Toxicology and Applied Pharmacology 238 (2009) 37ndash46

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Toxicology and Applied Pharmacology

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Effect of short-term exposure to dichlorvos on synaptic plasticity of rat hippocampalslices Involvement of acylpeptide hydrolase and α7 nicotinic receptors

Cristina Olmos a12 Rodrigo Sandoval a2 Carlos Rozas b Sebastiaacuten Navarro a Ursula Wyneken c Marc Zeise dBernardo Morales b Floria Pancetti aa Laboratory of Environmental Neurotoxicology Department of Biomedical Sciences Faculty of Medicine Universidad Catoacutelica del Norte Larrondo 1281 178-1421 Coquimbo Chileb Laboratory of Neurosciences Department of Biology Faculty of Chemistry and Biology Universidad de Santiago de Chile Alameda 3363 Santiago Chilec Laboratory of Neurosciences Faculty of Medicine Universidad de Los Andes San Carlos de Apoquindo 2200 Santiago Chiled School of Psychology Faculty of Humanities University of Santiago de Chile Alameda 3363 Santiago Chile

Corresponding author Fax +56 51 209837E-mail address pancettiucncl (F Pancetti)

1 Present address Laboratory of Neurosciences DepaSciences Universidad de Chile Las Palmeras 3425 Sant

2 Both authors contributed equally to this work

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Article historyReceived 19 May 2008Revised 2 April 2009Accepted 10 April 2009Available online 18 April 2009

KeywordsDichlorvosHippocampusSynaptic plasticityAcylpeptide hydrolaseNicotinic receptors

Dichlorvos is the active molecule of the pro-drug metrifonate used to revert the cognitive deficits associatedwith Alzheimers disease A few years ago it was reported that dichlorvos inhibits the enzyme acylpeptidehydrolase at lower doses than those necessary to inhibit acetylcholinesterase to the same extent Thereforethe aim of our investigation was to test the hypothesis that dichlorvos can enhance synaptic efficacy througha mechanism that involves acylpeptide hydrolase instead of acetylcholinesterase inhibition We used long-term potentiation induced in rat hippocampal slices as a model of synaptic plasticity Our results indicate thatshort-term exposures (20 min) to 50 μM dichlorvos enhance long-term potentiation in about 200 comparedto the control condition This effect is correlated with approximately 60 inhibition of acylpeptide hydrolaseactivity whereas acetylcholinesterase activity remains unaffected Paired-pulse facilitation and inhibitionexperiments indicate that dichlorvos does not have any presynaptic effect in the CA3rarrCA1 pathway noraffect gabaergic interneurons Interestingly the application of 100 nM methyllicaconitine an α7 nicotinicreceptor antagonist blocked the enhancing effect of dichlorvos on long-term potentiation These resultsindicate that under the exposure conditions described above dichlorvos enhances long-term potentiationthrough a postsynaptic mechanism that involves (a) the inhibition of the enzyme acylpeptide hydrolase and(b) the modulation of α7 nicotinic receptors

copy 2009 Elsevier Inc All rights reserved

Introduction

According to the ldquocholinergic hypothesisrdquo of Alzheimers diseasecognitive deterioration is due to the loss of cholinergic neurons in thebasal forebrain (Terry and Buccafusco 2003) In fact both clinical andexperimental studies have shown that therapies directed at restoringcholinergic deficit improve cognition (Buccafusco and Terry 2000)Many of the drugs used in these therapies are inhibitors ofacetylcholinesterase (AChE) the enzyme responsible for hydrolyzingacetylcholine at the synapse (Ivens et al 1998) Metrifonate is a drugoriginally used for the treatment of schistosomiasis and was recentlybeing used to revert the cholinergic deficit in Alzheimers diseaseAlthough the efficacy of metrifonate in reverting cognitive deteriora-tion has been proven at both experimental and clinical levels(Williams1999) the pharmaceutical companywithdrew their request

rtment of Biology Faculty ofiago Chile

ll rights reserved

for approval from the FDA due to reported cases of patients thatmanifested respiratory paralysis and problems in muscle neurotrans-mission (Lopez-Arrieta and Schneider 2006) The active compound inmetrifonate is the organophosphate dichlorvos (DDVP) which isreleased in the body bymeans of non enzymatic hydrolysis (Hinz et al1996Morris et al 1998 Ringman and Cummings1999) In addition toits pharmacological application in humans DDVP is widely used as aninsecticide for domestic purposes (Choudhary et al 2001)

There exists a large body of experimental evidence suggesting thatinhibitors of AChE used to potentiate cognition exert their effectthrough other targets (Richards et al 2000 Xie et al 2000 Duysen etal 2001 Ray and Richards 2001 Carter et al 2007) For example ithas been described that low doses of DDVP given to rats can potentiatecognitive abilities without causing a significant inhibition of brainAChE (Van der Staay et al 1996) In the same study whenwell knownAChE inhibitors such as paraoxon and physostigmine were adminis-tered to rats they failed to potentiate learning and memory Thisevidence demonstrate that the inhibition of AChE would not explainby itself the effect elicited on cognitive abilities

With the aim of identifying new targets for organophosphatesRichards et al (2000) studied the formation of adducts in pig brain

38 C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

homogenates and found that the enzyme acylpeptide hydrolase(ACPH) (Scaloni et al 1992 Polgar 2002) displayed a 6ndash10 timeshigher sensitivity to DDVP than AChE In light of these studies ACPH isbeing considered as a promising target for the development of newdrugs to potentiate cognition (Richards et al 2000 Rosenblum andKozarich 2003)

ACPH is a homomeric tetramer that belongs to the family of prolyloligopeptidases of the serine hydrolases (Rosenblum and Kozarich2003) and catalyzes the removal of N-acylated amino acids from shortpeptides to generate an acylated amino acid and a peptide with a freeN-terminal (Jones and Manning 1985 Polgar 2002) However therole that ACPH has in the central nervous system is unknownalthough it was recently discovered that the enzyme isolated fromconditionedmedia of neuroblastoma cell cultures was able to degradethe amyloid beta peptide 1ndash40 (Aβ1ndash40) in vitro (Yamin et al 2007) Inaccordance with the above the main goal of our study was todetermine a correlation between the inhibition of ACPH by DDVP andchanges in synaptic plasticity

