Human Islets Exhibit Electrical Activity on Microelectrode Arrays (MEA).

This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.

Biocompatible molecularly imprinted polymers for the voltage regulated uptake and release of L-glutamate in neutral pH solutions.

A biocompatible device for the voltage dependent uptake and release of the neural transmitter L-glutamate in neutral pH solutions is demonstrated. The device consists of a gold electrode coated with molecularly imprinted, overoxidised polypyrrole (oPPy). It is shown here that oPPy can behave as an anion exchanger in neutral pH. The voltage dependent uptake and release of glutamate from the oPPy as well as the enantioselectivity of the polymer layer for L-glutamate over D-glutamate are investigated in neutral pH solutions using electrochemical quartz crystal microbalance techniques. The biocompatibility of the oPPy layer is demonstrated using retinae from young rats. The retinae were isolated and the dissociated cells were kept in culture for up to 1-week. The cells were exposed to the oPPy layers for 3 days, and there is no significant difference in the survival rate between the cells cultured on the oPPy layers and the control samples. Additionally the cell-polymer interface from cells grown directly on the oPPy layers is investigated using electron microscopy.

Sexual selection on morphological and physiological traits and fluctuating asymmetry in the black scavenger fly Sepsis cynipsea.

Previous univariate studies of the fly Sepsis cynipsea (Diptera: Sepsidae) have demonstrated spatiotemporally variable and consequently overall weak sexual selection favouring large male size, which is nevertheless stronger on average than fecundity selection favouring larger females. To identify specific target(s) of selection on body size and additional traits possibly affecting mating success, two multivariate field studies of sexual selection were conducted. In one study using seasonal replicates from three populations, we assessed 15 morphological traits. No clear targets of sexual selection on male size could be detected, perhaps because spatiotemporal variation in selection was again strong. In particular, there was no (current) selection on male abdomen length or fore coxa length, the only traits for which S. cynipsea males are not smaller than females. Interestingly, copulating males had a consistently shorter fore femur base, a secondary sexual trait, and a wider clasper (hypopygium) gap, an external genital trait. In a second study using daily and seasonal replicates from one population, we included physiological measures of energy reserves (lipids, glucose, glycogen), in addition to hind tibia length and fluctuating asymmetry (FA) of all pairs of legs. This study again confirmed the mating advantage of large males, and additionally suggests independent positive influences of lipids (the long-term energy stores), with effects of glucose and glycogen (the short-term energy stores) tending to be negative. FA of paired traits was not associated with male mating success. Our study suggests that inclusion of physiological measures and genital traits in phenomenological studies of selection, which is rare, would be fruitful in other species.

Sexual selection on morphological and physiological traits and fluctuating asymmetry in the yellow dung fly.

Previous univariate studies of the yellow dung fly (Scathophaga stercoraria) have demonstrated strong sexual selection, in terms of mating success, on male size (estimated as hind tibia length). To identify specific target(s) of selection on body size and possible conflicting selection pressures on particular body parts, two multivariate field studies of sexual selection were conducted. In one study using point samples from three populations, we assessed several morphological traits, including genital traits and measures of fluctuating asymmetry (FA) of all paired traits. There was sexual selection for large male size in general, confirming previous, univariate studies. With the possible exception of thorax width, which was selected in the opposite direction, no main target of selection was identified, as most morphological traits were highly correlated. There was no detectable sexual selection on the male external genital structures assessed. In a second study using multiple samples from one population, we included physiological measures of energy reserves (lipids, glucose and glycogen) known to affect mating success, in addition to trait size and FA of wings and legs. Inclusion of physiological traits is rare in phenomenological studies of selection. This study again confirmed the mating advantage of large males, and additionally showed independent positive influences of lipid and glucose but not glycogen levels. FA in paired traits generally did not affect male mating success, but was negatively correlated with energy reserves. Our study suggests that inclusion of physiological measures and genital traits in phenomenological studies of selection would be fruitful in other species.

Geographical and altitudinal population genetic structure of two dung fly species with contrasting mobility and temperature preference.

Local adaptation of populations requires some degree of spatio-temporal isolation. Previous studies of the two dung fly species Scathophaga stercoraria and Sepsis cynipsea have revealed low levels of geographic and altitudinal genetic differentiation in quantitative life history and morphological traits, but instead high degrees of phenotypic plasticity. These patterns suggest that gene flow is extensive despite considerable geographic barriers and large spatio-temporal variation in selection on body size and related traits. In this study we addressed this hypothesis by investigating genetic differentiation of dung fly populations throughout Switzerland based on the same 10 electrophoretic loci in each species. Overall, we found no significant geographic differentiation of populations for either species. This is inconsistent with the higher rates of gene flow expected due to better flying capacity of the larger S. stercoraria. However, heterozygote deficiencies within populations indicated structuring on a finer scale, seen for several loci in S. cynipsea, and for the locus PGM (Phosphoglucomutase) in S. stercoraria. Additionally, S. cynipsea showed a tendency towards a greater gene diversity at higher altitudes, mediated primarily by the locus MDH (malate dehydrogenase), at which a second allele was only present in populations above 1000 m. This may be caused by increased environmental stress at higher altitudes in this warm-adapted species. MDH might thus be a candidate locus subject to thermal selection in this species, but this remains to be corroborated by direct evidence. In S. stercoraria, no altitudinal variation was found.

