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I. CARDIOVASCtfIAR, PIll1MfRCOLOGY and CHEbffSTRY II. EFIDSMOLOGY, STATISTICS• and;PSYCHOSObfATIC N0: 6- 1. Name of Inrestigator(s): (indude Title and Degrees). _ - - ' B.S., MyS,, Ph.D. 2. lnstitution & ~ddrEik-< = Sehoo•l-of Pharmacy Date: August 7, 1967 Carlton K. Erickson, Assistant Professor of Pharmacology and Toxicology, The University of Kansas Lawrence, Kansas. 66044! •,,;-ed alertness andilearning. Recent research, however, has demonstrated sorption on the subject's alertness and learning ability. Obviously, itg.`cattile `wott13•'be considered socially unacceptable if it seriously impair- COtIIdLTTEE: Dr. Cattell, Chm. Litbl e _. qpp~icatfon For Research Grant ts D J r. aco son For obvious social and medical reasons, nicotine is one of the common drugs which has been studied for its effect on learning and performance. In humans who smoke regularly and inhale a significant amount of nicotine, :°obvious questions arise concerning the effects of prolonged nicotine ab- C ; Mechanism of Learning Facilitation by Nicotine ; 1. Proposed Sthrting Data. November 1, 1967 S. Anticipated Duration of this Specific Studja 12 months 6. Brief Descripton of Objectives or Specifio Aims: that while large doses of nicotine depress learning, small doses can ; enhance perforr.tance in rats and mice (1,3)': Unfortunately, there has not been a concerted effort to study the true mechanism of nicotine's observed facilitatio7_pf.learning. McGaugh and Petrinovich. (9) have cautioned ~gain'st- assurting that changes in performance reflect actual cban;es in ' effic,iency of the neural processes involved in learning (i.e., the so- called formation of the memory traee). It is of special practical impor- tance with nicotine to differentiate a transient change in motivation or ---a2ertrcess fr'afn`t?ie relatively permanent alteration of neural processes involved in learnino. Er.perience has shown that in order to convincingly demonstrate that nicotine (or any centrally-active drug) is actuaLly affecting neural learnimg necnanisms, 3 important criteria must be fulfilled;: 1 The nhenn7eann of "drua disso^:.~.tion" r'>>:t t:r, rrle•_ out; ilcGauy~h `~aYih.Fetrinovic:i (9) have stated that "subsequent stLdies oE nicotine `on learnin.-) should ta.'.:e into account the possibility that dissociation 77ay occur with this eo :7-pound", Dru;; dissociation refers to• the-situation in which habits learned by subjects in a drugged state do not transfer to ( t the normal s tate, but can be evol;ed again when ever the sub ject is dru;-,~,_^,ed . ` In other e•+ords, nicotine may act as a secondary stimultRs dur ng, learnin~; 7. G"rveoariefSiatementofyourWarkingNkpothesis: tto pa"e la) hicotine enbances central nervous processes involved in Iearning, Er.d does not demonstrate deleterious ef.fects on, beaaviorai perform,ance.in doses comparable to those s_nvolved in snto'.:ir.g, 4N==K `_