Since cognitive potentiation is related to changes in synapticefficacy we used rat hippocampal slices as an in vitro model to studythe effect of DDVP on synaptic efficacy using two functionalapproaches i) the induction of long-term potentiation (LTP) toevaluate changes over a long time period and ii) facilitation andinhibition experiments of paired pulses (PPF and PPI respectively) toevaluate short-term effects that depend on presynaptic mechanismsor gabaergic interneurons

Briefly LTP is a cellular phenomenon that involves a steadyincrease in the strength of synaptic connections in response to a shortelectrical stimulation (Bear andMalenka 1994) LTP was described forthe first time a few decades ago in hippocampal dentate granule cellsof anesthetized rabbits after stimulation via the perforant path (Blissand Lomo 1973) Although LTP has been considered as a model ofsynaptic plasticity in vitro now it is known that learning induces LTPin the hippocampus in vivo (Whitlock et al 2006)

We have correlated our functional findings with the level of AChEand ACPH activity These measurements were carried out inhomogenates obtained from slices recovered after being electro-physiologically recorded With the goal of determine the mechanismby which DDVP could be exerting its effect on synaptic plasticity weused pharmacological tools such as methyllycaconitine (MLA) anantagonist of α7 nicotinic receptors (α7nAChRs) α7nAChRs arehomopentameric channels highly permeable to Ca+2 and desensitizequickly (Seguela et al 1993 McGehee 1999) They play an importantrole in normal and pathogenic cognitive processes (Alkondon andAlbuquerque 2004) and in the etiology of schizophrenia (Freedmanet al 1995 Freedman et al 1997) Functional and ultrastructuralstudies in the CA1 region of the hippocampus have determined thatα7nAChRs are located in both presynaptic and postsynaptic sites (Jiet al 2001 Fabian-Fine et al 2001) especially in gabaergicinterneurons (Wanaverbecq et al 2007) These receptors have alsobe found in excitatory synapses of other areas of the brain like thesomatosensorial cortex where are attached at the postsynapticdensities (Levy and Aoki 2002) We have focused our interest onthis receptor due to its susceptibility to be allosterically modulatedby peptides such as Aβ1ndash42 (Seguela et al 1993 Wang et al 2000a2000b Pettit et al 2001 Espinoza-Fonseca 2004) peptides derivedfrom apolipoprotein E (Klein and Yakel 2004) and a peptide derivedfrom the C-terminal of AChE (Greenfield et al 2004 Zbarsky et al2004) We propose that the inhibition of ACPH by DDVP could affectthe concentration of its peptide substrate and indirectly influence theactivity of α7nAChRs

Methods

Hippocampal slice preparation Transverse hippocampal slices wereprepared from 4-week-old SpraguendashDawley rats The animals were

anesthetized with halothane gas and then decapitated All theprotocols dealing with the maintenance and handling of the animalswere followed as stated in the Bioethical Guidelines of the UniversidadCatoacutelica del Norte and in the National Research Commissionguidelines Brains were rapidly removed and immersed in ice-colddissection buffer containing 2127 mM sucrose 5 mM KCl 125 mMNaH2PO4 3 mM MgSO4 1 mM CaCl2 26 mM NaHCO3 and 10 mMglucose pH 74 The hippocampus was dissected and transverse slices(400 μm thick) were obtained from the dorsal portion using avibratome (Model HA752 Campden Instruments Leicester UK) Theslices were transferred to an interface storage chamber containingartificial cerebrospinal fluid (ACSF) saturated with 95 O25 CO2 andwere left at least 1 h at 37 degC before recording ACSF contained 124mMNaCl 5 mM KCl 125 mM NaH2PO4 10 mM MgCl2 20 mM CaCl226 mM NaHCO3 and 10 mM glucose pH 74 Single slices were thentransferred to a recording chamber where they were kept completelysubmerged in ACSF and continually perfused (2 mlmin)

Extracellular field recording and LTP induction Field responses wereevoked with electrical stimulation (biphasic constant current 200 μsstimuli) delivered every 15 s to the Schaffer collateral pathway usingbipolar electrodes and recorded in the stratum radiatum formeasuring field excitatory postsynaptic potentials (fEPSP) slopes orstratum pyramidale for measuring population spike amplitudes ofthe CA1 hippocampal area Recording electrodes were glassmicropipettes (1ndash3 MΩ) filled with ACSF At the beginning of eachexperiment stimulusresponse curves were done by increasing theintensity of the stimulus in order to adjust it to elicit 50 of themaximum response LTP was elicited after 10ndash15 min of a stablebaseline by theta burst stimulation (TBS) consisting of 3 trains ofstimulus with an inter-train interval of 10 s Each train consisted of 10bursts at 5 Hz each burst having 4 pulses at 100 Hz After TBS dataacquisition lasted for 1 h Data were acquired using an extracellularamplifier (Model 1800 A-M systems USA) and a data acquisitionboard (National Instruments USA) controlled through Igor Prosoftware (Wavemetrics Inc USA) Cumulative probability plots andLTP values were constructed by averaging the percent of LTP observedbetween 30 and 60 min following TBS

Paired-pulse stimulation PPF experiments were performed in thestratum radiatum of the CA1 hippocampal area Biphasic constantcurrent 200 μs stimuli were delivered in pairs at 15 s intervals Pairedpulses were given 40 ms apart The stimulus intensity was adjusted insuch a way that the field response for the first pulse (P1) was 50 ofthe maximum amplitude PPI was performed in the stratumpyramidale of CA1 Stimuli were delivered in pairs at 15 s intervalsPaired pulses were given 13 ms apart

Application of DDVP and MLA DDVP (liquid analytical purity DrEhrenstorfer Lab Augsburg Germany) and MLA (Tocris Cookson LtdAvonmouth UK) were applied using a syringe pump and diluteddirectly to the perfusion solution The flux was adjusted in order toobtain the desired final concentration for each compound in therecording chamber After 10ndash15 min of a stable baselineconcentrated DDVP dissolved in ACSF was injected during 10 20 or70 min into the perfusion system at a proper flow in order to achievea concentration of 50 μM in the recording chamber TBS was elicitedin the middle of the application of DDVP for 10 and 20 min exposureFor 70 min TBS was elicited after the first 10 min of DDVP exposureA description of DDVP exposure times is shown in supplementaryfigure S1 In another set of experiments DDVP was applied in order toreach a final concentration of 75 μM for 20 min and TBS was appliedin the middle of the exposure In experiments where MLA wasapplied it was started 5 min before DDVP application and lasted by10 min Control experiments were performed following the sametime-schedules

