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To: • Subject: . PHILIP HORRIS U. S. A. I NTER-OFF I CE CORRESPONDENCE RICHMOND, VIRGINIA Dr. T. Osdene Date: October 14, 1980 V. J. DeNoble and L. Carron Progress Report The Behavioral Pharmacology Program DeNoble, Carron, and Allen Major objectives of the Behavioral Pharmacology Program are (I) To develop a better understanding of the reinforcing actions of nicotine and nicotine analogues, (2) To, gain insight into the neurobehavioral actions of nicotine, and (3) To develop and use animal behavior techniques to: s.creen nicotine analogues for their nicotine el icilting properties. NICOTINE SELF-ADMINISTRATION A major objective of the behavioral research program is to evaluate the most useful and powerful techniques devised for compound evaluation and comparison. This is a critical test for the nicotine analogues because it has been previously shown that reinforcing efficacy does not always correlate with interoceptive generalizati Two reports (Hanson' et a.l;. 1977 and Lang, et al. 1977). have shown that rats can be trained to lever press for intravenously, delivered nicotine. However, neither report used appropriate control measures to clearly show that nicotine was functioning, as a reinforcer. A first step in our laboratory was to demonstrate clearly that nicotine can function as an intravenously delivered reinforcer, reinforcing properties of nicotine and nicotine analogues. This is being accomplished by utilizing intravenous and intracerebral self-administration techn~iques. Intravenous self-administration has proven to be one of the

2 Rats were prepared with an indwelling venous catheter made of siliconized rubber. The catheter was anchored in the external jugular veiin and passed ~; subcutaneously until it exited through the animals back. This was connected " via protective t~bing and swivel joints to a remote iinjection pump. Responding on one arbitrarily selected response llever was automatically progra~ned to activate the injection, pump for 6 seconds, delivering an injection of 0.1376m£ of solution dlirectly into the animals bloodstream. Responses on, the control lever were recorded but had no programmed consequence. CONTROL STUDIES Nicotine self-administration was initially established at 32~/~/injection. Nicotine was available 24 hours per day, under a fixed ratioT 1 schedule. Generally 10-14 sessions are necessary for responwding to stabilize. After stable behavior is obtained, changes are made in the nicotine delivery procedure to determine if lever pressing is maintained by the contingency established between lever pressing and nicotine delivery. Changes include. substitution of saline fornicotine, reversal of nicotine-lever and control (action of)lever functions, and automatic non-contingent nicotine injections. The results from a small number of animals show that n~cotine self-administra- tion by rats is maintained by the responseznicotine contingency, rather than by other behavioral effects of the drug. Substitution of saline for nicotine failed to maintain responding. When nicotine (32u~/~g/injection) was reintroduced, the number of injections rose to previous levels. During the self-administration, responding occurred almost entirely on the lever delivering nicotine. Control lever ~esponses were. less than 10% of all total I'~ mesponses. When. nicotine injections were. deliivered non-contingently~-~ number of responding decreased as a function of the frequency of the non-contingent injection. " ....

These results show clearly that nicotine can function as a positive reinforcer for rats. INJECTION DOSE -~, Nicotine self-administration was established during ~4 hr/day access sessions at 32~g/~g/injection under FR 1 conditions. After stabilization, the effects of injection dose was determined on response rate and nicotine intake (mg/~g/day). Injection doses were presented in descending order (64,32,16,8.0, 4.0, and 2.0uzj/~g/injection). Rats were tested for a minimum of 7 days at each dose. The result shows that as the dose of nicotine was decreased from 64.0 to 4.0~g/~g/injection, the number of injections increased. At 2.0~g~g/injection, the response rate decreased. The mg/~g/~ntake was highest at 64.0u~/~g/injection (2.5mg/~g/day) and decreased with decreasing concentrations. . Future studies: 1) Examine a dlos.e response curve under various schedules. 2) Examine the effects of cholinerg~c antagoni, st and~of self-administration behavior. 3) Attempt direct substitution o.f nicotine analogues. 4) Demonstrate that nilcotine self-administration does not interfere with ongoing behavior. 5) Show that termination of self-administration does. not ?~o~c~ behavioral impairments. 6) Nicotine self-adminilstration does not alter self-administration of other reinforcers (food, water, saccharine, etc.). Mo~ ~

