Tetrahydroisoquinolines in the Brain: The Basis of an Animal M ode1 of Alcoholism R. D. Myers, Ph.0. Quite surprisingly, the direct introduction of THP into the brain induces a remarkable shift in voluntary alcohol intake.. . Even as much as 6 mo later, with no further infusions of THP, the remarkable voluntary selection of alcohol did not abate.
T
HE initial proposal that a condensation
product of a biogenic amine could be involved in the abnormal drinking of ethyl alcohol' was not greeted with great scientific enthusiasm.* The collective criticisms leveled at the notion at that time were in one sense logical, but nevertheless demanded some sort of a resolution. The following set of questions expresses the crucial points that have, until recently, remained in obscurity. Does the presence of a tiny amount of an amine metabolite in the brain alter the characteristics of alcohol ingestion? What is the nature of the strangely powerful, but missing, cellular link between the neurochemical mechanism underlying the addictive process and alcohol drinking? Of course, at this neonatal stage of our scientific perspective, one could not rule out any prospect concerning this disease, even an imaginative one. If one accepts the supposition, if not the fact, that the ingestion of an alcoholic beverage causes serious perturbations in the normal metabolism of biogenic amines, amino acids, cyclic nucleotides, cations, proteins, and other endogenous factors in the brain, then certain neurochemical hypotheses converging from different directions become worthy of test. A condensation product, such as a tetrahydroisoquinoline (TIQ) o r a tetrahydro-flcarboline (THB), could not only be related to an opiate, but also exert pharmacologic effects on nerve t i s ~ u e . ~Over - ~ the past 2 yr, we have investigated intensively several aspects upon which the veracity of this proposition hinges. Our findings thus far have left us impressed with the overall possibility of the intimate involvement of condensation or other metabolic by-products in the etiology of alcoholism. MATERIALS AND METHODS In conducting our experiments to test the condensation product hypothesis, we were guided at the outset by three
suppositions: First, the brain would have to be chronically exposed to the alkaloid metabolite. Second, the quantities of the metabolite would have to be present in only miniscule amounts in the brain. Third, the blood-brain barrier should be circumvented because of the possible impenetrability of the metabolite. if formed peripherally, into target sites within the central nervous system (CNS). Therefore, an experimental procedure had to be utilized that permitted us to deliver the condensation products around the clock, in only a trace amount, directly into the brain of our laboratory test animal. A standard alcohol preference test was conducted initially in which each rat was offered concentrations of alcohol that were increased in strength from 3% to 30% over 12 days.R Then, an infusion cannula was implanted stereotaxically to rest in the cerebral ventricle of each animal.' Two days later, a solution of tetrahydropapaveroline (THP), tryptoline (TLN; noreleagnine), or other metabolite was infused in 1.0 or 4.0 pl volumes every 15 or 30 min, respectively, according to standard procedures.' In one control group, artificial cerebrospinal fluid (CSF) was infused in corresponding volumes according to the same regimen; but in the other control group, T H P or TLN was infused chronically into the brain parenchyma rather than within the cerebral ventricle. In other experiments, the compounds were injected acutely into the ventricle once or twice a day. Figure I presents the structure of each compound thus far tested for its action when given chronically or acutely by the intracerebra1 route.
R ESU LTS
T/Q-lnducedAlcohol Drinking Quite surprisingly, the direct introduction of THP into the brain induces a remarkable shift in voluntary alcohol intake. As illustrated in Fig. 2, the preference for alcohol increases sharply during the actual 12-day period of chronic infusion of THP. Both the proportion of alcohol to water consumed (Fig. 2, top) as well as the actual intake in terms of gm/kg of alcohol (Fig. 2, bottom) are significantly higher than those values obtained for the control
From the Departmenrs of Psychological and Biological Sciences, Purdue University. Lafayeiie. Ind. Supported in par1 by NSF Grant BMS 75-18441 and US. Ogfce of Naval Research Contract N-ooO14-75-C-0203. Reprint requests should be addressed to R. D. Myers. Ph. D.. Departments of Psychological and Biological Sciences, Purdue University, Lafayette. lnd. 47907. 0 1978 by Grune & Stratton. Inc. 0145-6008/78/0202-00I 9$02.00/0
Alcoholism: Clinical and Experimental Research, Vol. 2. No.2 (April). 1978
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TlQs IN THE BRAIN: ANIMAL MODEL OF ALCOHOLISM
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group. A 1-mo interval elapsed, during which time the rats lived in the home cages in our colony room, and there were no intraventricular infusions of THP. When an identical retest for alcohol preference was done, once again, the patterns of excessive drinking persisted (Fig. 2). Even as much as 6 mo later, with no further infusions of THP, the remarkable voluntary selection of alcohol did not abate (Fig. 2). This result clearly indicates that the action of T H P in the rat's brain is perhaps irreversible. The induction of abnormal ingestion of alcohol far outlasts any immediate pharmacologic effect that is exerted by theTIQ.
investigation, tryptoline (noreleagnine) was infused chronically into the cerebral ventricle of a group of rats according to the same schedule and in the same range of doses as the group in which T H P had been given intraventricularly. Figure 3 demonstrates the potent effect of the minute amounts of noreleagnine, which likewise cause an augmented preference for alcohol by the rat. Again, in terms of both the proportion and gm/kg intake measures, the consumption of alcohol was significantly higher than that of the control level. In addition to the surprising duration of the effect of the condensation products, three other characteristics of these findings are noteworthy. First, during the course of a series of chronic infusions, many of the rats develop withdrawallike symptoms typical of animals made dependent on alcohol by ethanol vapor or other procedure. I z These signs include forelimb myoclonus, tail extension, hyperactivity, whisker twitching, rearing, and "wet-dog'' shakes. l x i r Second, T H P delivered directly into the brain is efficacious in picogram concentrations. The extraordinary potency of the TIQ molecule would NORELEAGNINE (pg/4.0
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p Carboline-lnduced A Icohol Drinking The P-carbolines, which are condensation products derived from the serotonin pathway,9 are also suspected of being compounds that t h e addictive might participate in phenomenon.lO However, a preliminary report indicated that a 0-car bol in e administered systemically does not necessarily affect the rat's free-choice selection of alcohol." In the present
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R. D. MYERS
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suggest that its in vivo presence in the alcoholtreated animal would be very difficult to detect.15 Third, in samples of blood collected for subsequent analysis during THP or TLN infusions, the level of alcohol was in a concentration sufficiently high to suggest that the rat was in an intoxication-like state. In fact, this result did correspond to the ataxia and incoordination often witnessed in those rats that drank unusual quantities of alcohol within a relatively short time. l6
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Since the amount of T H P and other products required to produce t h e shift in alcohol preference is so very small, we speculated that perhaps the brain need not be exposed to a given metabolite almost continuously. Therefore, a separate investigation was undertaken in which the compounds were infused acutely only once or twice a day, again in exceptionally minute doses. When either 0.1 or 1.0 pg of T H P was infused in the lateral ventricle of a rat once a day in a volume of 5.0 pl, the amount of alcohol selected by the animals was again significantly elevated.I7 Figure 4 illustrates the concomitant increases both in the proportion of alcohol consumed as well as the g/kg intakes for five rats. Of particular interest is the fact that after the third day of microinfusion, the intakes stabilized at approximately the 6.0 g/kg level even at gustatorily noxious concentrations such as 25% or 30% alcohol. Neither the higher 10.0 pg nor the lower 0.1 pg dose of THP, both infused once or twice daily, were as effective in inducing the shift in alcohol intake. A counteractive effect of a high dose of the metabolite, presumably due to some sort of a toxicologic property of the compound, could be responsible for this result. In still other experiments, T H P and tryptoline were mixed together in the same infusion solution. When they were injected in doses of 0.5 pg each in a carrier volume of 10.0 pl into the rat’s ventricle, the same sort of shift in alcohol consumption occurred. Similarly, the combination of the same doses of tryptoline and salsolinol, again infused simultaneously in the rat’s cerebral ventricle in the same solution, also evoked a substantial increase in the
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preference for and intake of the alcohol solutions offered in the range of 3%-30%. Thus, a whole group of metabolites could either synergise or mimic one another as they are taken up by receptors or other ultrastructural elements in the limbic system that helps form the walls of the cerebral ventricles.” WithdrawalSymptoms In many of the rats in which T H P or the other condensation products were infused chronically into the ventricle, or given by acute injection, signs of withdrawal became evident within the first 3 or 4 days. In some animals, stiffness of the tail, a broad-based gail, “wet-dog’’ shakes, whisker twitching, and even postural squirming were readily noted during a sequence of infusions. In several rats, convulsive episodes occurred intermittently without warning. An example of this sort of attack is shown in Fig. 5 , in which the animal had been given the alkaloid conjugate chronically according to the regimen previously described. Many of the withdrawal symptoms were observed in the daytime hours, during the interval when the rat drank little, if any, alcohol. Drinking, of course, always occurs
TlQs IN THE BRAIN: ANIMAL MODEL OF ALCOHOLISM
149
Fig. 5. Convulsive episode during withdrawal-like behavior of a rat in which 4.0 pg/4.0 pI of a TIC! was infused intraventricularly. (A.B) The tail is stiffened and raised up. (C.D) The rat is prone and undergoing a full-blown tonic and clonic seizure. Note the PE tubing line connected to the skull which carried the TIQ solution. (From Myers and Melchior. 1977.")
