DTIC ADA561712: Role of Acetylcholinesterase on the Structure and Function of Cholinergic Synapses: Insights Gained from Studies on Knockout Mice

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Role of acetylcholinesterase on the structure and function of cholinergic synapses:
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Adler, M, Sweeney, R., Hamilton, TA, Lockridge, O, Duysen, EG, Purcell, AL,
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Published in Cellular and Molecular Neurobiology, 31(6), 909-920, 2011.This research was supported by the Defense Threat Reduction
Agency-Joint Science and Technology Office, Medical S & T Division.
14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Neuromuscular junction , Cholinesterase, Knockout mouse, Diaphragm muscle, Electron microscopy, Endplate potential, Medical
chemical defense
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CellMolNeurobiol(2011)31:909–920
DOI10.1007/s10571-011-9690-5
ORIGINAL PAPER
Role of Acetylcholinesterase on the Structure and Function
of Cholinergic Synapses: Insights Gained from Studies
on Knockout Mice
Michael Adler • Richard E. Sweeney • Tracey A. Hamilton •
Oksana Lockridge • Ellen G. Duysen • Angela L. Purcell •
Sharad S. Deshpande
Received:3February2011/Accepted:6April2011/Publishedonline:3May2011
(cid:2)SpringerScience+BusinessMedia,LLC(outsidetheUSA)2011
Abstract Electrophysiological and ultrastructural studies assessment of functional alterations of the diaphragm
were performed on phrenic nerve-hemidiaphragm prepa- muscle resulting from the absence of AChE. Tension
rations isolated from wild-type and acetylcholinesterase measurements from AChE-/- mice revealed that the
(AChE) knockout (KO) mice to determine the compensa- amplitude of twitch tensions was potentiated, but tetanic
tory mechanisms manifested by the neuromuscular junc- tensions underwent a use-dependent decline at frequencies
tion to excess acetylcholine (ACh). The diaphragm was below 70 Hz and above 100 Hz. MEPPs recorded from
selectedsinceitistheprimarymuscleofrespiration,andit hemidiaphragms of AChE-/- mice showed a reduction in
must adapt to allow for survival of the organism in the frequency and a prolongation in decay (37%) but no
absence of AChE. Nerve-elicited muscle contractions, change in amplitude compared to values observed in age-
miniature endplate potentials (MEPPs) and evoked end- matched wild-type littermates. In contrast, MEPPs recor-
plate potentials (EPPs) were recorded by conventional ded from hemidiaphragms of wild-type mice that were
electrophysiological techniques from phrenic nerve- exposed for 30 min to the selective AChE inhibitor
hemidiaphragmpreparationsisolatedfrom1.5-to2-month- 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one
old wild-type (AChE?/?) or AChE KO (AChE-/-) mice. (BW284C51) exhibited a pronounced increase in ampli-
These recordings were chosen to provide a comprehensive tude (42%) and a more marked prolongation in decay
(76%). The difference between MEPP amplitudes and
decays in AChE-/- hemidiaphragms and in wild-type
M.Adler(&)(cid:2)A.L.Purcell(cid:2)S.S.Deshpande
hemidiaphragmstreated withBW284C51 representseffec-
NeurobehavioralToxicologyBranch,AnalyticalToxicology
Division,USArmyMedicalResearchInstituteofChemical tive adaptation by the former to a high ACh environment.
Defense,AberdeenProvingGround,MD21010-5400,USA Electronmicroscopicexaminationrevealedthatdiaphragm
e-mail:[email protected] musclesofAChE-/-micehadsmallernerveterminalsand
diminishedpre- andpost-synaptic surface contactsrelative
R.E.Sweeney
PharmacologyBranch,ResearchDivision,USArmyMedical to neuromuscular junctions of AChE?/? mice. The mor-
ResearchInstituteofChemicalDefense,AberdeenProving phological changes are suggested to account, in part, for
Ground,MD21010-5400,USA theabilityofmusclefromAChE-/-micetofunctioninthe
complete absence of AChE.
T.A.Hamilton
ComparativePathologyBranch,ResearchSupportDivision,
USArmyMedicalResearchInstituteofChemicalDefense, Keywords Neuromuscular junction (cid:2) Cholinesterase (cid:2)
AberdeenProvingGround,MD21010-5400,USA
Knockout mouse (cid:2) Diaphragm muscle (cid:2) Electron
microscopy (cid:2) Endplate potential
O.Lockridge(cid:2)E.G.Duysen
EppleyInstitute,UniversityofNebraskaMedicalCenter,
Omaha,NE68198-6805,USA Abbreviations
ACh Acetylcholine
PresentAddress:
AChE Acetylcholinesterase
A.L.Purcell
CooleyLLP,Washington,DC20001,USA AChR Acetylcholine receptor
123
910 CellMolNeurobiol(2011)31:909–920
AChE?/? 129SVJ wild-type mice conditions, BChE is thought to have little or no role in
AChE-/- AChE knockout mice hydrolysis of ACh due to its lower Kcat, higher Km, lower
BChE Butyrylcholinesterase density and less favorable localization (Chatonnet and
BW284C51 1,5-Bis(4-allyldimethyl- Lockridge 1989; Brimijoin and Koenigsberger 1999).
