- Email: [email protected]
Depression of the cat cortical visual evoked potential by soman
Life Sciences, Vol. 48, pp. 1269-1276 Printed in the U.S.A.
Pergamon Press
DEPRESSION OF THE CAT CORTICAL VISUAL EVOKED POTENTIAL BY SOMAN E J DeBruyn, G K Corbett and A B Bonds Department of Electncal Engineering Vanderb,t Umversity, Nashwlle, TN 37235 (Received in final form January 23, 1991)
Summary The effects of intravenous admmmtrat~on of the ant=chohnesterase agent soman (pmacolyl methytphosphonofluondate, 3-15 i~g/kg) on the visual evoked potential (VEP) were examined ,n cats using phase-reversed sine wave grating st=muh of diferent spat,al frequenc=es and contrasts Doses of 5-7 I~g/kg caused a depress=on of the VEP across all spatml frequencies ,n an abrupt, non-graded lash=on Stud=es m wh,ch contrast was vaned showed that VEP depression resulted pnmardy from a decrease ,n the system gain rather than a change =n the contrast sens~tMty, and that response depress,on increased with increasing contrast The dominant changes m gain revealed by these stud=es a r e consistent w=th a modulation of potass=um conductance in the cell membrane wh,ch prewous stud,es have shown to be dependent on a chohnerg~c mechanmm Antichohnesterase agents such as physost=gmine, pyndostigmme, d,sopropylfluorophosphate (DFP) and soman are thought to exert thmr tox,c actions by mact,vatmg acetylchohnesterase (ACHE) The resulting accumulation of acetylchohne (ACh) throughout the penpheral and central nervous systems produces symptoms of continuous chohnergm st=mulat=on Whde the general impact of these agents has been rev,ewed at length (t-4) and reformat=on is avadable on the=r act=on on many systems of the body, the effects of these agents on sensory systems have been less well stud,ed The visual system ,s the best understood sensory system, and as such prov,des a good model for the study of the actions of ant,chohnesterases Of special interest =s the act,on of these agents on the funct=on of pnmary vmual cortex, since chohnergic influences are apparently widespread in th=s area (5) Evoked potent=al studies (6-11) have shown that system=cally delivered ACh and chohnerg~c agonmts have a pnmardy depressant role in area 17, whde chohnerg=c antagonmts enhance responses Add=t,onally, Phdhs and York (12-14), and Sdhto and Kemp (5) have presented ev,dence for chohnerg,c modulation of single cell responses The latter authors found that 92% of the cells encountered in visual cortex responded m some way to ACh, suggest,ng that ACh may play a fundamental role m vmual cortmal processing We have reported ead=er (15,16) that intravenously administered physost,gmme, pyndost=gmme, DFP and soman depress the v,sual evoked potent,al (VEP) by altering the contrast gain of the system In the present report we extend our ongmal find,ngs on soman by examining ~s ,mpact on the response of the VEP to st=muh of different spatial frequenc,es and contrasts Methods Pr~parat=on - Bas=c procedures have been descnbed elsewhere (16) Ten cats (1 5-5 2 kg) w e r e anesthet,zed w,th halothane, and surg,cal anesthesm was subsequently maintained w=th intravenous (I V ) 2 5% thtamylal sodium Following tracheal cannulat=on, the animal was mounted ,n a stereotaxic headholder, the scalp was reflected and stainless steel screws were inserted at bregma (indiferent Aaaress correspondence to Dr Edward DeBruyn, Dept of Electncal Engmeenng, Vanderbdt Univers~y, Nashvdle TN 37235 0024-3205/91 $3.00 + 00 Copyrlght (c) 1991 Pergamon Press plc
1270
Soman and Vzsual Evoked Potentlal
Vol. 48, No. 13, 1991
electrode) and on the mldhne at H-C coordinate P4 (recording electrode) Mydr,asls and cycloplegla were induced with top=cal phenylephr,ne HCI (10%) and atropine sulfate (1%) and the corneas were fitted with contact lenses with 4 mm artificial pupds to optimize retinal amage quality The animal was refracted using direct ophthalmoscopy and auxiliary spectacle lenses were used to render the retinas conjugate w~h the stimulus plane For recording, the cats were paralyzed with gallamme tnethlod=de (10 mg/kg/hr) in lactated Ringers solution containing 1 mg/kg/hr Suntal to stabdlze anesthes=a The animals were artrf,clally respired with a mixture of N20 (75%), O2 (23 5%) and CO= (1 5%) and peak expired CO= was maintained at 4 2% Rectal temperature was maintained at 37°C with a servo controlled heat pad and heart acttv~ and blood pressure were monitored as Indicators of adequate anesthesia and drug effects The electroencephalogram (EEG) was mon~ored as an indicator of the animal's state of arousal St,mulatton and Data Processmq - One-dlmens=onal sine wave gratings were generated digitally on a Joyce Electronics display (mean luminance 280 cd/m =) with linear luminance modulation for contrasts up to 1 0 View,ng distance was 57 cm and displays were masked to a circular field subtending 18 degrees The grat,ngs were oriented horizontally and viewed monocularly Following the method of Snyder and Shapley (17), the results were Fourier transformed, and the first 15 even harmon=cs (which were temporally correlated with the test stimuli) were summed to give a figure for signal plus noise The first 15 odd harmonics provided a figure for noise alone A total of 100 seconds of averaging per datum yielded a best signal-to-noise ratio of t 5 1 Drugs - Soman was obtained from the USAMRDC (Fort Detnck, Frederick, MD), dduted to a fmal concentrat=on of 40 i~g/ml in sterile saline and stored at -80°C Immediately prior to ,nlect=on it was thawed in warm tap water, and stored on =cefor the remainder of the expenment A range of dosages (315 I~g/kg, dosages for each experiment are indicated below) was administered by slow I V anjectlon over a 1 minute period Exoenmental Deslqn - Up to six tnals