DTIC ADA540751: Glutamate Receptor Pathology is Present in the Hippocampus Following Repeated Sub-Lethal Soman Exposure in the Absence of Spatial Memory Deficits

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Glutamate receptor pathology is present in the hippocampus following repeated sub-lethal
soman exposure in the absence of spatial memory deficits 5b. GRANT NUMBER

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6. AUTHOR(S) 5d. PROJECT NUMBER
Johnson, EA, Daugherty, KS, Gallagher, SJ, Moran, AV, DeFord, SM
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US Army Medical Research Institute of Aberdeen Proving Ground, MD
Chemical Defense 21010-5400 USAMRICD-P07-003
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3100 Ricketts Point Road

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Published in NeuroToxicology, 29, 73–80, 2008. This work was supported by the U.S. Army Medical
Research and Materiel Command under Contract/Grant/Intergovernmental Project Order #DAMD17-00-2-0002.
14. ABSTRACT
See reprint.
15. SUBJECT TERMS
Soman; GD; NMDA receptor; AMPA receptor; Glutamate; Morris water maze; Sub-lethal; Spatial memory; GluR2; NMDAR1;
NMDAR2a; NMDAR2b
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OF ABSTRACT OF PAGES Erik A. Johnson
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NeuroToxicology29(2008)73–80
Glutamate receptor pathology is present in the hippocampus
following repeated sub-lethal soman exposure in the absence
of spatial memory deficits
Erik A. Johnsona,b,*, Kelly S. Daughertyb, Sarah J. Gallagherb,
Anita V. Moranb, S. Michelle DeFordb,c
aUSArmyMedicalResearchInstituteofChemicalDefense(USAMRICD),3100RickettsPointRoad,ComparativeMedicineDivision,
ComparativePathologyBranch,AberdeenProvingGround,MD21010-5400,USA
bUSArmyMedicalResearchInstituteofChemicalDefense(USAMRICD),AnalyticalToxicologyDivision,NeurobehavioralToxicologyBranch,
AberdeenProvingGround,MD21010-5400,USA
cDepartmentofPsychology,UniversityofMassachusettsatDartmouth,285OldWestportRoad,NorthDartmouth,MA02747,USA
Received7August2007;accepted6September2007
Availableonline12September2007
Abstract
Muchisstillunknownaboutthelong-termeffectsofrepeated,sub-lethalexposuretoorganophosphorus(OP)nerveagents,suchassoman
(GD),onlearningandmemorytasksandrelatedproteinexpressioninthehippocampus.Inthepresentstudy,guineapigswereexposedtosub-lethal
dosesofGDfor10daysandcognitiveperformanceassessedusingtheMorriswatermaze(MWM)upto88dayspost-exposuretoinvestigate
spatial learning. Additionally, hippocampal lysates were probed for cytoskeletal, synaptic and glutamate receptor proteins using Western blot
analyses. No significant difference in MWM performance was observed between repeated sub-lethal GD exposed and saline control groups.
However, Western blot analyses revealed significant changes in glutamate receptor protein immunoreactivity for subunits GluR2, NMDAR1,
NMDAR2aandNMDAR2binthehippocampiofGD-exposedguineapigs.LevelsofGluR2,NMDAR2aandNMDAR2bincreasedby3months
post-initial exposure and returned to control levels by 6 months while NMDAR1 decreased by 6 months. No significant differences in
neurofilamentmedium(NFM),neurofilamentlight(NFL)orsynaptophysindensitometryweredetectedanda-II-spectrinproteolyticbreakdown
wasalsoabsent.Theseresultsrevealthatrepeated,sub-lethalexposuretoGDaffectsglutamatereceptorsubunitexpressionbutdoesnotaffect
cytoskeletalproteinimmunoreactivityortheproteolyticstateinthehippocampus.Thoughthesechangesdonotaffectspatialmemory,theymay
contributetoother cognitivedeficits previouslyobservedfollowingsub-lethal OPexposure.
PublishedbyElsevier Inc.