during the night-time period because of the rat's diurnal cycle. I M The Roleof Taste It is entirely possible that T H P and other condensation products evoke alcohol drinking simply by interfering with the animal's taste discrimination. To investigate this idea, we screened a group of 6 animals on a similar 12day fluid preference test in order to establish acceptance-rejection curves for water versus quinine. The bitter tasting drug was offered in 12 concentrations of increasing strength. Once this was done, either THP or TLN in the same efficacious dose of 1.0 pg was infused into each rat's cerebral ventricle once a day. Figure 6 illustrates unequivocally that the preference-aversion curve for quinine as tested against water was unaffected by the intracerebra1 injections of the condensation products.
Thus, these metabolites do not impair or interfere with the animal's gustatory sensibility, which would incapacitate its discrimination of a noxious fluid such as alcohol. PossibleSynaptic Action of THPand THD
In order to obtain a better understanding of the possible synaptic mechanism whereby a condensation product could exert its effect, dopaminergic-rich structures have been examined with respect to the kinetics of the monoamine's activity in the presence of a metabolite. For this type of experiment, a perfusion cannula was first implanted so as to rest just above a dopamine-containing structure, such as the caudate nucleus, nucleus accumbens, or tuberculum olfactorium. Postoperatively, the intended site of perfusion in the rat's forebrain was prelabeled with "C-dopamine
R . D. MYERS
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way of some other mechanism is presently unknown. D ISCU SSION
Quinine Concentration ( mg/100ml) Fig. 6. Proportion of quinine to total fluid intake for each concentration of quinine offered over the 1 2 successivedays before infusion (control m-m) and during daily infusions of 1.0 pg of THP (A-A) in 10.0 pl (N 6 3) or 1.0 pg of TLN (N = 3). The proportion values were combined (N 5 6) since there were no differences between groups. (From Myers and Oblinger, 1977.")
(DA) by means of the injection of a I.0-pl droplet of the nuclide. A series of push-pull
perfusions of 5-min duration at 25.0 pllmin was begun 30 min after the label was injected, with each perfusion spaced at 10-min intervals (Melchior, Myers, and Simpson, unpublished observations). Figure 7 (top) illustrates the typical wash-out of '*C-DA activity that is always seen with repeated perfusion with an artificial CSF or a solution containing a compound that is inactive when added to the push-pull perfusate. During the course of the third perfusion, (Fig. 7, top), the addition of TIQ to the perfusate had no effect on D A release. However, on another day, when T H P was included in the perfusion medium in a dose of 0.2 pgll.0 pl, the efflux of dopamine was greatly enhanced (Fig. 7, middle) by the presence of the condensation product. On still another day, the addition of tryptoline, as shown in Fig. 7 (bottom), in the same dose and perfused at the same rate within the same site, likewise stimulated the release of dopamine during the course of the third perfusion. These results therefore suggest that the presence of only a minute amount of the metabolite dose causes an intense effect on the synaptic activity of dopaminergic neurons. Of great significance is the fact that the D A release is highly localized anatomically. Whether or not these two substances actually displace the catecholamine from the synaptic vesicles in the DA nerve endings, prevent its re-uptake, or act by
Alcohol is naturally manufactured in the alimentary canal of every person, 24 hr/day, by virtue of one's intestinal flora. With respect to the etiology of alcoholism, what does an excess plasma titer of alcohol do to the body's normal metabolic machinery? The theory that a family of metabolites, formed in vivo by excessive alcohol intake serves to stimulate alcohol drinking, leaves a major question in its wake. What is the explanation for the functional differentiation between an alcoholic and a nonalcoholic individual who can cope with the beverage? In other words, why can some individuals imbibe a large amount of alcohol over some period of time, without becoming addicted? Of course, the whole answer to this perplexing riddle lies in future research; yet even now, several alternatives are suggested by the present experiments. First, if the metabolites underwent formation 1.125
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151
TlQs IN THE BRAIN: ANIMAL MODEL OF ALCOHOLISM
peripherally in the presence of alcohol, but did not penetrate the blood-brain barrier, the nonalcoholic individual would be protected against its central action. Conversely, with repeated bouts of intensive drinking, selective damage to the blood-brain barrier could be sustained. The pathologic “holes” caused by ultrastructural lesions to the barrier would permit the abnormal entry of a group of amine metabolites into a dopaminergic-rich region of the brain. If their deposition would occur in a sufficiently high concentration, the immoderate intake of alcohol could then be triggered. Second, in contrast to the alcoholic, the nonalcoholic individual simply may not form the metabolites peripherally, either in the appropriate chemical profile or in an adequate concentration. Thus, the clinical distinction could simply rest in the overall difference in substrate availability or other mechanism entailed in metabolite synthesis. Third, the alcoholic may be endowed genetically with the neurochemical machinery that favors the synthesis of just a scant amount of the metabolites in the brain itself. Thus, if THP, TLN, salsolinol, and other condensation products were produced more readily in the presence of alcohol, which does pass the blood-brain barrier, an autocatalytic process could develop. This means that increased alcohol drinking would yield an augmented synthesis of metabolites, which leads to even more alcohol drinking, and so forth. Fourth, if the metabolites reach the brain or do form in vivo in brain tissue, they may not be degraded enzymatically at a sufficient rate in the alcoholic individual. Should this transpire, .they could sequester in the cerebral parenchyma, be taken u p intra-
neuronally, o r bound to nerve cell membranes. Such an explanation would account at least partially for the long-term effects on alcohol drinking of T H P and the other products when they are introduced directly into the brain. Fifth, the presence of a family of metabolites may be a characteristic of all individuals who ingest alcohol in excess, but the efficacy of the compounds may ultimately depend on their specialized interaction with endogenous molecules, such as monoamines or amino acids or perhaps calcium ion^,'^.^'' in the alcoholic patient. CONCLUSION
In view of the present results, we now propose that a family of metabolites could be s pecifical I y involved in the n eu roc h em ical mechanism that sustains the abnormal drinking of alcoholic beverages. Should such a family of compounds be formed in vivo in picomolar quantities at certain circumscribed sites in the brain, their sequestration could lead to the powerful pharmacologic effect that we have noted in these studies. Although the specific region in which their central action could take place is unknown, dopamine-rich structures, particularly in the limbic system of the forebrain, are likely morphological candidates. What is so encouraging now, is that the alternatives cited in the Discussion above are capable of being put to the experimental test. ACKNOWLEDGMENT We thank S. Teitel of Hoffman LaRoche for kindly sup plying us.with THP and its isomer and M. Collins of Loyola University for other condensation products. I am grateful to my colleagues, Christine L. Melchoir and Monica M. Oblinger, for their splendid contributions to this research. R. A. Nattermann provided excellent technical assistance.
REFERENCES I . Davis VE, Walsh MJ: Alcohol, amines, and alkaloids: A possible biochemical basis for alcohol addiction. Science 167:1005-1007, 1970 2. Seevers MH: Morphine and ethanol physical dependence: A critique of a hypothesis. Science 170: I 1 13-1 114, 1970 3. Brezenoff HE, Cohen G: Hypothermia following intraventricular injection of a dopamine-derived tetrahydroisoquinoline alkaloid. Neuropharmacol 12:1033-l038. 1973 4. Cohen G: Alkaloid products in the metabolism of alcohol and biogenic amines. Biochem Pharmacol 2511 123-1 128, 1976
5 . Holman RB, Elliott GR, Seagraves E, et al: Tryptolines: Their potential role in the effects of ethanol, in: Satellite Symposium of the Fifth International Congress on Pharmacology. Helsinki, Finnish Foundation for Alcohol Studies, 1975 6. Myers RD: Voluntary alcohol consumption in animals: Peripheral and intracerebral factors. Psychosom Med 28:484-497, 1966 7. Myers RD: Methods for chemical stimulation of the brain, in Myers RD (ed):Methods in Psychobiology, vol 1. London, Academic, 197 I, p 247 8. Myers RD: Alcohol consumption in rats: Effects of intracranial injections of ethanol. Science 142: 240-241, 1963
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9. Dajani RM. Saheb SE: A further insight into the metabolism of certain 6-carbolines. Ann NY Acad Sci 2 15: 120- 123. 1973 10. Walsh MJ: Biogenesis of biologically active alkaloids
from amines by alcohol and acetaldehyde. Ann NY Acad Sci 215:98-110, 1973 11. Geller I, h r d y R: Alteration of ethanol preference in rats; effects of Bcarbolines, in Gross M (ed): Alcohol Intoxication and Withdrawal, vol2. New York, Plenum, 1975, p 295 12. Goldstein DB: Physical dependence on alcohol in mice. Fed Proc 34:1953-1961, 1975 13. Majchrowicz E Induction of physical dependence upon ethanol and the associated behavioral changes in rats. Psychopharmacologia (Berl) 43:245-254,1975 14. Hunter BE, Riley JN, Walker DW. et al: Ethanol dependence in the rat: A parametric analysis. Pharmacol Biochem Behav 3:619-629, 1975 15. ONeill PJ, Rahwan RG: Absence of formation of brain salsolinol in ethanol-dependent mice. J Pharmacol Exp Ther 200:306-3 13,1977
16. Myers RD, Melchior CL: Alcohol drinking: Abnormal intake caused by tetrahydropapaveroline (THP) in brain. Science 196554-556, 1977 17. Myers RD, Oblinger MM: Alcohol drinking in the rat induced by acute intracerebral infusion of two TIQs and a Bcarboline. J Drug Alcohol Depend (in press) 18. Melchior CL, Myers RD: Preference for alcohol in the rat induced by chronic infusion of tetrahydropapaveroline (THP) in the cerebral ventricle. Pharmacol Biochem Behav 7:19-35, 1977 19. Ross DH. Medina MA, Cardenas HL: Morphine and ethanol: Selective depletion of regional brain calcium. Science 186: 63-65, 1974 20. Myers RD: Brain mechanisms involved in volitional intake of ethanol in animals, in: International Symposium Biological Aspects of Alcohol Consumption. Helsinki, Finnish Foundation for Alcohol Studies, 1972 21. Myers RD, Melchior CL: Differential actions of centrally infused tetrahydroisoquinolines or a Bcarboline in evoking alcohol drinking in the rat. Pharmacol Biochem Behav 7:38 1-392. 1977
DISCUSSION
,
Edward B. Truitt, Jr., Ph. D.. Professor of Pharmacology. Northeastern Ohio Universities, College of Medicine: I have always operated on the idea that the rats know what they are doing, and if you drive them to drink you may be driving them to drink for a good reason. The thing that has interested me about Dr. Myers’ observations is the possibility that he may be producing, in a localized area of the brain, a feeling or an effect that the rat finds he can correct through alcohol. I think that certainly this should be tested with a number of indices that are already available or will be available soon. 1 was thinking particularly of determining whether the release of catecholamines that is found is accompanied by increased turnover of catecholamines in that particular area. I realize that the microtechniques for small sample analysis of turnover are still being developed, but they may w o n be widely available. The other interesting thing, of course, would be to try some Antabuse and see what happens to the animals. Dr. Myers: In response to your first comment, we have examined selected samples of perfusate by means of twodimensional thin-layer chromatography, and find that HVA and other metabolites are elevated. Thus, the metabolic profile, as reflected by both alcohol and aldehyde metabolites, following the localized perfusion with THP in a dopaminergic-rich region does suggest that the turnover of the catecholamine is somewhat enhanced. Further, there doesn’t seem to be any differential shift in the metabolism of dopamine due to the presence of excess THPor tryptoline in the push-pull perfusate. However, I don’t want to make a firm statement on this as yet, because we have not fully analyzed all the data. Second, we have begun to treat the rats that drink alcohol, as a result of THP infused intracerebrally, with an opiate antagonist as well as agonist. Although the results are extremely exciting, it is too early to comment on them. Antabuse is also on our schedule of compounds to be investigated. Richard A. Deitrich. Ph. D., Professor of Pharmacology,
University of Colorado School of Medicine: My question somewhat relates to the “reason these animals are drinking so much ethanol.” I think maybe we should follow upon Dr. Truitt’s suggestion that perhaps what we have here is a technique whereby we can induce an animal to drink. If we look a t that separately from any possible connection between THP and alcoholism, per se, because those may be two different questions, then perhaps we really do have a valuable technique. The thing that bothers me about Dr. Myers’ data, and I have discussed it with him and several people in his laboratory, is that by and large the data from the other animal studies show that when the blood acetaldehyde rises, that is. conditions under which TIQs should form most readily, the animals stop drinking. This is based on a lot of genetic work, the Antabuse work, and other inhibitors of aldehyde dehydrogenase. and shows that, as w o n as the blood acetaldehyde rises for any reason, the animals do not continue to drink. I t is a paradox if we are going to try to relate T l Q formation to alcohol. The other thing that seems strange about this is, if we use the opiate kind of model, when you give an animal the compound he is s u p posed to be addicted to, h e will not select for it. In other words, it is another paradox. If you give the animal the compound he is supposed to be making, therefore addicted to, he will not select more of that same compound or more of the precursor. So there are two paradoxes that are perhaps trying to tell us something, and what we have to do is stop and listen. Dr. Myers: Your points are excellent. They serve to underscore the inordinate complexity of this entire field generally and theTHP problem specifically. Your first point really hits home with respect to why the rat, and most of the other animals that have been properly tested, reject alcohol in a free-choice situation. Possibly the rat simply does not have the genetic material to synthesize the TIQs. In fact, one alternative that might be useful to explain our results (Myers and Melchior, Science 196554556, 1977) is that we have unwittingly bypassed the whole protective ma-
TlQs IN THE BRAIN: ANIMAL MODEL
OF ALCOHOLISM
chinery against T H P formation and simply simulated the production of the TlQs in the animal. lnterestingly enough, if the rat is not permitted to drink alcohol during the course of a series of chronic infusions ofTHP into the brain, that is, if the fluid is withheld, the rat may enter into withdrawal within 3-6 days, having never tasted a drop of alcohol. At least two or three rats have exhibited this response. Again, it appears that we may have bypassed the whole etiologic mechanism underlying the neurochemistry of withdrawal and have stimulated the CNS mechanism, whatever it is, whereby these symptoms are generated. Kenneth Blunt. Ph.D.. Department of Pharmacology and Pathology, Universitji of Texas Healrh Science Cenrer a / San Anronio: I would like to make a comment concerning the paradox mentioned by Dr. Deitrich-that is, the reason why the TlQs maintain alcohol drinking in animals s u p posedly addicted to either alcohol or the TIQ alkaloids. Dr. Deitrich is quite correct with respect to the opiate model: animals will decrease their consumption or self-selection of opiates when an opiate is administered, and this has been found even for voluntary ethanol consumption in certain animal species. However, it has also been shown that hamsters increase their consumption of ethanol if they are injected with a narcotic antagonist such as naltrexone. These opiate antagonist properties may indeed result in continued alcohol drinking. Evidence is being gathered which may shed more light with reference to this point in that TIQ derivatives may possess opiate-agonist-antagonist properties. The real concern I have is, why do animals continue to drink ethanol following ethanol infusion? Boris Tabakoff, P h . D . , Professor of Physiology. Universiry of Illinois Medical Cenrer: Most of my comments have been covered by previous discussants, but I would like, however, to consider your suggestion that particular TlQs induce a withdrawal state. If this is the case, as several laboratories have found, why do animals made physically dependent on alcohol and going through withdrawal not select alcohol? Another point which I find quite interesting, which you mentioned in passing, is the possibility of a specific receptor or receptors for TlQs. I don’t think the TlQs would be formed preferentially in any part of the dopaminergic system; that is, assuming they are formed nonenzymatically from acetaldehyde and dopamine. As long as the concentration of the two precursors is sufficient, then they should be formed anywhere. However. your point about specific receptors would be an interesting one to follow up on. Do you have any specific comments about this? Dr. Myers: Not really, except to point out that the difficulty in detecting the presence of a TIQ in the brain (e.g., ONeill and Rahwan, 1977) may be due to the limitations in the physicochemical assay. After all, if a picomolar concentration is efficacious, then endogenously, one would expect to find far less of the metabolite. Maurice Hirsr. Ph. D . , Professor of Pharmacology, Universiry of Wesrern Onrario: I have a couple of questions. One is, have you attempted to determine whether the animals treated with T H P would also prefer CNS depressants other than alcohol, in a similar drinking paradigm? Paraldehyde, for example. My second question is, have you attempted to see whether the infusion of THP would have any effect on the withdrawal from alcohol?
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Dr. Myers: Do you mean to perform the experiment the other way around? First “addict” the rat to alcohol and then attempt to modify the subsequent withdrawal with an intraventricular infusion of THP? Dr. Hirsr: Yes. Dr. Myers: To answer the first question, these experiments are beginning this summer. As far as the second question is concerned, the closest I can come to an answer is an experiment in which we gave T H P intraventricularly for I2 days at a dose of 400 pg every 30 min without any alcohol present. Then the T H P infusions were discontinued; in a sense, we set up the conditions in the CNS as if T H P had been formed for 12 days. Having never tasted alcohol before and then being offered the choice of alcohol and water, the animals immediately chose to drink alcohol. This response is very difficult for me to comprehend, because one would expect that there should be an association between T H P and ethanol in the CNS, especially in view of Dr. Tabakoff’s second comment. So the question is: why, when the brain is initially exposed to THP, does an animal drink alcohol long after clearance of T H P has presumably taken place? Murra.v G. Hamilton, M S c . , University of Western Ontario: I have a simple statement concerning the pH of the infusion solution. I f it is around 7, as is artificial cerebrospinal fluid when it is made up freshly, I suspect T H P and the other isoquinolines would not be very stable under those conditions. I know this is trueof salsolinol. Dr. Myers: We always lower the pH of the infusion medium to 3.8-4.0 with ascorbic acid, depending on the compound being infused. Further, Dr. Peter Kissinger of our Department of Chemistry, using high-pressure liquid chromatography. has verified the fact that the T H P sample that w e have used so far has not been degraded. In practice, we change the solution every 24 hr, because if not, the T H P moiety could well degrade to a protoberberine. Dr. Truitt: Perhaps what we have here is a vicious cycle. You put in a pulse of T H P which may release biogenic amines and be pleasurable at first, and then following this, that is, the metabolism of the biogenic amines, a feeling of irritability may ensue. The animals find they can achieve the same thing by drinking ethanol, producing acetaldehyde which releases catecholamines and essentially produces the same thing-an alleviation of this irritability. Essentially, by this trigger (the THP), you are setting up the parameters for this type of cycle. Ari Cohen. Ph. D.. Eagleville Hospital and Rehabilitarion Center: Just a comment concerning a previous question. What would you speculate if barbiturates or a narcotic solution rather than alcohol was utilized in the free-choice selection? Dr. Myers: Dr. Truitt’s suggestion certainly is a plausible explanation, and is worthy of experimental test. As far as the opiates are concerned, this is a very difficult problem. Because of their bitter taste, it is difficult to induce an animal to self-select an opiate alkaloid. Of course, the same problem exists with the presentation of alcohol to an animal, since its taste is noxious as well. Khavari (1972) has undertaken studies on opiate self-selection whereby the solution is adulterated and made more palatable in order that the animal might select and drink an otherwise aversive opiate solution. Although it may be our only recourse, w e hate to use this sort of adulterated solution for a number of
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reasons. One really never knows the complicated interaction between the properties of the solution. Does the animal drink the solution because of its flavor or for the narcotic effect? With alcohol, one faces the additional variable of caloric value. When one uses Metrecal" or Sustagen" or other flavoring agents, the problem is compounded even more because of their high vitamin content and other nutritional benefits. Thus, when a rat drinks a Metrecal-flavored solu-
R . D. MYERS
tion of alcohol, the drinking response has no bearing on the question of alcohol selection for its pharmacologic or addictive property. Dr. Cohen: What about the barbiturates? Dr. Myers: I have not used barbiturates as yet in a freechoice situation, again becauseof their very bitter taste, so I really don't know whether an elevated THP level in the brain would evoke the voluntary ingestion of a barbiturate.
T
HE initial proposal that a condensation
product of a biogenic amine could be involved in the abnormal drinking of ethyl alcohol' was not greeted with great scientific enthusiasm.* The collective criticisms leveled at the notion at that time were in one sense logical, but nevertheless demanded some sort of a resolution. The following set of questions expresses the crucial points that have, until recently, remained in obscurity. Does the presence of a tiny amount of an amine metabolite in the brain alter the characteristics of alcohol ingestion? What is the nature of the strangely powerful, but missing, cellular link between the neurochemical mechanism underlying the addictive process and alcohol drinking? Of course, at this neonatal stage of our scientific perspective, one could not rule out any prospect concerning this disease, even an imaginative one. If one accepts the supposition, if not the fact, that the ingestion of an alcoholic beverage causes serious perturbations in the normal metabolism of biogenic amines, amino acids, cyclic nucleotides, cations, proteins, and other endogenous factors in the brain, then certain neurochemical hypotheses converging from different directions become worthy of test. A condensation product, such as a tetrahydroisoquinoline (TIQ) o r a tetrahydro-flcarboline (THB), could not only be related to an opiate, but also exert pharmacologic effects on nerve t i s ~ u e . ~Over - ~ the past 2 yr, we have investigated intensively several aspects upon which the veracity of this proposition hinges. Our findings thus far have left us impressed with the overall possibility of the intimate involvement of condensation or other metabolic by-products in the etiology of alcoholism. MATERIALS AND METHODS In conducting our experiments to test the condensation product hypothesis, we were guided at the outset by three
suppositions: First, the brain would have to be chronically exposed to the alkaloid metabolite. Second, the quantities of the metabolite would have to be present in only miniscule amounts in the brain. Third, the blood-brain barrier should be circumvented because of the possible impenetrability of the metabolite. if formed peripherally, into target sites within the central nervous system (CNS). Therefore, an experimental procedure had to be utilized that permitted us to deliver the condensation products around the clock, in only a trace amount, directly into the brain of our laboratory test animal. A standard alcohol preference test was conducted initially in which each rat was offered concentrations of alcohol that were increased in strength from 3% to 30% over 12 days.R Then, an infusion cannula was implanted stereotaxically to rest in the cerebral ventricle of each animal.' Two days later, a solution of tetrahydropapaveroline (THP), tryptoline (TLN; noreleagnine), or other metabolite was infused in 1.0 or 4.0 pl volumes every 15 or 30 min, respectively, according to standard procedures.' In one control group, artificial cerebrospinal fluid (CSF) was infused in corresponding volumes according to the same regimen; but in the other control group, T H P or TLN was infused chronically into the brain parenchyma rather than within the cerebral ventricle. In other experiments, the compounds were injected acutely into the ventricle once or twice a day. Figure I presents the structure of each compound thus far tested for its action when given chronically or acutely by the intracerebra1 route.
R ESU LTS
T/Q-lnducedAlcohol Drinking Quite surprisingly, the direct introduction of THP into the brain induces a remarkable shift in voluntary alcohol intake. As illustrated in Fig. 2, the preference for alcohol increases sharply during the actual 12-day period of chronic infusion of THP. Both the proportion of alcohol to water consumed (Fig. 2, top) as well as the actual intake in terms of gm/kg of alcohol (Fig. 2, bottom) are significantly higher than those values obtained for the control
From the Departmenrs of Psychological and Biological Sciences, Purdue University. Lafayeiie. Ind. Supported in par1 by NSF Grant BMS 75-18441 and US. Ogfce of Naval Research Contract N-ooO14-75-C-0203. Reprint requests should be addressed to R. D. Myers. Ph. D.. Departments of Psychological and Biological Sciences, Purdue University, Lafayette. lnd. 47907. 0 1978 by Grune & Stratton. Inc. 0145-6008/78/0202-00I 9$02.00/0
Alcoholism: Clinical and Experimental Research, Vol. 2. No.2 (April). 1978
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R . D. MYERS 146
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Fig. 1. Ring structures of ten of the compounds thus far tested on alcohol preference of the rat, following their intraven. tricular infusion either acutely or chronically.
TlQs IN THE BRAIN: ANIMAL MODEL OF ALCOHOLISM
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Fig. 2. The proportion of alcohol (ETOH) to total fluid intake (top) and g alcohol/kg body weight (bottom) consumed at each concentrationof alcohol offered during the intraventricular infusion, every 30 min. of 4.0 pg of THP given in a volume of 4.0 p l , 1 mo postinfusion, and 6 mo postinfusion. (Modified from Melchior and Myers. 1977.'")
group. A 1-mo interval elapsed, during which time the rats lived in the home cages in our colony room, and there were no intraventricular infusions of THP. When an identical retest for alcohol preference was done, once again, the patterns of excessive drinking persisted (Fig. 2). Even as much as 6 mo later, with no further infusions of THP, the remarkable voluntary selection of alcohol did not abate (Fig. 2). This result clearly indicates that the action of T H P in the rat's brain is perhaps irreversible. The induction of abnormal ingestion of alcohol far outlasts any immediate pharmacologic effect that is exerted by theTIQ.
investigation, tryptoline (noreleagnine) was infused chronically into the cerebral ventricle of a group of rats according to the same schedule and in the same range of doses as the group in which T H P had been given intraventricularly. Figure 3 demonstrates the potent effect of the minute amounts of noreleagnine, which likewise cause an augmented preference for alcohol by the rat. Again, in terms of both the proportion and gm/kg intake measures, the consumption of alcohol was significantly higher than that of the control level. In addition to the surprising duration of the effect of the condensation products, three other characteristics of these findings are noteworthy. First, during the course of a series of chronic infusions, many of the rats develop withdrawallike symptoms typical of animals made dependent on alcohol by ethanol vapor or other procedure. I z These signs include forelimb myoclonus, tail extension, hyperactivity, whisker twitching, rearing, and "wet-dog'' shakes. l x i r Second, T H P delivered directly into the brain is efficacious in picogram concentrations. The extraordinary potency of the TIQ molecule would NORELEAGNINE (pg/4.0
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p Carboline-lnduced A Icohol Drinking The P-carbolines, which are condensation products derived from the serotonin pathway,9 are also suspected of being compounds that t h e addictive might participate in phenomenon.lO However, a preliminary report indicated that a 0-car bol in e administered systemically does not necessarily affect the rat's free-choice selection of alcohol." In the present
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R. D. MYERS
148
suggest that its in vivo presence in the alcoholtreated animal would be very difficult to detect.15 Third, in samples of blood collected for subsequent analysis during THP or TLN infusions, the level of alcohol was in a concentration sufficiently high to suggest that the rat was in an intoxication-like state. In fact, this result did correspond to the ataxia and incoordination often witnessed in those rats that drank unusual quantities of alcohol within a relatively short time. l6
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Since the amount of T H P and other products required to produce t h e shift in alcohol preference is so very small, we speculated that perhaps the brain need not be exposed to a given metabolite almost continuously. Therefore, a separate investigation was undertaken in which the compounds were infused acutely only once or twice a day, again in exceptionally minute doses. When either 0.1 or 1.0 pg of T H P was infused in the lateral ventricle of a rat once a day in a volume of 5.0 pl, the amount of alcohol selected by the animals was again significantly elevated.I7 Figure 4 illustrates the concomitant increases both in the proportion of alcohol consumed as well as the g/kg intakes for five rats. Of particular interest is the fact that after the third day of microinfusion, the intakes stabilized at approximately the 6.0 g/kg level even at gustatorily noxious concentrations such as 25% or 30% alcohol. Neither the higher 10.0 pg nor the lower 0.1 pg dose of THP, both infused once or twice daily, were as effective in inducing the shift in alcohol intake. A counteractive effect of a high dose of the metabolite, presumably due to some sort of a toxicologic property of the compound, could be responsible for this result. In still other experiments, T H P and tryptoline were mixed together in the same infusion solution. When they were injected in doses of 0.5 pg each in a carrier volume of 10.0 pl into the rat’s ventricle, the same sort of shift in alcohol consumption occurred. Similarly, the combination of the same doses of tryptoline and salsolinol, again infused simultaneously in the rat’s cerebral ventricle in the same solution, also evoked a substantial increase in the
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preference for and intake of the alcohol solutions offered in the range of 3%-30%. Thus, a whole group of metabolites could either synergise or mimic one another as they are taken up by receptors or other ultrastructural elements in the limbic system that helps form the walls of the cerebral ventricles.” WithdrawalSymptoms In many of the rats in which T H P or the other condensation products were infused chronically into the ventricle, or given by acute injection, signs of withdrawal became evident within the first 3 or 4 days. In some animals, stiffness of the tail, a broad-based gail, “wet-dog’’ shakes, whisker twitching, and even postural squirming were readily noted during a sequence of infusions. In several rats, convulsive episodes occurred intermittently without warning. An example of this sort of attack is shown in Fig. 5 , in which the animal had been given the alkaloid conjugate chronically according to the regimen previously described. Many of the withdrawal symptoms were observed in the daytime hours, during the interval when the rat drank little, if any, alcohol. Drinking, of course, always occurs
TlQs IN THE BRAIN: ANIMAL MODEL OF ALCOHOLISM
149
Fig. 5. Convulsive episode during withdrawal-like behavior of a rat in which 4.0 pg/4.0 pI of a TIC! was infused intraventricularly. (A.B) The tail is stiffened and raised up. (C.D) The rat is prone and undergoing a full-blown tonic and clonic seizure. Note the PE tubing line connected to the skull which carried the TIQ solution. (From Myers and Melchior. 1977.")
during the night-time period because of the rat's diurnal cycle. I M The Roleof Taste It is entirely possible that T H P and other condensation products evoke alcohol drinking simply by interfering with the animal's taste discrimination. To investigate this idea, we screened a group of 6 animals on a similar 12day fluid preference test in order to establish acceptance-rejection curves for water versus quinine. The bitter tasting drug was offered in 12 concentrations of increasing strength. Once this was done, either THP or TLN in the same efficacious dose of 1.0 pg was infused into each rat's cerebral ventricle once a day. Figure 6 illustrates unequivocally that the preference-aversion curve for quinine as tested against water was unaffected by the intracerebra1 injections of the condensation products.
Thus, these metabolites do not impair or interfere with the animal's gustatory sensibility, which would incapacitate its discrimination of a noxious fluid such as alcohol. PossibleSynaptic Action of THPand THD
In order to obtain a better understanding of the possible synaptic mechanism whereby a condensation product could exert its effect, dopaminergic-rich structures have been examined with respect to the kinetics of the monoamine's activity in the presence of a metabolite. For this type of experiment, a perfusion cannula was first implanted so as to rest just above a dopamine-containing structure, such as the caudate nucleus, nucleus accumbens, or tuberculum olfactorium. Postoperatively, the intended site of perfusion in the rat's forebrain was prelabeled with "C-dopamine
R . D. MYERS
1 50
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way of some other mechanism is presently unknown. D ISCU SSION
Quinine Concentration ( mg/100ml) Fig. 6. Proportion of quinine to total fluid intake for each concentration of quinine offered over the 1 2 successivedays before infusion (control m-m) and during daily infusions of 1.0 pg of THP (A-A) in 10.0 pl (N 6 3) or 1.0 pg of TLN (N = 3). The proportion values were combined (N 5 6) since there were no differences between groups. (From Myers and Oblinger, 1977.")
(DA) by means of the injection of a I.0-pl droplet of the nuclide. A series of push-pull
perfusions of 5-min duration at 25.0 pllmin was begun 30 min after the label was injected, with each perfusion spaced at 10-min intervals (Melchior, Myers, and Simpson, unpublished observations). Figure 7 (top) illustrates the typical wash-out of '*C-DA activity that is always seen with repeated perfusion with an artificial CSF or a solution containing a compound that is inactive when added to the push-pull perfusate. During the course of the third perfusion, (Fig. 7, top), the addition of TIQ to the perfusate had no effect on D A release. However, on another day, when T H P was included in the perfusion medium in a dose of 0.2 pgll.0 pl, the efflux of dopamine was greatly enhanced (Fig. 7, middle) by the presence of the condensation product. On still another day, the addition of tryptoline, as shown in Fig. 7 (bottom), in the same dose and perfused at the same rate within the same site, likewise stimulated the release of dopamine during the course of the third perfusion. These results therefore suggest that the presence of only a minute amount of the metabolite dose causes an intense effect on the synaptic activity of dopaminergic neurons. Of great significance is the fact that the D A release is highly localized anatomically. Whether or not these two substances actually displace the catecholamine from the synaptic vesicles in the DA nerve endings, prevent its re-uptake, or act by
Alcohol is naturally manufactured in the alimentary canal of every person, 24 hr/day, by virtue of one's intestinal flora. With respect to the etiology of alcoholism, what does an excess plasma titer of alcohol do to the body's normal metabolic machinery? The theory that a family of metabolites, formed in vivo by excessive alcohol intake serves to stimulate alcohol drinking, leaves a major question in its wake. What is the explanation for the functional differentiation between an alcoholic and a nonalcoholic individual who can cope with the beverage? In other words, why can some individuals imbibe a large amount of alcohol over some period of time, without becoming addicted? Of course, the whole answer to this perplexing riddle lies in future research; yet even now, several alternatives are suggested by the present experiments. First, if the metabolites underwent formation 1.125
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151
TlQs IN THE BRAIN: ANIMAL MODEL OF ALCOHOLISM
peripherally in the presence of alcohol, but did not penetrate the blood-brain barrier, the nonalcoholic individual would be protected against its central action. Conversely, with repeated bouts of intensive drinking, selective damage to the blood-brain barrier could be sustained. The pathologic “holes” caused by ultrastructural lesions to the barrier would permit the abnormal entry of a group of amine metabolites into a dopaminergic-rich region of the brain. If their deposition would occur in a sufficiently high concentration, the immoderate intake of alcohol could then be triggered. Second, in contrast to the alcoholic, the nonalcoholic individual simply may not form the metabolites peripherally, either in the appropriate chemical profile or in an adequate concentration. Thus, the clinical distinction could simply rest in the overall difference in substrate availability or other mechanism entailed in metabolite synthesis. Third, the alcoholic may be endowed genetically with the neurochemical machinery that favors the synthesis of just a scant amount of the metabolites in the brain itself. Thus, if THP, TLN, salsolinol, and other condensation products were produced more readily in the presence of alcohol, which does pass the blood-brain barrier, an autocatalytic process could develop. This means that increased alcohol drinking would yield an augmented synthesis of metabolites, which leads to even more alcohol drinking, and so forth. Fourth, if the metabolites reach the brain or do form in vivo in brain tissue, they may not be degraded enzymatically at a sufficient rate in the alcoholic individual. Should this transpire, .they could sequester in the cerebral parenchyma, be taken u p intra-
neuronally, o r bound to nerve cell membranes. Such an explanation would account at least partially for the long-term effects on alcohol drinking of T H P and the other products when they are introduced directly into the brain. Fifth, the presence of a family of metabolites may be a characteristic of all individuals who ingest alcohol in excess, but the efficacy of the compounds may ultimately depend on their specialized interaction with endogenous molecules, such as monoamines or amino acids or perhaps calcium ion^,'^.^'' in the alcoholic patient. CONCLUSION
In view of the present results, we now propose that a family of metabolites could be s pecifical I y involved in the n eu roc h em ical mechanism that sustains the abnormal drinking of alcoholic beverages. Should such a family of compounds be formed in vivo in picomolar quantities at certain circumscribed sites in the brain, their sequestration could lead to the powerful pharmacologic effect that we have noted in these studies. Although the specific region in which their central action could take place is unknown, dopamine-rich structures, particularly in the limbic system of the forebrain, are likely morphological candidates. What is so encouraging now, is that the alternatives cited in the Discussion above are capable of being put to the experimental test. ACKNOWLEDGMENT We thank S. Teitel of Hoffman LaRoche for kindly sup plying us.with THP and its isomer and M. Collins of Loyola University for other condensation products. I am grateful to my colleagues, Christine L. Melchoir and Monica M. Oblinger, for their splendid contributions to this research. R. A. Nattermann provided excellent technical assistance.
REFERENCES I . Davis VE, Walsh MJ: Alcohol, amines, and alkaloids: A possible biochemical basis for alcohol addiction. Science 167:1005-1007, 1970 2. Seevers MH: Morphine and ethanol physical dependence: A critique of a hypothesis. Science 170: I 1 13-1 114, 1970 3. Brezenoff HE, Cohen G: Hypothermia following intraventricular injection of a dopamine-derived tetrahydroisoquinoline alkaloid. Neuropharmacol 12:1033-l038. 1973 4. Cohen G: Alkaloid products in the metabolism of alcohol and biogenic amines. Biochem Pharmacol 2511 123-1 128, 1976
5 . Holman RB, Elliott GR, Seagraves E, et al: Tryptolines: Their potential role in the effects of ethanol, in: Satellite Symposium of the Fifth International Congress on Pharmacology. Helsinki, Finnish Foundation for Alcohol Studies, 1975 6. Myers RD: Voluntary alcohol consumption in animals: Peripheral and intracerebral factors. Psychosom Med 28:484-497, 1966 7. Myers RD: Methods for chemical stimulation of the brain, in Myers RD (ed):Methods in Psychobiology, vol 1. London, Academic, 197 I, p 247 8. Myers RD: Alcohol consumption in rats: Effects of intracranial injections of ethanol. Science 142: 240-241, 1963
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9. Dajani RM. Saheb SE: A further insight into the metabolism of certain 6-carbolines. Ann NY Acad Sci 2 15: 120- 123. 1973 10. Walsh MJ: Biogenesis of biologically active alkaloids
from amines by alcohol and acetaldehyde. Ann NY Acad Sci 215:98-110, 1973 11. Geller I, h r d y R: Alteration of ethanol preference in rats; effects of Bcarbolines, in Gross M (ed): Alcohol Intoxication and Withdrawal, vol2. New York, Plenum, 1975, p 295 12. Goldstein DB: Physical dependence on alcohol in mice. Fed Proc 34:1953-1961, 1975 13. Majchrowicz E Induction of physical dependence upon ethanol and the associated behavioral changes in rats. Psychopharmacologia (Berl) 43:245-254,1975 14. Hunter BE, Riley JN, Walker DW. et al: Ethanol dependence in the rat: A parametric analysis. Pharmacol Biochem Behav 3:619-629, 1975 15. ONeill PJ, Rahwan RG: Absence of formation of brain salsolinol in ethanol-dependent mice. J Pharmacol Exp Ther 200:306-3 13,1977
16. Myers RD, Melchior CL: Alcohol drinking: Abnormal intake caused by tetrahydropapaveroline (THP) in brain. Science 196554-556, 1977 17. Myers RD, Oblinger MM: Alcohol drinking in the rat induced by acute intracerebral infusion of two TIQs and a Bcarboline. J Drug Alcohol Depend (in press) 18. Melchior CL, Myers RD: Preference for alcohol in the rat induced by chronic infusion of tetrahydropapaveroline (THP) in the cerebral ventricle. Pharmacol Biochem Behav 7:19-35, 1977 19. Ross DH. Medina MA, Cardenas HL: Morphine and ethanol: Selective depletion of regional brain calcium. Science 186: 63-65, 1974 20. Myers RD: Brain mechanisms involved in volitional intake of ethanol in animals, in: International Symposium Biological Aspects of Alcohol Consumption. Helsinki, Finnish Foundation for Alcohol Studies, 1972 21. Myers RD, Melchior CL: Differential actions of centrally infused tetrahydroisoquinolines or a Bcarboline in evoking alcohol drinking in the rat. Pharmacol Biochem Behav 7:38 1-392. 1977
DISCUSSION
,
Edward B. Truitt, Jr., Ph. D.. Professor of Pharmacology. Northeastern Ohio Universities, College of Medicine: I have always operated on the idea that the rats know what they are doing, and if you drive them to drink you may be driving them to drink for a good reason. The thing that has interested me about Dr. Myers’ observations is the possibility that he may be producing, in a localized area of the brain, a feeling or an effect that the rat finds he can correct through alcohol. I think that certainly this should be tested with a number of indices that are already available or will be available soon. 1 was thinking particularly of determining whether the release of catecholamines that is found is accompanied by increased turnover of catecholamines in that particular area. I realize that the microtechniques for small sample analysis of turnover are still being developed, but they may w o n be widely available. The other interesting thing, of course, would be to try some Antabuse and see what happens to the animals. Dr. Myers: In response to your first comment, we have examined selected samples of perfusate by means of twodimensional thin-layer chromatography, and find that HVA and other metabolites are elevated. Thus, the metabolic profile, as reflected by both alcohol and aldehyde metabolites, following the localized perfusion with THP in a dopaminergic-rich region does suggest that the turnover of the catecholamine is somewhat enhanced. Further, there doesn’t seem to be any differential shift in the metabolism of dopamine due to the presence of excess THPor tryptoline in the push-pull perfusate. However, I don’t want to make a firm statement on this as yet, because we have not fully analyzed all the data. Second, we have begun to treat the rats that drink alcohol, as a result of THP infused intracerebrally, with an opiate antagonist as well as agonist. Although the results are extremely exciting, it is too early to comment on them. Antabuse is also on our schedule of compounds to be investigated. Richard A. Deitrich. Ph. D., Professor of Pharmacology,
University of Colorado School of Medicine: My question somewhat relates to the “reason these animals are drinking so much ethanol.” I think maybe we should follow upon Dr. Truitt’s suggestion that perhaps what we have here is a technique whereby we can induce an animal to drink. If we look a t that separately from any possible connection between THP and alcoholism, per se, because those may be two different questions, then perhaps we really do have a valuable technique. The thing that bothers me about Dr. Myers’ data, and I have discussed it with him and several people in his laboratory, is that by and large the data from the other animal studies show that when the blood acetaldehyde rises, that is. conditions under which TIQs should form most readily, the animals stop drinking. This is based on a lot of genetic work, the Antabuse work, and other inhibitors of aldehyde dehydrogenase. and shows that, as w o n as the blood acetaldehyde rises for any reason, the animals do not continue to drink. I t is a paradox if we are going to try to relate T l Q formation to alcohol. The other thing that seems strange about this is, if we use the opiate kind of model, when you give an animal the compound he is s u p posed to be addicted to, h e will not select for it. In other words, it is another paradox. If you give the animal the compound he is supposed to be making, therefore addicted to, he will not select more of that same compound or more of the precursor. So there are two paradoxes that are perhaps trying to tell us something, and what we have to do is stop and listen. Dr. Myers: Your points are excellent. They serve to underscore the inordinate complexity of this entire field generally and theTHP problem specifically. Your first point really hits home with respect to why the rat, and most of the other animals that have been properly tested, reject alcohol in a free-choice situation. Possibly the rat simply does not have the genetic material to synthesize the TIQs. In fact, one alternative that might be useful to explain our results (Myers and Melchior, Science 196554556, 1977) is that we have unwittingly bypassed the whole protective ma-
TlQs IN THE BRAIN: ANIMAL MODEL
OF ALCOHOLISM
chinery against T H P formation and simply simulated the production of the TlQs in the animal. lnterestingly enough, if the rat is not permitted to drink alcohol during the course of a series of chronic infusions ofTHP into the brain, that is, if the fluid is withheld, the rat may enter into withdrawal within 3-6 days, having never tasted a drop of alcohol. At least two or three rats have exhibited this response. Again, it appears that we may have bypassed the whole etiologic mechanism underlying the neurochemistry of withdrawal and have stimulated the CNS mechanism, whatever it is, whereby these symptoms are generated. Kenneth Blunt. Ph.D.. Department of Pharmacology and Pathology, Universitji of Texas Healrh Science Cenrer a / San Anronio: I would like to make a comment concerning the paradox mentioned by Dr. Deitrich-that is, the reason why the TlQs maintain alcohol drinking in animals s u p posedly addicted to either alcohol or the TIQ alkaloids. Dr. Deitrich is quite correct with respect to the opiate model: animals will decrease their consumption or self-selection of opiates when an opiate is administered, and this has been found even for voluntary ethanol consumption in certain animal species. However, it has also been shown that hamsters increase their consumption of ethanol if they are injected with a narcotic antagonist such as naltrexone. These opiate antagonist properties may indeed result in continued alcohol drinking. Evidence is being gathered which may shed more light with reference to this point in that TIQ derivatives may possess opiate-agonist-antagonist properties. The real concern I have is, why do animals continue to drink ethanol following ethanol infusion? Boris Tabakoff, P h . D . , Professor of Physiology. Universiry of Illinois Medical Cenrer: Most of my comments have been covered by previous discussants, but I would like, however, to consider your suggestion that particular TlQs induce a withdrawal state. If this is the case, as several laboratories have found, why do animals made physically dependent on alcohol and going through withdrawal not select alcohol? Another point which I find quite interesting, which you mentioned in passing, is the possibility of a specific receptor or receptors for TlQs. I don’t think the TlQs would be formed preferentially in any part of the dopaminergic system; that is, assuming they are formed nonenzymatically from acetaldehyde and dopamine. As long as the concentration of the two precursors is sufficient, then they should be formed anywhere. However. your point about specific receptors would be an interesting one to follow up on. Do you have any specific comments about this? Dr. Myers: Not really, except to point out that the difficulty in detecting the presence of a TIQ in the brain (e.g., ONeill and Rahwan, 1977) may be due to the limitations in the physicochemical assay. After all, if a picomolar concentration is efficacious, then endogenously, one would expect to find far less of the metabolite. Maurice Hirsr. Ph. D . , Professor of Pharmacology, Universiry of Wesrern Onrario: I have a couple of questions. One is, have you attempted to determine whether the animals treated with T H P would also prefer CNS depressants other than alcohol, in a similar drinking paradigm? Paraldehyde, for example. My second question is, have you attempted to see whether the infusion of THP would have any effect on the withdrawal from alcohol?
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Dr. Myers: Do you mean to perform the experiment the other way around? First “addict” the rat to alcohol and then attempt to modify the subsequent withdrawal with an intraventricular infusion of THP? Dr. Hirsr: Yes. Dr. Myers: To answer the first question, these experiments are beginning this summer. As far as the second question is concerned, the closest I can come to an answer is an experiment in which we gave T H P intraventricularly for I2 days at a dose of 400 pg every 30 min without any alcohol present. Then the T H P infusions were discontinued; in a sense, we set up the conditions in the CNS as if T H P had been formed for 12 days. Having never tasted alcohol before and then being offered the choice of alcohol and water, the animals immediately chose to drink alcohol. This response is very difficult for me to comprehend, because one would expect that there should be an association between T H P and ethanol in the CNS, especially in view of Dr. Tabakoff’s second comment. So the question is: why, when the brain is initially exposed to THP, does an animal drink alcohol long after clearance of T H P has presumably taken place? Murra.v G. Hamilton, M S c . , University of Western Ontario: I have a simple statement concerning the pH of the infusion solution. I f it is around 7, as is artificial cerebrospinal fluid when it is made up freshly, I suspect T H P and the other isoquinolines would not be very stable under those conditions. I know this is trueof salsolinol. Dr. Myers: We always lower the pH of the infusion medium to 3.8-4.0 with ascorbic acid, depending on the compound being infused. Further, Dr. Peter Kissinger of our Department of Chemistry, using high-pressure liquid chromatography. has verified the fact that the T H P sample that w e have used so far has not been degraded. In practice, we change the solution every 24 hr, because if not, the T H P moiety could well degrade to a protoberberine. Dr. Truitt: Perhaps what we have here is a vicious cycle. You put in a pulse of T H P which may release biogenic amines and be pleasurable at first, and then following this, that is, the metabolism of the biogenic amines, a feeling of irritability may ensue. The animals find they can achieve the same thing by drinking ethanol, producing acetaldehyde which releases catecholamines and essentially produces the same thing-an alleviation of this irritability. Essentially, by this trigger (the THP), you are setting up the parameters for this type of cycle. Ari Cohen. Ph. D.. Eagleville Hospital and Rehabilitarion Center: Just a comment concerning a previous question. What would you speculate if barbiturates or a narcotic solution rather than alcohol was utilized in the free-choice selection? Dr. Myers: Dr. Truitt’s suggestion certainly is a plausible explanation, and is worthy of experimental test. As far as the opiates are concerned, this is a very difficult problem. Because of their bitter taste, it is difficult to induce an animal to self-select an opiate alkaloid. Of course, the same problem exists with the presentation of alcohol to an animal, since its taste is noxious as well. Khavari (1972) has undertaken studies on opiate self-selection whereby the solution is adulterated and made more palatable in order that the animal might select and drink an otherwise aversive opiate solution. Although it may be our only recourse, w e hate to use this sort of adulterated solution for a number of
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reasons. One really never knows the complicated interaction between the properties of the solution. Does the animal drink the solution because of its flavor or for the narcotic effect? With alcohol, one faces the additional variable of caloric value. When one uses Metrecal" or Sustagen" or other flavoring agents, the problem is compounded even more because of their high vitamin content and other nutritional benefits. Thus, when a rat drinks a Metrecal-flavored solu-
R . D. MYERS
tion of alcohol, the drinking response has no bearing on the question of alcohol selection for its pharmacologic or addictive property. Dr. Cohen: What about the barbiturates? Dr. Myers: I have not used barbiturates as yet in a freechoice situation, again becauseof their very bitter taste, so I really don't know whether an elevated THP level in the brain would evoke the voluntary ingestion of a barbiturate.