ammoniumphenyl)pentane-3-one) However, BChE has been suggested to participate in the
l-CTX l-Conotoxin GIIIB hydrolysisofAChinothersystems,suchasairwaysmooth
EPP Endplate potential muscle, that function on a slower time scale (Adler et al.
iso-OMPA Tetraisopropylpyrophosphoramine 1991). Moreover, BChE has been reported to play a role
KO Knockout in the hydrolysis of ACh in the brain of humans with
MEPP Miniature endplate potential Alzheimer’s disease, where selective loss of AChE may
m QuantalcontentoftheEPPbythemethodof lead to a more dominant role for BChE (Mesulam et al.
0
failure 2002; Lane et al. 2006).
m QuantalcontentoftheEPPbyratio ofEPP/ MuchofourinformationontheroleofAChEinsynaptic
r
MEPP in l-conotoxin GIIIB transmissionhascomefromuseofselectiveAChEinhibitors
PBS Phosphate-buffered saline suchas1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-
SSV Small synaptic vesicle 3-one (BW284C51), huperzine, galantamine, rivastigmine,
TEM Transmission electron microscopy and fasciculin (Bois et al. 1980; Rosenberry et al. 1999;
Albuquerqueetal.2006).Whilethesestudieshaveshedlight
ontheconsequencesofimpairedAChhydrolysis,interpre-
tationofdataisoftencomplicatedbyincompleteinhibition,
imperfect selectivity and unrelated ‘‘direct’’ actions of the
Introduction inhibitors(Boisetal.1980;Fiekers1985;Adleretal.1991).
An alternative approach is to study neuromuscular
Neuromuscular transmission is initiated by depolarization transmission in a line of AChE knockout (KO) mice
of the motoneuron nerve terminal and culminates in the (AChE-/-) developed in the laboratory of Dr. Oksana
synchronous release of acetylcholine (ACh) from small Lockridge(Xieetal.2000;Lietal.2000).Thesemicefail
synaptic vesicles (SSVs) docked at active zones (Heuser toexpressAChE,butproducenormallevelsofbothsoluble
etal.1979).AChthendiffusesacrossthesynapticcleftand andasymmetricformsofBChE.AChE-/-micehavebeen
combines with binding sites on two a subunits of the found to exhibit low body weight and an exaggerated
pentameric nicotinic ACh receptor (AChR), leading to the startle response. In addition they manifest fine motor
opening of the associated ligand-gated ion channel (Karlin tremors, pinpoint pupils, circling gait and generalized
2002). Channel opening triggers, in turn, the endplate muscle weakness (Duysen et al. 2001). In spite of these
current, endplate potential (EPP), muscle action potential abnormalities, AChE-/- mice have a life expectancy of
and muscle contraction (Miyazawa et al. 2003). Normal over 3 months, and some are able to live to adulthood,
operation of the neuromuscular junction requires removal when maintained on a nutritionally balanced liquid diet
of ACh within several milliseconds of receptor activation (Duysenetal.2002;Minicetal.2003).Theabilityofthese
to allow for sustained high frequency firing: otherwise mice to survive is remarkable since acute inhibition of
persistence of ACh would lead to membrane depolariza- AChE leads to death within several minutes. Inhibition of
tion, retrograde firing, receptor desensitization, muscle AChEis,infact,theprimarymechanismoftoxicityforthe
fasciculationandmuscleweakness(KatzandMiledi1973; highly lethal nerve agents (Taylor 1991).
Heffron and Hobbiger 1979; Magleby and Pallotta 1981; In a previous study, we found that endplates from
Adler et al. 1992). Removal of ACh is accomplished pri- AChE-/- mice exhibited a marked reduction of AChRs as
marily by hydrolysis to choline and acetate, neither of revealedbydecreasedbindingoffluorescenta-bungarotoxin
which has appreciable agonist action at the neuromuscular (Adleretal.2004).AdecreaseinAChRdensitywouldhave
junction(Akketal.2005),andtoamorelimitedextentby theeffectofreducingtransmitteraccumulationbyallowing
diffusion of ACh from the synaptic cleft (Katz and Miledi ACheliminationtoapproachfreediffusionlimits(Katzand
1973). Miledi1973;MaglebyandPallotta1981;Girardetal.2007).