Keywords: Soman; GD; NMDA receptor; AMPA receptor; Glutamate; Morris water maze; Sub-lethal; Spatial memory; GluR2; NMDAR1; NMDAR2a;
NMDAR2b
1. Introduction binds to acetylcholinesterase (AChE) in both the peripheral
andcentralnervoussystems.Atlethaldoses,accumulationof
Soman (pinacolyl methylphosphonofluoridate, GD) is a acetylcholinerapidlyleadstoseizures,respiratoryfailureand
potent organophosphorous compound (OP) that irreversibly death. Survivors of acute, sub-lethal doses can experience
long-term health and psychological effects (Kawana et al.,
2001; McCauley et al., 2001; Ohtani et al., 2004; Kawada
et al., 2005). However, past events have shown that most
Abbreviations: GD, soman; XGD, dilute soman; MWM, variable start
anterogradeMorriswatermazeparadigm;GluR,glutamatereceptor;AMPA, people, especially emergency and medical treatment person-
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDAR, N- nel, exposed during a mass nerve agent incident receive an
methyl-D-aspartic acid glutamate receptor; NFM, neurofilament medium; initial mild or asymptomatic level of exposure (single,
NFL,neurofilamentlight;PIE,post-initialexposure;AChE,acetylcholinester-
multiple or extended) as opposed to an acute symptomatic
ase.
exposure (Levin and Rodnitzky, 1976; Morita et al., 1995;
* Correspondingauthor.Tel.:[phone];fax:[phone].
E-mailaddress:[email protected](E.A.Johnson). Gray et al., 1999).
0161-813X/$–seefrontmatter.PublishedbyElsevierInc.
doi:10.1016/j.neuro.2007.09.002
74 E.A.Johnsonetal./NeuroToxicology29(2008)73–80
Previous studies have revealed changes in the brain carboxyesterase(CaE)levelsandturnoverratesfortheguinea
following sub-lethal nerve agent exposure (Burchfiel et al., pig more closely mimic humans than those for either rats or
1976;Duffyetal.,1979)thatinvolvenotonlytheacetylcholine mice (Atchison et al., 2004). Guinea pigs were exposed to
system, but also the glutamate system (Shih et al., 1990; multiple,sub-lethaldosesofGD(0.4(cid:2)LD )andassessedup
50
Lallement et al., 1991, 1992, 1994b; Sparenborg et al., 1992; to88dayspost-initialinjectionusingtheMorriswatermazeto
McDonoughandShih,1993;deGrootetal.,2001).Excitotoxic study long-term learning and memory deficits. The hippo-
injury caused by increased levels of glutamate has repeatedly campusofeachanimalwasthenprobedforalteredcytoskeletal,
been shown to cause cognitive dysfunction (Phillips et al., synaptic and glutamate receptor protein expression using
1998; O’Dell et al., 2000; Faden et al., 2001). Interestingly, Western blot analyses to correlate protein changes with any
studies show changes in the brain following sub-lethal nerve observed behavioral deficits.
agent exposurethat lead tomemory andattentiondeficits that
normally involve the hippocampus (Hatta et al., 1996; 2. Materials and methods
Nishiwaki et al., 2001; Miyaki et al., 2005).
The role of the hippocampus in complex visuo-spatial 2.1. Animals
learning and memory has been well established. The high
concentrations of N-methyl-D-aspartate glutamate (NMDA) Ten-week-olddietrestrictedmaleHartleyguineapigs(Crl:
and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (HA)BR) (Charles River Laboratories, Wilmington, MA)
acid (AMPA) glutamate receptors, which play a key role in weighing approximately 460g were used for the behavioral
hippocampal-mediated learning and memory (Izquierdo and andbiochemicalsectionsofthisstudy.Thepathophysiologyof
Medina,1997), alsomakethe hippocampus highlyvulnerable GD exposure varies depending on weight and fitness (Sipos
to glutamate-induced excitotoxic injury from GD poisoning etal.,2002)anddietcontrolisasimpleproceduretodetectGD
(Shih et al., 1990; Lallement et al., 1991, 1992, 1994b; exposure effects in long-term guinea pig studies (Nold et al.,
Sparenborgetal.,1992;McDonoughandShih,1993;deGroot 2001).Therefore,theseanimalsweredietcontrolledratherthan
et al., 2001). Repeated sub-lethal exposure to OP compounds free-fed and sedentary as a better correlate to the lifestyles of
canraiseextracellularglutamatelevelsinthebrain(Singhand active military and emergency personnel (Sipos et al., 2002).
