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Abstract To aid in the diagnosis, monitoring, and surveillance of bladder carcinoma, this study aimed to develop and test an algorithm that creates a referenceable bladder map rendered from standard cystoscopy videos without the need for specialized equipment. A vision-based algorithm was developed to generate 2D bladder maps from individual video frames, by sequentially stitching image frames based on matching surface features, and subsequently localize and track frames during reevaluation. The algorithm was developed and calibrated in a 2D model and 3D anthropomorphic bladder phantom. The performance was evaluated in vivo in swine and with retrospective clinical cystoscopy video. Results showed that the algorithm was capable of capturing and stitching intravesical images with different sweeping patterns. Between 93% and 99% of frames had sufficient features for bladder map generation. Upon reevaluation, the cystoscope accurately localized a frame within 4.5 s. In swine, a virtual mucosal surface map was generated that matched the explant anatomy. A surface map could be generated based on archived patient cystoscopy images. This tool could aid recording and referencing pathologic findings and biopsy or treatment locations for subsequent procedures and may have utility in patients with metachronous bladder cancer and in low-resource settings.

Background: We formed a self-funded hypertension treatment group in a resource-poor community in rural Honduras. After training community health workers and creating protocols for standardized treatment, we used group membership fees to maintain the group, purchase generic medications in bulk on the local market, and hire a physician to manage treatment. We then assessed whether participation in the group improved treatment, medication adherence, and hypertension control. Design: This is a program evaluation using quasi-experimental design and no control group. Using data from the 86 members of the hypertension treatment group, we analyzed baseline and follow-up surveys of members, along with 30 months of clinical records of treatment, medication adherence, and blood pressure readings. Results: Our initial hypertension needs assessment revealed that at baseline, community hypertensives relied on the local Ministry of Health clinic as their source of anti-hypertensive medications and reported that irregular supply interfered with medication adherence. At baseline, hypertension group members were mainly female, overweight or obese, physically active, non-smoking, and non-drinking. After 30 months of managing the treatment group, we found a significant increase in medication adherence, from 54.8 to 76.2% (p

Dyslipidemia is a primary driver for chronic cardiovascular conditions and there is no comprehensive literature about its management in South Africa. The objective of this study was to assess the prevalence, awareness, treatment, and control of dyslipidemia in rural South Africa and how they are impacted by different behaviors and non-modifiable factors. To fulfill this objective we recruited for this cohort study adults aged ≥40 years residing in the Agincourt sub-district of Mpumalanga Province. Data collection included socioeconomic and clinical data, anthropometric measures, blood pressure (BP), HIV-status, point-of-care glucose and lipid levels. Framingham CVD Risk Score was ascribed to patients based upon categories for 10 year cardiovascular risk of low (

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.

Palytoxin (PTX), isolated from a zoanthid of the genus Palythoa, is the most potent marine toxin known. Intoxication by PTX leads to vasoconstriction, hemorrhage, ataxia, muscle weakness, ventricular fibrillation, pulmonary hypertension, ischemia and death. In this study, clonal A7r5 rat aortic smooth muscle cells were used to study the mechanism of PTX-mediated cytotoxicity. A7r5 cells exposed to PTX for > or = 15 min exhibited surface granularities, vacuoles and rounding. These alterations culminated in a loss of viability as indicated by marked increases in the release of lactate dehydrogenase. Electrophysiological recording from A7r5 cells disclosed a profound membrane depolarization and an increase in conductance to Na+ and K+. PTX-mediated cytotoxicity could not be reversed by washout or by the addition of 10 microM verapamil but was antagonized by 100 microM ouabain or by removal of extracellular Na+ or Ca2+. In light of the involvement of vascular smooth muscle in PTX poisoning, A7r5 cells could serve as a useful model to test specific drugs for treatment of PTX intoxication., (2005 John Wiley & Sons, Ltd.)

