Electrophysiological identification of the sugar cell in antennal taste sensilla of the predatory ground beetle Pterostichus aethiops

By single sensillum tip recording technique, in addition to the salt and pH cells found in antennal taste sensilla of some ground beetles earlier, the third chemosensory cell of four innervating these large sensilla was electrophysiologically identified as a sugar cell in the ground beetle Pterostichus aethiops. This cell generated action potentials of considerably smaller amplitude than those of the salt and pH cells, and phasic-tonically responded to sucrose and glucose over the range of 1-1000 mM tested. Responses were concentration dependent, with sucrose generating more spikes than glucose. During the first second of the response, maximum rates of firing of the sugar cell reached up to 19 and 37 imp/s when stimulated with 1000 mM glucose and sucrose, respectively. Three to four seconds later, the responses decreased close to zero. Both sugars are important in plant carbohydrate metabolism. These ground dwelling insects may come into contact with live and decayed plant material everywhere in their habitat including their preferred overwintering sites in brown-rot decayed wood. In conclusion, we hypothesize that high content of soluble sugars in their overwintering sites and refugia is unfavourable for these ground beetles, most probably to avoid contact with dangerous fungi.     

The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile

A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca²⁺ fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs⁺-conducting, Ca²⁺-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca²⁺ increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx_a), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca²⁺ dependence of [³H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals¹. Though being a crucial part of the visual processing by shaping the retinal output², most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue^3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage⁵. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments⁶.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.     

Electrophysiological identification of cold receptors on the antennae of the ground beetle Pterostichus aethiops

Abstract.  In single-sensillum extracellular electrophysiological recordings, terminal campaniform sensilla at the tip of antennae of the ground beetle Pterostichus aethiops (Pz., 1797) show action potentials of three sensory cells, A-, B- and C-cells, distinguished by differences in their spike amplitudes. Only the A-cell, with the largest spike amplitude, is highly sensitive to temperature fluctuations, showing remarkable changes in its firing rate induced by changes in temperature of 0.1 °C. The firing rate of A-cells at 23 °C varies from 15–52 Hz among different beetles. Mean impulse frequency of A-cells is found to be a function of steady temperature, the firing rate decreasing with temperature increase. A-cells respond to a rapid temperature drop with a strong phasic-tonic reaction; larger decreases in temperature evoke higher peak frequency values. Maximum peak frequencies, varying from 380–630 Hz in different beetles, are induced by temperature decreases of 3–10 °C, whereas temperature rise strongly inhibits impulse activity of the A-cell. The first manifestation of rapid warming in the nerve impulse sequence is a very long interspike period, followed by diminished activity. Both the length of the long interspike period and the rate of following impulse activity are functions of temperature change; hence, A-cells respond to temperature changes as typical cold receptors, similar to coeloconic and short hair-like sensilla in other insects.     

Electrophysiological responses of the antennal campaniform sensilla to rapid changes of temperature in the ground beetles Pterostichus oblongopunctatus and Poecilus cupreus (Tribe Pterostichini) with different ecological preferences

Abstract.  Cold cells innervating antennal campaniform sensilla of the ground beetles Pterostichus oblongopunctatus (Fabricius, 1787) and Poecilus cupreus (Linnaeus, 1758) belonging to the tribe Pterostichini fire at a stationary rate of 22–23 imp s⁻¹ at 23 °C. In P. oblongopunctatus , there is a strong negative correlation between the stationary firing rate of the cold cell and temperature. By contrast, no relationship between the firing rate and temperature is observed in P. cupreus. Mean peak frequencies, reaching up to nearly 500 Hz, and first-second firing rates, reaching up to 140 imp s⁻¹, are observed at the beginning of the phasic-tonic response to rapid cooling of the cold cells of P. cupreus, which are significantly higher than those for P. oblongopunctatus . However, firing rates of the cold cells of the two ground beetles studied do not differ 3 s later, during the tonic stabilization period of the response. The length of the long interspike period, lasting up to several seconds, at the beginning of rapid warming, is a positive function of the extent of change in temperature, and is longer in P. oblongopunctatus than in P. cupreus . These differences in the responses of the cold cells are related to the ecological preferences of the two ground beetles.     

Autoantibodies Against an Extracellular Peptide of the GluR3 Subtype of AMPA Receptors Activate Both Homomeric and Heteromeric AMPA Receptor Channels

Autoantibodies to the GluR3-subtype of AMPA/glutamate receptors are found in the sera and cerebrospinal fluid of some individuals with epilepsy. They could possibly play a role in the pathophysiology of epilepsy since anti-GluR3 sera display glutamatergic agonist activity. We have investigated here the ability of affinity-purified antibodies (Abs) directed against the immunogenic peptide GluR3B (amino-acid 372–395) to interact with and activate recombinant GluR3-receptor channels expressed by Xenopus oocytes. We report here that the affinity-purified anti-GluR3B Abs directly activate GluR3-containing homomeric and heteromeric AMPA receptor complexes without the requirement of neuronal, glial or blood ancillary molecules. We present some of the properties of the purified anti-GluR3B Abs and discuss the possible physiological or pathological consequences of their activation of glutamate receptors.     

