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Studies of Nicotine Aetion upon Memory Consolidation 01.1 •.' A. propo:ed 5tarting Dbte~ Oatober l, lyb (;•=,,: _. ~, .- :. b. Mticipated Duration of this Specific Study: 11ia years. C 6. Brief Desuipton of ObjectivesorSpecificAimu ?.` The rpajor objective of this proposed research is to assess the possible role of =`-•' - aicotine in determining the tenporal character of memory conso lidation in experimaita7. '; animalls. The proposed e-Veriments represent an attempt to relate the effects of nicotine :=centrnl avents_ _ SnracifSeallv_ the effect of nicotine on lvain aminP coneentration and ->+ "Yn the central nervous system to the behavioral processes which are dependent upon sueh " s Y P Y s~,_. P s of msno consolidation (the rocess b tumover -Ul 5e' which the r5~ zelated, Yo the m ce r basis upon-whieh memory consolidation,may be facilitated. The parameters to be con- - ;idgred in th~,~ronosed studies are nicotine d~sage and the intensity of the amnesic event which, for the present experiments, will be electroconvulsive shock. A one-trial conditioning technique will be utilized to establish a passive avoidance response, the ,retention of which will be tested, based upon the interaction of the effects of nicotine : on the central nervous system and the effects of electnoconvulsive shock both on the -.,central nervous system as well as an 2rcnesic event specific to the behavior conrerned ~,..grop.osed- studies. : ~rax~ amzne levels, specifi.cally aerotonin, the studies proposed herein are based upon t?*~tf~e+assqmptian that nicotine-induced changes in brain anine levels will eonsti.tute the z~r~~a~y~~Eiais.a~ay be shortened or lengthenedy dependirg upon.changes brought about ia ; " "' previous work in this laboratory has demonstrated that the tenporal course for mmtory , fined in terms of tha time interval following learning within which. memory for an acquired :..„;.~ c.-event may be dLsnuuted by ttie introduction of external agents attd/or events) . Since the t. {, ~~menory trace• oecomes zixatea~ in tne central nervous systemj tnls may De emp2ri.caliy ae- -~10-= 7 Giveo6riefSlatementofyourWorking,Nypomhesis: Memory cons,lidation, as deflned• by the post-conditioning time int'erval, within which retention of a conditioned response may be, disrupted by electro- convulsive shock will be facilitated through. the prior administration of nicotine. Such facilitation wiS1 be defined by a reduction in the time interval fbllowing learning, wterein. eltctroconvulsive shock results in a subsequent retrograde annesia for the corziiti.oned behavior. _

C ~1. 6ioprophical sketches of all prineipa[ and professional personnel(append) See attached page 10. 12. List of puhlications: (Five most recent as pertinent) (append) See attached page 11,

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Detaf 1s of Experimental Design and Procedures: ' i ;Y ay { ._a* J _' ~"`All experiments will be based upon the use of a reliable technique which pro-~ vides for the establishment of a condit3•oned passive awidance response in mice, E;utilizing a single training trial (Essman & Alpern' 1965). The apparatus basically r•-' •~;;~consi sted of a small transparent lucite chamber f5 xed to a hole opening into a:,:;~ ~`arger.opaque chamber. The floor of the op.e ue chamber consisted of stainless sfi~eel aq -'~'•~grids which were wired in series through a cam-operated grid scrambler to a 95 volt °:power supply. Completion of the circuit by ~ anuaal stepping •firom,.the outer ch~amber~ ~ ~ :into the inner one, through any 2 adjacent grids? ~ovided for a 3.2 ma, shock '::,; 'i flelivered' thro u~ the pa~rs of the ani.mal for 1 sec. The interval between placement ~. . ~~: of the animal in the outer chanber and its entry into the larger ad3acent chambe r ~ ~ : was ne asured' (response latency}'-_ : w : .. ; r.. . .. ..,r:~!ii!• . . ..- . . ..,.; - - ..... . ...-_.... - .. :. __. .- _ ._.. . --. -•. .. _. - . . . -.. . .. - _ All animals utilized in the proposed exQeriments will be male, CF-i strain mice Nius Musculus ), to be obtained from a commerc3~a1l vendor (Carworth Farms, Inc .) at ; ; ; wening 2 days) ,, and fblloSSing adaptation to conditions of laboratory housing ~~ '` (1Q anim~ls t~er caaewill be utilized as experimental subjects at approximatnely- ~:' f~~~ ~:30 d~{~s of a ~'e ~jb. `.V The specific experiments will be concerned kd:th: 1: Dltihi fotf ose-response reaonspsr nicotine sulfate and rae o memory ;; consolidation; the latter will be determined from the incidence of ; reduced retrograde amnesia induced by electroconvulsive shock (ECS). . The temporal gradient for ECS-induced retrograde amnesia in animals pre-;: treated with nico tine sa! fate. ., The effect of nicotine sulfate on thr incidence of conditioned response acquisition as a funrtion of con3itioning stimulus intensity. The effect of nicotine sulfate on the incidence of conditioned response retention as a function of the intensity of thec stimulus (ECS) - - amnesi. ~; W ".