There are two enzymes capable of hydrolyzing ACh at To shed further light on the alterations in neuromuscular
cholinergic synapses: acetylcholinesterase (AChE, EC structure and function in AChE-/- mice, we studied the
3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8). consequencesoflossofAChEondiaphragmatictensions,on
AChEistheprimaryhydrolyticenzymeandproducesrapid spontaneous miniature endplate potentials (MEPPs) and
cleavage of ACh (Zimmerman and Soreq 2006; Colletier nerve-evoked endplate potentials (EPPs), and on synaptic
et al. 2006). At the neuromuscular junction, under normal ultrastructure.Repetitiveindirectstimulationofdiaphragm
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CellMolNeurobiol(2011)31:909–920 911
musclesfromAChE-/-miceledtotetanicfadeatfrequen- Contractility Measurements
cies B50 and C200 Hz but to sustained tension at 70 and
100 Hz. Electrophysiological recordings revealed that the Hemidiaphragms with attached phrenic nerves were
probabilityofreleasewasnotalteredinAChE-/-miceunder mounted in tissue baths at 37(cid:3)C and immersed in an oxy-
low use conditions, but was reduced transiently following genated Krebs-Ringer solution. To obtain single twitches,
highfrequencytrains.MEPPsinAChE-/-micehadnormal the phrenic nerve was stimulated supramaximally (5–7 V)
amplitudesandlowerfrequencies,neitherofwhichischar- with 0.2-mspulsesat 0.03 Hz. The response ofmusclesto
acteristicofacuteAChEinhibition(KatzandMiledi1973). repetitive stimulation was probed by eliciting 500-ms long
MEPP decays were prolonged, but to a much lower extent trainsatfrequenciesof20,50,70,100,200and400 Hz.To
than in wild-type mice inhibited acutely by BW284C51. avoid neuromuscular fatigue, trains were elicited at a low
Transmissionelectronmicroscopic(TEM)studiesindicated dutycycle(2trainspermin).Stimuliweredeliveredwitha
extensive synaptic remodeling consisting of reductions in Grass S88 stimulator (Astro-Med Inc., West Warwick, RI,
nerve terminal size and of post-junctional surface area. USA). Muscle twitches were measured with an isometric
These ultrastructural alterations, in conjunction with a force transducer (Grass FT03, Astro-Med Inc., West
reduced receptor density (Adler et al. 2004; Girard et al. Warwick,RI,USA)andanalyzedoff-linewithpClamp9.2
2007)andmaintainedBChEactivity(Lietal.2000)maybe software (Molecular Devices, Sunnyvale, CA, USA).
responsible for the ability of the diaphragm muscle to Resting tensions were adjusted to 0.5 g to obtain optimal
functioninthecompleteabsenceofAChE. twitch tensions. Parameters measured were peak tension,
frequency and 90–10% relaxation time.
Materials and Methods Intracellular Microelectrode Techniques
Animal Preparation MEPPs and EPPs were recorded at 35–37(cid:3)C from surface
fibers of AChE?/? and AChE-/- hemidiaphragms using
Wild-type 129SVJ mice (AChE?/?) and nullizygous standard electrophysiological techniques. Muscle fibers
AChE-/- mice of either sex were obtained from the Uni- were impaled near endplate regions with borosilicate glass
versity of Nebraska Medical Center and housed at the US microelectrodes filledwith 3 MKCl (15–20 MX).Criteria
Army Medical Research Institute of Chemical Defense for successful impalement consisted of MEPPs with rise
under an Association for Assessment and Accreditation of times B1 ms and stable resting potentials negative to
Laboratory Animal Care International (AAALAC) accred- –60 mV. EPPs were elicited by supramaximal rectangular
ited animal care and use program. AChE-/- mice were pulses of 0.1 ms using a Grass S88 stimulator applied to
maintained on Ensure fiberTM (Abbott Laboratories, phrenic nerves via suction electrodes.
Columbus,OH,USA)insterilepetridishes(8 ml/mouseper Quantal content of the EPP was determined under con-
24-h period), while AChE?/? mice were provided with ditions of low probability of release by the method of
commercial rodent ration. Both AChE?/? and AChE-/- failures (DelCastilloandKatz 1954)andunder conditions
mice had access to tap water ad libitum. Animal holding of normal probability by determining the ratio of EPPs to
rooms were maintained at 70 ± 2 (cid:3)F with 50 ± 10%rela- MEPPs in the presence of l-conotoxin GIIIB (l-CTX)
tivehumidityusingatleast10completechangesperhourof (Plomp et al. 1992). For the former, muscles were super-
100%conditionedfreshair.Themicewereona12-hlight/ fused with a modified Krebs-Ringer solution in which
darkfull-spectrumlightingcycle.Micewereeuthanizedby Mg2? was increased to 8 mM and Ca2? was reduced to
exposuretoexcessCO ,anddiaphragmswereexcisedand 1 mM.Thissolutionreducedtheprobabilityofreleaseand
2
pinned at resting length in SylgardTM-coated dishes con- allowed the quantal nature of the EPP to be determined
taining oxygenated Krebs-Ringer solution of the following accordingtothe expression m = ln(N/N ),whereN is the
0 0
composition(mM):NaCl,145;KCl,5.4;CaCl ,1.8;MgCl , totalnumberofstimuliandN isthenumberofstimulithat
2 2 0
1.0;NaHCO ,15;NaH PO ,1.0;glucose,11.0.Thesolution fail to produce an evoked response (Del Castillo and Katz
3 2 4
wasbubbledwithagasmixtureof95%O and5%CO and 1954).Atleast50MEPPsand50EPPswererecordedfrom
2 2
had a pH of 7.3–7.4. Diaphragm muscles were split at the each fiber for determining m .