were run and averaged to provide pre drug control values Soman was then administered and an experimental trial was run The dose of soman was tltrated m 2-3 I~g/kg increments until a reliable reduction of the VEP was noted, and the VEP was observed for up to 20 hours following drug administration In order to document any posstble spontaneous recovery Doses were given at either 12 or 35 minute intervals (depending on the length of trial being run), and blood samples for biochemical analysis were taken before each administration of soman, and at the begmmng of each tr,al following administration Chohnesterase actMty was determined by the EIIman (18) techn=que, and blood gas analysis (02, CO2 and pH) was performed Jn order to rule out the posslbdJty that changes ,n the VEP were due to hypoxla Results Normal VEP Response - At the beg,nmng of each expenment, the overall visual performance of each animal was tested by present,ng a series of gratings at different spatial frequenmes and contrasts spanning the normal operating range of the cat's vtsual system Values for high frequency cutoff and contrast sens~rvity were estabhshed as described elsewhere (19) High frequency cutoff values were usually between 3 and 4 cycles/degree (c/deg) with a maximum value of 5 6 c/deg, and the average contrast sensitMty at the optimum spatial frequency was 21, w~th a maximum value of 36 Both of these values are similar to (although somewhat lower than) those reported for the behaving cat (20,21), so we feel that the VEP =s a reasonably good md,cator of the overall performance of the visual system Effects of Soman - Admin=stratJonof soman (3-15 I~g/kg) usually resulted in a generahzed depression of the VEP and the endogenous cortical noise level over all tested parameters, along w=th desynchron,zation of the EEG The depression was readdy reversed by atropine, suggesting that ~tJs mediated by muscarinlc influences Spatial Sensltlv~y - Soman produced a depression of the response at all spat,al frequenc0es (FIG 1) For ten cats (doses ranged from 3-15 p.g/kg) the average depression at the lowest spatial frequency (0 1 c/deg) was 57 79% (+ 2 63 SEM), while that at the highest frequency (1 6 c/deg) was 53 78% (± 2 65) We found no s~gnificant rod=cation of a preferential depression at low spatial frequenmes as opposed to
Vol. 48, No. 13, 1991
Soman and Visual Evoked Potential
1271
high spatial frequencies, as reported by Harding et al (9,22) and K0rby, et al (10,11) Contrast Sensatrvrtv and Gain - The contrast senmtMty of each ammal was determined by presenting a series of contrasts at different spatial frequencnes spanning the normal range of the cat visual system In agreement wnth eadier results (19,21), the response ampl0tude was found to vary lineady with the logarithm of the contrast in both pre and post drug trials, and a least squares hnear fit was used to define threshold (the intercept with the noise level) and slope (equivalent to the system gain) As reported eadner (16), soman reduced the gain of the function to a greater extent than it changed the sensitrvlty Across 30 trials, the average decrease nn gain (slope) was 0 45 log umt, whde the average reduction in the contrast B 60
50 tO
40
O
Nil0 Contrast = 16%
Contrast = 6%
"10
I
I
i
I
n," (1)
¢O rr
D
C I
60
50
40 Nil0 Contrast = 40% 01
02
N=50 All Contrasts 05
10
01
02
05
10
Spatial Frequency (cycles/degree) FIG 1 Soman-mduced response depressnon at dufferent spatml frequenc0es The symbols represent the mean VEP depressnon for 10 cats for three different spatial frequencies (0 1, 0 4 and 1 6 c/deg) at contrasts of 6% (A), 16% (B), 40% (C), and at all contrasts combined (D) Although the plots tend to have a negatnve slope, the response depressnon at h~gh spatnal frequencies vs that at low spatial frequencies is not sugnfflcantly dnfferent sensit,vnty (the inverse of threshold) was 0 11 log un~ The decrease in slope approximated the average reduct0on in the VEP response amplitude (0 38 log umt), suggesting that the gain change accounts largely for VEP depression A second effect of soman on the response vs contrast relationship is illustrated tn FIG 2 When the data from all 10 cats are pooled, higher contrast levels are seen to produce proportionately greater depression of the VEP This trend is evident from the positive slopes of all the graphs, and is slgnsficant (p<0 05) at 1 6 cycles/degree and when all spatual frequencies are pooled The data suggest that soman is causing a saturation of the VEP at high contrast levels
1272
Soman and Vlsual Evoked Potential
Vol. 48, No. 13, 1991
Dose Response and Correlat00n w~th Blood Chohnesterase Levels - The dosage of soman was varied systematically m an effort to determine whether or not the magn=tude of the VEP depression was proportional to the amount of drug administered Soman showed a steep dose-dependent curve with a threshold between 5 and 7 i~g/kg body we=ght Below this threshold, the average reduct=on In VEP amplrtude was 10%, whde suprathreshold doses reduced the VEP an average of 85% Although some vartation was seen between cats, on the whole the threshold dose was far more consistent than results reported for physost=gmme or pyndost,gmlne (15) Comparison w=th blood cholinesterase data showed that blood chohnesterase levels were dose dependent and that the threshold for VEP reduction was reached when blood cholinesterase levels were reduced by approx=mately50% (mean = 50 4% ± 5 1 SEM, N = 9) Unhke physostmgmme (15) and DFP (10), no evidence of spontaneous recovery of either blood chohnesterase or VEP amplitude was seen, although all cats were momtored for up to 20 hours after an effecttve soman challenge
60
50 v
tO
40
N=10 S F = 0 1 cycles/degree
0
L
13
A
L
N=10
S F = 0 4 cycles/degree I
I
I
ntO 60 (3.