- In the first series of experiments nicatine salfate will be administered i.p. mice in dose ranges of 0.25 mg/kg to 2.00 mg/kg (larger doses would not be indicated in view of neurotoxic effects). Control groups will be given an equivalent i.p* volume of 0,~ saline. Each group will be given a single training trial one hr: ,~ l :post-in3ection (stable CvS stimulant effect reached) in the apparatus described above, =- utilizing a 3.0 mA, 3 sec. training shock (yielding approximately 100% conditioned e'~ ,±~~response acquisition)~, followed either iimnediately, 5, 10, 15, 20, 30, !~5, or 60 min ~•~~by a 10 mA electroconvulsi.v eve shock (ECS), applied transcorneall.y for 200 msc. (sufficient to produce approximately 100% retrograde amnesia in control mice, when applied immediately po st-trai,ning). A testing ~ trial. -will be given to all groups of mice 2!~ hrs. following the training trial. Conditioned response retention will be evaluated on the basis of response latencies in the testing trial (L y 30 sec.) and , retrograde amnesia wy~1l also be defined in terms of the absence of conditioned response as determined from latency measures (L ~ 5 sec.), Control groups, both, drug and. saline-treated:, as described above, will be given no conditioning shock followed at the appropriate intervals by ECS, conditioning shock with subsequent sham-ECS (corneall electrodes applied, but no current passed)~ and no, conditioning shock and sham-ECS. The outcome of the control group treatments is expected' to be _ very similar to that obtaired in pilot studies reported in the ~eliminary supporting data appended to this application. The data resulting from the proposed experiments :. wi11 allow for oonclusi•ons regardir~g the dose-response relationship for nicotine .,= • sulfate and the rate of memory co nsolidation, determn.ned from the incidence of :' reduced retrograde amnesia induced by ECS. Alro the tem oral , p gradients for ECS- ' ~~;... induced retrograde amnesia will be determined for nicotine sulfate. Since this ,~~:~ ~%~}» +u~ a.`. '~`:.ttn .'t~ ...f~•

compound, within the proposed dose range, does s, not alter the susceptibi,lity of mice =;to electroshock-induced convulsiont one may therefore assume that• ariy change in the„,~ P'.amnesic effect of electroshock is due to a central event rather than a threshold °= ,, haige. The usual tenporal gradient for electroshock-irduced amnesia has in our ., ~~~ : _ r~,~s experience, fallen witrii~ 1-hr. post-training, with; a reduced ircidence of ar~nesia ='~4ui ftif th tiilth occrrn as auncon oeranng eecrosoc ~ k time interval. From psel >!ra na: 4>,: data it appears as though the.gradient is appreciably shortened when• animals have : been cotinetdieshould "; ~':~~ ivle~n lusively establishlthe temporalboundaries of the amnesic ~fectSOf~S•~y `•-..A The second series of proposed experiments will concern the effects of nicotine 'slft t btilid i th d uaeoe uzeneose ran e ecified ab oveu the acauisition and Pon t;ak, , g' sp s r retention of conditioned avoidance behavior as a function of (a) conditionin Yg stimulus intensity (i.Q.. 2.0, or 3.0 mA) and (b) post-training ECS intensity (5.0, : ,~:10.0, or 20.0 mA). The incidence of oonditioned response acquisition and the dura- :-tion of stability of this response has been shown to increase as the conditioning• ushock intensity is increasedy and the incidence of electroshock induced amnesia is increased as the ECS intensity is increased. In the first experiment of this series animal.s will be conditioned., utilizing¢~ ,;_,the technique described above and will be given either a foot shock of 1.0s 2.0, or :-;3.0 mA, applied through the grid floor upon, successful completion of the response: .", ::.Control animals in this situation will be given no conditioning shock. Fbr each of ` these conditions a post-training electroconvulsive shock will be given at 5.0., 10.0, : o 200ih 6 r. mA, eter immediately following the training trial or at 10, 20. 30, or0z : minutes followi,ng the training trial All animals wi11 be tested for acquisition .. !