39C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

Preparation of tissue extracts and protein determination In order toperform AChE and ACPH enzymatic assays after each electro-physiological experiment the slice was recovered from the perfusionchamber and frozen at minus80 degC until each of themwere homogenizedat 4 degC in a buffer containing 50 mM TrisndashCl 1 M NaCl 50 mM MgCl2and 1 Triton X-100 pH 74 Protein concentrations were determinedusing the Bradford method with bovine serum albumin as thestandard

Acetylcholinesterase activity assays AChE activity present in brainslice homogenates was determined spectrophometrically moni-toring the hydrolysis of S-acetylthiocholine iodide at 30 degC (ɛ406=13300 Mminus1 cmminus1) as described by the Ellmans method (Ellmanet al 1961) Briefly acetylthiocholine iodide was used as asynthetic substrate for AChE The sample (7 μl) was mixed with1 ml of 025 mM dithiobisnitrobenzoate (DTNB) in 50 mMphosphate buffer at pH 79 and incubated at 30 degC in a water-

Fig 1 Effect of 50 μM DDVP on LTP elicited in the stratum pyramidale and stratum radiatumEffect of 20 min of application of 50 μM DDVP on LTP in the stratum pyramidale of rat hipporespect to the mean of baseline responses (pb005) B The traces correspond to fieldexperiments for each condition (control and DDVP treated) C Cumulative probability plots gto baseline for a given experiment 30ndash60 min (average) after TBS D The same protocol depotentiation were calculated with respect to the mean of baseline responses (pb001) Erepresentative experiments for each condition (control and DDVP treated) F Cumulative prchange relative to baseline for a given experiment 30ndash60 min (average) after TBS

jacketed cuvette holder The reaction was initiated by adding 30 μlsubstrate (5 μmol) The thiocholine released by the hydrolysis ofthe substrate reacts in the presence of DTNB yielding 5-thio-2-nitrobenzoate that was quantified at 406 nm in a SpectronicGenesys V spectrophotometer (Thermo Electron Corporation USA)The enzymatic activity was normalized in function of the proteincontent in the assay

Acylpeptide hydrolase activity assays ACPH activity was performedas described previously (Perrier et al 2002) Briefly ACPH activitywas assayed monitoring the hydrolysis of the synthetic substrate N-acetyl-L-alanine p-nitroanilide (AANA) The samples (10ndash100 μl) weremixed with 50 mM phosphate buffer at pH 80 in a total volume of1 ml and incubated at 37 degC in a water-jacketed cuvette holder Thereaction was initiated by adding 10 μl substrate (1 nmol) dissolved indimethylsulfoxide The p-nitroaniline released was determinedquantitatively in a Spectronic Genesys V spectrophotometer (Thermo

of rat hippocampus All recordings were made from at least three different animals Acampal slices For each experiment the percents of potentiation were calculated with

responses measured in the stratum pyramidale (population spikes) of representativeraphically summarize the data Each point represents the magnitude of change relativescribed in A was applied in experiments done in the stratum radiatum The percents ofThe traces correspond to field responses measured in the stratum radiatum (fEPSP) ofobability plots graphically summarize the data Each point represents the magnitude of

40 C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

Electron Corporation USA) by measuring the absorbance at 410 nmusing ɛ410=8800 Mminus1 cmminus1 The enzymatic activity was normalizedin function of the protein content in the assay

Injection of recombinant α7nAChR cDNA in Xenopus laevis oocytes Asmuch as 10ndash30 ng of rat recombinant α7nAChR cDNA suspended insterile distilled water (10ndash30 nL 1 mgmL) were injected intoXenopus oocytes Following injection the oocytes were kept for 72 h at16 degC in modified Barths solution [MBS 88 mM NaCl 1 mM KCl033 mM Ca(NO3)2 082 mMMgSO4 24 mMNaHCO3 10mMHEPESpH 74 supplemented with 100 IUmL penicillin and 01 mgmLstreptomycin] until the electrophysiological recordings wereperformed As controls oocytes from the same batch were injectedwith 10ndash30 nL of sterile distilled water

Electrophysiological recordings of α7nAChR expressed in Xenopus laevisoocytes Membrane currents were recorded from voltage-clampedoocytes by using two microelectrodes filled with 3 molL KCl Theoocytes were placed in a recording chamber containing 05 mL oocyteRingers solution (ORS 115 mM NaCl 2 mM KCl 18 mM BaCl2 5 mMHEPES adjusted to pH 74 with NaOH) at room temperature (20ndash22 degC) The oocyte membrane potential was held at minus60 mV andincreasing concentrations of acetylcholine (ACh) (05 to 2 mM) wereapplied continuously for 10 s Between each agonist applicationoocytes were perfused with oocyte Ringers solution for 10 min (5 mLat 05 mLmin) 50 μM DDVP dissolved in ORS was continuouslyperfused for 20 min before and during the application of ACh For all

Fig 2 Effect of the exposure time and 75 μM DDVP on LTP All recordings were obtained fromLTP in control slices (open circles) and slices exposed to 50 μM DDVP during 10 min (blackrepresent the exposure time to DDVP B The column graph summarizes the data showed in Aresponses at 50 min post TBS (pb005) C The graph represents the effect of 75 μMDDVP onof the graph represents the exposure time to DDVP D The column graph summarizes the datpost TBS (pb005)

the experiments the maximal amplitude was measured The WCPprogram provided by John Dempster (University of Strathclyde UK)was used for data acquisition and analysis Unless otherwise specifiedvalues given correspond to the averageplusmnstandard error of the mean(SEM) Data obtained in the same oocyte were compared using thepaired Students t-test

