Paul Kosterin

An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology.

We demonstrate that high power light-emitting diodes (LEDs) exhibit low-frequency noise characteristics that are clearly superior to those of quartz tungsten halogen lamps, the non-coherent light source most commonly employed when freedom from intensity variation is critical. Their extreme stability over tens of seconds (combined with readily selectable wavelength) makes high power LEDs ideal light sources for DC recording of optical changes, from living cells and tissues, that last more than a few hundred milliseconds. These optical signals (DeltaI/I(0)) may be intrinsic (light scattering, absorbance or fluorescence) or extrinsic (absorbance or fluorescence from probe molecules) and we show that changes as small as approximately 8 x 10(-5) can be recorded without signal averaging when LEDs are used as monochromatic light sources. Here, rapid and slow changes in the intrinsic optical properties of mammalian peptidergic nerve terminals are used to illustrate the advantages of high power LEDs compared to filament bulbs.

Low frequency noise and long-term stability of noncoherent light sources

Low frequency fluctuations in light intensity were measured from five different types of commercially available light emitting diodes with wavelengths from 375 nm to 740 nm and from two different halogen lamps. At low frequencies below 10–100 Hz some light emitting diodes (LEDs) can provide lower levels of noise than halogen lamps. A noise quality factor β is proposed in order to characterize noise behavior of LEDs.

Changes in FAD and NADH Fluorescence in Neurosecretory Terminals Are Triggered by Calcium Entry and by ADP Production

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1–2 s) trains of stimuli. With fluorescence excitation at either 350 ± 20 or 450 ± 50 nm, and with emission measured, respectively, at 450 ± 50 or ≥ 520 nm, ΔF/Fo was ∼5–8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by ∼100 ms and were eliminated by 1 μM tetrodotoxin (TTX). The signals were partially inhibited by 500 μM Cd2+, by substitution of Mg2+ for Ca2+, by Ca2+-free Ringer’s with 0.5 mM EGTA, and by 50 μM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 μM), FCCP (0.3 μM), and NaN3 (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO2) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH2 is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca2+]i triggers an increase in FAD and NAD as FADH2 and NADH are oxidized, but that elevation of [Ca2+]i, alone cannot account for the totality of changes in intrinsic fluorescence.

Microparticles generated by decompression stress cause central nervous system injury manifested as neurohypophysial terminal action potential broadening

The study goal was to use membrane voltage changes during neurohypophysial action potential (AP) propagation as an index of nerve function to evaluate the role that circulating microparticles (MPs) play in causing central nervous system injury in response to decompression stress in a murine model. Mice studied 1 h following decompression from 790 kPa air pressure for 2 h exhibit a 45% broadening of the neurohypophysial AP. Broadening did not occur if mice were injected with the MP lytic agent polyethylene glycol telomere B immediately after decompression, were rendered thrombocytopenic, or were treated with an inhibitor of nitric oxide synthase-2 (iNOS) prior to decompression, or in knockout (KO) mice lacking myeloperoxidase or iNOS. If MPs were harvested from control (no decompression) mice and injected into naive mice, no AP broadening occurred, but AP broadening was observed with injections of equal numbers of MPs from either wild-type or iNOS KO mice subjected to decompression stress. Although not required for AP broadening, MPs from decompressed mice, but not control mice, exhibit NADPH oxidase activation. We conclude that inherent differences in MPs from decompressed mice, rather than elevated MPs numbers, mediate neurological injury and that a component of the perivascular response to MPs involves iNOS. Additional study is needed to determine the mechanism of AP broadening and also mechanisms for MP generation associated with exposure to elevated gas pressure.