`f thditid ftif thtii dfiti oe conone response as auncon oe pre-ranngosage o ncone sulfate (0.25 mg/kg to 2.00- mg/kg), 24 hrs. follojing the training trial where the conditioning shock was varied. These data, for the effects of the drug upon re- ~~ tention as a fumtion of the conditioning shock intensity, will be available from :Y ~;~, ..... the non-convulsed control animals for which a saline-injected group will serve as a :oontrol for the drug dosage. Data relevant to the issue of post-trainingECS , ~-~y intenslty and the incidence of retrograde alnnesia as a flxraction thereof will be ._ determined from nicotine sulfate and saline-treated groups given these injections :=; one hour prior to the training trial. The testing trial will again be given 24 hours "• folloh2ng training, and on the basis of the incidence of retrognade amnesia obtained `y~ statements with respect to the temporal gradient far retrograde amnesia as a function of an intensity of ttae amnesic event, electroconvulsive shock, may be made. On the basis of lot studies it is antici ated that nicotine sulfate will reduce the Pi p temporal gradient for the amnesie effect of electroconvulsive shock, and that such :,a reduction will be a function of decreased ECS intensity as well. . --,, ..•,..:...: . . . ., . Another series of experiments will be concerned'with the biochemical effects of nicotine sulfate in the dose range which proves to be effective in facilitating memory cmnsolidation, as determined from the experiments outlined above. Those specific biochemical, changes with. which' the experiments will be concerned will deal primarily with, the biogenic amines serotonin (5-hydroxytryptamine) and norepinephrine, as we11 as the substrates to which thiltd Silii ese amnes are reae.nce premnary data has suggested that in the strain of mouse used • there are no appreciable changes over titae in norepinephrirE, this observation will again be verified experim_entally and the majority of the chemical determinations: to be carried out will concern themselves with serotonin metabolism; in this connectiony 5-hydro.Vtryptoghane, the serotonin precursor and 5-hydroxyindoleacetic acid, the serotonin metabolite, will be assayed, - ~'/ ,•.0® j, 3545969 f.7,`,~ = y . .:..kf 'Z.

1 :x. ~d.~C ,`~y `.. i ~{~ ,4 utili.zing spectrofluorometric procedures which have bren well standardized in this a ~'i laboratory. Through these means serotonin concentration can be determined and !.~.A ~ -_`t+ reliable estimates of serotonin turnover may be obtained. These chemical deteri+iina=.' .tions tiull be carried out under conditions of both, experimental as well as control ; `.:.C' . drug treatment and under conditions wherein electroconvulsive shock is administered`~ or controlled.for by sham treatment. •It is anticipated that as a result of electro=~ ~ `~~convulsive shock whole brain serotonin concentration in control animals will 'be : ••:.. ; z':.elevated. ~.:This hypothesis is based upon previous observations in this (Essman,B::- M. W~:~x Chan ges in memory consolidation with alterations in neural RNA. -Proc . Coll. ` Int: `~ Neuzo psychophaim., 1967, 108-113) and other laboratories (Kato, h., et al. . Histam epinephrine and norepinephrine in the rat brain, following convulsions.`~ ~~ ~° Int. J. Neurops3'c h.'1967, 3, l~6-51). It is further hYP.o-t,hesized that the de ee":' ---~ ~' rto wfuch eleetroshock elevates brain serotonin will be limited under conditions `~ .where nico tine sulfate results in decreased brain' serotonin levels as a fZnction . of dosage and post-injection time, and that under these conditions either seroton3ii =..~> ~,... ,. concentration or sero•to nin turnover may be statistically correlated with the be- ~. . .. "=?>.havioral data indicating the incidence under these conditions of conditioned respo~ ~,~.. . retention; i.e., facilitated memory consolidation,, inferred from nicotine sulfate- ~~ uiduced attenuation of electroshock-induced retrograde amnesia, will be Positivel :~ - Y :"~ correlated at, thos•e dosages and post-injection times where maxiimall changes in brain', ' serotonin concentration and/or turnover are observed. The proposed experiments may be summarized as an attempt to explore the relationship.between the effects of nicotine sulfate on amine metabolism 3n the .~' : ~.:. ~ .;. central nervous system and tihe process of iner• ory consolidation based upori the ~...;~~.; hypothesis that nicotine-induced alterati.ons in brain amine levels may account ~4. for a nicotine-induced facilitation of inemory consolidatiOn • .. , . . . . ... ..e .,_.. ra1.,.: .. . .~T" . . .. . . . - - . 11 7 Y~~ K ,. W'. 1 i y