Statistical analysis All the electrophysiological recordings that usedrat hippocampal slices were done using slices from at least threedifferent animals for each condition (control and DDVP exposedslices) The data were analyzed using the program Instat (GraphpadSoftware Inc USA) A probability level of 005 or less was consideredsignificant

Results

Effect of DDVP exposure on LTP induction in rat hippocampal slices

To test if a correlation exists between DDVP exposure and afavoring effect on LTP induction that account for the cognitiveimprovement seen in some animals administered with DDVP weperformed a series of experiments using different doses and exposuretimes As seen in Fig 1A when hippocampal slices were exposed to50 μM DDVP during 20 min we obtained a potentiation of 2191plusmn399 (SEM n=8 black circles) in the stratum pyramidale Thispotentiation was significantly different compared with the valueobtained in control slices of 1079plusmn233 (SEM n=7 open circles

at least three different animals A The graph represents the effect of exposure time oncircles) and 70 min (grey circles) The grey and black bars at the bottom of the graph

At longer exposures times to DDVP (70min) there is a significant reduction in the fEPSPLTP (black circles) compared to control slices (open circles) The grey bar at the bottoma showed in C Note that there is a significant reduction in the fEPSP responses at 20 min

Fig 3 Measurement of ACPH and AChE enzymatic activities All slices were frozenseparately and subsequently homogenized for enzymatic assays Determinations wereperformed in triplicate A A total number of 11 control and 12 exposed slices wererecovered from the recording chamber after being completed the LTP experiments inboth stratum pyramidale and stratum radiatum (Fig 1) Only ACPH activity shows asignificant inhibition of sim60 after 20 min of DDVP exposure (pb0001) B Thegraph displays the enzymatic activities of control (n=5) and slices exposed to 50 μMDDVP during 10 (n=4) and 70 min (n=5) (see Fig 2A) ACPH was inhibited by DDVPat both times of exposure whereas AChE showed a significant inhibition only at 70 minof DDVP exposure (pb001) C The graph displays the effect of a higher concentrationof DDVP (75 μM applied during 20 min) on enzymatic activities measured from slicesrecovered after being completed the LTP experiment showed in Fig 2C At this DDVPconcentration both enzymes are inhibited (pb0001)

41C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

pb005 unpaired t-test) Fig 1B corresponds to field potentialsmeasured in the stratum pyramidale (population spikes) of arepresentative experiment similar to that shown in Fig 1A Thecumulative probability distribution of the normalized responses isshown in Fig 1C Similar results were found in the stratumradiatum were the same protocol for DDVP exposure induced apotentiation of 2448plusmn126 (SEM n=5 black circles) comparedwith 1492plusmn145 (SEM n=5 open circles) for the controlcondition (pb001 unpaired t-test) (Fig 1D) Fig 1E corresponds tofield potentials measured in the stratum radiatum (fEPSP) of arepresentative experiment similar to that shown in Fig 1D Thecumulative probability distribution of the normalized responses isshown in Fig 1F

A shorter exposure time to DDVP (10 min) did not produce anenhancement of LTP as the observed with 20 min of exposure(compare Fig 1A and Dwith Fig 2A black circles) In fact there is no asignificant difference between both magnitudes of LTP (controlcondition and 10 min of DDVP exposure p=0629) By the otherhand a longer exposure time to DDVP (70 min) failed to produce asustained and significant LTP (p=077 Wilcoxon test) (Fig 2A greycircles)

In another set of experiments we tested the effect of a higher doseof DDVP (75 μM) applied for 20 min As it can be seen in Fig 2C thishigher dose of DDVP is detrimental for the establishment of LTPimmediately after TBS although at the end of the data acquisition(approximately 40 min post TBS) a recovery effect appeared towardcontrol values Taken together these results account for the existenceof a specific window of dose and exposure time where pharmacolo-gical instead of toxicological effects caused by DDVP are beingdetected

Effect of DDVP exposure on ACPH and AChE activities

ACPH and AChE enzymatic activities were measured in homo-genates prepared from control and DDVP exposed slices recoveredfrom the recording chamber and subsequently frozen In Fig 3A it canbe seen that at 20 min exposure of 50 μM DDVP only ACPH activityshows a significant inhibition of 593plusmn94 (pb0001 unpaired t-test) Interestingly AChE activity was not affected by DDVP under thisexposure protocol (p=0748 unpaired t-test) Similar results werefound at 10 min and 70min of DDVP exposure for ACPH (599plusmn144pb001 and 658plusmn35 pb001 respectively) For AChE we found asignificant inhibition of its activity only at 70 min of DDVP exposure(315plusmn135 for 70 min pb001) (Fig 3B) When slices were exposedto 75 μM DDVP for 20 min we found a significant inhibition of bothACPH and AChE activities (809plusmn116 pb0001 and 408plusmn142pb0001 respectively) (Fig 3C) Taken together these results indicatethat the inhibition of ACPH is not a requirement by itself for theenhancement of LTP Additionally AChE inhibition does not appear tocontribute a positive influence on LTP The correlation between ACPHinhibition by DDVP and the enhancement of LTP is seen only at aspecific window (range) of dose and exposure time (see Discussion)

Effect of DDVP on paired-pulse facilitation or inhibition protocols atglutamatergic synapses

In another set of slices different from those used for LTPexperiments PPF and PPI experiments were performed in order tostudy a possible effect of DDVP at the presynaptic level and thecontribution of gabaergic interneurons Briefly PPF is described as anincrease in a second postsynaptic response induced shortly after a firstresponse elicited by a conditioning stimulus The increase in thesecond response is due to the presence of residual presynaptic calciumfrom the first response that favors the release of neurotransmitterstherefore PPF is considered an example of presynaptic plasticity(Kleschevnikov et al 1997) On the other hand PPI is a phenomenon

that can be observed when two consecutive stimulation pulsesseparated by short time intervals (10ndash13 ms) are applied to theSchaffer collateral pathway and the responses are recorded at thestratum pyramidale of CA1 The amplitude of the population spikeresponse evoked by the second stimulus is decreased with respect tothe first evoked response This is due to inhibition of CA1 pyramidalneurons because of activation of inhibitory synapses located prefer-entially at the soma (Megias et al 2001) Therefore PPI is due to localactivation of inhibitory interneurons that release GABA as a neuro-transmitter (Higgins and Stone 1993)