The actions of botulinum neurotoxin (BoNT) were studied on evoked release of the neurotransmitter glycine in primary mouse spinal cord cells. 3[H]-glycine was taken up by cells in physiological solution and released by depolarization with 56 mM K+ in the presence of 2 mM Ca2+. Release of 3[H]-glycine was found to be inhibited by BoNT serotypes A, B and E with similar potency ratios to those observed in the acutely isolated mouse diaphragm muscle. When spinal cord cultures were exposed to BoNT/A for 24 h, inhibition of 3[H]-glycine release was detected at toxin concentrations as low as 10(-14) M, and complete inhibition was observed at concentration >or=10(-12) M. Preincubation of BoNT/A with polyclonal equine antiserum led to antagonism of toxin-induced inhibition of 3[H]-glycine release in spinal cord cells and to protection of mice from the lethal effects of BoNT/A. It is concluded that spinal cord neurons are a useful model for studying botulinum intoxication and for evaluating BoNT antagonists.

Nerve-evoked contractions were studied in vitro in phrenic nerve-hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE-/-) mice and their wild-type littermates (AChE+/+). The AChE-/- mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE-/- mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals. In AChE-/- preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW 284C51). The ability of diaphragm muscles from AChE-/- mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms--including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region--were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors., (Copyright 2004 Wiley Periodicals, Inc.)

An animal dosing model and related maximum tolerated dose (MTD) were developed for repeated exposures in guinea pigs to three organophosphorus chemical warfare nerve agents (CWNA). Male animals were injected subcutaneously with sarin (GB), soman (GD) or VX once a day (Monday through Friday) for 2-, 4-, or 13-weeks. An initial 13-week study for each CWNA employed doses of vehicle (normal saline), 0.2x, 0.4x, 0.6x, and 0.8x the previously established acute LD(50). A 2-week and 4-week exposure were also performed for each agent at doses less than the 13-week MTD to verify lack of toxicity. Animals dosed daily for 13 weeks with 0.4x LD(50) of GB or GD or with 0.2x LD(50) of VX did not display signs of acute cholinergic toxicity. In animals dosed daily for either 2- or 4-weeks, the MTDs were 0.4x the acute LD(50) for all three CWNA. There were no differences among these groups and their respective vehicle controls for weight gains, body temperature, complete blood cell counts, blood chemistries, nor by histopathology. At the MTD in all groups, red blood cell cholinesterase activity one hour after the last exposure was inhibited up to 90% compared with controls. The toxicity observed with repeated doses above the MTD for up to chronic exposure durations was not significantly different from symptoms observed after acute exposure. For all three nerve agents the MTDs for subacute exposure durations can be expressed by the same constant fraction of the acute LD(50), despite differences in the absolute amount of nerve agent administered.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in the docking and fusion of synaptic vesicles with the nerve terminal in preparation for neurotransmitter release. SNAP-25 is also a substrate for three of the seven serotypes of botulinum neurotoxin (BoNT). Intoxication by BoNT/A, /C1 or /E results in weakness and paralysis of skeletal muscle due to cleavage of SNAP-25 (and syntaxin la in the case /C1) at discrete serotype-specific sites. To elucidate the role of SNAP-25 in muscle function in more detail, contractility and neuromuscular transmission were studied in a mutant mouse model termed coloboma. The coloboma mutation results from a contiguous deletion of 1-2 centiMorgans on chromosome 2, which includes the entire SNAP-25 locus and three other identified genes. Homozygotes do not survive beyond gestation day 6; heterozygotes (Cm/+) have a normal life-span but express reduced levels of SNAP-25 mRNA and protein in the brain. The consequences of the Cm/+ mutation on twitch and tetanic tension, quantal release of neurotransmitter and spinal motoneuron expression of SNAP-25 were examined in the present study. Contrary to expectations, Cm/+ mice exhibited no alteration in twitch tension and generated normal tetanic tension even at the highest frequency examined (800 Hz). Microelectrode recordings revealed that MEPP amplitude and frequency were both within control limits. The ventral spinal cord of Cm/+ mice showed no deficiency in SNAP-25 content and immunohistochemical examination of nerve terminals in Cm/+ mice disclosed that SNAP-25 levels and distribution were similar to those of control mice. It is concluded that spinal motor neurons up-regulate SNAP-25 to preserve vital neuromuscular function.