0
central tendontoprovide 2hemidiaphragmswith indepen- To calculatem,preparations were bathedinl-CTXfor
r
dent nerve supply, and they were prepared for tension C1 h to suppress muscle action potentials, and full-sized
recordings or microelectrode studies, as appropriate. EPPs were recorded at 70 Hz for 1 s; m was assessed at
r
AChE?/? and AChE-/- mice were 58.7 ± 2.9 and the beginning(Initial) andat theend ofthe train (Final)to
51.6 ± 3.2 days old and weighed 33.9 ± 2.8 and 19.1 ± ascertainwhetheradecreaseintransmitterreleaseplayeda
0.81 g,respectively,atthetimeofeuthanasia. roleinthedeclineoftensionsduringrepetitivestimulation.
123
912 CellMolNeurobiol(2011)31:909–920
To calculate m , the first EPP during the train was Warrington, PA, USA. Glutaraldehyde, paraformaldehyde,
r Initial
divided by the mean amplitude of MEPPs obtained 1 min and osmium tetroxide (all TEM grade) were purchased
prior to the train; m was calculated by averaging the fromElectronMicroscopySciences,FortWashington,PA.
r Final
last 4 EPPs during the train and dividing these by MEPPs SylgardTM was obtained from Dow Chemical Co., Mid-
obtained 100 ms following the 1-s stimulus trains. The land, MI, USA. BW284C51, iso-OMPA, l-CTX and all
100-ms interval was selected to provide conditions where other reagents were from Sigma-Aldrich Corp. (St. Louis,
theEPPshadreturnedtobaselinebutwheredesensitization MO, USA).
was still close to maximum (Elenes and Auerbach 2002).
EPPswerecorrectedfornon-linearsummation(McLachlan
andMartin1981),andMEPPswerenormalizedtoaresting Results
potential of -70 mV.
MicroelectrodesignalswererecordedwithanAxoclamp- Contractility Studies
2Bamplifier.Theoutputsignalwasdigitizedat10,000sam-
ples/s(Digidata1332A),andanalyzedoff-linewithpClamp The characteristics of single twitches from isolated
9.2software(equipmentandsoftwarefromMolecularDevi- hemidiaphragm muscles of AChE?/? and AChE-/- mice
ces, Sunnyvale, CA, USA). The parameters measured were are summarized in Table 1. Twitch tensions in muscles
peakamplitude,frequencyand90–10%decaytime. fromAChE-/-micehadlargeramplitudesandslowerrise
and decay times than those observed in wild-type mice.
Data Analysis The larger amplitudes were unexpected, especially in light
of the lower body weight and overall weakness of these
Data were obtained from 38 to 42 muscle fibers in 8–10 animals (Duysen et al. 2002). Spontaneous muscle fascic-
AChE?/? and AChE-/- mice, and are expressed as ulations, which were commonly observed in the intact
mean ± SEM. Statistical analysis was performed by animal, were not detected in the isolated AChE-/-
ANOVAfollowedbytheBonferroniMultipleComparisons hemidiaphragm muscle, suggesting that these responses
Test,orthetwo-tailedStudent’st-test,asappropriate(InStat, may require a more complete neuronal circuitry.
GraphPad Software Inc., La Jolla, CA, USA). A The ability of diaphragm muscles from AChE-/- mice
Pvalue B 0.05wasconsideredtobestatisticallysignificant. to develop and maintain tetanic tension was probed by
recordingresponsestorepetitivestimulationofthephrenic
TEM Procedures nerve at frequencies ranging from 20 to 400 Hz. Muscles
from wild-type mice maintained tension at these frequen-
Diaphragm muscles were fixed by immersion in buffered cies with little or no decrement (Fig. 1a). Muscles from
1.6% formaldehyde/2.5% glutaraldehyde at 4(cid:3)C. After AChE-/- mice were able to generate sustained tetanic
incubation for 24 h, muscles were washed 3 times for responses at 70 and 100 Hz, but higher or lower stimula-
15 min each in 0.1 M Na cacodylate buffer (pH 7.4, tion frequencies led to progressive reductions in muscle
194 mOsm/Kg) and post-fixed for 30 min in buffered 1% tension during the 500-ms trains (Fig. 1b). The mainte-
osmiumtetroxide.Tissuesweredehydratedthroughgraded nance of tetanic tensions at 70 and 100 Hz may be a
ethanol and bathed in propylene oxide for two 15-min consequence of the relativelyslow time course oftwitches
periods. This was followed by addition of Poly/bed 812(cid:4) inAChE-/-mice(Table 1).Theslowertimecoursewould
resinandpropyleneoxideatratios(w/w)of1:1and1:3for allowformoreeffectivesummationoftensionbyenabling
2 h each and by incubation in pure Poly/bed resin over- tetanic fusion to occur at lower frequencies. This may
night under vacuum. Tissues were flat embedded and accountinpartforthefindingthatfunctionaladaptationin
polymerizedat60(cid:3)Cfor24 h.Semithinsectionswereused diaphragm muscle of AChE KO mice was more complete
for preliminary morphological assessment and to select
areas of interest for TEM. Ultrathin tissue sections for
Table1 Indirectly elicited single twitches in AChE?/? and
TEM were collected on copper mesh supporting grids and AChE-/-micea
counterstained using uranyl acetate and lead citrate.