ectives and Specific Aims (continued): ih: of a response •or other information; when the drug is not pr.,sent, the ..;,,'• :~,.learn•ed material may no-t be recalled. Peripheral actions of the drug must be ruled out. It is well knoc~n RP that changes in peripheral functiioning such as autonomic and ganglionic ~ ='transmission changes, sight and hearing changes, etc, can influence :~.learning, These must be shown to be inoperative in any suspected drug . ; .'- ; =;,effects on the learning process. The contribution of central "side effects" must be determined, . ; 1 , •1~• d t th th ff f t ti d b d t t ll i i t it or ve- er e e ec a cen ra y-ac rug e p npo n o s a o e °action on central-learning processes, other central effects such as changes ~in motivation (hunger, thirst); and excitability level (anxiety, confusion,, ':hyperesthesia), must be ruled, out. Contribution of these effects to :}"observed performance would not be considered part of true learning altera=-• :-':tion. since they are temporary and do not directly inf luence ermanent: p • ~memory. :This project, -then, is designed to fulfill the preceding criteria for •.:nicotine by aa series of approprilate pha.rmacologic and neurophysiolo~ic experiments, Since the hippocampus, a limbic system structure, has been ;.' imp licated by many workers as a ma j or bra in, area invo lved in lea1 Liino, .;--.thi,s as well as other suspected. structures (cortex, reticular activating system) will be conce.ntrated upon. It is interesting that low doses of ,:.~.nicotine which. enhance learned performance in animals also produce a :.characteristic theta rhythm (4-7 cps slow, wave activity) in the hippocampal , ELG (8). Conversely, high doses of nicotine which block learned behavior can elicit seizure activity in the hippocampus. Although these observations have never been corre.lated in the same animal, they provide an•interes•tino theoretical basis for studying the relations between nicotine, learning, : and the hippocanpus and other related brain structures, ~ A unique.technique to be used in this study concerns the "chronic" r administration of small doses of nicotine during a relatively lengthy avoidance training session•. Because of the short duration of action of nicotine, rats will be fittedi with:chronically-indwelling intravenous catneters-for repeated nicotine administration. It is felt that this will : also clo$e1y parall•al the effects of a chain smoker performing a• relatively ' l :co:apex_ mental or physical task.

, .. • "S. fletaNSs oE Expedmental Desi9n and Procedures: (Attach Separate Pages). Animals - Rats will be used throughout the study, in order to satis•fy' `:•,.eurrent housing requirements and to allow data accumulation on a large ;~eu h 'number of inexpensive subjects; ' Trainin- procedure - Since most of the published behavioral studies •':7::`:- avoidance response, it seems best to expand upon these results with with rats and,nicotine have utilized either the shuttlebox or pole-climb .similar behavioral situation. This worker is most familiar with the ;,' '4discriminated. lever-press avoidance 'resPonse and its interP retations , . ..;'_.: ; " ;-detail in an acqompanying reprint (Psychonoiri. Sci,, 1967), The pr.ocedure ~-;~ ~so this training situation will be used. The procedure is.described in ;~ is especiatiy aavantageous tor Learning studies because acquisition of :of changes in response rate due to drug treatment, °'the response is relatively slow, thereby allowing accurate observation• -.•Dru7 administration method - Since the purpose of this project is to, during training sessions only. That is, doses will be administered up to the time when a preset criterion is reached (either 85% avoidance averaoe study the effects of nicotine on learning rather than on previously- learned behavior, nicotine and saline (control) wi11 be administered -for 3 consecutive sessions, or 15 sessions, iehichever is reached first). • •Although doses, onsets, and durations will have to be determined for our The applicant has at his di.sposal for• tha.s project the following facilities and equipment: . : particular test conditions and animals, the literature does provide 9. Physica[ Faciities Availabl'e (Where Other than Administering Organization Indicate Geographical Location) ( t0 page 2a ) 1-Staff office (air conditioned) 1-Laboratory-office combination containing desks for 5 students • and animal surgery area (air conditioned) (to page 2,0 • 0. Additional Requirements: - - ; For completion of pro~ramming equipment in available test chambers: 2-Grason Stadler shocker-scrarb lers, Model E1064GS 1-Heavy-duty power supply, 28VDC (Grason, Stadler), Model E783DA 2-Lehigh Valley basic interval timer, Model 1309 1-Lehigh Valley session timer, Model 1350C 1-Lehigh Valley digital counters, Model 1425-10 Z- • 11. Biographical sketches of all principal and professional personnel (append) See page 2c? , .. • • = 12. List of puLrlications: (Five most recent as pertinent) (append) See page 2e. ._:~•,. . .- . . ~. .:~ .._ . _ .. : .:.: .. _: _._ _ ... -. .•_~_.......__-,... .. . ...:_