The neutralization of botulinum toxin serotype A with polyclonal equine antitoxin was studied in isolated mouse hemidiaphragms and compared to the same action in live mice. The biological activity of the toxin in the isolated muscle could be markedly reduced with excess antitoxin, estimated as 3:1 molar ratios of IgG Ab:toxin or better. Toxin neutralization in vivo required higher ratios of Ab:toxin, ranging from 30:1 at high toxin doses and increasing to 100:1 at 10xLD50 toxin. At equimolar Ab to toxin ratios in the isolated muscle, the biological activity of the toxin underwent a statistically significant increase. This paradoxical effect of the polyclonal antisera was serotype selective and independent of the presence or absence of hemagglutinin in the toxin. The enhancement of toxin activity was subsequently localized to occupancy of one of four epitopes on the toxin using monoclonal antibodies to mimic the effect of the antitoxin. The enhancement of toxin activity suggests that botulinum toxin may undergo a conformational change upon binding antibodies to certain domains. This phenomenon could contribute to the observed concentration dependent changes in neutralization efficacy with antitoxin in vivo.

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.

Measurements of the efficacy of novel botulinum toxin antagonists can be based on classical bioassays of toxin concentration. However, the relative sensitivities of in vivo and in vitro assays to the effects of antagonists are not necessarily correlated with the sensitivities of the assays to toxin. Comparisons of the sensitivity of an in vitro mouse muscle contraction assay with an in vivo mouse survival assay indicated that the in vivo assay was more sensitive to botulinum toxin serotype A by more than one order of magnitude at equivalent molar concentrations. However, in studies of toxin neutralization with equine antisera, the in vitro muscle assay was more than three times more sensitive to the presence of antisera than the equivalent mouse survival assay. For the development of new drugs to treat botulism, antagonist sensitivity is a primary consideration in determining relative efficacy during structure-activity studies. Thus, in studies of toxin antagonists, the in vitro assay appears to be superior for initial testing.

Botulinum neurotoxin (BoNT) is known to produce cationic channels in artificial bilayers. This study examined ion channels formed by BoNT in native membranes from cultured PC12 cells under conditions approximating those thought to occur during toxin internalization. Membrane patches were excised from PC12 cells using patch electrodes and exposed to symmetrical solutions containing either 200 mM CsCl, RbCl or KCl. The patch pipettes also contained 1-5 microg/ml BoNT buffered to pH 5.3 while the bath solutions were buffered to pH 7.0. In the presence of toxin, bursts of ion channel openings were observed. These toxin-induced channels were most active with a negative voltage applied to the same side as the toxin (cis). The increased activity at negative voltages was due to an increase in mean open time of e-fold per 120 mV and a decrease in mean closed time between bursts of e-fold per 110 mV. The shorter mean closed time within a burst was independent of membrane voltage. While BoNT-induced ion channels started as a single conductance level of 27 pS (KCl), 34 pS (RbCl) or 46 pS (CsCl) they typically increased in roughly equal steps to five or more times the original channel conductance. These higher conductance BoNT 'channels' opened and closed synchronously and could be distinguished from superposition of multiple independent channels. Despite differences in putative transmembrane sequences between BoNT/A and BoNT/E, both serotypes evidenced the same channel conductance and mean open time.