Fig 3 Measurement of ACPH and AChE enzymatic activities All slices were frozenseparately and subsequently homogenized for enzymatic assays Determinations wereperformed in triplicate A A total number of 11 control and 12 exposed slices wererecovered from the recording chamber after being completed the LTP experiments inboth stratum pyramidale and stratum radiatum (Fig 1) Only ACPH activity shows asignificant inhibition of sim60 after 20 min of DDVP exposure (pb0001) B Thegraph displays the enzymatic activities of control (n=5) and slices exposed to 50 μMDDVP during 10 (n=4) and 70 min (n=5) (see Fig 2A) ACPH was inhibited by DDVPat both times of exposure whereas AChE showed a significant inhibition only at 70 minof DDVP exposure (pb001) C The graph displays the effect of a higher concentrationof DDVP (75 μM applied during 20 min) on enzymatic activities measured from slicesrecovered after being completed the LTP experiment showed in Fig 2C At this DDVPconcentration both enzymes are inhibited (pb0001)

41C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

pb005 unpaired t-test) Fig 1B corresponds to field potentialsmeasured in the stratum pyramidale (population spikes) of arepresentative experiment similar to that shown in Fig 1A Thecumulative probability distribution of the normalized responses isshown in Fig 1C Similar results were found in the stratumradiatum were the same protocol for DDVP exposure induced apotentiation of 2448plusmn126 (SEM n=5 black circles) comparedwith 1492plusmn145 (SEM n=5 open circles) for the controlcondition (pb001 unpaired t-test) (Fig 1D) Fig 1E corresponds tofield potentials measured in the stratum radiatum (fEPSP) of arepresentative experiment similar to that shown in Fig 1D Thecumulative probability distribution of the normalized responses isshown in Fig 1F

A shorter exposure time to DDVP (10 min) did not produce anenhancement of LTP as the observed with 20 min of exposure(compare Fig 1A and Dwith Fig 2A black circles) In fact there is no asignificant difference between both magnitudes of LTP (controlcondition and 10 min of DDVP exposure p=0629) By the otherhand a longer exposure time to DDVP (70 min) failed to produce asustained and significant LTP (p=077 Wilcoxon test) (Fig 2A greycircles)

In another set of experiments we tested the effect of a higher doseof DDVP (75 μM) applied for 20 min As it can be seen in Fig 2C thishigher dose of DDVP is detrimental for the establishment of LTPimmediately after TBS although at the end of the data acquisition(approximately 40 min post TBS) a recovery effect appeared towardcontrol values Taken together these results account for the existenceof a specific window of dose and exposure time where pharmacolo-gical instead of toxicological effects caused by DDVP are beingdetected

Effect of DDVP exposure on ACPH and AChE activities

ACPH and AChE enzymatic activities were measured in homo-genates prepared from control and DDVP exposed slices recoveredfrom the recording chamber and subsequently frozen In Fig 3A it canbe seen that at 20 min exposure of 50 μM DDVP only ACPH activityshows a significant inhibition of 593plusmn94 (pb0001 unpaired t-test) Interestingly AChE activity was not affected by DDVP under thisexposure protocol (p=0748 unpaired t-test) Similar results werefound at 10 min and 70min of DDVP exposure for ACPH (599plusmn144pb001 and 658plusmn35 pb001 respectively) For AChE we found asignificant inhibition of its activity only at 70 min of DDVP exposure(315plusmn135 for 70 min pb001) (Fig 3B) When slices were exposedto 75 μM DDVP for 20 min we found a significant inhibition of bothACPH and AChE activities (809plusmn116 pb0001 and 408plusmn142pb0001 respectively) (Fig 3C) Taken together these results indicatethat the inhibition of ACPH is not a requirement by itself for theenhancement of LTP Additionally AChE inhibition does not appear tocontribute a positive influence on LTP The correlation between ACPHinhibition by DDVP and the enhancement of LTP is seen only at aspecific window (range) of dose and exposure time (see Discussion)

Effect of DDVP on paired-pulse facilitation or inhibition protocols atglutamatergic synapses

In another set of slices different from those used for LTPexperiments PPF and PPI experiments were performed in order tostudy a possible effect of DDVP at the presynaptic level and thecontribution of gabaergic interneurons Briefly PPF is described as anincrease in a second postsynaptic response induced shortly after a firstresponse elicited by a conditioning stimulus The increase in thesecond response is due to the presence of residual presynaptic calciumfrom the first response that favors the release of neurotransmitterstherefore PPF is considered an example of presynaptic plasticity(Kleschevnikov et al 1997) On the other hand PPI is a phenomenon

that can be observed when two consecutive stimulation pulsesseparated by short time intervals (10ndash13 ms) are applied to theSchaffer collateral pathway and the responses are recorded at thestratum pyramidale of CA1 The amplitude of the population spikeresponse evoked by the second stimulus is decreased with respect tothe first evoked response This is due to inhibition of CA1 pyramidalneurons because of activation of inhibitory synapses located prefer-entially at the soma (Megias et al 2001) Therefore PPI is due to localactivation of inhibitory interneurons that release GABA as a neuro-transmitter (Higgins and Stone 1993)

42 C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

Fig 4A shows that 50 μM DDVP applied for 20 min after 10 min ofstable baseline responses induces a non-significant and reversibleincrement in the slopes of fEPSPs in both the first pulse (open circlesp=007) and second pulse (black circles p=012) (ANOVAwith posttest) in the stratum radiatum The graph displaying the fEPSP sloperatio (P2P1) is shown in Fig 4C As can be observed DDVP does nothave any effect on the facilitation achieved with an inter-stimulusinterval of 40 ms In PPI experiments DDVP was applied in the sameway as for PPF experiments DDVP induced a significant and reversibleincrement in the population spike amplitude only in the first pulse(open circles pb005 ANOVA with post test) in the stratum pyrami-dale (Fig 4D) However this slight increase in the first pulse does notaffect the population spike amplitude ratio (P2P1) (Fig 4F)demonstrating that DDVP does not have any effect on the inhibitionachieved with an inter-stimulus interval of 13 ms

Involvement of α7 nicotinic receptors (α7nAChRs) in the enhancing effectof DDVP on LTP

In order to clarify if α7nAChRs are involved in the enhancingeffect of DDVP on LTP we performed a similar experiment to thatshown in Fig 1 but previously blocking α7nAChRs with the specificantagonist MLA The dose of MLA used in this study was chosenbased on existing literature on the in vitro effects of this antagonistin hippocampal slices (Fujii and Sumikawa 2001 Chen et al 2006Guan et al 2006) First in order to discard any effect of MLA alonein synaptic responses control experiments were done applying MLA

Fig 4 Effect of DDVP over paired-pulse facilitation or inhibition at glutamatergic synapseexperiments P1 corresponds to the first response (open circles) and P2 to the second responsthe duration of DDVP exposure B Representative field responses from a PPF experiment befoof the fEPSP slope ratio (P2P1) shown in Fig 1 A before during and after DDVP applicatexperiments P1 corresponds to the first response (open circles) and P2 to the second responthe duration of DDVP exposure E Representative field responses from a PPI experiment beforof the population spike amplitude ratio (P2P1) shown in Fig 1D before during and after D

at a concentration of 100 nM during 10 min to rat hippocampalslices As can be seen in Fig 5A 100 nM MLA did not induce anysignificant effect on the baseline responses Next we wanted tofurther explore if the application of MLA alone could have an effecton LTP induction Fig 5B shows that when TBS was applied after10 min of MLA exposure to rat hippocampal slices the effects onLTP did not differ significantly from the LTP induced in control slicesat approximately 30 and 60 min post TBS (p=0777 and p=02517respectively) indicating that MLA applied alone did not have anyeffect on LTP induction (Fig 5C) These results are not in agreementwith previously reported data that demonstrate that functionalα7nAChRs are necessary for LTP induction (Chen et al 2006)however contrary to our experimental design the protocoldescribed by Chen et al (2006) for the blockade of α7nAChRs isperformed during 20 min and high frequency stimulation is elicitedin the presence of MLA in the recording chamber

Finally we wanted to examine if α7nAChRs were involved in theeffects of DDVP on LTP To accomplish this we applied 100 nM MLAduring 10 min Halfway during this application (ie at 5 min) 50 μMDDVP was perfused into the recording chamber and left to act during20min similar to previous experiments The enhancing effect of DDVPon LTP disappeared when α7nAChRs were previously blocked withMLA (Fig 5D) At 20ndash30 min and 50ndash60 min post TBS the enhancingeffect of DDVP on LTP was completely abolished in the slices that werepreviously exposed to MLA (pb001 ANOVA with post test) (Fig 5E)The cumulative probability distribution of the normalized responses isshown in Fig 5F

s A The graph corresponds to the mean of the normalized field responses of 6 PPFe (black circles) of two consecutive stimulus applied 40ms apart The grey bar indicatesre during and after the application of 50 μMDDVP C The graph corresponds to themeanion D The graph corresponds to the mean of the normalized field responses of 6 PPIse (black circles) of two consecutive stimulus applied 13ms apart The grey bar indicatese during and after the application of 50 μMDDVP F The graph corresponds to the meanDVP application

Fig 5 Effect of the blockade of α7nAChRs on the enhancing effect of DDVP on LTP All the experiments were performed in the stratum pyramidale of the rat hippocampus A Effect of100 nM MLA applied during 10 min on baseline field responses (n=4) B The graph shows the effect of MLA (grey circles n=5) compared to control (open circles n=7) on LTPinduction in rat hippocampal slices C The columns in the graph represent the normalized magnitude of the responses before TBS and at 20ndash30 min and at 50ndash60 min after TBS Non-significant differences were found between the control and MLA treated slices at each time analyzed D The graph shows the effect of α7nAChR blockade on the enhancing effect ofDDVP on LTP (grey circles n=7) compared to DDVP alone (black circles n=8) and control slices (open circles n=7) E The columns in the graph represent the normalizedmagnitude of the responses before TBS and at 20ndash30 min and at 50ndash60 min after TBS for each experimental condition shown in D Significant differences were found at 20ndash30 minand 50ndash60 min post TBS as specified in the graph (pb005 and pb001 respectively) F Cumulative probability distribution of the normalized responses

43C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

Effect of DDVP on α7nAChRs expressed in Xenopus oocytes

In order to determine if DDVP has a direct effect on α7nAChRs weexpressed the rat recombinant protein in Xenopus laevis oocytes andrecorded the ACh induced currents using voltage clamp techniquesExposure to 50 μM DDVP during 20 min did not affect the maximalamplitude of the inward currents elicited by different concentrationsof ACh (05 1 and 2 mM ACh Fig 6A) The dose-dependent responseto ACh in the presence or absence of DDVP is shown in Fig 6B Theseresults indicate that DDVP does not have any direct effect on theactivity of α7nAChRs

Discussion

In the past some reports have shown that DDVP classicallydescribed as an anticholinesterase can induce opposite effects oncognitive function depending on the dose and exposure time Vander Staay et al (1996) demonstrated that dichlorvos can improvethe acquisition of the water escape task in rats when it isadministered at 003 mgkg po 60 min prior to the behavioraltest On the contrary well known AChE inhibitors like physostig-

mine and others did not affect learning and memory in this studysuggesting the involvement of an unknown mechanism of action(Van der Staay et al 1996) By the other hand it has beenreported that rats chronically exposed to dichlorvos (6 mgkg bwtday for 8 weeks) display memory impairment measured asconditioned avoidance response This effect was correlated with adecreased activity of AChE and others carboxylesterases (Sarin andGill 1998)

Our study was designed to test the hypothesis that non-AChE (ieACPH) might have a role in the cognitive enhancement elicited bysome drugs (ie AChE inhibitors) in certain well delimited range ofdose and exposure time Here we demonstrate for the first time thatacute in vitro exposure to DDVP (50 μM for 20 min) induces asignificant increase of LTP in glutamatergic synapses of rat hippo-campal slices through a mechanism that involves ACPH but not AChEinhibition Interestingly when we used other exposure times (10 or70 min) or a higher DDVP concentration (75 μM) the enhancementeffect on LTP is not observed These results agree with the idea thatACPH could be a promising target of action for the development ofdrugs that potentiate cognition (Richards et al 2000 Polgar 2002Rosenblum and Kozarich 2003)

Fig 6 Effect of 50 μMDDVP in ACh-activated currents in Xenopus oocytes expressingα7nAChR A Representative traces of inward currents at different ACh concentrations in absence(left column) and presence (middle column) of DDVP Right column represent the antagonistic effect of 20 nM MLA B The graph shows the dose-dependent response to ACh inabsence (black squares) and presence (black circles) of DDVP in 3 independent experiments

44 C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

Besides the effects of DDVP over long-term plasticity changes wefurther wanted to explore its effect over short-term plasticity changesExperiments on synaptic facilitation or inhibition by the application ofpaired pulses (PPF and PPI) suggested that DDVP did not inducefacilitation of the synaptic transmission since no changewas producedin the P2P1 ratio In addition gabaergic transmission was notaffected as shown by the data extracted from the PPI experiments

Finally our results show that the blockade of α7nAChRs with theantagonist MLA suppresses the positive effect of DDVP on LTP It isknown that several organophosphates and other AChE inhibitors caninteract directly with receptors of the cholinergic system or modulatetheir expression levels Specifically the global effect of thesecompounds on nicotinic receptors can be inhibition or potentiationof their activity (Smulders et al 2004 2005) For example in voltageclamp experiments performed in Xenopus oocytes expressing humanα4β2 nicotinic receptors it has been demonstrated that DDVP can actas a non-competitive antagonist when it is co-applied with a widerange of ACh concentrations (Smulders et al 2005) In theseexperiments the IC50 for DDVP was sim300 μM which is 6 times highercompared to the concentration used in our experiments (50 μM)Voltage clamp experiments performed in our laboratory with Xenopusoocytes expressing rat α7nAChRs discard that DDVP at the concentra-tion used in our LTP experiments could have a direct effect on theactivity of α7nAChRs (Figs 6A and B)

An important issue that should be taken into account deals withthe timing of the effect elicited by DDVP exposure The experimentsperformed with the aim to determine the timing of the effect showthat the specific inhibition of ACPH is not enough to trigger an

enhancement of LTP For example in Fig 3B it can be observed thatwhen DDVP is applied during 10 min we obtained a specific ACPHinhibition that is not accompanied by the enhancing effect on LTP(Fig 2A)

All together these results point to an indirect mechanism ofexcitatory neurotransmission modulation where the inhibition ofACPH by DDVP at the proper timing would block its activity towardsits endogenous N-acylpeptide substrate It is generally accepted thatN-acylation of peptides prevents their proteolysis so under theeffects of DDVP ACPH substrates would remain in the tissue Thepresence of these N-acylated peptides could have some kind ofallosteric effects on α7nAChRs preferentially those localized in thepostsynaptic membrane However the ACPH substrate should reacha critical concentration to trigger the α7nAChR-dependent enhance-ment of LTP In the results previously shown the critical concentra-tion of the ACPH substrate is probably achieved at 20 min but not at10 min

It is known that α7nAChRs are allosterically modulated in thehippocampus by peptides like β-amyloid1ndash42 (Seguela et al 1993Wang et al 2000a 2000b Pettit et al 2001 Espinoza-Fonseca 2004)apolipoprotein E-derived peptides (Klein and Yakel 2004) and apeptide derived from the C-terminus of AChE (Greenfield et al 2004Zbarsky et al 2004) We believe that regardless of the type ofpostsynaptic neuron (gabaergic or glutamatergic) the net effect in thehippocampal network is an increase in the excitability which favorsthe induction of LTP Our proposed model is shown in Fig 7 Fig 7Adepicts a situation where ACPH in the presynaptic terminal acts on itsN-acylpeptide substrate which is liberated together with

Fig 7General hypothesis of themechanism responsible for the effects of DDVP on synaptic plasticity The figure depicts two possible situations In panel A ACPH is being inhibited byDDVP in the presynaptic terminal In panel B the enzyme is being inhibited in the synaptic cleft The final effect in the intrahippocampal circuitry is the enhancement of excitabilityThe possible involvement of specific peptides like Aβ (as mentioned in the Discussion) is omitted

45C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

neurotransmitters from synaptic vesicles Another possibility isdisplayed in Fig 7B where ACPH in the synaptic cleft acts on itssubstrate released from the presynaptic terminal We eliminated thepossibility that ACPH is associated to the postsynaptic machinerysince the activity of ACPH is absent in isolated postsynaptic densities(data not shown) However we cannot discard the possibility of thepresence of ACPH contained in internal postsynaptic reservoirs but itscontribution to the mechanism described above needs to be furtherexplored in future experiments Moreover the subcellular localizationand distribution of ACPH in the brain still needs to be understood sincethere is little or no information on the subject

A strategy for the development of drugs for the treatment ofAlzheimers disease is to design ligands that specifically targetnicotinic receptors and elicit allosteric effects on them increasingthe probability of channel opening induced by ACh and nicotinicagonists and decreasing receptor desensitization in the case ofα7nAChRs (Maelicke et al 2000 Timmermann et al 2007) DDVPcould be indirectly exerting a similar effect through the putativemechanism described above Obviously this hypothesis and ourmodel proposal still need to be corroborated

An important issue that should be resolved is to find out theendogenous neuronal substrate of ACPH Some ACPH substrates havebeen identified in specific tissues For example it has been reportedthat in bovine and human lens ACPH catalytic subunit of 75 kDaremoves N-acetylated amino acid residues from theαA-crystallin anda truncated form of 55 kDa has an endoprotease activity that couldplay a role in the age-related cleavage of βB2-crystallins (Senthilk-umar et al 2001) Interestingly the total ACPH activity was found tobe decreased in human cataract lenses (Senthilkumar et al 2001)and this may contribute to the accumulation of N-terminally blockedpeptides in the lens nucleus (Sharma and Kester 1996)