c
•
(1)
rr"
50
40 J
S F = 1 6 cycles/degree I
I
10
20
A
30
N=30 All SpataalFrequencies I
I
I
10
20
30
Contrast (%) FIG 2 Pooled data showmg modtficatton of the response vs contrast funct0on by soman (3o15 i~g/kg) St=mulatlon with 5 contrast levels (6%,10%,16%,25% and 40%) at spatml frequencies of 0 1 c/deg (A), 0 4 c/deg (B), 1 6 c/deg (C), and at all spatml frequencies combined (D) Note that the slope of these curves ts poslttve, mdicating proportionately greater response depress=on with h=gher contrast levels Response depressaons marked with astensks are signtficantly (p<0 05) greater than those at 6% contrast levels
Vol. 48, No
13, 1991
Soman and Vzsual Evoked Potential
1273
Discussion Intravenous administration of antncholinesterases mod05es both the EEG and the VEP response, producing a desynchronnzatnon of the EEG and a reduction in power of the VEP response The response reductnon appears to stem more from a reduction in the system gain than from changes in contrast sensitivity, although the differential is not so pronounced for organophosphates as it is for carbamates (16) We have found that soman has the same impact on the VEP as other antlchohnesterases, although at lower doses Before proceeding further, it is important to note that since the VEP is believed to represent the summation of both excitatory and inhnb~ory slow potentials (23), a reductnon m VEP size could result from a decrease in either excatatlon or mhubRnon We are therefore unable to confirm unambiguously any spec~ic impact of these agents on inh,brtion or excrtatlon The present VEP results are consistent with previous studies showing that administration of antlcholmesterase agents can modnfy the EEG Grob et al (24) reported that administration of DFP to humans for 5 days resulted in an increase in the amphtude and frequency of the EEG for 3-4 weeks, and Metcalf and Holmes (25) described desynchronlzatlon and theta rhythms in volunteers treated w~h organophosphate agents In animals, increases in the frequency wnth concomitant decreases m the amplitude of the EEG have been reported in cats (9,10,11,15,26) monkeys (26) and rabbits (3,8) following admlnnstratlon of physostlgmine or DFP In all cases, atropine reversed the effects, suggesting a muscarmnc mechanism Antlchohnesterases also affect evoked potentmls Administration of physostogmlne or DFP depresses the VEP in the cat (7,9,10,11,15,22) and the rabbit (8) More specific comparisons between the present results and those of most eadler studies are dnff=cult because of dnfferences in methodology Where comparison is possible, our results differ somewhat from those prevnously reported Harding et al (10,22) and Kirby et al (10,11) describe a selective reduction on the responses to low (0 1-0 2 c/deg) spatnal frequencies as the predominant response to physost0gmme and DFP, whde ead0erresults from this laboratory (15,16) show equnvalent depression at all spatial frequencies in 72% of the cats recervnng physost,gmlne, 50% of the cats receiving pyndostugmlne and all cats receMng DFP The d0fference between our results and those of Hardnng and Kirby is apparently due to our use of low doses of barbiturate anesthesia while recording (27) Furthermore, these authors found that sufficiently high doses of DFP also abohshed low spatml frequency responses Our fadure to find any sele~t0ve low frequency depression with soman was also found with DFP, and may suggest that organophosphate agents have a mode of act=on that duffers from that of carbamates, perhaps because of non-chohnergoc factors (28) Our finding that contrast sens~ovity is decreased (although not as much as contrast gain) agrees with the results of Arthur C Ginsburg (USAF, Dayton Ohio, personal communication), who found decreases in contrast sensatMty at low and muddle spatial frequencies of human volunteers exposed to organophosphates However, this threshold elevataon ns not likely to be due to direct chohnerglc action of these compounds An eadler study (16) comparing the effects of physostigmlne, pyndostigmine, DFP and soman on the contrast response of the VEP found class-dependent dafferences in the degree to which contrast gain and contrast sensitive/were affected Organophosphates alter the sensltN~y to a greater extent than do carbamates DFP and soman reduced contrast sensutlwty an average of 0 09 log unit, while the average reduction wnth physostigmlne and pyndostlgmlne, was only 0 03 log unit These data suggest that agent-speclfuc (28), rather than chohnerg0c, actions may be the cause of changes in contrast sens=tnvRy Regardless of origin, however, 0t is clear from our fundnngsthat changes in contrast sensntrvCy are minor when compared to the changes on the system gain, and that suprathreshold responses, as opposed to the threshold measurements used for calculation of contrast sensatIvrty, are more appropriate for evaluating the impact of antncholinesterases on the visual system Soman has a proportionately greater effect at high contrasts than at low contrasts, indicating a saturataon of the VEP response Under normal condutlons, saturatnon of the VEP does not occur until relatively hagh contrast levels (19), although the responses of single visual cortical cells show saturation at even moderate contrast levels A possible reason that the VEP does not saturate until after single cells do is recruntment As contrast levels increase, the responses of cells less optimally driven by the stimulus increasingly inhibit the responses of the optnmally driven cells, thus decreasing saturation If this is the
1274
Soman and Vzsual Evoked Potentzal
Vol. 48, No.
13, 1991