Experimental Desien•and Procedures (continuedi)s ;.; . . 'information which can be used to design the dose schedule. Domino (3) has shown that nicotine in relatively small doses reaches its peak after minutes and has a duratioin of approximately 30 minutes. Since the ' avoidance training method to be used requires 4-hour sessions every other ;;'day, it is obvious that nicotine will have to. be given more than once in ;;f;`order to provide prolonged drug.action for an antire session. Thus animals wi11 be fitted with chronic-indwelling catheters vial the jugular vein according to a method described by Weeks and Davis (11L) and modified by us (5). The cannula will be attached•to a swivel fluid-lock assembly y mounted in the top of the test chamber which will allow the animal' -to ~:move freely without twisting the cannula. Injections will then be made . ;at desired intervals without physically interrupting the animalts ,training session. 4hi;.oDoses to be used - According to Johnston (6), 1/50, gr. of intravenous nicotine produces, in a habitual smoker, the same psychic effects as smoking one cigarette. Assuming this effect occurs in a 70 kg. man, the :!Ocigarette equivalent" of nicotine received is calculated to be 18.6 ug/kg. ~~ of body weight. Since our experimental design will utilize intravenous nicotine administration, the nicotine "cigarette equivaLent", as well as j~other,simi:lar small doses, will be thoroughly investigated. Along these Alines, it is interesting that the doses used by Domino (3) to facilitate avoidance behavior (40-80 ug/kg, subcutaneously) and by Broc•m (2) to. evoke hippocar.ipal responses (30-50 ug/kg. subcutaneously) are similar to !Four calculated cigarette equivalenr. It further seems very possible that i'; ur 186 ug/kg dose given intravenously may produce the same effects as ,, 0rthe Domino and Brown subcutaneous,doses, Experimental pLan, - The 3 criteria discussed•under Objectives will be `investigated'using nicotine as followss U~' ~~1.- Drug dissociation - In order to determine the presence or absence of ~Znicotine dissociation of learning, groups of rats will be trained in the conditioned avoidance situation under the effects of nicotine and saline. Rate of response acquisition to the preset criterion will be recorded to determine enhancement of response rate with nicotine. Later sessions will `' consist of performance measurement of both groups.treated with saline. Drug dissociation will be ruled out if the nicotine-trained rats continue to perform at the pre-saline level . Central action of nicotine - The usual method of determining whether g drug is acting centrally is to demonstrate a behavioral action with the s.•=k 'drug followed by loss of the action with a quaternary derivative which -- , _ - . . ll not pass the blood-brain barrier. In this re~ard, Domino (4) has ;reported that congeners such as nicotine methiodide are relatively ineffec- 4tive incontrast to nicotine in blocking conditioned, avoidance behavior _ ~kin rats. Nicotine methiodide will be used to confirm these results in our conditions, and to control for central specificity in other parts of the study. . . .- . . . • - .... Another method to test for specificity of drug effects in the brain is simpLy to place the:drug into various structures of interest by means of ~''`a cannula. This provides a. means of determining significant differences %';in drug effect from parenteral administration. Using apparatus similar to that described above for intravenous injections, rats with chronically- idllil ih biil bdihll dfi ,nweng cannuasn teran wle teste wt smaoses o nco- tine injected into the cortex, hippocampus, and reticular foi Vation; Intracerebral quantities of nicotine will apptoximate I x 10' moles, and C"solution volumes will be not more than 2 uL/injection. A modification of •the nylon-stainless steel assemblies.of Valenstein et a_1. (10) will be used for cannula construction, and nicotine will be administered• intra- cerebraLly at times comparable to parenteral administration. c Q C

