurons. The role of TREK-2-like channel control by adrenergic receptors is discussed.

Muscarinic receptor control of pyramidal neuron membrane potential in the medial prefrontal cortex (mPFC) in rats.

Damage to the cholinergic input to the prefrontal cortex has been implicated in neuropsychiatric disorders. Cholinergic endings release acetylcholine, which activates nicotinic and/or G-protein-coupled muscarinic receptors. Muscarinic receptors activate transduction systems, which control cellular effectors that regulate the membrane potential in medial prefrontal cortex (mPFC) neurons. The mechanisms responsible for the cholinergic-dependent depolarization of mPFC layer V pyramidal neurons in slices obtained from young rats were elucidated in this study. Glutamatergic and GABAergic transmission as well as tetrodotoxin (TTX)-sensitive Na(+) and voltage-dependent Ca(++) currents were eliminated. Cholinergic receptor stimulation by carbamoylcholine chloride (CCh; 100 µM) evoked depolarization (10.0 ± 1.3 mV), which was blocked by M1/M4 (pirenzepine dihydrochloride, 2 µM) and M1 (VU 0255035, 5 µM) muscarinic receptor antagonists and was not affected by a nicotinic receptor antagonist (mecamylamine hydrochloride, 10 µM). CCh-dependent depolarization was attenuated by extra- (20 µM) or intracellular (50 µM) application of an inhibitor of the ßγ-subunit-dependent transduction system (gallein). It was also inhibited by intracellular application of a ßγ-subunit-binding peptide (GRK2i, 10µM). mPFC pyramidal neurons express Nav1.9 channels. CCh-dependent depolarization was abolished in the presence of antibodies against Nav1.9 channels in the intracellular solution and augmented by the presence of ProTx-I toxin (100 nM) in the extracellular solution. CCh-induced depolarization was not affected by the following reagents: intracellular transduction system blockers, including U-73122 (10 µM), chelerythrine chloride (5 µM), SQ 22536 (100 µM) and H-89 (2 µM); channel blockers, including Ba(++) ions (200 µM), apamin (100 nM), flufenamic acid (200 µM), 2-APB (200 µM), SKF 96365 (50 µM), and ZD 7288 (50 µM); and a Na(+)/Ca(++) exchanger blocker, benzamil (20 µM). We conclude that muscarinic M1 receptor-dependent depolarization in mPFC pyramidal neurons is evoked by the activation of Nav1.9 channels and that the signal transduction pathway involves G-protein ßγ subunits.

Molecular techniques for the detection of granary weevil (Sitophilus granarius L.) in wheat and flour.

The granary weevil (Sitophilus granarius L.) is a stored grain pest that causes major economic losses. It reduces the quantity and quality of the grain by its feeding and excretion. Sequences of S. granarius mitochondrial cytochrome oxidase subunits genes mtCOI and mtCOII were analysed and compared with mtCOI/II sequences available in GenBank. The analysed genes displayed a high level of homology between corresponding subunits. Attempts were undertaken to develop detection methods for contamination by S. granarius in wheat and wheat flour based on the molecular biology techniques: standard and real-time polymerase chain reaction (PCR) with a TaqMan molecular probe. (TaqMan probes are dual-labelled hydrolysis probes) Specific primers designed based on available sequences for mtCOI and mtCOII genes were applied and optimal reaction conditions established. The specificity of both methods was studied by using a species closely related to S. granarius: S. oryzae and S. zeamais. It is shown that the sensitivity threshold was very high - we were able to detect the equivalent of one beetle per 100 kg of flour when the real-time PCR with TaqMan probe method was applied to model samples. The primer sets used turned out to be species specific, and the technique was rapid, reliable and very sensitive.