PROSTRATION SYNDROME The laboratory has been able to demonstrate the prostration syndrome reliably with both (-) nicotine and (+)-nicotine, with the latter being about 1/10 to 1/20 as active. Having gained a reliable data base with both (-)-nicotine and (+)-nicotine, it is now essential to examine ~elative potency of the nicotine analogues, most importantly analogues that have shown to be nicotine-like in the discrimination tests, yet have a different structural configuration. In addition, studies to locate sites of action J and determine the extent of the behavioral prostration are now in progress. Even without detailed knowledge of the underlying physiologicall mechanisms, the use of behavioral measures Will provide iimportant information about the sites of action in the brain. ~e are currently using scheduled cont~oliled behavior to evaluate the effects of ~ntraventricular injections of nicotine. Operant performance has been shown to be more sensitive than activity rating scales, and provides a more stable baseline fromwhich comparisons can be made. Although the prostrationsyndrome is a reliable screen for behaviorally active nicotine analogues, the ratingscale developed by Dr, Abood provides only a descriptive interpretation of the compounds' effects, and does not permit a determination of possiblle prolonged, changes in CNS activity. However, previous investigatilons (DeNoble & Begleiiter, 1976, DeNoblle & Caplan, 1977, Bowman, 1980, Mele and Caplan, 1980)have demonstrated that schedulle- controlled behavior is sensitive to CNS changes. Schedule-controlled behavior is a research technique that is based upon principles of operant conditioning. This technique produces a highl!y stable and reproduceable baseline of behavior which has been shown to. be

dependent.on the integrity of the CNS. Therefore, this technique was used to measure CNS recovery times in nicotine-infused rats. Twelve male albino rats weighing between 190 and 230 grams and 120 days old were used. They were gradually reduced to 80% of their free feed!ng body weight. The a~imals were then trained to press a ~ever in a standard operant chamber for a single delivery of milk. Subsequently, the contingency for reinforcement was increased to a fixed ratio 16 (FR 16). (Under an FR schedule, reinforcement is contingent upon completing "x" number of responses.) Animals were trained daily (Monday-Friday) during two successive 15-minute sessions with a 5-minute time out period after the first 15 minutes. After a stable baseline of behavior was obtained, the animals were anesthetized and implanted with a stainless steel cannula into the. left llateral ventricle o.f the brain. Following two or three days of recovery from surgery, rats were retested under the FR16 schedul!e. Intraventricular injections of saline or nicotine were administered when there was less than~ 10% variance iin daily response rate for 5 days. The animals were first tested with saline. Testing began at the 5-minute time out period during their daily session. Animals were infused with 5u~ of 0.9%1 saline solution and then placed imediately back ~n the box. The animals were tested in repetitive. 15-minute segments until (1) baselline behavior was recaptured: or (2)un~til their response rate decreased (satiation). Having. established a baseliine with the saline infusion, the anilmals were then infused with 5~g of (£)-nicotine (free base)in 5u~. Figure 1 shows that the infusion of saline producedl no major change in response rate. However, the same animals infused with 5~ showed a suppression in response rate. -

6 Using schedule-controlled behavior as a measure, rats continued to display behavioral disruptions 10-12 minutes post infustions. Observations of these animals via a one-way mirror revealed that typical locomoting and grooming behaviors were displayed 5 to 7 minutes before recovery under the FR schedule, The duration of suppression in response rate_w~s approximately 110% longer than that observed with Dr. Abood's ~cale where recovery from the prostration effects occurs 3-5 minutes post infusion. Recent electroencephalographic ~ecordings taken by Dr. Abood after intra- ventricular injections of nicotine into rats have demonstrated that recovery of baseline hippocampal activity occurs 10-12 minutes post infusion. These latencies in conjunction with the latencies found in the schedule-controlled behavi, oral task demonstrate that prolonged CNS changes are taking place. ~' ..... :'~ ~ ese anima.ls were then tested with twice their original dose of Z-nicotine (5~ to lOngin 5u~). The time between infusions for all animals was noless than 71days ~ndl daily response rate ~ showedless than a 15%.variance from day to day. Surprisingly,.the duration of the suppression in response rate produced by the infusion of lOng of Z-nicotine explanations of this result. First, it is possible that the rat devel:oped a metabolic tolerance. Metabolic tolerance can be divided into two major categories; peripheral and central. Some of the peripheral mechanisms include alteration in enzymatic was shorter than that produced! by 5~g. of Z-nicotine. There are several possible degradation, changes in absorption, or changes in lipid s.olubiility. Central ~ mechanisms refer to the neural adaptation to the presence of the substance such that "normal'" function occurs with the substance present. We can rul~ out

7 the peripheral mechanismbecause we are delivering the nicotine intravenou,sly and therefore bypassing the peripheral system. While the presence of central cellular adaptation cannot be fully ruled out as a pdssiblle contributing factor in the change in behavioral response, it is unlikely since the infusions were given-7 days apart. The other explanation, of the data is that the rats rapidly developed behavioral tollerance. Behavioral tolerance is a diminished effect (behavioral): of a compound with repeated exposure to that compound without the iinduction of metabolic tolerance. In order to examine the development of tollerance, we increased the requirement for reinforcement from 16 to 32. Once the rats were stabilized: under the FR 32 schedule, they were infused Ot~i'~.~. • the daily session, with 5~z3 of n~cotine on two occasions separated by a mean of 5 days. The secon~d infusion of nicotine had a diminished effect. Presently we are designing a ~eries bf studies which will more accurately characterize the development of tolerance. In addition, we are Sellectivelly manipullating neurotransmitter systems to better understand the central action of nicotine. We demonstrated that 2'-methylnicotine was behaviorallly active in discrimination and prostration tests and is equally as potent as (-)-nicotine. Using Dr. Abood's rating scale,. 2'methylnicotine did not produce some of the peripheral~signs of prostration. (Less motor control loss, no hyperventiliation, no piloerection, no excess urinatiion or defecation). In view of these findings, we tested 2'-methylnicotine using scheduled controlled behavior. Under FR 32, the rats were tested first with 5~g of ~-nicotine~ then 5 days later with 5ug of 2'-methylnicotineA The data shows that the 2'-methylnicotine produced less suppression of response, rate under the FR 32 schedule. This result ( 0001.2 593

could reflect 2'-methylnicotine's cross tolerance to ~.~nicotine and/or a diminished peripheral effect. In the next series of studies we willl separate these events. In the futurewe willi be conducting studies in which the selective blockade of neural structures will be evaluated on the behaviloral components o,f prostration. We think th,at this will provide evidence for the sit(~s of action of nicotine and nicotine analogues. DI:SCRIMINATION STUDIES As part of the ongoing nicotine discrimination program, we have completed initial testing on a series of additional compounds. These compounds included various dialkylaminomethylpyridines, metanicotine, its dihydro derivative, and several isomeri~ nicotines. Addition of the diimethylam~inomethyl substituent to the pyridine ring in the 3 position produced "n,icotine-li~e" responses from the animals whereas the isomeric 2- and 4- substituted compounds gave no. indication of activity. The 3-pyridyl derivatives yielded "nicotine" responses in, 55% of the animals at a 4.0 mg/kg dose. (This leveli represents lOx that of the training dose usediin the nicotine discrimination task.)i When the dose was increased to 8.0 mg/kg, 4 animals responded on the nicotine correct lever and 3 animals gave incompl!ete tests. Metanicotine, unlike its dihydro derivative, showed nilcotinic activity in the animals tested. Apparently, the unsaturation in the side chain, in this compound, is necessary to produce activity, since metaniicotine at a. dose of 41.Omg/kg produced: n,icotine cues in approximately half of the animals. When the dose was increased to 8.0 mg/kg, 100%1 of the animals responded on the nicotine correct l!ever.

Of the isonicotine derivatives, the 3,3~substltuted N-methylpyrrolidine showed activity while the 2,3~isonicotine did not. At a dose only 5x that of the normal discrimination training dose of h-nicotine (2.0 mg/kg), the 3~3~sonicotine was active in 9 out of 11 animals. When the dose was. reduced to 1.0 mg/kg, 63% of the animals tested responded with the nicotine correct lever. A series of open-chain nicotines were synthesized. A methylethyl- aminomethyl substituent or a diethylaminoethyl substituent was added to the pyridi!ne ring in the third position. Animals injected with a wide range of doses of each compound did not respond on the. nicotine correct lever. However, one open-chain compound, 3-dimethylaminomethyl! pyridine, dlid produce behavioral activity. At a 4.0 mg/kg/body weight dose, 75% of the animals responded on the nicotihe correct lever. Only 40% of the animals tested at a dose of 2.0mg/kg/body weight emittedl a nicotine response. Note. that these doses are 5 to 10 times higher than the daily dose of ~,-nicotine used " in the discrimination task. The most interesting finding was with the 2'-methylniicotine. At a 0.4 mg/kg/body weilght dose, 100% of the animals tested responded! on the nicotine correct lever. This is the same dose used during the daily nicotine training sessions. Doses of the 2':-methyllnicotine higher than, 0.4 mg/kg/body weight produced' incomplete responding. Presently, we are preparing to do a dose response curve usi.ng the 2'-methylnicotine. A dose response curve will allow us to access 2'-methyl- nicotine's relative potency to ~Z-nicotine and £-niicotine. The effectiveness of 2'methylnicotine in the discrimination task is now being tested with

10 preinjections of mecamylamine and hexame.thmni.um, These results should .... indicate whether the discrimination of the 2'-methylnicotine is centrally or peripherally mediated. M~jor Strengthsof the Behaviora|Pharmacology.SeCtion 1) The use of Sensitive and reliable behavioral m~asures for the evaluation of compounds. 2) To use behavioral events as indicators of nervous system activity. 3) To directly correlate behavioral and nervous system changes induced by nicotine in the intact organism. 4) To provide empirical evidence that nicotine is a positive reinforcer that does not produce dependence. Major Weakness of the Behavioral Pharmacology Program The rate of expansion of this program, has been great and two types of additional personnel are necessary. First, it is important to obtain a high level assistant (MS. or BS) and second, to increase our .......... staff by ~: .,;/ person. The depth of difficulity of the present research requires the Assistant (L. Carron). to continously monitor our technilcian, in addition to running her own studies. My time is divided as follows:: 50% running! animals; 40% data analysis a6d overseeing other studies; and 10% literature reading. We find that the lack of personnel is a maj,or failiure of this program.