Drewes, 1987) and increased glutamate availability can alter Animals were fed 60mg/kg of Harlan Teklad guinea pig diet
glutamatereceptorexpression(Piehletal.,1995;Kreutzetal., once daily following GD exposure and MWM trials. Each
1998; Cebers et al., 2001). Additionally, perturbations in animal was implanted with an IPTTTM-300 transponder chip
NMDA receptor subunit distribution on the neuronal cell (Bio Medic Data Systems, Seaford, DE) for identification
surface (Hardingham et al., 2002) or a rearrangement in the purposes and to record body temperature. Temperatures and
ratioofNMDAsubunitsinindividualreceptorscanchangethe weights were recorded twice daily just prior to morning GD
overall physiology of the receptor andthe functionality ofthe exposureandafternoonMWMtrials.Datawerecollectedpost-
hippocampus (Cebers et al., 1999).Manymodelsofcognitive initialexposure(PIE)anddesignatedasimmediate(7–11days
dysfunctionhaveshownalteredglutamatereceptorexpression PIE), 1 month (14–28 days PIE), 3 months (84–88 days PIE)
(Luthi-Carter et al., 2003; Mishizen-Eberz et al., 2004) and and 6 months (154 days PIE). The research environment and
NMDA subunit ratio rearrangement (Mikuni et al., 1998) protocols for animal experimentation were approved by the
suggesting that these mechanisms may also contribute to institutional animal care and use committee (IACUC) and
cognitive dysfunction following sub-lethal GD exposure. complieswiththeNationalInstitutesofHealthGuideforCare
Altered cytoskeletal structural protein [e.g., microtubule andUseofLaboratoryAnimals(PublicationNo.85-23,revised
associated protein 2 (MAP2), neurofilaments (NF), a-II- 1985). The animal care program at this Institute is fully
spectrin] immunoreactivity has been correlated with adverse accredited by the Association for Assessment and Accredita-
behavioral effects following CNS injury (Pike et al., 1998; tion of Laboratory Animal Care International.
Isaksson et al., 2001; Shaw et al., 2005; Briones et al., 2006;
Bruschettinietal.,2006)anddistinctlossesofMAP2havebeen 2.2. Soman administration
shown in the hippocampus following acute GD exposure
(Ballough et al., 1995). Though altered expression of these GD (GD-U-2323-CTF-N, purity 98.8wt%) was obtained
structuralproteinsalonedoesnotsufficeasevidenceforaltered and diluted at the US Army Medical Research Institute of
inter-neuroncommunication(Huhetal.,2003),inconjunction Chemical Defense (USAMRICD, Aberdeen Proving Ground,
with markers of synaptic architecture (e.g., synaptophysin), MD).Thehighestsub-lethaldoseofsoman(XGD;11.2mg/kg
thesemarkerscanbeagoodindicatorofmaladaptiveplasticity or 0.4(cid:2)LD ) that does not produce acute toxic signs
50
and altered inter-neuron communication (Phillips et al., 1994; following a single injection (Atchison et al., 2004) was
D’Ambrosio et al., 1998). administered subcutaneously in the scruff of neck once a day
The present study investigated the effects of repeated sub- (MON-FRI)for10exposuresat1.0mL/kgperinjection.Signs
lethal GD exposure on anterograde visuo-spatial learning and of GD toxicity were measured twice daily, in the morning
memory and correlated those results to cytoskeletal, synaptic within30minofexposureandintheafternoonwithin30min
andglutamatereceptorproteinalterationsinthehippocampus. of the last MWM trial. Weight and temperature along with
The guinea pig model was used for these studies due to either the presence or absence of lacrimation, salivation,
E.A.Johnsonetal./NeuroToxicology29(2008)73–80 75
fasciculations and hyperactivity were measured. No animals (Caframo;Wairton,ONT)andTissueTearer(Biospec;Racine,
died as the result of GD exposure. WI) on ice. Protein concentration was determined using
bicinchoninic acid (BCA) micro protein assays (Pierce, Inc.;
2.3. Morris water maze (MWM) visuo-spatial complex Rockford, IL). Forty micrograms of protein were loaded per
learning and memory task well and separated by SDS-PAGE, transferred to polyvinyli-
denedifluoridemembranesandprobedwithaprimaryantibody
The variable start Morris water maze (MWM) anterograde for either NFM (Encor Biotechnology; Alachua, FL), NFL
memorytaskhassuccessfullyandreliablymeasuredcognitive (Encor Biotechnology; Alachua, FL), synaptophysin (Sigma–
impairmentinothermodelsofCNSinjury(Hammetal.,1992), Aldrich; St. Louis, MO), a-II-spectrin (Biomol International;
includingrepeatedmildexcitotoxicbraininjury(DeFordetal., Plymouth Meeting, PA), NMDAR2a (Sigma–Aldrich; St.
