LD ratios and the entrainment of circadian activity in a nocturnal and a diurnal rodent

Summary The activity rhythms of 5 flying squirrels,Glaucomys volans, and 7 chipmunks,Tamias striatus, were examined under controlled conditions in the laboratory. Free-running, circadian rhythms were demonstrated using a total of 25 LL or DD experiments. With 46 LD schedules the limits of entrainment in a 24-hour day were determined, and the phase angle difference for each schedule measured.Glaucomys was able to synchronize to schedules ranging from 1 second of light per 24-hour day to at least 18 hours light per day with little or no change in the phase angle.Tamias showed an oscillatory type of entrainment when the photoperiod was less than 3 hours per 24-hour day or greater than 23 hours, but in the intervening region was capable of stable entrainment. A tendency was evident for the phase angle difference to become less positive as the LD ratio increased. InGlaucomys single, isolated light pulses of either one second or 24 hours duration were able to bring about relatively large shifts in the phase of the activity rhythm.Dedicated to Professor Jürgen Aschoff on the occasion of his 60th birthday.I wish to acknowledge the hospitality and assistance of the Zoology Department, University of Wisconsin, Madison, the Max-Planck Institute, Erling-Andechs, Germany, and the Belle W. Baruch Coastal Research Institute, University of South Carolina, Columbia. Special thanks are due Dr. John Emlen and Dr. William Reeder of Madison, Prof. J. Aschoff of Erling-Andechs, Dr. John Vernberg and Dr. Winona Vernberg of Columbia, and my husband, Dr. George DeCoursey, for their untiring encouragement and help with these experiments.     

Charge compensation during the phagocyte respiratory burst

The phagocyte NADPH oxidase produces superoxide anion (O(2)(.-)) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O(2)(*-) to H(2)O(2) and then to HOCl. A small contribution by K+ or Cl- fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.     

Strong glucose dependence of electron current in human monocytes

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that by neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even in the absence of glucose. Human peripheral blood monocytes (PBM), in contrast, are suspected not to be able to produce any ROS if glucose is absent from the media. Here we compare ROS production by monocytes and neutrophils, measured electrophysiologically on a single-cell level. Perforated-patch-clamp measurements revealed that electron current appeared after stimulation of PBM with phorbol myristate acetate. Electron current reflects the translocation of electrons through the NADPH oxidase, the main source of ROS production. The electron current was nearly abolished by omitting glucose from the media. Furthermore, in preactivated glucose-deprived cells, electron current appeared immediately with the addition of glucose to the bath. To characterize glucose dependence of PBM further, NADPH oxidase activity was assessed as hydrogen peroxide (H(2)O(2)) production and was recorded fluorometrically. H(2)O(2) production exhibited similar glucose dependence as did electron current. We show fundamental differences in the glucose dependence of ROS in human monocytes compared with human neutrophils.     

The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K(+) (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853-858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA(2) or the production of superoxide anion (O(2*)(-)). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn(2+) inhibition of NADPH oxidase activity assessed by H(2)O(2) production, thus validating previous studies showing that Zn(2+) inhibited O(2*)(-) production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.     

Idiosyncratic Gating of HERG-like K+ Channels in Microglia

A simple kinetic model is presented to explain the gating of a HERG-like voltage-gated K⁺ conductance described in the accompanying paper (Zhou, W., F.S. Cayabyab, P.S. Pennefather, L.C. Schlichter, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:781–794). The model proposes two kinetically distinct closing pathways, a rapid one favored by depolarization (deactivation) and a slow one favored by hyperpolarization (inactivation). The overlap of these two processes leads to a window current between −50 and +20 mV with a peak at −36 mV of ∼12% maximal conductance. The near absence of depolarization-activated outward current in microglia, compared with HERG channels expressed in oocytes or cardiac myocytes, can be explained if activation is shifted negatively in microglia. As seen with experimental data, availability predicted by the model was more steeply voltage dependent, and the midpoint more positive when determined by making the holding potential progressively more positive at intervals of 20 s (starting at −120 mV), rather than progressively more negative (starting at 40 mV). In the model, this hysteresis was generated by postulating slow and ultra-slow components of inactivation. The ultra-slow component takes minutes to equilibrate at −40 mV but is steeply voltage dependent, leading to protocol-dependent modulation of the HERG-like current. The data suggest that “deactivation” and “inactivation” are coupled through the open state. This is particularly evident in isotonic Cs⁺, where a delayed and transient outward current develops on depolarization with a decay time constant more voltage dependent and slower than the deactivation process observed at the same potential after a brief hyperpolarization.     

Phylogenetic significance of the kinethmoid‐associated Y‐shaped ligament and long intercostal ligaments in the Cypriniformes (Actinopterygii: Ostariophysi)