case, the saturat,on of the VEP under the influence of soman suggests that soman interferes with the effectiveness of recruitment v,a gain reduct,on and poss,bly threshold elevat,on Alternately, soman could be causing ton,c elevat,on of f,ring rate ,n all cells, in wh,ch case increased saturation would be apparent Poss,ble S,te of Act0on Whether the depressant act,on of systemically admm,stered chohnerg,c agonists seen m evoked potential stud,es (7-11,15,22) can be considered to be med,ated =n area 17 ,s not clear Changes in a global response such as the VEP could be effected at several levels of the v,sual system, and ACh involvement m v,sual function has been reported at the level of both the retina and dorsal lateral genlculate nucleus (29-33) However, topical application of chohnerg,c agon,sts to cortex produces changes m the EEG and VEPs s,mdar to those produced by system,c doses (3), md,cating that the reduct,on ,n VEP response ampl,tude seen m the present stud=es can be attnbuted, at least ,n part, to the intracortlcal action of ACh If the s,te of ant,chohnesterase-medJated VEP depress=on ,s pnmary visual cortex, apphcat,on of Ach on single cort,cal cells should produce s=mdarchanges, this ,s the case S,II=toand Kemp (5) have reported that ACh affects single cells In the vtsual cortex m a manner cons=stent w~th gain changes ,n the VEP They report that 92% of the,r cell sample responded to ,ontophoret,c apphcat,on of ACh, 61% of the responses were fac,htated and 31% were mh=b,ted The facilltatory responses revolved an enhancement of the cell's response to st,muh of opt,mal onentatlon or direct,on w,th no change in the spatml selectivity of the receptive f~eld or increase ,n the resting discharge of the cell, exactly the type of change that would be expected from an increase m the gain of the response Taken together, these data suggest that v,sual cortex may be one s=teof act,on of ant,cholmesterase agents However, =t =s important to note that these data do not rule out an extracort,cal ongm for th=s mechamsm, stud=es have reported that lesions of the basal forebra,n result m a depletion of cort=cal AChE and choline acetyltransferase (34-36), indicating that a large majority of cort=cal chohnerg,c mnervat=on is extnns,c Moreover, SdlJto and Kemp (5) have suggested that the excitatory act,on of ACh ,s due to extnns,c mnervat=on because of Its slow time course At f,rst glance, the increase m respons,veness at the cellular level reported by SdlJto and Kemp seems mcons=stent w~ththe decrease in the response of the VEP, however, since the VEP does not d,rectly reflect sp,ke activity (23), changes m the VEP from chohnerg,c drugs may d=fferfrom those reported at the single cell level (where responses are enhanced m the majority of cases) w,thout being in conflict The AC couphng inherent m the VEP would not reveal the steady (DC) component of a generalized elevation ,n f,rmg, which would only be apparent If ~ts instantaneous vanat~on contnbuted to an increase in the noise level Possible Mechan=sms Ant~chohnesterase agents can have excitatory or mh0bftory effects on d=fferent cells The changes ~nthe cells' responses to v~sual st,muh follow,ng admm=strat,on of physost=gmine are s,mdar to those seen using ACh (5), suggesting s,mdar modes of act=on Th,s argument ,s strengthened by the reversal of ant=cholmesterase-mduced VEP depression w=th atropine ACh action on cortical neurons is mediated through potass=um modulat,on at the cell membrane KrnJevIc and h=s co-workers (37-39) have reported that the ,ontophoretlc apphcatlon of ACh had a twofold effect (1) a slow depolarizing action, which enhanced the action of other inputs, and (2) a slowing of repolanzatJon following a spike, resulting in a tendency toward repet,t,ve f~nng The cell membrane resistance was found to r,se secondardy to a reduct,on ,n =onJcconductance, and voltage-current expenments indicated that EAcH was almost ,dentical to EK, md=catmg potassium was revolved Halhwell and Adams (40) have also reported that ACh excites h,ppocampal cells through a voltage-dependent potass,um channel The mh,b=tory act,on of ACh is apparently due to the same mechanism Cole and Shinn,ck-Gallagher (41) showed that ACh-med,ated mh,b~,on m rabb~t super=or cerv,cal ganghon cells ,s due to an increase m potass,um conductance with a resulting decrease ,n membrane resistance and Iowenng of the cell response Such changes m membrane permeab,hty would result m a shunting type of ,nh=b=t,on,the type of inhibit,on that ,s indicated by a change m the slope of the response vs log contrast function The antichohnesterase agents themselves could also have indirect effects in addition to ACho associated actJvuty Carbamates act almost exclus,vely on the contrast gain of the system, whde the act,ons
Vol
48, No
13, 1991
Soman and Vlsual Evoked Potential
1275
of soman and DFP are more balanced between gain and contrast sens~ivlty, suggesting that agent-specific dtfferences are present Add~ionally, antlchohnesterases are known to cause spectre changes at the level of the cell membrane (28) The exact relattonshlp of chohnerglc and non chohnerglc effects and their relattve impact on visual processing remain to be investigated Acknowledgments This study was supported by the U S Army Medical Research and Development Command, Contract No DAMD17-83-3186 The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
D GROB, Anttcholmesterase intoxication in man and its treatment, G B Kolle, (ed), Hndbch d Exper Pharmakol, Ergwk, 15, 989-1027, Sprlnger-Verlag, Bedm (1963) A SILVER, The Bioloqv of Chohnesterases Frontiers in Biology (Amsterdam), 36, 59, (1974) A G KARCZMAR, .A.cetvlchohneand Neuropsychlatnc Disease, K L Davis and P A Berger, (eds), 265-310, Plenum Publ Co, New York (1979) A G KARCZMAR, Report tO N A S Commtttee on Toxscoloqy, Antlchohnesterase Panel, 1981) A M SILLITOand J A KEMP, Brain Res, 289143-155 (1983) S MARRA7_.ZIand E R HART, Fed Proc, 985 (1950) P 0 CHATFIELD and D P PURPURA, Electroenceph Clan Neurophyslo16287-298 (1954) S RUMP, T RABSZTYN,and J KOPEC, Act Nerv Super (Praha), 160224-225 (1974) T H HARDING, R W WILEY and AW KIRBY, Science, 2211076-1078 (1983) AW KIRBY, R W WILEY and T H HARDING, Neurosci Abstr, 9370 (1983) A W KIRBY, T H HARDING and R W WILEY, Invest Ophthalmol Vis Scl Suppl, 2._5534(1984) J W PHILLIS and D H YORK, Brain Res, 5517-520 (1967) J W PHILLIS and D H YORK, Nature, 216922-923 (1987) J W PHILLIS and D H YORK, Brain Res, 10297-306 (1968) Y A GAJEWSKI, E J DEBRUYN and A B BONDS, Invest Ophthalmol Vis Scl Suppl, 2._5534(1984) E J DEBRUYN, Y A GAJEWSKI and A B BONDS, Neuroscl Lett, 7_! 311-316 (1986) A SNYDER and R M SHAPLEY, Exp Brain Res, 3_! 73-86 (1979) G L ELLMAN, K B COURTNEY, V ANDRES and R M FEATHERSTONE,Biochem Pharmacol, 7 88-95 (1961) A B BONDS, Invest Ophthalmol Vis Scl, 2._55640-646 (1984) R BLAKE, Percept Psychophys, 2.66423-448 (1979) F W CAMPBELL, L MAFFEI and M PICCOLINO, J Physiol (Lind), 22___99719-731 (1973) T H HARDING, AW KIRBY and R WWILEY, Brain Res 32_55357-361 (1985) P NUNEZ, Electric Fields of the Brain Oxford University Press, New York (1981) D GROB, A M HARVEY, R 0 LANGWORTHY and J L LILIENTHAL JR, Bulletin of Johns Hopkins Hospital 8.