Certain aminoquinoline antimalarial compounds, such as chloroquine, antagonize the paralytic actions of botulinum neurotoxins (BoNT). These studies have been extended to determine the critical structural groups necessary for synthetic aminoquinolines to have antagonist activity against BoNT. Isolated mouse hemidiaphragms were maintained at 36 degrees C and indirectly stimulated; the resulting isometric twitch tensions were recorded as a measure of synaptic function. The muscles were exposed to the test compounds before being treated with a challenge concentration of BoNT (typically 0.2 nM of serotype A). The time to onset of 50% muscle paralysis due to BoNT was used to assess quantitatively the efficacy of the test compounds, which were then ranked on the basis of the concentrations necessary to delay paralysis by a specified time increment. Of the compounds tested, those having a 7-chloro-4-aminoquinoline configuration, similar to chloroquine (or the structurally similar 6-chloro-9-amino acridine group in quinacrine), were most effective. Truncation of the alkyl-amino-alkyl group from chloroquine and conversion of the 4-amino nitrogen to a primary amine did not significantly alter its effectiveness as a BoNT antagonist. However, the 6-chloro- or 8-chloro- isomers of chloroquine were essentially ineffective. These results suggest that aminoquinolines antagonize the paralytic actions of BoNT through interaction with a selective, stereospecific site that is not well correlated with antimalarial activity.

Various 4- and 8-aminoquinolines, which are effective antimalarial agents, were examined as potential pretreatment compounds for prolongation of the time to 50% block of nerve-elicited muscle twitches in isolated mouse diaphragms exposed to botulinum type A neurotoxin. The 4-aminoquinolines (chloroquine, amodiaquine) and quinacrine, an acridine derivative similar to chloroquine, prolonged the time required for botulinum type A neurotoxin to block neuromuscular transmission by more than 3-fold; 8-aminoquinolines (primaquine and WR242511) had no antibotulinum type A neurotoxin activity. Pyrimethamine, an antimalarial drug lacking the quinoline ring structure, was also ineffective. Rank order potencies based on equimolar effective concentrations for the test compounds were quinacrine > amodiaquine > chloroquine > quinine or quinidine. Maximum protection from botulinum type A neurotoxin-induced neuromuscular block was achieved when muscles were exposed to drug prior to or simultaneously with the toxin. A delay of more than 20 min abolished the protective ability of the antimalarial agents, presumably owing to the release of the toxin from endosomes in quantities sufficient to initiate neuromuscular block. All of the test compounds except quinine and quinidine depressed muscle contractions when concentrations exceeded 20 microM. In addition, amodiaquine at 50 microM induced muscle contracture. A combination of agents at low concentrations that act at different steps of botulinum type A neurotoxin poisoning potentiated the prolongation of time to 50% block in an approximately additive fashion. Thus N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (2 microM) and quinacrine (5 microM), when administered in combination, produced up to a 4-fold increase in time to 50% block. A similar level of protection with quinacrine alone required a 4-fold increase in the aminoquinoline concentration. Although the mechanism of protection by these antimalarial agents is probably through the raising of endosomal pH, the possibility that some of these drugs could also act by inhibiting toxin-induced channel formation cannot be ruled out.

Botulinum neurotoxins (BoNT) are thought to enter cells through endocytotic vesicles where acidification is required for release of these toxins into the cytoplasm. Two ionophores, nigericin and monensin, that increase membrane permeability to H+ and K+ or H+, Na+ and K+, respectively, block vesicle acidification by acting as H+ shunts to neutralize pH gradients. Nanomolar concentrations of nigericin or monensin delayed development of blockade in BoNT-A or BoNT-B treated muscles two-to threefold over onset times in unprotected muscles. However, higher concentrations of the ionophores directly blocked synapses. Thus, nigericin and monensin could delay onset of BoNT paralysis only over a narrow range of concentrations.