More relevant to our results is the finding reported by Yamin etal (2007) indicating that ACPH degrades Aβ1ndash40 in vitro and thatAlzheimers disease brains express lower levels of ACPH mRNA thanbrains of age-matched controls (Yamin et al 2007) This finding isin agreement with the idea that an active ACPH is necessary toavoid the accumulation of Aβ and the formation of plaquesHowever supposing that Aβ1ndash40 could be one of the putativeACPH substrates in our model this contradicts with our data sincewe demonstrate that inhibition of ACPH by acute exposure to DDVP

increases LTP which would not explain the memory loss associatedwith Alzheimers disease One possible explanation deals with thebiphasic characteristic of the allosteric effect elicited by peptides onion channels For example it is known that different Aβ concentra-tions produce opposite effects in the cell system under study Whilelow concentrations are neurotrophic high concentrations areneurotoxic inducing neuronal death (Yankner et al 1990) Webelieve that the biphasic effect described above would apply for theendogenous substrate of ACPH and its accumulation could beexerting a positive modulation of synaptic plasticity A longerinhibition of ACPH would have the opposite effect For example ourresults show that LTP cannot be induced in slices exposed during70 min to DDVP (Fig 2A)

In conclusion our study demonstrates that a compound consid-ered to be an inhibitor of AChE is able to exert its effects on synapticplasticity through a different target in this case ACPH We postulatethat this effect would be through an indirect mechanism that dependson the presence of the peptidic substrate of ACPH In order tocorroborate our model more experiments need to be performed todetermine the endogenous substrate for ACPH as well as determinethe subcellular localization of this enzyme and its function in synapticplasticity

Conflict of interest statementAll the authors state that there are no actual or potential conflicts of interest

Acknowledgments

This work was supported by grants from Direccioacuten General deInvestigacioacuten y Postgrado (DGIP) from Universidad Catoacutelica del Norteto FP from Programa Bicentenario en Ciencia y Tecnologiacutea PSD-11 toFP and RS and from Fondecyt grant No 1030220 to BM and FP Wethank to Dr Fernando Saacutechez-Santed for his helpful reading of themanuscript and to Dr Luis G Aguayo for providing us with theα7nAChR cDNA clone

Appendix A Supplementary data

Supplementary data associated with this article can be found inthe online version at doi101016jtaap200904011

46 C Olmos et al Toxicology and Applied Pharmacology 238 (2009) 37ndash46

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Greenfield SA Day T Mann EO Bermudez I 2004 A novel peptide modulatesalpha7 nicotinic receptor responses implications for a possible trophicndashtoxicmechanism within the brain J Neurochem 90 325ndash331

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Levy RB Aoki C 2002 Alpha7 nicotinic acetylcholine receptors occur at postsynapticdensities of AMPA receptor-positive and -negative excitatory synapses in ratsensory cortex J Neurosci 22 5001ndash5015

Lopez-Arrieta JM Schneider L 2006 Metrifonate for Alzheimers disease CochraneDatabase Syst Rev Issue 2 Art Ndeg CD003155 DOI10100214651858CD003155pub3

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Morris JC Cyrus PA Orazem J Mas J Bieber F Ruzicka BB Gulanski B 1998Metrifonate benefits cognitive behavioral and global function in patients withAlzheimers disease Neurology 50 1222ndash1230

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sensitive site for reaction with organophosphorus compounds and a potentialtarget for cognitive enhancing drugs Mol Pharmacol 58 577ndash583

Ringman JM Cummings JL 1999 Metrifonate update on a new antidementia agentJ Clin Psychiatry 60 776ndash782

Rosenblum JS Kozarich JW 2003 Prolyl peptidases a serine protease subfamilywithhigh potential for drug discovery Curr Opin Chem Biol 7 496ndash504

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Terry Jr AV Buccafusco JJ 2003 The cholinergic hypothesis of age and Alzheimersdisease-related cognitive deficits recent challenges and their implications for noveldrug development J Pharmacol Exp Ther 306 821ndash827

Timmermann DB Gronlien JH Kohlhaas KL Nielsen EO Dam E Jorgensen TDAhring PK Peters D Holst D Chrsitensen JK Malysz J Briggs CAGopalakrishnan M Olsen GM 2007 An allosteric modulator of the alpha7nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivoJ Pharmacol Exp Ther 323 294ndash307

Van der Staay FJ Hinz VCH Schmidt BH 1996 Effects of metrifonate itstransformation product dichlorvos and other organophosphorus and referencecholinesterase inhibitors on Morris water escape behaviour in young-adult ratsJ Pharmacol Exp Ther 278 697ndash708

Wanaverbecq N Semyanov A Pavlov I Walker MC Kullmann DM 2007Cholinergic axonsmodulate GABAergic signaling among hippocampal interneuronsvia postsynaptic alpha 7 nicotinic receptors J Neurosci 27 5683ndash5693

Wang HY Lee DH DAndrea MR Peterson PA Shank RP Reitz AB 2000a beta-Amyloid(1ndash42) binds to alpha7 nicotinic acetylcholine receptor with high affinityImplications for Alzheimers disease pathology J Biol Chem 275 5626ndash5632

Wang HY Lee DH Davis CB Shank RP 2000b Amyloid peptide Abeta(1ndash42)binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholinereceptors J Neurochem 75 1155ndash1161

Whitlock JR Heynen AJ Shuler MG Bear MF 2006 Learning induces long-termpotentiation in the hippocampus Science 313 1093ndash1097

Williams BR 1999 Metrifonate a new agent for the treatment of Alzheimers diseaseAm J Health Syst Pharm 56 427ndash432

Xie W Stribley JA Chatonnet A Wilder PJ Rizzino A McComb RD Taylor PHinrichs SH Lockridge O 2000 Postnatal developmental delay and super-sensitivity to organophosphate in gene-targeted mice lacking acetylcholinesteraseJ Pharmacol Exp Ther 293 896ndash902

Yamin R Bagchi S Hildebrant R Scaloni A Widom RL Abraham CR 2007Acyl peptide hydrolase a serine proteinase isolated from conditioned medium ofneuroblastoma cells degrades the amyloid-beta peptide J Neurochem 100458ndash467

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Zbarsky V Thomas J Greenfield S 2004 Bioactivity of a peptide derived fromacetylcholinesterase involvement of an ivermectin-sensitive site on the alpha 7nicotinic receptor Neurobiol Dis 16 283ndash289