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.'17in: Cr>cNciL IFoie Toii:ccco Rrait.~r,~cl[-II.S.~I., Iac: March 110, 1978 NLEM©RAN DUM TO: Addison Yeaman FROM; DL H. Ford SUBJECT: The Rationale for Nicotine or Smoking Studies in Relation to the Central Nervous System (CNS). Regardless of the discipline of the various investigators dealing with the nervous system~ the ultimate question asked is: how does it work,, how do we think, store or recall memory2 How are these nrocesses influenced by drugs or disease? Thus, it is necessary to know the nature of the anatomical components and how they are linked together, how they synthesize or degrade their end products (neurotransmitters or hormones), how they are influenced by outside events (i.e., phar.macoliog~c agents)' andihow the CNS controls,the rest of the body? An inescapablie fact concerning the CNS is that it does controli much of what goes on -'n the rest of the body, either directly (respiration, cardiovascular function, water metabolism, growth at one point in life, reproductive activity, and the function of the endocrine system, or imdirectly through its control of endocrine function,. Further, what occurs in some of these systems will influence brain activity. Thus, defective cardiovascular or respiratory function r.;ay disrupt normali Ctis activity. Iniaddition, the hormones liberated from the adrenals, thyroid, and gonads all very markedly influence brain function in the adult and differentiation in the fetus. Thus, there i~s an intricate series of feedback control systems such that changes in function of one system influence functions in a second or third systems which, in turn, then influence the function of the first system. A comment made by many neuroscientists is to the effect that any agent (hormone, drug, etc.) which influences any aspect of the overall interaction of C:.S metabolism or transmitter activity may ultimately infl'uence the subsequent res©onses which it makes. Thus, dpfecti've or altered synthesis,, degradation or release of one of the many transmitters of the C:4S, delayed action~of a hormone involvedi in,differentiation, or synthesis of an enzyme involved in maintaining cell respiration might well lead~to an alteration in CNS function which ceulid impair health. The degree to ::hich:any pharmacological agent (nicotine),might do this can only be determined by experimentation. Acetylcholiine was the first transmitter dilscoverediin the 1'~9th century. This was in relation to its function with one of the peripheral nerves,, the vagps. A substance was found coming from the vacus nerve which, when~ applied to the heart, slowsd~its beat. This vacwssUO_`f turned out to be acetylcholine. In later years it turned out that acetylcholi~ne caused two distinctly different kinds of response. One was mi~id:e1 by the nharaacologic agent, muscarine, and hence came to be called a muscarinilc response. The other response was mimicked by nicotine. Hence, nicotinic. The action of acetylcholine at the neuromuscular junction,was found to be nicotinic. The subsecuent discovery

that this response could be blocked by curare and the snake toxin x-bungar~tor.in led to literally hundreds of neurophysiologic studies defining the properties of this interaction. aore recentlbf investigators have observed CNS cholinergic systems which are both muscarinic and'nicotinic. Thus it is now well established that nicotine (and perhaps cotinilne) have effects on the CNS. For example: the inhibitory effects of Renshaw cells on motor neurons in the spi!na1 cord are mediated through nicotinic effects of the transmitter acetylcholine; the release of the antidiuretic-vasopressin hormone in the hypothalamus is facilitated by nicotine lieading to a decrease i'n water secretion and an increase in blood pressure; there are numberous specific effects, both excitatory and inhibitory, in~the electrical activity (EEGY responses of neurons in different areas of the brain; there are inhibitory effects of nicotine on the release of pituitary hormones which reguliate ovulation; and there are effects of nicotine on pain perception (the threshold for oerception is eievated). In view of these andlother respcnses to ni~cotine, it would be useful to know the precise sites at which these events are mediated, what is the nature of the receptor for nicotine, is there a:nicotine responsive system in the CNS which is integrated in its,function with~other transmitter systems -- that is, do nicotinic receptors modulate the resnonses of other systems? -- i~s there a;normal transmitter other than acetylcholine in the CNS which nicotine mimi,cs, and is there a relationship between the hycothesized nicotine system and the enkaphalin-optiate system which is responsible for the bl!urring of pain perception caused by sr,oking? There may be other auestions which might be asked!, but these should suffice to indicate that nicotine (or a substance other than.acetylcholine which nicotine mimics)i may play a role in normal brain function to the degree that its presence or absence might influence brai~n, regulatory function and, in so doing, inf_-uence health. What nrojects are currently sunported by CTR which are germaine to this issue? 1. L. Abood (Rochester)! is working on the dilstribution and characterization of nicotine receptors which are unique and not associated with the acceflted cholinercic-nicotinic receotor interaction of acetv_lcholiine. He has performed studies demonstrating the presence of such,a receptor which may, under normal circumstances, be stimulated by an as yet ur.identified peptide transmitter in the brain whose activity is mimickediby nicotine. When this nicotine responding receptor is activated the ani:nals becor,e flaccid. 2'. M. Aceto (Richmond) has just com.m..enced working on the distribution of sterosoecific receptors for nicotine. Nicotine,,like many biologically activee compounds, exists in two molecular configurations which are mirror images of each~ other. The form wriich~rotates polarized light to the left (levoi-1) is the active form, whilie the form which rotates poliarized light to the right (+ dextro) is pharmacologically inactive. The two forms are called iso-ners. So far it would appear that the number of pharrnacologicalliy active (1) spec_fic receptors for nicotine constitute only about 3-4% of the total num:er of niaotine receptors. 47hL,t then is the role of the nicotine which bi:nds to rece_ot_ors not associated withh pharmacologic activation (dextro form) of a neuron? Does it relate to other `ziochemical activities within the cell?, 3. L. Hall (MIT) has been working,to~chasacterize the nicotine receptor of acetylcholine responding neurons i~nithe drosophila fruit fLy. The fruit fl}^ is normally killed by nicotine. However, she has develocediseveral 4-=

C 3 C mutant strains by feeding the larvae nicotine at doses which kill most of the worms. The few which survive are allowed'to attain adulthood and then~mated. The final offspring are then used to investigate the biochemical characteristics of the receptors which~bind nicotine. 4. A. Lajtha (NYS Res. Inst. for Neurochemistry), has demonstrated the uptake characteristics of nicotine in vitro and in vivo into rat brain and shown it to be taken up more rapidly than any other molecule yet studied in brain tissue. He willl be investigating the distributioniof nicotine into:the various brain~metabolic pools and will determine its effect on protein~synthesis and~degradation in different brain areas. Future investigations woul~d'deal with its effect on some of the braim-speci!fic proteins, such as the S-100 protein (ifound in supporting cells) and synaotin (a protein found at the junctional membrane between neurons). Blockage of the action of these proteins appears to interfere wi'thimemory consolidation. 5. J. Rosecrans (Richmond), while interested in the behavioral effects of nicotine, is also interested in~some specific pharmacologic effects,, which he has just started to consi~d'er. Specifically, he is examining the effect of systemically applied nicotine on the synthesis and degradation of dGpamine (an inhibitory transmitter): in the caudate nucleus (a deficiency of dopamine at this site will lead to Parkinsonism). He is also investigating similar parameters in relation to the transmitter serotonin in the hiopocanpus. (The hippocampus is an important part of a division of the CNS concerned with~regulating emotional resnonses and is involved with reproduction, pain percention~, fear, etc.) Thus, while Abood, Aceto, Hall ('and Schmidt and Tometsko) are interested in recePters, one aspect of the Rosecrans program is to determine what reay be happening to transmitters which may be active i~n,moduliating the function of these receptors. At the same time, Lajtha and'his group is investigating mechanisms which may be involved in the synthesis of receot,or proteins and of the various transmitters. 6. J. Schmidt (Stony Brook)', has been involved in studies dealing with acetylcholine receptors for many years. His current interest is to identify sites of nicotine receptors by causing a-Bungarotoxin (a snake toxin) labeled with 1251 to be bound to the receptor proteins on a tissue section. The section is then coated with a;photographic emulsion which will be exposed at those sitess where electrons are given off by the radioactive iodide (radioautography). By such a procedure he has identified some receptors in a part of the olfiactory, systen,in the optic tectum (a regionlreceiving an input frcm~the retina) and in the retina. He has further noted that the di!stribu*_ion of radioactivity in brains after exposure to the labeled snake toxin is not equivalent to that seen using, labeled nicotine, suggesting that the toxiln binding may be non-specific. He is also interested imicharacterizing the molecular nature of the receptor. 7. A Tometsko (Rochester) i's basically an organic chemist who has been synthesizing analogues of nicotine (:as well as oreoarinc the d-isomer of nicotine which is not pharmacolbgicalLy, active andiwhich may, be resconsiblefos most of the non-specific binding of nicotine seen in CNS). He is binding these a.:alogues. (which are radioactive) too comoounds w:2ic:7 uncergoa transformation wnemexposed to light. (Tnis is called photo-a_`finity labeling.) This transformation serves to bind:the molecule to the structures adjacent to it, thus locking it in to the site where it was _oresent. This radioactilve bound molecule can then be detected by radi1oautcgraehy and! the anatomic site Located.

The general thrust of these studies is to locate sites of nicotine action, characterize the receptors and to determine if nicotine has any effect on those mechanisms involived in the synthetic and dhgradative mechanisms which regulate receptors and transmitters. Prosoective Studies 1. F. B1oomi(Sa1k Institute) has submitted a oreposal for a study which,plans to map the organization of a nicotine receptor system using an immunocytochemical procedure. Once this is accomplished, he plans to correlate this map with those existing for other transmitter systems,, using,existing maps. (Bloom"s past work has contributed significantly to the creation of these other maps.) 2. W. Stumpf (Chapel Hill) is the world's leading investigator in using,radioautographiic procedures to map sites of localization of water and lipid soluble compounds, such as nicotine. He ils proposing to utilize this procedure to localize nicotine (not the receptor) throughout the entire CNS and to correlate it directly with the distribution of neurons containing transmitters of the amine systems, using the same sections to d'emonstrate both localization of nicotine and localization of amine transmitter. By this approach one can discern if the same cell is associated with both nicotine andithe other transr.itters, or whether they are merely adjacent to each~other. 3. A. Vernadakis (Denver) plans to use an in vitro approach to~ determine if nicotine or cotinine influence the dif_°.erentiation~of irmna*_c:re neurons obtai~ned from develooing,chick brain into mature neurons, as we1Q as to determine if they establish contact with other neurons or muscle. She will also be analyzing the development of transmitter systems within the culture dishes. 4. P. Timiras (Berkeley) is a neuroendocrinologist who has submitted a proposal with D. Hudson on the effect of in utero exuosure to nicotine on the develo_oment of neurcendocrine parameters in the brains of rats in rclation to the normal increases in R,VA, DNA and protein to be exoected in such anorga.n. Thus, it is a study of growth andidevelooment in relation to the differentiation of the neuroendocrine system. 5. R. Michael (Emory U. ) has submitted a proposali for a seri'es of studies which~will start with the rat and finishiwith the rhesus r.onkev. He plans to implant stimulating electrodes into~the so-called "reward" centers of the brain and determine the effect of nicotine on the rate at which the animals will selif-stimulate (or reward themselves). These respcnses wi1'1, be coordinated with circadian events and aLarge number of hormonal factors which maybe influenced by the self-stimulation. Both the Timiras and Vernaddkis studies may provide some information about the develo^r..ent of recectors for nicotine (ene of Timiras' students has already de:eonstrated that hypothyroidism depresses the devel!cpment or differentiatiom of nicotine rece^tors in the rat) . Vernadakis will be able to stsdl* develcpr:ental effects related'to receotors in a manner which will be free of any contributory effect which might be made by nicotine on the placenta. S4h-ile the study pro:osedd by Michaell will not deal directly with the proble7s of receptors, it wi11, deal with effects of nicotine on responses occurring follo-.%ing stimu1'~ation of specific brain sites and thus nay serve to deli!neate nicotine responsive areas, not only in rat, but imithe monkey.

6. A somewhat different approach has been suggested for consideration by K. Brizzee and M. Ordy (Delta Primate Center, New Orleans). They have proposed a study of squirrel monkeys exposed in utero to nicotine. The subsea_uent dpvelopment of reflex, cognitive and oculomotor (eye response to light and roving objects)', responses would be studiied in conjunction with l'evels of protein, RNA and DNA in various parts of the brain; morphological development and differentia- tion of cortical neurons; and an analysis of the develiop^ent and differentiation of the neuroendocrime system. This study to a degree parallels that of Timiras, but in, monkeys. It also includes behavioral studies plus a somewhat broader group of neurochemical correlates. Thus, it appears that CTR is aLready sunporti:ng,several' studies dealing with the nicotine receptor. With the addition of those currently to be reviewed in April and at the October meeting ( which would include Stumpf and Brilzzee if approved by sta£f), we should have a fairly com.prehensive series of studies being conducted by welli known knowledaable people. Hope°ully,, within the next few years a nicotinic system will have been defined which might be im part a cholinergic systemiand part purely nicotinic. Once this is achievedione could proceed to determine how it interacts with other CNS .°unctional systems and contributes to normal' brain function in health and disease. D. H.. Ford DH°':ek

ui I A C ~ Receptive zone of cell body and Axon (Rc), zone for Zone CLthe membrane junctionpoint where information dendrite(D) lelectrical transmit- is tra sfcrred chemically by the release of a cherlieal al of information I•mediat (transmitter) to the next cell. I I I l Sites at which nicotine (ectinine) may effect neuronal function 1. At the level of the nucleus (PI) xhcrein the translation of the genetic code mbe disturbed. might 2. At the level of structures in the cell body (SN) involved in synthesis of protcinaceeus enz-~r,as, structural proteins or transraitters.. 3. In the axon (ax) where the movement of protein precursors or formed proteins which may be or erhich may contribute to formation of recePtorsmay be occurring along the ncurotuLules (T). - - - !t. At the synaptic junction (C) rrhere there is a junction Frith the terminal end of one neuron •.aith the effector region of the next neuron, muscle or secretory cell. iielease of information via the chemical tr nsmitter mediators stored within vesicles (SV) mil;ht be altcred. Further, there miett be induced altered functicnal states on recel>tcrn at R' vhich influencc the rclr::rse of the trmsmitter. There also may be efi'ects on reulatal:e of the tran>mit.tcr into the oril;inal n:~iircnal ending for degradation o2 there may be effects on the receptor (R) of the reccti.vu unit (D) of the next cell which influence the degree of re.:ponse. There may also be indirect effects mediated by the hormones of the endocrine system which alter neuronal activ3ty at all of the aLove levels. Csszs9EO I

",Tiii: Cou.-;cir, holz ToB.,icco R]at:Ar,ci1~-U.S.11., INC. March 10, 1978' After one has seriously considered the various already demonstrated reliationships between nicotine (or smoking) and the CNS, one may stilil ask the question as to where or how does this all fit into contemporary living? In other words,,.what may smoking contribute to individuals which is based on its interactions within the CNS? The answer may well be, as suggested in a recent NEw YORK TIPiES editorial that some agent contained within the smoke of a cigarette makes it possible for some individuals to cope with the problems of present day society. That is, for some it is the "'picker-upper" in the morning,; for others it is the pause that relaxes in the evening. The suggestion is, of course that nicotine, as an active pharmacologic action, serves to stimulate in the morning,hours andidecrease nervous tension through inhibitory aspects in the evening;. Ignoring other effects on CNS hormones, which are also significant,, it may well be that it is these aspects of smoking,, mediated through theilr effects on neurons and their receptors and transmitters, which make smoking (nicotine) the single most effective "crutch" in our particular society. Indeed, it is a truly remarkable pharmacological agent which can, at different times of day, act as both stimulator and tranquilizer. The mechanisms for this action of smoking,(nicotine) are unknown. It would be a goal of GT'R studies in the CNS to provide an understanding of how nicotine achieves its remarkable excitatory (stimulating),and inhibitory (tranquilizing) effects. D. H. Ford DHR:ek