A.L. Obaid

Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis

The submucous plexus of the guinea pig intestine is a quasi-two-dimensional mammalian neural network that is particularly amenable to study using multiple site optical recording of transmembrane voltage (MSORTV) [Biol. Bull. 183 (1992) 344; J. Neurosci. 19 (1999) 3073]. For several years the potentiometric dye of choice for monitoring the electrical activity of its individual neurons has been di-8-ANEPPS [Neuron 9 (1992) 393], a naphthylstyryl-pyridinium dye with a propylsulfonate headgroup that provides relatively large fluorescence changes during action potentials and synaptic potentials. Limitations to the use of this dye, however, have been its phototoxicity and its low water solubility which requires the presence of DMSO and Pluronic F-127 in the staining solution. In searching for less toxic and more soluble dyes exhibiting larger fluorescence signals, we first tried the dienylstyryl-pyridinium dye RH795 [J. Neurosci. 14 (1994) 2545] which is highly soluble in water. This dye yielded relatively large signals, but it was internalized quickly by the submucosal neurons resulting in rapid degradation of the signal-to-noise ratio. We decided to synthesize a series of naphthylstyryl-pyridinium dyes (di-n-ANEPPDHQ) having the same chromophore as di-8-ANEPPS and the quaternary ammonium headgroup (DHQ) of RH795 (resulting in two positive charges versus the neutral propylsulfonate-ring nitrogen combination), and we tested the di-methyl (JPW3039), di-ethyl (JPW2081), di-propyl (JPW3031), di-butyl (JPW5029), and di-octyl (JPW5037) analogues, all of them soluble in ethanol. We found that the di-propyl (di-3-ANEPPDHQ) and the di-butyl (di-4-ANEPPDHQ) forms yielded the best combination of signal-to-noise ratio, moderate phototoxicity and absence of dye internalization.

Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon.

The characteristics of transmittance and fluorescence changes of 4-(p-aminostyryl)-1-pyridinium dyes in response to voltage-clamp pulses on the squid giant axon were examined. A zwitterionic styryl dye displays transmittance and excitation spectra on the voltage-clamped squid axon with shapes similar to those previously measured on a model membrane system and consistent with a postulated electrochromic mechanism. The speed of the transmittance response is faster than 1.2 microseconds. The size of the fluorescence change is a factor of 40 lower than on the model membrane; this diminution can be rationalized in terms of the background fluorescence from Schwann cells and the nonoptimal geometric arrangement of the axon membrane. When the emission spectrum is dissected from the excitation response, a nonelectrochromic component is found. This component might result from molecular motion during the excited state lifetime. A positively charged dye permeates the axon membrane and displays complex response waveforms dependent on the method of application and the axon holding potential. This contrasts markedly with model membrane results where the behavior of the cationic and zwitterionic dyes were indistinguishable.

Action Potentials and Frequency-Dependent Secretion in the Mouse Neurohypophysis

The frequency-dependence of secretion of arginine vasopressin (AVP) from the mouse neural lobe in vitro was studied and found to be comparable to that reported for the rat neural lobe in vitro. For a stimulus train of 600 pulses, the secretion of AVP per pulse (i.e., facilitation) increased to a maximum at 20 Hz. Compound intracellular action potentials were recorded from the mouse neural lobe using optical recording methods and potentiometric dyes. These extrinsic optical signals reflect the true time courses of transmembrane potential changes (e.g., action potentials), and the action potentials recorded from mouse neural lobes had a duration of 5 ms; at half-maximum peak height. Optical recordings during repetitive stimulation showed that significant spike broadening occurred in each subsequent spike at 10 and 16 Hz stimulation. These data are consistent with a spike broadening hypothesis of frequency-dependent facilitation in the neural lobe. However, 4-aminopyridine, a drug which causes spike broadening in neural tissues by blocking potassium channels, did not produce an increase in secretion of AVP per stimulus from the mouse neural lobe.

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.

Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.

SUMMARY Nicotinic transmission in the enteric nervous system (ENS) is extensive, but the role of individual nicotinic acetylcholine receptor (nAChR) subtypes in the functional connectivity of its plexuses has been elusive. Using monoclonal antibodies (mAbs) against neuronal α3-, α4-,α 3/α5-, β2-, β4- and α7-subunits, combined with radioimmunoassays and immunocytochemistry, we demonstrate that guinea-pig enteric ganglia contain all of these nAChR-subunits with the exception ofα 4, and so, differ from mammalian brain. This information alone, however, is insufficient to establish the functional role of the identified nAChR-subtypes within the enteric networks and, ultimately, their specific contributions to gastrointestinal physiology. We have used voltage-sensitive dyes and a high-speed CCD camera, in conjunction with specific antagonists to various nAChRs, to elucidate some of the distinct contributions of the individual subtypes to the behaviour of enteric networks. In the guinea-pig, the submucous plexus has the extraordinary advantage that it is virtually two-dimensional, permitting optical recording, with single cell resolution, of the electrical activity of all of its neurones. In this plexus, the block of α3β2-, α3β4- and/or α7-nAChRs always results in a decrease in the magnitude of the synaptic response. However, the magnitude of the fast excitatory post-synaptic potentials (epsps) evoked by electrical stimulation of a neighbouring ganglion varies from cell to cell, reflecting the differential expression of subunits already observed using mAbs, as well as the strengths of the activated synaptic inputs. At the same time, we observe that submucous neurones have a substantial mecamylamine (Mec)-insensitive (non-nicotinic) component to their fast epsps, which may point to the presence of purinergic or serotonergic fast epsps in this system. In the myenteric plexus, on the other hand, the antagonist-induced changes in the evoked synaptic response vary depending upon the location of the stimulating electrode with respect to the ganglion under study. The range of activity patterns that follows sequential pharmacological elimination of individual subtypes suggests that nAChRs may be capable of regulating the activity of both excitatory and inhibitory pathways, in a manner similar to that described in the central nervous system.

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.

HCO 3 − /Cl− exchange across the human erythrocyte membrane: Effects of pH and temperature

SummaryChanges in extracellular pH (pHo) in red cell suspensions were monitored in a stopped-flow rapid reaction apparatus under conditions wheredpHo/dt was determined by the rate of HCO3−/Cl− exchange across the membrane. Experiments were performed at 5°C<T<40°C using either untreated cells or cells exposed to 0.11mm SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid). Although SITS exposure reduced the rate of exchange by 90%, both untreated and SITS-treated cells are similarly affected by changes in pH0 and temperature. The rate of HCO3−/Cl− exchange exhibits a minimum at about pHo 5 and a maximum at about pH0 7.4 at all temperatures. A transition temperature of 17°C was observed in the Arrhenius relationship for all pH0. The activation energies (Ea) in kcal/mol are 19.6 below and 11.7 above 17°C for 5<pH0<8. These findings, similar to those reported for Cl− self-exchange, suggest that: (i) a change in the rate-limiting step for HCO3−/Cl− exchange occurs at 17°C, possibly due to an altered interaction between the transport pathway and membrane lipids; (ii) the carrier system can be titrated by either H+ or SITS from the outside of the membrane, but the untitrated sites continue to transport normally; (iii) the pH0 dependence of the rate of exchange is consistent with the titratable carrier having its most alkaline pK in the range expected for amino groups; and (iv) below pH0 5, the nature of the exchange is markedly altered.

The effects of maleic anhydride on the ionic permeability of red cells

SummaryMaleic anhydride (MA) has been shown to react specifically and rapidly with amino groups of proteins; the maleyl amino groups are negatively charged and completely stable at neutral pH. Treatment of human red cells with this reagent results in a significant increase in K+ permeability which is associated with a much smaller increase in Na+ permeability. Opposite effects are observed on anion permeability, the SO4−− and Cl− permeability being decreased to an approximately similar extent upon treatment with MA.Studies on the distribution of MA between membrane lipids and proteins shows that most of the membrane-bound MA is associated with membrane proteins. These results suggest that the observed effects of MA on the ion permeability of the red cell are caused by its combination with amino groups of cell membrane proteins.

Bicarbonate-Chloride Exchange in Erythrocyte Suspensions: Stopped-Flow pH Electrode Measurements

A pH-sensitive glass electrode was used in a temperature-controlled stopped-flow rapid reaction apparatus to determine rates of pH equilibration in red cell suspensions. The apparatus requires less than 2 ml of reactants. The electrode is insensitive to pressure and flow variations, and has a response time of < 5 ms. A 20% suspension of washed fresh human erythrocytes in saline at pH 7.7 containing NaHCO(3) and extracellular carbonic anhydrase is mixed with an equal volume of 30 mM phosphate buffer at pH 6.7. Within a few milliseconds after mixing, extracellular HCO(3) (-) reacts with H(+) to form CO(2), which enters the red cells and rehydrates to form HCO(3) (-), producing an electrochemical potential gradient for HCO(3) (-) from inside to outside the cells. HCO(3) (-) then leaves the cells in exchange for Cl(-), and extracellular pH increases as the HCO(3) (-) flowing out of the cells reacts with H(+). Flux of HCO(3) (-) is calculated from the dpH/dt during HCO(3) (-)-Cl(-) exchange, and a velocity constant is computed from the flux and the calculated intracellular and extracellular [HCO(3) (-)]. The activation energy for the exchange process is 18.6 kcal/mol between 5 degrees C and 17 degrees C (transition temperature), and 11.4 kcal/mol from 17 degrees C to 40 degrees C. The activation energies and transition temperature are not significantly altered in the presence of a potent anion exchange inhibitor (SITS), although the fluxes are markedly decreased. These findings suggest that the rate-limiting step in red cell anion exchange changes at 17 degrees C, either because of an alteration in the nature of the transport site or because of a transition in the physical state of membrane lipids affecting protein-lipid interactions.

Exchange of HCO 3 − for monovalent anions across the human erythrocyte membrane

SummaryA stopped-flow rapid reaction apparatus was used for measuring changes in extracellular pH (pHo) of red cell suspensions under conditions wheredpHo/dt was determined by the rate of HCO3−/X− exchange across the membrane (X−=Cl−, Br−, F−, I−, NO3− or SCN−). The rate of the exchange at 37°C decreased forX− in the order: Cl−>Br−>F−>I−>NO3−>SCN−, with rate constants in the ratios 1∶0.86∶0.77∶0.55∶0.52∶0.31. When HCO3− is exchanged for Cl−, Br−, F−, NO3− or SCN−, a change in the rate-limiting step of the process takes place at a transition temperature (TT) between 16 and 26°C. In I− medium, however, no transition temperature is detected between 3 and 42°C. AlthoughTT varies withX−, the activation energies both above and belowTT are similar for Cl−, Br−, NO3− and F−. The values of activation energy are considerably higher whenX−=I− or SCN−. The apparent turnover numbers calculated for HCO3−/X− exchange (except forX−=I−) at the correspondingTT ranged from 140 to 460 ions/site ·sec for our experimental conditions. These findings suggest that: (i) HCO3−/X− exchange for allX− studied takes place via the rapid anion exchange pathway; (ii) the rate of HCO3−/X− exchange is influenced by the specific anions involved in the 1∶1 obligatory exchange; and (iii) the different transition temperatures in the Arrhenius diagrams of the HCO3−/X− exchange do not seem to be directly related to a critical turnover number, but may be dependent upon the influence ofX− on protein-lipid interactions in the red blood cell membrane.