Condition Amplitude(g) Risetime(ms) Relaxation(ms)
Observations were performed using a JEOL 1200EX
electron microscope. AChE?/? 0.9±0.1 7.7±0.5 17.4±0.6
AChE-/- 4.7±0.4b 15.9±0.8b 24.0±0.9b
Reagents
a Dataareexpressedasmean±SEMandwereobtainedfrom8–21
AChE?/? and 9–11 AChE-/- mice. Tensions were elicited by
Poly/Bed 812(cid:4), propylene oxide (electron microscopy supramaximalstimulationofthephrenicnerve(5–7V,0.2ms)
grade)andsodiumcacodylatewerefromPolysciencesInc., b DifferssignificantlyfromcontrolAChE?/?values,PB0.05
123
CellMolNeurobiol(2011)31:909–920 913
Fig.1 Muscle tensions in response to repetitive stimulation of the
phrenic nerve in AChE?/? (a) or AChE-/- (b) hemidiaphragms.
Tensions were generated by applying 500-ms long stimuli to the
phrenic nerve at 20, 50, 70, 100, 200 and 400Hz. The muscle was Fig.2 a Traces showing MEPPs recorded from surface fibers of
allowedtorestfor30sbetweenstimulustrains.Recordswerejoined diaphragm muscles under control conditions and 30min after
atbaselinetofacilitatepresentation.Profilesillustratedaretypicalof exposure to the selective AChE inhibitor BW284C51 (1lM),
diaphragmatic tensions recorded in muscles isolated from 8–21 or the selective BChE inhibitor iso-OMPA (10lM) in AChE?/?
AChE?/?and9–11AChE-/-mice.Notethatat100Hz,thetetanus/ andAChE-/-mice.NotethatBW284C1increasedtheamplitudeand
twich ratio is over 5 in AChE?/? muscle, but only 2.3 in AChE-/- decay of MEPPs in AChE?/? but not in AChE-/- diaphragms, and
muscle,andthattetanicfusionisachievedat50Hzinthelatterbut thatiso-OMPAhadnoeffectineithermuscle.Calibrationbarsapply
onlyat70Hzandaboveintheformer toalltraces.b,cHistogramsshowingMEPPamplitudeanddecayin
hemidiaphragmsfromAChE?/?(openbar)andAChE-/-(filledbar)
mice.Valuesrepresentthemean±SEMof38–42fibersfrom8–10
than in the Tibialis anterior muscle, where pronounced muscles. *Differs significantly from values in control AChE?/?
tetanic fade was observed at frequencies above 50 Hz hemidiaphragm; **Differs significantly from decay in AChE?/?
(Mouiseletal.2006).Superioradaptationofthediaphragm hemidiaphragm, P\0.05. MEPP decays in AChE-/- hemidia-
phragms were slightly reduced in BW284C51 and iso-OMPA, but
is consistent with its critical role in the survival of the
thiswasnotsignificantbyANOVA
animal (Nguyen-Huu et al. 2009).
Synaptic Transmission from the absence of AChE. MEPPs recorded from
hemidiaphragm muscle of AChE?/? mice exhibited an
Representative MEPPs recorded from AChE?/? and amplitude of 1.18 ± 0.11 mV, a decay time of 2.85 ±
AChE-/-miceareshowninFig. 2a,andquantalresponses 0.16 ms and a frequency of 2.18 ± 0.21/s, which are typ-
averaged from 38 to 42 fibers in 8–10 muscles are sum- ical values for control MEPPs in mammalian skeletal
marized in Fig. 2b, c. MEPPs represent spontaneous muscle (Del Castillo and Katz 1954; Yu and Van der
release of ACh from active zones, and their amplitude, Kloot 1991). MEPPs recorded in hemidiaphragm muscle
frequency and time course reveal important information of AChE-/- mice had an amplitude similar to control
about alterations of neuromuscular transmission that result (1.09 ± 0.10 mV), but a lower frequency (1.38 ± 0.15/s)
123
914 CellMolNeurobiol(2011)31:909–920
and a longer decay time (3.87 ± 0.14 ms). MEPPs in conjunction with the report that BChE can hydrolyze ACh
AChE-/- mice were less severely altered, however, than in the hippocampus of AChE KO mice (which have high
those of wild-type mice following acute inhibition of resting levels of ACh), suggests that BChE is effective
AChE. Thus, addition of 1 lM BW284C51 to diaphragm underconditionswhereAChismaintainedatasteadyhigh
muscles of AChE?/? mice resulted in a 97.8 ± 0.7% level (Hartmann et al. 2007), but not during the brief time
inhibition of AChE (Adler et al. 2004), leading to a 76% course of a single synaptic event.
increaseinMEPPdecaytimeanda42%increaseinMEPP TodeterminewhetherAChE-/-miceexhibitalterations
amplitude (Fig. 2). Addition of BW284C51 to muscles in evoked release, we examined the quantal content of the
fromAChE-/-micewaswithouteffect,consistentwiththe EPP under conditions where EPPs are reduced to small
absence of AChE in this preparation (Fig. 2). multiplesofunitresponses,aswellasunderconditionsthat
Since AChE-/- mice are able to express normal levels allow for recording of full-sized EPPs. Figure 3 shows
ofBChE(Xieetal.2000;Lietal.2000;Minicetal.2003; superimposed traces of EPPs from hemidiaphragm mus-
Girard et al. 2007), hydrolysis of ACh by BChE may cles of AChE?/? and AChE-/- mice in a high Mg2?/low
contribute to the maintenance of cholinergic function and Ca2? solution, which reduced the probability of release.
thus enable the survival of these animals. To examine this From the ratio of the number of stimulations/number of
possibility, we recorded MEPPs in the presence of the failures,itispossibletodeterminem ,themeannumberof
0
selective BChE inhibitor iso-OMPA (10 lM). At this quanta per nerve impulse (Del Castillo and Katz 1954). In
concentration, iso-OMPA inhibits 89% of BChE but only AChE?/?mice,m wascalculatedtobe2.14 ± 0.19under
0
12% of AChE (Adler et al. 2004). If BChE contributes to the present experimental conditions. In muscles from
thehydrolysisofACh,MEPPswouldbeexpectedtoshow AChE-/- mice, m was determined to be 1.90 ± 0.13, a
0
increasedamplitudesandprolongeddecaysinthepresence reduction of only 11%, which was not statistically signifi-
of iso-OMPA. The actions of iso-OMPA would be espe- cant. An absence of change in quantal content was previ-
cially prominent in muscles from AChE-/- mice, where ously reported by Minic et al. (2003) in AChE-/- mice.
BChEistheonlyenzymecapableofhydrolyzingACh. As Further examination of EPPs in diaphragms of AChE-/-
indicated in Fig. 2, however, iso-OMPA failed to alter andAChE?/?micerevealedthattherewerenodifferences
MEPP amplitudes or decay times in diaphragm muscle in latency (time from nerve stimulation to the onset of
from either AChE-/- or wild-type animals. This is in response)andinrisetime,whilethedecayoftheEPPwas
agreement with the findings of Girard et al. (2007), who prolonged (similar to that of the MEPP). These results
sawnoeffectof100 lMiso-OMPAonminiatureendplate suggest that evoked synaptic transmission in diaphragm
current amplitudes or decays. These results make it unli- muscle is well-maintained in AChE-/- mice.
kely that BChE is responsible for the relatively normal
synaptic responses in AChE-/- mice. However, BChE Recordings of EPPs and MEPPs in l-CTX
does appear to contribute to the hydrolysis of ACh during
repetitive stimulation as evidenced by the finding that iso- To examine neuromuscular transmission under conditions
OMPA markedly enhanced tetanic fade in hemidiaphragm approximating those of the contractility experiments,
muscles from AChE-/- mice during high frequency stim- recordings were made in the presence of 2.5 lM l-CTX,
ulation (C70 Hz) (Adler et al. 2004). This finding, in whichselectivelyinhibitsmuscleNa?channelsandallows
Fig.3 SuperimposedtracesshowingevokedEPPsrecordedfromthe potentialsandtwitches.FailureofEPPgenerationisreflectedbythe
endplate region of hemidiaphragm muscles isolated from AChE?/? horizontal traces lacking responses. Four superimposed traces are
(a) and AChE-/- (b) mice. EPPs were elicited by supramaximal shownforeachcondition.ThetriphasicresponsesprecedingtheEPPs
nerve stimulation at 0.5Hz in a modified Krebs-Ringer solution arestimulusartifacts
containing 8mM Mg2? and 1mM Ca2? to suppress muscle action
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CellMolNeurobiol(2011)31:909–920 915
fortherecordingoffull-sizedEPPs.Representativerecords musclesexposedacutelytoAChEinhibitors.Forthelatter,
elicited at 70 Hz in AChE?/? and AChE-/- hemidia- theamplitudesgenerallydiminishwithstimulationwithout
phragmsareillustratedinFig. 4.EPPsinwild-typemuscle reaching a plateau (Kuba et al. 1974).
showed a typical pattern consisting of initial facilitation At the conclusion of the train, two MEPPs were detec-
followed by moderate depression and then a gradual but tible in the AChE-/- muscle (arrows); the one occurring
incomplete recovery for the duration of the train (Moyer 100 ms from the end of the last EPP being considerably
and Van Lunteren 1999) (Fig. 4a). Transient depression of smaller (0.7 mV) than the one observed 50 ms later
EPPs during high frequency stimulation has been ascribed (1.6 mV) (Fig. 4d). Low amplitude MEPPs were typically
toadepletionofimmediatelyreleasableSSVsattheactive found during and immediately following repetitive stimu-
zone of the presynaptic membrane (Elmqvist and Quastel lation and are likely the result of desensitization of the
1965). The last four EPPs are also displayed on an AChRs from accumulation of ACh during the train. The
expanded time base in Fig. 4b, which shows MEPPs of subsequent increase in MEPP amplitude presumably
normal amplitude (1.0–1.3 mV) during the last 30 ms of reflects recovery from desensitization during the ensuing
thetrainandintheimmediatepost-tetanicperiod(arrows). post-train period (Magleby and Pallotta 1981; Elenes and
The record from the AChE-/- diaphragm depicts a Auerbach 2002).
similar pattern but with two notable differences: EPP Histograms of EPPs and MEPPs obtained prior to,
amplitudes were considerably more depressed than those during and following 70 Hz trains are shown in Fig. 5.
from muscles of wild-type mice, and a prominent under- MEPPsrecordedinthepresenceof2.5 lMl-CTXpriorto
lying depolarization was consistently observed, especially stimulation had similar amplitudes to those obtained in
during the initial 200 ms of stimulation (Fig. 4c). In spite control Krebs-Ringer solution (Fig. 2b), and no significant
of the marked frequency-dependent reduction in EPP difference between AChE?/? and AChE-/- mice was
amplitude, however, these responses were better main- observed(Fig. 5a,left).MEPPamplitudesrecordedduring
tained in muscles of AChE KO mice than in control the first 100 ms after the train were unaltered in AChE?/?
Fig.4 EPPs elicited by repetitive stimulation at 70Hz in the in baseline in (a) is likely the consequence of weak contraction of
presence of 2.5lM l-CTX in diaphragms from AChE?/? (a) or underlying muscle fibers, and was only infrequently observed,
AChE-/-mice(c);thelast4EPPsandpost-tetanicMEPPs(arrows) whereas that in (c) is due primarily to depolarization of the post-
are shown on an expanded time base in (b) and (d). Most muscle synaptic membrane during the high frequency train and was always
fibersfromAChE?/?miceand*60%fromAChE-/-miceexhibited observed.Trainswereelicitedforadurationof1sat3-minintervals.
facilitationofEPPsduringthesecondorthirdstimulation.Theshift Restingpotentialswere-61mVin(a)and-63mVin(c)
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916 CellMolNeurobiol(2011)31:909–920
mice, but underwent a significant reduction in AChE-/-
mice of nearly 40% relative to post-tetanic MEPPs in the
wild-type mice (Fig. 5a, right, P B 0.05).
EPP amplitudes determined from the first response in
the train showed no alteration between AChE?/? and
AChE-/- mice (Fig. 5b, left), whereas amplitudes at the
endofthetrain(averageoflastfour)exhibitedareduction
of59%inAChE-/-mice (P B 0.05; Fig. 5b,right). From
the values of EPPs and MEPPs, we calculated the quantal
content of the EPP prior to and following the 70 Hz train.
Using the ratio of the first EPP and the mean value of
MEPPsobtainedduring1-minperiodspriortothetrain,no
significant difference in quantal content (m ) was
r Initial
found between values in AChE?/? and AChE-/- mice
(Fig. 5c, left). Using the ratio of the last four EPPs of the
trainandMEPPamplitudesrecorded100 msfollowingthe
train, a 34% reduction in quantal content (m ) was
r Final
observed (P B 0.05; Fig. 5c, right).
These data indicate that during high frequency stimu-
lation, a combination of presynaptic (decrease in m) and
r
post-synapticfactors(depolarization/desensitization)ledto
transient reduction of EPP amplitudes. Of importance was
the finding that the amplitudes reached a plateau, and did
not decline progressively with stimulation (Fig. 4). At the
plateau level, it would still be possible to trigger action
potentials (in the absence of l-CTX). The ability of
AChE-/- diaphragm muscle to maintain firing arises from
the high safety factor of neuromuscular transmission, such
that EPPs need only reach a potential of approx. -55 mV
to achieve threshold for spike generation.
Synaptic Ultrastructure
To investigate morphological alterations of endplates
resulting from the absence of AChE, we examined neuro-
muscular junctions of AChE?/? and AChE-/- hemidia-
phragms by TEM. Typical neuromuscular junctions from
Fig.5 Histograms of MEPP amplitude (a), EPP amplitude (b) and an AChE?/? mouse and an age-matched AChE-/- mouse
quantalcontent(c)obtainedpriorto,duringandfollowing70Hztrains
(1s)inmuscle fibersfrom AChE?/? andAChE-/-mice. aMEPPs are shown in Fig. 6a and b, respectively. Neuromuscular
wererecordedfora1-minperiodpriortostimulation(Beforetrain)and junctions from AChE-/- mice displayed marked abnor-
for100msafterthelastEPP(Aftertrain).bAmplitudesofthefirstEPP malities, including alterations of presynaptic membranes,
andtheaverageofthelastfourEPPsweredeterminedforeachfiber.
synaptic clefts and junctional folds (Fig. 6b). Nerve ter-
cQuantalcontentwascalculatedbytheratioofEPP/MEPPamplitudes.
minals exhibited a smaller surface area and appeared to
InitialwascalculatedfromthefirstEPPinthetrainandMEPPsthat
wererecordedduring1minpriortothetrain(m ).Finalvalues contain fewer SSVs. Junctional folds were reduced in
rInitial
wereobtainedfromtheaverageofthelastfourEPPamplitudesdivided number and irregular in shape. In addition, Schwann cells,
byMEPPamplitudesrecordedfor100msaftertheendofthetrain(m
r whicharenormallyfoundontheoutersurfaceofthenerve
).Averagingoffourresponsesservedtoreducetheeffectofrandom
Final terminal, were often observed extending into the synaptic
fluctuationindeterminingthefinalEPPamplitude.Forallhistograms,
EPPs were corrected for non-linear summation, and MEPPs were cleft. This is clearly illustrated in the inset to Fig. 6b,
referenced to a resting potential of -70mV. Thedata represent the where a Schwann cell process is positioned between the
mean±SEMofvalues from12to 16muscle fibersfromat least 3
nerve terminal and the primary synaptic cleft, and in the
AChE?/?and3AChE-/-mice.*Differssignificantlyfromvaluesin
correspondingcontrolAChE?/?diaphragm,P\0.05.Allrecordings upper right section of Fig. 6b, where a nerve terminal is
wereinthepresenceof2.5lMl-CTX completely engulfed by a Schwann cell.
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CellMolNeurobiol(2011)31:909–920 917
Fig.6 aMotorendplatefroma
diaphragmmuscleofan
AChE?/?mouse.Structures
indicatedaresynapticvesicles
(sv),presynapticmembrane
(pm),primarycleft(pc)and
junctionalfolds(j).bMotor
endplatefromadiaphragmofan
AChE-/-mouse.Note
Schwanncellprocess
(s)occupyingtheprimarycleft.
Inset37,0009.Endplatesshown
herearerepresentativeofover
106examined.AChE?/?and
AChE-/-micewerefromthe
sameagegroupasthoseusedin
theelectrophysiologicalstudy
The cumulative effect of these alterations would result since acute inhibition of AChE leads to a pronounced
in reduction of pre- and post-synaptic contacts and the tetanic fade,which becomes moresevere with increasesin
opening of additional pathways for diffusion of ACh. stimulation frequency (Heffron and Hobbiger 1979; Adler
These findings are consistent with morphological changes et al. 2004).
described in patients with a genetic impairment of colla- Animportantadaptationthatweidentifiedinaprevious
gen-tailed AChE (Hutchinson et al. 1993). Significantly, study was a reduction in AChR density at the AChE-/-
AChE-/- endplates did not show swelling of organelles endplate (Adler et al. 2004). Reduction of AChRs would
(mitochondria, endoplasmic reticulum) or supercontrac- have the effect of accelerating the removal of ACh by
tions,whicharecommonultrastructuralchangesfollowing diffusion,sincethehighdensityofreceptorsattheendplate
acute inhibition of AChE (Laskowski et al. 1977; Adler acts to retard the efflux of ACh from the synaptic cleft
et al. 1992). (KatzandMiledi1973;MaglebyandPallotta1981).Since
downregulationofnicotinic AChRs has alsobeen reported
to be triggered by chronic exposure to cholinesterase
Discussion inhibitors (Costa and Murphy 1983), the loss of AChRs is
likely to be a consequence of ACh accumulation. More-
The present study was undertaken to determine the char- over, Hutchinson et al. (1993) have found similar reduc-
acteristics of neuromuscular physiology and ultrastructure tions in AChRs in muscles from patients with congenital
in AChE-/- mice. Examination of muscle contractility human AChE collagen tail deficiency.
revealed that AChE-/- mice were able to generate sus- Our current TEM study suggests that there is also a
tained neurally elicited tetanic tensions at 70 and 100 Hz, reduction in the fraction of the post-junctional membrane
but produced decremental responses at lower and higher that is in contact with the nerve terminal and an overall
stimulation frequencies (Fig. 1). These findings suggest simplification of the junctional folds (Fig. 6b). It has been
that AChE KO mice had adapted to the absence of AChE, estimated that the normal junctional folds, such as those
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