Efficacy and stability of quantal GABA release at a hippocampal interneuron-principal neuron synapse.

We have examined factors that determine the strength and dynamics of GABAergic synapses between interneurons [dentate gyrus basket cells (BCs)] and principal neurons [dentate gyrus granule cells (GCs)] using paired recordings in rat hippocampal slices at 34 degrees C. Unitary IPSCs recorded from BC-GC pairs in high intracellular Cl(-) concentration showed a fast rise and a biexponential decay, with mean time constants of 2 and 9 msec. The mean quantal conductance change, determined directly at reduced extracellular Ca(2+)/Mg(2+) concentration ratios, was 1.7 nS. Quantal release at the BC-GC synapse occurred with short delay and was highly synchronized. Analysis of IPSC peak amplitudes and numbers of failures by multiple probability compound binomial analysis indicated that synaptic transmission at the BC-GC synapse involves three to seven release sites, each of which releases transmitter with high probability ( approximately 0.5 in 2 mm Ca(2+)/1 mm Mg(2+)). Unitary BC-GC IPSCs showed paired-pulse depression (PPD); maximal depression, measured for 10 msec intervals, was 37%, and recovery from depression occurred with a time constant of 2 sec. Paired-pulse depression was mainly presynaptic in origin but appeared to be independent of previous release. Synaptic transmission at the BC-GC synapse showed frequency-dependent depression, with half-maximal decrease at 5 Hz after a series of 1000 presynaptic action potentials. The relative stability of transmission at the BC-GC synapse is consistent with a model in which an activity-dependent gating mechanism reduces release probability and thereby prevents depletion of the releasable pool of synaptic vesicles. Thus several mechanisms converge on the generation of powerful and sustained transmission at interneuron-principal neuron synapses in hippocampal circuits.

Dentate gyrus basket cell GABAA receptors are blocked by Zn2+ via changes of their desensitization kinetics: an in situ patch-clamp and single-cell PCR study.

Although GABA type A receptors (GABAARs) in principal cells have been studied in detail, there is only limited information about GABAARs in interneurons. We have used the patch-clamp technique in acute rat hippocampal slices in combination with single-cell PCR to determine kinetic, pharmacological, and structural properties of dentate gyrus basket cell GABAARs. Application of 1 mM GABA (100 msec) to nucleated patches via a piezo-driven fast application device resulted in a current with a fast rise and a marked biexponential decay (time constants 2.4 and 61.8 msec). This decay could be attributed to strong receptor desensitization. Dose-response curves for the peak and the slow component yielded EC50 values of 139 and 24 microM, respectively. Zn2+ caused a marked blocking effect on both the peak and the slow component via a noncompetitive mechanism (IC50 values of 8 and 16 microM). This led to an acceleration of the slow component as well as a prolongation of recovery from desensitization. Zn2+ sensitivity was suggested to depend on the absence of gamma-subunits in GABAARs. To test this hypothesis we performed single-cell reverse transcription PCR that revealed primarily the presence of alpha2-, beta2-, beta3-, gamma1-, and gamma2-subunit mRNAs. In addition, flunitrazepam increased the receptor affinity for its agonist, indicating the presence of functional benzodiazepine binding sites, i.e., gamma-subunits. Thus, additional factors seem to co-determine the Zn2+ sensitivity of native GABAARs. The modulatory effects of Zn2+ on GABAAR desensitization suggest direct influences on synaptic integration via changes in inhibition and shunting at GABAergic synapses.

Functional properties of AMPA and NMDA receptors expressed in identified types of basal ganglia neurons.

AMPA- and NMDA-type glutamate receptors (AMPARs and NMDARs) mediate excitatory synaptic transmission in the basal ganglia and may contribute to excitotoxic injury. We investigated the functional properties of AMPARs and NMDARs expressed by six main types of basal ganglia neurons in acute rat brain slices (principal neurons and cholinergic interneurons of striatum, GABAergic and dopaminergic neurons of substantia nigra, globus pallidus neurons, and subthalamic nucleus neurons) using fast application of glutamate to nucleated and outside-out membrane patches. AMPARs in different types of basal ganglia neurons were functionally distinct. Those expressed in striatal principal neurons exhibited the slowest gating (desensitization time constant tau = 11.5 msec, 1 mM glutamate, 22 degrees C), whereas those in striatal cholinergic interneurons showed the fastest gating (desensitization time constant tau = 3.6 msec). The lowest Ca2+ permeability of AMPARs was observed in nigral dopaminergic neurons (PCa/PNa = 0.10), whereas the highest Ca2+ permeability was found in subthalamic nucleus neurons (PCa/PNa = 1.17). NMDARs of different types of basal ganglia neurons were less variable in their functional properties; those expressed in nigral dopaminergic neurons exhibited the slowest gating (deactivation time constant of predominant fast component tau1 = 150 msec, 100 microM glutamate), and those of globus pallidus neurons showed the fastest gating (tau1 = 67 msec). The Mg2+ block of NMDARs was similar; the average chord conductance ratio g-60mV/g+40mV was 0.18-0.22 in 100 microM external Mg2+. Hence, AMPARs expressed in different types of basal ganglia neurons are markedly diverse, whereas NMDARs are less variable in functional properties that are relevant for excitatory synaptic transmission and neuronal vulnerability.