Liao, T.Y. & Kullander, S.O. (2012). Phylogenetic significance of the kinethmoid‐associated Y‐shaped ligament and long intercostal ligaments in the Cypriniformes (Actinopterygii: Ostariophysi). —Zoologica Scripta, 42, 71–87. The phylogenetic significance of the Y‐shaped and long intercostal ligaments in the Cypriniformes is examined using character optimization in 184 species representing 20 non‐ostariophysan teleost species, five ostariophysan orders, seven cypriniform families and 14 cyprinid subfamilies. Character states were optimized on the phylogenetic trees of previous studies. Given the topology of Saitoh et al. (2011) , the Y‐shaped ligament, connecting the kinethmoid to the ethmoid complex, is shown to be a synapomorphy for the Cyprinidae, with reversals observed in the Cyprininae, Danioninae, Gobioninae and Psilorhynchinae. The condition of the Y‐shaped ligament is consistent within most subfamilies with a few exceptions. Despite the exceptions, the Y‐shaped ligament may be considered as a diagnostic character distinguishing cyprinid subfamilies with otherwise similar morphology, that is, the Danioninae and Opsariichthyinae. The long intercostal ligament, connecting five to eight ribs and ascending from the subdistal end of the fifth rib, is present in the Catostomidae and all cyprinid subfamilies, except for the Psilorhynchinae and two developmentally truncated genera, Danionella and Paedocypris. In addition to these two cypriniforme families, the long intercostal ligament is homoplastically present in some catfishes. Given the topology of Saitoh et al. (2011) , presence of the long intercostal ligament is a synapomorphy of Cyprinidae+Catostomidae. Some shorter ligaments are also present in the Cypriniformes and Chilodus gracilis (Characiformes), near the base of the anterior ribs and only occurring anterodorsally to the putative line of the long intercostal ligament even when it is absent.     

Hydrogen ion currents in rat alveolar epithelial cells.

Alveolar epithelial cells isolated from rats and maintained in primary culture were studied using the whole-cell configuration of the "patch-clamp" technique. After other ionic conductances were eliminated by replacing permeant ions with N-methyl-D-glucamine methanesulfonate, large voltage-activated hydrogen-selective currents were observed. Like H+ currents in snail neurons and axolotl oocytes, those in alveolar epithelium are activated by depolarization, deactivate upon repolarization, and are inhibited by Cd2+ and Zn2+. Activation of H+ currents is slower in alveolar epithelium than in other tissues, and often has a sigmoid time course. Activation occurs at more positive potentials when external pH is decreased. Saturation of the currents suggests that diffusion limitation may occur; increasing the pipette buffer concentration from 5 to 120 mM at a constant pH of 5.5 increased the maximum current density from 8.7 to 27.3 pA/pF, indicating that the current amplitude can be limited in 5 mM buffer solutions by the rate at which buffer molecules can supply H+ to the membrane. These data indicate that voltage-dependent H+ currents exist in mammalian cells.     

Light-sampling behavior in photoentrainment of a rodent circadian rhythm

Summary Behavioral aspects of photoentrainment of circadian locomotor activity rhythms were recorded for a nocturnal den-dwelling rodent, the flying squirrel,Glaucomys volans. Methods included both telemetric monitoring and infrared observations of animals under constant dark (DD) or light-dark (LD) schedules in either standard wheel cages or in newly developed simulated den cages. By means of the den cages, several aspects of a circadian activity cycle could be simultaneously measured emphasizing the arousal from rest, the light-sampling behavior by which a squirrel assessed the environmental photoregimen, and the phase-shifting by which photoentrainment was achieved. Each animal in a den cage remained for 12 or more hours of its rest period almost exclusively in the darkened nest box, then at an abrupt arousal time moved to the light-sampling porthole. In darkness each animal initiated wheel activity immediately after arousal; light at arousal time, however, induced a return to the nest box for a nap and a delay phase-shift in onset of activity of approximately 40 min. On subsequent days, each animal appeared to be free-running ( _FR< 24 h) until onset again advanced into the light period. A squirrel usually viewed only a few minutes light per day, and on free-running days occasionally saw none of the 12-h light period. The significance of these data for theories of circadian photoentrainment is discussed.Abbreviations CT circadian time - PRC phase response curve - SCN suprachiasmatic nucleus     

Rejecting strictly allopatric speciation on a continental island: prolonged postdivergence gene flow between Taiwan (Leucodioptron taewanus,Passeriformes Timaliidae) and Chinese (L. canorum canorum) hwameis

Allopatry is conventionally considered the geographical mode of speciation for continental island organisms. However, strictly allopatric speciation models that assume the lack of postdivergence gene flow seem oversimplified given the recurrence of land bridges during glacial periods since the late Pliocene. Here, to evaluate whether a continental island endemic, the Taiwan hwamei (Leucodioptron taewanus, Passeriformes Timaliidae) speciated in strict allopatry, we used weighted‐regression‐based approximate Bayesian computation (ABC) to analyse the genetic polymorphism of 18 neutral nuclear loci (total length: 8500 bp) in Taiwan hwamei and its continental sister species, the Chinese hwamei (L. canorum canorum). The nonallopatry model was found to fit better with observed genetic polymorphism of the two hwamei species (posterior possibility = 0.82). We also recovered unambiguous signals of nontrivial bidirectional postdivergence gene flow (N_em » 1) between Chinese hwamei and Taiwan hwamei until 0.5 Ma. Divergence time was estimated to be 3.5 to 2 million years earlier than that estimated from mitochondrial cytochrome b sequences. Finally, using the inferred nonallopatry model to simulate genetic variation at 24 nuclear genes examined showed that the adiponectin receptor 1 gene may be under divergent adaptation. Our findings imply that the role of geographical barrier may be less prominent for the speciation of continental island endemics, and suggest a shift in speciation studies from simply correlating geographical barrier and genetic divergence to examining factors that facilitate and maintain divergence, e.g. differential selection and sexual selection, especially in the face of interpopulation gene flow.