LD, Conficmation of cannula placements•will be performed histologically. by this drug. each session wouZd•strongly indicate facilitation of memory trace formation nhancement of conditioned avoidance learning when nicotine is given•after Dom ino, E.F., "Some behavioral actions ofnicotine!', in Tobacco (1967). W ' .- Browm, B'.B, , "Relationship between evoked response changes and behavior ~ following,small doses of nicotine", Ann. Y•.Y. Acad; Sci: 142r19•0-200 , a~~ y .avoidance conditioning of inbred:strains of mice", Psychopharmr. 10t : 5 (1966) References : Bovet, D,,, Bovet-Nitti, F:, and Oliverio, A•,,'"Effects of nicotine on : ReLated Comnoarzds, ed. Von EuZer, The i4aci•i211an• Company, .":e~. Yark 965), ;''kUts optical isomer•, and• related: compounds", in Tbhacco AZ'_:aloidts and omino, S.F,, "Some comparative pharmacological actions of (-)-nicouine, '= .. . . .. . . ; . . • . ., .. _ ... .. . ;r., ,_. .. . ... ... . ' . . - . .. .; ' : . .. .. ... . . . . . : , , New York (1965), pp • 4 -Q . - .. . : _ . _ Alklidd RLd CM C,x aos aneateomnounds The b;acillanorapany r, ed. Von Euler • 3 = 3. ~ .:::;.;,~ ' Frozen sk:ctid•ns of 50 micron;thickness will be cut, placed on slides, and stainediwithtcres,vlechtvioletl and methyLene blue according to the :•',storage effects by ruling out motivational changes and excitability factors. fi ~;•~;"•r••adopted by a r.tunber of investigators as a means o assessng memory ;j method' of i:luver and Barrera•(7)•. 3i Direct action on neural learning processes - The procedure of ;:;:admnistering drugs after trainino,rather• than'before training•has been . " --~;and the stronger the incoming stimulus, the'more deeply the memory trace " '.ing the CNS'initiate reverberating neural pathways'which•must continue for `_'A current popular theory'of learning and memory states that stimuli enter- fixed time to foum a memory trace; The longer the pathway reverberates i ld: Thidiil (:lhki Usanteus any overrng stmuuse,g, eectrosoc or certan ` =:.drug~ tahich, disrupts the reverberating neural pathway (assuming the path- . way is'eLectrical-chemical in nature) will inhibit learning and trace in this manner (9) On the other handl stilats suh as ~~., centramunc ~:-rmphetasnine and strychnine given after training trials in aninals have a c e~ q g pro "" '= ?~formation: Post-trial eLectroshocl: and various dru $ such as barbiturates g f,,, :"=y"', and tran uilizers have indeed been shoxm to fife rnin cess t the l ~ { y u a e ve w or rugs are max ma y e ect n m nutes an c t :~g 's<which ~re metabolized Prior• to retention tests, as is ni.cotine, Thus ~ a "been shown to enhance the learning process (9). This technique is especi- s 11 it bl f d ll ff d whi h i i i hi i C ; Johnston, L,it,, "Tobacco smol:ing and nicotine", Lancet 243:742 (1942). y. ... . . . . -- ' Erickson, C,'_{,, uapublished data on chronic intravenous alcohol infusion in rats: . - . : . - . . ' . . . . ~ .. . ... . . . •7: Kluver, I;. and Barrera, E.A., "A method for the combined study of cells• and fibers in the nervous system~~, J. I'europzthol. exp. heuroi. 12s •:•400-3 (1953), • 8. Lon_-o, V.G., r3vnta, F., and Scotti de Carolis, A., "Effects of nicotine on the electroencephalo--ram of, the rabbit", Y?:.Y. Acad. Sci. 142:159-69• (1967), , Q C

~ c, ~ t•1cu^augh, J.L•: 'and Petrinovich, L.F., "'Effects of drugs menory", Intern: Pev; Neurobiol. 8s 139-96 (1965) ; k'Gn... . ~ . . _._ . : Valenstein, E.S., Hados, jV:, and Stein, L,;, "A simplified electrode- ~h1assembly for implanting chronic electrodes in the brains of small animalsrt Am. J. Psychol. 7<<:125-5 (1961). ; Weeks, J.i<.. and Davis, J.D,., "Chror:ic intravenous AppL. Physiol. 19:540-1 (1964); Physical Facilities Available (continued): _1-Behavioral laboratory (air conditioned) containing 9 rat test .be available for this project; so*:e prooranmin(-n equipment is chambers and relay progranr.ling equipment (3 test chambers will required, to complete the char,~bers - see Addi tior_ai Recuirements) work), with Microto~:~e equipped for carbon dioxide and paraffin 1-Histoloo-i.cal laboratory ( for confirmation of cannula placer.:ent sectioning -

1 Z ::4i' , ...,._ . . :°~'~ • ti: _ halmers, R.K. and Eric'..tson, C.K., "Central choliner~ic blockade of the••"`~~"~~. ~~~"~~.~ ~ ;jt ~;. ~, conditioned avoidance response in rats'~. Psychopharmacoloooia 6t 31-+1 ;`_~~:-•; s~a..F:,t t, r ~:,c t'FE faC~ » ~~ ~Erickson, C.IL, and, Chalmers. P.,K,, 11l~ippocampal theta rhythrt•involvement hZ.,kin cholinergic-induced blocicade ot discriininated avoidance responding ` Ran rats", Arch. int, Pharm.acodyn; 163: 70-8• (1966), ~Ericnson, C.v., "Facilitated responding in a discriminated lever press avoidance situation"~ Psychonom. Sci, 8s37-8 (19b7). :•'Erickson, C,K,,. "A reliable lever-press avoidance training method in artthe rat", Fed, Proc. 25:738 (1967). :- ---• ~ ; ., •. ... atel, J: and Erickson, C.K., "Enhanced avoidance acquisition by loio- ;intensity hippocarnpal stimuli", Pharmacologist 9:200 (1967). .........x:......=...:.._r..V.. ~~.' d5.'•=-~awr.•:w:..«.~...-.~.::.+.+s.....,.:......

R: REDACTED MATERIAL A. Salades (Personnel by names) _. % time Professional • -. ~ Carlton,I:. Erickson, Prin, Invest. 9 months academic year 207. 3 months st.trmter 1007. Fringe benefits Jitendra B-. Patel, Graduate Student 12 months calendar year 50% None Sub•Total Sab-Total D. Permanent Equipment Qtemize) Amount 400 100 100 50 2-Grason Stadler Shockers with Grid Scrambler, Model E10G4GS ? $ 285.00 $ 570 1-Grasorn Stadler Heavy-duty Power Supply, 281JDC, Model E783DA 175 2-Lehigh- Valley Basic Interval Timers, Model 1309 0 ; ~o ~ $ 95. 0: 3 _ - - a) (to page .. Total-$ 1,214: E. Orerhead(16%•ofA-}s6+C)' $ 1.0m Total q 5n $ Q Estimated Future RequlPements: NO:+E s Salories Consumable SuppU Other Expenses Permanent Equip. Overhead` ' Total C Animals and feed Cannula materials Dru--s: and••histologlcal chemicals Recorder paper, notebooks C. Other Expenses (temize) Yean2 Year 3 Signature t•tiTx1~C C1Y,_ K C[/La.GI': So-r~ It is undlntood thotlhe applicant and institutional iofficers oi-+...f r..i«t 913 UN4-4004 in,applying for a gronthavs read and found acceptable , n Q Te[ephone & _ ~/36 the Council's "Statement of Paliry Conlaining Conditionz Signaturt ~~LL !~T_16L t i( f f!~ L~ t/ 1 14 and.Terms Under Which Projed Grants Are Made." 4.4- Oflkrr ef,the InitAUkon LIYFCH-{'vfFC'e_ a(~Telephone . r=att 1 lnrAx,cr:j

... • `? -- s ... .a Permanent Equipment ( continued•) 1-Lehigh Valley Digital Counters, Model 14•:25-10 1-i.ehigr Valley Session Tiner, Model 1350C

i a ^ List finandal support for research from all wynp, indudlnp own Instttutlon, for this ond/ort.lated nsearch projects. Thle of Project "Addictive Ability of Self-injected Intravenous Alcohol in P,a:s" ~. "Behavioral Hyperexcitability Induced by Alcohol ?lithdravra1 in P.ats" "Enhancement of Learning by Limbic System Stimulation" VSO4tScOOI fi i A