~1257-266 (1947) D R METCALF and J H HOLMES, Ann N Y Acad Scl, 160357-365 (1969) W C WESCOE, R E GREEN, B P MCNAMARA and S KROP, J Pharmacol Exp Ther, 9263-72 (1948) AW KIRBY and AT TOWNSEND, Sic for Neuroscl Abstr,l._55118 (1989) E X ALBUQUERQUE, A AKAIKE, K P SHAW and D L RICKETT, Fund Appl Toxicol, 4 $27-$33 (1984) A M SILLITO, J A KEMP and N BERARDI, Brain Res, 280299-307 (1983) W SINGER, Physiol Rev, 57386-442 (1977) M STRASCHILL and J PERWEIN, Pflugers Arch, 33__..~9289-298(1973) M STRASCHILL, Vis Res, 83547 (1968) G NIEMEYER and L CERVETTO, Invest Ophthalmol VIs Sci Suppl, 1.__7755(1976) P H KELLY and K E MOORE, Exp Neurol, 6_!1479-484 (t 978) M V JOHNSON, M MCKINNEY and J T COYLE, Exp Brain Res, 4_33159-172 (1981) K M CARNES, M F BEAR and F F EBNER, Invest Ophthalmol Vis Sct Suppl, 2._55212(1984) K KRNJEVIC, Physlol Rev, 5.~4418-540 (1974)
1276
38 39 40 41
Soman and Visual Evoked Potentlal
Vol. 48, No. 13, 1991
K KRNJEVIC and J W PHILUS, J Phys=ol (Lond), 166 296-327 (1963) K. KRNJEVIC, R PUMAIN and L RENAUD, J Physlol (Lond), 215 247-268 (1971) J V HALLIWELL and P R ADAMS, Brain Res, 250 71-92 (1982) A E COLE and P SHINNICK-GALAGHER,Nature, 307 270-271 (1984)
Pergamon Press
DEPRESSION OF THE CAT CORTICAL VISUAL EVOKED POTENTIAL BY SOMAN E J DeBruyn, G K Corbett and A B Bonds Department of Electncal Engineering Vanderb,t Umversity, Nashwlle, TN 37235 (Received in final form January 23, 1991)
Summary The effects of intravenous admmmtrat~on of the ant=chohnesterase agent soman (pmacolyl methytphosphonofluondate, 3-15 i~g/kg) on the visual evoked potential (VEP) were examined ,n cats using phase-reversed sine wave grating st=muh of diferent spat,al frequenc=es and contrasts Doses of 5-7 I~g/kg caused a depress=on of the VEP across all spatml frequencies ,n an abrupt, non-graded lash=on Stud=es m wh,ch contrast was vaned showed that VEP depression resulted pnmardy from a decrease ,n the system gain rather than a change =n the contrast sens~tMty, and that response depress,on increased with increasing contrast The dominant changes m gain revealed by these stud=es a r e consistent w=th a modulation of potass=um conductance in the cell membrane wh,ch prewous stud,es have shown to be dependent on a chohnerg~c mechanmm Antichohnesterase agents such as physost=gmine, pyndostigmme, d,sopropylfluorophosphate (DFP) and soman are thought to exert thmr tox,c actions by mact,vatmg acetylchohnesterase (ACHE) The resulting accumulation of acetylchohne (ACh) throughout the penpheral and central nervous systems produces symptoms of continuous chohnergm st=mulat=on Whde the general impact of these agents has been rev,ewed at length (t-4) and reformat=on is avadable on the=r act=on on many systems of the body, the effects of these agents on sensory systems have been less well stud,ed The visual system ,s the best understood sensory system, and as such prov,des a good model for the study of the actions of ant,chohnesterases Of special interest =s the act,on of these agents on the funct=on of pnmary vmual cortex, since chohnergic influences are apparently widespread in th=s area (5) Evoked potent=al studies (6-11) have shown that system=cally delivered ACh and chohnerg~c agonmts have a pnmardy depressant role in area 17, whde chohnerg=c antagonmts enhance responses Add=t,onally, Phdhs and York (12-14), and Sdhto and Kemp (5) have presented ev,dence for chohnerg,c modulation of single cell responses The latter authors found that 92% of the cells encountered in visual cortex responded m some way to ACh, suggest,ng that ACh may play a fundamental role m vmual cortmal processing We have reported ead=er (15,16) that intravenously administered physost,gmme, pyndost=gmme, DFP and soman depress the v,sual evoked potent,al (VEP) by altering the contrast gain of the system In the present report we extend our ongmal find,ngs on soman by examining ~s ,mpact on the response of the VEP to st=muh of different spatial frequenc,es and contrasts Methods Pr~parat=on - Bas=c procedures have been descnbed elsewhere (16) Ten cats (1 5-5 2 kg) w e r e anesthet,zed w,th halothane, and surg,cal anesthesm was subsequently maintained w=th intravenous (I V ) 2 5% thtamylal sodium Following tracheal cannulat=on, the animal was mounted ,n a stereotaxic headholder, the scalp was reflected and stainless steel screws were inserted at bregma (indiferent Aaaress correspondence to Dr Edward DeBruyn, Dept of Electncal Engmeenng, Vanderbdt Univers~y, Nashvdle TN 37235 0024-3205/91 $3.00 + 00 Copyrlght (c) 1991 Pergamon Press plc
1270
Soman and Vzsual Evoked Potentlal
Vol. 48, No. 13, 1991
electrode) and on the mldhne at H-C coordinate P4 (recording electrode) Mydr,asls and cycloplegla were induced with top=cal phenylephr,ne HCI (10%) and atropine sulfate (1%) and the corneas were fitted with contact lenses with 4 mm artificial pupds to optimize retinal amage quality The animal was refracted using direct ophthalmoscopy and auxiliary spectacle lenses were used to render the retinas conjugate w~h the stimulus plane For recording, the cats were paralyzed with gallamme tnethlod=de (10 mg/kg/hr) in lactated Ringers solution containing 1 mg/kg/hr Suntal to stabdlze anesthes=a The animals were artrf,clally respired with a mixture of N20 (75%), O2 (23 5%) and CO= (1 5%) and peak expired CO= was maintained at 4 2% Rectal temperature was maintained at 37°C with a servo controlled heat pad and heart acttv~ and blood pressure were monitored as Indicators of adequate anesthesia and drug effects The electroencephalogram (EEG) was mon~ored as an indicator of the animal's state of arousal St,mulatton and Data Processmq - One-dlmens=onal sine wave gratings were generated digitally on a Joyce Electronics display (mean luminance 280 cd/m =) with linear luminance modulation for contrasts up to 1 0 View,ng distance was 57 cm and displays were masked to a circular field subtending 18 degrees The grat,ngs were oriented horizontally and viewed monocularly Following the method of Snyder and Shapley (17), the results were Fourier transformed, and the first 15 even harmon=cs (which were temporally correlated with the test stimuli) were summed to give a figure for signal plus noise The first 15 odd