Carbon monoxide inhalation increases microparticles causing vascular and CNS dysfunction

We hypothesized that circulating microparticles (MPs) play a role in pro-inflammatory effects associated with carbon monoxide (CO) inhalation. Mice exposed for 1h to 100 ppm CO or more exhibit increases in circulating MPs derived from a variety of vascular cells as well as neutrophil activation. Tissue injury was quantified as 2000 kDa dextran leakage from vessels and as neutrophil sequestration in the brain and skeletal muscle; and central nervous system nerve dysfunction was documented as broadening of the neurohypophysial action potential (AP). Indices of injury occurred following exposures to 1000 ppm for 1h or to 1000 ppm for 40 min followed by 3000 ppm for 20 min. MPs were implicated in causing injuries because infusing the surfactant MP lytic agent, polyethylene glycol telomere B (PEGtB) abrogated elevations in MPs, vascular leak, neutrophil sequestration and AP prolongation. These manifestations of tissue injury also did not occur in mice lacking myeloperoxidase. Vascular leakage and AP prolongation were produced in naïve mice infused with MPs that had been obtained from CO poisoned mice, but this did not occur with MPs obtained from control mice. We conclude that CO poisoning triggers elevations of MPs that activate neutrophils which subsequently cause tissue injuries.

Measuring Intrinsic Optical Signals from Mammalian Nerve Terminals

Intrinsic optical changes (light scattering signals) occur in mammalian nerve terminals during and immediately following the arrival of the action potential. In the neurohypophysis (posterior pituitary gland), the action potential is coupled to calcium-mediated secretion of the neuropeptides oxytocin and vasopressin. This excitation-secretion coupling is intimately related to extremely rapid changes in light scattering. These optical signals provide a millisecond-time-resolved monitor of events in the terminals that follow the arrival of the action potential and the entry of calcium. Light scattering procedures are designed to measure intrinsic optical signals from mammalian nerve terminals. In practice, these signals are remarkably simple to record from any of the mammalian neurohypophyses that have been studied. To date, this approach has been used successfully in mouse, rat, and guinea pig. This protocol provides instrumentation requirements and a method for preparation of the neurohypophysis so that intrinsic optical signals can be measured from nerve terminals. It also includes a discussion of the interpretation of the signals that are obtained.

GABA UPTAKE BY GAT1 MODULATES LONG-TERM OPTICAL CHANGES FOLLOWING ELECTRICAL STIMULATION OF THE PITUITARY GLAND NEUROINTERMEDIATE LOBE

Intrinsic optical changes that follow infundibular stalk stimulation of the neurointermediate lobe of the mouse pituitary gland exhibit three different phases that reflect three distinct physiological events. The first (E-wave) is the rapid light-scattering increase that is associated with a nerve terminal volume increase (mechanical spike), and that accompanies excitation of the neurohypophysial terminals by the invading action potential; the second (S-wave) is the slower light-scattering decrease that is tightly correlated with the secretion of the peptide hormones oxytocin and arginine vasopressin, and the third is the long-duration response (R-wave) that reflects cell volume changes in the pars intermedia. We have studied the E-wave and the S-wave in earlier publications. The R-wave, considered here, is sensitive to chloride replacement as well as to blockade of chloride channels. By blocking GABAA receptors (which are ligand-gated chloride channels) with pharmacological agents, and by applying GABA directly into the bathing solution, or evoking its release from GABAergic inputs, we have demonstrated that this long-duration optical response is sensitive to chloride movements and reflects GABA-induced changes in the intrinsic optical properties of the pars intermedia. The full time-course of this optical response takes minutes and, therefore, has to embody some other process (or processes) related to the restoration of resting physiological chloride concentrations, following the opening and closing of GABAA-receptor channels. Here we demonstrate that the shape of the R-wave, the long-lasting light-scattering signal, is indeed affected by the activity of GAT1, one of the sodium- and chloride-dependent GABA transporters.

INTRINSIC FLUORESCENCE CHANGES IN NERVE TERMINALS, EVOKED BY ACTION POTENTIALS, ARE MODULATED BY KREBS CYCLE SUBSTRATES

Electrical stimulation of the mammalian neurohypophysial infundibular stalk evokes the entry of Na+ and Ca2+ into the neurosecretory terminals during the action potential. These events, in turn, increase intracellular Ca2+ and activate NaK- and Ca-ATPases, prompting the mitochondria to increase oxidative phosphorylation which can be monitored by recording the changes in FAD and NADH fluorescence. This paper reflects our efforts to determine whether or not modulating the capacity of mitochondria to produce ATP, by changing the concentrations of two important substrates of the Krebs cycle of the nerve terminal mitochondria, pyruvate and glucose, has an effect on the intrinsic fluorescence changes triggered by action potential stimulation.