Exposure of isolated mouse hemidiaphragms to botulinum neurotoxins, 0.1 nM BoNT-A or BoNT-B, at 36 degrees C reduced nerve-elicited peak isometric twitch tension to 50% of control values at 55 min (BoNT-A) to 68 min (BoNT-B) after application. Either coincubation of BoNT with the heavy metal chelator TPEN, preincubation with TPEN followed by BoNT, or application of TPEN after BoNT but before neuromuscular block, delayed the onset of muscle failure in a dose-dependent manner by up to five-fold. TPEN doses between 2 and 10 microM were required to antagonize significantly the muscle block produced by BoNT, and the delay in onset was maximal between 10 and 50 microM TPEN. Treatment of muscles with a Zn(2+)-TPEN coordination complex, rather than TPEN alone, eliminated any beneficial effects of TPEN on BoNT intoxication, indicating that these effects were mediated by chelation of Zn2+. Other metal chelators that were not as membrane permeant as TPEN were ineffective in delaying BoNT paralysis, suggesting that TPEN acts by chelating intraterminal Zn2+. In the absence of BoNT, TPEN caused a dose-dependent increase in nerve-elicited twitch tension with a half-maximal concentration at 8 microM. There was no corresponding change in twitches from direct electrical stimulation of the muscle. After BoNT (A or B serotype) had reduced the muscle twitch by 20 to 70%, however, subsequent application of TPEN rapidly depressed nerve-elicited twitches. The shift from potentiation to depression after BoNT treatment suggests that presynaptic vesicle mobilization and/or release involve Zn(2+)-dependent enzymes and that BoNTs interact with these enzyme pathways.

Zinc-dependent metalloprotease inhibitors phosphoramidon, captopril and a peptide hydroxamate were studied as potential pretreatment compounds by examining their ability to delay the onset or to prolong the time to 50% block of nerve-elicited muscle twitch tension in the mouse phrenic-nerve diaphragm (in vitro at 36 degrees C) after botulinum neurotoxin serotypes A and B (BoNT-A, BoNT-B). Addition of BoNT-A or BoNT-B (1 x 10(-10) M) produced 50% block of the twitch response at 56 +/- 9 min and 76 +/- 4 min, respectively. Preincubation (45 min) of muscles with phosphoramidon (0.2 mM) prolonged the time to 50% block by 15 min in BoNT-B-poisoned muscles with no effect on the time-course of paralysis in BoNT-A exposed muscles. When the same quantities of BoNT-A or BoNT-B (equivalent to 1 x 10(-10) M bath concentration) were preincubated for 2 hr with phosphoramidon (equivalent to 0.2 mM final bath concentration), and the incubation mixture was added to the muscle chamber, the times to 50% block were prolonged by 38 min and 18 min for BoNT-B and BoNT-A, respectively. Preincubation of diaphragms with captopril (up to 10 mM) or peptide hydroxamate (75 microM) failed to antagonize BoNT-A or BoNT-B-induced neuromuscular block. Among the three metalloprotease inhibitors examined here, only phosphoramidon showed a significant protection against both serotypes of BoNT. A search for better inhibitor compounds specifically tailored to match the active site on BoNT molecule deserves attention.

The mechanism of inhibition of skeletal muscle function by brevetoxin (PbTX-3) was examined in vitro in the rat phrenic nerve-diaphragm preparation. PbTX-3 in low concentrations (< 0.06 microM) preferentially blocked conduction in the phrenic nerve without altering the resting membrane potential of the muscle fibers. Endplate potential failure occurred in an all-or-none fashion in the presence of PbTX-3 (> 0.06 microM). An increase in the frequency of miniature endplate potentials resulting from nerve terminal depolarization was observed only after endplate potential failure. Higher concentrations of toxin (> 0.3 microM) depressed directly-elicited muscle twitches and produced significant muscle membrane depolarization. Tetrodotoxin was effective in reversing membrane depolarization and alterations in MEPP frequency caused by PbTX-3. These findings suggest that diaphragmatic failure in PbTX-3 is primarily caused by a block of impulse conduction in the phrenic nerve due to a higher sensitivity of nerve than muscle membrane to the toxin.