~ ~- -. , _:. ....<• , .,. .,- . .: _ Several experiments coinpleted in this laboratory have indicated the feasibility :; :of the experi-ments propos id. The first of these studies was a series concerned with ;~ :the acquisition by mice of a simple maze response requiring a choice di.scrimination~;£ -, 3n order to escape from a water maze. Gmups of an3ma3 s were given either 0.9% salane, ~ 2?;;, 0.25 mg/kg~ 0.50 mgf kg, or 1.00 m gf kg of nico tine sulfate, intraperitoneal?'y. ,t 15 min . K•following injection all animals were given 4 training trials spaced at 15-min. 3nter=; ~va1s in a water maze. Their escape'times and incidence of errorless performance in:~ ~ Vthe acquisition of the escape task were recorded. When the esc~e timA s for all 4.r, , ±-~ = groups o£ animals were linearly transformed across all trials in order to derive a ~~--y ;,decreraent score indicative of the rate of maze acquisition, the only group reachin stati.stical significance was that comprised of those mice which were given 1.00 m ; of nicotine sulfate 15 min. prior to the first training, trial (t - 1.92; E-C.05) : A co,riparimn between groups. for thee incidence of errorless performance throughout , ~ ;' .the acquisition of the escape response indicated that there was a significant differ-".j Swtence between drou s in errorless erformance and that this sionificance was ]ar el b P P o g y:: %..'accounted for by the high incidence of errorless performance among those animals "`;ii 100/kfitilft ('2 195 1 ~02) Th~ -recevr.g. mgg o ncone ssae -1., df = , E..ere was a ~ incidence of errorless performance a~nong animals treated with 1.00 mg/kg of : . nicotine sulfate, as compared with an averabe of 28% errorless performance among ~ `th i thitd Tt/e e other groupsns suy.hese findings suggesed that 1.00 mgkg of nicotin " sulfate exerted a significant effect in facilitating the acquisition of a simple escape response by mice. - i:- - ~ s:~:r:' • - .. . . .. .. . . _ . . . . =' Subsequent studies were concerned withe the effect of a 1.00 mg/kg dose of nieotine sulfate in relation to the memory consolidation process. In these studies animals were treated with either nicotine s~.ilfate or physiological saline 60 min. :1 ~=" prior to being given a single training trial to establish a conditioned avoidance ; ='~,response. The training trial was followed immediately by a 10 mA electroconvulsive ~~ shock administered transcorneally for 200 msec.; 24 hrs. following the training . trial all animals were tested for the incidence of retention and/or retrograde ~";. arnnasia. Saline-treated, animals showed between 90% and 100% incidence of retrograde ' amnesia, as defined by the absence of any conditioned avoidance behavior, whereas Dapproximately 66% of the nicotine sulfate-treated 2nimals showed retention of the r conditioned avoidanae response, with the remaining 34% showing the same retrograde ~ ;: annesia for the conditioned avoidance behavior, as was shozr2 by almost, all of the ~1 saline-treated co ntrol animals. This finding suggested that the amnesic effect of -.electroconvulsive shock was either attenuated by nicotine sulfate treatment or that : the time interval required for fixation of the memory trace was appreciably shortened through the effects of nicotine sulfate and was thereby less vulnerable to the• amnesic °. effects characteristically exerted by post-learning electroconvulsive shock. The first alternative suggested by these data does not seem to appear dependent upon any alteration in the threshold to con,.m]sion produced by electroshock since, for the dosage used, independent experiments established that for electroshock, varied from 5 through 20 mA, there was no alteration, in the incidence of a full clonic-tonic : convulsion following such electroshock administration at intervals from 15 mi.m. ta 60 min after injection of 100 mg/kg oflft .. nicotine suae. 1003546971 In another study where the interval between administration of 1.00• mg/kg of nicotine sulfate and the training trial, i*nmediatelyy folloUred by electroshock, was - varied from 15 min. thruuph 60 tmi.n., the data indicated that only when the co und ` - ... 7 was ad~inistered 60 min. prior to co nditioning trial did a si~ificant (n t.01~ ,:.~..: : _. . =;°tinddenee of conditiored response retention oecur. These data were consistent with "those:{those obtained in the previous study in that comparable results emerged, further ,~ . _ ` .. . .-- ~ - ,~~.