harmonics provided a figure for noise alone A total of 100 seconds of averaging per datum yielded a best signal-to-noise ratio of t 5 1 Drugs - Soman was obtained from the USAMRDC (Fort Detnck, Frederick, MD), dduted to a fmal concentrat=on of 40 i~g/ml in sterile saline and stored at -80°C Immediately prior to ,nlect=on it was thawed in warm tap water, and stored on =cefor the remainder of the expenment A range of dosages (315 I~g/kg, dosages for each experiment are indicated below) was administered by slow I V anjectlon over a 1 minute period Exoenmental Deslqn - Up to six tnals were run and averaged to provide pre drug control values Soman was then administered and an experimental trial was run The dose of soman was tltrated m 2-3 I~g/kg increments until a reliable reduction of the VEP was noted, and the VEP was observed for up to 20 hours following drug administration In order to document any posstble spontaneous recovery Doses were given at either 12 or 35 minute intervals (depending on the length of trial being run), and blood samples for biochemical analysis were taken before each administration of soman, and at the begmmng of each tr,al following administration Chohnesterase actMty was determined by the EIIman (18) techn=que, and blood gas analysis (02, CO2 and pH) was performed Jn order to rule out the posslbdJty that changes ,n the VEP were due to hypoxla Results Normal VEP Response - At the beg,nmng of each expenment, the overall visual performance of each animal was tested by present,ng a series of gratings at different spatial frequenmes and contrasts spanning the normal operating range of the cat's vtsual system Values for high frequency cutoff and contrast sens~rvity were estabhshed as described elsewhere (19) High frequency cutoff values were usually between 3 and 4 cycles/degree (c/deg) with a maximum value of 5 6 c/deg, and the average contrast sensitMty at the optimum spatial frequency was 21, w~th a maximum value of 36 Both of these values are similar to (although somewhat lower than) those reported for the behaving cat (20,21), so we feel that the VEP =s a reasonably good md,cator of the overall performance of the visual system Effects of Soman - Admin=stratJonof soman (3-15 I~g/kg) usually resulted in a generahzed depression of the VEP and the endogenous cortical noise level over all tested parameters, along w=th desynchron,zation of the EEG The depression was readdy reversed by atropine, suggesting that ~tJs mediated by muscarinlc influences Spatial Sensltlv~y - Soman produced a depression of the response at all spat,al frequenc0es (FIG 1) For ten cats (doses ranged from 3-15 p.g/kg) the average depression at the lowest spatial frequency (0 1 c/deg) was 57 79% (+ 2 63 SEM), while that at the highest frequency (1 6 c/deg) was 53 78% (± 2 65) We found no s~gnificant rod=cation of a preferential depression at low spatial frequenmes as opposed to
Vol. 48, No. 13, 1991
Soman and Visual Evoked Potential
1271
high spatial frequencies, as reported by Harding et al (9,22) and K0rby, et al (10,11) Contrast Sensatrvrtv and Gain - The contrast senmtMty of each ammal was determined by presenting a series of contrasts at different spatial frequencnes spanning the normal range of the cat visual system In agreement wnth eadier results (19,21), the response ampl0tude was found to vary lineady with the logarithm of the contrast in both pre and post drug trials, and a least squares hnear fit was used to define threshold (the intercept with the noise level) and slope (equivalent to the system gain) As reported eadner (16), soman reduced the gain of the function to a greater extent than it changed the sensitrvlty Across 30 trials, the average decrease nn gain (slope) was 0 45 log umt, whde the average reduction in the contrast B 60
50 tO
40
O
Nil0 Contrast = 16%
Contrast = 6%
"10
I
I
i
I
n," (1)
¢O rr
D
C I
60
50
40 Nil0 Contrast = 40% 01
02
N=50 All Contrasts 05
10
01
02
05
10
Spatial Frequency (cycles/degree) FIG 1 Soman-mduced response depressnon at dufferent spatml frequenc0es The symbols represent the mean VEP depressnon for 10 cats for three different spatial frequencies (0 1, 0 4 and 1 6 c/deg) at contrasts of 6% (A), 16% (B), 40% (C), and at all contrasts combined (D) Although the plots tend to have a negatnve slope, the response depressnon at h~gh spatnal frequencies vs that at low spatial frequencies is not sugnfflcantly dnfferent sensit,vnty (the inverse of threshold) was 0 11 log un~ The decrease in slope approximated the average reduct0on in the VEP response amplitude (0 38 log umt), suggesting that the gain change accounts largely for VEP depression A second effect of soman on the response vs contrast relationship is illustrated tn FIG 2 When the data from all 10 cats are pooled, higher contrast levels are seen to produce proportionately greater depression of the VEP This trend is evident from the positive slopes of all the graphs, and is slgnsficant (p<0 05) at 1 6 cycles/degree and when all spatual frequencies are pooled The data suggest that soman is causing a saturation of the VEP at high contrast levels
1272
Soman and Vlsual Evoked Potential
Vol. 48, No. 13, 1991
Dose Response and Correlat00n w~th Blood Chohnesterase Levels - The dosage of soman was varied systematically m an effort to determine whether or not the magn=tude of the VEP depression was proportional to the amount of drug administered Soman showed a steep dose-dependent curve with a threshold between 5 and 7 i~g/kg body we=ght Below this threshold, the average reduct=on In VEP amplrtude was 10%, whde suprathreshold doses reduced the VEP an average of 85% Although some vartation was seen between cats, on the whole the threshold dose was far more consistent than results reported for physost=gmme or pyndost,gmlne (15) Comparison w=th blood cholinesterase data showed that blood chohnesterase levels were dose dependent and that the threshold for VEP reduction was reached when blood cholinesterase levels were reduced by approx=mately50% (mean = 50 4% ± 5 1 SEM, N = 9) Unhke physostmgmme (15) and DFP (10), no evidence of spontaneous recovery of either blood chohnesterase or VEP amplitude was seen, although all cats were momtored for up to 20 hours after an effecttve soman challenge
60
50 v
tO
40
N=10 S F = 0 1 cycles/degree
0
L
13
A
L
N=10
S F = 0 4 cycles/degree I
I
I
ntO 60 (3.
c
•
(1)
rr"
50
40 J
S F = 1 6 cycles/degree I
I
10
20
A
30
N=30 All SpataalFrequencies I
I
I
10
20
30
Contrast (%) FIG 2 Pooled data showmg modtficatton of the response vs contrast funct0on by soman (3o15 i~g/kg) St=mulatlon with 5 contrast levels (6%,10%,16%,25% and 40%) at spatml frequencies of 0 1 c/deg (A), 0 4 c/deg (B), 1 6 c/deg (C), and at all spatml frequencies combined (D) Note that the slope of these curves ts poslttve, mdicating proportionately greater response depress=on with h=gher contrast levels Response depressaons marked with astensks are signtficantly (p<0 05) greater than those at 6% contrast levels
Vol. 48, No
13, 1991
Soman and Vzsual Evoked Potential
1273
Discussion Intravenous administration of antncholinesterases mod05es both the EEG and the VEP response, producing a desynchronnzatnon of the EEG and a reduction in power of the VEP response The response reductnon appears to stem more from a reduction in the system gain than from changes in contrast sensitivity, although the differential is not so pronounced for organophosphates as it is for carbamates (16) We have found that soman has the same impact on the VEP as other antlchohnesterases, although at lower doses Before proceeding further, it is important to note that since the VEP is believed to represent the summation of both excitatory and inhnb~ory slow potentials (23), a reductnon m VEP size could result from a decrease in either excatatlon or mhubRnon We are therefore unable to confirm unambiguously any spec~ic impact of these agents on inh,brtion or excrtatlon The present VEP results are consistent with previous studies showing that administration of antlcholmesterase agents can modnfy the EEG Grob et al (24) reported that administration of DFP to humans for 5 days resulted in an increase in the amphtude and frequency of the EEG for 3-4 weeks, and Metcalf and Holmes (25) described desynchronlzatlon and theta rhythms in volunteers treated w~h organophosphate agents In animals, increases in the frequency wnth concomitant decreases m the amplitude of the EEG have been reported in cats (9,10,11,15,26) monkeys (26) and rabbits (3,8) following admlnnstratlon of physostlgmine or DFP In all cases, atropine reversed the effects, suggesting a muscarmnc mechanism Antlchohnesterases also affect evoked potentmls Administration of physostogmlne or DFP depresses the VEP in the cat (7,9,10,11,15,22) and the rabbit (8) More specific comparisons between the present results and those of most eadler studies are dnff=cult because of dnfferences in methodology Where comparison is possible, our results differ somewhat from those prevnously reported Harding et al (10,22) and Kirby et al (10,11) describe a selective reduction on the responses to low (0 1-0 2 c/deg) spatnal frequencies as the predominant response to physost0gmme and DFP, whde ead0erresults from this laboratory (15,16) show equnvalent depression at all spatial frequencies in 72% of the cats recervnng physost,gmlne, 50% of the cats receiving pyndostugmlne and all cats receMng DFP The d0fference between our results and those of Hardnng and Kirby is apparently due to our use of low doses of barbiturate anesthesia while recording (27) Furthermore, these authors found that sufficiently high doses of DFP also abohshed low spatml frequency responses Our fadure to find any sele~t0ve low frequency depression with soman was also found with DFP, and may suggest that organophosphate agents have a mode of act=on that duffers from that of carbamates, perhaps because of non-chohnergoc factors (28) Our finding that contrast sens~ovity is decreased (although not as much as contrast gain) agrees with the results of Arthur C Ginsburg (USAF, Dayton Ohio, personal communication), who found decreases in contrast sensatMty at low and muddle spatial frequencies of human volunteers exposed to organophosphates However, this threshold elevataon ns not likely to be due to direct chohnerglc action of these compounds An eadler study (16) comparing the effects of physostigmlne, pyndostigmine, DFP and soman on the contrast response of the VEP found class-dependent dafferences in the degree to which contrast gain and contrast sensitive/were affected Organophosphates alter the sensltN~y to a greater extent than do carbamates DFP and soman reduced contrast sensutlwty an average of 0 09 log unit, while the average reduction wnth physostigmlne and pyndostlgmlne, was only 0 03 log unit These data suggest that agent-speclfuc (28), rather than chohnerg0c, actions may be the cause of changes in contrast sens=tnvRy Regardless of origin, however, 0t is clear from our fundnngsthat changes in contrast sensntrvCy are minor when compared to the changes on the system gain, and that suprathreshold responses, as opposed to the threshold measurements used for calculation of contrast sensatIvrty, are more appropriate for evaluating the impact of antncholinesterases on the visual system Soman has a proportionately greater effect at high contrasts than at low contrasts, indicating a saturataon of the VEP response Under normal condutlons, saturatnon of the VEP does not occur until relatively hagh contrast levels (19), although the responses of single visual cortical cells show saturation at even moderate contrast levels A possible reason that the VEP does not saturate until after single cells do is recruntment As contrast levels increase, the responses of cells less optimally driven by the stimulus increasingly inhibit the responses of the optnmally driven cells, thus decreasing saturation If this is the
1274
Soman and Vzsual Evoked Potentzal
Vol. 48, No.
13, 1991
case, the saturat,on of the VEP under the influence of soman suggests that soman interferes with the effectiveness of recruitment v,a gain reduct,on and poss,bly threshold elevat,on Alternately, soman could be causing ton,c elevat,on of f,ring rate ,n all cells, in wh,ch case increased saturation would be apparent Poss,ble S,te of Act0on Whether the depressant act,on of systemically admm,stered chohnerg,c agonists seen m evoked potential stud,es (7-11,15,22) can be considered to be med,ated =n area 17 ,s not clear Changes in a global response such as the VEP could be effected at several levels of the v,sual system, and ACh involvement m v,sual function has been reported at the level of both the retina and dorsal lateral genlculate nucleus (29-33) However, topical application of chohnerg,c agon,sts to cortex produces changes m the EEG and VEPs s,mdar to those produced by system,c doses (3), md,cating that the reduct,on ,n VEP response ampl,tude seen m the present stud=es can be attnbuted, at least ,n part, to the intracortlcal action of ACh If the s,te of ant,chohnesterase-medJated VEP depress=on ,s pnmary visual cortex, apphcat,on of Ach on single cort,cal cells should produce s=mdarchanges, this ,s the case S,II=toand Kemp (5) have reported that ACh affects single cells In the vtsual cortex m a manner cons=stent w~th gain changes ,n the VEP They report that 92% of the,r cell