Extracellular recordings were obtained from area CA1 of guinea pig hippocampal slices. PbTx-3, a brevetoxin fraction isolated from the red tide dinoflagellate Ptychodiscus brevis, was applied by bath perfusion. The toxin produced a concentration-dependent depression of the orthodromically evoked population spike with an EC50 of 37.5 nM. Brevetoxin concentrations below 10 nM were without effect, and concentrations above 100 nM led to total inhibition of evoked responses. PbTx-3 did not produce spontaneous synchronous discharges but did induce afterdischarges following evoked responses in about 50% of the slices tested, particularly at concentrations between 10 nM and 100 nM. Orthodromically evoked responses were more sensitive to PbTx-3 than were those elicited by antidromic stimulation. High-Ca2+ solution, 4-aminopyridine, and tetraethylammonium failed to antagonize either orthodromic or antidromic effects of the toxin. Although the precise mechanism by which PbTx-3 depresses evoked responses is not certain, depolarization of the presynaptic nerve terminals leading to failure of transmitter release could explain the toxin's actions. This is the first report of the effects of brevetoxin applied directly to central nervous system tissue.

Electrically evoked sodium currents were recorded under whole-cell patch clamp from undifferentiated HCN-1A cells. Peak sodium currents had a half-maximal activation, Vm0.5, of -22.6 +/- 1.0 mV with a voltage dependence, km, of 7.28 +/- 0.39 mV-1. Steady-state inactivation indicated the presence of two types of sodium channel. One type inactivated with Vh0.5 = -93.8 +/- 1.2 mV and kh = -6.8 +/- 0.4 mV-1. The second type of sodium channel inactivated with Vh0.5 = -44.6 +/- 1.5 mV and kh = -7.3 +/- 0.4 mV-1. The occurrence of each channel type varied from cell to cell and ranged from 0 to 100% of the total sodium current. No variation in the rate of inactivation was seen when the holding potential was adjusted to eliminate the more negative of the two inactivation components. Application of tetrodotoxin (TTX) or saxitoxin (STX) revealed channel types with two different affinities for each toxin. TTX blocked peak sodium conductance with apparent IC50s of 22 nM and 5.3 microM. STX was more potent, with apparent IC50s of 1.6 nM and 1.2 microM. There was no statistical correlation between toxin sensitivity and steady-state inactivation voltage, suggesting that these properties varied independently among sodium channel types.

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.

1 The rates at which tubocurarine associates with, and dissociates from, the nicotinic receptor, while exerting its classical competitive effect, are still in doubt. We have investigated this problem by observing the effect of low concentrations of tubocurarine on the re-equilibration rate, following a step change in membrane potential, of the current produced by carbachol in voltage-clamped endplates of frog muscle. 2 It is expected, and observed, that in order to see the effects of competition (as opposed to ion channel block), sufficiently high agonist concentrations must be used so that the relaxation rate becomes faster than that seen at low agonist concentrations. 3 Small concentrations of tubocurarine were found to reduce this relaxation rate, towards a value appropriate to a lower agonist concentration. 4 The results suggest that tubocurarine equilibrates very rapidly with the nicotinic receptor. 5 Some of the possible technical problems of this sort of experiment are discussed. The results are similar to those already published for nicotinic receptors in eel electric tissue.

The action of gallamine, a classical competitive neuromuscular blocking agent, has been examined on voltage-clamped endplates of frog skeletal muscle fibres. Gallamine produces a parallel shift of the equilibrium log (concentration)--response curves in concentrations of up to about 40 microM. At a membrane potential of -70 mV the Schild plot of the dose ratios so measured has a gradient of slightly less than the theoretical value, for a competitive antagonist, of unity. The apparent equilibrium constant for 'competitive' block is about 2 microM, and is approximately independent of the membrane potential. Fluctuation analysis of the endplate current shows two components in the presence of gallamine. The results can be fitted, over the range tested, by a mechanism that involves block of open ion channels by gallamine in a manner similar to that by procaine or quaternary local anaesthetic analogues. The rate constants for this action are strongly dependent on the membrane potential. At -100 mV the association rate constant is about 4 x 10(7) M-1S-1, the dissociation rate constant is about 600 s-1, and the equilibrium constant about 15 microM. Other kinetic measurements (voltage-jump relaxation, and nerve-evoked endplate currents) give results consistent with this conclusion, but apparently these results are valid over a range of conditions narrower than that for fluctuation analysis.