Glomerular targets of Heliothis subflexa male olfactory receptor neurons housed within long trichoid sensilla

We used single-sensillum recordings to characterize male Heliothis subflexa antennal olfactory receptor neuron physiology in response to compounds related to their sex pheromone. The recordings were then followed by cobalt staining in order to trace the neurons' axons to their glomerular destinations in the antennal lobe. Receptor neurons responding to the major pheromone component, (Z)-11-hexadecenal, in the first type of sensillum, type-A, projected axons to the cumulus of the macroglomerular complex (MGC). In approximately 40% of the type-A sensilla, a colocalized receptor neuron was stained that projected consistently to the posterior complex 1 (PCx1), a specific glomerulus in an 8-glomerulus complex that we call the Posterior Complex (PCx). We found that receptor neurons residing in type-B sensilla and responding to a secondary pheromone component, (Z)-9-hexadecenal, send their axons to the dorsal medial glomerulus of the MGC. As in the type-A sensilla, we found a cocompartmentalized neuron within type-B sensilla that sends its axon to a different glomerulus of the PCx4. One neuron in type-C sensilla tuned to a third pheromone component, (Z)-11-hexadecenol, and a colocalized neuron responding to (Z)-11-hexadecenyl acetate projected their axons to the anteromedial and ventromedial glomeruli of the MGC, respectively.     

Responses of antennal campaniform sensilla to rapid temperature changes in ground beetles of the tribe platynini with different habitat preferences and daily activity rhythms

Responses of temperature sensitive (cold) cells from the antenna of ground beetles (tribe Platynini) were compared in species with different ecological preferences and daily activity rhythms. Action potential rates were characterized at various temperatures (ranges 23-39 degrees C) and during rapid changes in it (Deltat=0.5-15 degrees C). The stationary firing frequencies were nearly twice as high in eurythermic open field ground beetles Agonum muelleri and Anchomenus dorsalis (firing rates ranging from 22 to 47imp/s) than in a stenothermic forest species Platynus assimilis. In the eurythermic species, the firing rate did not significantly depend on temperature (Anchomenus dorsalis range of 23-27 degrees C and Agonum muelleri range of 23-33 degrees C) but plots of firing rate versus temperature showed rapid declines when lethally high temperatures were approached. In contrast, a nearly linear decline of the firing rate/temperature curve was observed in Platynus assimilis. Responses to rapid temperature decreases were also considerably higher in eurythermic species. Both the peak frequency of the initial burst (maximum 420-650Hz) as well as the sustained discharge in the first 4s of the response were higher than in Platynus assimilis. Long silent periods, lasting up to several seconds, that occurred at the beginning of the response to rapid warming were significantly shorter in Agonum muelleri and Anchomenus dorsalis compared to Platynus assimilis. These findings suggest that the responses of thermoreceptors to temperature changes may be correlated with specific ecological preferences.     

Characterization of Recombinant Human Cardiac KCNQ1/KCNE1 Channels (I Ks) Stably Expressed in HEK 293 Cells

The present study was designed to characterize pharmacological, biophysical and electrophysiological properties of the recombinant human cardiac I _Ks (KCNQ1/KCNE1) channels at physiological temperature. Human cardiac KCNQ1 and KCNE1 genes were cotransfected into HEK 293 cells, and a cell clone stably expressing both genes was selected. Membrane currents were recorded using a perforated patch-clamp technique. The typical I _Ks was slowly activated upon depolarization voltages in HEK 293 cells stably expressing human cardiac KCNQ1 and KCNE1 genes, and the current was inhibited by I _Ks blockers HMR 1556 and chromanol 293B, with 50% inhibitory concentrations (IC₅₀s) of 83.8 nM and 9.2 μM, respectively. I _Ks showed a significant temperature-dependent increase in its magnitude upon elevating bath temperature to 36°C from room temperature (21°C). The current was upregulated by the β-adrenoceptor agonist isoproterenol, and the effect was reversed by H89. In addition, I _Ks was inhibited by Ba²⁺ in a concentration-dependent manner (IC₅₀ = 1.4 mM). Action potential clamp revealed a “bell-shaped” time course of I _Ks during the action potential, and maximal peak current was seen at the plateau of the action potential. A significant use- and frequency-dependent increase of I _Ks was observed during a train of action potential clamp. These results indicate that the recombinant human cardiac I _Ks stably expressed in HEK 293 cells is similar to native I _Ks in drug sensitivity and regulated by Ba²⁺ and β-adrenoceptor via the cyclic adenosine monophosphate/protein kinase A pathway. Importantly, the current exhibits significant temperature dependence, a bell-shaped time course during action potential and prominent use- or frequency-dependent accumulation during a train of action potentials.