;41,. ~_~, ?::=..,- >- .. .. .. - . ;. . .~ ... _ . _..., . : ... ~ .- . ...,. _ . . ..: _ .. , the observation that nicotine s.ilfate exerts a peak effect upon menory ns~ lidation when its central action occurs ona hr. follo-A ng, administration of •.supportiflg c o This ohservation is supported by some biochemical data in which~ to mice /k 100 m : . g g ; . :; icotine sulfate at ` f /k 00 1 g o n m g v for saline-injected 2nimals or• mice treated with i . . who7:~e brain serotonin and the renoval of brain tissue ecedin l t i ' , g s pr erva n various i ~1o waarr~ c7Ct:rM[i aSSdVed snectrofluorometrically. Contrary to a c~~~~a.~ -.--r----r------- ~ ct of nicvtine and related substaxices ;,:~ Eff 1 t T C ~ e . a . ., e ecent report (Westfall,, 83-100) no changes ]I~2 d Sci 1967 i NY A . , , ca An n the brain ine levels 9:~.....9 upon an 6 i 0 m n. in norepinephrine were observed, as fLintion of pst-in3ection time up to • ~ a trend to~rard tl biti didllhow tion whereas whoeran seroonn generay s ' s ec o ~~.:p J I ~ These data and the bazavioral data previously : n of time ti f t ~ ' . o unr as a decremen ~ . ~ ~rreferred to are summarized in Table 1. ~ " Mean Whole Brain, Serotonin Concen-hration. and Per Cent Incidence of Conditioned Response Retention after Electro- ~'~}~~~~'?=~f~ shock for Mice given 0.9% Saline or 1.00 mdkg of Nicotine - StLl.fate at. Varyinfz Intervals Prior to Cond3,tioning. Per Cent Incidence ' Mpan Whole of cK xetention Brain, Seroto nd.n ~' after ECS• .,[.ev.. - . _._.....- : _ - ,. - . -.. kT. , an ges • w°~These data are consistent with a previous suggestion Essman `~~P , ~ :: ?~~F~~~mPmnrv ns~lidation with alterations in neural RNA. Proc. Coll. Int. Neuro- m :..,..: .. s•ti~• ~ 1= sP ychopharm., 1967, 108-113) that a reduction in brain serotonin level or pharnaco -:" ` - ~ d aA: ~~ ; ~'illid liittin rte of serotonin turnover in the brain ten oecay-.mposemaons o thea- ~~ 'limit the degree to which amnesic agents or events become eriectizve zn zmposa.ng 3, ' a retso grade amnesia, and~ that these observations may be interpreted as a facilita- time interval fo llos~ ng learning th r b h lid ti i '` , e y e e on p rocess w tion of the menory co ns~ a :. ;: -; .r <? r = ; :, tive effects of knocai di t th ll l bl t , ~ srup o e :I, y vu nera e race is norma ~. wherein tt~e msnory amnesic agents or events, is appreciably shortened by those conditions which tend : to lead to a reduc tion in or limitation on the activity of bra3,n s erotonin. It is• `:; 1i_.• felt• that nicotine sulfate is one such agent which, at the dosage uZisIzea, proviaes for a wide margin of safety insofar as neurotoxic effects are concerned.

Ph.D. - University of North Dakota, 1957 A. - University of North Dakota, 1955 B. A. - New York University, 1954 eriencel _ . .. . . . . , _~•.,,.,. _ ~,. . .. < : . . . .. . . ,Instructor, Dept. of Neuropsychiatry,'U.S. Army Medical Service School, 1958-59.-' ,, Director, Psychophysiological Research, U.S. Army Surgical Research Unit, '?~;~~,, j. , 1958-59 Senior Post-Doctoral Fellow, Neurophysiology, Albert Einstein College of . Medicine, 1959"61 Research Associate, Dept. of Physiolog5*, Albert Einstein Co].lzge of Medicine~`° . . . . . : ', . ~ . -_ . ~ . . .: _. . .. .... , . . ' :'^~•.'... '' c'a ' ee.! 1961-62. Assistant Professor,' Queens College, 1962-64. Associate Professor, Queens College, 1965-66. :'. Professor, Queens College, 1967 - Present. ;: Research Fellowy Laboratory of Neurochemistry, Nt. Sinai Ho spitali, New York,y .~ 1964-66.. .Research, Associate, Laboratory of Neurochemistry,,Mt. Sinai Hospits7, :; New'York, 1966 - Present. - Lecturer (Neurochemistry), Coltunbi•a University, 1964 - Present. Areas of Major Research Interest: ' 1. Biochemistry of the central nervous system. 2. - Neurochemieal correlates of behavior. 3. Memory processes. Member•ships: :Amera.can, Psychological Association. Physiological Society : American Society of Zoologists New York Academy of Sciences American Association for the Advzncement of Science Psi Chi • " • Phi Delta Kappa Alpha Pi Zeta Sigma Xi .: „

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