When solutions containing agonists are applied to the innervated face of an Electrophorus electroplaque, the membrane's conductance increases. The agonist-induced conductance is increased at more negative membrane potentials. The "instantaneous" current-voltage curve for agonist-induced currents is linear and shows a reversal potential near zero mV; chord conductances, calculated on the basis of this reversal potential, change epsilon-fold for every 62-mV change in potential when the conductance is small. Conductance depends non-linearly on small agonist concentrations; at all potentials, the dose-response curve has a Hill coefficient of 1.45 for decamethonium (Deca) and 1.90 for carbamylcholine (Carb). With agonist concentrations greater than 10(-4) M Carb or 10(-%) M Deca, the conductance rises to a peak 0.5-1.5 min after introduction of agonist, then declines with time; this effect resembles the "desensitization" reported for myoneural junctions. Elapid alpha-toxin, tubocurarine, and desensitization reduce the conductance without changing the effects of potential; the apparent dissociation constant for tubocurarine is 2 X 10(-7) M. By contrast, procaine effects a greater fractional inhibition of the conductance at high negative potentials.

This paper compares the conductance induced by bath-applied acetyl-choline (ACh) and by the same transmitter released from nerve terminals at Electrophorus electroplaques. For the former case, dose-response relations are characterized by the maximal agonist-induced conductance, rgamma (130 mmho/cm2), and by the concentration which induces half this conductance; this concentration is termed Kapp and equals 50 micron at -85 mV. For the latter case, neurally evoked postsynaptic currents (PSCs) are characterized by the peak conductance during strongly facilitated release, gPSC, and by the rate constant for decay, alpha. Since gPSC roughly equals rgamma, it is concluded that the PSC activates nearly all available receptor channels. These and other data agree with recent estimates that during the growth phase of the quantal response, (a) the ACh concentration is at least several hundred micromolar; and (b) most nearby channels are activated. However both alpha and Kapp increase during depolarization, at a rate of about e-fold per 86 mV. These observations on voltage sensitivity suggest that a suprathreshold synaptic event is rapidly terminated because the action potential abruptly releases ACh molecules from receptors.

The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.

The actions of brevetoxin (PbTX-3) were studied on single, voltage-dependent sodium channels and whole-cell currents from the neuroblastoma x glioma cell line NG108-15. Purified PbTX-3 shifted the activation of sodium channels to membrane potentials negative to normal. PbTX-3 did not alter the single-channel mean open lifetime, suggesting that the toxin does not change the rate of sodium channel inactivation from the open state. There was also no change in single-channel conductance. These results indicate that brevetoxin increases sodium current at rest by shifting the voltage dependence of channel activation and that the resulting depolarization is limited by channel inactivation.

1. The action of tubocurarine on voltage-clamped frog muscle end-plates has been re-examined by means (a) equilibrium dose-ratio measurements, (b) current fluctuation measurements and (c) voltage-jump relaxation measurements. 2. The equilibrium measurements can be interpreted as implying that tubocurarine has (a) a competitive blocking action, with a dissociation constant of 0.34 microM, which is not dependent on membrane potential, and (b) an additional voltage-dependent blocking action. 3. In the presence of tubocurarine two kinetic components can be seen. The faster one is similar to, but rather faster than, the normal ion channel closing rate. The other is much slower (1--3 sec), and, in relaxation experiments it is in the opposite direction to the fast relaxation. 4. A number of alternative explanations for the results are discussed. The mechanism that fits them best appears to be a combination of competitive block (or block of shut channels), with a strongly voltage-dependent block of open ion channels by tubocurarine. Estimates of the rate constants for channel blocking (and their voltage dependence) are derived. From these estimates the dissociation constant for the binding of tubocurarine to open channels appears to be roughly 0.12 microM at --70 mV and 0.02 microM at --12 mV. 5. Several potential sources of error in the experiments, and in their interpretation, are discussed. The most serious of these are problems associated with diffusion in the small volume of the synaptic cleft, viz. (a) changes in cleft concentration consequent on changes in binding, and (b) ionophoretic flux of antagonist and agonist into the synaptic cleft.