sample responded to ,ontophoret,c apphcat,on of ACh, 61% of the responses were fac,htated and 31% were mh=b,ted The facilltatory responses revolved an enhancement of the cell's response to st,muh of opt,mal onentatlon or direct,on w,th no change in the spatml selectivity of the receptive f~eld or increase ,n the resting discharge of the cell, exactly the type of change that would be expected from an increase m the gain of the response Taken together, these data suggest that v,sual cortex may be one s=teof act,on of ant,cholmesterase agents However, =t =s important to note that these data do not rule out an extracort,cal ongm for th=s mechamsm, stud=es have reported that lesions of the basal forebra,n result m a depletion of cort=cal AChE and choline acetyltransferase (34-36), indicating that a large majority of cort=cal chohnerg,c mnervat=on is extnns,c Moreover, SdlJto and Kemp (5) have suggested that the excitatory act,on of ACh ,s due to extnns,c mnervat=on because of Its slow time course At f,rst glance, the increase m respons,veness at the cellular level reported by SdlJto and Kemp seems mcons=stent w~ththe decrease in the response of the VEP, however, since the VEP does not d,rectly reflect sp,ke activity (23), changes m the VEP from chohnerg,c drugs may d=fferfrom those reported at the single cell level (where responses are enhanced m the majority of cases) w,thout being in conflict The AC couphng inherent m the VEP would not reveal the steady (DC) component of a generalized elevation ,n f,rmg, which would only be apparent If ~ts instantaneous vanat~on contnbuted to an increase in the noise level Possible Mechan=sms Ant~chohnesterase agents can have excitatory or mh0bftory effects on d=fferent cells The changes ~nthe cells' responses to v~sual st,muh follow,ng admm=strat,on of physost=gmine are s,mdar to those seen using ACh (5), suggesting s,mdar modes of act=on Th,s argument ,s strengthened by the reversal of ant=cholmesterase-mduced VEP depression w=th atropine ACh action on cortical neurons is mediated through potass=um modulat,on at the cell membrane KrnJevIc and h=s co-workers (37-39) have reported that the ,ontophoretlc apphcatlon of ACh had a twofold effect (1) a slow depolarizing action, which enhanced the action of other inputs, and (2) a slowing of repolanzatJon following a spike, resulting in a tendency toward repet,t,ve f~nng The cell membrane resistance was found to r,se secondardy to a reduct,on ,n =onJcconductance, and voltage-current expenments indicated that EAcH was almost ,dentical to EK, md=catmg potassium was revolved Halhwell and Adams (40) have also reported that ACh excites h,ppocampal cells through a voltage-dependent potass,um channel The mh,b=tory act,on of ACh is apparently due to the same mechanism Cole and Shinn,ck-Gallagher (41) showed that ACh-med,ated mh,b~,on m rabb~t super=or cerv,cal ganghon cells ,s due to an increase m potass,um conductance with a resulting decrease ,n membrane resistance and Iowenng of the cell response Such changes m membrane permeab,hty would result m a shunting type of ,nh=b=t,on,the type of inhibit,on that ,s indicated by a change m the slope of the response vs log contrast function The antichohnesterase agents themselves could also have indirect effects in addition to ACho associated actJvuty Carbamates act almost exclus,vely on the contrast gain of the system, whde the act,ons
Vol
48, No
13, 1991
Soman and Vlsual Evoked Potential
1275
of soman and DFP are more balanced between gain and contrast sens~ivlty, suggesting that agent-specific dtfferences are present Add~ionally, antlchohnesterases are known to cause spectre changes at the level of the cell membrane (28) The exact relattonshlp of chohnerglc and non chohnerglc effects and their relattve impact on visual processing remain to be investigated Acknowledgments This study was supported by the U S Army Medical Research and Development Command, Contract No DAMD17-83-3186 The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
D GROB, Anttcholmesterase intoxication in man and its treatment, G B Kolle, (ed), Hndbch d Exper Pharmakol, Ergwk, 15, 989-1027, Sprlnger-Verlag, Bedm (1963) A SILVER, The Bioloqv of Chohnesterases Frontiers in Biology (Amsterdam), 36, 59, (1974) A G KARCZMAR, .A.cetvlchohneand Neuropsychlatnc Disease, K L Davis and P A Berger, (eds), 265-310, Plenum Publ Co, New York (1979) A G KARCZMAR, Report tO N A S Commtttee on Toxscoloqy, Antlchohnesterase Panel, 1981) A M SILLITOand J A KEMP, Brain Res, 289143-155 (1983) S MARRA7_.ZIand E R HART, Fed Proc, 985 (1950) P 0 CHATFIELD and D P PURPURA, Electroenceph Clan Neurophyslo16287-298 (1954) S RUMP, T RABSZTYN,and J KOPEC, Act Nerv Super (Praha), 160224-225 (1974) T H HARDING, R W WILEY and AW KIRBY, Science, 2211076-1078 (1983) AW KIRBY, R W WILEY and T H HARDING, Neurosci Abstr, 9370 (1983) A W KIRBY, T H HARDING and R W WILEY, Invest Ophthalmol Vis Scl Suppl, 2._5534(1984) J W PHILLIS and D H YORK, Brain Res, 5517-520 (1967) J W PHILLIS and D H YORK, Nature, 216922-923 (1987) J W PHILLIS and D H YORK, Brain Res, 10297-306 (1968) Y A GAJEWSKI, E J DEBRUYN and A B BONDS, Invest Ophthalmol Vis Scl Suppl, 2._5534(1984) E J DEBRUYN, Y A GAJEWSKI and A B BONDS, Neuroscl Lett, 7_! 311-316 (1986) A SNYDER and R M SHAPLEY, Exp Brain Res, 3_! 73-86 (1979) G L ELLMAN, K B COURTNEY, V ANDRES and R M FEATHERSTONE,Biochem Pharmacol, 7 88-95 (1961) A B BONDS, Invest Ophthalmol Vis Scl, 2._55640-646 (1984) R BLAKE, Percept Psychophys, 2.66423-448 (1979) F W CAMPBELL, L MAFFEI and M PICCOLINO, J Physiol (Lind), 22___99719-731 (1973) T H HARDING, AW KIRBY and R WWILEY, Brain Res 32_55357-361 (1985) P NUNEZ, Electric Fields of the Brain Oxford University Press, New York (1981) D GROB, A M HARVEY, R 0 LANGWORTHY and J L LILIENTHAL JR, Bulletin of Johns Hopkins Hospital 8.~1257-266 (1947) D R METCALF and J H HOLMES, Ann N Y Acad Scl, 160357-365 (1969) W C WESCOE, R E GREEN, B P MCNAMARA and S KROP, J Pharmacol Exp Ther, 9263-72 (1948) AW KIRBY and AT TOWNSEND, Sic for Neuroscl Abstr,l._55118 (1989) E X ALBUQUERQUE, A AKAIKE, K P SHAW and D L RICKETT, Fund Appl Toxicol, 4 $27-$33 (1984) A M SILLITO, J A KEMP and N BERARDI, Brain Res, 280299-307 (1983) W SINGER, Physiol Rev, 57386-442 (1977) M STRASCHILL and J PERWEIN, Pflugers Arch, 33__..~9289-298(1973) M STRASCHILL, Vis Res, 83547 (1968) G NIEMEYER and L CERVETTO, Invest Ophthalmol VIs Sci Suppl, 1.__7755(1976) P H KELLY and K E MOORE, Exp Neurol, 6_!1479-484 (t 978) M V JOHNSON, M MCKINNEY and J T COYLE, Exp Brain Res, 4_33159-172 (1981) K M CARNES, M F BEAR and F F EBNER, Invest Ophthalmol Vis Sct Suppl, 2._55212(1984) K KRNJEVIC, Physlol Rev, 5.~4418-540 (1974)
1276
38 39 40 41
Soman and Visual Evoked Potentlal
Vol. 48, No. 13, 1991
K KRNJEVIC and J W PHILUS, J Phys=ol (Lond), 166 296-327 (1963) K. KRNJEVIC, R PUMAIN and L RENAUD, J Physlol (Lond), 215 247-268 (1971) J V HALLIWELL and P R ADAMS, Brain Res, 250 71-92 (1982) A E COLE and P SHINNICK-GALAGHER,Nature, 307 270-271 (1984)