2002). Guinea pigs were trained on the variable start Louis, MO), NMDAR2b (Sigma–Aldrich; St. Louis, MO),
anterogradeMWMparadigmwithexternalmazecues(Morris GluR2/3 (Chemicon; Temecula, CA) or GluR3 (Chemicon;
et al., 1982)with modification: a 15cm platform replaced the Temecula, CA). Actin (Sigma–Aldrich; St. Louis, MO) was
10cmplatformtoaccommodatetheguineapig(deGrootetal., labeled as a loading control. The membranes were then
2001). Briefly, a light colored fiberglass pool with a 160cm thoroughly rinsed in Tris-buffered saline with Tween 20,
diameterandaheightof60cmwaslocatedina2.4m(cid:2)2.1m incubated with anti-rabbit (Sigma–Aldrich; St. Louis, MO) or
room.Thetankwasrefilleddailywith258Cwatertoaheightof anti-mouse (Zymed/Invitrogen; Carlsbad, CA) alkaline phos-
50cmtooptimizetheobservanceofvisualcuesfortheguinea phatase-conjugated secondary antibody and developed using
pig. Extra-maze cues were positioned around the pool and Enhanced Chemifluorescence reagents (ECF, Amersham;
remainedconstantthroughouttheexperiment.Theguineapigs Arlington Heights, IL). The membranes were then imaged
weretrainedtolocatetheclearplexiglassplatformsubmerged using the STORM 860phosphorimager/fluorimager (Molecu-
2cmbelowthewatersurfaceandfurtherobscuredbynon-toxic larDynamics;GEHealthcare,Piscataway,NJ).Salinecontrols,
whiteTemperapaint.Foreachtrial,theguineapigwasplaced experimentalgroups(1,3and6monthsPIE)andna¨ıvecontrol
facingthewallofthepoolinoneofthefourpoolquadrants.The (unexposed) groups each had an n=6. Transformed data
pool quadrant was determined randomly for each day and (exposed or saline densitometry value/na¨ıve control densito-
separatelyforeachanimal.Eachtrialbeganwiththeguineapig metryvalue(cid:2)100)wereevaluatedbyANOVAandapost-hoc
beingplacedinthepoolandendedwhentheanimalclimbedon Bonferroni-test for selected pairs (i.e., saline v GD for each
the platform. The maximum duration allowed was 120s. For timepoint)wasapplied.Valuesareexpressedaspercentageof
eachsession,theanimalwasmarkedwithalargeblackinkdot naive controls and are given as the mean(cid:5)S.D. Differences
on its back for video tracking purposes. Each animal was were considered significant at the level of p(cid:3)0.05.
trackedusingaPanasonicBP334Digitalvideocameraanddata
were recorded and analyzed using WatermazeTM version 2.6 3. Results
software (Coulbourn Instruments; Allentown, PA). If the
platform was not found after 120s, the animal was placed 3.1. Physiological measurements
directly on the platform for 15s. At the end of each trial, the
animal was toweled dry and placed in a transfer cage with a Guinea pigs were injected for 10 days (MON-FRI for 2
heatinglamp.AssessmentofMWMperformanceconsistedof weeks)witheithersalineor0.4(cid:2)LD GD.Changesinbody
50
four trials per day (2–5min inter-trial interval) for 5 mass from the first to the last exposure (10 day mass change)
consecutivedays.Animalswere assessedat3-week-longtime was significantly different between the sub-lethal GD and
points(7–11, 14–18and 84–88 days PIE). At each week-long saline-treated groups (saline: +32.2g versus GD: +19.5g,
time point, the platform was randomly placed in a new p<0.05). Total average body temperature readings were also
quadrant.Trialduration(s),totalpathdistance(cm),andswim significantly different between the two groups (saline:
speed (cm/s) were recorded for analysis. The number of 101.1(cid:5)1.68CversusGD:102.0(cid:5)0.88C,p(cid:3)0.001)though
animalsusedforeachtrialwasn=20(days7–11,14–18)and tolerance was not observed (XGD day 1 versus XGD day 12,
n=10(days84–88).Datawereevaluatedbyrepeatedmeasures datanotshown).Lacrimationwasabsentinbothgroups.Excess
ANOVA analysis with significance set at p(cid:3)0.05. salivation, fasciculations and hyperactivity were absent in the
saline group but appeared sporadically, though not on
2.4. Western blot analyses consecutivemeasurements, in the GD group. Out of 200 total
observations for the GD group, only 5% were marked as
Western blot analyses of hippocampal tissue lysates were hyperactive,14.5%asfasciculatingand2%ashavingsignsof
conducted as previously described (Johnson et al., 2004). excess salivation.
Briefly, guinea pigs were deeply anesthetized using a lethal
dose of pentobarbital, hippocampus tissues were rapidly 3.2. Learning and memory
excised, frozen in liquid nitrogen and stored at (cid:4)808C. The
tissuewashomogenizedinice-coldtripledetergentlysisbuffer To determine whether repeated sub-lethal GD administra-
containing a CompleteTM protease inhibitor cocktail (Roche tion affected visuo-spatial complex learning and memory,
Biochemicals; Indianapolis, IN) using a motorized pestle guinea pigs were assessed using the MWM task as described
76 E.A.Johnsonetal./NeuroToxicology29(2008)73–80
Fig.1. NolearningandmemorydeficitsweredetectedinGD-exposedguinea
pigsusingtheMWMtask.Nosignificantdifferenceswereobservedbetween
Fig.2. NMDAR2adensitometryissignificantlyincreasedfollowingrepeated
saline-treatedandGD-treatedgroupinlatencytimesatanypost-initialexposure
sub-lethal GD exposure. (A) Representative western blots of NMDAR2a
(PIE)time.Additionally,nodifferenceswereobservedfordistancetraveledand
(170kDa)in hippocampusrevealedanincreaseinNMDAR2aimmunoreac-
averagevelocityusingaone-wayANOVAanalysis(notshown).Eachdatapoint
tivityfollowingrepeatedsub-lethalGDexposurecomparedtosalinetreatment.
representstheaveragetrialsperday(20trialspertimepointperanimal,)for
(B)DensitometricanalysisoftheNMDAR2abandshowedsignificantincreases
each group from an n=20 (days 7–18) or n=10 (days 84–88). Values are (*p<0.05)at3monthspost-initialexposure(PIE).Actin(43kDa)wasusedas
reportedasmean+S.D.
aloadingcontrol.Dataaregivenaspercentofthenaivecontrols;eachtime
pointrepresentsdatafromann=6andisreportedasmean(cid:5)S.D.
above. Repeated measures ANOVA revealed no significant
differences at anyMWM time point between the salinegroup increase from saline controls of 28 and 32%, respectively
andtheGD-treatedgroupinlatency(Fig.1),totalpathdistance (Figs. 2 and 3). Analysis of the NMDAR1 subunit revealed a
or swim speed (data not shown). significant decrease in immunoreactivity in the GD-exposed
groupcomparedtothesalinegroupby6monthsPIE(p(cid:3)0.01)
3.3. Cytoskeletal and synaptic protein immunoreactivity representing a reduction of 75% from saline controls (Fig. 4).
For AMPA receptors, antibodies for GluR2/3 (which
To determine whether repeated sub-lethal GD administra- recognizes both GluR2 and GluR3) and GluR3 were used.
tionledtochangesinneurocytoskeletalintegrity,densitometric Densitometric analyses for GluR3 revealed no significant
valuesfromWesternblotswereobservedforNFM,NFL,full-
lengtha-II-spectrinandsynaptophysin.Nosignificantchanges
were observed in the hippocampus of the GD-exposed group
for any structural protein compared to saline controls at any
timepoint.Similarly,thecytoskeletalproteinactinrevealedno
significantchangesinthehippocampusconfirmingitsuseasa
consistent loading control.
We also observed no significant differences in the
immunoreactivity of a-II-spectrin proteolytic breakdown
products, indicative of cell death pathway activation (Pike
et al., 1998), at 150kDa (calpain and caspase-3), 145kDa
(calpain-specific) or 120kDa (caspase-3-specific) at any time
point (data not shown).
3.4. Glutamate receptor immunoreactivity
To determine whether repeated sub-lethal GD administra-
tion affected NMDA or AMPA glutamate receptor immunor-
Fig.3. NMDAR2bdensitometryissignificantlyincreasedfollowingrepeated
eactivity, Western blot analyses were performed on
sub-lethal GD exposure. (A) Representative western blots of NMDAR2b
hippocampal lysates for NMDAR2a, NMDAR2b, NMDAR1, (180kDa)inhippocampusrevealedanincreaseinNMDAR2bimmunoreac-
GluR2/3andGluR3.DensitometricanalysesoftheNMDAR2a tivityfollowingrepeatedsub-lethalGDexposurecomparedtosalinetreatment.
and NMDAR2b subunits revealed significantly greater immu- (B) Densitometric analysis of the NMDAR2b band in hippocampus shows
significantincreases(*p<0.05)at3monthspost-initialexposure(PIE).Actin
noreactivity of both subunits in the GD-exposed group
(43kDa)wasusedasaloadingcontrol.Dataaregivenaspercentofthenaive
compared to saline controls in the hippocampus at 3 months
controls; each time point represents data from an n=6 and is reported as
PIE(p(cid:3)0.05;forbothNMDAR2aandb)andrepresentingan mean(cid:5)S.D.
E.A.Johnsonetal./NeuroToxicology29(2008)73–80 77
Fig.4. NMDAR1densitometryisdecreasedincreasedfollowingrepeatedsub-
lethalGDexposure.(A)RepresentativewesternblotsofNMDAR1(116kDa) Fig.5. GluR2densitometryissignificantlydecreasedfollowingrepeatedsub-
inhippocampusrevealedadecreaseinNMDAR1immunoreactivityfollowing lethalGDexposure.(A)RepresentativewesternblotsofGluR2/3((cid:6)106kDa)
repeatedsub-lethalGDexposurecomparedtosalinetreatment.(B)Densito- in hippocampus obtained from repeated sub-lethal GD-treated and saline-
metric analysis of the NMDAR1 band showed significant decreases treatedguineapigsrevealedadecreaseinGluR2/3immunoreactivityfollowing
(**p<0.01)at6monthspost-initialexposure(PIE)comparedtosalinetreat- repeatedsub-lethalGDexposurecomparedtosalinetreatment.(B)Densito-
ment.Actin(43kDa)wasusedasaloadingcontrol.Dataaregivenaspercentof metric analysis of the GluR2/3 bands in hippocampus shows significant
thenaivecontrols;eachtimepointrepresentsdatafromann=6andisreported decreases(**p<0.01)at3monthspost-initialexposure(PIE)thoughttobe
asmean(cid:5)S.D. due to decreases in GluR2 alone as analysis of GluR3 alone showed no
significantchanges(notshown).Actin(43kDa)wasusedasaloadingcontrol.
Dataaregivenaspercentofthenaivecontrols;eachtimepointrepresentsdata
difference between the GD-exposed and saline-treated groups
fromann=6andisreportedasmean(cid:5)S.D.
(data notshown). However, therewas a significantdecreasein
GluR2/3 immunoreactivity in the hippocampal lysates of the
GD-treated group at 3 months PIE (p(cid:3)0.01) representing a 1990), conditioned avoidance response and passive avoidance
decreasefromsalinecontrolsof43%(Fig.5).This,takenwith tasks following repeated, sub-lethal GD-exposure (Russell
theGluR3data,suggestsadecreaseinGluR2immunoreactivity. et al., 1986). Additionally, MWM performance following
repeated sub-lethal exposure to the OP compound, methami-
4. Discussion dophos, revealed no learning impairments (Temerowski and
vanderStaay,2005).However,thedosingparadigmitselfmay
The present study investigated visuo-spatial memory and preclude our ability to detect behavioral changes due to OP
cytoskeleton, synaptic membrane and glutamate receptor compound tolerance following repeated administrations (Rus-
subunit immunoreactivity in the hippocampus of guinea pigs sell et al., 1986; Hymowitz et al., 1990).
exposed to repeated sub-lethal doses of GD. This GD-dosing Previous studies of repeated sub-lethal GD exposures have
regime revealed significant changes in glutamate receptor demonstrated pathological and electroencephalographic
immunoreactivitydespitenoobservationofsignificantchanges changes following GD exposure in the absence of behavioral
invisuo-spatialmemoryorimmunoreactivityofstructuraland changes (Burchfiel et al., 1976; Duffy et al., 1979; Hymowitz
synaptic proteins in the hippocampus. et al., 1990). To determine whether the current GD dosing
This GD dosing paradigm has been shown to significantly paradigm led to cytoskeletal and synaptic protein alterations,
reduceredbloodcell(RBC)AChElevelsto9%ofcontrolsat suggestiveofneuronaldysfunctionordamage,weinvestigated
theendof2weeksofexposure.However,RBCanddiaphragm long-termproteinchangesinthehippocampusupto6months
AChEactivitysignificantlyrecoverby3daysfollowingthelast PIE by examining the expression patterns of three neuron-
GDexposure(Atchisonetal.,2004)whichsuggestsanylong- specific cytoskeletal proteins; NFM, NFLanda-II-spectrinas
term changes observed are not likely due to continued AChE well as the synaptic vesicle protein synaptophysin. No
inhibition.Followingrepeated,sub-lethalexposurestoGD,no significant changes in NFL, NFM, full-length a-II-spectrin
statistical difference was observed for latency, swim speed or or synaptophysin immunoreactivity were detected suggesting
total path distance between GD-exposed and saline-treated that the neuronal cyto-architecture remained unaltered.
groups in the MWM task despite the successful use of this Additionally,nosignificantchangesinthecaspase-3orcalpain
modelbyothergroups(deGrootetal.,2001;Filliatetal.,2002; generated breakdown products of a-II-spectrin were observed
Byrnes et al., 2004; Iqbal et al., 2004). These results are indicating that cell death pathways were not activated in
consistentwithpreviousworkshowingnolong-termbehavioral agreement with previous work (Churchill et al., 1985;
effects for schedule-controlled behavior (Hymowitz et al., Lallement et al., 1994a; Baille et al., 2001; Carpentier et al.,
78 E.A.Johnsonetal./NeuroToxicology29(2008)73–80
2001; Thomson et al., 2005). However, cell death is not subunit ratio alterations may have been functionally insuffi-
necessary for the manifestation of cognitive deficits (Lyeth cient, and in response, the expression of the entire receptor
et al., 1990). Altered immunoreactivity of other proteins, complex(asevidencedbyNMDAR1)islikelydown-regulated
such as glutamate receptors, may also produce cognitive (Cebers et al., 2001).
dysfunction. The ramifications of altered NMDAR subunit distribution
BoththeAMPA-andNMDA-typeglutamatereceptorsplay followingGDexposurearenumerous.IncreasesinNMDAR2a
anintegralroleinlearningandmemoryfunctionaswellasOP- and b subunit expression have been linked to aberrant
induced neural pathology. Specifically, AMPA-typeglutamate hippocampal mossy fiber sprouting and epiliptogenesis
receptor(GluR)activationisimplicatedinhippocampalhyper- (Mathern et al., 1996; Mikuni et al., 1998, 1999) and can
excitabilityobservedfollowingGDexposure(Sheardownetal., make NMDAR-expressing neurons particularly vulnerable to
1990; Lallement et al., 1991; Wood and Tattersall, 2001). excitotoxic insults (Kotapka et al., 1991; Hamm et al., 1993;
Densitometry revealed a significant long-term but transient Backetal.,2004;Mattsonetal.,2005).Additionally,decreases
decreaseinGluR2immunoreactivityinGDexposedanimalsat in NMDA1 receptors can lead to deficits in areas such as
3 months that disappeared by 6 months PIE. The increases in associativememoryrecall(Nakazawaetal.,2002)andshortto
GluR2levelsinthesalinecontrolgroupat3monthsPIEmaybe long-term memory conversion (Shimizu et al., 2000). Inter-
reflective of normal developmental changes in glutamate estingly, spatial memory may be a more resilient cognitive
receptordistributionastheanimalsagedfrom10to35weeks,a faculty as decreases in hippocampal NMDAR1 expression by
phenomenonthatmaybesuppressedinXGDexposedanimals. as much as 30% do not induce spatial memory deficits
Though GluR2 immunoreactivity normally decreases in the measurablebytheMWM(Inadaetal.,2003).Thoughthislevel
hippocampuswithage(Gazzaleyetal.,1996),largerdecreases ofdecreasedimmunoreactivitywasexceededat6monthsPIE
areseeninAlzheimer’sdisease(Hofetal.,2002),aswithour when compared to saline controls ((cid:4)75%), it was not if
model, and other neurodegenerative disorders including comparedtona¨ıvecontrols((cid:4)10%).Thismayhavecontributed
amyotrophic lateral sclerosis, epilepsy and brain ischemia to our failure to detect behavioral changes, though the
(Pellegrini-Giampietro et al., 1997; Weiss and Sensi, 2000; successful use of this model by others and the significant
Carter et al., 2004; Soundarapandian et al., 2005; Peng et al., reductionofAChEbyourdosingregimethatproducedsimilar
2006;Tortaroloetal.,2006).Thissuggeststhatrepeated,sub- reductions in NMDAR1 indicate otherwise.
lethalexposuretoGDmayrendersubjectsmorevulnerableto In conclusion, the Morris water maze did not detect
cognitive dysfunction development especially by exposure to complex visuo-spatial learning and anterograde memory
otherwise benign excitotoxic insults (Pellegrini-Giampietro deficits following repeated sub-lethal 0.4(cid:2)LD GD expo-
50
etal.,1997;MunirathinamandBahr,2004).Thesedatasuggest sures in guinea pigs. Additionally, Western blot analyses of
a transient pathological role for decreasing GluR2 subunit neuronal cytoskeletal and synaptic membrane proteins
expression in response to repeated sub-lethal GD-exposure revealed no abnormalities in the cyto-architecture or the
though this role is not well defined by this study and requires presenceofpathologicalproteolysis.Resultsdemonstratethat
further investigation. repeated sub-lethal GD-related leads to alteration of both
NMDA-type glutamate receptors, especially in the hippo- AMPA and NMDA glutamate receptor protein expression
campus, are integral to learning and memory but are also within the hippocampus up to 6 months PIE. Though altered
involved in pathological processes following acute OP expression of glutamate receptor protein has been linked to
poisoning (Churchill et al., 1985; Lallement et al., 1991; cognitive dysfunction, the ramifications of these changes
McDonough and Shih, 1997). A typical NMDA receptor is a following repeated sub-lethal GD exposure require further
tetramer comprised of a ratio of NMDAR2 subunits, which investigation.
contribute to the functionality of the receptor, and the main
obligatorysubunit,NMDAR1(Waffordetal.,1993;Chazotand
Acknowledgments
Stephenson,1997;Luoetal.,1997)witheachcomplexhaving
its own distinct pharmacological properties. Significant
This work was supported by the U.S. Army Medical
increases in NMDAR2a and b subunit immunoreactivity
Research and Materiel Command under Contract/Grant/
occurred in the hippocampus at 3 months PIE but returned
Intergovernmental Project Order #DAMD17-00-2-0002. The
to saline-control levels by 6 months PIE. Conversely,
findingscontainedhereinaretheprivateviewsoftheauthor(s)
NMDAR1 immunoreactivity was comparable between the
andarenottobeconstruedasofficialorasreflectingtheviews
GD-exposed and saline-treated groups at 3 months but
of the U.S. Army or Department of Defense.
significantly declined by 6 months PIE in the GD-exposed
group. This observation may reflect a redistribution of
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