The cis and trans isomers of the photoisomerizable compound, 3,3'-bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), were purified by high-performance liquid chromatography using the ion-pair partitioning technique on a reverse-phase column. Solutions of cis-Bis-Q are stable at -20 degrees; at 25 degrees, thermal isomerization proceeds at a rate of 0.65%/day. cis-Bis-Q is less than 1% as potent a nicotinic agonist as the trans configuration. At concentrations of 1.5 microM or less, cis-Bis-Q exerts little or no blockade of the conductances induced by agonists. In voltage-clamped Electrophorus electroplaques exposed to cis-Bis-Q, laser flashes induce cis leads to trans photoisomerizations and increase the agonist-induced current by a factor of 20 within a few milliseconds.

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, alpha, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/tau as the sum of the rate constant alpha for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate alpha, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to alpha-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).

In Electrophorus electroplaques, the agonist-induced postsynaptic conductance depends on membrane potential. During steady exposure to agonists, after a voltage step the conductance relaxes on a millisecond time scale, exponentially approaching a new equilibrium value. The relaxation rate constant k is an instantaneous function of voltage, insensitive to the past or present conductance. Two components sum to form k. A concentration-sensitive component increases linearly with agonist concentration and decreases during desensitization or exposure to curare. Thus this component reflects the average frequency at which acetylcholine receptors are opening. The voltage-sensitive component, obtained by extrapolating k to zero agonist concentration, increases at more positive potentials. For acetylcholine, the voltage-sensitive component equals the rate constant for the exponential decay of postsynaptic currents; it thus seems to be the closing rate for active receptors. The voltage-sensitive component has the relative amplitudes acetylcholine less than carbamoylcholine less than decamethonium, and for each agonist equals the closing rate determined from "noise" measurements at neuromuscular junctions. The kinetic data explain several aspects of the steady-state conductance induced by agonists, but shed no light on apparent cooperative effects.

These programs were written for the IBM personal computer equipped with a Tecmar Labmaster analog interface board. The software operates under the MS-DOS operating system; it is written in compiled BASIC and employs short machine-language subroutines for crucial functions. Details are presented on the analog interface routines which make special use of the 9513 counter/timer chip on the Labmaster. Time resolution is about 32 microseconds per sample on the PC and 15 microseconds on the PC/AT. One series of programs performs traditional voltage- and current-clamp experiments on macroscopic currents in whole cells. A second series interfaces with a hardware window discriminator to capture single-channel events in a recirculating buffer; a stimulus can be triggered to fire during an open channel. Off-line programs perform standard analyses, and further processing can be performed with standard spreadsheet programs.

The construction of a microscope stage temperature controller is described that works equally well with upright or inverted microscopes. The control circuit directly regulates the bath temperature near the physiological preparation from 0.0 degree C to 40.0 degrees C with a stability of +/- 0.1 degrees C; it allows biophysical studies of whole-cell or single channel currents to be carried out at different temperatures without introducing additional electrical noise into the measurements. The device may also be of interest to neurobiologists who work with tissue-culture preparations.

These experiments examine changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed. Voltage-clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-Bis-Q. A 1-microsecond laser flash photoisomerizes some trans-Bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans leads to cis photoisomerization. Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of Bis-Q solutions caused by flashes. At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. As a partial control for changes in the photochemistry caused by binding of Bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule.